Varki EssentialsofGlycobiologyLibro

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Contributors
Maarten Chrispeels, Professor of Biology Member Glycobiology Research and Training Center
University of California, San Diego, La Jolla, California
Richard Cummings, Professor of Biochemistry Ed Miller Endowed Professor of Biochemistry

and Molecular Biology The University of Oklahoma, Oklahoma City, Oklahoma
Jeffrey Esko, Professor of Cellular Molecular Medicine Associate Director, Glycobiology

Research and Training Center University of California, San Diego, La Jolla, California
Hudson Freeze, Professor of Glycobiology The Burnham Institute, La Jolla, California
Gerald Hart, Director and Professor of Biological Chemistry Johns Hopkins University School
of Medicine, Baltimore, Maryland
Ole Hindsgaul, Professor of Chemistry University of Alberta, Edmonton, Canada
John Lowe, Professor of Pathology, HHMI Investigator University of Michigan, Ann Arbor,
Michigan
Adriana Manzi, Director, Analytical Research Development/Quality Control Nextran Inc., an

affiliate of Baxter Healthcare Corporation, San Diego, California
Jamey Marth, Professor of Medicine HHMI Associate Investigator Member, Glycobiology

James C. Paulson, Professor of Molecular Biology The Scripps Research Institute, La Jolla,
California
Leland Powell, Adjunct Professor of Medicine University of California, Los Angeles, California
Herman van Halbeek, Director, Glycotechnology Core Resource Member, Glycobiology

Ajit Varki, Professor of Medicine Director, Glycobiology Research and Training Center
University of California, San Diego, La Jolla, California
Dedication
To the giants who created the field of Glycobiology on whose shoulders we have tried to stand
Foreword
The ESSENTIALS OF GLYCOBIOLOGY could not appear at a more opportune time, for the field
is in a period of enormous progress and the prospects for future advances are even greater.
Glycobiology has its roots in the nineteenth century, when chemists first began to analyze sugars
and polysaccharides. Perhaps the first glycoprotein to be studied was the "glycogenous matter"
of liver which the famous French physiologist Claude Bernard identified in 1855 as a storage
form of glucose. (Interestingly, the evidence that glycogen is a glycoprotein was not obtained
until more than 100 years later.) Advances in this area continued at a steady rate during most of
this century, but the past 20 years has witnessed an unparalleled explosion of new knowledge
that has transformed the field. There are many reasons for this acceleration of progress, including
great technical advances in establishing oligosaccharide structures, but by far the most important
is the application of recombinant DNA technology to the field. This has resulted in the molecular
characterization of the enzymes involved in the assembly, processing, and degradation of
oligosaccharides and proteoglycans. It has also allowed the identification of numerous families
of plant and animal lectins that recognize carbohydrate structures. The surprising finding to
emerge is the vast number of enzymes and proteins that are devoted to glycoprotein and
glycolipid synthesis and function. The understanding of the biologic roles of glycans has also
increased to a great extent, and we now know that these molecules serve multiple functions,
ranging from assisting the folding of nascent proteins to determining the trafficking of
lymphocytes and granulocytes in the circulation. The important role of glycans is underscored by
the growing list of human diseases that are the result of defects in glycan assembly. The
challenge for the future is to further define the biologic functions played by glycans. In this
regard, recombinant DNA technology has provided another valuable tool for the glycobiologist:
the ability to disrupt genes of interest in mice and other organisms. This presents an unparalleled
opportunity for the scientist interested in elucidating the biologic roles of sugars. Without a
doubt, the future in this area is the brightest it has ever been.
Essentials of Glycobiology provides an ideal entry into the field. It contains the basic information
needed to understand this area along with the most current work at the forefront of the field. The
authors are to be commended for assembling a broad, comprehensive, well-organized overview
of this burgeoning field. They have also been successful in conveying the excitement in this area
of research.
Stuart Kornfeld
Washington University School of Medicine
March 1999
Preface
Emerging From Its Roots in classical carbohydrate chemistry and biochemistry, glycobiology
has become a vibrant, expanding, and important extension of modern molecular biology. Over
the years, many outstanding monographs and books have documented important advances in this
area and summarized critical methods and concepts (see listing following this preface). These
volumes continue to serve as excellent resources for those interested in glycobiology. Why then
should one publish an additional book on the subject? Most of these prior volumes have been
directed at the specialist, assuming a substantial level of technical sophistication and expertise
and a working knowledge of the relevant jargon. We present here a book that seeks to fulfill a
somewhat different need: to summarize the current state of the art for the expert and yet serve as
a resource for the novice wishing to explore the essentials of glycobiology.
This book had its origins from some independent lines of effort. For several years, some of us
have been teaching a short elective course in glycobiology for graduate students at the University
of California, San Diego. With the recent arrival of additional faculty with expertise in this field,
it was decided to present a more comprehensive course on the subject, to be supplemented by a
course book that could be then converted into a formal text. Meanwhile, other experts elsewhere
in the country had put forward independent proposals to fill the perceived need for a basic
textbook in glycobiology. Following a discussion over a beer after a glycobiology conference,
we decided to pool all our efforts in this direction.
Since a major goal was to produce a text that would be accessible to students and other trainees,
we used the 1998 UCSD Spring Quarter Graduate Course in Glycobiology as the basis for
creating the text. By recruiting several additional experts as lecturers, we could present a
comprehensive course that covered most aspects of the field. Each lecturer was asked to provide
handouts for the students that were essentially the first drafts of chapters for the book. In turn,
each student was required to provide anonymous critiques of some chapters as a part of the
course requirement. This approach ensured not only that the draft chapters were written early on,
but also that they underwent in-depth evaluation by bright young minds with an expressed
interest in the field. Additional rounds of internal review by the group of six editors served to
produce what we hope will be a valuable resource not only for the expert in the field, but also for
the novice who wants to learn about glycobiology. We have tried to be as accurate and up to date
as possible and to present a balanced point of view on controversial subjects. Given the current
breadth of knowledge, it was not possible to do full justice to all aspects of the field, nor to
comprehensively reference the extensive literature that exists. The relative emphasis on
vertebrate biology bespeaks the greater volume of information currently available in this area of
glycobiology.
The Editors are indebted to many others who made this book possible. Besides the students who
took the course for credit, several other trainees audited the course and provided very useful
feedback. Although the editors wrote the majority of the chapters in the book, the efforts of the
other lecturer/authors were crucial in assuring the depth of expertise needed to cover the field
effectively. Special thanks are due to John Inglis and Kaaren Janssen at Cold Spring Harbor
Laboratory Press for realizing the potential of this book, and for putting up with our many
demands and idiosyncrasies. We also thank the Press staff, Jan Argentine, Inez Sialiano, Mary
Cozza, Denise Weiss, Dotty Brown, and Danny deBruin, who deserve much credit for keeping
us on track and converting our efforts into an attractive product. Last but not least, we
acknowledge our families, lab members, and administrative assistants who supported us through
all of the hard work needed to create this text. It now remains for the reader to decide if we have
achieved our goals in producing this book.
Ajit Varki
for
The Editors
General Principles
1. Historical Background and Overview
Primary contributions to this chapter were made by A. Varki (University of California at San
Diego).
THIS CHAPTER PROVIDES HISTORICAL BACKGROUND to the emergence of the field of

glycobiology, as well as an overview of this book. General terms and definitions found
throughout the volume are also considered. The common monosaccharide units of
glycoconjugates are mentioned and a uniform symbol nomenclature used for structural
depictions throughout the book is presented. The general oligosaccharide classes to be discussed
in the book are mentioned, and an overview of the general pathways for their biosynthesis is
provided. Topological issues relevant to biosynthesis and function are also considered.
What Is Glycobiology? (1 4)
The central paradigm of modern molecular biology is that biological information flows from
DNA to RNA to protein. The power of this concept lies not only in its template-driven precision,
but also in the ability to manipulate any one class of molecules based on knowledge of another,
and in the patterns of sequence homology and relatedness that predict function and reveal
evolutionary relationships. With the upcoming completion of the genomic sequences of humans
and several other commonly studied model organisms, even more spectacular gains in the
understanding of biological systems are anticipated. However, there is often a tendency to
assume the following extension of the central paradigm:
In actual fact, creating a cell requires two other major classes of molecules: lipids and
carbohydrates. These molecules can serve as intermediates in generating energy, as signaling
molecules, or as structural components. The structural roles of carbohydrates become
particularly important in constructing complex multicellular organs and organisms, which
requires interactions of cells with one another and with the surrounding matrix. Indeed, all cells
and many macromolecules in nature carry a dense and complex array of covalently attached
sugar chains (called oligosaccharides or glycans). In some instances, these glycans can also be
free-standing entities. Since most glycans are on the outer surface of cellular and secreted
macromolecules, they are in a position to modulate or mediate a wide variety of events in cell-
cell and cell-matrix interactions crucial to the development and function of a complex
multicellular organism. They are also in a position to mediate interactions between organisms
(e.g., between host and parasite). In addition, simple, highly dynamic protein-bound glycans are
abundant in the nucleus and cytoplasm, where they appear to serve as regulatory switches. An
extended paradigm of molecular biology can thus be rendered as follows:
In the first part of this century, the chemistry, biochemistry, and biology of carbohydrates were
very prominent matters of interest. However, during the initial phase of the modern revolution in
molecular biology, studies of glycans lagged far behind those of other major classes of
molecules. This was in large part due to their inherent structural complexity, the difficulty in
easily determining their sequence, and the fact that their biosynthesis could not be directly
predicted from the DNA template. The development of a variety of new technologies for
exploring the structures of these sugar chains has opened up a new frontier of molecular biology
which has been called glycobiology. This word was first coined in 1988 by Rademacher, Parekh,
and Dwek to recognize the coming together of the traditional disciplines of carbohydrate
chemistry and biochemistry with modern understanding of the cellular and molecular biology of
glycans. The term glycobiology has gained wide acceptance, with a major biomedical journal, a
growing scientific society, and a Gordon Research Conference now bearing this name.
Defined in the broadest sense, glycobiology is then the study of the structure, biosynthesis, and
biology of saccharides (sugar chains or glycans) that are widely distributed in nature. It is one of
the more rapidly growing fields in the biomedical sciences, with relevance to basic research,
biomedicine, and biotechnology. Indeed, several biotechnology, pharmaceutical, and laboratory
supply companies have invested heavily in the area. The field ranges from the chemistry of
carbohydrates and the enzymology of glycan-modifying proteins to the functions of glycans in
complex biological systems, and their manipulation by a variety of techniques. Research in
glycobiology requires a foundation not only in the nomenclature, biosynthesis, structure,
chemical synthesis, and functions of complex glycans, but also in the general disciplines of
molecular genetics, cellular biology, physiology, and protein chemistry. This volume provides an
overview of the field of glycobiology, with a particular emphasis on the glycans of higher animal
systems, about which the greatest amount is currently known. It is assumed that the reader has a
basic background in graduate-level chemistry, biochemistry, and cell biology.
Monosaccharides Are the Basic Structural Units of Glycans (5)

Carbohydrates are defined as polyhydroxyaldehydes or polyhydroxyketones, or larger
compounds that can be hydrolyzed into such units (for examples, see below and for more details,
see Chapter 2). A monosaccharide is a carbohydrate that cannot be hydrolyzed into a simpler
unit. It has a potential carbonyl group at the end of the carbon chain (an aldehyde group) or at an
inner carbon (a ketone group). These two types of monosaccharides are therefore named aldoses
and ketoses. Free monosaccharides can exist in open chain or ring forms (Figure 1.1).
Ring forms of the monosaccharides are the rule in oligosaccharides, which are branched or linear
chains of monosaccharides attached to one another via glycosidic linkages (the term
polysaccharide is typically reserved for large glycans that are composed of repeating
oligosaccharide motifs). The ring form of a monosaccharide generates a chiral (anomeric) center
(at C-1 for aldo sugars or at C-2 for keto sugars) (for details, see Chapter 2). A glycosidic
linkage involves the attachment of a monosaccharide to another residue, typically via the
hydroxyl group of this anomeric center, which can be α linkages or β linkages depending on the
relationship of the oxygen to the anomeric carbon (see Chapter 2). It is important to realize that
these two types of linkages confer very different structural properties and biological functions
upon sequences that are otherwise identical in composition. A glycoconjugate is a compound in
which one or more monosaccharide or oligosaccharide units (the glycone) are covalently linked
to a noncarbohydrate moiety (the aglycone). An oligosaccharide that is unattached to an
aglycone usually retains the potential reducing power of the aldehyde or ketone in its terminal
monosaccharide component. This end of a sugar chain is therefore often called the reducing
terminus or reducing end (this term tends to be used even when the sugar chain is attached to an
aglycone and thus has actually lost its reducing power). Correspondingly, the outer end of the
chain tends to be called the nonreducing end (note the analogy to the 5 and 3 ends of nucleotide
chains or the amino and carboxyl termini of polypeptides).
Figure 1.1. Open chain and ring forms of galactose. Changes in the orientation of hydroxyl
groups around specific carbon atoms result in new molecules that have a distinct biology and
biochemistry (e.g., glucose is the 4-epimer of galactose).
Glycans Can Constitute a Major Portion of a Glycoconjugate (2)

In naturally occurring glycoconjugates, the portion of the molecule comprising the glycans can
vary greatly, from being very minor in amount to being the dominant component. Indeed, it is
striking that sugar chains make up a substantial portion of the mass of most glycoconjugates (for
a typical example, see Figure 1.2). For this reason, the surfaces of most types of cells (which are
heavily decorated with different kinds of glycoconjugates) are effectively covered with a dense
coating of sugars, giving rise to the so-called glycocalyx. This cell surface structure was first
observed by electron microscopists many years ago as an anionic layer external to the
plasmalemma, which could be decorated with polycationic reagents like cationized ferritin (for
an example, see Figure 1.3).
Figure 1.2. Schematic representation of the Thy-1 glycoprotein including the three N-glycans
and a glycophospholipid (GPI-glycan) anchor whose acyl chains would normally be embedded
in the membrane bilayer. Note that the polypeptide represents only a relatively small portion of
the total mass of the protein. (Modified, with permission, from [2] Rademacher et al. 1988 [©
Annual Reviews].)
Figure 1.3. Electron micrograph of endothelial cells from a blood capillary in the diaphragm
muscle of a rat, showing the lumenal plasmalemma of the cells (facing the blood) decorated with
particles of (pI 8.4) cationized ferritin (see arrowheads). These particles are binding to acidic
residues (sialic-acid-containing glycans and sulfated glycosaminoglycans) contained in the cell
surface "glycocalyx." Note that the particles are several layers deep, indicating the remarkable
thickness of this layer of glycoconjugates. (Courtesy of George E. Palade.)
Monosaccharides Generate More Linkage Variation Than

Amino Acids or Nucleotides (1, 6)
Nucleotides and proteins are linear polymers that can each have only one basic type of linkage.
In contrast, each monosaccharide can theoretically generate an α or a β linkage to any one of
several positions on another monosaccharide in a chain or to another type of molecule. Thus, it
has been pointed out that although three nucleotide bases or amino acids can only generate six
variations, three hexoses could produce (depending on which factors are considered) anywhere
from 1,056 to 27,648 unique trisaccharides. As the number of units in the polymer increases, this
difference in complexity becomes even greater. For example, a hexasaccharide with six hexoses
could have more than 1 trillion possible combinations. Thus, an almost unimaginable number of
possible saccharide units could be theoretically present in biological systems. Fortunately, for the
student of glycobiology, naturally occurring biological macromolecules contain relatively few of
the possible monosaccharide units in a limited number of combinations.
Common Monosaccharide Units of Animal Glycoconjugates (5, 7)

The common monosaccharides found in higher animal oligosaccharides are listed below, along
with their standard abbreviations (for more details regarding their structures, see Chapter 2).
• Sialic Acids: Family of nine-carbon acidic sugars (generic abbreviation is Sia), of which
the most common is N-acetyl neuraminic acid (Neu5Ac, also sometimes called NeuNAc,
NeuAc, or NANA) (for more details, see Chapter 15).
• Hexoses: Six-carbon neutral sugars, including glucose (Glc), galactose (Gal), mannose
(Man).
• Hexosamines: Hexose with an amino group at the 2-position, which can be either free or,
more commonly, N-acetylated: N-acetylglucosamine (GlcNAc) and N-
acetylgalactosamine (GalNAc).
• Deoxyhexoses: Six-carbon neutral sugar without the hydroxyl group at the 6-position,
fucose (Fuc).
• Pentoses: Five-carbon sugar, xylose (Xyl).
• Uronic Acids: Hexose with a negatively charged carboxylate at the 6-position, glucuronic
acid (GlcA) and iduronic acid (IdA).
This limited set of monosaccharides seems to dominate the glycobiology of higher

animals, but several others can be found in lower animals (e.g., tyvelose; see Chapter 36),
bacteria (e.g., keto-deoxyoctulosonic acid, rhamnose, heptose, and muramic acid; see
Chapter 21), or plants (e.g., apiose and galacturonic acid; see Chapter 20).
A variety of modifications of oligosaccharides enhance the diversity of oligosaccharides

in nature and frequently serve to mediate specific biological functions. The hydroxyl
groups of different monosaccharides can be subject to phosphorylation, sulfation,
methylation, O-acetylation, or fatty acylation. Amino groups can remain free or be N-
acetylated or N-sulfated. Carboxyl groups are occasionally subject to lactonization to
nearby hydroxyl groups.
Details regarding the structural depiction of monosaccharides, linkages, and

oligosaccharides are discussed in Chapter 2. Many figures in this book utilize a
simplified style of depiction of sugar chains (as outlined in Figure 1.4). This figure
(reproduced on the inside front cover) also indicates a uniform system of symbols that is
used in several figures through this book.
Figure 1.4. Recommended symbols and conventions for drawing glycan structures. The example
used is a typical branched "biantennary" N-glycan with two types of outer termini. This symbolic
system for representing monosaccharides is used throughout this book (the figure is also
reproduced on the inside front cover). The monosaccharides assigned these symbols are those
most commonly found in higher animal glycoconjugates. Unless otherwise indicated, all are
assumed to be in the d-configuration, except for l-Fuc and l-IdoA; all glycosidically linked
monosaccharides are assumed to be in the pyranose (p) form (six-membered ring); and all
glycosidic linkages are assumed to originate from the C1 hydroxyl group except for the sialic
acids, which are linked from the C2 hydroxyl group.
Major Classes of Glycoconjugates and Oligosaccharides (8 12)
The common classes of oligosaccharides found on eukaryotic cells are primarily defined
according to the nature of the linkage (core) regions to the aglycone (protein or lipid) (see Figure
1.5). An N-glycan (N-linked oligosaccharide, N-(Asn)-linked oligosaccharide) is a sugar chain
covalently linked to an asparagine residue of a polypeptide chain within the consensus peptide
sequence: Asn-X-Ser/Thr. N-glycans share a common pentasaccharide core region and can be
generally divided into three main classes: high-mannose-type, complex-type, and hybrid-type
(see Chapter 7). An O-glycan (O-linked oligosaccharide, O-(Ser/Thr)-linked oligosaccharide) is
typically linked to the polypeptide via N-acetylgalactosamine (GalNAc) to a serine or threonine
residue and can be extended into a variety of different structural core classes (see Chapter 8).
Other types of "O-linked oligosaccharides" do exist (e.g., O-linked mannose). However, since
the O-GalNAc linkage is the best known, it is often described by the generic term O-glycan. A
glycophospholipid anchor is a glycan bridge between phosphatidylinositol and a
phosphoethanolamine in amide linkage to the carboxyl terminus of a protein. This structure
typically constitutes the only anchor to the lipid bilayer membrane for such proteins (see Chapter
9). A glycoprotein is a glycoconjugate in which a protein carries one or more oligosaccharide
chains covalently attached to a polypeptide backbone, usually via N- or O-linkages (see above).
A proteoglycan is a glycoconjugate having one or more covalently attached glycosaminoglycan
chains (see definition below). The distinction from a glycoprotein is otherwise arbitrary, since
some polypeptides can carry both glycosaminoglycan chains and N- or O-linked chains (see
Chapter 10). A mucin is a large glycoprotein that carries many O-glycans that are often closely
spaced (clustered). A glycosphingolipid (often called glycolipid) is an oligosaccharide usually
attached via glucose or galactose to the terminal primary hydroxyl group of the lipid moiety
ceramide, which is itself composed of a long chain base (i.e., sphingosine) and a fatty acid (see
Chapter 6). Glycolipids can be neutral or anionic. A ganglioside is an anionic glycolipid
containing one or more residues of sialic acid. It should be emphasized that these represent only
the most common classes of glycans reported in eukaryotic cells. There are several other less
common types found on both sides of the cell membrane in animal cells (see Chapters 12 and
13).
Figure 1.5. Basic core structures of the common classes of animal glycans. (For the
monosaccharide symbol code used for the depictions, see Figure 1.4.)
Topological Issues Relevant to the Biosynthesis of Glycans (10,12 22)
Most well-characterized pathways for the biosynthesis of different classes of glycans occur
within the ER-Golgi-plasmalemma pathway and its other ramifications. Thus, for example,
newly synthesized proteins or lipids originating from the ER are either cotranslationally or
posttranslationally modified with sugar chains at various stages in their itinerary toward their
final destinations. Most glycosylation reactions utilize activated forms of monosaccharides
(sugar nucleotides) as donors for reactions that are catalyzed by enzymes called
glycosyltransferases (for details about their biochemistry, molecular genetics, and cell biology,
see Chapter 17). These nucleotide donors are synthesized within the cytosolic compartment from
monosaccharide precursors of endogenous or exogenous origin (see Chapter 6). To be available
to carry out glycosylation reactions within the lumen of the ER-Golgi pathway, these donors
must be actively transported across a membrane bilayer. Much effort has therefore gone into
understanding the mechanisms of glycosylation within the ER and the Golgi apparatus, and it is
clear that a variety of factors determine the final outcome of glycosylation reactions.
Some bulky sugar chains are made on the cytoplasmic face of these intracellular membranes and
flipped across to the other side, but most are added to the growing chain on the inside of the ER
or the Golgi. Regardless, whatever portion of a molecule faces the inside of the ER or Golgi will
ultimately face the inside of a secretory granule or lysosome, but it is topologically considered to
be outside of the cell. The biosynthetic enzymes (mostly glycosyltransferases) responsible for
these reactions are well studied (see Chapter 17), and their location has helped to define various
functional compartments of the ER-Golgi pathway. The commonly held model envisions these
enzymes as being physically lined up along this pathway in the precise sequence in which they
actually work. This appears to be an oversimplified view, since there is considerable overlap
among these enzymes, and the actual distribution of a given enzyme probably depends on the
cell type. The low-molecular-weight sugar nucleotides that act as donors for most of the
biosynthetic steps are made in the cytosol (see Chapter 6) and specifically transported into the
lumen of the organelles. Another consequence of this topological asymmetry is that many classes
of glycans are designed to be involved in cell-cell and cell-matrix interactions. Of course, these
topological considerations do not apply to nuclear and cytoplasmic glycosylation (see below),
since the active sites of the relevant glycosyltransferases face the cytosol.
Nuclear and Cytoplasmic Glycosylation Is Common (10)

Until the mid 1980s, a commonly stated dogma was that glycoconjugates, such as glycoprotein
and glycolipids, occur exclusively on the outer surface of cells, on the internal (luminal) surface
of intracellular organelles, and on secreted molecules. As discussed above, this fit well with
knowledge of the topology of the biosynthesis of the classes of glycans known at the time, which
took place within the lumen of the Golgi-ER pathway. Thus, despite several clues to the
contrary, the cytosol and nucleus (which are topologically semicontinuous because of the
existence of nuclear pores) were assumed to be devoid of glycosylation capacity. However, in
the last two decades, it has become clear that certain types of glycoconjugates are synthesized
and reside within the cytosol and nucleus. Indeed, one of them (O-linked GlcNAc; see Chapter
14) may well be numerically the most common type of glycoconjugate in many cells. The fact
that this major form of glycosylation was missed by so many investigators for so long
emphasizes the relatively unexplored state of the whole field of glycobiology.
Outer Structures Are Often Shared among Classes of Glycans

(14,16)
In contrast to the unique core regions of different classes of glycans, certain outer structural
sequences are often shared among different classes of glycans. For example, N- and O-linked
glycans and GSLs often carry the subterminal disaccharide Galβ1-4(3)GlcNAcβ1 (lactosamine
or LacNAc units) which can sometimes be repeated (polylactosamines, polylactosaminoglycans,
or poly-N-acetyllactosamines), or less commonly, GalNAcβ1-4GlcNAcβ1-(LacdiNAc) units.
These chains may be modified by fucosylation or branching and are typically capped by sialic
acids, fucose, α-Gal, β-GalNAc, or β-GlcA units (see Chapter 16). A glycosaminoglycan is a
linear copolymer of acidic disaccharide repeating units, each containing a hexosamine and a
hexose (Gal) or a hexuronic acid (GlcA or IdoA). These are the chains whose presence defines a
proteoglycan (see Chapter 11). The type of disaccharide unit defines the glycosaminoglycans as
chondroitin or dermatan sulfate (GalNAcβ1-4GlcA/IdoA), heparin or heparan sulfate
(GlcNAcα1-4GlcA/IdoA), or keratan sulfate (Galβ1-4GlcNAc). Keratan sulfate is actually a 6-
O-sulfated form of a polylactosamine and is therefore attached to a N- or O-glycan core rather
than a typical proteoglycan core region. One type of glycosaminoglycan, hyaluronan
(GlcNAcβ1-4GlcA)n, appears to exist primarily as a free sugar chain, unattached to any
aglycone. The glycosaminoglycans (except for hyaluronan) also typically have sulfate esters
substituting either hydroxyl or amino groups (N- or O-sulfate groups). Polysialic acid is a
homopolymer of sialic acid selectively expressed on a few mammalian proteins and on the
capsular polysaccharides of certain pathogenic bacteria.
Microheterogeneity: A Common Feature of Protein

Glycosylation (2, 4, 8, 23)
One of the most fascinating and yet frustrating aspects of protein glycosylation is the
phenomenon of microheterogeneity. This term indicates that at any given glycosylation site on a
given protein synthesized by a particular cell type, a range of variations can be found in the
precise structure of the glycan. Even the extent of this heterogeneity can vary considerably from
glycosylation site to glycosylation site, from protein to protein, and from cell type to cell type.
Thus, a given glycoprotein can exist in numerous glycoforms, each effectively being a distinct
molecular species. Mechanistically, this heterogeneity might be explained by the rapidity with
which multiple, sequential, partially competitive glycosylation reactions must take place in the
Golgi apparatus through which the newly synthesized glycoprotein is passing. An alternate
possibility is that each individual cell is in fact exquisitely specific in the details of glycosylation
which its Golgi apparatus produces, but intercellular variations result in the observed
heterogeneity of samples from natural multicellular sources.
From the practical point of view, microheterogeneity explains the anomalous behavior of
glycoproteins in various forms of chromatography (such as the diffuse bands observed on SDS-
PAGE gels) and makes the complete structural analysis of most glycoproteins a difficult task.
From a functional point of view, the meaning of this heterogeneity remains unclear. It is possible
that this is a type of "diversity generator" intended for either diversifying endogenous
recognition functions and/or for evading microbes and parasites that can bind with high
specificity to certain glycan structures.
Turnover and Degradation of Glycans (24, 25)

Like all components of living cells, glycans are constantly being created and degraded (see
Chapter 18). The latter reactions are mediated by enzymes that cleave sugar chains either at the
outer (nonreducing) terminal end (exoglycosidases) or internally (endoglycosidases). Some outer
units can also be removed and then reattached without degradation of the underlying chain. The
final complete degradation of most glycans is generally carried out by a series of glycosidases in
the lysosome. Once broken down, their individual unit monosaccharides are then typically
exported from the lysosome into the cytosol so that they can be reutilized again (see Figure 1.6).
In contrast to the relatively slow turnover of glycans derived from the ER-Golgi pathway,
glycans of the nucleus and cytoplasm may be more dynamic and rapidly turned over (see
Chapters 13 14).
Figure 1.6. Biosynthesis, utilization, and turnover of a common monosaccharide. This schematic
shows the biosynthesis, fate, and turnover of one common monosaccharide constituent of animal
glycans, galactose. Although small amounts of galactose can be taken up from the outside of the
cell, most is either synthesized de novo from glucose or recycled from degradation of
glycoconjugates in the lysosome. The schematic presents a simplified view of the generation of
the UDP sugar nucleotide, its equilibrium state with UDP-glucose, and its uptake and utilization
in the Golgi apparatus for synthesis of new glycans. (Solid lines) Biochemical pathways; (dashed
lines) pathways for the trafficking of membranes and glycans.
Tools Used to Study Glycosylation (26 36)
Unlike oligonucleotides and proteins, glycan chains are rarely expressed in a linear, unbranched
fashion, and even when they are, such chains are often subject to various modifications. Thus,
the complete sequencing of oligosaccharides is difficult to accomplish by a single method and
therefore requires iterative combinations of physical and chemical approaches that eventually
yield the details of the structure under study (for a discussion of the various forms of low- and
high-resolution separation and analysis, including mass spectrometery and NMR, see Chapter
38). Likewise, glycosylation can be perturbed in a variety of ways to explore the biology of
glycans. Other tools and methods used to study glycosylation include enzymes
(endoglycosidases and exoglycosidases), lectins (carbohydrate-binding proteins) (see Chapter
30), chemical modification or cleavage, metabolic radioactive labeling, glycosylation inhibitors
and primers (Chapter 40), antibodies, molecular cloning of glycosyltransferases (Chapter 17),
and the genetic manipulation of glycosylation in intact cells and organisms (Chapters 31 and 32).
The directed in vitro synthesis of glycans using chemical and enzymatic methods has also taken
great strides forward in recent years, providing many new tools for exploring glycobiology
(Chapter 39). The generation of complex oligosaccharide libraries by a variety of routes has
further enhanced this interface of chemistry and biology (Chapter 39).
Genetic Glycosylation Defects in Cultured Cells and Intact

Animals (12,37 40)
A variety of specific genetic defects have been defined in mutant variants of cultured cell lines
that express specific defects in glycan biosynthesis. Although there are some exceptions, defects
have been obtained at many steps of almost all of the pathways of glycan biosynthesis in
cultured animal cells. This has been of great value in elucidating the details of glycan
biosynthetic pathways (see Chapter 31). However, it also implies that the specific details of
biosynthesis of many types of glycans are not crucial to the housekeeping activities of single
cells living in the environment of the tissue culture dish. Rather, they may be more important in
mediating cell-cell and cell-matrix interactions in intact multicellular organisms and/or in
mediating interactions between organisms. In keeping with this, genetic defects of glycosylation
in intact animals have been found with relative rarity (see Chapter 32). On the other hand, such
naturally occurring mutants may simply have complex or unexpected phenotypes that may not
have lent themselves to easy elucidation with the limited diagnostic tools currently available.
Regardless, there is clearly much to be learned by generating genetic defects in intact animals
and examining the consequences. In recent years, this has become an important new frontier in
glycobiology (see Chapter 33).
Biological Roles of Glycans Appear to Be Diverse (4, 7,41 50)
A major theme of this volume is the exploration of the biological roles of glycans. As with any
biological system, the best approach carefully considers the relationship of the structure and
biosynthesis of glycans to their actual functions. As might be imagined from their ubiquitous and
complex nature, the biological roles of glycans are quite varied. Thus, all of the proposed
theories regarding glycan function appear to be partly correct, but exceptions to each can be
found. As might be expected for such a diverse group of molecules, the biological roles of
glycans span the spectrum from those that are trivial to those that are crucial for the
development, growth, function, or survival of an organism (for further discussion, see Chapter
5).
The diverse biological functions ascribed to glycans can be more simply grouped into two
general classes: (1) structural and modulatory functions involving the glycans themselves or their
modulation of molecules to which they are attached and (2) specific recognition of glycans by
lectins (carbohydrate-binding proteins). Such lectins can be either endogenous to the organism
that synthesized the glycans (e.g., see Chapters 22 27 concerning animal lectins) or exogenous
(Chapter 28 concerns microbial lectins that bind to specific glycans on host cells). In several
instances, the detailed kinetics and the atomic details of these carbohydrate-protein interactions
have been elucidated (see Chapter 4). The following is a common emerging theme: Monovalent
carbohydrate-lectin binding tends to be of relatively low affinity, and such systems typically
achieve their specificity and function by creating multivalent arrays of carbohydrate and lectin to
enhance avidity (see Chapters 30 and 40).
Glycosylation Changes in Development, Differentiation,

Malignancy, and Phylogeny (12,51 57)
Whenever a new probe (e.g., antibody or lectin) specific for a particular glycan is developed and
used to study its expression in intact organisms, it is typical to find temporal and spatial patterns
of expression in relation to cellular activation, embryonic development, organogenesis, and
differentiation (see Chapter 33). Changes in expression of glycans are also often found in the
setting of transformation and progression to malignancy (see Chapter 35). These spatially and
temporally controlled patterns of glycan expression imply the mechanistic involvement of the
glycans in many processes.
Remarkably little is known about the evolution of glycosylation. There are clearly shared and
unique features of glycosylation in different kingdoms and taxa, and among animals, an
increasing complexity is often seen in higher forms. Intra- and interspecies variations in
glycosylation are also relatively common. It has been suggested that the more specific biological
roles of oligosaccharides are often mediated by unusual glycan structures, unusual presentations
of common structures, or further modifications of the saccharides themselves. Such structures
likely result from the unique expression patterns of the relevant glycosyltransferases. However,
such glycans are also more likely to be targets for recognition by pathogenic toxins and
microorganisms. Thus, at least a portion of the diversity in oligosaccharide expression must be
related to the evolutionary selection pressures generated by interspecies interactions (e.g., host-
pathogen or host-symbiont interactions). In other words, the two different classes of glycan
recognition mentioned above are in constant competition with each other with regard to any
specific glycan-receptor interaction. Of course, the specialized glycans expressed by parasites
and microbes (see Chapters 36 and 37) are themselves presumably subject to evolutionary
selection pressures and are of great interest from the biomedical point of view. These
evolutionary issues are considered further in Chapter 3, which also considers how various glycan
biosynthetic pathways appear to have evolved and diverged in different life forms.
Glycobiology in Biotechnology and Medicine (23,58 61)
Many natural bioactive molecules are glycoconjugates, and the attached glycans can have
dramatic effects on the biosynthesis, stability, action, and turnover of these molecules in intact
organisms. For this reason alone, glycobiology and carbohydrate chemistry have become of
increasing importance in modern biotechnology. In addition, many important biological
interactions and functions mediated by glycans are potentially amenable to manipulation in vivo.
Furthermore, several human disease states are characterized by changes in glycan biosynthesis
that can be of diagnostic and/or therapeutic significance. The emerging importance of
glycobiology in biotechnology and medicine is further considered in Chapters 37 and 41.
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2. Saccharide Structure and Nomenclature
Primary contributions to this chapter were made by A.E. Manzi (Nextran Corporation, San
Diego, California) and H. van Halbeek (University of California at San Diego).
FOLLOWING AN INTRODUCTION ON GENERAL CARBOHYDRATE

NOMENCLATURE, this chapter provides an overview of the structure and chemistry of
monosaccharides, focusing on their stereochemical features. The generation of complex
carbohydrate structures from their monosaccharide constituents is described, pointing to the
importance of the glycosidic linkage, including its position and configuration (anomericity). The
final section of the chapter introduces CarbBank, a computer program that facilitates the search
of the CCSD, a database of carbohydrate sequences.
Nomenclature
Suggested as a name more than 100 years ago without knowledge of detailed structure,
carbohydrate is not an exact term. It applies to a very large number of materials and includes a
wide spectrum of chemical structures. Originally referring to those naturally occurring
substances that have a composition according to the formula (C H2O)n, the meaning of the term
carbohydrate today is far more general than "carbon hydrates" and includes any substance that
satisfies this criterion and many derived substances.
In general, carbohydrates contain a number of monosaccharides linked together as oligomers or

polymers. The latter are referred to as oligosaccharides, polysaccharides, or, more generically,
saccharides, sugar chains, or glycans. The term saccharide (derived from the Greek sakchar,
meaning sugar or sweetness) is related to the characteristic taste of many of the mono- and
disaccharides. Monosaccharides are the major, but not the only, components of glycans. The
relationship of monosaccharides to complex carbohydrates is similar to the relationship between
amino acids and proteins or between nucleotides and nucleic acids. The monosaccharide residues
within complex carbohydrates may contain noncarbohydrate moieties, such as phosphate and
sulfate groups.
The term glycoconjugate is often used to describe any macromolecule that contains a
(mono)saccharide covalently linked to another building block of nature, such as an amino acid
(peptide) or a lipid. Complex carbohydrates occur in such glycoconjugates as glycoproteins,
glycolipids, and proteoglycans. The prefix glyco- or the suffix -saccharide or -glycan in those
terms indicates the presence of monosaccharide constituents. Thus, the major classes of
biological macromolecules may be designated as nucleic acids, proteins, (complex)
carbohydrates (including glycoconjugates), and lipids.
The designation "complex" is an even less exact term than carbohydrate. A carbohydrate is often
termed complex if it contains more than one type of monosaccharide building unit. Thus, the
glucose-polymer cellulose would be a "simple" carbohydrate, whereas a galactomannan
polysaccharide is an example of a complex carbohydrate. (However, so-called simple glycans,
such as cellulose and starch, may have very complex molecular structures in three dimensions.)
In the description of glycoprotein N-glycans (see Chapter 7), complex is used more specifically
as a synonym for N-acetyllactosamine-containing chains, implying that high-mannose chains are
characterized by a simpler monosaccharide composition. Finally, the term complex
carbohydrates includes glycoconjugates, whereas the term carbohydrates per se would not.
Additional nomenclature issues will be covered in the various sections of this chapter. For more
detailed information on the topics covered in this chapter, see References 1 6.
Monosaccharides: Basic Structures and Stereoisomerism
Our understanding of carbohydrate structure has its origins in the latter part of the nineteenth
century with the pioneering studies of Emil Fischer, who was the first to establish the structure of
several of the monosaccharides. From a chemical standpoint, monosaccharides can be described
as polyhydroxy aldehydes, polyhydroxy ketones, and derivatives thereof. All simple
monosaccharides have the general empirical formula (CH2O)n, where n is an integer number
ranging from 3 to 9. Regardless of the number of carbon atoms, all monosaccharides can be
grouped into one of two general classes: aldoses or ketoses. (The -ose ending is characteristic in
carbohydrate nomenclature.) Aldoses contain a functional aldehyde group ( CH=O), whereas
ketoses contain a functional ketone group (C=O). Subclasses are then distinguished based on the
number of carbon atoms according to the following terms: aldotriose, ketotriose, aldotetrose,
ketotetrose, and so on.
Glyceraldehyde is the simplest aldose and dihydroxy acetone is the simplest ketose (Figure
2.1.a), and each can be conceived of as the parent compound of higher (CHOH) homologs in
their class. The structures of glyceraldehyde and dihydroxy acetone are also distinct in that
glyceraldehyde contains an asymmetric (chiral) carbon atom (Figure 2.1.b), whereas dihydroxy
acetone does not. With the exception of dihydroxy acetone, all monosaccharides have at least
one asymmetric center, the total number being equal to the number of internal (CHOH) groups
(n 2 for aldoses, n 3 for ketoses with n carbon atoms). The number of stereoisomers
corresponds to 2k, where k equals the number of asymmetric carbons. For example, an
aldohexose with the general formula C6H12O6 and four asymmetric carbons, i.e., four (CHOH)
groups, can exist in any one of 16 possible isomeric forms. Eight of these are d forms, and the
other eight are l forms.
With d-glyceraldehyde as the parent compound, Figure 2.2 illustrates the structures of all d-
aldoses through the aldohexose group. The numbering of the carbon atoms follows the rules of
organic chemistry nomenclature, such that the aldehyde carbon is referred to as C-1 and the
carbonyl group in ketoses is C-2. The overall configuration (d or l nature) of each sugar is
determined by the orientation of the CHOH group most distant from the aldehyde functional
group (i.e., with the highest numbered asymmetric carbon atom; this would be C-5 in hexoses,
C-4 in pentoses). The d configuration, with the OH at the aforementioned carbon atom projecting
to the right, predominates in nature. The "linear" structural representations shown in Figure 2.2
are referred to as Fischer projection formulae.
Any two sugars that differ only in the configuration around a single chiral carbon atom are called
epimers. For example, d-mannose is the C-2 epimer of d-glucose, whereas d-galactose is the C-4
epimer of d-glucose (cf. Figure 2.2). The names of monosaccharides are frequently abbreviated;
most common are three-letter abbreviations for simple monosaccharides (e.g., Gal, Glc, Man,
Xyl, Rib). Unless specified otherwise, the d configuration is implied in these abbreviated names.
Furthermore, a symbolic notation for the monosaccharides that are most abundant in vertebrate
glycoconjugates is used in this book (see Chapter 1, Figure 1.4). In solution, very few sugar
molecules exist with free aldehyde or ketone groups; rather, they exist as cyclic hemiacetals or
hemiketals, respectively. The hemiacetal linkage is formed from the condensation of an aldehyde
group and a hydroxyl group (Figure 2.3). If the reaction is intra-molecular, as it is in
monosaccharide, the resultant hemiacetal is cyclic. For reasons of chemical stability, five- and
six-membered rings are most common. Generally, aldohexoses form six-membered rings via a
C-1 O C-5 ring closure; ketohexoses form five-membered rings via a C-2 O C-5 cyclization
to yield hemiketals; aldopentoses form five-membered rings through the C-1 O C-4 linkage, or
six-membered rings through a C-1 O C-5 ring closure. Formation of a cyclic hemiacetal or
hemiketal generates an additional asymmetric center at the original carbonyl atom. The new
asymmetric center is termed the anomeric carbon. Two stereoisomers exist because the anomeric
hydroxyl group can assume either one of two possible spatial orientations. In the linear Fischer
representations, the structure with the anomeric hydroxyl group directed to the same side as the
hydroxyl group at the highest numbered asymmetric carbon atom (C-5 for hexoses) is termed the
α form and that with the opposite orientation ( OH at C-1 and C-5 going in different directions)
is termed the β form. Anomeric isomers exist for all sugars with free "reducing ends" (see
below), i.e., with a potentially free aldehyde group; in solution, these anomers are
interconvertible and exist in an equilibrium mixture (see below, Mutarotation).
From Figure 2.4, it is obvious that a Fischer projection formula of a cyclic hemiacetal is an
awkwardly looking and inaccurate representation of the ring structure. A slightly more realistic
structural representation is the Haworth projection formula in which both five-membered and
six-membered cyclic structures are depicted as planar ring systems, with the hydroxyl groups
oriented either above or below the plane of the ring (Figure 2.5). Although it does not really
represent the actual three-dimensional structure of a sugar, the Haworth representation has been
used since the late 1920s as an easy-to-draw formula that permits a quick evaluation of the
relative orientation of the OH groups in the structure. Because of the structural similarity to the
organic compounds furan and pyran, a five-membered cyclic hemiacetal is called a furanose and
a six-membered hemiacetal ring is called a pyranose. (These ring sizes may be included with the
abbreviated name of the monosaccharide, as f or p, in italics, for example, Glcp or Galf.) The
Haworth representations are preferably drawn with the ring oxygen atom in the top (for
furanose) or top right corner (for pyranose) of the structure; the numbering of the ring carbons
increases in clockwise direction.
For any d sugar, the conversion of a Fischer formula into a Haworth formula proceeds as
follows: (1) any groups (atoms) that are directed to the right in the Fischer structure are given a
downward orientation in the Haworth structure, (2) any groups (atoms) that are directed to the
left in the Fischer structure are given an upward orientation in the Haworth structure, and (3) the
terminal CH2OH group is given an upward orientation in the Haworth structure. For an l sugar,
1 and 2 are the same, but the terminal CH2OH group is projected downward. The structures of
α-d-glucopyranose and β-d-fructofuranose illustrate the conversion (Figure 2.6). Note the
shorthand form, in which only dashes are used to represent the positions of the OH groups and
all C-linked H atoms are omitted.
Figure 2.1. (a) Structures of glyceraldehyde and dihydroxy acetone in Fischer projection; (b) d-
and l-glyceraldehyde in quasi three-dimensional representation. The chiral nature of the central
carbon in glyceraldehyde gives rise to two possible configurations of the molecule, termed d and
l.
Figure 2.2. Acyclic forms of the d series of aldoses, ranging from triose to hexose.
Figure 2.3. Hemiacetal formation.
Figure 2.4. The α and β anomers (C-1 epimers) of d-glucopyranose (in cyclic hemi-acetal
structure). The dashed lines represent a distorted bond, projecting toward the rear.
Figure 2.5. Haworth representations of furanose and pyranose structures. The simplified
Haworth form with carbons omitted from the ring is routinely used. Note their apparent
similarity to the furan and pyran rings.
Figure 2.6. Conversion from Fischer to Haworth projection formula. Each hydroxyl (or
hydroxymethylene) group projected to the right in the Fischer projection points down in the
Haworth formula.
Ring Conformation of Sugars

The planar Haworth structures are distorted representations of the actual molecules. The furanose
ring is rather flexible and not entirely flat in any of its energetically favored conformations; e.g.,
it has a slight pucker when viewed from the side, as seen in the representations of the so-called
envelope and twist (or skew) conformations (Figure 2.7). The preferred conformation of a
pyranose ring is the chair conformation, similar to the structure of cyclohexane, and thus far
from flat. In the chair conformation, the OH groups exist in either axial (vertical) or equatorial
(nonvertical) positions (Figure 2.7). The conversion from Haworth projection to chair
conformation leaves the downward or upward orientation of ring substituents unaltered. Two
chair conformations can be distinguished, designated 4C1 and 1C4 or 4C1 and 1C4, respectively
(Figure 2.7). The first numeral (sometimes written as superscript) indicates the number of the
ring carbon atom above the "seat of the chair (C)," and the second numeral (subscript) indicates
the number of the ring carbon atom below the plane of the seat (spanned by C-2, C-3, C-5, and
the ring O). Chair conformations are designated from structures with the ring oxygen atom in the
top right corner of the ring "seat," resulting in the clockwise appearance of the ring numbering.
The boat conformation is energetically unfavorable for almost any pyranose.
Figure 2.7. (a) α-d-glucose in Haworth projection and in its 4C1 and 1C4 chair conformations; (b)
definitions of axial and equatorial orientations in a chair conformation (atoms C-2, C-3, C-5, and
the ring oxygen form the seat of the chair); (c) envelope and twist conformations for five-
membered ring structure.
Chemical Reactions of Monosaccharides

Because of the presence of several functional OH groups in a ring structure and the potential for
the existence of a free CH=O (aldehyde) or C=O (ketone) group, monosaccharides can undergo
reactions that are common to alcohols (especially those of polyhydroxy ring structures),
aldehydes, and ketones. The scope of our consideration is limited to only a few of these, with a
focus on reactions of biological significance.
Mutarotation
The interconversion of the α and β anomeric forms of a monosaccharide (mentioned above)

occurs as the hemiacetal ring opens and recloses, yielding the opposite anomeric configuration.
This process, which is catalyzed by dilute acid, involves the "open chain" structure with the free
aldehyde or ketone group as intermediate (see Figure 2.4).
Mutarotation refers to the rapid change of the optical rotation (denoted [α]d) of a freshly
prepared aqueous solution of a single, pure anomeric form of a monosaccharide. The α and β
forms of d-glucopyranose are stereoisomers and thus, by definition, rotate the plane of plane-
polarized light an equal amount but in opposite directions. β-d-glucopyranose shows an initial
rotation of 19° (in water). The initial [α]d of α-d-glucopyranose (+112° in water) changes upon
dissolution, until it reaches a constant value of +52.5°. Mutarotation arises from the complex
equilibria set up on dissolution of monosaccharides, as shown schematically in Figure 2.8.
Figure 2.8. Mutarotation of d-glucose. The transformations are catalyzed by mildly acidic
conditions.
The proportions of the five possible forms (α-p, β-p, α-f, β-f, and open chain) depends on the
thermodynamic stabilities of each and varies widely from sugar to sugar. Generally, the acyclic
(aldehyde) form is only present in minor amounts (<0.01%). Nevertheless, the important aspect
of this transformation is that sugars, even in the absence of catalytic amounts of H+, can exist in
the open-chain structure with a free carbonyl group. Consequently, a sugar can participate in a
chemical reaction as either the open-chain form or the cyclic form. Thus, depending on the
reaction, the sugar is depicted in the appropriate structure.
Esterification
Alcohols readily form esters when reacted with acids, anhydrides, or acyl halides. The most
important types of sugar esters that occur in nature are (1) phosphate esters (including
diphosphate esters), (2) acyl esters (with acetic acid or fatty acids), and (3) sulfate esters.
Oxidation
Three different acid derivatives of aldoses can be produced by oxidizing terminal groups to
COOH groups. Acids arising from the oxidation of the terminal CH=O group are called
glyconic acids. If the terminal CH2OH group is oxidized, a glycuronic acid is produced, and if
both terminal groups are oxidized, the product is a glycaric acid. The three acids derived from d-
glucose are shown in Figure 2.9. Note that these acids have a tendency to undergo intramolecular
esterification reactions, preferably yielding six-membered cyclic lactones. Two examples of such
lactones are shown in Figure 2.9.
Figure 2.9. Oxidized forms of d-glucose.
When being oxidized to a COOH group, the free aldehyde group formally functions as the
reducing agent. This type of oxidation reaction provides the foundation for the use of the term
reducing terminus or free reducing end. The reducing power of a saccharide is traditionally
tested by a colorimetric reaction: Reducing sugars readily reduce Fehling's solution and also
ammoniacal silver nitrate.
Reduction
Two important reduced forms of sugars are polyhydroxy alcohols (e.g., alditols, i.e., reduced
aldoses) and deoxy sugars. An example of each is shown in Figure 2.10a. Please note that an
alditol has lost the ability to form a hemiacetal, i.e., to undergo ring closure. Myo-inositol
(Figure 2.10b), the typical constituent of GPI anchors, has a cyclohexane-derived ring structure,
but is not a sugar. Figure 2.11 shows the structures of a number of "special" monosaccharides
(most of them deoxy monosaccharides) that occur in the glycoconjugates and polysaccharides to
be covered in the following chapters.
Figure 2.10. (a) Examples of an alditol and a deoxy monosaccharide. (b) Structure of myo-
inositol.
Figure 2.11. A few non-(CH2O)n building blocks of glycoconjugates.
Glycosides
When a hemiacetal (ROH) reacts with an alcohol (R OH), the product is a full acetal (ROR ). The
acetal product is called a glycoside; one distinguishes pyranosides and furanosides. In either
case, the newly formed linkage of Canomeric-OR is called a glycosidic bond. The monosaccharide
portion of the glycoside is termed the glycone, the R -group the aglycone (except when the R
group is another sugar; see below). The reaction as shown in Figure 2.12 is catalyzed by H+.
Figure 2.12. Glycoside formation: conversion of hemiacetal into acetal.
Oligosaccharides, Polysaccharides, and Glycoconjugates

When the alcohol R OH in the previous section is one of the OH groups in another
monosaccharide, the resulting ROR acetal product is a disaccharide. The glycosidic linkage
represents the covalent bond of all monosaccharide-monosaccharide interactions. The
relationship of the glycosidic bond to oligo- and polysaccharides is the same as the relationship
of the peptide bond to oligo- and polypeptides, and the phosphodiester bond to oligo- and
polynucleotides. The glycosidic linkage involves the anomeric hydroxyl group, in α or β
configuration, of one monosaccharide and any available hydroxyl group in a second
monosaccharide. The formation of α(1 4) and β(1 6) glycosidic linkages between two
glucose molecules is depicted in Figure 2.13. The symbolism identifies the OH sites (linkage
positions) that are involved in the bond. It should be noted that the reducing-end sugar in an
oligosaccharide in solution can undergo mutarotation as described for monosaccharides, the
exception being those saccharides that end in a nonreducing disaccharide such as sucrose, i.e.,
Fru-β-(2 1)-α-Glc (Figure 2.13), and trehalose, Glc-α-(1 1)-α-Glc.
Figure 2.13. Structures of disaccharides.
Two disaccharides that differ only in the position and/or configuration of their glycosidic linkage
may have significantly different conformations, which in turn account for distinctive physical
properties (compare maltose and gentiobiose in Figure 2.13). This statement is readily
extrapolated to oligo- and polysaccharides. Moreover, the glycosidic linkage is the potentially
most flexible part of a disaccharide structure. Whereas the chair conformation of the constituting
monosaccharides is relatively rigid, the torsion angles around the glycosidic bond (φ, ψ, and ω;
Figure 2.14) may vary to some extent. Thus, a disaccharide of well-defined primary structure can
adopt multiple conformations in solution, differing in the relative orientation of the two
monosaccharides with respect to each other. The combination of structural rigidity and flexibility
is typical of complex carbohydrates and, more than likely, essential to their biological functions.
Figure 2.14. Torsion angles involved in glycosidic linkages. Definition of the dihedral angles φ,
ψ and ω. (a) Newman projection along the O-1 C-1 bond with the definition of the φ angle of a
1 4 linkage. (b) Newman projection along the C-4 O-1 bond with the definition of the ψ angle
of a 1 4 linkage. (c) Newman projection along the C-6 O-1 bond with the definition of the ψ
angle of a 1 6 linkage. (d) Newman projection along the C-5 C-6 bond with the definition of
the ω angle of a 1 6 linkage.
Depending on the number of monomers linked to each other via glycosidic bonds, an
oligosaccharide is termed a disaccharide, a trisaccharide, and so on, with an upper limit of ten
residues as the arbitrary distinction from polysaccharides. Structures are commonly written from
the nonreducing end toward the reducing end; this polarity is similar to the convention to write
protein sequences from the amino to carboxyl terminus. When a monosaccharide constituent of
an oligosaccharide is involved in more than two glycosidic linkages, it serves as a branchpoint in
the structure. The ability to form branched structures (as opposed to linear sequences, as
commonly found in peptides) is an important feature of carbohydrates.
The term homopolysaccharide is used to indicate a carbohydrate polymer that is composed of

identical monosaccharide residues; e.g., cellulose and amylose (both of which are glucose
polymers) are homopolysaccharides. The presence of two or more different types of monomers
characterizes a heteropolysaccharide. Oligo- and polysaccharides can be hydrolyzed by dilute
aqueous acid to the constituent monosaccharides (see Chapter 38; Monosaccharide Composition
Analysis).
Finally, glycoconjugates are molecules that contain one or more carbohydrate groups covalently
linked to a peptide/protein, a lipid, or another biological or nonbiological molecule. The
carbohydrate group may be as small as a single monosaccharide or as large as a high-molecular-
weight polysaccharide. Often, as in typical glycoproteins, the carbohydrate moiety consists of
several oligosaccharides.
CarbBank
A given number of monosaccharides have the potential to generate, through formation of
glycosidic linkages, a large number of oligosaccharides of different primary structures. Most of
these combinations do in fact occur in nature in various classes of complex carbohydrates. To
assist the scientific community in keeping track of the primary structures of carbohydrates
elucidated from different sources, the compilation of these structures in a computer-searchable
database was begun in 1986. The occurrence of branched structures required specialized
algorithms for searching the database, totally unlike those used for linear sequences as found in
GenBank.
The complex carbohydrate structure database (CCSD) contains close to 50,000 records of
carbohydrate structures and associated text (including literature references). The database
includes more than 22,000 unique structures, derived from 14,000 different articles in the
literature. CarbBank is the computer software that allows the user to access the information in
the CCSD; the program (version 3.2; Spring 1999) runs on IBM-compatible personal computers
under Microsoft Windows 95/98 or NT. It may be obtained free of charge, e.g., from
ftp://ncbi.nlm.nih.gov/repository/carbbank. No version for Apple Macintosh computers is
available. However, the CarbBank web site
(http://www.ncbi.nlm.nih.gov/books/20http://www.ccrc.uga.edu) allows on-line searches of the
CCSD using a standard browser. Scientists are encouraged to use the web site to submit records
from their (accepted) publications.
CarbBank has an editor to create database records and a search facility that allows the user to
retrieve records based on text (author name, phrase in title of publication, trivial name), citation,
molecular weight, composition, types of residues and linkages, complete structures (or fragments
thereof), and a number of other criteria. Extensive search help is provided.
References
1. Allen H.J. and Kisailus E.C., eds. 1992. Glycoconjugates: Composition, structure, and
function. Marcel Dekker, New York.
2. Collins P.M. and Ferrier R.J. 1995. Monosaccharides Their chemistry and their roles in
natural products. Wiley, Chichester.
3. El Khadem H.S. 1988. Carbohydrate chemistry Monosaccharides and their oligomers.

Academic Press, San Diego.
4. Guthrie R.D. and Honeyman J. 1974. Introduction to carbohydrate chemistry (4th edition).
Oxford University Press, United Kingdom.
5. Morrison R.T. and Boyd R.N. 1992. Organic chemistry (6th edition). Prentice Hall,
Englewood, New Jersey.
6. Rao V.S.R., Qasba P.K., Balaji P.V., and Chandrasekaran R. 1998. Conformation of
carbohydrates . Harwood, Singapore.
3. Evolution of Glycan Diversity
Diego).
THIS CHAPTER PROVIDES A COMPARATIVE OVERVIEW of the patterns of glycosylation

in different types of organisms and discusses the complexity and diversity of glycans from an
evolutionary perspective. Because most of the available information to date concerns higher
vertebrates, much of the chapter is presented in the form of comparisons between the glycans of
vertebrates and those of other taxa. The selective forces that might determine the generation of
glycan diversity are briefly considered, including endogenous host-lectin functions and
exogenous host-pathogen interactions.
Relatively Little Is Known about Glycan Diversity in Nature (1 8)
Available data indicate that considerable diversity of glycan structure and expression exists in
nature. However, partly because of the inherent difficulties in studying glycan structure,
relatively little is known about the details of this diversity (there are very few published reviews
on this subject). For many taxa, there is essentially no information at all. Sufficient data indicate
that, unlike the case with nucleic acids and the Genetic Code, there is no universal "glycan
structure code." Indeed, the glycans expressed by free-living nonpathogenic prokaryotes (see
Chapter 21) have relatively little in common with glycans of eukaryotes, and each of these broad
groups encompasses organisms with considerable glycan diversity. On the other hand, most of
the major classes of glycans that have been identified in higher animal cells seem to be
represented in some related form among other eukaryotes.
Evolution of the N-Glycan Processing Pathway (9 18)
Perhaps the broadest base of structural information about glycans in living organisms concerns
N-glycans (see Chapter 7). Although Eubacteriae do not express N-glycans, there is evidence
that Archaebacteriae do. However, the linkage involved can be different (e.g., GalNAc-Asn and
Glc-Asn). All plants and animals studied to date seem to reproduce perfectly the early stages of
the classic N-glycan processing pathway (see Chapter 7), including the transfer of the
Glc3Man9GlcNAc2 from a dolichol-linked precursor to asparagine residues in newly synthesized
proteins (a few parasitic trypanosomes have been reported to transfer a very similar glycan
without the glucose residues). Thereafter, the trimming and extension along the classic N-glycan
processing pathway seen in vertebrates seem to be recapitulated somewhat in evolution. Yeast
and vegetative slime molds do not appear to complete the trimming of mannose residues and are
thus unable to generate typical "complex-type" N-glycans. Yeast often further specialize their
high-mannose glycans by extending them into large mannans. In contrast, developing slime
molds trim down the high-mannose form to some extent, but they do not extend them (see
Chapter 19). In insects, mannose trimming appears to be generally completed, down to a
Man3GlcNAc2 structure. The subsequent addition of GlcNAc residues is frequently followed by
removal of these residues by a very active β-hexosaminidase. Thus, the final structure in insects
often has only the three mannose residues (Figure 3.1). Prior to the removal of the GlcNAc
residues in insect cells, an α1 3-linked fucose unit is often added to the Core GlcNAc residue
(in addition to the α1 6-linked fucose typically found in vertebrate N-glycans). Controversy
exists as to whether or not further extensions with galactose and sialic acid typical of vertebrates
occur in insect cells. Plants follow a pathway similar to that in vertebrates in the initial stages,
but then often add a bisecting β1 2Xyl residue on the β-linked mannose residue (see Chapter 20
and Figure 3.1). The latter structure is also present in some invertebrates, but it appears to be
immunogenic in vertebrates. Some plants have even been reported to add outer-chain fucose
residues (Lewis X-like structures) to their outer antennae. Certain molluscs seem to prefer an
outer GalNAcβ1 4GlcNAc structure (the so-called LacDiNAc or LDN units) in place of the
typical Galβ1 4GlcNAc (LacNAc) structure more commonly seen in vertebrates.
In keeping with the above findings, the early-processing α-mannosidases of the N-glycan
pathway have a wide evolutionary distribution. In contrast, the endo-α-d-mannosidase
processing enzyme that provides an alternate deglucosylating pathway for N-glycans (see
Chapter 7) appears to be limited to members of the chordate phylum, with the single exception of
the Mollusca, where it was detected in three distinct classes. The absence of this enzyme in all
other invertebrates examined, as well as in yeast, various protozoa, and higher plants, suggests
that the need for an alternate deglucosylation route paralleled the development of complex N-
glycans in higher animals. Regarding outer terminating structures, the SO4-4-GalNAcβ1
4GlcNAc units of pituitary glycoprotein hormones have been conserved throughout vertebrate
evolution, suggesting that they are critical for biological activity. Very limited information exists
regarding the presence and distribution of most other N-glycan pathway enzymes and genes in
most animals and plants. Overall, it appears that the N-glycan pathway is evolutionarily ancient
and found throughout the eukaryotes. However, there is insufficient information to paint a clear
picture of exactly how it has been diversified and specialized during evolution.
Figure 3.1. Dominant pathways of N-glycan processing among different taxa.
Evolutionary Distribution of O-Glycans (19 21)
Homologs of the O-GalNAc:Ser/Thr transferases that initiate O-glycans in vertebrates (see

Chapter 8) have been found throughout the animal kingdom. Evidence suggests that six ancestral
isoforms of this GalNAc transferase may exist in the nematode Caenorhabditis elegans. The
classic Core 1 Galβ1 3GalNAc of vertebrates is clearly present in insects, where it also forms
part of a mucin-like protective layer in the gut. In contrast, plants do not appear to have O-linked
GalNAc. Instead, they express arabinose O-linked to hydroxyproline and Gal O-linked to serine
and threonine. Far less is known about bacterial O-glycosylation, although it is clear that novel
O-glycans can be found within bacterial "S" layers. For example, the crystalline surface layer (S
layer) glycoproteins of a Thermoanaerobacter sp. were found to contain glycan structures with a
Galβ1-OTyr core structure.
Evolutionary Distribution of Glycosphingolipids (4, 5, 8,22 24)
Glucosylceramide is found in both plants and animals. However, the classical core structures of
higher-animal glycosphingolipids, e.g., based on Galβ1 4Glc-Cer, can be varied in other
organisms (e.g., Manβ1 4Glc-Cer in certain invertebrates). Other variations are inositol-1-O-
phosphorylceramide, e.g., mannosyl-diinositolphosphorylceramide, the most abundant
sphingolipid of yeast, and GlcNAcα1 4GlcAα1 2-myo-inositol-1-O-phosphorylceramide found
in tobacco leaves. Galactosylceramides and sulfatides seem to be limited to the nervous system
of deuterostomes, whereas all protostome nerves contain mainly glucocerebrosides. An
evolutionary trend is suggested, from gluco- to galactocerebrosides, which corresponds with
changes in the nervous system from loosely structured to highly structured myelin. With regard
to the complex gangliosides of the nervous system, some general trends are seen from reptiles to
fish to mammals: an increase in sialic acid content, a decrease in the complexity of ganglioside
composition, and a decrease in "alkali-labile" molecules (bearing O-acetylated sialic acids). A
general rule has also been suggested that "the lower the environmental temperature, the more
polar is the composition of brain gangliosides." Thus, poikilothermic (cold-blooded) animals
tend to express many polysialylated gangliosides in the brain.
Diversity of Sialic Acids in Evolution (25 27)
Sialic acids are typically found at the outer termini of vertebrate N- and O-glycans as well as
glycosphingolipids. With rare exceptions that may have alternate explanations (e.g., convergent
evolution), sialic acids have only been found in animals of the deuterostome lineage (see Chapter
15). Regarding diversity, the most complex sialic acids seem to be found in invertebrates such as
echinoderms, whereas the simplest sialic acids are found in humans. Likewise, complex
substituted polysialic acids are found in echinoderms and fish, but simpler polysialic acids tend
to be found in humans. Although there is a tendency for some types of sialic acids to be
dominant in certain mammalian species (e.g., Neu5Gc in pigs and 4-O-acetylated sialic acids in
horses), careful investigation usually reveals the presence of lower quantities in other species. As
mentioned above, polysialosyl groups and sialic acid O-acetylation in gangliosides seem to be
particularly enriched in poikilothermic animals. A recent interesting finding is that humans may
be considered as "knockout" primates for the enzyme CMP-Sia hydroxylase. Thus, unlike the
closely related great apes, humans are markedly deficient in expression of N-glycolyl-
neuraminic acid (see Chapter 15).
Evolutionary Distribution of Glycosaminoglycan Chains (1, 2)

Structures typical of higher-animal heparan sulfate and chondroitin sulfate chains have been
found in many invertebrates, including insects and molluscs. The most widely distributed and
evolutionarily ancient class appears to be the chondroitin sulfate chains. The more highly
sulfated and epimerized forms of heparin and dermatan sulfate tend to be found only in higher
animal species. In the simplest multicellular animals (e.g., sponges), novel glycosaminoglycans
are also seen that have uronic acids, but these do not have the typical repeats of chondroitin
sulfate and heparan sulfate. Plants do not have typical animal glycosaminoglycans; instead, they
have acidic pectin polysaccharides, characterized by the presence of galacturonic acid (see
Chapter 20). Bacteria have completely distinct polysaccharides (Chapter 21), although certain
pathogenic bacteria can mimic mammalian GAG chains (see below).
Evolutionary Distribution of Glycophospholipid Anchors (6, 7, 28, 29)

GPI-anchored proteins sharing the core motif Manαl-4GlcNαl-6-myo-inositol-1-HPO4 lipid are
distributed ubiquitously in eukaryotes. In some species, e.g., yeast and slime molds, the lipid tail
can be a ceramide (see above). GPI anchors are generally thought not to occur in prokaryotes.
However, a recent study showed that the archaebacterium Sulfolobus sp. contained at least one
GPI-anchored protein. GPI-anchored lipids and proteins can constitute the major components of
the outer membranes of some parasitic protozoa, e.g., Leishmania.
Evolutionary Distribution of O-linked N-Acetylglucosamine (30, 31)

The O-β-GlcNAc modification of cytosolic and nuclear proteins is known to be widely
expressed in higher animals and plants. The recently cloned O-GlcNAc transferase responsible
for creating this structure has highly conserved homologs in the nematode C. elegans. No clear
homolog is evident in the yeast genome. Although the structure has been claimed to be detected
in Dictyostelium, this is actually a distinct α-linked GlcNAc. It is as yet unclear whether bacteria
can express this modification.
Molecular Mimicry of Animal Glycans by Pathogens (32 40)
From the above information, it is evident that great differences exist between the pathways
generating the glycan structures of microbes and those of mammals. Despite this, occasional
microbial surface structures are found to be strikingly similar to those of mammalian cells.
Interestingly, most examples of this type of molecular mimicry occur in pathogenic
microorganisms. This presumably adapts them to survive better in the host. Some examples are
listed in Table 3.1.
The initial hope of scientists trying to clone vertebrate glycosyltransferases was that these
represented instances of lateral gene transfer and that the microbial genes would provide a back
door approach to isolating the corresponding ones from higher organisms. However, in most
cases where genetic information has become available, convergent evolution rather than gene
transfer seems to have been the dominant mechanism involved. For example, the genes involved
in synthesizing polysialic acids in bacteria seem to have been derived from the preexisting
bacterial synthesis and transfer of keto-deoxyoctulosonate, a bacterial sugar with a structural
resemblance to sialic acids.
Interspecies and Intraspecies Differences in Glycosylation (41 52)
Does the same glycoprotein have the same type of glycosylation between different related
species? Relatively little data are available concerning this issue, but examples of both extreme
conservation and extreme diversification can be found. A reasonable explanation is that
conservation of glycan structure is only required when there are very specific functions for the
glycans. In other instances, considerable drift in the details of glycosylation might be tolerated,
as long as the underlying protein is able to carry out its primary function.
It is also clear that there can be significant variation in glycosylation between members of the
same species, particularly with regard to terminal glycan sequences. The classic example is that
of the ABO blood group system, which has persisted for tens of millions of years of primate
evolution, despite making no obvious difference to the intrinsic biology of individuals (see
Chapter 16). Another unexplained phenomenon is the complete loss of the very common
terminal Galα1 3Galβ1 4GlcNAc structure in Old World primates and humans. The fact that
both of these systems are associated with natural antibodies against the missing type of Galα1
3Gal determinant raises the possibility that such antibodies are protective, perhaps causing
complement-mediated lysis of enveloped viruses generated within other individuals who can
express the structure.
As discussed in Chapter 5, two classes of biological roles involve recognition of specific

glycans: recognition by endogenous receptors within the same organism and exogenous
recognition by other organisms, such as pathogenic or symbiotic microbes and parasites. Most
pathogenic organisms must first bind to their target cells via recognition of specific glycan
sequences (see Chapter 28). Thus, these two classes of glycan recognition must be under very
different types and rates of evolutionary selection pressure. The situation becomes more
complicated if two exogenous microbial selection pressures are competing for two different
outcomes of glycan recognition. Overall, it is likely that at least some of the intra- and
interspecies variation in glycosylation is the consequence of such ongoing host-pathogen
interactions during evolution. A more detailed discussion of these issues can be found in the
literature cited.
Why Do Widely Expressed Glycosyltransferases Have Limited

Endogenous Functions? (52 56)
Prior to the recent production of glycosyltransferase-deficient mice, it was popular to suggest
that every single glycan on every single cell type must have a critical endogenous function.
Recently available gene disruption data indicate that this may not be the case. For example, the
ST6Gal-I α2 6 sialyltransferase is the only enzyme known to produce Siaα2 6Galβ1 4GlcNAc
termini on vertebrate glycans. Although this sequence serves as a specific ligand for the B-cell
regulatory molecule CD22, it is also found on many other cell types, as well as on many soluble
molecules. Furthermore, ST6Gal-I mRNA varies markedly among cell types, and its
transcription is regulated by several cell-type-specific promoters which in turn are modulated by
hormones and cytokines. Despite all these data suggesting very diverse and complex roles for
this enzyme and its product, the functional consequences of eliminating its expression in mice
seem so far to be restricted to the B cell, with decreased signaling and proliferative responses,
and markedly impaired antibody production. No other obvious abnormalities have yet been
found in physiology, morphology, or behavior. If the specific endogenous functions of the
ST6Gal-I oligosaccharide product are in fact restricted to B cells, why bother to express it in so
many other locations? Even more puzzling, why up-regulate its expression so markedly in the
liver and endothelium during a so-called "acute phase" inflammatory response? The answers to
these questions must be explored, taking into account the evolutionary selection pressures
(endogenous and exogenous recognition) on such a terminal glycosyltransferase product.
Future Directions
There is obviously too little information available today to allow a comprehensive exposition of
the evolution of even the major classes of glycans. It is reasonable to suggest that glycan
diversification in complex multicellular organisms was driven by evolutionary selection
pressures of both endogenous and exogenous origin. Given the rapid evolution of exogenous
pathogens, it is possible that a significant portion of the overall diversity in vertebrate glycan
structure is derived from pathogen-mediated selection processes. Obviously, more gene
disruption studies in intact animals would be helpful to differentiate between these endogenous
and exogenous glycan functions. More systematic comparative glycobiology could also
contribute, by making predictions about endogenous glycan function (the consistent expression
of the same structure in the same cell type across several species would imply a critical
endogenous role). Such work might also help define the rate of oligosaccharide diversification
during evolution, better define the relative roles of the exogenous and endogenous selective
forces, and eventually lead to a better understanding of the functional significance of glycan
diversification during evolution.
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4. Protein-Glycan Interactions
Primary contributions to this chapter were made by O. Hindsgaul (University of Alberta,
Canada) and R.D. Cummings (The Oklahoma Health Sciences Center, Oklahoma).
NEARLY 100 CRYSTAL STRUCTURES of glycan-protein complexes have been solved, and
several complexes have been deduced by NMR spectroscopy. The common features of these
complexes are that the glycan-binding sites are small (spanning ~2.5 sugar residues), and
interactions with proteins involve both hydrogen bonds to hydroxyl groups and hydrophobic
interactions. Most glycans are bound in or near the minimum energy conformations observed in
solution, but there are exceptions. Understanding the role of water in complex formation remains
a major challenge. This chapter focuses on what is known about the detailed molecular
mechanisms of these interactions and on the methods of analyzing glycan-protein interactions.
Many different glycan-binding proteins have been described so far (see Chapters 22 30), and
their interactions with glycans have been studied by a wide variety of approaches.
Historical Background (1 10)
Much of the early history related to glycan-protein interactions revolved around recognition of
glycans by enzymes, such as the endoglycosidase lysozyme, which can degrade bacterial cell
walls, and enzymes involved in intermediary metabolism, such as glycogen and starch synthases
and phosphorylases. For the discovery of the antibacterial action of lysozyme and penicillin,
Fleming, Chain, and Corey received the Nobel prize in 1945. Lysozyme was subsequently
shown to be a highly specific endoglycosidase, capable of specifically cleaving β1 4 linkages in
bacterial peptidoglycan (see Chapter 21). Glycogen synthetase was found to generate α1 4
glucosyl residues in glycogen, whereas other branching and debranching enzymes recognized
α1 6-branched glucose residues. Studies on the metabolism of glycogen by these enzymes led to
award of the Nobel prize in 1970 to Luis F. Leloir for his discovery of sugar nucleotides and
their role in the biosynthesis of glycoconjugates and polysaccharides.
The concept of glycans being specifically recognized by proteins dates back to Emil Fischer,
who used the phrase "lock and key" to refer to enzymes that recognize specific glycan substrates.
The dream of specific interactions between sugars and proteins in three-dimensional space was
realized by the determination of the crystal structure of lysozyme, which was the first
"carbohydrate-binding protein" to be crystallized. Its structure was solved in complex with a
tetrasaccharide in an elegant series of studies by Phillips and coworkers in the late 1960s.
Lysozyme is an ellipsoidal protein which has a long cleft that runs for most of the length of one
surface of the protein. This cleft is astonishingly large, considering that lysozyme has only 129
amino acids, and is capable of accommodating a hexasaccharide and cleaving it into a
disaccharide product and a tetrasaccharide product. Other glycan-binding proteins whose three-
dimensional structures are of historical significance are concanavalin A (crystal structure
reported in 1972) and influenza virus hemagglutinin (crystal structure reported in 1981). In
addition, critical information to the development of this field was gathered by Lemieux and
Kabat and coworkers in studies on the combining sites of lectins and antibodies toward specific
blood group antigens. From these and many recent studies, it is clear that the specific recognition
of sugars by proteins occurs by several mechanisms. In discussing these interactions, the term
lectin is now generally used to denote proteins with glycan-binding activity (see Chapters 22
30).
Recognition of Glycans by Proteins (11 13)
In today's terminology, the lock and key concept can be rephrased as follows: "What is the origin
of the specificity seen in glycan-protein recognition?" In other words, why does a given bacterial
adhesin, toxin, plant lectin, viral hemagglutinin, antibody, or selectin bind to only a very limited
number of glycans among the thousands that are present on a cell surface? The fundamental
question at the molecular level is why does a specific glycan leave the aqueous phase to enter a
protein combining site? The forces involved in the binding of a glycan to a protein are the same
as those for the binding of any ligand to its receptor: hydrogen bonding, van der Waals
interactions, and charge and dipole attraction. So why are the dissociation constants (Kd)
normally only in the 100 µm range and so difficult to calculate or predict?
The "Water Problem" (12)

Few questions have generated such intense debate in the area of glycobiology. Unfortunately,
most attempts to understand the energetics of glycan-protein binding have focused on calculating
single contributing factors, such as determining the preferred conformation of a ligand or
estimating the short-range interactions between the glycan and functional groups in the protein-
combining site. Each such calculation is illuminating. However, as shown in Figure 4.1, the
overall process of binding involves the meeting of a solvated polyhydroxylated glycan and a
solvated protein-combining site. If a surface on the glycan is complementary to the protein-
combining site, water can be displaced and binding occurs. When the complex finally forms, it
presents a new surface to the surrounding medium which will also be hydrated.
Solvation/desolvation energies are very large due to entropy and cannot be reliably calculated for
hydrophilic compounds such as sugars. Thus, even though the energetic contributions of van der
Waals and hydrogen-bonding interactions in the combining site can be estimated, errors in the
estimation of the attendant solvation energy changes can be much larger, making the overall
calculations of binding energy difficult.
Figure 4.1. Schematic of the binding of a glycan to a protein in water, resulting in displacement
of water.
Atomic Details of Cholera Toxin Binding to the Pentasaccharide
from GM1 (13)
Many complexes of glycans bound to proteins have been solved by X-ray crystallography. A
consensus binding pattern has emerged where glycan surfaces of approximately 300 400 Å2
make contact with the protein, corresponding to about 2.5 sugar residues. The complex of
cholera toxin, a pentameric adhesion protein, bound to the GM1 pentasaccharide illustrates the
consensus binding pattern (Figure 4.2) (also see Chapter 28). Although GM1 is a pentasaccharide,
only three monosaccharides make any contact with the protein. The terminal β-linked galactose
residue is almost completely buried in the protein-combining site, making numerous hydrogen-
bonding interactions with protein functional groups, some via bound water molecules. Also very
typical of glycan-protein complexes is the "stacking" interaction of an aromatic amino acid (Trp-
88) with the hydrophobic "underside" of one of the sugar residues, in this case the terminal β-
linked galactose residue. The penultimate β-linked GalNAc residue makes only minor contacts
with the protein, whereas the Galβ1 4Glc reducing end disaccharide makes no contact at all.
However, the purpose of this disaccharide is to hold the branching sialic acid residue in the
proper orientation to make further contacts with the protein, as well as to the β-linked GalNAc
residue. The net result is a very tight complex with a Kd value near 0.1 µm, one of the tightest
known for monovalent glycan-protein binding.
Figure 4.2. Simplified structure of pentasaccharide GM1 binding to cholera toxin. The amino acid
residues in cholera toxin that contact the glycan directly or through H bonds with water are
indicated.
Kinetics of Glycan-Protein Interactions

In addition to defining the three-dimensional structure of a glycan-binding protein, it is important
to also define its specific interactions with a variety of glycans. The binding of a lectin (L) or an
antibody to a glycan (G) is governed by the simple Equation 4.1. The affinity constant, K, is
defined as an association constant (or Ka) by Equation 4.2; thus, K is also equal to k1/k2. Like any
equilibrium constant, K is related to the standard free-energy change of the binding reaction at
pH 7 (∆Go) in kcal per mole, as shown by Equation 4.3, where R is the gas constant (0.00198
kcal/mol-degree) and T is the absolute temperature (298°K).
The affinity constant K is related to thermodynamic parameters by Equation 4.4, where ∆G, ∆H,
and ∆S represent the changes in free energy, enthalpy, and entropy of binding, respectively.
Although it is obviously important to define K, k1, k2, ∆G, ∆H, and ∆S for each binding
phenomenon under consideration, many times investigators simply try to define K. Data are
usually discussed in terms of the Kd (1/Ka) and terms of mm, µm, or nm are used. There are
many different ways to study binding of glycans to proteins, and each approach has its
advantages and disadvantages in terms of thermodynamic rigor, amounts of protein and glycan
needed, and speed of analyses. A discussion follows of some of the major ways in which the
binding between a glycan and protein is studied.
Since the affinity of single glycan-protein interactions is generally low, many naturally occurring
animal lectins bind in a multivalent fashion. Multivalency raises questions of affinity versus
avidity. Binding of a monovalent lectin to a monovalent ligand is easily defined by the
equilibrium kinetics described in Equations 4.1 and 4.2. However, with multivalent ligands or
lectins, multiple affinities occur and a more complex binding equilibrium, more accurately
described by a set of equilibrium constants, must be used. The term avidity refers to the strength
of multivalent ligand binding and obviously has complex kinetics. Typically, for multivalent
ligands and lectins, the values reported for affinity are apparent affinity constants and usually
measure the avidity. For divalent antibodies, where the situation has been more carefully
analyzed, the avidity for antigen is typically 10 100 times that of the monovalent antibody. A
similar situation may be true for most multivalent lectins. Another complication in studying
some lectins is their membrane association. In the pure form, they probably occur as oligomers
in detergent solutions. Another complication is that ligands for some lectins may be
glycoproteins (e.g., mucins) or polysaccharides (e.g., heparin) with potentially multiple binding
sites for the lectin. The density of binding sites on the ligand and the valency of the lectin may
dramatically alter the apparent affinity of binding. All of these issues should be taken into
account when studying the interactions of a lectin with glycans.
To understand the interactions between a glycan and a protein, it is important to define many
parameters, including the precise molecular interactions and interatomic distances within a
complex of glycan and protein, the equilibrium binding constant, the on and off rates of the
interaction, and the entropy and enthalpy of the interaction. Such issues are usually related to a
free glycan or small glycoconjugate interacting with a protein. However, in real biological
situations, a glycan-binding protein may be interacting with a glycolipid or a glycoprotein that
may be present at high density in the plane of the membrane, a polysaccharide with multiple
repeating determinants, or a glycoprotein with many clustered determinants. Because of this
complexity, most of our information about glycan-protein interactions derive from studies on
relatively small glycan ligands interacting with a protein. In examining these interactions, there
are three broad categories of approaches: biophysical methods, such as X-ray diffraction and
NMR; kinetic methods, such as equilibrium dialysis and titration calorimetry; and indirect
methods, such as hapten inhibition and ELISA (enzyme-linked immunosorbent assay)-based
approaches.
Biophysical Methods: X-ray and NMR (14 18)
Two biophysical approaches for examining protein-glycan interactions at the molecular level are
X-ray crystallography and nuclear magnetic resonance. Crystal complexes with glycans have
been defined for a number of antibodies, plant and animal lectins, bacterial toxins, and enzymes
that utilize carbohydrate. Approximately 100 three-dimensional structures of carbohydrate-
binding proteins are listed in the Brookhaven Protein Data Bank. In addition, more than 1000
three-dimensional structures of mono- and oligosaccharides are stored in the Cambridge
Structural Database. A resolution of at least 2 2.5 Å is required to accurately identify the
positions and mode of binding of glycans. Such resolution is often difficult to obtain. In some
cases, a crystal structure can be obtained for a glycan-binding protein independent of ligand, and
its structure and potential binding site can be predicted using information from the three-
dimensional structure of a homologous protein.
For the determination of the structure of a biomolecule in solution, NMR is the preferred
technique. In general, monosaccharides are small and relatively rigid molecules, but an
assembled glycan has a higher degree of flexibility, due to the general freedom of rotation about
glycosidic bonds. In NMR, the proton-proton distances can be obtained following assignment of
the proton resonances, through multidimensional techniques, such as the nuclear Overhauser
effect (NOE). This information coupled with computational methods employing computer
modeling allows the prediction of the free-state glycan conformation in solution. It is possible to
extend this information to examine the interaction of a glycan with a protein in solution, using
transfer NOE (TRNOE). This approach is based on the premise that the tumbling and rotation of
a glycan in complex with a protein are much slower than that of free unbound glycans. In
TRNOE, a small protein (<30 kD) is dissolved with a large excess of glycan that has been
equilibrated with 2D2O, and the resonances of the bound glycan can then be resolved
independently from the free glycan. Available computer-assisted modeling strategies and
information from glycan solution conformations and protein three-dimensional structures can be
combined with NMR to provide even more information about the nature of the molecular details
of the interactions between glycans and proteins. Although these approaches are highly
informative, they are limited by the degree to which small glycans structurally mimic the larger
macromolecule to which they are usually attached and by the problem that many of these
interactions are often performed under nonequilibrium conditions.
Equilibrium Dialysis (19 23)
In equilibrium dialysis, a concentrated solution of a glycan-binding protein (e.g., lectin and

antibody) is placed in a dialysis chamber that is permeable to a ligand (glycan or other small
hapten). The chamber is then placed in equilibrium with a known volume of buffer that contains
the glycan in the concentration range of 1/K. At equilibrium, the concentration of bound plus free
glycan inside the bag [In] and free glycan outside the bag [Out] will depend on the concentration
and affinity of the protein inside the bag. From this information, both the association constant K
and the lectin valence n can be determined from the relationship shown in Equation 4.5, where r
is the ratio of mole of glycan bound per mole of lectin and c is the concentration of unbound
glycan [Out]. The mole of glycan bound is determined by simply subtracting [Out] from [In].
A plot of r/c versus r for different hapten concentrations (Scatchard analysis) will approximate a
straight line with a slope of -K. The association constant K can be determined as the negative of
the slope, and the valence of binding is defined by the r intercept at an infinite hapten
concentration.
A number of important assumptions are made in these experiments and their validity must be
demonstrated. These include demonstrating that the protein and its hapten are stable and active
over the course of the experiment, the hapten is freely diffusible, the complex is at equilibrium,
and structurally unrelated haptens, not expected to bind, show no apparent binding in the
experimental setup. The following are several advantages to equilibrium dialysis: (1) The
approach is not complicated and sophisticated equipment is not necessary; (2) if the affinity is
high, then relatively small amounts of protein are needed ( 1 µmole); (3) if the affinity is high
enough, small amounts of hapten may be required; (4) if the protein and haptens are very stable,
they may be recovered and reused; (5) radioactive haptens may be used; and (6) reliable
equilibrium measurements can be made. The following are some drawbacks of the approach: (1)
It can only provide K; (2) if the affinity of the lectin or antibody for the hapten is low, then
relatively large amounts of both may be required; and (3) many different measurements must be
made and this may require many days or weeks to complete for a single experiment.
A variation of this equilibrium technique is illustrated by equilibrium gel filtration developed by

Hummel and Dreyer. In the Hummel-Dreyer method, a protein (e.g., lectin) is applied to a gel-
filtration column that is preequilibrated with a ligand (e.g., glycan) of interest that is easily
detectable, e.g., by radioactive or fluorescent tagging. As the protein binds to ligand, a complex
is formed that emerges from the column as a "peak" above the baseline of ligand alone, followed
by a "trough," where the concentration of ligand is decreased below the baseline, which extends
to the included or salt volume of the column. The amount of complex formed is easily
determined by the known specific activity of the ligand. Because the amount of complex formed
is directly proportional to the amount of protein (or ligand) applied, it is easy to calculate a
binding curve from several different equilibrium gel filtration column profiles at different
concentrations of either protein or ligand. This binding curve allows the calculation of the
equilibrium constant of the interaction. The advantages and drawbacks of this technique are
generally the same as those for equilibrium dialysis, except that Hummel-Dreyer analyses are
often quicker to perform and can be used with ligands of many different sizes. Although this
approach has not been used historically for lectin-glycan interactions, recent studies suggest that
it is extremely useful in defining the interactions of soluble selectins with small ligands.
Affinity Chromatography (24 27)
In this approach, a lectin is immobilized to an affinity support, such as Affi-GelTM, cyanogen-

bromide-activated Sepharose, UltraLinkTM, or some other activated support. The immobilized
lectin may be placed in a variety of column formats, ranging from old-fashioned gravity flow
columns to HPLC. In the simplest approach, a lectin or other glycan-binding protein is
immobilized to a support in a concentration range of 0.1 to 10 mg/ml. A buffer containing a
glycan is added to the immobilized lectin, and binding to the lectin is measured, by virtue of its
elution profile. If the glycan binds to the immobilized lectin relatively tightly, buffer containing a
known hapten may be added to force dissociation of the complex. For example, high-mannose-
type and hybrid-type N-glycans will bind avidly to a column of ConA-agarose and 500 mmα-
methyl-Man will be required to elute the bound material efficiently (see Chapter 30). In contrast,
many highly branched complex-type N-glycans will not bind. Biantennary complex-type N-
glycans bind to ConA-agarose, but their elution can be effected by using 10 mmα-methyl-Man.
In this manner, the binding specificity and approximate binding affinity of the lectin can be
assessed, since the concentration of immobilized lectin is known and its capacity can also be
defined. This approach is rather crude, and although it gives valuable practical information about
the use of an immobilized lectin to bind specific glycans, it does not provide detailed affinity
measurements. A variant of this method is to immobilize the glycan ligand and measure lectin
binding. However, this is especially difficult to do with multiple ligands at multiple
concentrations.
In a more sophisticated version of this approach, commonly termed frontal affinity

chromatography, a solution containing a glycan of known concentration is applied to a column of
immobilized glycan-binding protein, and the elution of the glycan from the column (the elution
front) is monitored. Thus, the ligand is continuously applied to the column. The volume
representing the elution front, i.e., when the ligand begins to present in the elution buffer, is a
measure of the binding affinity. This can be directly calculated by Equation 4.6, where the
binding capacity of the column is Vf[T]/Vt; Vf is the frontal volume; Vt is the total column
volume; [O] is the concentration of the glycan in the added solution; and [L] is the lectin
concentration in the matrix.
Various glycan concentrations are used, a series of graphs are obtained, and the 50% saturation
points are derived (Figure 4.3). The data are plotted as 1/[O]o (Vf-Vt) versus 1/[O]o. From this
plot, the value of the Kd can be derived from the intercept on the abscissa, which corresponds to -
1/Kd and the intercept on the ordinate provides the concentration of active lectin [L]. This
approach offers many advantages, which are similar to those discussed for equilibrium dialysis:
(1) The approach is not complicated; (2) if the affinity is high, then relatively small amounts of
protein are needed ( 1 µmole), and in fact a single column is required; (3) correspondingly small
amounts of hapten may be used if the Kd is rather low (in the range of 10 nm to 10 µm); (4) if the
haptens are stable, they may be recovered and reused; (5) radioactive haptens may be used; and
(6) reliable equilibrium measurements can be made. However, the drawbacks to this approach
are that (1) only K can be derived, (2) the conjugation of the glycan-binding protein to the matrix
must be stable and the protein must retain reasonable activity for many different column runs, (3)
many different column runs must be made with a single glycan, (4) if the Kd is high (>10 µm),
then relatively large amounts of glycan may be required, and (5) the entire analysis can take
weeks to months to complete.
Figure 4.3. Example of frontal affinity chromatography, where different concentrations of a

glycan are applied to a column of immobilized lectin. At the lowest concentration (D), the
elution of the glycan is retarded and the chromatographic front is indicated by the vertical line.
At increasing concentrations (C A), the glycan elutes in the earlier fractions. The Vf is the
"frontal volume" as defined in the text.
Titration Calorimetry (28 30)
The binding of a glycan to a lectin can be measured as a change in free energy through
isothermal titration microcalorimetry using a commercial microcalorimeter. In this approach, a
solution containing a glycan of interest is added via a syringe into a fixed concentration of lectin
solution in a microcalorimetry cell (1 2 ml). Control experiments are done in which glycans are
added to cells lacking protein. The 1 3 µl of glycan is added at many intervals and the heat
evolved from binding is measured as µcal/sec. During the course of the experiment, the
concentration of glycan is increased in the mixing cell over the range of a molar ratio of glycan
to lectin from 0 to 10. The change of heat capacity of binding is determined, and the data are
plotted as kcal/mole of injectant versus the molar ratio. These data are then analyzed by a
Scatchard plot to obtain the Kd. In addition, the heat change is directly related to the enthalpy of
reaction ∆H and is the heat of reaction (heat absorbed or given off) at constant temperature and
pressure. From the knowledge the Kd and ∆H, and using Equation 4.4, it is possible to define the
binding entropy ∆S.
The major advantage of this approach is that it can provide all major thermodynamic information
about the binding of a glycan to a glycan-binding protein, and thus, it is highly superior to
equilibrium dialysis and affinity chromatography. The disadvantages of this approach are that (1)
relatively large amounts of protein are typically required (>10 mg), (2) relatively large amounts
of glycans are required, and (3) because of the foregoing problem, these analyses do not typically
use a wide range of different glycans.
Surface Plasmon Resonance (31 36)
SPR is a technique in which the association of a free molecule (termed the analyte) with an
immobilized ligand on a sensor chip induces a change in the refractive index of the chip surface.
In SPR technology, light is reflected from the side of the surface not in direct contact with the
sample, and the change in SPR causes a change in the reflected light intensity at a specific
combination of angle and wavelength. This is measured as a change in the refractive index at the
surface layer and is recorded as the SPR signal or resonance units (RU) (Figure 4.4A). In
general, changes in refractive index for a given change of mass concentration at the surface layer
are largely independent of the molecule of interest and are the same for proteins, glycans, lipids
(liposomes), and nucleic acids. Typically, 1 RU approximates 1 pg/mm2. Measurements may be
conducted in a BIACORETM instrument from Biacore AB. Binding is measured in real time in
terms of 1 10 counts/sec (Figure 4.4B), and thus, information about the association and
dissociation kinetics of the binding, and the overall Kd is obtainable.
In a typical example, a solution of a protein (analyte) is passed continuously over a sensor chip
microsurface containing the immobilized ligand (glycan), using a microfluidic system that
precisely controls analyte delivery, all washing steps, etc. The BIACORETM 2000 allows up to
four channels or flow cells to be used simultaneously across a single sensor surface. One of these
channels is used as the in-line reference or control. If high throughput is required, then different
ligands can be immobilized in each channel. There are a variety of chemistries available for
coupling of the ligand, including coupling by amine, thiols, aldehydes, and biotin capture. In
some approaches, a glycoprotein ligand for a lectin is immobilized and the binding of the lectin
is directly measured. It is also possible to sequentially degrade the immobilized glycoprotein
ligand on the chip by passing through solutions containing exoglycosidases and reexamining the
binding at each step to different lectins, thereby obtaining information about the glycosylation of
the immobilized protein. The immobilized ligand is usually quite stable and can be used
repeatedly for hundreds of runs over a period of months.
The advantages of this approach are that (1) affinities in the range from millimolar to picomolar
can be measured; (2) complete measurements of k1 and k2 (Equation 4.1) are routine; (3) for
immobilization of a molecule using amine coupling, 1 5 µg is normally sufficient; (4) typically,
the concentration range of analyte is 0.1 100 × Kd and the typical volumes needed are in the
range of 50 150 µl; and (5) measurements are extremely rapid, and complete experimental
results can be obtained within a few days. The drawbacks of this approach are that analytes must
have sufficient mass (>2000 daltons) to cause a significant change in SPR upon binding (thus,
the glycan is usually immobilized instead of the protein) and coupling of free glycans to the chip
surface is inefficient; thus, neoglycoproteins or some other type of large conjugate must be
immobilized. Another drawback of SPR is that there may be some nonhomogeneity in conditions
on the BIACORETM due to mass transport effects, allowing rebinding of analyte to the solid-
phase surface, although this may in fact mimic some biological conditions, such as those
occurring in cell-cell interactions. At high analyte concentrations, this problem could
considerably lower the dissociation rate k2 (Equation 4.2), and thus provide a Kd that may be low.
This problem is usually addressed by performing many measurements at different analyte
concentrations and relying on those in the low analyte concentration range.
Figure 4.4. Example of surface plasmon resonance. (A) In SPR, the reflective light is measured
and is altered in response to binding of the analyte to the immobilized ligand. (B) An example of
a sensorgram showing the binding of the analyte to the ligand and the kinetics of binding and
dissociation. RU indicates resonance units.
ELISA-type Assays (37 40)
The conventional ELISA has been adapted for studying glycans and glycan-binding proteins in a
variety of formats. Of course, many glycans are antigens, and antibodies to them can be analyzed
in the conventional ELISA format. In the first adaptation of this approach by Saul Roseman's
group, sugars were attached to a polyacrylamide matrix and actual cell adhesion through the
hepatocyte Gal/GalNAc (asialoglycoprotein) receptor was measured. In most adaptations, an
antibody or a glycan-binding protein of interest is immobilized and the binding of a glycan to the
protein is measured. For this to succeed, the glycans are conjugated in some way, such as to
biotin or another protein with an attached reporter group (e.g., fluorescent moiety or enzyme,
such as peroxidase). Competition ELISA-type assays have recently been developed to probe the
binding site of a lectin or an antibody. In this approach, a glycan is coupled to a carrier protein
(the target) and its binding to an immobilized protein in the solid phase is detected directly.
Competitive glycans or antibodies to components in the assay are added to the wells and their
competition for the target is observed and defined as an IC50, i.e., the concentration at which they
inhibit by 50% the binding of the target. The major advantage of this approach is its ease and the
ability to define the relative binding activity of a panel of glycoconjugates. The major
disadvantage is that it does not provide direct information about affinity constants or other
thermodynamic parameters.
IC50-Hapten Inhibition(41 46)
In this approach, the ability of a soluable hapten to block the binding of a lectin or antibody to a
target glycoconjugate is measured. (In glycobiology, the term hapten is often used to denote a
small glycan that competitively binds to a lectin and competes for its binding to a more complex
ligand.) The target may be cells, as in a visual agglutination assay, or immobilized, as in ELISA-
type formats discussed above. The concentration of added hapten that provides 50% inhibition of
binding of the lectin or antibody is taken as the inhibitory concentration (IC50). Such approaches
have been used for many years in studies on lectin agglutination of cells and were useful in
elucidating the nature of the human blood group substances. If a sufficiently diverse panel of
soluble haptens is used, then the relative efficacies of each glycan can be measured and defined
as the IC50. The IC50 does not, however, relate directly to the binding affinity, since what is being
measured is inhibition, and the amount of inhibitor needed is determined by the affinity of the
lectin or antibody to the immobilized target. The actual binding affinity must be defined by other
techniques described earlier in this chapter. However, the ease of deriving the IC50 and the direct
comparisons that arise from using a large panel of haptens make this an attractive method for
many in the field.
Precipitation (47 51)
The interaction of a multivalent lectin or antibody with a multivalent ligand allows for the
formation of cross-linked complexes. In many cases, these complexes are insoluble and can be
identified as precipitates. The formation of precipitates using antibodies to multivalent ligands
was of great historical significance in the development of the field of immunology. In this
technique, a fixed amount of lectin or antibody (receptor) is titrated with a glycoprotein or a
glycan. At a precise ratio of ligand to receptor, a precipitate is formed. Such precipitation is
highly specific to the affinity constant of the ligand to the receptor. The amount of protein or
ligand in the precipitate can be measured directly by chemical means, using assays for glycan or
protein. The technique of precipitation is still useful for studying those potentially multivalent
ligands and has been used to demonstrate recently that each branch of terminally galactosylated
complex-type di-, tri-, and tetraantennary N-glycans is independently recognized by galactose-
binding lectins. Another precipitation approach takes advantage of the fact that a complex
between a lectin and a glycan can be "salted" out or precipitated by ammonium sulfate. A
variation of this approach was used in early studies on the characterization of the hepatocyte
Gal/GalNAc receptor (asialoglycoprotein receptor), in which the ligand (in this case 125I-labeled
asialoorosomucoid) was incubated with a preparation of receptor. The sample was treated with
an amount of ammonium sulfate capable of precipitating the complex but not the free unbound
ligand. The precipitated complex was captured as a precipitate on a filter and the amount of
ligand in the complex was directly determined by γ-counting.
Electrophoresis (52 54)
In this approach, a glycoprotein (or ligand) is mixed with a glycan-binding protein or antibody,
and the mixture is electrophoretically separated in polyacrylamide. For glycosaminoglycans, this
technique is termed affinity coelectrophoresis (ACE) (see Chapter 29). This method is
particularly useful in defining the apparent Kd of the interaction and allows for identification of
subpopulations of glycosaminoglycans that differentially interact with the glycan-binding
protein. In another method, termed crossed affinity immunoelectrophoresis, a second step of
electrophoresis is conducted in the second perpendicular dimension in the presence of a
precipitating monospecific antibody to the glycoprotein or ligand in 1% agarose gels. The gel is
then stained with Coomassie brilliant blue and an immunoelectrophoretogram is obtained.
Glycoproteins not interacting with the lectin or antibody have faster mobility than the complex.
The amount of glycoprotein or ligand is determined by the area under the curves obtained in the
two-dimensional analysis. This method is useful for studying glycoforms of proteins and has
been particularly valuable in analyzing glycoforms of α1-acid glycoprotein, an acute phase
glycoprotein, in serum and changes in its α1 3-fucosylation.
Expression of cDNAs for Ligands and Receptors (55 58)
Another modern approach to studying glycan-protein interactions is to express the cDNA

encoding either a glycosyltransferase or a lectin in an animal or bacterial cell. The adhesion of
the modified cell to a target is then measured. For example, such approaches have been highly
valuable in studying selectins and their ligands. It helped lead to the identification of sialyl Lewis
X and sialyl Lewis A as important recognition determinants for selectins and the expression
cloning of the cDNA encoding the P-selectin glycoprotein ligand-1 (see Chapter 26). The
expression of glycan-binding proteins, such as selectins or I-type lectins, on the cell surface of
transfected cells has been helpful in evaulating the specific role of lectins in cell adhesion under
physiological flow conditions (see Chapters 24 and 26).
Future Directions
The detailed interactions of glycans and proteins allow for "decoding" glycan structural
information. However, the complex mechanistic details of these interactions are just beginning to
be unraveled. Furthermore, increasing numbers of glycan-binding proteins identified to date
suggest that each glycan may have a functional receptor, either in the animal or in a pathogenic
organism attacking the animal. Understanding the molecular interactions between glycans and
proteins is essential for the development of a real molecular-level appreciation of the biology of
glycoconjugates. There are many approaches currently being used, and each has its advantages
and disadvantages. Obviously, a combination of methods provides a better overall description
than any single method alone. It is anticipated that new synthetic approaches to glycans will
allow more detailed studies in the future, since the amounts of naturally occurring glycans of
interest may be low and difficult to obtain in sufficient quantity. In addition, the increasing
availability of recombinant forms of glycan-binding proteins will enhance the evolution of this
area of glycobiology. These recent developments in understanding the molecular details of
glycan-protein interactions may allow for the generation of specifically tailored compounds that
mimic a glycan-binding site, which may allow formulation of new families of pharmaceutical
agents. Finally, much of the present information derives from studies on small glycans and
lectins, and little is known about the very complex interactions between a glycan-binding protein
and its macromolecular glycoconjugate ligand. Although there are many methods of studying
glycan-protein interactions, great difficulty still exists in translating the "one-dimensional"
information based on free diffusion to the two- and three-dimensional nature of cell-cell
adhesion or cell-matrix adhesion. In addition, exciting new developments in several fields
suggest that higher-order structures of glycoconjugates may also be important in regulating
glycan-protein interactions. As examples, glycosphingolipids may cluster in "rafts" and some
glycoproteins have important carbohydrate determinants adjacent to noncarbohydrate
determinants, such as tyrosine sulfate. The coordinated interactions between such organized
glycan determinants may promote high-affinity interactions and present an even greater
challenge to future research in this field.
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5. Exploring the Biological Roles of Glycans
Diego).
THIS CHAPTER PROVIDES AN OVERVIEW concerning the biological roles of the major
classes of glycans in eukaryotic cells and makes an attempt to synthesize some general principles
for understanding and exploring these roles. For more details regarding many of the specific
biological roles mentioned, see the the reviews cited, the other chapters in this book, and the
original literature cited therein.
General Principles and Conclusions about Biological Roles of

Glycans (1 42)
The commonly heard question what is the function of glycosylation? is not actually a
reasonable one. As with most other classes of macromolecules, the biological roles of glycans
span the complete spectrum, from those that are relatively unimportant to those that appear
crucial for the development, growth, functioning, or survival of the organism. Over the years,
many theories have been advanced regarding the biological roles of glycans. Although the
available evidence provides support for essentially all of these theories, exceptions to each can
be easily found. Given the enormous diversity of glycans in nature, this should perhaps not be
surprising. An added level of complexity arises from the fact that glycans are very frequently
targets for the binding of microbes and pathogens. The biological roles of glycans can be broadly
divided into two groups (Figure 5.1). One group relies on the structural and modulatory
properties of glycans and the other relies on specific recognition of glycan structures by other
molecules (generally receptor proteins or lectins). The latter category can be further subdivided
into two major groups: those involving recognition by endogenous receptors within the same
organism and those resulting from recognition by exogenous agents. The last group consists
mostly of pathogen receptors and toxins, but it can also include symbiotic agents. As discussed
in Chapter 3, these two types of recognition are likely to represent opposing selective forces
during evolution.
Figure 5.1. General classification of the biological roles of glycans. A simplified and broad
classification is presented, emphasizing the roles of endogenous and exogenous lectins in
recognizing glycans. There is some overlap between the groups, e.g., some structural properties
involve specific recognition of glycans. (Reprinted, with permission, from [42] Gagneux and
Varki 1999 [© Oxford University Press].)
Other general principles should also be mentioned. The biological consequences of altering
glycosylation in various systems seem to be highly variable and unpredictable (see discussion
below). A given glycan can also have different roles in different tissues or at different times in
development. As a broad generalization, it can be stated that terminal sequences, unusual
structures, and modifications of glycans are more likely to be found to mediate specific
biological roles within the organism. However, such unusual glycans or modifications are also
more likely to be targets for pathogens and toxins. Perhaps as a consequence (for discussion, see
Chapter 3), intraspecies and interspecies variations in glycosylation are relatively common, and
at least some of the diversity of glycans seen in nature may represent evolutionary "junk."
Finally, since genetic defects in glycosylation are easily obtained in cultured cells, but often have
limited consequences, many major functions of glycans may be operative only within an intact
organism. Each of these principles is discussed briefly below.
Biological Consequences of Altering Glycosylation Are Variable

Approaches taken to understand the biological roles of glycans include the prevention of initial
glycosylation, alteration of oligosaccharide processing, enzymatic or chemical deglycosylation
of completed chains, genetic elimination of glycosylation sites, and the study of naturally
occurring variants and genetic mutants in glycosylation. In reviewing many such studies, the
consequences of altering glycosylation range from being essentially undetectable to the complete
loss of particular functions, or even loss of the entire glycoprotein itself. Even within a particular
class of proteins (e.g., cell surface receptors), the effects of altering glycosylation are highly
variable and unpredictable. Moreover, the same glycosylation change can have markedly
different effects when studied in vivo or in vitro. The answer obtained may depend on the
structure of the glycan, the biological context, and the specific biological question being asked.
Overall, it is difficult to predict a priori the functions that a given oligosaccharide on a given
glycoconjugate might mediate or its relative importance to the organism.
Structural and Modulatory Roles of Glycans

There is little doubt that glycans have many protective, stabilizing, organizational, and barrier
functions. As discussed in Chapter 1, the glycocalyx that covers most cells can represent a
substantial physical barrier. The glycans attached to matrix molecules such as collagens and
proteoglycans are important for the maintenance of tissue structure, porosity, and integrity. Such
molecules also contain binding sites for specific types of glycans, which in turn help with the
overall organization of the matrix. The external location of glycans on most glycoproteins can
provide a general shield, protecting the underlying polypeptide from recognition by proteases or
antibodies. Glycans are also involved in the proper folding of newly synthesized polypeptides in
the ER and/or in the subsequent maintenance of protein solubility and conformation. Indeed, if
some proteins are incorrectly glycosylated, they may fail to fold properly and/or fail to exit the
ER. Conversely, there are also examples of glycoproteins whose synthesis, folding, trafficking,
sensitivity to proteolysis, or immune recognition seem quite unaffected by altering their
glycosylation. Overall, these types of functions are obviously of importance to the intact
organism. However, they cannot explain the need to evolve the enormous structural complexities
found in glycans in nature. In keeping with this view, inhibitors that affect only the later steps of
glycan processing usually do not interfere with such basic structural functions.
There are many examples wherein glycosylation can modulate the interaction of proteins with
one another. Some growth factor receptors seem to acquire their binding abilities in a
glycosylation-dependent manner. This may limit unwanted early interactions of a newly
synthesized receptor with a growth factor that is synthesized in the same cell. Glycosylation of a
polypeptide can also mediate an on-off or switching effect. For example, when the hormone β-
human chorionic gonadotrophin (β-HCG) is deglycosylated, it is still able to bind to its receptor
with similar affinity. However, it fails to stimulate adenylate cyclase. The mechanism by which
this agonist:antagonist conversion occurs remains unknown. In most instances, such effects of
glycosylation are incomplete, i.e., glycosylation appears to be tuning the primary function of the
protein. For example, the activity of some glycosylated growth factors and hormones can be
modulated over a wide range by the extent and type of their glycosylation. This becomes
particularly evident when recombinant forms of such molecules are produced, bearing different
types and extents of glycosylation. A striking example of this "tuning" function is demonstrated
by polysialic acid chains attached to the N-CAM. This adhesion receptor normally mediates
homophilic binding between neuronal cells. In the embryonic state, or in other states of neural
"plasticity," these anionic chains tend to be very long, thereby interfering with homophilic
binding (see Chapter 15). There are also instances where protein functions can be tuned by
glycans attached to other neighboring structures. Thus, the polysialic acids of embryonic N-
CAM mentioned above can interfere with the interactions of other unrelated receptor-ligand
pairs, simply by physically separating the cells. Another example is the tyrosine phosphorylation
activity of the EGF receptor and the insulin receptor, which can be modulated by endogenous
gangliosides (see Chapter 9). Although the precise mechanism of these effects is uncertain, some
specificity is implied by the requirement for a defined oligosaccharide sequence in the
ganglioside. Perhaps the most dramatic example of a tuning function of a glycan is the effect of
heparin/heparan sulfate chains on the action of the natural anticoagulant antithrombin III (see
Chapter 11). A specific pentasaccharide sequence within the polysaccharides converts this weak
antithrombin into a potent anticoagulant.
Since most such tuning effects of specific glycan sequences are partial, their overall importance
might be questioned. However, it should be realized that the sum total of several such partial
effects can be a dramatic effect upon the final biological outcome. Thus, glycosylation may be a
mechanism for generating important functional diversity while utilizing a limited set of basic
receptor-ligand interactions dictated by the size of the genome. Of course, as with most other
functions of glycans, exceptions to these concepts can be easily found. There are many receptors
whose ligand binding is not acquired in a glycosylation-dependent manner, and there are many
peptide ligands whose binding and action are not obviously affected by glycosylation.
Another structural/modulatory function of glycans is to act as a protective storage depot for

biologically important molecules. A particularly interesting case is that of the heparin-binding
growth factors (see Chapter 11) that are bound to the GAG chains of the extracellular matrix
adjacent to cells that need to be stimulated (e.g., the basement membrane underlying endothelial
cells). This prevents diffusion of the factors away from the site, protects them from nonspecific
proteolysis, prolongs their active lives, and allows them to be released under specific conditions.
Likewise, the GAG chains found in secretory granules seem to bind and protect protein contents
of the granule and to modulate their functions. There are several other such examples wherein
glycans act as sinks or depots for biologically important molecules, ranging from water, to ions,
to complement regulatory proteins.
Glycans as Specific Ligands for Exogenous Receptors

As discussed in Chapter 28, certain glycans act as specific receptors for a variety of viruses,
bacteria, and parasites. They are also receptors for many plant and bacterial toxins and can serve
as antigens for autoimmune and alloimmune reactions. In most instances, there is excellent
specificity for the sequence of the oligosaccharide involved. Thus, for example, influenza virus
hemagglutinins specifically recognize the type of sialic acid, its modifications, and its linkage to
the underlying sugar chain, whereas various toxins bind with great specificity to certain
gangliosides and not to related structures (see Chapter 28). Some incompletely synthesized
oligosaccharides such as the Tn antigen (GalNAc-O-Ser/Thr on mucins) can also act as
autoantigens. There is little doubt about the structural specificity of this group of functions of
glycans. Indeed, the relevant binding proteins have even been used as specific tools for studying
the expression of the sugar chains. However, as far as the organism that synthesized such glycans
is concerned, there is no clear value resulting from providing such traitorous signposts, which
can aid the access of pathogenic microorganisms or permit damaging autoimmune reactions.
Perhaps to counter these deleterious consequences, the addition of specific monosaccharides or
modifications can mask sequences recognized by microorganisms, toxins, or antibodies. Glycan
sequences on soluble glycoconjugates, such as the secreted mucins, can also act as decoys for
microorganisms and parasites. Thus, a pathogenic organism or toxin seeking to bind to mucosal
cell membranes might first encounter the specific oligosaccharide ligand attached to a soluble
mucin, which can then be washed away, removing the potential danger to the cells below. In
contrast, there are instances in which symbiosis between species is mediated by specific glycan
recognition. Examples of these include some commensal bacteria in the gut lumen of animals
and bacteria involved in forming plant root nodules.
Glycans as Specific Ligands for Endogenous Receptors

The earliest endogenous receptors for glycans to be recognized were those mediating clearance,
turnover, and intracellular trafficking (for examples, see Chapters 23 and 25). However, even the
most elegantly precise examples, such as the role of Man-6-P in the trafficking of lysosomal
enzymes, include some exceptions. Thus, Man-6-phosphorylation is not absolutely required for
the trafficking of lysosomal enzymes in certain cell types, nor is it operative at all in some lower
eukaryotes. There are also endocytic receptors recognizing specific glycan sequences whose
functions have yet to be assigned. There are several examples wherein free oligosaccharides can
have hormonal actions inducing specific responses in a highly structure-specific manner.
Examples include the action of oligosaccharins in plants (see Chapter 20) and the bioactive
properties of fragments of hyaluronan in mammalian systems, both of which can induce
biological responses in a size- and structure-dependent manner. Likewise, free heparan sulfate
fragments released by certain cell types have effects in complex situations such as wound
healing. In many of these instances, the putative receptors for these molecules and their precise
mechanisms of action have yet to be defined.
Many examples are now available of specific biological roles of oligosaccharides in cell- cell
recognition and cell-matrix interactions. Perhaps the best-documented case is that of the selectin
family of adhesion molecules that mediate critical interactions among blood cells in a wide
variety of normal and pathological situations (see Chapter 26). There is also now substantial
evidence that sperm:egg binding involves the O-glycans on the egg zona pellucida glycoprotein
ZP3 (see Chapter 34). Other clear examples include CD44 recognition of hyaluronan (Chapter
29) and the interactions of bone marrow cells with sialoadhesin on macrophages (Chapter 24).
As indicated above, there are also specific interactions between lectins and carbohydrates present
on cell surfaces, with molecules in the matrix. In many such instances, the specific biological
significance of recognition has yet to be conclusively demonstrated in the intact animal.
Carbohydrate-carbohydrate interactions may also play a specific role in cell-cell interactions and
adhesion. The most dramatic example is the species-specific interactions of marine sponges,
which are mediated via homotypic binding of the glycans on a large cell-surface proteoglycan.
Another example is the compaction of the mouse embryo at the morula stage, which seems to be
due to an Lex-Lex interaction. The single-site affinities of such interactions are not very strong
and are difficult to measure. However, if the molecules in question are present in very high copy
numbers on the cell surface, a large number of relatively low-affinity interactions can collaborate
to produce a high-avidity "velcro" effect, which may be clearly sufficient to mediate biologically
relevant interactions.
The Same Glycan Can Have Different Roles within An

Organism
The expression of specific types of glycosylation on different glyconjugates in different tissues at
different times of development implies that these structures have diverse roles within the same
organism. For example, Man-6-P-containing oligosaccharides were first found on lysosomal
enzymes and are involved in lysosomal trafficking (see Chapter 23). However, Man-6-P-
containing glycans are found on a variety of apparently unrelated proteins, including proliferin,
thyroglobulin, the EGF receptor, and the TGF-β precursor. Likewise, the sialylated fucosylated
lactosamines critical for selectin recognition (see Chapter 26) are found in a variety of unrelated
cell types, and the polysialic acid chains that play such an important part in embryonic N-CAM
(Chapter 15) are found on egg jelly coat proteins and a sodium channel protein. Since
oligosaccharides are added posttranslationally, these observations should not be surprising. Once
a new oligosaccharide or modification has been expressed in an organism, several distinct usages
could evolve independently in different tissues and at different times in development. If any of
these situations provide a function vital to the survival of the organism, then the gene responsible
for expression of the glycan would remain conserved in evolution.
Are There "Junk" Glycans?

Since microorganisms and parasites that bind glycans evolve in parallel with their multicellular
hosts (see Chapter 3), they have to adapt to bind to any new "masking" glycan structure
presented by the host. In response, the host may find it easiest to generate new masking
structures, especially if the primary structure had meanwhile evolved a vital function elsewhere
within the organism. Thus, there would be no choice but to preserve the underlying scaffolding
upon which the latest "mask" was placed, adding yet another layer of complexity to its glycans.
Such cycles of interaction between microbes and hosts could perhaps explain some of the
complex and extended sugar chains found on mucosal surfaces and secreted mucins. In this
manner, "junk" glycans could develop, akin to "junk" DNA. Although such structures may still
function as a scaffolding, they may have no other specific role in the organism.
Intraspecies and Interspecies Variations in Glycosylation

Species-specific variations in glycan structure also indicate that some glycans do not have
fundamental and universal roles (see Chapter 3). Such diversity in glycosylation could be
involved in generating the obvious differences in morphology and function observed between
species. These variations could also simply reflect differing selection pressures resulting from
exposure to different pathogens. Furthermore, substantial intra-species polymorphism in
oligosaccharide structure can exist without obvious functional value. The potential role of such
polymorphisms in the interplay between parasites and host populations is discussed in Chapter 3.
Of course, it should be noted that extensive interspecies variability in primary sequence also
occurs in proteins, without any obvious consequences to essential functions (e.g., some yeast
proteins are functional when transfected in mammalian cells, and vice versa, despite relatively
limited sequence homology).
Importance of Terminal Sequences, Modifications, and Unusual
Structures
Given all of the above, it is a challenge to predict which glycan structures are likely to mediate
the more specific or crucial biological roles within an organism. The available data suggest that
terminal sugar sequences, unusual structures, or modifications of the glycans are more likely to
be involved in such specific roles. The predictive value of this observation is reduced by the fact
that such terminal sequences, unusual glycans, or modifications are also more likely to be
involved in interactions with microorganisms and other noxious agents, i.e., the balance between
the traitorous and masking functions of glycans discussed above tends to involve such structures.
The challenge then is to predict and sort out which of these two distinct roles is to be assigned to
a given glycan structure.
Approaches to Uncovering Specific Biological Roles of Glycans

Some functions of glycans are discovered serendipitously. In most cases, the investigator who
has elucidated complete details of the structure and biosynthesis of a specific glycan is still left
without knowing its functions. It is necessary to design experiments that can differentiate
between the trivial and crucial functions mediated by each glycan. Various approaches that can
be considered are discussed below, emphasizing the pros and cons of each. These approaches are
also presented in schematic form in Figure 5.2 (the figure assumes that the investigator is
exploring a glycan function mediated by a specific receptor:ligand pair).
Figure 5.2. Approaches toward uncovering biological roles of glycans. The figure assumes that a
specific biological role is being mediated by recognition of a certain glycan structure by a
specific receptor lectin. Clues to this biological role could be obtained by a variety of different
approaches (for discussion of each approach, see text).
Localization of or Interference with Specific Glycans Using Lectins or
Antibodies
The availability of numerous lectins and antibodies that are apparently specific for certain
glycans has permitted the exploration of the regional and cell-type-specific localization of these
structures. Once a specific glycan has been localized in a interesting context, it is natural to
consider introducing the same lectin or antibody into the intact system, hoping that it will
interfere with a specific function, generating an interpretable phenotype. This approach has
proven to be highly successful when investigating the function of proteins by introduction of
specific antibodies. However, with some possible exceptions, the same approach is likely to give
confusing results with regard to glycan function. Most antibodies against glycans are of the IgM
variety and hence tend to have weak affinity and show cross-reactivity between species
(although high-affinity IgG antibodies are preferred, they are hard to obtain, because glycans
tend to be T-independent antigens and often do not generate high-titer immune responses).
Likewise, although some plant lectins seem to be very specific for animal glycans, it must be
remembered that the lectins originated from organisms that typically do not contain the cognate
ligand. Thus, their apparent specificity may not be as reliable when introducing them into
complex animal biological systems where unknown cross-reacting glycan structures are
potentially present. Finally, both antibodies and lectins are multivalent and their cognate ligands
(the glycans) tend to be present in multiple copies on multiple glycoconjugates. Thus,
introduction of a lectin or antibody into a complex biological system is likely to cause
nonspecific aggregation of various molecules and cell types, and the effects seen may have
nothing to do with the biological functions of the glycan in question. In the long run, it would
seem more worthwhile to develop recombinant monovalent high-affinity lectin modules that are
derived from the same system being investigated. The effects of introducing such monovalent
lectins into a complex system may yield more interpretable clues.
Metabolic Inhibition or Alteration of Glycosylation
As outlined in Chapter 40, a large number of pharmacological agents can metabolically inhibit or
alter glycosylation in intact cells and animals. Although metabolic inhibitors are powerful tools
to elucidate biosynthetic pathways, they can sometimes yield confusing results in complex
systems. One concern is that the inhibitor may have effects on other unrelated pathways (e.g., the
inhibitor tunicamycin that blocks N-linked glycosylation can also inhibit UDP-Gal uptake into
the Golgi). The second is that the inhibitor may result in such massive changes in glycan
synthesis that the physical properties of the glycoconjugates and/or membranes are altered,
making it difficult to interpret the results. Somewhat more useful results can be obtained by
introducing low-molecular-weight primers of glycosylation, which can act as alternate substrates
for Golgi enzymes, diverting synthesis away from the endogenous glycoproteins. However, this
approach runs the risk of simultaneously generating incomplete glycans on the endogenous
glycoconjugates, as well as producing secreted glycan chains that could have their own
biological effects.
Finding Natural Glycan Ligands for Specific Receptors
With the ability to recognize different types of lectin motifs within a primary amino acid
sequence (see Chapter 22), many newly cloned proteins may be predicted to bind glycans. If the
potential lectin protein can be produced in large enough quantities, techniques such as red cell
agglutination, flow cytometry, surface plasmon resonance, and affinity chromatography can then
be used to look for the presence of specific ligands. It should be kept in mind that the
monovalent affinity of the putative lectin for its ligand may not be high, and high densities
and/or multivalent arrays may be needed, to avoid missing a biologically relevant interaction.
The question also arises as to where to look for the biologically relevant ligands. Furthermore,
since many glycan structures can be expressed in different tissues at different times in
development and growth, the recombinant lectin may detect a cognate structure in a location and
time where it is not actually of biologically relevance. Careful consideration of the natural
occurrence and expression profile of the lectins should lead to a rational decision as to where to
look for the glycan ligands.
Finding Receptors Recognizing Specific Glycans
The converse situation arises when an unusual glycan is found to be expressed in an interesting
context and is hypothesized to be a ligand for a specific receptor. It is possible to search for such
a receptor by techniques similar to those mentioned above, such as hemagglutination, flow
cytometry, and affinity chromatography. Of course, to facilitate the search, it is necessary to
have reasonable quantities of the pure defined glycan in question, as well as a variety of closely
related structures that can act as negative controls. Since many biologically relevant lectin-like
interactions can be of low affinity, it is probably also advisable to use a multivalent form of the
glycan as the probe. Finally, it may not necessarily be obvious where to look for receptor. For
example, the receptor that specifically recognizes the sulfated glycans of pituitary glycoprotein
hormones was eventually found not in the pituitary itself, nor in any of the target tissues for the
hormones, but in the endothelial cells of the liver, where it regulates the circulating half-life of
the hormones. Indeed, the most biologically relevant receptor for a particular glycan might even
be found in another organism (a pathogen or a symbiont).
Interference by Soluble Glycans or Structural Mimics
The addition of soluble glycans or their structural mimics into the system can cause interference
with the interaction between an endogenous lectin and a specific glycan. If a sufficient
concentration of the specific inhibitor can be achieved, the resulting phenotypic changes can be
instructive. When studying in vitro systems, even monosaccharides can be used to advantage in
such studies (e.g., the exploration of the Man-6-P receptor pathway, Chapter 23). However, is
often necessary to use pure glycans in somewhat large quantities to block the relatively low-
affinity interactions. Effective blockade may also require multivalency of the cognate glycan.
Finally, especially when studying complex multicellular systems, the glycans introduced could
cross-react with other as yet unknown receptors, giving a phenotypic readout that is confusing.
Eliminating Specific Glycan Structures by Glycosidases
A powerful approach to understanding the biological roles of glycans is to make use of the many
known degradative enzymes that are highly specific for certain glycan sequences. The advantage
of this approach is that one is not interfering with the basic biosynthetic cellular machinery, but
simply eliminating the structures selectively after normal synthesis has been completed. Thus,
for example, sialidase treatment abolished lymphocyte binding to the high endothelial venules of
lymph nodes and provided the first prediction of the ligands for l-selectin: Injection of
endoneuraminidase into the developing retina suggested specific roles for polysialic acids, and
injection of heparanase into developing embryo resulted in a randomization of left-right axis
formation. In all such studies, the purity of the enzyme being used is obviously critical, and
appropriate controls are necessary. A genetic approach can be used to avoid problems of
contamination, by expressing a cDNA for the glycan-modifying enzyme in the intact cell or
animal. For example, transgenic expression in mice of an influenza sialic-acid-specific C 9-O-
acetylesterase gave either early or late abnormalities in development, depending on the promoter
used.
Studying Natural or Genetically Engineered Glycan Mutants
This is intuitively a powerful approach to understanding glycan function. Technically, it is

easiest to study glycosylation mutants in cultured cell lines (see Chapter 31). However, although
genetic or acquired defects in glycosylation are easily obtained in cultured cell lines, these
defects may have limited or not easily discernible consequences. This may be because of the lack
of other factors or cell types that would be present in the intact organism; e.g., the cognate
receptor for the glycan may not be present in the same cell type. Of course, such mutants can still
be used to analyze basic structural functions of the glycans and their relevance to the physiology
of a single cell. Furthermore, one can add back external factors or other cell types thought to
interact with the modified glycan. Some mutants can also be reintroduced into intact organisms
(e.g., to study tumorigenicity or metastatic behavior). Overall, much useful information can be
gained by this approach, but many of the more specific roles of glycans need to be uncovered by
studying mutations in the intact multicellular organism.
Genetic defects in glycosylation in intact organisms seem to be relatively uncommon and have
highly variable consequences. Looking back on the mutants that have been discovered to date in
mice and humans (see Chapters 32 and 33), it is clear that glycan changes often affect multiple
systems and that the phenotypes are unpredictable and highly variable. The relative rarity of
naturally occurring mutations can be explained in several ways. Perhaps they do occur frequently
but have little detectable biological consequence. Alternatively, many may cause lethal
aberrations that prevent completion of embryogenesis. Another possibility is that genetic
glycosylation abnormalities remain undetected because of redundant or fail-safe pathways.
Regardless of the reasons, the value of constructing glycosylation mutants in intact animals is
evident. Given the complex phenotypes that can result and the potential for early developmental
lethality, the ability to disrupt genes in a temporally controlled and cell-type-specific manner can
be particularly valuable (for details and examples, see Chapter 32).
Studying Natural or Genetically Engineered Glycan Receptor Mutants
Eliminating a specific glycan receptor can yield a phenotype that may be very instructive with
regard to the functions of the glycan. As with genetic modification of the glycan, the results are
more likely to be useful if studied in the intact organism. However, the receptor protein may
have other functions that are unrelated to glycan recognition. Conversely, the glycan in question
may have other functions that are not mediated by the receptor. Thus, for example, the genetic
elimination of the CD22 receptor and the ST6Gal I enzyme that generates its ligand gave
complementary, but not identical, phenotypes.
Future Directions
The future now appears to be very bright for elucidating many new biological roles of glycans.
These roles are likely to become more obvious in the upcoming "post-genomic era," when more
and more attention is going to be focused upon the molecular basis of the development and
physiology of whole organs or organisms. The various approaches described in this chapter must
be combined as needed to elucidate these biological roles.
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Biosynthesis, Metabolism, and
Function
6. Monosaccharide Metabolism
Primary contributions to this chapter were made by H.H. Freeze (The Burnham Institute, La
Jolla, California).
GLYCOCONJUGATE BIOSYNTHESIS REQUIRES THE CONVERSION of monosaccharides

to activated sugar nucleotides which then donate the sugars to various acceptors using specific
glycosyltransferases. To achieve this, monosaccharides must be imported into the cell, salvaged
from degraded glycoconjugates, or derived from other sugars within the cell. Although most
glycosylation reactions occur in the Golgi, sugar import, interconversion, and activation occur in
the cytoplasm. Sugar nucleotide-specific transporters carry the activated donors into the Golgi.
This chapter explores various pathways that eukaryotic cells use to accomplish these tasks, with
an emphasis on the metabolism of the major monosaccharides found in higher animal cells.
Plants and microbes synthesize complex glycoconjugates with additional monosaccharides such
as rhamnose, arabinose, and galacturonic acid to name just a few; however, these have not (yet)
been found in animal cells, and their biosynthetic pathways are not discussed here. The same
general biosynthetic principles apply to their synthesis.
General Principles
Glucose and fructose are the major carbon and energy sources for organisms as diverse as yeast
and human beings. Both organisms can derive the other monosaccharides needed for
glycoconjugate synthesis from these major suppliers. It is important to appreciate that all of the
biosynthetic pathways are not equally active in all types of cells. However, there are some
guideposts for this bedrock of biochemistry. Glycoconjugate synthesis requires activated sugar
nucleotide donors. The role of nucleoside diphosphate sugars was first discovered in 1950 by
Nobel Laureate L.F. Leloir in Buenos Aires. He showed that a nucleotide triphosphate such as
UTP reacts with a glycosyl-1-P to form the high-energy donor that could participate in
glycoconjugate synthesis. There are a few variations on this theme, but regardless of the sugar
and its origin, all must be either activated by a kinase (reaction 1 below) or generated from a
previously synthesized activated sugar nucleotide (reactions 2 and 3 below):
The most common sugar nucleotide donors are shown in Table 6.1. Sialic acid is the only
monosaccharide that is activated as a mononucleotide, CMP-Sia. Iduronic acid does not have a
sugar nucleotide parent since it is formed by epimerization of glucuronic acid after this sugar is
incorporated into the glycosaminoglycan chains. In some instances, rarer nucleotides can be
formed from the more common ones by a nucleotide exchange reaction (reaction 3 above). These
rarer forms (e.g., ADP-Glc) tend to occur in plants and microbes, but there are few examples in
animal cells.
Table 6.1. Activated sugar donors
Source of Monosaccharides
External Sources and Transporters (1 4)
There are three types of sugar transporters. One is an energy-independent facilitated diffusion
transporter. A good example is the family of hexose transporters found in mammalian cells, e.g.,
the family of GLUT (glucose transporter) transporters. They have Km values for glucose uptake
in the 2 20 mm range. Yeast also have a wide range of such transporters with similar Kmvalues.
A second type of transporter is an energy-dependent variety such as the sodium-glucose
transporters (SGLT). These are typically found in epithelial cells of the intestine and in kidney
tubules where they absorb monosaccharides derived from the diet or retrieve glucose from the
kidney filtrates, respectively. They are more efficient than the GLUT transporters and have Km
values of less than 1 mm (see Table 6.2). The last type of sugar transporter is a large group of
energy-dependent transporters that couple ATP-dependent phosphorylation with sugar import.
These transporters have Kuptakes in the micromolar range so they can scavenge traces of available
nutrients from the environment. This highly efficient and diverse group of transporters is found
in bacteria, but not in yeast or animal cells, and is not covered in this chapter.
Table 6.2. Sugar transporters in mammalian cells
Type Primary location Km (glucose) (mm)
Ion-coupled
(Na+/glucose) epithelial cells
SGLT-1 small intestine 0.1 0.8
some kidney
SGLT-2 kidney cortex 1.6
Facilitated diffusion
GLUT-1 erythrocytes, blood tissue barrier 5
GLUT-2 liver, small intestine 6 12
GLUT-3 neurons, placenta 1 2
GLUT-4 adipose tissue, skeletal muscle,
insulin-regulated 5
GLUT-5 small intestine 6 (fructose)
GLUT-7 microsomal, remove Glc from ER
Mannose many cell types 30 70 µm (mannose)
Fucose several cell types ~250 µm (fucose)
Lower Eukaryotes
The yeast Saccharomyces cerevisiae typically lives in grape juice, which is an equimolar mixture
of glucose and fructose (1 1.5 m in total sugar). These sugars serve as both carbon and energy
sources. The yeast plasma membrane contains energy-independent facilitated diffusion
transporters. Yeast have at least 18 genes that code for such sugar transporters, and although they
are not energy-dependent, their expression levels are coordinated with those of sugar kinases to
avoid ATP depletion. Although many of them transport glucose, others are specific for galactose
and fructose and some for disaccharides. Yeast also secrete hydrolases to break down
disaccharides, such as sucrose (Glcα1-2Fru) or maltose (Glcα1-4Glc), and then use the hexose
transporters to take in the resulting monosaccharides. All of these transporters are about the same
size (40 55 kD) and have similar structures with 12-membrane spanning domains and only a
minority of amino acids facing the outside of the cell. Their structure is typical of many other
sugar and nonsugar transporters found in eukaryotes. The 12 domains probably form a barrel and
a small pore for the sugar to pass through. Once inside the cell, sugars are mostly catabolized for
energy or used to generate other sugars, amino acids, etc. For an organism composed of more
than 15% by weight complex carbohydrates (mannans and chitin), deriving the component
sugars Man and GlcNAc from glucose is vital. Other eukaryotic microorganisms living in less
carbohydrate-rich environments also rely on hexose transporters and sugar interconversions for
glycoconjugate synthesis.
Higher Organisms
Higher organisms such as vertebrates have a cadre of carbohydrate-degrading enzymes (such as

α-amylase and disaccharidases) that are secreted into the digestive tract or localized to the brush
border of intestinal epithelial cells. These generate monosaccharides for sugar transporters.
Glucose and fructose account for the great bulk of monosaccharide traffic in higher organisms,
and most attention has been focused on them. Glucose is carried out of the gut by sodium-
dependent energy-requiring transporter (SGLT-1). Fructose transport is carried out by one of the
facilitated diffusion GLUT transporters, GLUT-5.
Except for GLUT-5, the other members of the GLUT family transport glucose. They have been
intensively studied, and their general structure in mammals is highly homologous to those found
in yeast. The five members of this family have different distributions in mammalian cells and
different Km values that enable them to respond to the availability of glucose. Although most of
the GLUT family members are located on the cell surface, a portion of GLUT-4 is associated
with intracellular vesicles, and it is recruited to the cell surface in response to insulin. Even
though the GLUT transporters can recognize other hexoses, such as mannose and galactose, the
only monosaccharides present in sufficiently high concentrations to be physiologically relevant
are glucose or fructose (Table 6.2).
Other sugar-specific transporters have been found in higher organisms and may have
physiological importance. Two types of mannose transporters are known. One is an energy-
dependent transporter(s) analogous to the SGLT variety for glucose. It is located on the brush
border of enterocytes and the surface of kidney tubule epithelial cells. It presumably transports
mannose liberated from digested macromolecules and retrieves mannose from the kidney
filtrates, much like those used for glucose transport. The second type is a highly mannose-
specific, energy-independent transporter found on the surface of many types of mammalian cells.
It is relatively insensitive to glucose inhibition and its Kuptake is near the concentration of
mannose found in the blood (50 100 µm) of many mammals. Intravenous injections of mannose
in rats show that it is primarily taken up by the liver and intestine. In cultured human hepatoma
and fibroblasts, mannose transporters actually supply up to 80% of the mannose used for
glycoprotein synthesis. Direct conversion from glucose is relatively minor. This is quite striking
considering that glucose in the blood is 100-fold more abundant than mannose. These studies
were done in cell culture using physiological concentrations of mannose and glucose. At present,
the relative contributions of mannose and glucose in different tissues and organs in an intact
animal are not known.
A fucose-specific transporter has also been reported in several types of mammalian cells. Its
Kuptake is approximately 250 µm, which is probably much higher than the fucose concentration in
blood. Much of what is taken up can be converted into GDP-Fuc and incorporated into
glycoproteins, but its contribution to glycosylation compared to synthesis from GDP-Man (see
below) is unknown. No cell surface transporters specific for galactose, amino sugars, or sialic
acids are known in higher organisms.
Intracellular Sources of Sugars

Salvage Pathways (5 10)
Monosaccharides can also be salvaged from glycoconjugates degraded within the same cell.
Details of specific degradation pathways are presented in Chapter 18. Most of them occur at low
pH in the lysosomes. Although salvage pathways have not been systematically explored and
have received relatively little attention, their contribution to glycosylation may be quite
substantial (see Table 6.3). For example, 80% of the radioactively labeled GlcNAc from a
glycoprotein degraded in liver lysosomes is converted into UDP-GlcNAc, and at least one-third
is used to synthesize secreted glycoproteins. In addition, fibroblasts endocytosing labeled
glycoconjugates reutilize about 50% of the amino sugars for new glycoprotein synthesis. Thyroid
glands blocked in de novo synthesis of UDP-GlcNAc from glucose can easily fulfill their
glycosylation needs by degrading and reusing the sugars derived from thyroglobulin. Efficient
salvaging is not limited to GlcNAc. It can be seen in glycosylation-impaired mutant ldlD CHO
cells that require supplements of Gal and GalNAc for normal O-glycosylation. Catabolism of
glycoproteins present in 5 10% fetal bovine serum can also provide sufficient salvaged sugar for
glycosylation. Multiple studies suggest that 30 90% of sialic acids released in the lysosome are
reutilized for new glycoproteins.
Table 6.3. Evidence favoring carbohydrate salvage pathways
Amino sugars
more than 50% of label reused for new glycoconjugates (GalNAc, GlcNAc)
approximately 70% of [3H]GlcNAc incorporated into sugar nucleotides in rat liver
15 90% sialic acids recycled in some cells
GlcNAc/ManNAc kinase present in many tissues
GalNAc-1-kinase distinct from Gal-1-kinase
Hexose and fucose
mannose transporter, uses physiological concentrations of mannose
cells lacking UDP-Gal-4-epimerase, can use serum glycoproteins for galactose
fucose transporter identified
correction of GDP-Man GDP-Fuc deficiency with exogenous fucose
Reutilization of salvaged sugars requires that the monosaccharides exit the lysosome to gain
access to the cytosol where the biosynthetic enzymes are located. Separate lysosomal carriers for
neutral hexoses, N-acetylated amino sugars, and acidic hexoses have been identified. The neutral
hexose carrier has a Km value of 50 75 mm and can also carry fucose and xylose. Although these
sugars can diffuse through the lysosomal membrane, their efflux rate may not be high enough.
The N-acetylhexosamine carrier (4.4 mmKm) will not use non-acetylated amino sugars. The
sialic acid and glucuronic acid carrier (~300 550 µmKm) is obviously important, since its loss
leads to an accumulation of these sugars in the lysosome and secretion in the urine and causes a
human lysosomal storage disease.
Once the sugars have reached the cytoplasm, they are available for activation to sugar nucleotide
donors as described below. A small amount of GlcNAc kinase is found in lysosomal preparations
from rat liver, suggesting that GlcNAc transport and phosphorylation could be coupled. The
relative contributions made by exogenous sugar, salvage pathways, and interconversions
probably vary depending on the cell type and amount of glycoprotein synthesized. This is clearly
the situation seen in the case of CMP-Sia synthesis described later in this chapter.
Most of the work on monosaccharides and sugar nucleotide metabolism has focused on the direct
activation or interconversions of various sugars, regardless of their source. In nearly all cell
culture studies, growth media contain only glucose and serum. It is usually assumed that glucose
generates all of the other sugars, but contributions from degraded serum glycoproteins may also
be important. The relative contributions of diet, salvaging, and glucose interconversion for
glycoprotein synthesis is really not known. Recent studies suggest that some key biosynthetic
enzymes are quite restricted in their distribution among different organs.
Activation and Interconversion of Monosaccharides
The major pathways for sugar activation and monosaccharide interconversions are shown in
Figure 6.1.
Figure 6.1. Biosynthesis and interconversion of monosaccharides. The relative contributions of

each under physiological conditions are unknown. (Shadowed rectangles and shadowed ovals)
Donors; (open ovals) monosaccharides; (stars) control points. Glycogen (11 13)
Glycogen is the major storage polysaccharide in animal cells.Its biosynthesis is covered in

Chapter 13. This megadalton size molecule contains up to 100,000 glucose units, arranged in
Glcα1,4Glc repeating disaccharides with periodic α1,6Glc branches. Glycogen synthesis and
degradation (glycogenolysis) are of course highly regulated for energy utilization. Some of the
glucose used to synthesize other sugars may be derived from glycogen as well as from glucose
transported into the cell. Glycogen is synthesized by addition of single glucose units from UDP-
Glc, and it is degraded by glycogen phosphorylase. The non-ATP-dependent reaction forms Glc-
1-P, which can be used directly to form UDP-Glc or epimerized to Glc-6-P, leading to
glycolysis. Glycogen is actually an unusual glycoprotein called glycogenin.
Green fluorescent protein fused to the amino terminus of rabbit muscle glycogenin showed a
nuclear and cytosolic distribution. Disruption of the cytoskeleton with cytocholasin D changed
the distribution of glycogen within the cell, but a point mutation (K324A) in a carboxy-terminal
actin-binding motif (DNIKKKL) gave a uniform distribution throughout the cytosol that was not
affected by the drug. Although the distribution was not correlated with functional localization,
the findings suggest that the cytoskeleton may be important in glycogen metabolism.
Glucose
Glucose is the central monosaccharide in carbohydrate metabolism. It is convertible into all other
sugars needed for glycosylation (see Figure 6.1). As part of the glycolytic pathway, glucose is
converted to Glc-6-P by hexokinase and then either to Fru-6-P by phosphoglucose isomerase or
to Glc-1-P by phosphoglucomutase. Reaction of Glc-1-P with UTP forms the high-energy donor
UDP-Glc. The UDP-Glc pool is quite large, and it is used for synthesizing glycogen and for
other glucose-containing molecules such as glucosylceramide (see Chapter 9) and Dol-P-Glc
which is used in the N-glycan biosynthetic pathway (see Chapter 7).
Glucuronic Acid
UDP-GlcA is synthesized directly from UDP-Glc by oxidation at the C-6-OH, which requires
NAD. This acidic sugar is used primarily for glycosaminoglycan biosynthesis, but also for
adding GlcA to N- and O-glycans and glycolipids.
UDP-GlcA is also used for glucuronidation of bile acids and for detoxification of xenobiotic
compounds in the ER. A large class of glucuronosyl transferases are devoted to such reactions.
The addition of GlcA increases the solubility of bile acids and xenobiotics. There is some
evidence that UDP-GlcA is derived from glycogenolysis rather than gluconeogenesis.
Iduronic Acid
IdoA is the 5-epimer of GlcA, and it is also found in glycosaminoglycans. Unlike all other
monosaccharides found in glycoconjugates, IdoA is not directly synthesized from a sugar
nucleotide donor. Instead, IdoA is created by epimerizing GlcA after it has been incorporated
into the growing GAG chain (see Chapter 11).
Xylose (14, 15)
Decarboxylation of UDP-GlcA gives UDP-Xyl, which is used primarily to initiate

glycosaminoglycan synthesis (see Chapter 11), but UDP-Xyl is also found in several clotting
proteins (Chapter 12). The decarboxylation reaction to generate UDP-Xyl appears to occur in the
lumen of the ER or early in the Golgi, as well as in the cytoplasm.
Mannose (2, 16, 17)
Mannose is a key sugar used for N-linked oligosaccharide and GPI-anchor synthesis. GDP-Man
can be produced in two ways. The first is by direct phosphorylation of mannose via hexokinase.
In some invertebrates, there is a suggestion that a specific mannokinase exists. The second, and
by far the best known way, is by conversion of Fru-6-P to Man-6-P through the enzyme
phosphomannose isomerase (see Figure 6.1). Genetic loss of a majority of this enzyme produces
a potentially fatal human disease called carbohydrate-deficient glycoprotein syndrome (type 1b)
(see Chapter 32). In yeast, loss of phosphomannose isomerase is lethal. This is easy to
understand since free exogenous mannose is not commonly available, and this enzyme is the key
link between mannose and glucose. Nearly 14% of the dry weight of yeast is mannose. Both
yeast and human phosphomannose isomerase deficiencies can be rescued by providing
exogenous mannose.
Although mannose is essential, in the "honeybee syndrome" it is lethal! This curious

phenomenon arises when honeybees are allowed to feed on mannose instead of sucrose or
glucose. At first, the bees continue to behave normally for several minutes, but then they
suddenly deteriorate and die. The reason is that mannose enters the cells and is phosphorylated
by the abundant hexokinase using ATP. Since Man-6-P is now the sole energy source for the
bees, it must be converted into Fru-6-P to enter glycolysis. The problem is that honeybees have a
relatively low phosphomannose isomerase activity. This creates a bottleneck and excess Man-6-
P accumulates. It is quickly degraded by phosphatase to free mannose but is again
phosphorylated using the diminishing supply of ATP. Multiple futile cycles deplete the ATP
pool and death results. For similar reasons, very high concentrations of mannose are teratogenic
in rats. This is because during early development, the embryo relies more on glycolysis rather
than on oxidative phosphorylation. At high concentrations, mannose enters cells via the typical
glucose transporters, but once inside, the low amount of phosphomannose isomerase creates a
bottleneck, restricting energy metabolism and causing abnormal or arrested development.
Once Man-6-P is formed, it is then converted to Man-1-P by phosphomannomutase. For this

conversion, two isozymes are known in humans, and the loss of one of them produces another
form of carbohydrate-deficient glycoprotein syndrome (type 1a) that results from
underglycosylation of proteins (see Chapter 32). Of course, failure to make both Man-6-P and
Man-1-P depletes the formation of GDP-Man, the critical donor for glycoconjugate synthesis.
GDP-Man can be used directly for the formation of the lipid-linked oligosaccharide on the
cytosolic face of the ER. GDP-Man can also transfer mannose to dolichol phosphate to form
Dol-P-Man in the ER membrane. This molecule then flips to the inside of the ER where it is used
for adding the last four mannose residues to the N-linked sugar chain precursor (see Chapter 7).
Dol-P-Man is also the donor for synthesis of glycophospholipid anchors (Chapter 10) and the
recently identified C-mannosylation reaction (Chapter 12). One form of carbohydrate-deficient
glycoprotein syndrome results from defective Dol-P-Man synthesis (see Chapter 32).
Fucose (18, 19)
GDP-Fuc can be derived from GDP-Man by reduction of the CH2OH at the C-6 position of
mannose to a CH3. This reaction is catalyzed by the sequential action of two enzymes involving
three steps. In the first step, the C-4 mannose of GDP-Man is oxidized to a ketone, GDP-4-
dehydro-6-deoxy-mannose, by the enzyme GDP-Man 4,6-dehydratase along with the reduction
of NADP to NADPH. The next two reactions are catalyzed by a single polypeptide that has
epimerase and reductase activity and is well-conserved from bacteria and mammals. Thus, GDP-
4-keto-6-deoxy-mannose is epimerized at C-3 and C-5 to form GDP-4-keto-6-deoxyglucose and
then reduced with NADPH at C-4 to form GDP-Fuc (see Figure 6.2). These last two reactions
are catalyzed by a single polypeptide that is well conserved from bacteria to mammals. The first
dehydration step is feedback inhibited by GDP-Fuc. Fucose can also be used directly by
formation of Fuc-1-P and then conversion to GDP-Fuc. Mutant CHO cells that cannot convert
GDP-Man to GDP-Fuc form underfucosylated proteins, but they can be corrected with
exogenous fucose to yield normal glycoproteins. A rare human disease called leukocyte adhesion
deficiency type II (LADII), may result from reduced synthesis of GDP-Fuc from GDP-Man or
reduced efficiency of GDP-Fuc utilization. Regardless of the actual mechanism, this creates
underfucosylated glycoproteins that are especially important for the synthesis of fucose-
containing selectin ligands (see Chapter 26). Patients with LADII have chronic infections since
they cannot effectively extravasate leukocytes to the sites of inflammation (see Chapter 26). In
addition, to synthesize GDP-Fuc, the fucose transporter in mammalian cells may directly provide
fucose for biosynthesis, but the quantitative contribution of this pathway is not known. The free
fucose concentration in the blood is very low, but one LADII patient has responded to fucose
supplementation therapy. Neutrophil extravasation rapidly became normal (see Chapter 32).
Figure 6.2. Conversion of activated sugar donors. Galactose (20, 21)
Galactose can be activated to UDP-Gal in several ways. The first is by direct phosphorylation at
the 1-position, to give Gal-1-P, which can react with UTP to form UDP-Gal. Alternatively, Gal-
1-P can be converted to UDP-Gal via a uridyl transferase exchange reaction with UDP-Glc that
displaces Glc-1-P. A deficiency in the uridyl transferase activity results in a severe human
disease called galactosemia, leading to mental retardation, liver damage, and eventual death (see
Chapter 32). Finally, UDP-Gal can be formed from UDP-Glc by the NAD-dependent reaction
catalyzed by UDP-Gal-4-epimerase. The only difference between the two sugar nucleotides is at
the 4-position. In galactose, the hydroxyl group has an axial orientation and in glucose it has an
equatorial orientation. NAD first converts the hydroxyl group to a 4-keto derivative forming
NADH. In the next step, the NAD reforms and the hydroxyl group is converted to the other
monosaccharide (Figure 6.2). The same enzyme converts UDP-GalNAc to UDP-GlcNAc.
N-acetylglucosamine (22, 23)
Synthesis of UDP-GlcNAc begins with the formation of GlcN-6-P from Fru-6-P by

transamidation using glutamine as the NH2 donor. Glucosamine-6-P is then N-acetylated via
acetyl-CoA-mediated reaction to form GlcNAc-6-P and then isomerized to GlcNAc-1-P via a
1,6-bis-phosphate intermediate. Similar to the other activation reactions, GlcNAc-1-P then reacts
with UTP to form UDP-GlcNAc and pyrophosphate. Alternatively, GlcNAc can be directly
phosphorylated to form GlcNAc-6-P via a kinase that can use either GlcNAc or ManNAc. A
mutase then converts this to GlcNAc-1-P. This route may account for the efficient salvage of
GlcNAc from lysosomal degradation. GlcN can be very efficiently used for liver glycoprotein
synthesis in intact animals, showing that direct phosphorylation can be important. Not all GlcN
is necessarily used for glycoprotein synthesis. It depends on the needs of the cell. A recent study
on the enzyme glucosamine-6-P deaminase (GlcN-6-P Fru-6-P + NH4+) (GNPDA) suggests
that high-energy-requiring tissues such as neurons and transporting epithelial cells in the kidney
and intestine use this enzyme to generate Fru-6-P for glycolysis. Note that this is not simply the
reverse of the transamidation reaction using glutamine. The GNPDA reaction proceeds in the
forward direction, since the concentration of NH4+ required for GlcNH2 formation would be
extremely high. This enzyme is essentially absent from the liver where other sources of energy
are available. Thus, UDP-GlcNAc can be formed from Fru-6-P or from GlcNAc via kinase.
N-acetylgalactosamine (24)
UDP-GalNAc can arise from two routes. One is the direct reaction of GalNAc-1-P with UTP.
GalNAc-1-P is formed by a specific kinase that probably uses ATP, but it can also use ITP. This
enzyme is distinct from Gal-1-kinase. UDP-GalNAc can also be formed by epimerization of
UDP-GlcNAc using the same NAD-dependent epimerase that converts UDP-Glc to UDP-Gal.
Sialic Acids (22, 25)
The term sialic acid is the name given to a group of more than 30 different variations of the
parent compound. The modifications, including oxidation, single and multiple acetylations,
sulfation, and methylation, are discussed more fully in Chapter 15. Except for the formation of
the glycolyl derivative as an activated sugar nucleotide, all of the other modifications of sialic
acid probably occur in the Golgi after transfer to acceptors. CMP-N-acetyl (or
glycolyl)neuraminic acid is the immediate donor. Its biosynthetic pathway is more complicated
compared to those of the other activated sugars. First, UDP-GlcNAc is converted to N-
acetylmannosamine by a single enzyme that has two catalytic activities. The first involves
epimerization at the 2-position and cleavage of the UDP to yield N-acetylmannosamine. In the
next step, this enzyme acts as a kinase using ATP to form N-acetylmannosamine-6-P. In the next
step, this compound condenses with phosphoenolpyruvate to form N-acetylneuraminic acid-9-P.
The phosphate is then cleaved by a phosphatase. Activation with CTP yields CMP-N-
acetylneuraminic acid. All of the other steps occur in the cytosol, but the last step occurs in the
nucleus with subsequent export of the activated precursor to the cytoplasm.
There appears to be an alternate pathway as well. The epimerase/kinase enzyme activity can be
detected only in relatively few tissues such as the liver, salivary gland, and intestinal mucosa.
Northern blot analyses of rat tissues show that its expression is restricted to the liver, but clearly,
many other tissues contain sialylated glycoproteins and glycolipids. This finding suggests that
other pathways probably exist. Salvage of sialic acids from other glycoproteins is possible and is
known to occur, but another possibility is the epimerization of GlcNAc to ManNAc using a
widely distributed 2-epimerase. However, no ManNAc-specific kinase has been described,
although the GlcNAc kinase is known to occur in several rat tissues and can also use ManNAc to
form ManNAc-6-P.
Fructose
Although fructose is not known to occur in glycoproteins, it is a major dietary component and
plays an important part in the regulation of energy metabolism. Fructokinase in the liver
produces Fru-1-P, but this compound cannot be mutated to Fru-6-P or phosphorylated to Fru-1,6
diphosphate. Instead, Fru-1-P aldolase converts it to dihydroxyacetone phosphate (which enters
glycolysis) and glyceraldehyde. This compound must be reduced to glycerol and then
phosphorylated and oxidized to dihydroxyacetone phosphate before it is ready for glycolysis.
Sugar Nucleotide Transporters (26 29)
Once the sugar nucleotides are synthesized in the cytosol (or in the case of CMP-Sia in the
nucleus), they are topologically mislocalized, since most glycosylation occurs in the ER and
Golgi. Their negative charge prevents them from simply diffusing across membranes into these
compartments. To overcome this problem, eukaryotic cells have devised a set of nonenergy-
requiring sugar nucleotide transporters, actually antiporters, that deliver sugar nucleotides into
the lumen of these organelles with simultaneous exit of nucleotide monophosphates which must
first be derived from the nucleotide diphosphates (Figure 6.3). A large body of work has led to
our current biochemical understanding of these transport processes. Most of the studies used
isolated Golgi and ER vesicle preparations, but analysis of mutant cells with defective
transporters underscores the importance of these proteins. The Km of the transporters ranges from
1 to 10 µm. Using in vitro systems, the transporters have been shown to increase the
concentration of the sugar nucleotides within the Golgi lumen by 10 50-fold. This is usually
sufficient to reach or exceed the calculated Km of most glycosyltransferases for sugar
nucleotides.
Figure 6.3. Transporters for sugar nucleotides, PAPS, and ATP are located in the Golgi
membranes of mammals, yeast, protozoa, and plants. These proteins are actually antiporters, and
the corresponding nucleoside monophosphate is carried into the cytosol with sugar nucleotide
transport. Since most glycosylation reactions produce a nucleoside diphosphate, this requires
conversion to the nucleoside monophosphate. For PAPS, the corresponding exiting molecule is
unknown, and for ATP, it is AMP, ADP, or both. The phosphate (Pi) transporter is hypothetical.
(Reprinted, with permission, from [26] Abeijon et al. 1997[©Elsevier Science].)
Most of the antiporters are found in the Golgi, but some are also found in the ER; thus, they are
organelle-specific and their location usually corresponds to the location of the known
transferases (see Table 6.4). Nucleotide import into the Golgi is not energy-dependent or affected
by ionophores. However, it is competitively inhibited by the corresponding nucleoside mono-
and diphosphates, but not by the monosaccharides. In addition to sugar nucleotide-specific
transporters, separate transporters exist for ATP and PAPS, the donor for protein and
carbohydrate sulfation.
Table 6.4. Nucleotide transport in Golgi and ER
Nucleotide ER Golgi
CMP-Sia +++
GDP-Fuc ++++
UDP-Gal ++++
PAPS ++++
GDP-Man ++++
UDP-GlcNAc ++ ++++
UDP-GalNAc ++ ++++
UDP-Xyl ++ ++++
ATP +++ ++++
UDP-GlcA ++++ ++++
UDP-Glc ++++ +
The relative distribution of the nucleotide transporters in the ER and Golgi is indicated by the
number of plus (+) signs. A minus ( ) indicates that the transporter is not found in that
compartment. For the types of organisms that contain these nucleotide transporters, see Figure
6.3.
Glucuronidation of bile and xenobiotic compounds in the ER is consistent with the presence of
the UDP-GlcA transporter in both the ER and Golgi, and the identification of reglycosylation of
misfolded glycophorins in the ER (see Chapter 7) can explain the need for an ER UDP-Glc
transporter. The existence of UDP-GlcNAc and UDP-GalNAc transporters in the ER is more
difficult to explain, since most glycosyltransferase reactions utilizing these sugar nucleotides
occur in the Golgi apparatus. UDP-Xyl may be used to initiate GAG chain synthesis in the ER.
Since the Km of the transporters is in the 1 10 µm range, cytosolic enzymes such as O-GlcNAc
transferase or the fucosyl transferase seen in Dictyostelium (see Chapter 19) would have to
compete with the transporters for available substrate. They do so by having low micromolar Km
values.
For most glycosylation reactions, the sugar nucleotide donates the sugar, resulting in the
formation of nucleoside diphosphate, which must be converted into a monophosphate by the
nucleoside diphosphatase that occurs in the Golgi lumen. Exchange through the antiporters is
electroneutral, since the sugar nucleotide with two negative charges (one on each
phosphodiester) enters and the nucleoside with a single phosphomonoester exits. Preloading of
Golgi vesicles with UMP at pH 7.5 stimulates uptake of UDP-GlcNAc, whereas loading at a pH
of approximately 5.5, where UMP has only a single charge, was slower. In yeast, a GDPase
converts GDP to GMP. Disrupting the gene that codes for this essential type II membrane
protein reduces glycosylation of all the mannosylated glycoconjugates.
The antiporter system has the advantage of coupling the rate of sugar nucleotide utilization with
its import. However, the Golgi can have a pool of transported but unutilized sugar nucleotides
that participates in glycosylation. This can be seen in in vitro Golgi preparations that are capable
of glycosylating partially completed endogenous glycoproteins as well as freely diffusible
glycoside acceptors. The artificial acceptors enter the Golgi where they encounter
glycosyltransferases, and, in some cases, glycosylation proceeds even though no exogenous
donors have been added. Another advantage of using the antiporter system is that the precursor
monophosphate is returned to the cytosol where it becomes available for another round of
activation. This creates a highly efficient salvage system for the precursors.
Several transporters including CMP-Sia, UDP-Gal, and UDP-GlcA have been reconstituted into
proteoliposomes and used for functional assays and purification of the proteins. Several
transporters have been cloned from mammals (CMP-Sia, UDP-Gal), yeast (UDP-GlcNAc), and
protozoa (GDP-Man). All are very hydrophobic and appear to be capable of spanning the
membrane many times. They appear to be functional homodimers. Several mutant mammalian
cell lines lack specific sugar nucleotide transporters, e.g., UDP-Gal and CMP-Sia, and as a
result, they make incomplete sugar chains. However, there is some "leakiness" in such mutants.
For instance, loss of the UDP-Gal in the Golgi of mutant MDCK cells decreases the synthesis of
galactosylated glycoproteins and glycolipids and of keratan sulfate, but leaves heparan and
chondroitin sulfates unaffected. This is probably because the galactosyl transferases that
synthesize the core region tetrasaccharide common to GAG chains have low Km values for their
sugar nucleotide donors (see Chapter 11).
Theoretically, glycosylation may be controlled in part by regulating availability of sugar

nucleotides within the Golgi, presumably by regulating the transporters. At this time, the
subcompartmental location (cis, medial, trans) of the transporters in the Golgi is not known nor
are their physical relationships to the various glycosyltransferases they service. Clearly, a
functional Golgi compartment requires both the sugar nucleotide donor and acceptor with a
colocalized transferase. Most of the attention on Golgi localization has focused on
glycosyltransferases. Cloning of nucleotide transporters will probably stimulate exploration of
their localization in the Golgi as well. However, there have been few revealing studies about how
the actual glycosylation reactions occur within the Golgi. Is it more like solution chemistry or
like solid-state transfers? Are there really "soluble pools" of sugar nucleotides? There is physical
evidence for complexes containing several mannosyl transferases in yeast as well as
circumstantial evidence for association of selected glycosyltransferases in complexes in
mammalian cells, but no studies have yet been able to determine whether the transporter is part
of a complex.
Dramatic time-lapse videos of glycosyltransferase chimeras with green fluorescent protein show
that the proteins are highly mobile within the Golgi. This contrasts sharply with the static view of
transferases neatly packaged into separate compartments. In addition, emerging evidence from
cell biology suggests that the entire Golgi may be a continuum with transient interconnections.
Regardless of the conceptual constraints and assumptions about Golgi itself, glycosylation
proceeds with relatively high fidelity. A "freeze-frame" picture of potential glycosylations in the
dynamic Golgi has been developed using purified, sealed, and correctly oriented Golgi
preparations. These studies show that endogenous acceptors, sugar nucleotide transporters, and
many glycosyltransferases are located in a single compartment. Simply supplying a radioactively
labeled exogenous sugar nucleotide tracks its transport into the Golgi and incorporation of the
monosaccharides into colocalized endogenous acceptors using the various glycosyltransferases.
Interestingly, small glycosides with a single monosaccharide can substitute for endogenous
acceptors when added to permeabilized cells that have intact Golgi, but lack ATP and cytosol
required for vesicular transport. The glycosyltransferases within the Golgi use previously
transported sugar nucleotides and those delivered by transporters to glycosylate the acceptors as
they diffuse within the confines of the Golgi. Surprisingly, within a few minutes, the great
majority of these itinerant acceptors in the Golgi are fully glycosylated with up to seven
additional monosaccharides using glycosyltransferases that are thought to reside in different
Golgi compartments. This glycosylation is as extensive and even more complete than when these
glycosides are incubated with intact cells. These results suggest that the small glycosides freely
travel between Golgi compartments, perhaps using transient connections that normally restrict
the movement of cargo proteins.
Control of Sugar Nucleotide Levels (30 34)
This topic is still murky, but it may be physiologically very important. Table 6.5 shows that
several key synthetic enzymes in sugar nucleotide biosynthesis are inhibited by their final
products when tested in vitro. A human genetic disorder called sialuria validates the importance
of at least one in vitro prediction. In this condition, massive amounts of sialic acid (5 7 g/day)
are secreted into the urine along with various intermediates in the UDP-GlcNAc CMP-Sia
pathway. This was confirmed to be due to a defective feedback inhibition of the 2-
epimerase/ManNAc kinase, the first step in the pathway.
Table 6.5. Some control points for sugar nucleotide synthesis
Enzyme Inhibitor
UDP-Glc dehydrogenase UDP-Xyl

GDP-Man 4,6-dehydratase GDP-Fuc
Glutamine:fructose-6-P acetyltransferase UDP-GlcNAc
UDP-GlcNAc epimerase/kinase CMP-Sia
Most of the precursor pools turn over within a matter of a few minutes, and the sizes of various
sugar nucleotide pools have been determined by methods with different reliabilities. But even
with the best methods, it is hard to interpret the measured numbers and translate them into a clear
picture, because the relative distribution of the precursors in the cytosol and Golgi is not known.
The average cellular concentrations of sugar nucleotide precursors may not be very meaningful,
since "cytosol" is an operational definition (100,000g supernatant) that may not detect
compartments of cytoplasmic organization.
Many experiments show that providing moderate amounts (<1 mm) of glucosamine to muscle
cells increases the size of their UDP-hexosamine pools and creates the biochemical phenotype of
noninsulin-dependent diabetes (see Chapter 14). Glucosamine-incubated cells do not increase
glucose uptake or form glycogen in response to insulin. Glucosamine can induce a similar
response when given to animals. The pathway and mechanism for this effect are not known, but
finding O-GlcNAc on many regulatory proteins may suggest a link. Alternatively, since the
UDP-GlcNAc concentration is second only to ATP (1 2 mm), some of the effects could simply
be due to consumption of ATP in producing UDP-GlcNAc.
In whole-animal studies, the GDP-Fuc pool and the fucosylated glycans in the intestine can be
regulated by the diet and time of weaning. Considering that resident bacteria in the small
intestine participate in the induction of fucosylation pathways in the enterocytes, dietary
manipulation of glycosylation introduces another level of complexity that has barely been
explored. The relationship of amino acid and nucleotide metabolism to sugar nucleotide
metabolism is also potentially important, but remains largely unexplored.
Future Directions
Some of the material presented in this chapter can be found in traditional biochemistry texts.
However, the perspective presented here stresses that the production and utilization of the
various precursors needed for complex glycan synthesis are more complex than the simple
metabolic chart indicates (see Figure 6.1). Future studies should address various sources of
monosaccharides, the organization of metabolic enzymes within the cells, and the influence of
diet and environment on the metabolic control of complex sugar chain synthesis. This will be
especially important in translating the large numbers of sequenced genomes into meaningful
physiology.
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7. N-Glycans
Primary contributions to this chapter were made by J.D. Marth (HHMI, University of California
at San Diego).
THE BIOSYNTHESIS OF ASPARAGINE (N)-linked oligosaccharides (N-glycans) is described

in this chapter, with an emphasis on vertebrate systems. Included is an overview of the
mechanisms in N-glycan formation, processing, control of protein folding, and structural
diversification. Also presented is a biological overview of N-glycan structure-function
relationships that have been defined by genetic lesions in intact vertebrates.
Background
The biosynthesis of N-glycans was elucidated in the 1960s and 1970s by using cell-free systems
and lectin-resistant cell lines. A surprising finding was the requirement for production of a lipid-
linked oligosaccharide precursor structure that is transferred en bloc to nascent proteins in the
ER. N-glycan addition was noted to occur on asparagines in the sequence context Asn-X-
Ser/Thr. All eukaryotic cells produce N-glycans and have conserved the earliest biosynthetic
steps involving the synthesis of this dolichol-oligosaccharide precursor as well as several
subsequent processing reactions in the ER. These conserved processing reactions include cycles
of glucose removal and readdition, which have been found to participate in protein-folding
mechanisms. Another unexpected finding in N-glycan biosynthesis is the extent of structural
diversification in the Golgi apparatus from high-mannose N-glycans into a large repertoire of
hybrid and complex N-glycan subtypes that are secreted or positioned at the vertebrate cell
surface. Of the organisms studied, including those within the largest phylum Arthropoda, only
vertebrates appear to produce a diverse and dynamic repertoire of hybrid and complex N-glycan
subtypes (see Chapter 3). This emanates from the presence of a large number of enzymes
encoded in the vertebrate genome, which include glycosyltransferases and glycosidases that
reside in the ER and Golgi apparatus (Chapter 17). Variations in glycan structures among plants,
invertebrates, and various microbes are described elsewhere (see Chapters 20, 21, and 36).
The molecular cloning of vertebrate glycosyltransferase and glycosidase genes involved in N-

glycan biosynthesis began in the 1980s and continues to the present. The encoded enzymes show
remarkable substrate specificity and their unique expression patterns contribute to the variation
of N-glycan structures noted among particular cell types. Observations that N-glycan structures
change in a variety of cellular and organismic events, including embryogenesis, cell activation,
and cancer (see Chapters 34 and 35), have led to many hypotheses regarding N-glycan function.
With the isolation and manipulation of genes that regulate the N-glycan diversification pathways
in the intact organism, investigations on the physiologic functions of N-glycans have become
possible and productive (see Chapters 5, 32, and 33).
Synthesis of the Dolichol Oligosaccharide Precursor (1 12)
There are many excellent reviews that describe pathways in N-glycan biosynthesis. The first
events involve assembling an oligosaccharide precursor structure linked to the lipid Dol-P
(Figure 7.1). The majority of the dolichol lipid exists in cellular compartments other than the ER
and is either esterified or in the form of free dolichol, indicating that most of the dolichol lipid is
not participating in the biosynthesis of N-glycans but is involved in the formation of cellular
membranes. Evidence from multiple systems indicates that if cellular levels of Dol-P are reduced
by treatment with compactin, exogenous cholesterol, or coenzyme Q, then N-glycosylation is
significantly impaired. Abolition of N-glycosylation in intact cells and embryos has been
observed following treatment with tunicamycin, an analog of UDP-GlcNAc that blocks the first
step in dolichol oligosaccharide precursor formation by inhibition of GlcNAc-1-
phosphotransferase activity.
Figure 7.1. Structure of the Dol-P lipid used in constructing the dolichol oligosaccharide
precursor in N-linked oligosaccharide biosynthesis. The number of isoprene repeats (n) varies
between 15 and 19.
The dolichol oligosaccharide precursor consists of dolichol lipid bearing a pyrophosphate

linkage to an oligosaccharide composed of 14 specific monosaccharide linkages. The structure
has been conserved among all eukaryotes (see Chapter 6). The cellular topology of the
biosynthetic pathway is also remarkable. Several enzymes operating in its biosynthesis have not
yet been isolated and characterized, although their activities appear evident by inference to
precursor structures experimentally defined (see below). Cytosolic and luminal fractions have
both been found to contain various intermediates in dolichol oligosaccharide biosynthesis,
suggesting that certain assembly reactions occur at one or the other side of the membrane.
Various ER and Golgi sugar nucleotide transporters exist that serve to transport various
monosaccharide donors across the membrane bilayer for glycan biosynthesis by luminal
glycosyltransferases. Recent molecular cloning and characterization of these transporters
indicate that they are essential in the formation of various glycans bearing the relevant
monosaccharide (see Chapter 6). In the production of the dolichol oligosaccharide precursor,
however, these transporters are not necessary.
Biochemical analyses of dolichol oligosaccharide intermediates have provided a relatively clear

outline of the details regarding assembly of the dolichol oligosaccharide precursor (Figure 7.2).
Evidence from multiple studies involving protease and glycosidase sensitivity indicates that the
initial linkages involving the two core GlcNAc monosaccharides (inhibited by tunicamycin) and
the first five mannose residues occur on the cytosolic side of the ER membrane. The
monosaccharide donors include UDP-GlcNAc and GDP-Man for the first seven linkage
reactions that occur on the cytosolic side of the ER membrane. In the first two steps, GlcNAc
residues are added to the Dol-P lipid by GlcNAc-1-phosphotransferase and then by a GlcNAc-
transferase. Next, five mannose residues are added sequentially using GDP-Man as donor (step
III). By a mechanism not fully understood, the Man5GlcNAc2-Dol precursor must "flip" across
the membrane bilayer to become oriented in the lumen of the ER. Next, four mannoses are added
in rapid succession using the Dol-P-Man donor (step IV). The assembly of the dolichol
oligosaccharide precursor is completed with the addition of three glucose residues donated by
Dol-P-Glc (step V). It appears that Dol-P-Man and Dol-P-Glc are also formed on the
cytoplasmic side of the ER and then are flipped across the bilayer to become available to
enzymes in the lumen of the ER. The completed dolichol lipid-linked oligosaccharide precursor
is now ready for transfer to asparagine residues on nascently translated proteins.
Figure 7.2. Biosynthesis of the dolichol oligosaccharide precursor.
Transfer of the Dolichol Oligosaccharide Precursor to Nascent

Proteins (13 17)
A multisubunit protein complex in the ER membrane of eukaryotes transfers the lipid-linked
oligosaccharide precursor to asparagine residues on nascently translated proteins. This complex
is termed the oligosaccharyltransferase (OST). The composition of the OST complex remains
under investigation. At least nine nonidentical subunits comprise the heteromeric OST complex
in yeast, where most data have been gathered from mutational studies. Members of the yeast
OST complex (homologous proteins in higher eukaryotes in parentheses) include Ost1p
(ribophorin I), Stt3p, Wbp1p (OST48), Ost3p, Ost6p, Swp1p (ribophorin II), Ost2p (DAD1),
Ost5p, and Ost4p. Ost1p is essential for OST activity. Proteins associated with mammalian OST
include DAD1 (defender against apoptotic cell death), which was initially isolated as a negative
regulator of programmed cell death (apoptosis). All OST subunits are transmembrane proteins,
with one to eight transmembrane domain regions. In a mechanism not fully understood, the OST
complex binds to the lipid-linked oligosaccharide and transfers it to nascently translated proteins
by cleavage of the high-energy GlcNAc-P bond, releasing Dol-P-P in the process (Figure 7.3).
Early studies of peptides modified by N-glycosylation resolved that the minimal sequence
requirement surrounding the acceptor asparagine is Asn-X-Thr/Ser, where X is any amino acid
except for proline. (In some rare cases, Asn-X-Cys is also utilized for N-glycosylation.) Recent
studies of a model rabies virus glycoprotein suggest that the identity of the amino acid following
the Ser/Thr residues can affect the efficiency of glycosylation. Only about 30% of potential N-
glycosylation sites appear to be used among all polypeptides bearing this consensus sequence,
although this percentage can vary significantly among specific glycoproteins. Studies with model
glycoproteins indicate that transfer of the dolichol oligosaccharide precursor occurs
approximately 30 amino acids from the active ribosome, consistent with results involving
protease treatment of microsomal fractions and spacing requirements for transfer in the luminal
compartment of the ER.
Figure 7.3. The OST complex residing in the ER membrane transfers the dolichol
oligosaccharide precursor to asparagine residues on nascently translated proteins. In yeast, at
least nine distinct subunits comprise the complex, including Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p,
Swp1p, Ost2p, Ost5p, and Ost4p. Ribosomes (60S and 40S subunits) bound to an mRNA and
involved in translation are depicted on the cytosolic side of the ER. The signal recognition
particle (SRP) is not shown.
Initial Steps in N-Glycan Processing and Control of Protein

Folding (18 29)
Following the covalent attachment of the oligosaccharide from the dolichol oligosaccharide
precursor to asparagine residues, a series of processing reactions occurs. The first several steps
appear to be conserved among all eukaryotic cells and are now known to play key roles in
regulating vertebrate glycoprotein folding and lysosomal trafficking.
Glucosidases I and II act first on the protein-linked oligosaccharide precursor to remove all three
glucoses sequentially. These glycosidases are present in the lumen of the ER with glucosidase I
acting specifically on the single α1 2-linked terminal glucose. Where it has been examined (e.g.,
using tunicamycin), the rate-limiting step in N-glycosylated protein secretion appears to be due
in large part to the time a glycoprotein spends in the ER. Removal of the first two outer glucoses
occurs rapidly and is followed minutes later by removal of the third. Removal of glucose
residues is associated with protein folding mechanisms, and this process contributes to the ER
retention time of a given glycoprotein (Figure 7.4). Improperly folded proteins are
reglucosylated by an α-glucosyltransferase located in the lumen of the ER, which acts on the
Man9GlcNAc2-Asn oligosaccharide of improperly folded proteins. The glucosyltransferase may
function as a sensor for improperly folded proteins; however, the nature of this sensor is
presently unresolved. Re-glucosylated N-glycans are retained in the ER where they are either
refolded into a proper conformation, or they are deglucosylated and degraded.
An understanding of how glucose-bearing N-glycan precursors modulate protein folding began

with the discovery of molecular chaperones that bind to newly translated proteins in the ER. One
such chaperone, termed calnexin, was first identified in association with the signal sequence
receptor (SSRα) and also with newly translated major histocompatibility complex class I heavy
chain. Subsequent studies indicated that calnexin selectively binds to newly synthesized proteins
that are incompletely folded. Moreover, it was determined that calnexin is a lectin and binds
specifically to glycoproteins in the secretory pathway bearing N-glycans, but not to
nonglycosylated secreted proteins. In studies to refine the lectin-binding specificity, the presence
of the innermost glucose-linked α3 to mannose was found to be crucial.
The major points in the stepwise mechanism by which glucosidase I, glucosidase II, calnexin,
and α-glucosyltransferase act are relatively clear. The initiating event is the processing of the N-
glycan precursor by glucosidases I and II to yield Glc1Man9GlcNAc2. Studies using ribonuclease
B as a model glycoprotein indicate that calnexin binds independently of protein conformation by
virtue of the single remaining α1 3-linked glucose on the Glc1Man8GlcNAc2-Asn
oligosaccharide structure and that this binding is in a dynamic state of equilibrium. Calnexin
appears to be acting purely as a lectin and may be best described as a retention factor in the
protein-folding pathway involving molecular chaperones. In this model, calnexin binding does
not prevent protein folding, but rather acts as a "docking" molecule during the protein-folding
process.
A second lectin with partially overlapping specificity has been identified and termed calreticulin,
which exists as a soluble lectin in the lumen of the ER. Both calnexin and calreticulin appear to
promote the proper folding of N-glycans in part by inhibiting inappropriate or premature protein
oligomerization and by reducing N-glycan degradation. Interestingly, the specificity of N-
glycans bound appears somewhat different between calnexin and calreticulin; however, the
biological relevance of this is presently unclear. A few glucose-containing N-glycan precursors
have been occasionally observed in the Golgi of some cells, possibly having escaped regulation
by this pathway. However, an endo-mannosidase activity is able to act on these precursors,
thereby generating the Man8GlcNAc2-Asn oligosaccharide structure that can then continue in the
N-glycan processing and secretory pathways (see below). In cells treated with glucosidase
inhibitors castanospermine and 1-deoxynojirimycin, or deficient in either glucosidase I or II,
calnexin does not associate with glycan substrates, and increased misfolding of certain proteins
has been observed in some cases. It has been suggested that only a subset of glycoproteins is
conformationally affected by loss of calnexin function. It is also possible that only a subset of
cellular glycoproteins may be influenced during folding in the ER by the role of glucosylation
and deglucosylation as outlined above.
Figure 7.4. Calnexin functions during glycoprotein folding in the ER. With removal of glucose
residues by glucosidase (Glcase) I and II, the glycoprotein either is properly folded and ready for
further processing or is reglucosylated by a glucosyltransferase (Glc-T). Calnexin binds
preferentially to the glucose α1 3 linked to underlying mannose and retains the glycoprotein in
the ER for folding. (Modified, with permission, from Hebert et al. 1995 [© Cell Press].)
N-Glycan Processing and Trafficking Involving Terminal

Mannose Linkages (30 34)
Following glucose trimming and release from the ER, N-glycans become available for
glycosidase reactions in the ER and Golgi. These N-glycans are referred to as the high-mannose
subtype, indicating that they terminate in unsubstituted mannose residues. Following the
production of the Man8GlcNAc2-Asn structure in the ER, this N-glycan is found in the cis-Golgi
where two avenues exist in the processing pathway (Figure 7.5). On certain glycoproteins, the
Man8GlcNAc2-Asn N-glycan is modified by a GlcNAc-phosphotransferase, and the GlcNAc
residues are subsequently removed yielding Man-6-P. Although the precise location in the
ER/Golgi of these reactions is not presently established, this modification is the key structural
determinant in targeting glycoproteins to the lysosomal compartment and forms the basis of the
first human malady to be recognized as a defect in glycosylation (I-cell disease, Chapter 23).
In most cases, however, distinct α-mannosidase enzymes located in the ER and Golgi
sequentially process the high-mannose N-glycans (Figure 7.5). These enzymes are termed class I
α-mannosidases and include α-mannosidase 1A and 1B which act specifically on α1 2-linked
mannose residues. Although each is capable of generating the Man5GlcNAc2-Asn structure from
the Man9-containing precursor in vitro, differences in their subcellular distributions exist. N-
glycan processing in mammals is initially similar to that in yeast in that a Man8GlcNAc2-Asn
structure is generated from the Man9-GlcNAc2-Asn structure in the ER by removal of an α1 2-
linked mannose on the middle chain. Next, and in contrast with vertebrate cells, yeast add
mannose to this structure to produce high-mannose N-glycans with up to 15 mannose residues.
In vertebrates, cell surface and secreted high-mannose N-glycans (Man9 and smaller) can
normally be found, generally at low levels, as most often processing has resulted in a
Man5GlcNAc2-Asn glycan that becomes a substrate in the Golgi for the diversification of
extracellular N-glycans (see below).
An exception to the above processing reactions exists as the result of a unique endo-mannosidase
in vertebrates that acts on glucosylated N-glycan precursors in the Golgi that have escaped
glucosidase action in the ER. The product of the endo-mannosidase reaction is Man8 3GlcNAc2-
Asn, and this is the enzyme that may account for the inability of glucosidase inhibitors to
completely block the production of further processing events in N-glycan diversification.
Figure 7.5. Processing of vertebrate N-linked oligosaccharides in the ER and Golgi. Most N-
glycan processing takes place through the action of an ER-localized α-mannosidase I followed
by a Golgi-resident isozyme. Some that have escaped into the Golgi with a Glc1Man5GlcNAc2-
Asn structure are acted upon by an endo-α-mannosidase. Glycoproteins bound for the lysosome
are modified by a GlcNAc phosphotransferase (GlcNAc-PT) and subsequently a GlcNAcase to
carry the Man-6-P signal.
The Diversification of N-Glycans (35 42)
An understanding of substrate specificities regarding the glycosyltransferases and glycosidases

acting in N-glycan biosynthesis has been obtained during the last two decades by analyses of
lectin-resistant cell lines deficient in specific glycosyltransferases and glycosidases, as well as
from the use of cell-free systems that process N-glycans in vitro. These specificities involve the
oligosaccharide moieties from the previous enzymatic step. The processed high-mannose
Man5GlcNAc2-Asn N-glycan serves as a substrate for the diversification of N-glycans in the
Golgi. Extracellular N-glycans in vertebrates exist as high-mannose, hybrid, or complex
subtypes (Figure 7.6). Hybrid structures are defined as those with both substituted (GlcNAc
linkage) and unsubstituted mannose residues. Complex N-glycans refer to those in which both
the α3- and α6-linked mannose residues are substituted with GlcNAc moieties. When total N-
glycans are analyzed from various cells, most vertebrate extracellular N-glycans are found to be
of the complex subtype.
The first enzyme needed in building diverse N-glycan structures is termed GlcNAcT-I and is
encoded by a single gene in mammals (Mgat1). GlcNAcT-I adds a GlcNAc in β1 2 linkage to
produce a hybrid N-glycan structure that is the substrate for α-mannosidase II activity. α-
Mannosidase II acts specifically on the GlcNAc1Man5GlcNAc2-Asn hybrid N-glycan in the
medial Golgi to remove the α1 3-linked and α1 6-linked mannose residues as depicted (Figure
7.6). The resulting processed hybrid N-glycan product (GlcNAc1Man3GlcNAc2-Asn) is the
specific substrate for the Mgat2-encoded enzyme GlcNAcT-II that catalyzes the conversion of
hybrid to complex N-glycans. It was initially thought that the route to complex N-glycans always
required α-mannosidase II processing; however, studies of α-mannosidase-II-deficient mice
have indicated the presence of an alternate pathway independent of α-mannosidase II function.
The candidate enzyme in this alternate pathway is a distinct α-mannosidase found enriched in
Golgi fractions (termed α-mannosidase III) that acts on the processed high-mannose N-glycan
Man5GlcNAc2-Asn, producing Man3GlcNAc2-Asn. This latter N-glycan is also found to be an
excellent substrate for GlcNAcT-I in vitro, yielding GlcNAc1Man3GlcNAc2-Asn by this alternate
route.
Figure 7.6. Vertebrate N-glycan diversification in the Golgi as shown generates three N-glycan
subtypes: high-mannose, hybrid, and complex. Most secreted and cell surface N-glycans are of
the complex type and are generated by one of two possible routes. α1 6 fucose can be added
earlier than indicated. The vertical arrows depict locations of branch formation in N-glycan
diversification, not all of which occur on any single N-glycan.
N-glycans of the hybrid and complex type may exist with two or more GlcNAc-bearing branches
that are referred to as antennae. In forming multi-antennary N-glycan structures, GlcNAc
residues may be added to the trimannosyl core by six different GlcNAc transferases (I VI)
(Figure 7.7). Up to five branches have been observed on N-glycans of some vertebrate
glycoproteins. GlcNAcT-I and GlcNAcT-II must both act to produce a complex N-glycan. All
GlcNAc residues except that added by GlcNAcT-III can be extended with additional
monosaccharide linkages. Hybrid N-glycans can be mono- or biantennary; the latter have two
branches on the α1 3-linked mannose as a result of GlcNAcT-I and GlcNAcT-IV action. Some
reactions preclude others, so that only a subset of linkages performed by the GlcNAc transferases
can occur on any one N-glycan structure. This may be due to competition between enzymes for
the same substrate. For example, GlcNAcT-III can act on the hybrid N-glycan
GlcNAc1Man5GlcNAc2-Asn, and if it does, α-mannosidase II cannot cleave the two outer
mannose residues; thus, the N-glycan is forever of the "unprocessed hybrid" subtype. GlcNAcT-
IV branching is inhibited by GlcNAcT-III action but appears optimal on N-glycans bearing the
GlcNAcT-V branch. GlcNAcT-V requires the prior activity of GlcNAcT-II. GlcNAcT-III and
GlcNAcT-V are sometimes exclusive, as the action of GlcNAcT-III inhibits the action of
GlcNAcT-V. GlcNAcT-VI action is uncommon, but it may require prior branching by
GlcNAcT-II and GlcNAcT-V.
Figure 7.7. UDP-GlcNAc transferases isolated and characterized in N-glycan branching and the
GDP-α1 6 fucosyltransferase involved in core fucosylation in animal cells. Not all reactions
shown occur on any given N-glycan as some glycosyltransferases compete for identical substrate
and products may inhibit the action of other glycosyltransferases (see text).
Core fucosylation is a common structural feature among N-glycans. After processing reactions in
the ER and early Golgi, many vertebrate N-glycans are modified by a fucosyltransferase that
adds fucose in an α1 6 linkage to the GlcNAc residue that is linked to asparagine (Figures 7.6
and 7.7). This modification is found on hybrid and complex N-glycan subtypes following
GlcNAcT-I action and is inhibited by the bisecting GlcNAc added by GlcNAcT-III. Another
core fucosylation reaction commonly occurs in plant and invertebrate N-glycans in which fucose
is added in α1 3 linkage also to the asparagine-linked GlcNAc residue (see Chapter 20).
Multiple N-glycosylation sites on the same protein may contain different glycan structures, a
finding referred to as microheterogeneity. It appears that factors other than protein sequence may
influence N-glycan diversification. Such factors may include sugar nucleotide metabolism,
transport rates in the ER and Golgi, and the localization of glycosyltransferases in the "assembly
line" model of their action in the Golgi which can determine which enzymes encounter glycan
substrates first. As the N-glycan transits through the medial- and trans-Golgi, it becomes
available to glycosyltransferases increasingly localized toward the end of the assembly line and
which therefore control more distal structural modifications (e.g., the sialyltransferases and
sulfotransferases in the trans-Golgi; see Chapter 15). Therefore, varied expression levels of
glycosyltransferases can affect the repertoire of N-glycan structures produced in a given cell
type. Some glycosyltransferase and glycosidase genes contain promoter regions bearing
transcription-factor-binding elements that function in growth regulatory and oncogene
transformation pathways, including motifs used by the ets gene family (see Chapters 34 and 35).
Another mechanism controlling N-glycan diversity may include cellular regulators of
glycosyltransferase activity. For example, α-lactalbumin in milk has been found to alter the
acceptor specificity of a β1 4 galactosyltransferase to recognize glucose instead of N-
acetylglucosamine (see Chapter 17).
The General Biology of N-Glycans (43 70)
Variation in N-glycan diversity occurs during vertebrate embryogenesis, cell activation,

morphogenesis, cell cycle entry, and cellular transformation by oncogenes (see Chapters 34 and
35). In early studies to define N-glycan function, tunicamycin treatment or site-directed
mutagenesis of the Asn-X-Ser/Thr motif was accomplished, precluding all N-glycan formation.
It is now known that the oligosaccharide precursor and core structure can be necessary early in
protein folding in the ER, thus precluding an analysis of specific linkages in N-glycan function.
Nevertheless, those proteins still secreted may exhibit alterations in half-life in serum (e.g.,
erythropoietin), and such proteins may also show aberrant or defective binding properties. An
interesting example of such a role for an N-glycan involves a sulfated GalNAc residue present on
hormones including lutropin, thyroid-stimulating hormone, and pro-opiomelanocortin (see
Chapter 16). This unique N-glycan structure controls glycoprotein half-life. A receptor for
sulfated GalNAc is located in the liver endothelium and rapidly removes glycoproteins bearing
this N-glycan from circulation. This is an example of a glycan structure on an extremely
restricted subset of cellular glycoproteins that appears focused to provide a specific function.
Most N-glycan functions have been difficult to detect in cultured animal cells, however, as
lectin-resistant cell lines that are deficient in specific glycosyltransferases and glycosidases are
commonly viable and do not provide good clues of glycan function (see Chapter 31). Therefore,
studies of enzyme deficiencies in intact organisms have become a focus for understanding N-
glycan function.
Both naturally occurring and experimentally induced mutations in N-glycan biosynthesis have
been identified and studied in vertebrates (Table 7.1). The first recognized defect involved
absence of the Man-6-P signal resulting in loss of lysosomal trafficking, and a severe syndrome
termed I-cell disease (see Chapter 23). Some naturally occurring variations are species-specific,
such as the α1 3Gal terminal structure. Its induced deficiency causes cataracts in mice and
generates a major xenotransplantation barrier in humans. The carbohydrate-deficient
glycoprotein syndromes (CDGS) comprise a number of relatively severe genetic diseases in
humans (see Chapter 32). These syndromes are caused by incomplete or failed production in
early steps of N-glycan biosynthesis. As a result, glycoproteins exist with fewer Asn-X-Ser/Thr
sequons bearing N-glycan moieties. Several CDGS types exist and have been termed types I V,
with subtypes being present (e.g., Ia and Ib, see Table 7.1). All CDGS typing thus far has been
based on the isoelectric focusing pattern of serum transferrin obtained in the clinic. A unique
transferrin pattern representing an abnormal N-glycan profile can be characteristic of more than
one specific enzyme deficiency. The etiology and nomenclature of CDGS type I variants are still
evolving. All cases of CDGS type I have been found to be deficiencies in enzymes operating
early in the production of the dolichol oligosaccharide precursor, and the syndromes differ
somewhat in their clinical presentation. This may be due to the nature of the structural defect in
the precursor and how well it is able to be attached and processed in the ER and early Golgi.
However, all CDGS type I cases present as childhood diseases with failure to thrive.
Interestingly, phosphomannose isomerase deficiency (often termed type 1b) is clinically cured
by mannose ingestion. The defect in CDGS type II involves loss of Mgat2 gene function with an
absence of GlcNAcT-II activity and complex N-glycans. Few patients presently have been
identified and they are more severely affected than CDGS type I individuals. Symptoms are
many and include mental retardation, locomotor dysfunction, and susceptibility to infection.
Genetic diseases of glycosylation in humans continue to be found as medical awareness
increases.
Table 7.1. Enzyme deficiencies and effects in vertebrate N-glycan biosynthesis
Enzyme Species Phenotype/Disease
GlcNAc-1- mouse embryonic lethality (E4.5)

phosphotransferase (GPT)
Phosphomannomutase-2 human CDGS type Ia; multiple defects including thrombosis
(PMM-2) and neurological manifestations
Phosphomannose isomerase human CDGS type Ib, multiple defects including hypoglycemia,
(PMI) thrombosis, and protein losing enteropathy
Dol-P-Man synthase human CDGS type IV
Glucosyltransferase-1 human CDGS type Ic
Lysosomal:GlcNAc- human I-cell disease
phospho-T
GlcNAcT-I mouse embryonic lethality (E9.5) with defects in heart
development, neural tube formation, and vascularization
α-Mannosidase IIa mouse dyserythropoiesis and glomerulonephritis
human CDA type II (?)
(?)
GlcNAcT-II mouse CDGS type II syndrome with frequent postnatal lethality
human CDGS type II
GlcNAcT-III mouse viable/under study
α1 3 galactosyltransferase mouse cataracts
(αGalT)
human xenotransplantation barrier
β1 4 GalTb
mouse multiple defects including epithelial and endocrine
abnormalities with frequent postnatal lethality
GlcNAcT-V mouse immune dysfunction and intestinal hyperplasia
FucT-IVb mouse partial inflammation response deficit, collaboration with
FucT-VII
FucT-VIIb mouse general leukocytosis, lymphoid homing defect, and
inflammatory response deficit
ST3Gal-III mouse viable/under study
ST8Sia-II (STX)b mouse viable/under study
ST3Gal-IVb mouse viable/under study
ST6Gal-I mouse immunodeficiency with attenuated B-cell function
a
Examples of α-mannosidase II deficiency in human CDA type II are rare and are not presently
linked to germ line mutations in the gene for α-mannosidase II.
b
These glycosyltransferases may not act only on N-linked oligosaccharides.
Experimentally induced glycosyltransferase and glycosidase deficiencies in the intact organism
are providing much information on N-glycan function (see Chapter 33). Mouse embryos
deficient in Mgat1-gene-encoded GlcNAcT-I die between embryonic days 9 and 10 (equivalent
to the end of the first trimester in human gestation time). In comparison, the GlcNAcT-I-
deficient Lec 1 CHO cell line is viable and proliferates normally in vitro. Morphogenic
abnormalities were present in embryos lacking GlcNAcT-I, including defects in neural tube
formation, situs inversus of the heart, and impaired vascularization. Two examples of embryonic
lethality exist in models of N-glycan biosynthetic deficiency as of this writing. The other is a
deficiency in GlcNAc-1-phosphotransferase which is required to build the dolichol
oligosaccharide precursor and without which N-glycosylation cannot occur. Both mutations
inhibit early biosynthetic steps where no overlap exists with another isozyme, and thus the
glycan product cannot be made by an alternate pathway. With other studies, the presence of
alternate pathways may be evident. Studies of mice lacking α-mannosidase II revealed that this
enzyme is essential for complex N-glycan biosynthesis only in erythroid cell types where a
dyserythropoiesis develops. Such studies may aid in understanding the etiology of some cases of
human CDA type II where this enzyme is sometimes deficient.
Hematopoiesis, immune function, and inflammation responses are frequently affected in

vertebrates lacking specific extracellular N-glycan linkages. For example, loss of FucT-VII
results in a profound leukocytosis with deficits in inflammatory responses and lymphoid homing
to the lymph nodes (see Chapter 26). This appears to reflect the essential role of selectins and
their carbohydrate ligands in regulating these processes. Genetic deficiency of the ST6Gal-I
sialyltransferase ablated formation of the Sia6LacNAc trisaccharide (Siaα2-6Galβ1 4GlcNAc-)
and resulted in an immunodeficiency with defective B-lymphocyte activation and attenuated
antibody production upon immunization. Such model systems provide a means to investigate
how glycans are capable of modulating biological processes and will likely lead to an
understanding as to why the hundreds of glycosyltransferase and glycosidase genes have been
conserved throughout vertebrate phylogeny.
Future Directions
As N-glycan biosynthesis is becoming relatively well resolved, studies of induced and sporadic
deficiencies of N-glycan biosynthesis in intact organisms will provide a rich source of
information on the physiologic processes in which N-glycans are involved. Such studies may
further disclose the presence of alternate biosynthetic pathways, or additional isozymes, present
in various cell types. N-glycan function may also encompass aspects of pathogen-host
interactions during infection and the immune responses that normally follow (see Chapters 3, 28,
33, and 36). Addressing the mechanism of how N-glycans regulate physiology will involve
identifying those glycoproteins bearing the targeted or modified glycan structure, and assessing
the function of these altered glycoconjugates. Recent studies indicate that a restricted set of
glycoproteins may be generated in an organism from any one glycosyltransferase, especially if
that enzyme is part of an isozyme family and is specifically expressed in certain cell types.
Structural analyses of oligosaccharides associated with mutant genotypes and phenotypes will be
necessary in fully characterizing N-glycan structure-function relationships. Understanding the
role of N-glycans in physiology is of increasing relevance to cellular biologists in various
disciplines who study glycoproteins yet lack information regarding the function of the attached
glycans.
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8. O-Glycans
at San Diego).
THE BIOSYNTHESIS AND BIOLOGY OF SERINE/THREONINE (O)-linked glycans (O-

glycans) involving the GalNAc-peptide linkage are detailed in this chapter with an emphasis on
vertebrate systems. Pathways involving the formation of the various O-glycan core structures are
further indicated. This chapter also provides an overview of the biological functions of O-
glycans.
Background (1 11)
The modification of serine or threonine residues on proteins by addition of a GalNAc residue

results in an O-linked oligosaccharide or O-glycan. This definition is thus distinct from other
types of protein glycosylation at serine and threonine residues (see below and Chapters 11 14).
O-glycan biosynthesis is simpler than asparagine (N)-linked oligosaccharide generation in that a
lipid-linked oligosaccharide precursor for transfer to protein is not required. The initiating event
is the addition of the monosaccharide GalNAc (from UDP-GalNAc) to serine and threonine
residues catalyzed by a polypeptide GalNAc transferase (GalNAcT). Also in contrast to N-
glycosylation, a consensus sequence for GalNAc addition to polypeptides has not been found,
although predictive algorithms do exist.
Many O-glycans are extended into long chains with variable termini that may be similar to the
termini of N-glycans (see Chapter 16). However, O-glycans are less branched than most N-
glycans and are commonly biantennary structures. O-glycosylation can result in the formation of
mucin-type molecules (Figure 8.1). Mucins are defined as cell surface or secreted glycoproteins
with large numbers of clustered O-glycans. Many mucins cross-link in solution by disulfide
bonding and this can promote gelation. The clustering of O-glycans on mucins is due in part to
the presence of a large number of serine and threonine residues in an uncharged and often
proline-rich peptide context (the apomucin). As a result, mucins are generally constrained in
extended peptide conformations. Mucin expression is enriched in epithelial cells specialized for
mucus production and residing at the interface with the external environment. Not all O-glycans
are found on classical mucins, as some proteins contain relatively few scattered O-glycans that
can be either short chains with few residues or elongated bi-antennary structures. In fact, on
some cells, such as certain circulating blood cells, O-glycans may be as abundant as N-glycans.
Figure 8.1. Human small intestinal mucin. Large numbers of O-glycan chains (wavy lines) are
found throughout the polypeptide (encoded MUC2 gene), also termed the apomucin. A few N-
glycosylation sites are indicated (tridents). Disulfide bridges may occur in an intra- or
intermolecular fashion. (Reprinted with permission, from [56] Toribara et al. 1991.)
O-Glycans as Compared to Other Types of Serine and

Threonine Glycosylation
Serine and threonine glycosylation of proteins occurs in the secretory pathway of all eukaryotic
cells studied. O-glycosylation as used in this book refers to a GalNAc monosaccharide α-linked
to the hydroxyl group (the "O" linkage) of serine or threonine residues on proteins. However,
other types of glycosylation also involve modification of serine and threonine residues in
proteins. For example, many intracellular proteins are modified by O-linked GlcNAc addition at
serine and threonine residues that are also sites of phosphorylation (see Chapter 13). Yeast
commonly glycosylate serine by adding mannose, and vertebrate cells can produce both O-linked
fucose and O-linked mannose (see Chapter 12). Plant glycosylation of serine and threonine
residues includes GalNAc addition but is also more varied in comparison (Chapter 20).
Glycosylation involving linkage of xylose to serine residues initiates proteoglycan formation
(Chapter 11). In contrast to these modifications, the formation of vertebrate O-glycans as defined
herein requires the activity of a polypeptide GalNAcT.
Predicting Sites of O-Glycosylation (12 20)
Polypeptide GalNAcT activity in vertebrates is widespread and can be detected in cell extracts
using acceptors that include deglycosylated mucins or various peptide sequences. In efforts to
define a sequence motif directing O-glycan formation, amino acid sequences surrounding known
sites of O-glycosylation have been compared. No consensus sequence has yet emerged; however,
some predictive ability has been achieved. This ability has emerged in part from comparative
studies, including the establishment of a database (O-Glycbase) containing the analysis of known
O-glycosylation sites among various proteins.
A preponderance of adjacent proline residues has been associated with sites of O-glycosylation.
Enrichment for proline residues is noted at the -1 and +3 positions. Charged residues are not
favored at the -1 and +3 positions. The prolines appear to influence protein conformation by
breaking helix formation and promoting the formation of β turns and β sheets. It is at the
predicted β turns where most O-glycosylation is found. Alanine, serine, and threonine are
commonly found adjacent to the glycosylated residue. It would appear that charge distribution is
a crucial factor and not charge per se. For example, acidic residues substituted at the -2, +1, and
+2 positions did not reduce the O-glycosylation of von Willebrand factor.
A Family of Polypeptide GalNAc Transferases (21 28)
Purification and characterization of polypeptide GalNAcT activity was first achieved from
bovine sources which led to the cloning and characterization of a gene encoding polypeptide
GalNAcT activity, termed polypeptide GalNAcT-1. The deduced amino acid sequence of bovine
polypeptide GalNAcT-1 did not exhibit much homology with other characterized
glycosyltransferases, although the enzyme, like all glycosyltransferases, appeared to be a type II
transmembrane protein with a small cytoplasmic-oriented segment. RNA expression for
polypeptide GalNAcT-1 appeared to be widespread and abundant in the human and the mouse,
as would be expected considering the ubiquitous presence of O-glycans among vertebrate cell
types. However, subsequent studies have revealed that at least eight homologous polypeptide
GalNAcT genes are present in a variety of organisms (polypeptide GalNAcT-1-8; Figure 8.2).
Most, if not all, polypeptide GalNAcT isozymes share some structural features at the catalytic
site, and O-glycan formation can be abrogated in various cell types (without major deleterious
consequences to the cell) by the addition of the substrate analog benzyl-αGalNAc.
Figure 8.2. Multiple polypeptide GalNAcTs initiate O-glycosylation in all multicellular

organisms studied. At least eight highly homologous genes exist in the genomes of
Caenorhabditis elegans, Mus musculus, and Homo sapiens. (Adapted, with permission, from
[55] Marth 1996 [© Oxford University Press].)
Understanding the functions of O-glycans will likely require an understanding of the reason for
the large number of genes present that can each initiate O-glycan biosynthesis. They are
generally homologous to each other, with some evidence for highly related pairs. At least four
mammalian isozymes (polypeptide GalNAcT-1-4) have been assayed in recombinant studies in
vitro and have been found to have polypeptide GalNAcT activity. Of 11 homologous genes
found in Caenorhabditis elegans, at least 5 have been shown to encode polypeptide GalNAcT
activity in vitro. In amino acid sequence comparisons of polypeptide GalNAcTs from multiple
mammalian species, homologs generally exhibit between 97% and 100% identity. Nucleic acid
homology is also substantial among homologs usually at the level of 95% identity within the
coding sequence. In isozyme comparisons among human sequences, polypeptide GalNAcT-2
and polypeptide GalNAcT-3 exhibit 60% and 67% amino acid identity, respectively, to
polypeptide GalNAcT-1 in the catalytic domain, and only 44% and 49% identity, respectively,
throughout the entire amino acid sequence. However, the mouse polypeptide GalNAcT-8
sequence thus far analyzed has over 93% amino acid identity with the corresponding region of
mouse polypeptide GalNAcT-1. Additionally, two processed polypeptide GalNAcT-1
pseudogenes have been found thus far, one in human DNA and a structurally distinct pseudogene
in the mouse germ line that harbors various mutations including frameshift and termination
signals.
How many functional polypeptide GalNAcTs are there? An estimate suggests the presence of at
least eight enzymes encoded in the mammalian genome. RNA expression studies indicate that
polypeptide GalNAcT-1 is found ubiquitously in vertebrate cells, whereas other polypeptide
GalNAcT genes are generally expressed in a subset of tissues and cell types. Substrate
preference studies using recombinant enzymes and peptide acceptors in vitro have shown
differences among isozymes. However, preferences for GalNAc transfer to either serine or
threonine are not obvious at this time.
The large number of polypeptide GalNAcTs may reflect individual roles in tissue- and cell-type-
specific glycosylation of one or few glycoproteins. It is also possible that the various polypeptide
GalNAc transferase isozymes are differentially located in subcellular compartments including
the ER and Golgi. This might then explain the apparent contradictory observations that indicate
that both the ER and Golgi are initial sites of O-glycan formation.
O-Glycan Diversification in Vertebrates: Core Subtype

Formation (29 35)
O-glycan structures generated following polypeptide GalNAcT action include four common
subtypes based on differential monosaccharide linkage reactions to the unsubstituted GalNAc
(GalNAcα-Ser/Thr). Most O-glycans contain the Core 1 subtype structure formed by the
addition of galactose in a β1 3 linkage to the GalNAc. The glycosyltransferase responsible is
known as the Core 1 β1 3 galactosyltransferase (Core 1 GalT) (Figure 8.3). None of the cDNAs
encoding the Core 1 GalTs have been identified, although it appears based on enzyme activities
that multiple Core 1 GalT enzymes may exist in vertebrates. Like the polypeptide GalNAcT
family, the Core 1 GalTs appear to be differentially expressed among specific tissues and cell
types, and some also exhibit substrate preferences in vitro involving peptide sequence
determinants. Whether these enzymatic properties reflect a role for various Core 1 GalTs in the
glycosylation of specific proteins in vivo remains to be established.
Figure 8.3. Core 1 and Core 2 O-glycan subtype formation is controlled by the activity of Core 1
GalT and Core 2 GlcNAc-T enzymes. Additional biosynthesis can yield O-glycans bearing
fucosylated and sialylated lactosamines.
Core 2-type O-glycans can be generated by addition of GlcNAc to the GalNAc in a β1 6

linkage. The production of Core 2 O-glycans requires the Core 1 structure as a substrate, so the
Core 2 structure also contains the Core 1 structure. For this reason, use of the term Core 1 O-
glycans refers to an O-glycan in which Core 2 GlcNAcT has not acted. Recent studies in which
the Core 2 GlcNAcT gene was deleted from the mouse germ line have revealed the presence of
at least one other Core 2 GlcNAcT enzyme in certain cell and tissue types, including the colon.
This second Core 2 GlcNAcT enzyme also appears to exhibit Core 4 GlcNAcT activity and thus
may use either the unmodified Core 1 or Core 3 O-glycan as a substrate (see below). Core 2 O-
glycan may become elongated into either a mono- or biantennary form with the presence of
multiple lactosamine (Galβ1-4GlcNAc) units and terminal linkages of fucose and sialic acid (see
Chapter 16). In myeloid cell types, Core 2 O-glycans appear to function as a scaffold for the
production of selectin ligands that act in regulating inflammation.
Core 3 O-glycan production is controlled by Core 3 GlcNAcT activity which, like Core 1 GalT,
uses the GalNAcα-Ser/Thr substrate (Figure 8.4). Thus, Core 3 O-glycan formation may depend
on a competition between Core 1 GalT and Core 3 GalT enzymes in some cell types or among
certain glycoproteins. The action of Core 3 GalT inhibits the ability of Core 2 GlcNAcT to act,
since Core 2 GlcNAcT requires a galactose residue in β1 3 linkage to the underlying GalNAc.
Nevertheless, this Core 3 O-glycan can also be a building block for the formation of biantennary
O-glycans by virtue of Core 4 GlcNAcT activity (Figure 8.4). Relatively few tissues show high
levels of Core 3 GlcNAcT and Core 4 GlcNAcT activities, except for the intestinal tract, a site
where mucin production is normally high.
Figure 8.4. Core 3 and Core 4 O-glycan subtype formation is controlled by the activity of the
Core 3 GlcNAcT and Core 4 GlcNAcT enzymes. Further O-glycan biosynthesis can yield bi-
antennary forms sometimes similar to those of the Core 2 subtypes. Additional linkages may
subsequently occur (vertical arrow).
Less Common O-Glycan Core Subtypes (29 30)
Core 1 to 4 O-glycans comprise the majority of O-glycan structures produced in vivo, with most
being of the Core 2 subtype. However, other modifications to the GalNAcα-Ser/Thr structure
have been reported (Figure 8.5). Core 5 O-glycans (GalNAcα1 3GalNAcα-Ser/Thr) have been
found on glycoproteins from several vertebrate species. This structure exists on some human
adenocarcinomas and on cells comprising the embryonic gut. Core 6 O-glycan formation
(GlcNAcβ1-6GalNAcα-Ser/Thr) has been reported on human embryonic gut and mucins from
ovarian cysts. However, it remains possible that Core 6 O-glycans are a degradation product of
β-galactosidase acting on Core 2 O-glycans during preparation and analysis. Core 7 O-glycans
(GalNAcα1-6GalNAcα-Ser/Thr) have been described in a study of submaxillary O-glycans from
bovine sources. In addition, linkage of fucose (α1 2) to the GalNAcα-Ser/Thr has been
observed. This modification does not appear to block formation of other Core O-glycan
subtypes. Further complexities in O-glycan diversification continue to arise with novel core
subtypes still being discovered, although most of these are found at relatively low levels and
perhaps are segregated to specific tissue or cell types.
Figure 8.5. Oligosaccharide structures of less common Core O-glycan subtypes.
O-Glycan Biosynthesis and T Antigen Formation (29, 35, 36)

The biosynthesis of O-glycans can be modified and terminated with the addition of sialic acid
residues relatively early in biosynthesis (Figure 8.6). For example, certain sialyltransferase
enzymes are capable of acting on GalNAcα-Ser/Thr, or early O-glycan core subtypes after Core
1 GalT action. These sialic acid additions give rise to a series of O-glycan structures that
generally restrict further biosynthetic steps and have been commonly referred to as tumor-
associated (T) antigens (see below). The use of the term T antigen began with the assignment of
the Galβ1 3GalNAcα-Ser/Thr disaccharide as the Thomsen-Friedenreich antigen, or the TF
antigen, which has subsequently been referred to simply as T antigen. Production of T antigens
may involve a competition among glycosyltransferases for the same substrate. As with the
biosynthesis of other oligosaccharides, including N-linked oligosaccharides, the expression
levels and subcellular distributions of glycosyltransferases within the Golgi apparatus may
determine the outcome. A hypothetical example involves GalNAcα-Ser/Thr as a known
substrate for either Core 1 GalT or ST6GalNAc-I. In cells expressing both enzymes, only the
Core 1 GalT pathway may appear to operate as sialyltransferases are commonly found in the
trans-Golgi and therefore generally acquire substrates after other cis- and medial-Golgi-localized
glycosyltransferases in the "assembly line" process. In contrast, inaction of the Core 1 GalT may
promote sialyl Tn (Siaα2 6GalNAcα-Ser/Thr) expression. The relative expression levels of
various glycosyltransferases may thereby affect the structural outcome in O-glycan biosynthesis
and diversification.
Figure 8.6. Early and alternate pathways in O-glycan biosynthesis leading to the formation of
tumor-associated (T) antigens. Specific sialyltransferases can act to produce the sialyl Tn and
disialyl T antigens. These appear to be biosynthetic "dead ends" (boxed) that cannot be further
modified. High levels of various T antigens are frequently associated with cancer cells (see
Chapter 35).
Mucin Polypeptide Genes (37 39)
More than eight genes exist in humans that encode an apomucin highly modified by O-glycans in
forming epithelial mucins. These are genetically designated MUC1, MUC2, etc., and are variably
expressed by epithelial cells of the gastrointestinal, tracheobronchial, and reproductive tracts.
They may be found either membrane-associated or secreted. The first characterized mucin,
encoded by the MUC1 gene, is membrane-associated by virtue of a transmembrane sequence, but
it may be secreted as the result of alternative RNA splicing or by protease action at the cell
surface. Increased expression of MUC1 has been noted in many carcinomas (see Chapter 35).
Four human mucin genes are found clustered on chromosome 11p15.5. Although not all mucin
genes have been fully sequenced at this time, their O-glycosylation sites have been localized to
tandem repeats in the polypeptide that are rich in prolines, serines, and threonines, as would be
predicted. Each mucin gene appears to have its own unique repeat sequence, which may be
reiterated a dozen times to more than 100 times. Mucins in humans are also polymorphic and the
number of repeats varies among individuals.
Where they have been defined, mucin genes are not highly conserved among vertebrate species.
Although the transmembrane and cytoplasmic sequences are more than 80% identical in
comparison to MUC1 sequences between the mouse and humans, the repeat sequences in the
"mucin" domain are much less conserved except for the presence of high levels of proline,
serine, and threonine as is common among sites of O-glycosylation. Although the classic mucin
genes (MUC series) are primarily found in epithelial cells, glycoproteins expressed in other
systems can have mucin-like features involving clustered O-glycans, with or without tandem
repeat sequences (e.g., PSGL-1, CD34, CD43, and the alternate exons of CD45).
Physical and Structural Functions of O-Glycans (40 43)
Upon secretion, mucins generally remain at the apical surface of epithelial cells and are capable
of providing a gelation function by their abilities to retain water ions and by their entanglement
through intra- or intermolecular disulfide linkages. Mucins function as protective barriers for
epithelial cells, and intestinal ulcers have been found to be associated with reduced mucin levels
in goblet cells. Goblet cells are specialized to secrete mucins and may do so by both single
granule and compound exocytotic processes. The latter type of secretion involves the fusion of
central granules in series with those at the plasma membrane and also results in the temporary
cavitation of the cell. Stimulation of mucus secretion by what are termed mucus secretagogues is
a function mediated by macrophages and lymphocytes. Endogenous mucus secretagogues known
at present include oxygen-free radicals and interleukin-1.
Salivary mucins also provide lubrication for epithelial surfaces and may modulate the infectious
nature of oral microbes. Humans lacking salivary gland function have abnormal mucosal
surfaces with chronic low-level inflammation. Salivary mucins also adhere to tooth enamel,
thereby protecting against demineralization by organic acids. Some salivary mucins can
agglutinate certain oral streptococcal strains. This is observed in studies of the salivary mucin
MG2. The interaction is not well elucidated, but it is known that the Siaα2 3Galβ1 3GalNAcα-
Ser/Thr glycopeptide competes for MG2-streptococcal interactions. Some mucin-type O-glycans
act as barriers against freezing of living tissues. Such "anti-freeze" glycoproteins have been
characterized from various species of Antarctic fish, and they have been shown to inhibit the
formation of "nucleation centers" at temperatures where water would otherwise freeze. The
characteristic property of these proteins is their use of the sequence Ala-Ala-Thr, in some cases
repeated more than 40 times. Each threonine residue carries the O-glycan structure Galβ1
3GalNAcα- and chemical elimination of these O-glycans ablates the antifreeze function.
O-glycans perform a role in the formation of the ABO blood group antigens, a system that has
clinical relevance in blood transfusions, although glycolipids are also known to carry these
antigens (see Chapter 16). The blood group antigens are enriched on the erythrocyte surface and
their diversity is a product of mutations in a glycosyltransferase encoded by the H locus.
Mutations in another glycosyltransferase generate either null alleles (O) or actual changes in
substrate specificity (A and B) (see Chapter 16). Antibodies to specific blood group
oligosaccharides have previously defined blood types by agglutination activities, but the
endogenous roles of these O-glycan structures are not presently known.
Various pathogens can use oligosaccharide structures found on cell surface O-glycans as
receptors for binding and infection of the host (see Chapters 28 and 36). In some cases, secreted
mucins contain these very same oligosaccharide structures at high densities and have been found
to be able to bind such pathogens, thereby saturating pathogen receptors. For this reason, O-
glycans on secreted mucins have been suggested to act as a "decoy" in an organism's effort to
evade pathogen infection.
O-Glycan Functions in Endogenous Lectin-Ligand Interactions

(44 57)
O-glycans have been reported to function in sperm binding to the egg. The mammalian egg coat
(the zona pellucida) contains a large number of O-glycans, as well as some N-glycans. Removal
of egg N-glycans by glycosidase treatment does not destroy sperm binding, but loss of O-glycans
following mild alkali treatment ablates sperm binding. Of those O-glycans found on the
mammalian egg, the ZP3 glycoprotein of the mouse zona pellucida has been reported to have the
primary role of the sperm receptor. Analyses of the O-glycan structures on ZP3 have revealed
terminal α1 3Gal residues on O-glycans. Although this structure was previously believed to be
responsible for sperm-egg binding, mice rendered deficient in α1 3 galactosyltransferase
activity were found to be fully fertile and in vitro fertilization was unaffected. A role for O-
glycans in sperm-egg binding has not been ruled out; however, the specific O-glycan structures
responsible and the level of their involvement are not defined at present. Other species appear to
use different glycan specificities in sperm-egg binding, leading to the view that glycans may play
a major part in providing the species specificity commonly observed in fertilization (and see
Chapter 34).
The hematopoietic system of vertebrates makes use of O-glycan structures during its
development and function. Lymphocyte homing and the inflammatory response involving
leukocyte intravasation are regulated by glycans that function as selectin ligands (see Chapter
26) and that may exist among O-glycans, N-glycans, glycolipids, and proteoglycans. Loss of
selectin ligand expression by FucT-VII deficiency, an enzyme that generates α1 3 fucose
linkages on a variety of glycan types, results in a widespread leukocytosis with deficits in both
neutrophil recruitment in peritoneal inflammation and lymphocyte homing to the high
endothelial venules of the lymph nodes. In contrast, absence of the Core 2 GlcNAcT enzyme
which produces Core 2 O-glycans results in a partial deficiency in E-, L-, and P-selectin ligand
biosynthesis, yet disrupts neutrophil recruitment to inflamed peritoneum without altering selectin
function in lymphocyte homing. A second Core 2 GlcNAcT isozyme has been isolated which
also harbors Core 4 activity and may be responsible for mucin production among epithelial cell
types. The role of O-glycans in selectin-mediated trafficking and adhesion processes may be
usurped to induce pathologic interactions in some situations. A role of selectin ligands in cancer
cell metastasis has been proposed from studies revealing reduced metastasis in selectin-ligand-
deficient systems (see Chapter 35).
O-glycans also have functions in lymphocytes and during immune responses. The thymus tissue
of all vertebrates studied from lizards to humans exhibits a change in peanut agglutinin lectin
reactivity that marks the cortical-medullary boundary. During T-cell development in the thymus,
loss of peanut agglutinin reactivity occurs as thymocytes mature into T cells and enter the
medullary compartment. This is due to the induction of the sialyltransferase ST3Gal-I that masks
the peanut-agglutinin-binding epitope, converting Galβ1 3GalNAcα-Ser/Thr to Siaα2 3Galβ1
3GalNAcα-Ser/Thr. By inactivating ST3Gal-I function, it has been discovered that this
modification regulates T-cell viability. Mice lacking ST3Gal-I are deficient in cytotoxic T cells
as a result of enhanced apoptosis. These and other studies that alter the formation of O-glycans in
vivo by genetic methods are providing useful information on the function of O-glycans (Table
8.1). It appears that specific O-glycan linkages have unique roles in various physiologic systems.
Table 8.1. Enzyme deficiencies in vertebrate O-glycan biosynthesis and effects
Enzyme Species Phenotype/Disease
Polypeptide GalNAcT-1 mouse viable/under study

Polypeptide GalNAcT-8 mouse viable/under study
Core 2 GlcNAcT mouse myeloid leukocytosis and inflammatory response deficit
FucT-IVa mouse partial inflammation response deficit; collaboration with
FucT-VII
FucT-VIIa mouse general leukocytosis, lymphoid homing defect, and
β1 4 GalTa mouse multiple defects including epithelial and endocrine
α1 3 mouse cataracts
galactosyltransferasea
Old World major xenotransplantation rejection antigen
primates
H locus human ABO blood grouping, transfusion relevance
glycosyltransferase
ST3Gal-I mouse cytotoxic-T-cell deficiency by apoptosis
ST3Gal-IIb mouse viable/under study
ST3Gal-IVa mouse viable/under study
a
These enzymes may not act only on O-glycans.
b
This enzyme prefers glycolipid-based substrates in vitro.
Future Directions
The most common biosynthetic steps in vertebrate O-glycan biosynthesis are relatively well
characterized, although additional core structures are still being reported. The number of
glycosyltransferases participating in O-glycan biosynthesis is rapidly increasing with multiple
glycosyltransferases found to generate the same linkage, as exemplified by the recently disclosed
families of polypeptide GalNAcTs, Core 1 GalTs, and at least a second Core 2 GlcNAcT. Why
this complexity exists is a fascinating question for study. It is possible that O-glycan formation is
under the control of glycosyltransferases that operate in relatively protein-specific glycosylation
reactions in vivo, which could imply that the function of some glycoproteins may be dependent
on the O-glycan structure attached. Additional regulation in O-glycan production may also be
achieved by segregating the genetic transcription of multiple isozymes to various tissue types by
unique promoter elements. Methods allowing the determination of O-glycan structures in vivo
among specific tissues and cell types will be needed to refine O-glycan structure-function
properties in genetically modified systems. A better understanding of O-glycan function will also
likely come from comparative studies of various organisms, including invertebrates, and those
that have been engineered to contain multiple deficiencies in glycosyltransferase genes that
operate in O-glycan biosynthesis.
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9. Glycosphingolipids
Diego).
THIS CHAPTER CONSIDERS PATHWAYS FOR THE BIOSYNTHESIS of the

glycosphingolipids, describes the structure and classification of the different core series of these
molecules, and outlines general principles regarding their biological roles in membrane structure,
modulation of membrane protein function, cell-cell recognition, and host-pathogen interactions.
The major emphasis is on higher animal glycosphingolipids, about which the most information is
currently available.
The glycosphingolipids were originally discovered in the 1940s as lipid-rich substances from
normal tissues that accumulated in the tissues of patients with so-called "storage disorders" such
as Tay-Sachs disease. Other investigators also extracted glycosphingolipids from the stroma of
the erythrocytes of various animals. Their complex and unusual structure as well as their as yet
unknown functions caused them to be called sphingolipids, after the enigmatic Egyptian Sphinx.
Further structural work showed that these amphipathic molecules consisted of two components:
an outer glycan portion attached to an inner lipid tail consisting of a sphingolipid called
ceramide. The hydrophobic nature of the sphingolipid component causes these molecules to be
embedded in the outer leaflet of cell membranes, along with phospholipids, cholesterol, and
glycerolipids. More than 200 structurally distinct glycosphingolipids have been reported from a
wide variety of eukaryotic sources.
Defining Structures, Major Classes, and Nomenclature (1 12)
The minimal motif that defines the glycosphingolipids is a monosaccharide attached directly to a
ceramide unit. In higher-animal cells, this monosaccharide is usually glucose or galactose, giving
glucosylceramide or galactosylceramide (Figure 9.1). Each of these basic units can then be
further extended by the stepwise addition of further monosaccharides (Figures 9.2 and 9.3). The
number of variations in extension of galactosylceramide is somewhat limited. Glucosylceramide
typically has an additional galactose added, giving lactosylceramide, which is then extended in
many ways, giving rise to several defined core structures (Table 9.1).
Some of these classes of molecules tend to be enriched in certain tissues types, e.g., the ganglio-
series in the brain and the neolacto-series in leukocytes. Although all existing classifications of
glycosphingolipids are based on these variations in core glycan structure, it is important to note
that the ceramide moiety can also show substantial structural heterogeneity. For example, the
typical sphingosine base (Figure 9.1) can vary in the number of double bonds and in the length
of the acyl chain. Likewise, the fatty acid attached to the sphingosine via an amide linkage
(Figure 9.1) can vary in length from C14 to C24, and in its degree of unsaturation and/or
hydroxylation. These variations in ceramide structure are also expressed in a cell-type-specific
and developmentally regulated manner, implying that they have some specific purpose. In this
regard, one interesting fact is that certain types of glycans are found to be enriched on certain
subclasses of ceramides; e.g., in brain gangliosides, the C20 homolog is much more likely to be
found in glycosphingolipids with more than one sialic acid residue. Another instance is the
ceramide galactosyltransferase that synthesizes GalCer, which displays a marked preference for
hydroxy fatty-acid-containing ceramides.
Figure 9.1. Pathways for the biosynthesis of ceramide and monohexosylceramides. The hexose
shown is glucose, but the same pathway applies for galactosylceramide. All of the enzymatic
steps take place on the cytosolic leaflet of the ER or Golgi membranes. The fatty acyl chain can
vary in length (thus, x = 14 24). Note that sphingosine itself is not a defined intermediate in the
pathway, since its desaturation is completed only after the fatty acylation step. However,
sphingosine or ceramide can be recycled into this pathway following the hydrolysis of
sphingomyelin or the turnover of previously synthesized glycosphingolipids.
Figure 9.2. Pathways for the biosynthesis of the core structures of the glucosylceramides and
galactosylceramides. Initiation takes place on the membranes of the ER or the Golgi. The types
of possible outer chains and extensions are shown in Figure 9.3, and details regarding the
ganglio-cores are discussed later in this chapter (Figure 9.4). Note that many of these extensions
are generally similar to those of N- and O-glycans (see also Chapters 7, 8, and 16).
Figure 9.3. Pathways for the extension of various glycosphingolipids. The GlcNAc, Gal, or
GalNAc residues indicated with an asterisk in Figure 9.2 can be extended in a variety of ways
which are shown in this figure in composite form. The boxed structures on the right side of the
line represent common extensions that are susceptible to different types of further extension.
Note that many of these extensions are generally similar to those of N-glycans and O-glycans
(see also Chapters 5, 6, and 15).
Glycosphingolipids are often simply referred to as glycolipids. This is because the other major
class of lipids with attached glycans (the glycosylinositolphospholipids or GPI lipids; see
Chapter 10) were only discovered and characterized much later, in the early 1980s. The term
ganglioside defines a glycosphingolipid with one or more sialic acid residues (sulfated
glycosphingolipids are also called gangliosides by some investigators). There is a well-known
nomenclature system suggested by Svennerholm for the ganglio-series gangliosides (Figure 9.4).
Despite the subsequent introduction of a more systematized official nomenclature for these
molecules, the original system has persisted and prospered, not just because of its historical
value, but also because investigators prefer to call specific molecules by a common name that is
easy to remember. For example, the ganglioside with the structure:
2
Neu5Acα3Galβ3GalNAcβ4Galβ4Glcβ1Cer would be called II Neu5Ac-GgOSe4-Cer in the
official IUPAC-IUB designation or simply GM1a in the Svennerholm nomenclature. For similar
historical reasons, there also remain some other idiosyncracies in the details of glycosphingolipid
nomenclature. For example, the structure called sialylparagloboside
(Neu5Acα3Galβ4GlcNAcβ3Galβ4Glcβ1Ceramide) is actually based on a neolacto-core (Table
9.1) and not on a globoside core.
Figure 9.4. Pathways for the biosynthesis of the ganglio series glycosphingolipids. Note that the
action of the GalNAc transferase commits the molecules to further extension along one of four
different pathways; the consequences of genetic disruption of this enzyme are discussed in the
text. Further additions of sialic acids occur in some cell types and species, giving
polysialogangliosides (not shown).
The glycosphingolipids are clearly significant contributors to the structure of the outer leaflet of
most eukaryotic cell membranes. The molar ratio of these molecules relative to the other major
membrane lipids (phospholipids, cholesterol, and glycerolipids) varies from being rather minor
(e.g. <5% in erythrocytes) to being a very major component (e.g., 30% of the total lipids in
neuronal plasma membranes). Although glycosphingolipids are found throughout all eukaryotes,
the nature of the core structures can vary in different taxa. For example, invertebrates such as
molluscs express a series of glycosphingolipids based on the core structure Manβ1 4Glcβ1 1
Ceramide, and inositolphosphate-ceramides are major sphingolipids in fungi and plants. For
further discussion of this matter, see Chapter 3, which considers the evolution of glycan
diversity.
Table 9.1. Names and abbreviations for major core structures of vertebrate
glycosphingolipids
Name (Series) Abbreviation Core Structure
Lacto (LcOSe4) Galβ3GlcNAcβ3Galβ4Glcβ1Ceramide

Lactoneo (LcnOSe4) Galβ4GlcNAcβ3Galβ4Glcβ1Ceramide
Globo (GbOSe4) GalNAcβ3Galα4Galβ4Glcβ1Ceramide
Isoglobo (GbiOSe4) GalNAcβ3Galα3Galβ4Glcβ1Ceramide
Ganglio (GgOSe4) Galβ3GalNAcβ4Galβ4Glcβ1Ceramide
Muco (MucOSe4) Galβ3Galβ3Galβ4Glcβ1Ceramide
Gala (GalOSe2) Galα4Galβ1Ceramide
Sulfatide 3-O-Sulfo-Galβ1Ceramide
Isolation, Purification, and Analysis (3, 4,13 15)
Most glycosphingolipids can be obtained in good yield from cells and tissues by sequential
organic extractions of increasing polarity. Subsequently, they can be fractionated away from
other lipids in the extract and from each other using techniques such as DEAE ion-exchange
chromatography and silica gel on either thin-layer plates or columns. High-performance liquid
chromatography adaptations of these traditional methods have been particularly powerful in
obtaining complete separations of these complex mixtures. For further details regarding the
approach to the structural characterization of these molecules, see Chapter 38. It is noteworthy
that the base treatment, which is traditionally used to eliminate phospholipids from crude
extracts, can damage labile O-acetyl and O-acyl groups in glycosphingolipids. The availability of
specific endoglycoceramidases that cleave the glycan portion away from the ceramide has
introduced an alternative approach to the their analysis.
Biosynthesis (2,3, 6, 11,16 26)
The de novo biosynthesis of glycosphingolipids begins on the inner leaflet of membranes in the
ER-Golgi pathway. Ceramide is first synthesized by the acylation and desaturation of d-erythro-
sphinganine (Figure 9.1) and then glucosylated by a ceramide-specific glucosyltransferase or
galactosylated by a specific galactosyltransferase. The glucosylation reaction has been shown to
occur on the cytosolic face of ER and Golgi membranes, indicating that there must be a
mechanism to "flip" the glucosylceramide into the lumen of the ER-Golgi pathway, where
further extension takes place. Galactosylceramide appears to be synthesized with a more
conventional topology, primarily within the ER. The next step for glucosylceramides is always
the addition of a β-linked galactose residue, giving lactosylceramide. Thereafter, the molecules
can be elongated in a stepwise fashion, giving a wide variety of different cores (see above and
Figure 9.2). Some of these biosynthetic steps involve mutually exclusive branching events that
"commit" the molecule to proceed along certain defined pathways. The classic example is the
ganglio series pathway (Figure 9.4) in which the action of a single enzyme (GalNAc transferase)
can restrict the subsequent extension of the molecule into the A, B, C, or O series and their
subpathways. The outer extensions of glycosphingolipids, including the addition of sialic acids,
fucose, blood group structures, or glucuronic acid residues share much in common with those of
N- and O-glycans (Figure 9.3; see Chapters 7, 8, and 16). Thus, although the initial steps in
glycosphingolipid biosynthesis are catalyzed by unique glycosyltransferases that recognize the
hydrophobic nature of the target substrate, many of the outer extensions are thought to be
catalyzed by shared glycosyltransferases that may also be involved in glycoprotein biosynthesis
(see Chapter 16). However, this sharing of glycosyltransferases has not been formally proven in
most instances. In addition, as with N- and O-glycans, the outer monosaccharide residues of
glycosphingolipids are targets for additional modifications such as 9(7)-O-acetylation of the
sialic acids, O-acylation of galactose residues in galactosylceramide, de-N-acetylation of sialic
acids, and O-sulfation.
The organization of the glycosphingolipid biosynthetic steps in the Golgi apparatus has not been
fully resolved. There is evidence for sequential subcompartmentation of the enzymatic activities
in the order in which they actually work. However, as with the other Golgi glycosylation
pathways, evidence also exists for considerable overlap between the compartments. In this
regard, drugs that change the organization of the Golgi apparatus (like monensin and Brefeldin
A) have yielded very useful, but not completely conclusive, results. Other drugs that can affect
glycosphingolipid biosynthesis include glycoside primers (which penetrate the Golgi and act as
alternate substrates for glycosphingolipid-modifying enzymes), N-butyldeoxynojirimycin, and
the family of ceramide-related compounds that were rationally synthesized by Radin to be
specific inhibitors of glucosylceramide synthesis. The first of these was a mixture of d- and l-
threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). These were potent
inhibitors of glycosphingolipid synthesis, but each had somewhat different effects and also
affected sphingomyelin biosynthesis. Recently, d,l-threo-1-phenyl-2-hexadecanoylamino-3-
pyrrolidino-1-propanol HCl (PPPP) has been reported to be a much more specific inhibitor of
glucosylceramide synthesis. An interesting fact is that genetic mutations in the glycosidases
involved in glycosphingolipid degradation (see Chapter 18) can be ameliorated by some of these
biosynthetic inhibitors (presumably by reducing the load of glycosphingolipids that need to be
degraded).
Recent studies have indicated that in addition to de novo synthesis (pathway 1), ceramide for
glycosylation can also come from hydrolysis of other sphingolipids (pathway 2) or from
recycling of glycosphingolipids in an endosomal pathway (pathway 3). The ratio of these
different pathways can vary greatly between cells. It is suggested that when the need for
synthesis is low, e.g., in slowly dividing cells, synthesis may predominantly originate from
ceramide salvaged from the hydrolytic pathways, whereas the need for increased synthesis is met
by up-regulating the de novo biosynthesis pathway.
Trafficking, Turnover, and Degradation (2, 7, 16, 18, 20, 22,27 39)
Theoretically, glycosphingolipids can enter into and traffic through any post-Golgi membrane
compartment. In fact, they have a strong tendency to cluster together (by as yet uncertain
physical principles) in the late part of the Golgi and be delivered primarily to the outer leaflet of
the plasma membrane. Together with some GPI-anchored proteins, these clusters are proposed to
develop into specialized "rafts" that tend to migrate to the apical domain of polarized epithelial
cells. Evidence also indicates the existence of separate glycosphingolipid-enriched
microdomains (GEMs). In a few cell types, certain glycosphingolipids have been reported to
have a primarily internal location (e.g., lactosylceramide is concentrated in the granules of blood
neutrophils). Some studies have claimed a truly unusual localization for glycosphingolipids,
namely, an association with the cytoskeleton. How the glycosphingolipids are able to flip across
the membrane to achieve such a cytosolic localization remains a mystery. In this regard,
vimentin-deficient cells have been shown to have a defect in glycolipid synthesis via pathway 2
(see section on biosynthesis above).
Particularly in cell types with a high concentration of glycosphingolipids, the molecules are
known to be shed from the surface and can be found in body fluids, often in mixed micelles or
apolipoprotein complexes. In some instances, such shed gangliosides have been shown to be
taken up by other cells and incorporated into their own membranes, in a manner that would be
indistinguishable from that of molecules that were endogenously synthesized. The extent to
which such intercellular transfer can take place in vivo is not known. When fluorescently labeled
ceramide is added to culture medium, it is taken up into the cells, where it tends to accumulate in
the Golgi apparatus, by unknown mechanisms.
Regardless of their original source, glycosphingolipids can be internalized from the cell surface,
and then enter endosomal compartments, sometimes becoming part of the inner membranes of
multivesicular bodies. Experiments with cultured cell lines suggest that such internalized
glycosphingolipids can be remodeled to some extent in late Golgi compartments. However, the
extent to which such remodeling actually occurs in vivo is not known. The bulk of the
internalized glycosphingolipids eventually undergoes terminal degradation in the lysosomes, via
a series of stepwise reactions catalyzed by specific lysosomal enzymes. Some of the final steps
involve cleavages that are close to the cell membrane and require facilitation by specific
sphingolipid activator proteins. These proteins are thought to "lift" the glycosphingolipids up
from the membrane, giving certain glycosidases the ability to act more efficiently. Four of these
activator proteins are derived by proteolysis of a single gene product, the sap precursor. In
addition to these, the GM2-activator protein is a distinct gene product. For further information
regarding the steps involved in lysosomal degradation of glycosphingolipids, as well as about
human diseases in which these degrading enzymes are known to be genetically deficient, see
Chapter 18. Eventually, glycosphingolipids are broken down to their individual components,
which are then available for reutilization in various pathways. An exception may be the final step
in removing glucose from glycosyl-ceramide. Some glucosylceramide generated from
degradative pathways may be recycled for use in a new round of biosynthesis of
glycosphingolipids. In this regard, inhibitors of glycosphingolipid biosynthesis are suggested as
agents to reduce the total load of glycosylceramides that accumulate in patients with lysosomal
storage disorders.
Relationships to Other Sphingolipids (8, 29, 30, 32, 40)

There is a tendency for glycobiologists to focus primarily on the structure and function of the
glycan component of the glycosphingolipids. However, by virtue of their ceramide component,
glycosphingolipids are also at the crossroads of a variety of other cellular pathways that are of
potential biological significance (Figure 9.5). Besides glycosphingolipids, the other major
ceramide derivative in most cells is sphingomyelin. This molecule is synthesized by the reaction
of phosphorylcholine with ceramide, giving essentially a ceramide with a choline headgroup.
Sphingomyelin can constitute the major pool of ceramide in a cell, exceeding even the
glycosphingolipids as a whole group. Thus, the ceramide precursor pool may be subject to
competition between sphingomyelin synthesis and the initiation of glycosphingolipid synthesis.
In this regard, it is of particular interest that a number of extracellular agents and insults (e.g.,
tumor necrosis factor, Fas ligands, and cancer chemotherapeutic agents) cause the activation of
endogenous sphingomyelinases, which then act on sphingomyelin to release ceramide. Many
studies indicate an important role for this released ceramide in regulating responses such as cell
cycle arrest, apoptosis, and cell senescence. In vitro, ceramide can also activate a serine-
threonine protein phosphatase. In intact cells, it seems to regulate protein phosphorylation and
some downstream targets such as interleukin converting enzyme (ICE)-like proteases, stress-
activated protein kinases, and the retinoblastoma gene product. Thus, there is increasing
evidence that ceramide is a key component of the intracellular stress response pathway.
Figure 9.5. Metabolic interrelationships among glycosphingolipids and other ceramide and
sphingosine derivatives. Some known inhibitors of the pathways are also shown. (PC)
Phosphorylcholine; (DAG) diacylglycerol; (PDMP) d-threo-1-phenyl-2-decanoylamino-3-
morpholino-1-propanol; (MAPP) d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol.
Another derivative of ceramide thought to have a signaling function is ceramide-1-phosphate. In

addition, a partial breakdown product of galactosylceramide (psychosine, derived by fatty N-
deacylation) is known to be a cytotoxic compound that is a strong inhibitor of protein kinase C
and an activator of the src kinase. A further variation is the addition of different plasma
substitutions to the galactosyl moiety of psychosine. Finally, the sphingosine moiety itself can
also be a substrate for formation of a variety of other bioactive compounds that are thought to be
involved in cell signaling, including di- and trimethylsphingosine and sphingosine-1-phosphate.
In vitro experiments have suggested that several of these compounds can negatively or positively
affect the signaling functions of several well-known cellular kinases. However, a coherent and
conclusive picture has yet to emerge, and the in vivo significance of these observations remains
mostly unproven. Likewise, the metabolic relationship of the ceramide moiety of
glycosphingolipids to the rest of the sphingolipids in intact cells is still being worked out (see
discussion of the three pathways of biosynthesis above).
Antibodies against Glycosphingolipids (8, 29,41 43)
During the early days following the development of monoclonal antibody technology, a
concerted effort was made by many investigators to see if such antibodies could differentiate
between normal and cancer cells. Of the many "tumor-specific" antibodies that were raised, it
was noted that more than half were directed against carbohydrates (see Chapter 35), and the
majority of these reacted best with glycosphingolipids. Further studies indicated that most of
these antibodies were actually detecting "onco-fetal" antigens, which are expressed at high levels
in fetal and cancer tissues and at low levels in normal adult cells. Since that time, a variety of
additional monospecific antibodies have been raised by other investigators using
glycosphingolipids themselves as immunogens. It should be kept in mind that the immune
system of vertebrates tends to have difficulties in generating high-affinity monospecific
antibodies against carbohydrate structures. Thus, several of these antibodies (particularly of the
IgM variety) may not be as monospecific as they were initially thought to be. Regardless, some
of them have been successfully used for diagnostic and prognostic applications in human
diseases, and a few are being exploited for attempts at therapy of tumors. Many have also been
used to demonstrate cell-type regulation or expression of specific glycosphingolipid structures in
a temporal and spatial manner during development. The precise meaning of these findings for the
biology of cancer and development is still being explored.
From the pathological point of view, there are some situations wherein humans generate
autoimmune antibodies against nervous system glycosphingolipids, associated with the
corresponding pathology (see Chapter 37). For example, the Guillian-Barre and Miller-Fisher
syndromes that occur following Campylobacter infections are associated with circulating
antibodies against certain gangliosides, thought to result from molecular mimicry by bacterial
lipooligosaccharide glycans. In addition, in some patients with human multiple myeloma, the
clonal population of tumor cells secretes a monoclonal antibody with specificity directed against
the sulfoglucuronosyl epitope of nervous system glycosphingolipids. This is associated with a
demyelinating peripheral neuropathy which can prove to be worse than the primary tumor itself.
Another proposed pathological role for gangliosides occurs in various cancers, which can
produce and shed a large amount of these molecules. These shed molecules have been shown to
have a strong immunosuppressive effect, via as yet unknown mechanisms.
Biological Roles (3, 5, 8, 26, 29, 39, 40, 42,44 50)
There are many putative biological roles of glycosphingolipids, of which only a few have been
conclusively established. From a physical point of view, glycosphingolipids may have an
organizing role in the cell membrane. For example, glucosylceramides and ceramides are
thought to be critical components of the epidermal (skin) permeability barrier. In this regard, it is
noteworthy that they are similar to glycophospholipid (GPI) anchors in tending to have long acyl
chains (24 26 carbons). It is thought that this common structural feature allows both types of
lipids to extend into the inner leaflet of the phospholipid bilayer of cell membranes and also
causes them to associate with each other and with cholesterol, forming putative "rafts" in the
micromembrane (some investigators report the existence of distinct glycosphingolipid-enriched
domains free of cholesterol that are thought to be involved in signal transduction via cytosolic
proteins). Such aggregations of glycosphingolipids and GPI-anchored proteins are thought to
form in the trans-Golgi region and then get targeted to the apical domains of polarized epithelial
cells that line the lumen of various body cavities. Here, they can represent as much as 50% of the
total lipid concentration of the outer leaflet of the apical membrane, likely providing physical
protection against the hostile environment often encountered in such lumens, as well as specific
binding sites for the adhesion of symbiotic bacteria. In the same location, other structural aspects
of the same molecule can be utilized as highly specific receptor targets for a variety of bacteria,
toxins, and viruses. The classic and well-established finding is that the B subunit of cholera toxin
binds to the ganglioside GM1, triggering a conformational change and delivering the toxic A
subunit to the interior of the cell (for additional specific examples, see Table 9.2). These types of
interactions are obviously not of benefit to the organism that originally synthesized the
glycosphingolipid in question.
Table 9.2. Examples of reported interactions between defined
glycosphingolipids and specific binding proteins
Binding protein Ganglioside(s) Proposed function(s)
Cholera toxin GM1 B subunit binds; facilitates delivery of the toxic A

subunit into the cytosol
Tetanus toxin GD1b/GT1b same as above?
Botulinum toxin GT1b/GQ1b same as above?
EGF receptor GM3 diminishes tyrosine phosphorylation upon receptor
activation; possibly by inhibiting dimerization
EGF receptor De-NAc-GM3 activates tyrosine phosphorylation upon receptor
activation; mechanism unknown
Insulin receptor SPG diminishes tyrosine phosphorylation upon receptor
activation; mechanism unknown
Thyroid-stimulating GD1a-lactone unusual structure selectively associated with this
hormone receptor receptor; biological significance unknown
NGF receptor GQ1b thought to induce/facilitate neurite outgrowth in certain
neuronal cells; mechanism unknown
Vitronectin receptor GD3 , GD2 thought to alter the adhesive action of receptor;
mechanism unknown
Src family tyrosine GD3 regulatation of Lyn tyrosine kinase activity in a
kinase Lyn caveolae-like domains on brain cell membranes
A recurring theme in the literature is the specific physical association of certain

glycosphingolipids with certain cell membrane receptors (Table 9.2 gives several examples).
Although the physical mechanism of such interactions is not certain, there is considerable
evidence indicating that they can alter the biology of these receptors. For example, the tyrosine
phosphorylation of the EGF receptor is specifically down-regulated by adding the ganglioside
GM3, whereas the closely related structure sialylparagloboside has a similar effect only on the
insulin receptor. A small modification of the GM3 molecule (de-N-acetylation) results in exactly
the opposite response (stimulation of EGF receptor tyrosine phosphorylation). Related studies
demonstrate the opposing effects of GM3 and lactosylceramide on cell growth. However, many of
these effects are demonstrated following the exogenous addition of glycosphingolipids to cells.
Thus, although at least a portion of the added glycosphingolipids are incorporated into the
membrane, one cannot rule out other effects caused by the micellar forms of the added
glycosphingolipids that can also be bound to cell surface proteins. In balance, the
stereospecificity of these effects for the fine structure of the glycan portion of the
glycosphingolipids implies that defined mechanisms must be involved.
Similar findings have been reported for the specific effects of ganglioside GQ1b in inducing
neuritogenesis, and a suggestion has been made that this involves the NGF receptor and/or an
ectoprotein kinase. Again, final mechanistic conclusions are lacking. Regardless, these findings
have encouraged other investigators to try ganglioside infusions in a variety of central nervous
system disorders, including stroke and demyelinating disorders. Particularly in stroke, there are
several reports of beneficial effects of infusing gangliosides into the bloodstream of patients.
Likewise, direct topical applications of conduritol B epoxide, a specific irreversible inhibitor of
β-glucocerebrosidase, increased GlcCer levels in the basal, proliferative cell layer (fourfold
increase) and stimulated proliferation. Simultaneous treatment with conduritol B epoxide plus
GlcCer resulted in an additive increase in DNA synthesis, strongly suggesting that GlcCer
directly stimulates epidermal mitogenesis.
It has also been suggested that gangliosides are involved in thermal adaptation of neuronal
membranes. The data suggest a general rule that "the lower the environmental temperature the
more polar is the composition of brain gangliosides." Studies with model bilayer membranes also
indicate that gangliosides can modulate basic membrane properties in a thermosensitive manner.
Hakomori and colleagues have also provided evidence that glycosphingolipids can mediate low-
affinity but high-specificity carbohydrate-carbohydrate interactions between different cell types.
In particular, they propose that glycolipids bearing the Lewis X motif may help to mediate
compaction of the early mouse embryo at the stage of morula formation (see Chapter 34). In
most other instances, the biological significance of such carbohydrate-carbohydrate interactions
has yet to be formally proven.
Natural and Induced Disorders in Biosynthesis (36,51 56)
As mentioned above, genetic defects in a variety of lysosomal enzymes and in the sphingolipid
activator proteins result in distinct storage diseases that are characterized by the accumulation of
specific intermediates that cannot be further degraded. These diseases are considered further in
Chapter 18. The existence of a cultured cell line that is completely deficient in glucosylceramide
synthase indicates that the glucosylceramide series of glycosphingolipids are not essential for the
growth of single cells in a culture dish and that the complexities of glycosphingolipid structures
have their critical functions primarily in the intact multicellular animal. In fact, in contrast to the
large number of metabolic errors found in the glycosphingolipid degradative pathways, there are
very few naturally defined genetic defects in the biosynthesis of glycosphingolipids. On the other
hand, dramatic changes in glycosphingolipid expression occurring during vertebrate ontogeny
and cellular activation indicate that the complexities of glycosphingolipid biosynthesis may be
required in the intact animal.
Targeted genetic mutations in an intact mammal are obviously required to define the true in vivo
biological roles of glycosphingolipids (see Table in Chapter 33). Some of the gene "knockouts"
affecting the outer chains of N- and O-glycans (e.g., FucT-VII and α1 3 galactosyltransferase;
see Chapters 17, 18, 32, and 33) may well affect outer-chain structures on glycosphingolipids as
well; this has yet to be investigated systematically. So far, only two gene disruptions that
selectively affect glycosphingolipid synthesis have been performed. Elimination of the ceramide
galactosyltransferase has given the most interesting and provocative results. Since the proximate
products of this enzyme reaction (GalCer and sulfatides) are major components of the myelin
sheaths of axons in the vertebrate nervous system, investigators expected to see a complete
disorganization of myelin. Surprisingly, the mice retained the ability to form myelin. However,
this myelin contained GlcCer, which had apparently substantially replaced the GalCer. Despite
myelin of relatively normal ultrastructural appearance, the mice showed generalized tremors and
mild ataxia, and electrophysiological analysis showed conduction deficits consistent with
diminished insulation capacity of the myelin sheath. Furthermore, with increasing age, the
mutant mice developed progressive hindlimb paralysis and severe vacuolation of the ventral
region of the spinal cord. These results indicate that GalCer and sulfatide have important roles in
myelin function and stability.
The biggest surprise in this field has arisen from the genetic ablation of the GalNAc transferase
(GM2/GD2 synthase) that is required for the formation of most of the complex gangliosides of the
ganglio series (see Figure 9.4). Despite the lack of these major gangliosides, the mice showed no
major defects in their nervous systems and no gross functional changes, only a slight reduction in
neural conduction velocity in some peripheral nerves. An increase in GM3 and GD3 occurs in the
brains of these mutant mice and seems to be sufficient to compensate for the lack of complex
gangliosides. These findings indicate that contrary to all prior expectations, the complex
gangliosides are not required for the primary morphogenesis and organogenesis of the brain. As
is often the case in such studies, further analyses have revealed more subtle defects in these
animals, e.g., sporadic axonal degeneration and a major defect in spermatogenesis. An
alternative approach is the overexpression of the same gene in transgenic mice. In this case, the
mice showed about tenfold higher expression of the GM2/GD2 synthase gene in tissues such as the
skin, and the endogenous gangliosides were dramatically converted away from GM3 toward GM1.
Although no gross morphological changes were observed, much stronger inflammatory reactions
involving neutrophils were observed in the transgenic mice, suggesting an increased sensitivity
of neutrophils to chemotactic factors in the transgenic mice.
Overall, one must conclude that the complexities of at least the ganglio series of
glycosphingolipids may not be required for most normal organ formation in the mouse embryo.
Supporting this notion has been the lack of effects of inhibitors of glucosylceramide synthesis on
the in vitro development of embryos of the medaka fish and the mouse. In the latter experiments,
it was even shown that the level of glycosphingolipid production had fallen to approximately
90% of that in the control. These data indicate either that very small levels of glycosphingolipids
are sufficient to mediate their critical roles in development or that these dramatic patterns of
selective expression have some other as yet undefined purpose. One possibility (discussed
further in Chapter 3) is that some of this diversity is actually meant to evade recognition by
exogenous pathogens and toxins. In this scenario, the extensive diversity of glycosphingolipid
structures would assure that pathogens or toxins with exquisite specificity for recognition of
glycosphingolipids could only affect a few cells within a given host or host organ.
Future Directions
Glycosphingolipids possess a remarkable degree of structural diversity, and numerous enzymes
are involved in their synthesis, recycling, and turnover. Ultimately, the understanding of their
specific functions will require the genetic dissection of each step in their synthesis. The cloning
of these enzymes, and the generation and analysis of mutant animals, is a formidable but
necessary task. Moreover, such animals will have to not only be analyzed for intrinsic
pathophysiology, but also be exposed to a variety of specific pathogens, to see if they show
altered disease susceptibility.
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10. Glycophospholipid Anchors
Primary contributions to this chapter were made by G.W. Hart (The Johns Hopkins University
School of Medicine, Baltimore, Maryland).
THIS CHAPTER FIRST REVIEWS THE DISCOVERY that many membrane proteins are
anchored to the membrane via novel glycolipid structures, termed glycosyl
phosphatidylinositols, which were not known to exist prior to the mid 1980s. An overview of the
species and protein distribution of GPIs is presented, and common features and structural
diversity of GPI anchors are described. Also summarized are the major features of the
biosynthetic pathway for GPIs and their attachment to polypeptides. Finally, the current
hypotheses for GPI anchor functions are critically evaluated.
Historical Background of the Discovery of GPI Anchors (1 4)
The first data suggesting the existence of protein-lipid anchors appeared in 1963 with the finding
that crude bacterial phospholipase C selectively releases alkaline phosphatase from mammalian
cells. Inositol-containing phospholipid protein anchors were first postulated by Hiro Ikezawa's
group in Japan and by Martin Low's group in the United States in the mid 1970s. Their
predictions were based on the ability of highly purified bacterial phosphatidylinositol
phospholipase C to release certain enzymes, such as alkaline phosphatase, from cell surfaces.
However, without supporting structural data to validate their hypothesis, the existence of such
lipid anchors was not widely accepted. Alan William's group in the United Kingdom had also
independently noted that the cell surface antigen Thy-1 displays both the attributes of a
glycolipid and the properties of a glycoprotein. The carboxyl terminus of the Thy-1 glycoprotein
was subsequently found to contain both fatty acids and ethanolamine. In 1981, Tony Holder's
and George Cross's groups showed that the soluble form of the variant surface glycoprotein
(termed sVSG) of African trypanosomes contains an immune cross-reactive carbohydrate
attached to its carboxyl terminus via an amide linkage involving ethanolamine. Concomitantly,
Mervyn Turner's group showed that trypanosomes contain an enzyme that rapidly releases the
normally membrane-associated VSG (mfVSG) upon cellular damage. In fact, conversion of
mfVSG on living cells to the water-soluble sVSG is so rapid that the membrane form could only
be detected by rapidly boiling live trypanosomes in SDS prior to gel electrophoresis. In 1985,
Bangs and colleagues at Johns Hopkins made use of Holder's findings to demonstrate that the
lipid anchor on VSG is added within 1 minute of the polypeptide's synthesis in the ER. On the
basis of its rapid attachment to nascent VSG, these authors postulated that the membrane anchor
might be first preassembled in the ER and then attached en bloc. Later in 1985, Michael
Ferguson and colleagues at Oxford published a tour de force structural elucidation of the
glycolipid attached to the mfVSG of trypanosomes. These studies were the first to define
structurally the term glycosylphosphatidylinositol. Subsequent studies in several laboratories on
Torpedo ACHe, Thy-1, and erythrocyte ACHe demonstrated the covalent association of these
proteins with GPI components.
Since 1985, hundreds of GPI-anchored proteins have been identified in organisms ranging from
archeabacteria to humans (e.g., see Table 10.1). However, GPIs have not yet been found in
eubacteria. In 1989, Masterson and colleagues at Johns Hopkins used pulse-chase radioactive
labeling of live trypanosomes with GPI lipid precursors, in combination with product
characterization, to elucidate the major steps of GPI anchor biosynthesis. Many laboratories have
since elaborated on the specific steps in GPI anchor biosynthetic pathways in yeast and
mammalian organisms. These studies have also relied on mutations in enzymes of the
biosynthetic pathway first defined in lymphocytes and later in yeast. However, even today, very
little is known with respect to the enzymology, structures, or regulation of the enzymes involved
in these complex biosynthetic pathways.
Table 10.1. Examples of proteins with GPI anchors
Hydrolytic Cell adhesion Mammalian Protozoal Miscellaneous

enzymes molecules antigens antigens
Alkaline LFA-3 Thy-1 Ssp-4 decay-accelerating

phosphatase (trypanosomal) factor
5 -Nucleotidase glypican family of RT-6 variant surface 130-kD placental
heparan sulfate glycoprotein growth factor
proteoglycans
Trehalase neural cell Qa surface proteins Scrapie prion
adhesion molecule (Paramecium) protein
Alkaline contact site A Ly-6 195-kD antigen GP-2 (zymogen
phosphodiesterase I (Dictyostelium) (Plasmodium) granule)
p63 protease PH-20 (guinea pig carcinoembryonic Antigen 117 tegument protein
(Leishmania) sperm) antigen (Dictyostelium) (Shistosoma)
Renal dipeptidase Blast-1 FcIII receptor
(human
neutrophils)
Merozoite protease CD14 oligodendrocyte-
(Plasmodium) myelin protein
Aminopeptidase P 125-kD
glycoprotein
(Saccharomyces)
Lipoprotein lipase homologous
restriction factor
Diversity of Proteins Anchored via GPIs(1 3)
Table 10.1 illustrates the functional and structural diversity of proteins that are GPI-anchored.
Features of GPI-anchored proteins found to date reveal several important points: (1) GPI anchors
are widely distributed among many different organisms and are particularly abundant in
protozoa, (2) GPI-anchored proteins occur in virtually all mammalian cell types, and (3) GPI-
anchored proteins are functionally diverse, including hydrolytic enzymes, adhesion proteins,
complement regulatory proteins, receptors, prion proteins, and antigens. In addition, several
proteins have been found in which the same gene product is alternatively attached to the
membrane by a peptide transmembrane domain or by a GPI anchor. These differences in modes
of attachment usually arise by differential RNA splicing. Thus, the functions of GPI anchors do
not correlate with any particular biological role or class of cell surface protein. The specific
functions of GPI versus polypeptide anchors largely remain an enigma. However, several
popular hypotheses are discussed later in this chapter.
GPI Anchor Structures (5, 6)
GPIs share a common core structure (Figure 10.1). Phosphatidylinositol is glycosidically linked
through carbon 6 of the inositol ring to the reducing end of a nonacetylated glucosamine moiety.
GPIs are one of the rare instances in nature where glucosamine is found without either an acetyl
group (most glycoconjugates) or a sulfate moiety (heparin) modifying the amine group at the 2-
position. Thus, the nonacetylated glucosamine is a universal hallmark of GPI anchors (see
below). Three mannosyl residues, linked α1 4, α1 6, and α1 2, respectively, are glycosidically
attached to the glucosamine. The terminal α1 2- linked mannose is linked to
phosphoethanolamine by a phosphodiester linkage. The GPI is attached to the carboxy-terminal
carboxyl group by an amide linkage to the amino group of phosphatidylethanolamine.
Figure 10.1. Structure of GPI anchors. All characterized GPI anchors share a common core
consisting of ethanolamine-PO4-6Manα1-2Manα1-6Manα1-4GlcNα1-6myo-Ino-1-PO4-lipid.
Heterogeneity in GPI anchors is derived from various substitutions of this core structure and are
represented as R groups. R1 and R2 may be long-chain fatty acyl or alkyl groups or ceramide. R3
is frequently palmitate positioned at either C2 or C3 of the inositol ring. R4 and R7 are additional
phosphoethanolamines. R5, R6, and R8 may be various glycan substitutions. The few known
substituted glycan structures are indicated. (Reprinted, with permission, from [1] Cole and Hart
1997 [© Elsevier Science].)
As more GPI anchor structures are elucidated, it is clear that they not only share a common core,
but also display extraordinary structural diversity that depends on both the organism and cell
type in which they are synthesized. Figure 10.1 also summarizes the structural diversity of GPI
anchors. Considerable variability exists in both the glycan and the lipid portions of GPI anchors.
For example, the first mannose in the glycan core may contain a branched chain of α-linked
galactosyl residues (e.g., VSG) or a β-linked N-acetylglucosamine moiety (e.g., Thy-1). The
inner mannosyl residues may be modified by phosphoethanolamine (e.g., human erythrocyte
acetylcholinesterase). The mannosyl core can be extended by additional α-linked mannosyl
residues. Recently, GPI structures that contain sialic acids and more complex glycan structures
have also been described. The fatty acid chains attached to the phosphoinositol also vary in chain
length and saturation, occurring as diacylglycerol (e. g., VSG or Torpedo ACHe), alkyl-
acylglycerol (e.g., human erythrocyte ACHe or decay accelerating factor), stearoyl-lysoglycerol
(e.g., trypansome procyclic acidic repetitive protein), or ceramide (e.g., slime mold and yeast
GPIs). In addition, the inositol ring can be acylated (generally by palmitate) at positions 2 or 3.
The biological significance of these structural variations is unknown. One possibility is that they
may be important for controlling the lateral associations of GPI-anchored proteins in the plasma
membrane.
GPI Anchor Biosynthesis (7 12)
The biosynthesis of GPI anchors occurs in two major steps: (1) Preassembly of the donor GPI in
the ER membrane and (2) attachment of the GPI with concomitant cleavage of the carboxy-
terminal peptide from the newly synthesized protein. Analysis of GPI anchor precursor
biosynthesis was first made possible by the development of a cell-free system in African
trypanosomes. Each trypanosome has about 1 × 107 molecules of GPI-linked mfVSG on its
surface. Therefore, intermediates in the GPI-anchoring pathway are particularly abundant in
microsomal membrane preparations produced from this organism. Similar cell-free GPI anchor
synthetic systems have now been developed in Toxoplasma, yeast, and mammalian cells.
Although some variations in the pathways have been documented among different organisms,
particularly in the addition of acyl chains and phosphoethanolamines, the basic pathway has been
conserved throughout evolution. Figures 10.2 and 10.3 summarize the major steps in the
biosynthesis of GPI anchor precursors which involves the simple stepwise assembly of the
anchor on phosphatidylinositol lipids in the ER membrane (Figure 10.2). First, GlcNAc is added
from its donor UDP-GlcNAc, and the GlcNAc is then rapidly deacetylated. Mannosyl residues
are sequentially added, but instead of using GDP mannose as the donor, the immediate donor is
dolichol-phosphomannose, the same high-energy isoprenoid glycolipid that is involved in N-
linked glycan biosynthesis (see Chapter 7). In trypanosomes, the mannose residues are added
directly to the GlcN-PI. However, in yeast and mammalian cells, the inositol ring must first be
acylated to produce GlcN-PI(acyl) before the mannose residues can be added. In addition,
although the pathway is a simple stepwise assembly in trypanosomes, it appears somewhat more
complicated in mammalian cells. For example, in mammalian cells, phosphoethanolamine
residues must be added to the first mannose before the other mannose residues can be added
(Figure 10.3). Recent studies have shown that the phosphoethanolamine moiety attached to C6 of
the terminal mannose in the core is added en bloc to the GPI precursor with
phosphatidylethanolamine serving as the donor.
Figure 10.2. Linear pathway for biosynthesis of trypanosomal GPI anchors. (Dol-P- ) Dolichyl-
phosphoryl-mannose; (EthN) phosphoethanolamine; ( NH2) glucosamine; ( ) N-
acetylglucosamine; ( ) mannose; (Ac) acetate; (UDP) uridine 5 -diphosphate. (Modified, with
permission, from [1] Cole and Hart 1997 [© Elsevier Science].)
Figure 10.3. Proposed branched pathway for biosynthesis of mammalian GPI anchors.
Abbreviations are the same as those in Figure 10.2. (acyl) Acylation of inositol ring; (EP)
phosphoethanolamine. (Modified, with permission, from [1] Cole and Hart 1997 [© Elsevier
Science].)
Some organism-specific variations in the GPI-biosynthetic pathway are worth noting.

Bloodstream trypanosomes assemble their GPI precursors on phosphatidylinositol with stearic
acid in the sn-1 position and a mixture of fatty acids including 18:0, 18:1, 20:4, and 22:6 in the
sn-2 position. However, after assembly of the GPI, trypanosomes sequentially "re-model" the
GPI so that all of the fatty acids are myristic acid (14:0) at both positions. This is particularly
surprising, since myristic acid is found in comparative low abundance in their mammalian hosts,
and the parasites do not have the ability to synthesize it. Another feature unique to trypanosomes,
perhaps due to their need to make so much GPI, is the reversible acylation of the inositol ring of
the GPI precursor, which appears to provide a storage form of the GPI that is used for synthesis
only after it is deacylated. The biosynthesis of yeast GPI differs in at least two ways from that in
trypanosomes or mammals. First, in yeast, inositol acylation occurs at the level of GlcN-PI and is
an obligate step in the pathway. Second, in yeast and in Dictyostelium, many of the GPIs are
ceramide-based rather than glycerolipid-based. Current data suggest that yeast exchange the
glycerolipid moiety of the GPI anchor for a ceramide-based lipid component after the GPI
anchor is transferred to the protein. The function of this switch is unclear, but it does not appear
to be required for viability in culture. Mammalian GPI anchor biosynthesis also differs in some
respects from that in other species. Inositol acylation occurs early in biosynthesis, analogous to
yeast. However, the enzymology of the process appears to differ. In addition, the attachment of
additional phosphoethanolamine moieties to the inner mannosyl residues in the GPI core appears
to be unique to mammalian cells.
Studies on the topology of GPI assembly indicate that most if not all of the preassembly of the
GPI precursor occurs on the cytoplasmic face of the ER. In contrast, protease protection
experiments have shown that actual transfer of the anchor to the protein takes place in the
luminal compartment of the ER. Very little is known about the process of translocating the GPI
across the membrane or about the regulation of any of the enzymes involved in these pathways.
Attachment of the GPI anchor to the polypeptide is a posttranslational modification that involves
a transamidation reaction resulting in the cleavage of a carboxy-terminal GPI signal sequence
and the concomitant en bloc transfer of the GPI anchor to the newly formed carboxy-terminal
amino acid. Figure 10.4 illustrates the nature of this transamidation reaction. Two peptide signal
sequences are required for GPI anchor addition. First, the protein must have an amino-terminal
signal peptide directing the nascent chain into the ER, and second, it must also have a carboxy-
terminal signal peptide directing GPI anchor attachment. Like the amino-terminal signal peptide,
the carboxy-terminal GPI signal does not have a canonical sequence, but rather, it has
characteristic features that have become evident from examination of numerous GPI-anchored
protein sequences. The residue to which the anchor is attached (termed the ω site) and the
residue that is two amino acids on the carboxyl side (ω + 2 site) always have small side chains,
whereas the ω + 1 site can have large side chains. The ω + 2 site is followed by 5 10 hydrophilic
amino acids and then by 15 20 hydrophobic amino acids at or near the carboxyl terminus. Many
studies have shown that artificial fusion of such a GPI-signal peptide to the carboxyl terminus of
proteins causes them to become attached to GPI anchors. Table 10.2 shows some examples of
these GPI signal sequences.
Figure 10.4. Model for transfer of the GPI anchor precursor to the newly synthesized
polypeptide. See text for details. (Note: Nonstandard symbols were used in this figure, which
have not been changed from the original). (purple pentagon) Inositol; (blue circle) GlcN; (green
circle) mannose; (black circle) phosphate; (pink square) ethanolamine. (Reprinted, with
permission, from [8] Doering et al. 1990.)
Table 10.2. Examples of carboxy-terminal sequences signaling the addition of
GPI anchors
Protein GPI signal sequence
Acetylcholinesterase NQFLPKLLNATA C DGELSSSGTSSSKGIIFYVLFSILYLIFY

(Torpedo)
Alkaline phosphatase TACDLAPPAGTT D AAHPGRSVVPALLPLLAGTLLLLETATAP
(placenta)
Decay accelerating HETTPNKGSGTT S GTTRLLSGHTCFTLTGLLGTLVTMGLLT
factor
PARP (T. Brucei) EPEPEPEPEPEP G AATLKSVALPFAIAAAALVAAF
Prion protein (hamster) QKESQAYYDGRR S SAVLFSSPPVILLISFLIFLMVG
Thy-1 (rat) KTINVIRDKLVK C GGISLLVQNTSWLLLLLLSLSFLQATDFISI
Variant surface
glycoprotein
(T. Brucei) ESNCKWENNACK D SSILVTKKFALTVVSAAFVALLF
Boldfaced amino acid is the site of attachment of the GPI. Sequence to the right of the space is
cleaved from the protein by the transpeptidase upon anchor addition.
Mutations in the GPI Anchor Biosynthetic Pathway (9, 10)

Mutant cell lines and yeast have proven to be valuable in the study of GPI biosynthetic
pathways. For example, the simple production of GlcN-PI appears to require three different
genes, only one of which has been identified as the GlcNAc transferase. Paroxysmal nocturnal
hemoglobinuria (PNH) is a human disease in which patients suffer from hemolytic anemia. The
condition arises from improper expression of several GPI-anchored proteins that protect their
blood cells from lysis by the complement system (e.g., decay accelerating factor and CD59). The
defect in PNH cells is the inability to synthesize GlcNAc-PI due to a somatic mutation in the
PIG-A gene, which is an X-linked gene likely encoding the enzyme that transfers GlcNAc to
phosphatidylinositol. The mutation appears to occur in a bone marrow stem cell. Unlike other
enzymes in the pathway, which are encoded by autosomal genes, PNH caused by PIG-A
mutations is thought to arise at a higher frequency because of X-inactivation. In a PNH
heterozygote, X-inactivation of the one active allele of PIG-A results in the complete loss of a
functional transferase.
Identification of GPI-anchored Proteins (1, 3, 6)

GPI-anchored proteins can be identified by their solubilization after specific enzymatic or
chemical cleavage, in conjunction with detergent partitioning (e.g., in Triton X-114), antibody
recognition, and metabolic radioactive labeling. Figure 10.5 indicates the specific cleavage sites
or structural features that are useful in the identification of GPI anchors. Perhaps the most
commonly used preliminary demonstration that a protein has a GPI anchor is its release from the
cell surface or its solubilization by treating with bacterial PI-PLC or trypanosome-derived GPI-
specific phospholipase C (GPI-PLC). These enzymes leave a diacylglycerol in the membrane
and produce the immunoreactive glycan epitope (CRD) on the protein, which can be detected by
Western blotting with antibodies produced against the GPI of trypanosomes. One common
problem with this approach especially encountered in mammalian cells is that the lipases cannot
cleave a GPI anchor in which the inositol is acylated. These require prior treatment with mild
alkali to remove the fatty acid on the inositol ring. Alternatively, serum-derived GPI-specific
phospholipase D may be used to cleave GPI anchors. This enzyme cleaves between the inositol
ring and the phosphatidic acid moiety and is not inhibited by inositol acylation. Hydrofluoric
acid cleaves GPI anchors between the inositol ring and phosphatidic acid and also cleaves the
phosphodiester linkages between any phosphoethanolamines and mannosyl residues. Dilute
nitrous acid is particularly useful in the study of GPI anchors because it cleaves specifically
between the nonacetylated glucosamine and the inositol ring, releasing the protein-bound glycan
(now containing a diagnostic anhydromannose moiety) and phosphatidylinositol. In combination
with CRD antibodies, composition analyses, radioacitve labeling with myo-inositol,
ethanolamine, glucosamine, mannose, or fatty acids and chromatographic or detergent
partitioning methods, these degradation methods represent a powerful set of tools to study GPI
anchors on proteins.
Figure 10.5. Enzymatic and chemical cleavage sites of GPI anchors useful in identifying GPI-
anchored membrane proteins. (GPI-PLC) GPI-specific phospholipase C; (GPI-PLD) GPI-
specific phospholipase D; (HF) hydrogen fluoride; (HONO) nitrous acid; (NaOH [NH3]) mild
alkali treatment with either sodium hydroxide or ammonia; (PI-PLC) phosphatidylinositol-
specific phospholipase C. (Reprinted, with permission, from [1] Cole and Hart 1997 [© Elsevier
Science].)
Putative Functions of GPI Anchors (1,13 15)
The physiological functions of GPI anchors are still largely unknown. Studies using mice in
which the PIG-A gene was knocked out indicate that germ-line GPI anchor deficiency in mice is
lethal, suggesting that GPI anchors are required for normal development to occur. GPI anchors
obviously serve to anchor proteins to the extracellular surface of plasma membranes. However,
there is much discussion regarding other specific functions for this highly conserved, multiple
step and complex alternative mechanisms for anchoring membrane proteins. GPI anchors may
provide subtle functions, such as influencing the overall characteristics of the cell membrane.
The fatty acid content of the anchor contributes to the lipid composition of the membrane and
can determine the membrane-packing characteristics of the protein. In fact, as documented for
the mfVSG of trypanosomes, the glycan substituents might serve an important function in
organizing the proteins within the lipid bilayer. Membrane-anchored proteins that do not require
transmembrane or cytoplasmic domains will, by default, reduce "protein clutter" at the
cytoplasmic face of the membrane by not interfering with other molecules in the region.
Functions suggested for GPI anchors include (1) allowing proteins an increased lateral mobility,
(2) mediation of the release or secretion of proteins by activation of a lipase, (3) targeting protein
to apical surfaces, (4) regulation of endocytosis or protein turnover, and (5) involvement in
signal transduction of receptor-mediated events.
The lack of a transmembrane domain precludes interactions of GPI-anchored proteins with the
cytoskeleton; thus, their lateral mobility is not restricted by cytoskeletal structures. Many GPI-
anchored proteins are receptors or adhesion molecules, and freedom of movement in the
membrane may be advantageous for their interactions with ligands. As attractive as this
hypothesis is, however, there is no strong evidence supporting increased lateral mobility due to a
GPI anchor. In fact, some GPI-anchored proteins have a somewhat higher mobility in the
membrane, but others appear to have a lower than average mobility. Clearly, complex
interactions with other components of the membrane are also involved. Direct comparison of the
lateral mobility of chimeric proteins, where the GPI anchor is replaced with a transmembrane
domain, has experimentally demonstrated little change in the mobility of the protein.
Cleavage of GPI anchors by highly specific phospholipases suggests a potential mechanism for
rapid protein release or secretion mediated by GPI anchors. This hypothesis is supported by the
presence of soluble and GPI-anchored forms of proteins, by the rapid shedding of GPI-linked
proteins from the surface of cells in culture, and by the presence of GPI-PLD in mammalian
serum. It is unclear, however, to what extent GPI-linked protein shedding occurs in vivo, and
whether it generally involves proteolytic cleavage or lipase activity. In some cases, proteins may
be released from cells without the removal of their membrane anchors. To ascertain whether GPI
anchors are directly involved in protein secretion, anchor removal mediated by lipase activity
must be demonstrated in vivo. One physiologically significant example is the rapid enzymatic
release of mfVSG to sVSG in damaged trypanosomes, which appears to play a part in the
parasite's defense against immune attack. mfVSG is also released upon differentiation of the
parasite from its bloodstream to the insect infective form.
Localization of GPI-anchored proteins to apical surfaces of epithelial cells has led to the
suggestion that GPI-anchored proteins contain an apical sorting or targeting signal. Two lines of
evidence support this hypothesis. First, GPI-anchored proteins are targeted to the apical surface
when transfected into epithelial cells in culture or in transgenic mice. Second, targeting of GPI-
linked and transmembrane proteins is altered by replacement of their membrane anchors. For
example, replacing the transmembrane and cytoplasmic regions of vesicular stomatitis virus
glycoprotein G or herpes simplex virus glycoprotein D with a GPI anchor re-routes the proteins
from a normally basolateral location to a new apical position. In contrast, apical proteins, such as
placental alkaline phosphatase, can be re-routed to basolateral locations by the addition of a
peptide transmembrane domain. Recent evidence indicates that the signal possessed by GPI-
anchored proteins may not be specific solely for apical surfaces, but for polarized surfaces in
general. For example, GPI-anchored Thy-1 is expressed specifically on axonal membranes,
rather than evenly distributed on the cell. Thus, the possession of a GPI anchor may signal the
sorting of the protein into cell-specific pathways, localizing the protein to a polarized surface.
The signal for targeting to a polarized surface is unknown. However, the sorting of GPI-linked
protein may involve the coclustering of GPI anchors with apical glycosphingolipids. This
hypothesis has gained support by the observation that GPI-anchored proteins form insoluble
complexes with glycosphingolipids (see Chapter 9). A transmembrane protein is postulated to be
necessary for sorting of GPI-anchored proteins, because it is likely that the glycolipid moiety of
GPI-anchored proteins cannot interact directly with the cytoplasmic sorting machinery.
GPI-anchored proteins appear to have a role in a specialized form of endocytosis, termed

potocytosis. Potocytosis involves the capture and import of scarce extracellular molecules or ions
against their concentration gradient through membrane invaginations, called caveolae,
independent of the lysosomal pathway. Caveolae are 50-nm-wide flask-shaped structures coated
with the 22-kD transmembrane protein, caveolin. Caveolae contain clusters of GPI-anchored
proteins, the most well studied of which is the folate receptor. However, evidence also exists for
the presence of other GPI-anchored proteins, such as alkaline phosphatase, Thy-1, and prion
PrP(C) in caveolae. High-density clusters (30,000 molecules/µm2) of a mixed population of GPI-
anchored proteins can reside in caveolae. The structural integrity of these clusters and that of
caveolae appear to be mediated by the interactions between GPI anchors and cholesterol. In
contrast, GPI-anchored proteins are poorly represented in clathrin-coated pits, the main pathway
for receptor-mediated endocytosis. GPI anchors may extend the half-life of cell surface proteins
whose functions do not involve internalization. This is consistent with their putative function in
caveolae. In this scenario, ligands are bound by GPI-anchored receptors in open caveolae.
Caveolae then close and the ligand is released enzymatically or by low pH. A large concentration
gradient results from the small volume within the caveolae. The trapped molecules or ions flow
down their concentration gradient into the cytoplasm through membrane carriers or transporters.
GPI-anchored proteins are not internalized, and when caveolae reopen, they are presented for the
next round of potocytosis.
Activation of lymphocytes may be mediated by GPI anchors. T cells are activated normally by
antigen receptors binding to antigenic peptides presented in association with major
histocompatibility proteins. Antibodies to GPI-anchored proteins on T cells mimic T-cell
activation by inducing cell proliferation, interleukin-1 and -2 production, and other metabolic
changes in T cells. In contrast, most antibodies to other membrane components of T cells do not
activate the cells. Moreover, pretreatment of lymphocytes with PI-PLC, thereby releasing GPI-
anchored proteins, reduces the response of T cells to antibody mitogens. Fusion proteins of H-2
or Qa-2 histocompatibility antigens have been engineered to assay the importance of GPI
anchors to T-cell activation. Under normal conditions, antibodies to Qa-2 are mitogenic, whereas
antibodies to H-2 are not. T-cell activation is induced by Qa-2- or H-2-specific antibodies
binding to the GPI-anchored forms of Qa-2 or H-2, respectively, and not by binding to the same
polypeptides bearing peptide transmembrane sequences. Likewise, the normal GPI-anchored
form of Ly-6E, transiently transfected into lymphocyte cell lines, mediates T-cell activation,
whereas the peptide transmembrane form does not. The in vivo functional significance of these
observations is not known.
The signal transduction mechanisms involving GPI-anchored proteins that lack intracellular
domains are not understood. GPI anchor degradation products, inositol phosphate glycan and
diacylglycerol, derived from phospholipase activity, have been suggested to mediate the action
of hormones such as insulin, insulin-like growth factor-1, nerve growth factor, interleukin-2, and
thyroid-stimulating hormone. These hormone-sensitive glycolipids have chemical compositions
similar to those of GPIs, but detailed structural data are lacking. Moreover, inositol glycans
derived from trypanosomal GPI mimic metabolic actions of insulin, whereas anti-inositol glycan
antibodies block the actions of insulin. Insulin-sensitive GPIs also appear to mediate T-cell
activation in T-cell mutants that are unable to link proteins to GPI anchors. An early step in T-
cell activation is stimulation of tyrosine kinase activity. Interestingly, protein tyrosine kinases
coimmunoprecipitate with antibodies against GPI-anchored proteins and colocalize with GPI-
anchored proteins in large noncovalent complexes. These studies suggest that protein tyrosine
kinases are part of the signal transduction mechanism by which GPI-anchored proteins mediate
T-cell activation. Clearly, we know very little about the mechanisms involved in GPI-mediated
signal transduction or indeed even how such signals are transduced across the plasma membrane.
Future Prospects and Directions

Only about 13 years ago, GPI anchors were not even known to exist. Now it is known that they
are a major mechanism by which membrane proteins are anchored to the membrane and indeed
are the dominant mechanism in protozoa. Several challenges are faced in understanding these
modifications. First, the function of GPI anchoring in most instances is not clearly understood.
Why is the same protein anchored by a peptide domain in some instances and by a GPI anchor in
others? What are the specific functions of any GPI at a mechanistic level? Second, very little is
known about the enzymology or regulation of GPI anchor biosynthesis. With the rapid cloning of
the genes encoding the enzymes in the biosynthetic pathway, this area of GPI anchor research
will be very active in the next 10 years. Finally, no understanding now exists of the functional
significance of the structural diversity of either the glycan or the lipid components of GPI
anchors. Clearly, GPI anchors will remain a "hot" area of glycobiology for years to come.
References
1. Cole R.N. and Hart G.W. 1997. Glycosyl-phosphatidylinositol anchors: Structure,

biosynthesis and function. In Glycoproteins II (ed. Montreuil J. et al.), pp. 69 88. Elsevier,
Amsterdam.
2. P.T. Englund. 1993. The structure and biosynthesis of glycosyl phosphatidylinositol protein
anchors Annu. Rev. Biochem. 62: 121-138. (PubMed)
3. M.A.J. Ferguson. 1992. Glycosyl-phosphatidylinositol membrane anchors: The tale of a tail

4. M.G. Low. 1987. Biochemistry of the glycosyl-phosphatidylinositol membrane protein

anchors Biochem. J. 244: 1-13. (PubMed) (Full Text in PMC)
5. M.A. Ferguson, S.W. Homans, R.A. Dwek, and T.W. Rademacher. 1988. Glycosyl-
phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the
membrane Science 239: 753-759. (PubMed)
6. A.K. Menon. 1994. Structural analysis of glycosylphosphatidylinositol anchors Methods

Enzymol. 230: 418-442. (PubMed)
7. T.L. Doering, W.J. Masterson, P.T. Englund, and G.W. Hart. 1989. Biosynthesis of the
glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein.
Origin of the non-acetylated glucosamine J. Biol. Chem. 264: 11168-11173. (PubMed)
8. T.L. Doering, W.J. Masterson, G.W. Hart, and P.T. Englund. 1990. Biosynthesis of glycosyl
phosphatidylinositol membrane anchors J. Biol. Chem. 265: 611-614. (PubMed)
9. V.L. Stevens. 1995. Biosynthesis of glycosylphosphatidylinositol membrane anchors

Biochem. J. 310: 361-370. (PubMed) (Full Text in PMC)
10. J. Takeda and T. Kinoshita. 1995. GPI-anchor biosynthesis Trends Biochem. Sci. 20: 367-
371. (PubMed)
11. A.M. Tartakoff and N. Singh. 1992. How to make a glycoinositol phospholipid anchor
12. S. Udenfriend and K. Kodukula. 1995. How glycosylphosphatidylinositol-anchored

membrane proteins are made Annu. Rev. Biochem. 64: 563-591. (PubMed)
13. R.G.W. Anderson. 1993. Potocytosis of small molecules and ions by caveolae Trends Cell
15. M. Sargiacomo, M. Sudol, Z. Tang, and M.P. Lisanti. 1993. Signal Transducing Molecules
and Glycosyl-phosphatidylinositol-linked Proteins Form a Caveolin-rich Insoluble Complex in
Mdck Cells J. Cell Biol. 122: 789-807. (PubMed)
11. Proteoglycans and Glycosaminoglycans
Primary contributions to this chapter were made by J.D. Esko (University of California at San
Diego).
THE FOCUS OF THIS CHAPTER IS on the structure, biosynthesis, and general biology of
proteoglycans. Topics include a description of the major families of proteoglycans, their
characteristic polysaccharide chains (glycosaminoglycans), and the pathways involved in their
biosynthesis.
Historical Perspective (1 4)
The study of proteoglycans dates back to the turn of the twentieth century with investigations of
"chondromucoid" from cartilage and anticoagulant preparations from liver (heparin). From 1930
to 1950, great strides were made in analyzing the chemistry of the polysaccharides of these
preparations (also known as "mucopolysaccharides"), notably by Karl Meyer and his associates,
who described the structure of hyaluronic acid, dermatan sulfate, keratan sulfate, and different
isomeric forms of chondroitin sulfate. During this period, Jorpes and Gardell in Sweden
described the chemical structure of heparin and heparan sulfate. These polysaccharides
eventually came to be known as glycosaminoglycans to indicate the presence of amino sugars
and other sugars in a polymeric form. Subsequent studies by Rodén and Lindahl provided
insights into the linkage of the chains to core proteins, and these structural studies together paved
the way for the biosynthetic studies that followed.
The 1970s marked a turning point in the field, when improved isolation and chromatographic
procedures were developed to purify and analyze tissue proteoglycans and glycosaminoglycans.
Sajdera and Hascall developed a density gradient method for separating the large aggregating
proteoglycans, which revealed that the extracellular matrix was much more organized than
previously appreciated (Figure 11.1). Also during this period, P.F. Kraemer showed that the
production of proteoglycans was a general property of animal cells and that proteoglycans and
glycosaminoglycans were present on the cell surface. This observation led to a rapid expansion
of the field and the eventual appreciation of proteoglycan function in cell adhesion and signaling,
as well as a host of other biological activities (for greater details, see Chapter 29). Today, studies
using somatic cell genetics, molecular cloning, and gene knockouts are under way using
organisms as diverse as flies, worms, and mice to better understand the physiological role of
proteoglycans. In turn, human diseases associated with aberrant biosynthesis of proteoglycans
have been discovered (see Chapters 31 37). By understanding the pathways in greater detail,
novel ways to intervene with proteoglycan assembly may emerge that may have therapeutic
value (see Chapters 40 and 41).
Figure 11.1. The large cartilage CS proteoglycan (aggrecan) forms an aggregate using
hyaluronic acid as a scaffold and link protein to stabilize the complex. In addition, cartilage
matrix contains collagen (not shown) and various glycoproteins (partially filled circles).
(Reprinted, with permission, from [7] Rodén 1980.)
Proteoglycans and Glycosaminoglycans Are Components of

Extracellular Matrices and Cell Surfaces (3,5 12)
Proteoglycans consist of a core protein and one or more covalently attached GAG chains (Figure
11.2). GAGs are linear polysaccharides, whose building blocks (disaccharides) consist of an
amino sugar (either GlcNAc or GalNAc) and an uronic acid (GlcA and IdoA). Virtually all
mammalian cells produce proteoglycans and either secrete them into the ECM, insert them into
the plasma membrane, or store them in secretory granules. The matrix proteoglycans include
small interstitial proteoglycans (decorin, biglycan, fibromodulin), a proteoglycan form of type IX
collagen, and one or more members of the aggrecan family of proteoglycans (aggrecan, brevican,
neurocan, or versican). Some of these proteoglycans contain only one GAG chain (e.g., decorin),
whereas others have more than 100 chains (e.g., aggrecan). The matrix proteoglycans typically
contain the GAGs known as CS or DS. Exceptions to this generalization exist, since the HS
proteoglycans perlecan and agrin are major species found in basement membranes.
Figure 11.2. Glycosaminoglycans consist of repeating disaccharide units. HA lacks any sulfate
groups, but the rest of the GAGs contain sulfates at various positions. As described in the text,
considerable variations occur in the positions of sulfation and IdoA.
The ECM determines the physical characteristics of tissues and many of the biological properties
of cells embedded in it. The major components of the ECM are fibrous proteins that provide
tensile strength (e.g., various collagens and elastin), adhesive glycoproteins (e.g., fibronectin,
laminin, and tenascin), and proteoglycans that provide a hydrated gel which resists compressive
forces.
Cells also elaborate a diverse group of membrane proteoglycans. These typically have type I
orientations and have either single membrane-spanning domains or a GPI anchor (see Chapter
10). Membrane proteoglycans tend to contain mostly HS (e.g., the glypicans), but many are
hybrid structures containing both HS and CS (e.g., the syndecans and betaglycan). A few
membrane proteoglycans contain exclusively CS (e.g., CD44 and NG2).
In addition, cells with storage granules concentrate proteoglycans along with other secretory
products. These proteoglycans typically contain highly sulfated forms of CS, although the
secretory granules of connective tissue mast cells contain mostly heparin. Secretory granule
proteoglycans are thought to help sequester and regulate the availability of positively charged
components, such as proteases and bioactive amines.
It should be kept in mind that most proteoglycans also contain O- and N-glycans typically found
on glycoproteins (see Chapters 7 and 8). The GAG chains are much larger than these other types
of glycans (e.g., a 20-kD GAG chain contains ~80 sugar residues, whereas a typical biantennary
N-glycan may contain 10 12 residues). Therefore, the properties of the GAGs tend to
predominate, but the other glycans may also have biological activities.
Proteoglycans Interact with a Variety of Ligands (11 14)
Table 11.1 lists some of the proteins known to interact with GAGs (see Chapter 29). Most of the
proteins bind to HS, which may reflect the greater chemical diversity and capacity of HS to
interact with proteins through varied arrangements of sulfated sugar residues. These interactions
have profound physiological effects. For example, injection of heparin into the bloodstream
results in rapid anticoagulation due to binding and activation of antithrombin. Ligand binding to
GAGs may result in (1) immobilization of proteins at sites of production, (2) regulation of
enzyme activity, (3) binding to a signaling receptor, (4) protection of ligands against
degradation, and (5) a reservoir of ligands for future mobilization. In some cases, the interaction
depends on a very specific sequence of modified sugars in the GAG chain. The best-studied
example is antithrombin-heparin, which depends on a specific pentasaccharide sequence (Figure
11.3)
Figure 11.3. The antithrombin-binding sequence in heparin consists of a very specific

arrangement of sulfated sugar residues and uronic acid epimers.
Table 11.1. Examples of proteins that bind to GAGs
Cell/Matrix interactions Coagulation/Fibrinolysis Lipolysis Inflammation/Growth
Laminin antithrombin III lipoprotein lipase FGFs and FGF

Fibronectin heparin cofactor II hepatic lipase receptors
Thrombospondin tissue factor pathway apoE scatter factor (HGF)
Type I collagen inhibitor LDL VEGF
Type III collagen thrombin IL-8/MIP-1β
Type V collagen protein C inhibitor TGF-β
Vitronectin tPA and PAI-1 L and P selectins
Tenascin superoxide dismutase
Proteoglycans Exhibit Great Structural Diversity (14 17)
Proteoglycans exhibit tremendous structural variation due to a number of factors. First,

proteoglycans that contain more than one GAG chain usually exhibit variation in the number of
attached chains. For example, syndecan-1 has five attachment sites for GAGs, but not all of the
sites are used equally. In addition, some sites contain either CS or HS. The length of the chains
also varies and the arrangement of sulfated residues along the chains differs. Thus, a preparation
of syndecan-1 represents a diverse population of molecules. Finally, syndecan-1 from different
cell types exhibits differences in the number of chains, their lengths, and their fine structures.
This characteristic of all proteoglycans presumably reflects the way GAGs are made, as
described below.
Hyaluronan Is the Simplest Glycosaminoglycan (18 24)
The HA disaccharide consists of GlcNAcβ1-3GlcAβ1-4 and is repeated many times in each

chain. HA is distributed widely in nature, from the capsules of Streptococcus to tissues of
invertebrate and vertebrate organisms. In mammals, HA is abundant in skin, skeletal tissues, the
vitreous of the eye, umbilical cord, and synovial fluid. A typical polymer might contain 104
disaccharides (mass 105 to 107 daltons). In solution, HA has an extended structure when
stretched end to end, a polymer of 106 daltons is about 2 µm. Because of its length, it tends to
entangle into a mesh-like structure. At a concentration of 10 mg/ml, its viscosity (η) is about
5000 that of water, which confers rigidity to tissues when HA is present at high concentrations
(e.g., rooster combs and the vitreous of the eye). Under shear stress, the viscosity drops rapidly,
but it remains quite elastic. Thus, HA has the property of a biological lubricant, reducing friction
during movement and providing resiliency under static conditions. The uniform structure of HA
would seem to obviate specific biological interactions, but in fact, several HA-binding proteins
have evolved. For more details, see Chapter 29.
The biosynthesis of HA involves copolymerization of GlcNAc and GlcA from of their respective
high-energy nucleotide donors, UDP-GlcNAc and UDP-GlcA. Unlike all the other GAGs, HA is
never covalently linked to protein. Its synthesis appears to occur at the plasma membrane in
cells, which is an exception to the rule that glycosylation generally occurs in the Golgi.
Furthermore, the polymer is thought to assemble from the reducing end, resulting in its extrusion
from the cell surface. Cells expressing an HA-binding protein on its surface (e.g., CD44) will
retain the extruded material as a pericellular coat one or two cell diameters thick.
Three HA synthetases have been cloned, and these constitute a family of homologous enzymes
(HAS 1 3). Isozymes have been identified in a number of organisms, including DG42 in
Xenopus, a homolog in a virus that infects a Chlorella-like green algae, and Streptococcus. The
HA synthetases vary in size from 42 to 65 kD, depending on isoform and species. Hydropathy
plots indicate that the enzyme may span the membrane as many as 12 times, but the functional
form of the enzyme is unknown. Its catalytic activity is impressive, since it can polymerize about
100 monosaccharides/sec in vitro, or about 106 daltons of polysaccharide in less than 1 minute.
Hyaluronidases are enzymes that degrade HA. Several types of hyaluronidases are known that
generate either tetrasaccharides (testicular hyaluronidase) or disaccharides (bacterial) as end
products. In addition, endothelial cells express a receptor that facilitates clearance of HA from
the blood. After entering lysosomes, the polymer can be completely degraded to GlcNAc, which
can be recycled (see Chapter 18), and GlcA, which is catabolized by the pentose pathway.
Keratan Sulfate, a Sulfated Polylactosamine (25 30)
KS is a sulfated polylactosamine chain identical to the type found on conventional glycoproteins

and mucins (see Chapters 7 and 8). Their linkage to protein distinguishes two types of KS. KS I,
originally described in cornea, is linked through a core glycan structure found in the N-
glycosylated glycoproteins. KS II (skeletal KS) is an O-glycan linked through GalNAc to
Ser/Thr, like the linkage found in mucins (see Chapter 8). Examples of KS proteoglycans are
shown in Figure 11.4 and Table 11.2. In the cornea, KS proteoglycans maintain the even spacing
of type I collagen fibrils, allowing the passage of light without scattering. Defects in sulfation
(macular corneal dystrophy) or chain formation (keratoconus) cause distortions in fibril
organization and corneal opacity. In cartilage, the function of the KS II is unclear. In humans and
cows, the large CS proteoglycan found in cartilage (aggrecan) contains a segment of 4 23
hexapeptide repeats where the KS chains are located (E-E-P-S,F-P-S), but rats and other rodents
lack this motif and do not contain KS.
Table 11.2. Examples of keratan sulfate proteoglycans
Proteoglycan Type Core mass (kD) Distribution
Lumican KS I 37 broad
Keratocan KS I 37 broad, but sulfated only in cornea
Fibromodulin KS I 59 broad
Mimecan KS I 25 broad, but sulfated only in cornea
SV2 KS I 80 synaptic vesicles
Claustrin KS II 105 CNS, membrane proteoglycan
Aggrecan KS II 200 cartilage
Figure 11.4. Keratan sulfates consist of a sulfated polylactosamine linked either to Asn or
Ser/Thr residues. The actual order of the various sulfated and nonsulfated disaccharides occurs
somewhat randomly along the chain. Not shown are sialic acid and fucose residues that may be
present at the termini of the chains.
The polylactosamine of KS I can be quite long (~50 disaccharides, 20 25 kD) and contain a
mixture of nonsulfated, monosulfated (Gal-GlcNAc6S), and disulfated disaccharides (Gal6S-
GlcNAc6S).
The biosynthesis of the polylactosamine and the underlying linkage structure is covered in
Chapters 7 and 8. At least two sulfotransferases, GlcNAc 6-O-sulfotransferase, and Gal 6-O-
sulfotransferase, both of which have been cloned, catalyze the sulfation reactions. These
enzymes, like other sulfotransferases, utilize activated sulfate, PAPS (3 -phosphoadenyl-5 -
phosphosulfate), as a high-energy donor (see Chapter 6). Studies of lung mucin biosynthesis
have suggested a scheme for coordinating polymer elongation and sulfation: GlcNAc 6-O-
sulfotransferase will act only on terminal GlcNAc residues, whereas Gal transferase will act on
both sulfated and nonsulfated GlcNAc (Figure 11.5). Thus, failure to add sulfate to a terminal
GlcNAc residue results in a disaccharide unit devoid of sulfate or having at most one sulfate
group located on the GlcNAc residue. The relationship of enzymes involved in KS I and KS II
sulfation is unclear at this time.
Figure 11.5. The pathway depicts a mechanism for generating fully sulfated (left) or partially
sulfated disaccharides (right) during KS biosynthesis. The inability of the GlcNAc 6-O-
sulfotransferase to act on internal residues means that polymerization and sulfation occur
simultaneously.
Bacterial keratanases degrade KS at characteristic positions (Table 11.3). In animals, KS is

degraded in lysosomes by the sequential action of exoglycosidases (β-galactosidase and β-
hexosaminidase) after removal of the sulfate groups on the terminal residue by sulfatases (see
Chapter 18).
Table 11.3. Keratanases
Enzyme Specificity
Endo-β-galactosidase (Flavobacterium) Gal-GlcNAc (no sulfate tolerated)

Keratanase I (Pseudomonas species) Gal-GlcNAc6S
(endo β-galactosidase)
Keratanase II (Gram-negative organisms) GlcNAc-Gal6 ± S
(endo β-glucosaminidase)
Heparan Sulfate and Chondroitin Sulfate Are Linked by Xylose

to Serine (31 33)
Two classes of GAG chains, CS and HS, are linked to serine residues in core proteins by way of
xylose (Figure 11.6). Xylosyltransferase initiates the process using UDP-xylose as donor. A
glycine residue almost invariably lies to the carboxy-terminal side of the serine, but a perfect
consensus sequence does not exist for the attachment site. In addition, at least two acidic amino
acid residues are usually present, and they can be located on one or both sides of the serine,
usually within a few residues. Several proteoglycans contain clustered GAG attachment sites,
raising the possibility that xylosyltransferase could act in a processive manner. Xylosylation is
an incomplete process in some proteoglycans, which may explain why proteoglycans with
multiple attachment sites contain different numbers of chains in different cells. Variation in the
degree of GAG substitution also might result from low levels of UDP-xylose, low activity of
xylosyltransferase, or competing reactions, such as phosphorylation, acylation, or other forms of
glycosylation.
Figure 11.6. Chondroitin and heparan sulfate biosynthesis initiates by the formation of a linkage
region tetrasaccharide. Addition of the first hexosamine residue commits the intermediate to
either CS or HS.
After xylose addition, a linkage tetrasaccharide is generated (Figure 11.6), which can undergo
phosphorylation at C-2 of xylose and sulfation of the galactose residues. In general,
phosphorylation and sulfation occur substoichiometrically. The lack of chain specificity for
phosphorylation would seem to exclude it as a signal for controlling composition. However,
phosphorylation may be transient, suggesting a role in processing or sorting. Additional studies
are needed to determine whether any relationship exists between galactose sulfation and chain
initiation, polymerization, and turnover.
The linkage tetrasaccharide lies at a bifurcation in the biosynthetic pathway. Three types of
reactions have been detected: addition of β-GalNAc (initiation of CS), addition of α-GlcNAc
(initiation of HS), and addition of α-GalNAc (Figure 11.6). These reactions are thought to be
catalyzed by three independent transferases. The α-GalNAc reaction is unusual and gives rise to
a pentasaccharide or heptasaccharide containing one CS disaccharide that has not yet been found
in a natural proteoglycan. These enzymes are important control points because they ultimately
regulate the type of GAG chain that will assemble. Control over the addition of β-GalNAc or α-
GlcNAc appears to be manifested at the level of enzyme recognition for linear amino acid
sequences immediately adjacent to the attachment site in the core protein. α-GlcNAc addition
involves recognition of the amino acid sequences flanking attachment sites for HS. These usually
contain a cluster of acidic residues within seven to nine residues of the site. In the example
shown below (syndecan-2), the underlined sequence indicates the sites of GAG attachment and
the boldface letters refer to the clustered acidic residues.
-SSIEEASGVYPIDDDDYSSASGSGADEDIESPVLTTS-
Although CS and HS chains usually assemble on the linkage region tetrasaccharide described
above, some cells also can generate these GAGs as N-glycans. These were detected in
oligosaccharide preparations released with N-glycanase, which liberates glycans linked to
asparagine. However, the structure of the "linkage fragment" (i.e., the underlying core glycan) is
not known.
Chondroitin Sulfate/Dermatan Sulfate Biosynthesis (2,34 36)
CS consists of repeating units of GalNAc-GlcA disaccharides (see Figure 11.1) polymerized into
long chains with an average size of 20 kD (~40 disaccharides). On the basis of the structure of
CS, at least five enzyme activities could be predicted, including three transferases (the initiating
GalNAc transferase described above and the polymerizing GalNAc and GlcA transferases) and
two sulfotransferases (GalNAc 4-sulfotransferase and GalNAc 6-sulfotransferase). Additional
enzymes exist for epimerization of GlcA to IdoA in DS, sulfation at C-2 of the uronic acids, and
other patterns of sulfation found in unusual species of chondroitin. The conversion of GlcA to
IdoA in DS is unusual since it takes place after the polymer has formed (cf. Chapter 6). To date,
only the GalNAc 6-sulfotransferase has been purified and cloned, although progress in this area
is expected to accelerate with recent advances in genome analysis and cloning techniques.
CS assembly can occur on virtually all proteoglycans, depending on the cell in which the core
protein is expressed. The major proteoglycans that typically contain CS or DS chains in vivo are
shown in Table 11.4. CSs from different sources vary in the location of sulfate groups. This is
easily assessed using bacterial chondroitinases, which cleave the chains into disaccharides.
Separation of the products reveals that many types of CS exist in nature (Table 11.5), but many
chains are hybrid structures containing more than one type of disaccharide. For example, DS
refers to a glycan that contains one or more IdoA-containing disaccharides (chondroitin sulfate
B) as well as GlcA-containing disaccharides. Animal cells also degrade CS in lysosomes using a
series of exoglycolytic activities (see Chapter 18).
Table 11.4. Examples of chondroitin sulfate proteoglycans
Proteoglycan Core protein (kD) Number of chains Tissue
Aggrecan 208 220 ~100 secreted, cartilage

Versican 265 12 15 secreted, fibroblasts
Neurocan 145 1 2 secreted, brain
Brevican 96 0 4 secreted, brain
Decorin 36 1 secreted, connective tissue cells
Biglycan 38 1 2 secreted, connective tissue cells
Bamacan 138 1 3 basement membranes
α2(IX) collagen 68 1 cartilage, vitreous humor
Thrombomodulin 58 1 endothelial membrane
CD44 37 1 4 lymphocytes, membrane
NG2 251 2 3 neural cells, membrane
Invariant chain 31 1 antigen-processing cells
Serglycin 10 19 10 15 myeloid cells, granules
Table 11.5. Types of chondroitin sulfates
Chondroitin type Disaccharide repeat Source
A GlcA-GalNAc4S cartilage
B IdoA2S-GalNAc4S dermatan sulfate, skin
C GlcA-GalNAc6S cartilage
D GlcA2S-GalNAc6S dermatan sulfate
E GlcA-GalNAc4,6diS squid, secretory granules
Heparin and Heparan Sulfate Biosynthesis (3,14, 17,37 39)
Heparin and HS consist of the disaccharide unit, GlcNAcα1-4GlcAβ1-4 (see Figure 11.1).
Heparin is produced exclusively by mast cells and differs from HS in the degree of modification
of the sugar residues, as described below. Virtually all cells express HS proteoglycans, and
several major families of core proteins have been cloned and analyzed biochemically,
genetically, and biologically (Table 11.6).
Table 11.6. Examples of heparan sulfate proteoglycans
Proteoglycan Core protein (kD) Number of chains Tissue
Perlecan 400 1 3 HS basement membranes

Agrin 212 2 3 HS basement membranes
Syndecans 1 4 31 45 1 3 CS epithelial cells and
1 2 HS fibroblasts
Betaglycan 110 1 HS fibroblasts
1 CS
Glypicans 1 5 ~60 1 3 HS epithelial cells and
fibroblasts
Serglycin 10 19 10 15 heparin/CS mast cells
As the polysaccharides polymerize, they undergo a series of modification reactions catalyzed by

at least four families of sulfotransferases, and one epimerase (Figure 11.7). A GlcNAc N-
deacetylase/N-sulfotransferase acts on a subset of GlcNAc residues in a cluster along the chain.
Generally, the enzyme deacetylates and rapidly adds sulfate to the free amino groups to form
GlcNSO3, but some of the deacetylated GlcN residues can escape N-sulfation. An epimerase,
such as the one involved in DS synthesis, then acts on GlcA residues immediately adjacent to the
GlcNS, followed by 2-O-sulfation of the IdoA that is generated. Next, a sulfotransferase adds
sulfate groups to the 6-OH of the GlcN residues adjacent to the uronic acid. Finally, certain
arrangements of sulfated sugar residues and uronic acid epimers act as a target for the 3-O-
sulfotransferase. The modifications tend to occur in clusters along the chain, with regions devoid
of sulfate separating the modified tracts. In general, the reactions proceed in the order indicated,
but they fail to go to completion, resulting in tremendous chemical heterogeneity within the
modified regions. The specific arrangement of sulfated residues and uronic acid epimers in
heparin and HS gives rise to specific binding sequences for ligands. A major question concerns
how the enzymes and pathway of HS/heparin biosynthesis are regulated to achieve tissue-
specific expression of ligand-binding sequences.
Figure 11.7. HS biosynthesis involves a series of modification reactions including sulfation and
epimerization of GlcA. Chain polymerization and modification are thought to occur
simultaneously. (PAPS) 3 -phosphoadenyl-5 -phosphosulfate, the high-energy donor of sulfate
groups.
During the last decade, nearly all of the enzymes involved with HS synthesis have been purified
or molecularly cloned. Several important features have emerged from these studies, which may
shed light on how different binding sequences arise.
• Several of the enzymes appear to have dual catalytic activities. Thus, a single protein
bearing two catalytic domains catalyzes N-deacetylation of GlcNAc residues and
subsequent N-sulfation. The same is true of the copolymerase, which transfers GlcNAc
and GlcA from the corresponding UDP sugars to the growing polymer. In contrast, the
epimerase, 2-O-sulfotransferase, and 6-O-sulfotransferase(s) appear to be unique
properties of independent enzymes.
• In several cases, multiple isozymes exist that can catalyze a single or pair of reactions.
Thus, four N-deacetylase/N-sulfotransferases exist, three 6-O-sulfotransferases have been
described, and five 3-O-sulfotransferases have been identified. Their tissue distribution
varies, which may cause differences in the pattern of sulfation. However, some overlap
exists as well, suggesting that individual isozymes may act on different sub-sequences
within the chain.
• The polymer modification reactions probably colocalize in the same stacks of the Golgi
complex. Thus, the enzymes may form a supramolecular complex that coordinates the
modification reactions. The composition of these complexes may play a part in regulating
the fine structure of the chains.
• In general, the composition of HS varies more between cell types than between core
proteins expressed in the same cell. This observation suggests that each cell type may
express a unique array of enzymes and potential regulatory factors.
The Difference between Heparin and Heparan Sulfate (14,34)

Considerable confusion exists over the definition of heparin and HS. The major differences are
summarized in Table 11.7. Heparin is produced by mast cells and sold by pharmaceutical
companies as an anticoagulant. In contrast, HS is made by virtually all cells. It also can contain
anticoagulant activity, but the crude preparations have much less activity than heparin. During
biosynthesis, heparin undergoes more extensive sulfation and uronic acid epimerization, such
that more than 85% of the GlcNAc residues are N-deacetylated and N-sulfated and more than
70% of the uronic acid is converted to IdoA.
Table 11.7. Major differences between heparin and heparan sulfate
Characteristic Heparan sulfate Heparin
Soluble in 2 m potassium acetate (pH 5.7, 4°C) yes no

Size 10 70 kD 10 12 kD
Sulfate/hexosamine 0.8 1.8 1.8 2.4
GlcN N-sulfates 40 60% 85%
IdoA content 30 50% 70%
Binding to antithrombin 0 0.3% ~30%
Site of synthesis virtually all cells mast cells
Another way to distinguish heparin from HS is by susceptibility to bacterial (Flavobacterium)

heparin lyases. These enzymes, like the bacterial chondroitinases, are eliminases and produce an
unusual unsaturated uronic acid on the nonreducing end of the oligosaccharide products.
Proteoglycans Turn Over Continuously (9, 40, 41)

Cells secrete proteoglycans directly into the extracellular environment, and some are shed from
the cell surface through proteolytic cleavage of the core protein. Cells also internalize a large
fraction of cell surface proteoglycans by endocytosis (Figure 11.8). These internalized
proteoglycans first encounter heparanases that cleave the chains at a limited number of sites,
probably dependent on the sequence. These smaller fragments eventually appear in the lysosome
and undergo complete degradation by way of a series of exoglycosidases and sulfatases (see
Chapter 18). The purpose of intracellular heparanases is not clear, but they may be involved in
release of bound ligands from the internalized proteoglycan prior to the lysosome. CS
proteoglycans follow a similar route, but endoglycosidases that degrade the chains prior to the
lysosome have not been described.
Figure 11.8. HS proteoglycans turn over both by shedding from the cell surface and by
endocytosis and step-wise degradation inside lysosomes. (Adapted, with permission, from [9]
Yanagishita and Hascall 1992.)
Genetic Studies of Proteoglycan Structure, Function, and

Metabolism (42 48)
A variety of mutant cell lines altered in GAG biosynthesis have been isolated and biochemically
characterized (see Chapter 31). In addition, Drosophila mutants with lesions in growth-factor-
dependent signaling have been described that turn out to have defects in GAG formation or
nucleotide precursors. With cDNA clones available for many core proteins and biosynthetic
enzymes, targeted disruption of genes is now a possibility. For greater detail of these subjects,
see Chapters 31 33.
Future Prospects
The last two decades have seen a tremendous increase in the number of glycoproteins found to
contain glycosaminoglycans. Furthermore, many of the details of the biosynthetic pathways have
been described. This information in turn has led to the purification and cloning of genes
encoding the core proteins and the biosynthetic enzymes, which have revealed that the relevant
proteins belong to families of presumably related function. Recombinant proteins can be
produced readily, which should permit the analysis of enzyme structure and function. The
availability of these reagents will revolutionize the way we study the biology of these
glycoconjugates in the future. With the genes in hand, genetic ablation experiments can now be
undertaken to study how proteoglycans participate in normal and pathophysiology. Already,
mutants have been characterized in cultured cell lines (see Chapter 31), in lower organisms (see
Chapter 19), and to a lesser extent in mice (see Chapter 33). Such models provide insights into
inborn errors in proteoglycan assembly in humans, and they define targets for potential
pharmaceutical intervention.
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12. Other Classes of Golgi-derived Glycans
Jolla, California).
EACH MAJOR FORM OF GLYCOSYLATION in eukaryotic cells is defined by the linkage

between the first sugar and the aglycone, the noncarbohydrate moiety, most of which are
synthesized in the ER and Golgi. This chapter explores examples of unusual glycosylation events
in the ER/Golgi pathway, the systems in which they occur, specific proteins modified,
glycosyltransferases involved, and a few proposed functions. Some of these modifications have
only been seen in lower eukaryotes, others only in higher eukaryotes, and still others span the
evolutionary spectrum.
Two types of single sugar modifications are not covered here. One is the addition of sugars such
as glucuronic acid to bile acids and xenobiotics. Their function is to increase the water solubility
of the conjugates. The other type is called glycation (not be confused with glycosylation). This is
the nonenzymatic addition of sugars to proteins in which the aldehyde group of a sugar such as
glucose reacts with free amino groups on proteins.
O-linked Modifications
The term O-linked glycosylation is typically assumed to represent the common addition of
GalNAc to serine or threonine residues (see Chapter 8). However, there are many other forms of
O-glycosylation (for a summary, see Table 12.1). Since O-(β)GlcNAc is the established term for
GlcNAc bound to serine and threonine hydroxyl groups in cytosolic and nuclear proteins, the
same nomenclature is adopted for the O-linked modifications described in this chapter.
Table 12.1. Less common types of glycosylation in the Golgi
Modification Proteins Location
O-(α)Fucose
Fucα-Thr/Ser urokinase, t-PA, factor XII, factor VII, EGF-like module
human notch-1
Sia2 6Gal1 4GlcNAc1 factor IX EGF-like module
3Fucα1-Thr/Ser
O-(β)Glc
Xylα1 3Xylα1 3Glcβ- factors VII, IX, protein 2, human EGF-like module
Ser/Thr Notch-1
O-(β)Gal
Glcα-1 2Gal-O-Hyl collagen, surfactant protein, collagen repeat
(hydroxylysine) complement factor Clq, mannan-
binding proteins
O-Man
Man-R-O-Ser/Thr brain proteoglycan, α-dystroglycan, ?
others in brain and neuronal tissue
O-(α)GlcNAc
GlcNAcα-Thr cell adhesion molecule gp80, PTVT-mucin repeats
extracellular matrix protein PST
Phosphoglycosylation
GlcNAcα-1-P-Ser Dictyostelium lysosomal proteins serine-rich domain of
cysteine protein (SGSG,
SSSS motifs)
Fucβ-1-P-Ser extracellular matrix proteins, cysteine probably mucin domains
proteinases
Manα-1-P-Ser Leishmania proteophosphoglycan and serine-rich domain
filamentous acid phosphatase
Glc-(β)Asn laminin ?
C-Mannosylation RNase 2, interleukin-12 WXXW motif
Proteins with EGF-like Modules Can Have Glc β-O-Ser and Fuc α-O-Ser/Thr
(1)
A select group of mammalian proteins with EGF-like modules is modified by two rare forms of
glycosylation. The proteins include the mammalian version of the widely distributed notch-1 and
several proteins involved in the coagulation cascade.
O-(α)Fucose(2 4)
The O-fucosylation pathway is defined by the linkage Fucα-Ser/Thr. The first evidence
suggesting the existence of this type of pathway was the identification of a glycopeptide, Glcβ1
3Fucα-Thr, in human urine. This discovery generated little interest at the time, but interest
increased when O-fucose was identified in human urokinase. This led to its discovery in other
clotting proteins (Table 12.1) such as tissue plasminogen activator (t-PA) and clotting factor VII.
All of the O-fucosylated proteins identified so far have an EGF-like module and glycosylation
occurs at a similar serine or threonine in each one, as shown in Figure 12.1. In factor VII, the
glycan is extended with three additional sugars to form Siaα2 6Galβ1 4GlcNAcβ1 3Fucα-.
This sialyl-lactosamine triad is the familiar signature of many serum glycoproteins with N-
glycans (see Chapter 7). Addition of sialic acid and galactose may use sialyl and
galactosyltransferases employed by other pathways, but formation of the GlcNAcβ1 3Fuc bond
probably requires a transferase unique to this pathway. Until recently, fucose was usually
considered to be a terminal sugar, so there was no inclination to search for glycosyltransferases
that use fucose as an acceptor. As more structures are identified, some of the old "rules" change.
For example, N-glycans in octopus rhodopsin have a nonterminal fucose (Galβ1 4 Fucα1
6GlcNAc) in the core region, and SKP1 in Dictyostelium has a nonterminal fucose in a
hydroxyproline-linked oligosaccharide synthesized in the cytosol (see Chapter 13).
Figure 12.1. Consensus sequences for Xyl-Glc and O-Fuc addition in EGF-like modules.
O-(α)Fuc is also found on glycoproteins made by CHO cells. This cell line is a favorite with
glycobiologists because it can be used for making glycosylation-defective mutants (see Chapter
31). A subset of the O-fucosylated glycoproteins in CHO cells also carries the Glcβ1 3
extension similar to the original glycopeptide found in human urine. Again, forming this
disaccharide structure probably involves a specific β-glucosyltransferase. The CHO proteins
have not been identified, and thus it is not known if they have an EGF-like module. Small
synthetic peptides that resemble parts of the EGF-like module are poor acceptors for in vitro O-
fucosyl transferase assays, which may mean that all the proper disulfide bonds of the EGF-like
module are required or that other aspects of the protein contribute to the specificity.
The function of O-Fuc is not known, but a fucose lectin-type receptor on the hepatocyte surface
was suggested to be involved in clearance of t-PA from the circulation. Fucose and fucosyl-
albumin conjugates inhibited binding, and α-fucosidase digestion of t-PA reduced binding.
Lectin-type receptors are known to control the half-life of other circulatory glycoproteins, as
discussed in Chapter 22, but the influence of fucose on the half-life of t-PA has not yet been
determined. The human mannose receptor (see Chapter 25) has a much higher affinity for t-PA
than other proteins containing high-mannose-type sugar chains. Could O-(α)Fuc have an
enhancing role in mediating t-PA clearance? Probably not, since mutating the O-(α)Fuc
attachment site has no effect on its binding to the mannose receptor. The addition of other sugars
to O-(α)Fuc on other proteins suggests that those sugars could have other functions. One
contribution could be in cell fate determination through the widely distributed protein, notch.
Mammalian notch-1 has a series of 36 tandem EGF-like modules, and 12 of them have the
consensus sequence for O-fucosylation. When expressed in CHO cells, this molecule is modified
with mono- and oligosaccharide versions of O-(α)Fuc.
O-fucosylation has been preserved through phylogeny, since it seems to occur on selected
proteins expressed during development of the primitive eukaryote, Dictyostelium discoideum.
Only a few proteins are modified, but they also occur in EGF-like modules with mucin-like
domains and form part of the coat surrounding spores made during multicellular development of
this organism (see Chapter 19).
O-(β)Glucose (5)
An O-(β)-linked glucose residue defines this pathway. Factors VII, IX, protein Z, and
thrombospondin from human or bovine sources (Figure 12.1) have Xylα1 3+/- Xylα1 3Glcβ-
O- at a similar serine residue in their EGF-like modules. The human notch-1 protein also has O-
glucosylation. The consensus site for addition occurs only nine amino acids away from the O-
fucosylation site of these proteins. Glucosylation occurs on all molecules, but the addition of
both xylose residues occurs in only a portion of the molecules. Each of the xylose residues is
added sequentially by a separate xylosyltransferase. No model peptides have been used to study
acceptor specificity. Recombinant factor VII without the glycosylation site has only slightly
reduced catalytic activity. The functional significance of this unusual modification remains
unknown, but its identification in notch-1 offers a new avenue of investigation.
Glcα1 2Galβ-O-Hydroxylysine(6 8)
This pathway was first recognized in the 1960s as acting on collagen and has since been
extended to a few other proteins that have collagen-like modules. The first step in the pathway is
the obligate hydroxylation of lysine to hydroxylysine. Once the acceptor sites are created, the
two-step glycosylation rapidly follows on most of the available acceptor sites. Several other
proteins with conserved collagen-like domains that form triple-helical structures also carry the
disaccharide, including pulmonary surfactant proteins, complement factor C1q, and rat and
human mannan-binding proteins made by the liver. The mannan- binding protein found in the
serum assembles into large aggregates by interchain disulfide bonds that depend on the creation
of hydroxylysine, and presumably on glycosylation. Triple-helix formation of collagen can also
prevent the addition of galactose to the glucosylated form, perhaps by sterically blocking the
saccharide residue.
Primitive forms of collagen in sponges and sea anemone also have disaccharide-modified
hydroxylysine, so these modifications have probably partnered with collagen for a long time.
These glycosylations are thought to be involved in mediating, or in some cases preventing,
aggregations of the triple helix in these molecules.
O-Mannose (9 11)
O-αMan was identified in yeast in the 1950s and is discussed in more detail in Chapter 19.
Briefly, mannose is α-linked to serine/threonine and may be elongated by several additional
mannose residues using a battery of mannosyltransferases that extend similar structures on N-
linked chains in yeast.
Mannose was first identified as the protein-linked sugar in a rat brain proteoglycan in 1979.
Because it was released by mild base hydrolysis with simultaneous reduction of mannose to
mannitol, the anomeric linkage could not be determined. Gal(β1 4)[Fucα1 3]GlcNAcβ1
2Man, GlcNAcβ1 2Man, and mannose by itself were found (Figure 12.2). More recently, O-
mannose-based glycans containing four to eight sugars have been found in total brain
glycopeptides and a tetrasaccharide derived from α-dystroglycan. This protein is found on
Schwann cell membranes and links another membrane protein, β-dystroglycan, to laminin in the
extracellular matrix. The oligosaccharide Sia2 3Galβ1 4GlcNAc-β1 2Man-Ser/Thr accounts
for two thirds of the O-glycans of α-dystroglycan and is probably involved in the important α-
dystroglycan-laminin binding, since sialidase prevents the interaction, but removal of N-glycans
does not. In addition, dystroglycan isolated from sheep brain has been shown to contain a
fucosylated, nonsialylated O-mannose-based structure with the Galβ1 4GlcNAcβ1 2Man
linkage. This Lewis X (Lex) determinant is well known on N-glycans and on more typical
GalNAc(α)-O-glycans. Some of the structures are shown in Figure 12.2. The activated mannose
donor used to initiate this pathway is unknown. The O-mannose biosynthetic pathway may be
more diverse and extensive than expected and may prove to be important for specific cell
adhesion processes.
Figure 12.2. O-mannose-based oligosaccharides.
A small portion of total rat brain glycopeptides has O-mannose-linked glycans and carries the
HNK-1 antigenic epitope, an unusual 3-O-sulfated glucuronic acid found at the nonreducing end
of the chain. This epitope is implicated in neuronal cell adhesion. Some of the O-mannose-based
chains with HNK-1 antigen are substituted with a second GlcNAc creating a branched structure.
These GlcNAc transferases are probably unique to the O-mannose pathway and different from
those used for the addition of GlcNAc to N-linked chains.
The key to finding these O-mannose-linked chains was the HNK-1-specific antibody, since it
recognizes the nonreducing end of a chain, but it is indifferent to the sugar found at the reducing
terminus. Selective recognition of a portion of the sugar chain is often found with both
antibodies and glycosyltransferases. Other proteins besides α-dystroglycan that carry these
interesting O-glycans have not been identified, but they must be abundant since the estimated
ratio of mannose-terminated to GalNAc-terminated O-glycans in total brain glycopeptides is
approximately 1:3 (A.M. Lawson and T. Feizi, pers. comm.).
O-αGlcNAc
Chapter 14 describes a broad range of functions of nuclear and cytoplasmic proteins modified by
O-(β)GlcNAc. However, O-(α)GlcNAc can be found in some proteins traversing the ER/Golgi
pathway. So far, it has only been recognized in a group of glycoproteins found in D. discoideum.
It was initially defined by a monoclonal antibody reactivity and later the structure was solved. α-
GlcNAc is added within a series of multiple mucin-like PTVT peptide repeats. Mutants lacking
this Golgi-added O-αGlcNAc have impaired multicellular development because an adhesion
protein called contact site A or gp80 is more sensitive to proteolysis when it lacks this
modification. The adhesion molecule has multiple mucin-like domain repeats, and the cluster of
α-GlcNAc residues is thought to cause this region to form a rod-like structure. This
conformation is not maintained without glycosylation, and a cell surface protease is able to
cleave it.
Phosphoglycosylation in Lower Eukaryotes (12)

Dictyostelium adds GlcNAcα-1-P and Fucβ-1-P directly to serine residues of selected proteins.
Leishmania adds Manα-1-P, which is sometimes extended with more sugars. None of these
modifications have yet been found in other organisms.
GlcNAcα-1-P-Ser and Fucβ-1-P-Ser inDictyostelium(13 16)
Dictyostelium is a unicellular amoeba that can grow on bacteria or in synthetic growth media.
When the food source is exhausted, up to 100,000 amoeba aggregate to form a multicellular
organism (see Chapter 19). During vegetative growth, GlcNAcα-1-P is directly added from
UDP-GlcNAc to serine residues of a select group of lysosomal proteins. Several papain-like
cysteine proteinases are the major carriers of this modification. Although cysteine proteinases are
highly conserved enzymes throughout plants and animals, the Dictyostelium enzymes have
additional serine-rich domains containing 20 110 amino acids where GlcNAc-1-P is added.
Small peptides that resemble the serine motifs from these enzymes are good artificial acceptors
in in vitro assays, but the native conformation of the cysteine proteinases imparts an added level
of recognition specificity. In fact, mammalian cysteine proteinases that lack the serine domain
and cannot be phosphoglycosylated still bind to GlcNAcα-1-P transferase and can act as
inhibitors. This probably means that GlcNAcα-1-P transferase recognizes a conformation-
dependent structural feature on the proteinase as well as the local glycosylation site, analogous to
that seen with the GlcNAcα-1-P transferase responsible for the first step in the addition of Man-
6-P to mammalian lysosomal enzymes (see Chapter 23).
The function of GlcNAcα-1-P-Ser in Dictyostelium is unproven, but it may help to segregate two
groups of lysosomal proteins from each other. Lysosomal proteins with GlcNAcα-1-P and those
with Man-6-P sort to different endosomal-lysosomal vesicles. These vesicles are also
functionally distinct since they sequentially fuse with bacteria phagocytosed by the amoeba as
seen in Figure 12.3. The cysteine proteinase-containing vesicles fuse first, followed by those
containing enzymes with Man-6-P. Surprisingly, pulse chase labeling with bacteria shows that
proteins with the two modifications hardly ever reside in the same vesicle, suggesting that the
first set of GlcNAcα-1-P-containing proteins is retrieved before the second wave of Man-6-P-
containing proteins attacks the bacterial carcass (Figure 12.3). Why do this? It may be because
the Dictyostelium endosomal-lysosomal system is constantly changing pH, initially dropping
from 6.5 to approximately 5 and then back to 6.5 during the course of 1 hour. The GlcNAcα-1-
P-containing proteins, such as the cysteine proteinases in the first wave, have higher pH optima
than those in the next wave. In this way, the amoeba can efficiently digest the cargo by taking
advantage of the full pH range. Retrieval of the lysosomal enzymes is also highly efficient.
Figure 12.3. (Left panel) Dictyostelium amoeba were stained with an antibody against GlcNAc-
1-P (FITC, green) or Man-6-P (TRITC, red). There is no colocalization of the two types of
modifications. The yellow areas suggesting colocalization resolve into separate green and red
vesicles when the cell image is rotated. These results indicate that the two different sets of
proteins with mutually exclusive modifications are sorted into two different sets of vesicles.
(Right panels) FITC (green)-labeled bacteria were phagocytosed by the amoeba for 3 15 min,
and then the cells were stained for either GlcNAc-1-P or Man-6-P (red in both cases). Vesicles
containing GlcNAc-1-P fuse with bacteria immediately (yellow). Man-6-P-containing vesicles do
not fuse with the phagosomes at 3 min, but only begin to fuse after about 15 min. Longer chases
(not shown) produce bacterial fragments in phagosomes that do not have either GlcNAc-1-P- or
Man-6-P- containing proteins in them. Since these modifications hardly ever colocalize in the
same vesicle even though they both attack the same phagocytosed bacteria, the enzymes seem to
be independently retrieved and probably recycled. (Adapted, with permission, from [13] Souza et
al., 1997[© Company of Biologists Ltd.].)
Proteins carrying Fucβ-1-P-Ser were first recognized because they bound to a carbohydrate-
specific antibody. Fucβ-1-P occurs on only a limited set of mucin-like proteins that include two
lysosomal cysteine proteinases, as well as a set of proteins that are packaged into vesicles
containing unassembled spore coat components. Small serine-containing peptides are substrates
for Fucβ-1-P transferase. The function of this modification is unknown, but mutants without
Fucβ-1-P have more porous spore coats and may have decreased survival in nature.
Manα-1-P-Ser/Thr in Leishmania (17)
The protozoan parasites of the Leishmania genus synthesize a family of phosphorylated glycans
important in the life cycle of the organism as it resides in both the insect and mammalian hosts
(see also Chapter 39). One component called the proteophosphoglycans (PPG) is composed of
large repeating polymers of Man-1-P and galactose (Galβ1 4 Manα1-P) that are O-linked to the
mucin-like core proteins through Manα-1-P residues (see Chapter 36). Some of the Manα-1-P-
initiated chains are also extended by only an additional mannose residue. Several species of
Leishmania synthesize a filamentous secreted acid phosphatase. This molecule contains a
Ser/Thr-rich domain that carries chains initiated through Man-1-P and then extended with
various combinations of Man, Manα-1-P, Glc, and Gal. It is unknown whether the same Manα-
1-P transferase initiates both types of glycans. The function of these chains and the reason they
are initiated with Manα-1-P are unknown.
N-linked Modifications (18 19)
There is only one other type of N-glycosylation besides the familiar GlcNAc-β-Asn. Technical
barriers to the release, biosynthetic labeling, and identification of putative N-linked sugars may
explain the limited membership in this group.
Glcβ-Asn was first identified in Halobacteria where it initiates the major N-glycosylation
pathway that involves transfer of a preformed lipid-linked oligosaccharide to the acceptor
proteins with Asn-X-Thr/Ser sequences. The pathway has features in common with the N-linked
pathway in eukaryotes. This modification is documented on only one vertebrate cell
glycoprotein, the basement glycoprotein laminin. Once again, a specific antibody against this
structure was the key in finding it in a mammalian glycoprotein. The antigen is localized to the
kidney glomerular basement membrane. The presence in laminin of the antigen defined by the
Glcβ-Asn antibody was seen only after acid treatment. The acid presumably removed other
monosaccharides and exposed Glcβ-Asn to the antibody. The remaining structure was confirmed
to be Glcβ-Asn by GC-MS. This is important because it rules out the trivial explanation that the
antibody simply cross-reacted with a similar structure. The number of peripheral
monosaccharides removed by the acid treatment is unknown, but the results suggest that this
pathway could be more complex than the addition of a single glucose unit. The function of Glcβ-
Asn is unknown.
C-Glycosylation (20 22)
Another novel type of glycosylation is called C-mannosylation. The C-1 atom of a single
mannose residue is added in an α linkage to the C-2 atom of the indole moiety of Trp-7 in RNase
2 (see Figure 12.4). Note that this is not a typical glycosidic linkage, but rather is a C-C bond.
This molecule was first detected in human urine. Structural analysis, biosynthetic studies in
mammalian cell lines, and a specific antibody against the modification show that C-
mannosylation is not a chemically induced artifact. Many mammalian cells transfected with
RNase 2 can C-mannosylate this protein, showing that they already contain the necessary
transferase. Plant protoplasts and bacteria do not C-mannosylate expressed RNase 2. This may
mean that it is a relatively recent form of glycosylation or that the transferase has a different
recognition specificity. Site-directed mutagenesis shows that the critical glycosylation sequence
in RNase 2 is Trp-X-X-Trp, with the first Trp being modified and X being any amino acid.
Synthetic peptides with this motif can be C-mannosylated in vitro, and a database search shows
that more than 300 mammalian cell proteins have this sequence. Two of these were examined
directly for C-mannosylation. One, the β-chain of fibrinogen B, was not modified, but human
interleukin 12β produced in CHO cells was C-mannosylated. These findings suggest that other
proteins with the Trp-X-X-Trp motif may be similarly modified. Eight proteins that have
thrombospondin type I repeats are also modified by C-mannose. The modification appears to
compete with protein folding since already folded proteins are poor substrates in vitro. This
finding suggests that the reaction may occur in the ER. The extent of C-mannosylation is
variable in different cells. Dol-P-Man produced from GDP-Man is the biosynthetic donor for C-
mannosylation. C-mannosylation was only discovered recently, and thus the search for a
function of this highly specific modification is only just beginning.
Figure 12.4. C-mannosylation biosynthetic pathway and structure details of Trp-7 in RNase 2.
Glycolipids (23 25)
Some nonvertebrates have "unusual" glycosphingolipids compared to those described for

vertebrates in Chapter 9. The glycans are attached to ceramide via Glcβ1 linkage but are often
extended with β1 4 mannose. In some cases, the Manβ1 4Glcβ1Cer core can be extended by
"typical" structures based on vertebrate standards or by unusual ones that can include internal
fucose and sialic acids, methylated monosaccharides, and multiple phosphoethanolamines or
phosphonates. Very little is known about the functions of these glycosphingolipids, but a
Drosophila cell surface molecule called gliolectin, which is expressed during embryonic
development on a subset of glial cells, binds specifically to some neutral and zwitterionic
(phosphoethanolamine-containing) glycolipids.
Glycosaminoglycans
The known proteoglycans (except for the keratan sulfate proteoglycans) are initiated by β-xylose
to serine, but it is important to remember that nearly all of the studies of proteoglycans, even in
nonvertebrates, are focused on examining for a particular type of glycosaminoglycan, e.g.,
chondroitin or heparan sulfate, in the organism. Few studies have yet focused on the structure of
the linkage region itself in nonvertebrate species.
Future Directions
Many unusual forms of glycosylation occur in the Golgi of eukaryotes. In some cases, the
modification is limited to a certain class of proteins that share a particular module or have
specific conformational features. Some of these forms first appeared in lower organisms and are
preserved through evolution. Biological roles are only suggested and the search for their
functions continues. The high selectivity for protein targets plus the maintenance of these
glycosylations in diverse organisms suggests specific function(s). Other unusual forms of
glycosylation will probably be identified in higher organisms by using specific antibodies or
rigorous structural analysis. Some of them may be modified versions of those already found in
the "treasure chest" of lower eukaryotes and invertebrates.
References
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13. Nuclear and Cytoplasmic Glycosylation
THIS CHAPTER CRITICALLY REVIEWS THE EVIDENCE for complex glycoconjugates in

the nucleoplasmic and cytoplasmic compartments of the cell. First, it discusses the evidence for
nuclear glycosyltransferases, cytosolic N-linked glycans, and sialic-acid-containing saccharides
on nucleoporins. Several structurally verified forms of cytosolic and nuclear glycoconjugates are
then described, such as glycogenin, O-linked mannose, O-linked fucose, and nuclear
glycosaminoglycans (O-linked GlcNAc is covered separately in Chapter 14). Finally, it
summarizes current views with respect to the glycosyltransferase alpha toxins of Clostridium, the
putative functions of nuclear lectins, and the possible roles of saccharides as nuclear localization
signals. Some of these relatively unexplored areas may represent important areas of focus for the
future of glycobiology.
Background (1 3)
Despite the large amount of data available with respect to the widespread O-GlcNAcylation of
nuclear and cytoplasmic proteins (see Chapter 14), most general biochemistry texts still persist in
the view that protein glycosylation is largely restricted to the addition of complex glycans to
proteins or lipids localized on the cell surface or within luminal compartments of subcellular
organelles, such as the Golgi or ER. In fact, more than 100 papers in the biochemistry literature
present data with respect to the presence of complex glycoconjugates within the nucleus and
cytoplasm. Many of the authors of these papers have used plant lectins (see Chapter 30) as tools
to detect saccharide-binding molecules in the nucleoplasmic (Table 13.1) or cytoplasmic (Table
13.2) compartments. These lectin-binding studies have largely been ignored by mainstream
biochemists for several reasons: (1) The known glycosyltransferases involved in complex glycan
biosynthesis are type II membrane proteins with their active sites within the lumen of the ER or
Golgi. Thus, the "enzymatic machinery" is on the "wrong" side of the membrane for the
biosynthesis of complex nuclear or cytoplasmic glycoconjugates. (2) Most of the lectin-binding
studies that make claims of cytosolic or nuclear glycosylation present no supporting structural
data to establish the nature of the putative cytoplasmic or nuclear glycoconjugates. (3) Plant
lectins, such as concanavalin A, for example, can, under some circumstances, bind to molecules
by nonspecific hydrophobic interactions. In addition, it is possible that the competing saccharide
ligand can alter the conformation of such lectins to induce a loss of this hydrophobic binding.
Therefore, although lectins are important tools in glycobiology, conclusions based on their use,
particularly those not compatible with known pathways and established concepts of cell biology,
must be followed up by rigorous structural analyses. Nevertheless, these many lectin-binding
studies indicate that GlcNAc-, mannose-, and fucose-containing glycans are abundant in the
nucleus and cytoplasm, suggesting that further analysis is warranted (Tables 13.1 and 13.2).
Table 13.1. Demonstration of lectin-binding sites within the nucleus
Binding site Lectin Competitive sugar Number of papers
Nuclear membrane ConA α-glucosides, α-mannosides 10

Nuclear membrane WGA d-GlcNAc and sialic acids 8
Nuclear membrane RCA d-Gal 2
Chromatin ConA α-glucosides, α-mannosides 12
Chromatin WGA d-GlcNAc and sialic acids 7
Chromatin RCA-I d-Gal 2
Chromatin LCA α-d-Man 2
Chromatin UEA-I α-l-Fuc 2
Chromatin RCA-II 1
Chromatin GSA α-l-Fuc 1
Chromatin APA α-l-Fuc 1
Chromatin TTA 1
Chromatin SBA 1
Chromatin PHA 1
Table 13.2. Demonstration of lectin-binding sites in the cytoplasm
Lectin Competitive sugar Number of papers
ConA α-mannosides
α-glucosides 4
RCA d-Gal 2
WGA d-GlcNAc
sialic acids 2
Nuclear Glycosyltransferases? (4 7)
In recent years, there have been several reports of the presence of glycosyltransferase activities
in highly purified preparations of rat liver nuclei, which were judged to be more than 99% pure
by marker enzyme analysis. These studies document the transfer of GlcNAc from UDP-GlcNAc
into endogenous acceptors and show that at least 80% of the activity is blocked by low
concentrations of the antibiotic tunicamycin, suggesting the involvement of N-linked
biosynthetic intermediates, such as N-acetylglucosaminyl-pyrophosphoryldolicohol (GlcNAc-
PP-Dol). Later studies demonstrated the direct transfer of chitobiose (GlcNAcβ1 4GlcNAc)
from chitobiosyldolichol to endogenous nuclear acceptors by these nuclear preparations,
suggesting a novel pathway of N-glycosylation. The products of these in vitro reactions were
found to be N-linked chitobiosyl moieties, based on their sensitivity to peptide N-glycosidase F
and hydrazinolysis, but insensitivity to alkali-induced β-elimination (see Chapter 38). Similar
studies have documented the presence of nuclear mannosyltransferases. Although these studies
are provocative, they must also be interpreted with caution. The ER, which is the widely
accepted site of N-glycosylation (see Chapter 7), is functionally contiguous with the outer
nuclear envelope. Thus, even a minor contamination of the nuclear envelope could lead to
misinterpretation of these findings. In addition, it is very difficult to purify nuclei such that other
cellular components do not nonspecifically adhere to the nuclei during their preparation. Given
these potential problems, widespread acceptance of the existence of these nuclear
glycosyltransferases must await independent confirmation by more direct criteria. Thus far, of
the cloned glycoprotein glycosyltransferases, only the O-GlcNAc transferase has been reported
to have a potential nuclear localization signal (see Chapter 14). Sugar nucleotide donor
substrates are synthesized in the cytosolic compartments, but CMP-NeuAc synthesis takes place
within the nucleus (see Chapter 6).
Cytoplasmic Complex Glycans? (8 11)
The α-subunit of the sodium pump (Na+, K+-ATPase) from dog kidney, a transmembrane
protein, was reported to contain N-linked glycans with terminal GlcNAc residues in its cytosolic
domain. This conclusion was based on the enzymatic attachment of radioactive galactose to
GlcNAc residues by galactosyltransferase labeling of permeabilized right-side-out membrane
vesicles. Peptide-N-glycosidase sensitivity of the radioactively labeled products suggested that
the acceptors are N-linked glycans (see Chapters 7 and 38). In such vesicles, the extracellular
components would be expected to be protected from enzymatic probes by being inside sealed
membranes. Like earlier studies, these provocative claims have been largely ignored because of
the lack of any site mapping of the putative cytosolic glycans and the absence of any structural
data, even though the primary sequence of this putative cytosolic glycoprotein is known. Several
studies have also strongly indicated that the so-called HMG proteins, which are important
structural components of chromatin, are bone fide "classical-type" glycoproteins. Highly purified
preparations of HMG 14 and HMG 17 were found to contain GlcNAc, mannose, galactose,
glucose, fucose, and possibly xylose. The fucose-specific lectin, Ulex europeus agglutinin I,
bound both to HMGs and to isolated nucleosomes. Alkali resistance suggested that the glycans
were N-linked. The purified HMGs were also found to be positive when analyzed by standard
colorimetric assays for sugars. In addition, these HMGs could be metabolically radioactively
labeled with tritiated fucose, galactose, mannose, or GlcNAc. Later studies suggested that the
glycans on HMG 14 and HMG 17 were required for their binding to the nuclear matrix, where
the HMGs have a role in modulating chromatin structure at active sites of gene transcription.
Even though these HMG studies are widely cited as evidence for nuclear N-glycans, they also
suffer from a lack of definitive structural data for the existence of such glycans. Required
structural data include physical proof of the linkage to protein and physical definition of the
structure of the putative N-glycan(s). In fact, very recent studies by Haltiwanger and colleagues
have not been successful in verifying the presence of complex glycans of any type on the HMG
proteins. However, these later studies did report that the HMGs are modified by O-GlcNAc (see
Chapter 14).
Sialic-acid-containing glycoproteins have been reported on the cytoplasmic face of the nuclear
envelope. The sialic-acid-specific lectin, Sambucus nigra agglutinin, was shown to bind to
several proteins, previously shown to also contain O-GlcNAc. Two of these proteins were
identified as major nucleoporins: p62 and p180. Prior sialidase treatment blocked binding of S.
nigra agglutinin to these nuclear pore proteins. On the basis of peptide-N-glycosidase sensitivity,
the sialic acids on the p180 appeared to be on N-linked glycans and those on p62 appeared to be
on O-linked glycans. The authors also demonstrated that S. nigra agglutinin blocked nuclear
protein import in neuroblastoma cells, suggesting that the sialic acids might have functional
importance on the nuclear pore proteins. Again, the significance of these provocative findings
must await systematic structural proof of the nature of the putative sialylated glycans on these
nucleoporins.
Glycogenin (12 18)
Glycogen is a large homopolysaccharide of glucose. It serves as a short-term storage form of

glucose and can be broken down to provide glucose to liver and muscle during times of stress.
Glycogen stores are also important in maintaining glucose homeostasis in the blood. Constant
levels of blood glucose are absolutely required, particularly by brain and blood cells. Several
reports have unequivocally established that glycogen is covalently attached to a polypeptide, and
thus is a glycoprotein. As early as 1886, it was suggested that glycogen might be bound to
protein. In 1975, Krisman and Barengo demonstrated that liver extracts could synthesize a
glycogen-like product from UDP-Glc and that this product was precipitated by trichloroacetic
acid. In vitro maltotetraose [(Glcα1 4)]4 is the smallest saccharide that primes the synthesis of
glycogen elongation. Therefore, it was proposed that a protein, not carbohydrate, was the
original primer for glycogen synthesis. In 1985, Whelan's group identified the protein primer of
glycogen and named it glycogenin. Cohen and colleagues purified glycogenin in a complex with
glycogen synthase and determined glycogenin's complete 322-amino-acid sequence. They also
established that the first glucosyl residue of glycogen is attached glycosidically to the hydroxyl
group of Tyr-194 of the protein. In muscle, each molecule of glycogen is covalently attached to
one molecule of glycogenin with a ratio of 1 glycogenin/Mr = 107 glycogen (107 is the
approximate maximum length of a glycogen molecule in muscle). In liver, the ratio of
glycogenin to glycogen is about 0.0025%. This small ratio reflects both the enormous size of
liver glycogen (Mr = up to 5 × 109 daltons) and the existence of large numbers of free glycogen
chains in liver glycogen granules.
Glycogenin is autocatalytic, attaching glucose to itself from the donor, UDP-Glc (Figure 13.1),
and is classified as a hexosyltransferase (EC 2.4.1.186). This self-glucosylation yields a malto-
octaose (eight glucose units) and both p-nitrophenyl α-glucoside and α-malto-oligosaccharides
are competitive substrates. Biochemically, glycogenin may have a key role in the regulation of
glucose homeostasis. Glycogenin has a Km for UDP-Glc about 1000 times lower than glycogen
synthase's Km for the sugar nucleotide. Production of the glycogenin primer, especially in muscle
cells, can be the rate-limiting step in glycogen formation. Thus, levels of glycogenin could be
capable of overriding the better-understood hormonally controlled mechanisms that involve
protein phosphorylation/dephosphorylation and regulate glycogen elongation. Figure 13.1
illustrates a model of glycogen synthesis by glycogenin, glycogen synthase, and branching
enzyme, as proposed by Smythe and Cohen. Glycogenin is found in a wide range of both plant
and animal species. Escherichia coli also appears to have a glycogenin-like molecule, but it is
incapable of glucosylating recombinant mammalian glycogenin. Crystal structure analyses and
the ability of glycogenin mutants to transfer glucose to exogenous substrates have suggested that
priming occurs by one subunit of a glycogenin dimer transfering glucose to Tyr-194 on an
adjacent subunit (intersubunit reaction), rather than by self-glucosylation of the same
polypeptide (intrasubunit reaction). These opposing models are depicted in Figure 13.2. To date,
glycogenin is the only known example of the glycosylation of tyrosine residues in animals.
Figure 13.1. Model for glycogen biosynthesis via glycogenin (GN), glycogen synthase (GS),
and branching enzyme, as proposed by Smythe and Cohen. (Step 1) Glycogenin (GN) is self
priming, attaching Glc to Tyr-194. (Step 2) Glycogenin elongates the Glc polymer up to eight
monosaccharide units. (Step 3) GS binds. (Step 4) GS elongates the polysaccharide (glycogen)
using the primer produced by GN. (Step 5) Branching enzyme attaches α6 branches to the α4
cores to produce highly branched glycogen polymers. (Closed triangles) Glucose
monosaccharides. (Modified, with permission, from [14] Alonso et al. 1995.)
Figure 13.2. Opposing models for the self-glucosylation of glycogenin. Structural data support
the hypothesis of intersubunit priming rather than intrasubunit priming. (Top) Active site of one
subunit of the glycogenin dimer adding α1 4Glc residues to the adjacent subunit. (Bottom)
Model where each subunit adds Glc to itself.
O-linked Mannose (19 27)
In 1982, Marchase and colleagues demonstrated the transfer of α-Glc-1-P from UDP-Glc to O-
linked mannosyl residues to a predominant 62-kD protein, later shown to be
phosphoglucomutase. Phosphoglucomutase is a key enzyme of energy metabolism that
interconverts Glc-1-P to Glc-6-P. This interconversion is a crucial branch step that determines
the utilization of glucose for energy or its incorporation into macromolecules. Latency studies
established that both the novel glucose phosphotransferase acting on phosphoglucomutase and
the Glc-1-P phosphodiesterase that cleaves glucose from phosphoglucomutase have their active
sites within the cytoplasm. The modification of phosphoglucomutase is highly responsive to
calcium levels and appears to regulate the association of phosphoglucomutase with the ER
membrane. There is a growing literature suggesting that Glc-6-P, and thus phosphoglucomutase,
might have a key role in the regulation of calcium levels in cells. Unfortunately, nothing is
known about the mannosyltransferases that modify phosphoglucomutase and little is known
about the underlying mannosyl oligosaccharide structures modified by Glc-1-phosphorylation.
Nevertheless, these findings suggest that both the O-mannosylation and Glc-1-phosphorylation
of phosphoglucomutase may be very important regulatory forms of cytoplasmic glycosylation.
O-linked Fucose-containing Glycans (28 31)
West and colleagues identified a small fucosylated protein (SKP1) in the cytoplasm of the
cellular slime mold, Dictyostelium. Found in a multiprotein complex (SCF complex) with cell
cycle regulatory proteins, SKP1 is involved in the ubiquitination of proteins important to the cell
cycle. Ubiquitination targets these proteins for rapid degradation. The SCF complex is also
important for certain phosphorylation events in the cell cycle. Early studies indicated that fucose
was a component of an O-linked saccharide equivalent in size of up to 4.8 glucose units. A
cytosolic α1 2-fucosyltransferase that transfers fucose from GDP-Fuc to SKP1 (formerly FP21)
was identified and partially purified. Mass spectrometric structural analyses have established that
SKP1 is glycosylated at Pro-143, which had also been hydroxylated. The cytoplasmic sugar
structure on SKP1 was found to be Galα1 6-Galα1Fucα1 2Galβ1 3GlcNAc-(HyPro). In these
studies, Pro-143 was found to be only partially hydroxylated, but all of the hydroxylated form
was glycosylated. The linkage to protein, localization of this type of structure in the cytoplasm,
and the structure itself are all novel, which suggests a very atypical glycosylation pathway in
these and perhaps other eukaryotes. Since SKP1 is widely distributed in eukaryotes, it will be of
interest to see if it is similarly glycosylated in mammalian species.
Nuclear Glycosaminoglycans? (32 39)
As early as 1964, Yamashina and colleagues proposed the presence of glycosaminoglycans in

purified nuclei. In a fascinating series of studies from 1971 to 1983, Kinoshita and colleagues
studied what they proposed were nuclear heparan sulfates in developing sea urchin embryos.
These workers found that heparin stimulated mRNA synthesis in pregastrula embryos, but not in
postgastrula embryos. Other glycosaminoglycans, such as chondroitin sulfates and hyaluronans,
were without effect. Pulse-chase studies suggested that the nuclear glycosaminoglycans appeared
first in the cytoplasm and were transported to the nucleus. β-xylosides, which are primers of
chondroitin sulfate and heparan sulfate biosynthesis (see Chapter 40), blocked development of
sea urchins at the blastula stage. Addition of postgastrula proteoglycans to the cultures prevented
this block in development. Pregastrula proteoglycans did not prevent the β-xyloside-induced
block to development. Microinjection of proteoglycans into embryos blocked development at the
stage from which they were isolated. Clearly, given the recent advances in the tools to study
proteoglycans, these potentially exciting observations warrant a critical reexamination.
The first structural data proposing nuclear glycosaminoglycans, which were not easily dismissed
on the basis of potential contamination with extracellular glycosaminoglycans, were published in
1989 by Fedarko and Conrad. These workers radioactively labeled rat hepatocytes with 35SO4,
isolated subcellular fractions by standard procedures, purified heparan sulfates, cleaved the
heparan sulfates with nitrous acid, and determined the structures of the fragments. Nuclear
fractions were found to contain 11% of the total cellular heparan sulfate, but they were greatly
enriched with a unique molecular species with a high content of a novel structure: β-d-
glucuronosyl(2-SO4) d-glucosamine-N,O-(SO4) disaccharides (GlcA-2-SO4). Given the unique
nature of these structures, it is hard to conceive how they could be derived by contamination of
nuclear preparations with cell surface heparan sulfate molecules, where such structures are not
present. Later pulse-chase studies suggested that cell surface heparan sulfate is taken into the
nucleus and modified to the unique nuclear molecular species. Uptake of heparan sulfate appears
to have a lag time of 2 hours with a t1/2 of 8 hours. The uptake of heparan sulfate into the nucleus
was not affected by chloroquine. In support of the uptake model, when unlabeled heparan sulfate
proteoglycan that contains no GlcUA-2-SO4 was incubated with cells, about 10% ended up
within the nucleus and resulted in free heparan sulfate chains containing the unusual
disaccharide. Given the ability of heparan sulfates to influence gene transcription in vitro, these
studies could eventually prove to be highly significant. In contrast, Hascall and colleagues used a
new nuclear isolation method to prepare nuclei from rat ovarian granulosa cells and found
dermatan sulfates, but not heparan sulfates, associated with the nucleus. These authors
emphasized the difficulties in isolating pure nuclei and in proving the existence of
glycosaminoglycans in native nuclei. Furthermore, all of the known glycosyltransferases
involved in the biosynthesis of glycosaminoglycans have their active sites in luminal
compartments, and pathways for nuclear uptake of such large and negatively charged molecules
have not been described.
Alpha Toxins of Clostridium (40 44)
Small cytoplasmic G proteins (GTP-binding proteins) of the Rho family are involved in
regulating the cytoskeleton. As shown in Figure 13.3, Cdc42 is involved in the formation of
filopodia, Rac regulates membrane ruffling, and Rho regulates focal adhesions and stress fibers.
Certain clostridial toxins were found by Aktories and colleagues to be glycosyltransferases that
attach a glycosyl moiety to a threonine residue in the GTP-binding site of these proteins (in Rho,
glycosylation occurs at Thr-37; Figure 13.4). The enterotoxins from Clostridium difficile (ToxA)
and Clostridium sordellii are glucosyltransferases that use UDP-Glc as the donor. In contrast, a
similar toxin from Clostridium novyi is an O-GlcNAc transferase that uses UDP-GlcNAc as the
donor. The C. novyi toxin has no primary sequence relationship to the cloned mammalian
endogenous O-GlcNAc transferase (see Chapter 14). However, there may be related mammalian
transferases that have not yet been identified. These glycosyltransferase toxins should prove to
be valuable as tools to study the functions of these small G proteins, and they represent a unique
example of abnormal cytosolic protein glycosylation.
Figure 13.3. Rho family proteins regulate receptor-mediated actin cytoskeleton perturbations
and are targets for bacterial toxins. Note how the different Rho family members are involved in
regulating different cytoskeletal structures. The figure depicts transmembrane receptors
mediating cytoskeletal structures via different Rho family members. The large X indicates
blockage of small G-protein-mediated cytoskeleton-mediated assembly of the structures
indicated.
Figure 13.4. Thr-37 is the site of glycosylation of Rho proteins by toxins and has a key role in
GTP/GDP nucleotide exchange by these G proteins. The cartoon depicts the GTP-binding site of
the proteins. Note the essential role of Thr-37 in the active site of the Rho family G proteins.
Nuclear Lectins (45 53)
Many studies with labeled neoglycoproteins by Monsigny, Hubert, and colleagues suggest the
existence of a number of nuclear lectins. However, very little is known about the molecular
nature of these putative carbohydrate-binding proteins. For example, BSA derivatized with l-
rhamnose, d-GlcNAc, d-Glc, lactose, Man-6-P, or l-Fuc binds to nuclei at threefold higher
affinity than underivatized BSA. In 1980, Feizi and colleagues demonstrated the specific binding
of BSA-lactose to cryostat sections of nuclei. Wang and colleagues have shown that CBP35
(now called galectin 3; see Chapter 27) is present in the nucleus as part of the heterogeneous
nuclear ribonucleoprotein complex and appears to be required for normal mRNA splicing.
Interestingly, a neoglycoprotein composed of blood group A tetrasaccharide attached to BSA
was found to specifically inhibit mRNA splicing in vitro. However, at present, it is unclear
whether the carbohydrate-binding activity of galectin 3 has any role in the functions of the
protein in mRNA splicing.
Saccharides as Nuclear Localization Signals? (54 57)
Molecules larger than approximately 40 kD cannot diffuse freely through nuclear pores and must
be specifically and actively transported into and out of the nucleus. Generally, nuclear
localization sequences consist of stretches of positive amino acids (e.g., YPKKKRKVEDPRC),
as are found on proteins, e.g., SV40 large T antigen and nucleoplasmin. During the past several
years, Monsigny and colleagues have presented evidence that sugars may also serve as nuclear
localization signals. The neoglycoproteins BSA-glucose, BSA-fucose, and BSA-mannose are
rapidly transported into the nucleus of HeLa cells, whereas BSA itself is not. The sugar-mediated
nuclear transport appears to be distinct from the basic peptide-mediated nuclear localization
sequence pathway. Like the classical pathway, the sugar-mediated nuclear transport requires
energy and is blocked by the lectin, wheat-germ agglutinin. However, unlike the basic peptide
system, the sugar-mediated pathway does not require cytosolic factors and is not blocked by
sulfhydryl-reactive chemicals, such as N-methylmaleimide. More recently, Monsigny and
colleagues have shown that BSA substituted with β-di-N-acetylchitobiosides
(GlcNAcβ4GlcNAc) is rapidly localized to the nucleus by a pathway distinct from the classically
defined nuclear localization sequence systems. Validation of these fascinating results awaits
characterization of the components involved and the identification of natural counterparts to the
neoglycoproteins.
Prospects and Future Directions
Overall, there are many tantalizing clues for the existence and importance of complex
glycoconjugates in the nucleus and cytoplasm. However, except for a few well-described
specific examples, definitive structural data are lacking. Particularly problematic are the reports
of "classical" type N- or O-glycans within the nucleoplasmic or cytoplasmic compartments. Such
claims fall short, since they are going against the overwhelming data for the localization of most
enzymes involved in the synthesis of these molecules. Proving an exception to the "dogma" of
the luminal or cell surface localization of complex glycans therefore requires detailed structural
proof that the proposed structures are indeed attached to a region of the protein known to be
cytosolic or nuclear. Some of these studies may point to exciting and important areas of
glycobiology research for the future.
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14. The O-GlcNAc Modification
UNTIL THE MID 1980S, IT WAS WIDELY BELIEVED that proteins within the nucleus and
cytoplasm were not glycosylated. We now know that many proteins within these compartments
are dynamically modified at their serine and threonine hydroxyl groups by the attachment of O-
linked N-acetylglucosamine monosaccharides. Figure 14.1 shows the structure of O-GlcNAc,
which is dynamically attached in a β-linkage to proteins. This chapter first presents a historical
view of the discovery and early studies on O-GlcNAc. The presence of O-GlcNAc on key
nuclear and cytoskeletal proteins and on infectious agents is then described. Finally, the role of
O-GlcNAc as a dynamic regulatory modification and a description of what is known about the
enzymes catalyzing its addition and removal are presented.
Figure 14.1. Schematic diagram of the structure of O-GlcNAc. Arrows indicate the highly
dynamic nature of the modification. O-GlcNAc is rapidly added and removed by O-GlcNAc
transferase(s) and O-GlcNAcase(s), respectively. The peptide sequence shown represents a
typical O-GlcNAc attachment site; however, there is no well-defined consensus sequence.
Historical Background of O-GlcNAc (1 6)
A few early studies in viruses and plants, which generally performed alkali treatments followed
by monosaccharide analyses, hinted at the presence of GlcNAc as a linking sugar to hydroxy
amino acids in proteins. Unfortunately, these early studies did not identify the protein-saccharide
linkage nor validate the existence of O-GlcNAc by structural analysis. O-GlcNAc was first
shown to be a major form of intracellular glycosylation in murine lymphocytes in 1984, during
studies designed to probe terminal GlcNAc on complex glycans of surface receptors. These
studies used highly purified glycosyltransferases and their corresponding radioactively labeled
sugar nucleotides as enzymatic probes of terminal saccharides on living cells. O-GlcNAc was
found by using radioactively labeled UDP-Gal and bovine milk galactosyltransferase as a probe.
Figure 14.2 illustrates the principle of this approach, which is still widely used to detect the O-
GlcNAc modification. In 1986, the O-GlcNAc modification was found to be abundant in the
cytoplasm and in virtually all subcellular organelles of rat liver, except for mitochondria. The
highest concentration (moles of sugar per mole of protein) of O-GlcNAc proteins was found in
the nuclear envelope. However, these same studies found that the nucleoplasm contains the
largest number of the O-GlcNAcylated (O-GlcNAc-modified) proteins. Rapidly, during the next
2 years, several different groups showed that nuclear pore proteins, which regulate trafficking of
molecules into and out of the nucleus, are extensively O-GlcNAcylated.
Figure 14.2. Galactosyltransferase labeling of O-GlcNAc. Purified bovine milk
galactosyltransferase, together with tritiated UDP-Gal, can be used to place a "tag" on O-
GlcNAc residues.
Also in 1987, several cytoplasmic O-GlcNAcylated proteins were identified in human

erythrocytes, including the cytoskeletal protein Band 4.1, which bridges the spectrin/actin
cytoskeleton to the cytoplasmic tail of glycophorin. In 1989, O-GlcNAcylated proteins were
shown to occur in very large amounts along the length of polytene chromosomes from the
salivary glands of third-instar Drosophila larvae. FITC-WGA staining of the O-GlcNAc-bearing
proteins shows a banding pattern similar to those of chromatin-binding dyes used by cytologists.
O-GlcNAc levels appear to be markedly reduced at active sites of gene transcription. Direct
biochemical analyses of Drosophila chromatin demonstrated that a very large number of
different chromatin proteins are O-GlcNAcylated.
Since these early studies, O-GlcNAc has been reported on a large number of proteins. Table 14.1
lists most of the O-GlcNAcylated proteins identified to date. Even though these O-GlcNAcylated
proteins have a large functional diversity, they have two common features: (1) They are also
phosphorylated and (2) they form reversible multimeric complexes with other polypeptides or
structures, and these associations are often regulated by phosphorylation. Nevertheless, at the
time of this writing, the majority of the hundreds of O-GlcNAc-modified proteins detected by
two-dimensional gel analyses of nuclear and cytosolic fractions have not been identified.
Table 14.1. Identified O-GlcNAcylated proteins
Nuclear proteins Cytoskeletal proteins Other proteins
Nuclear pore proteins cytokeratins 13, 8, 18 92-kD Ser protein

RNA polymerase II neurofilaments H, M, L p43/hnRNP
Many transcription factors human erythrocyte Band 4.1 adenovirus fiber
c-Myc oncoprotein synapsin I human CMV UL32
v-Erb-a oncoprotein many synaptic vesicle proteins rotavirus NS26 protein
p53 tumor suppressor MAPs baculovirus gp41 tegument protein
SV40 T antigen tau p67 translation regulation protein
Tyrosine phosphatase talin malarial proteins
Many chromatin proteins vinculin trypanosome proteins
Estrogen receptors clathrin assembly protein AP3 giardia proteins
Fungal DNA-binding proteins Aplasia E. histolytica proteins
neuron proteins
α-crystallins schistosome proteins
β-amyloid precursor proteins
O-GlcNAc Is Ubiquitous in Eukaryotes (7, 8)

During the past several years, O-GlcNAc-modified proteins have been shown to be almost
exclusively restricted to the cytoplasm and nucleus (except for α-linked O-GlcNAc saccharides
in slime molds, which are extracellular) and are abundant in virtually all eukaryotes examined,
including protozoa and fungi, as well as in viruses (Table 14.1). In addition, the recently cloned
O-GlcNAc glycosyltransferase is highly conserved evolutionarily, with more than 85% similarity
between the primary sequence of the enzyme derived from the nematode, Caenorhabditis
elegans, and humans. Homologous open reading frames occur in the public databases as far
down the evolutionary scale as cyanobacteria.
Nuclear O-GlcNAc Proteins (1, 5, 9, 10)

Nuclear Pore Glycoproteins (Nucleoporins)
The importance of the nuclear pore O-GlcNAcylated proteins in nuclear transport has been
shown by the abilities of either O-GlcNAc-specific monoclonal antibodies or the GlcNAc-
binding lectin WGA to block nuclear transport. Studies from several laboratories suggest a
model in which the nuclear pore glycoproteins play a part in the initial peripheral binding of
transport molecules, which is followed by the docking of the transported molecule at the center
of the pore complex, and its translocation. Direct evidence for the role of nuclear pore
glycoproteins in nuclear transport has been obtained from reconstitution studies. Xenopus egg
nuclear envelopes reassemble to form grossly morphologically normal nuclear pore complexes
from extracts that have been depleted of the WGA-binding nuclear pore glycoproteins. However,
these reassembled nuclear pores are defective in both binding and nuclear transport. Nuclear
transport is restored by the readdition of the isolated nuclear pore glycoproteins. Pores depleted
of the O-GlcNAcylated proteins contain "holes" in their central regions. This localization
suggests that these glycoproteins have a central role in nucleocytoplasmic transport.
Several of the nucleoporins have been cloned and sequenced, including nucleoporin (p62), which
is one of the most heavily O-GlcNAcylated proteins. Interestingly, Hanover and colleagues have
shown that when p62 is transcribed/translated in a coupled reticulocyte lysate system, it is
efficiently O-GlcNAcylated. Therefore, reticulocyte lysates contain both the O-GlcNAc
glycosyltransferase and the UDP-N-acetylglucosamine sugar donor. Immobilized nucleoporins
are able to deplete reticulocyte lysates of soluble proteins required for protein import into the
nucleus, suggesting that these glycoproteins interact with key proteins involved in nuclear
transport.
A direct role for the O-GlcNAc saccharide moieties in nuclear transport was evaluated by
reconstituting Xenopus nuclear pores with O-GlcNAcylated pore proteins in which the GlcNAc
residues had been previously enzymatically capped by the attachment of galactose. Capping of
many of the O-GlcNAc moieties with galactose residues did not appear to prevent nuclear
transport. Since "capping" GlcNAc would be predicted to prevent binding by GlcNAc-specific
carbohydrate-binding proteins, these studies suggest that the saccharides on nuclear pore
glycoproteins are not functioning as ligands in lectin-like interactions that might be involved in
nuclear transport.
Chromatin-associated Proteins and Transcription
A myriad of different chromatin proteins, with a broad range of physical associations with
chromatin, are O-GlcNAcylated. Virtually every RNA polymerase II transcription factor
examined to date contains O-GlcNAc. Functions proposed for O-GlcNAcylation of transcription
factors have included nuclear transport, assembly into multimeric complexes, and regulation of
phosphorylation. Recent findings suggest that O-GlcNAc on transcription factors in fungi is
important to transcription even in lower eukaryotes.
In addition to transcription factors, the catalytic subunit of RNA polymerase II is also multiply
O-GlcNAcylated on its carboxy-terminal domain repeat sequences. RNA polymerase II, which
synthesizes messenger RNA, is a complex enzyme composed of at least ten distinct polypeptide
subunits. The catalytic subunit in eukaryotes contains a unique seven-amino-acid sequence (-
Tyr-Ser-Pro-Thr-Ser-Pro-Ser-) at its carboxyl terminus (CTD), which is tandemly repeated up to
50 times and is essential for viability in yeast. During transcriptional elongation, the CTD is
extensively phosphorylated. A subset of the nonphosphorylated form of RNA polymerase II
(form IIA) is extensively and multiply glycosylated along the length of the CTD sequences. O-
GlcNAc is not detectable on the phosphorylated form of RNA polymerase II (form IIO),
indicating that O-GlcNAcylation and phosphorylation of the CTD are mutually reciprocal
events. These data and studies on transcriptional initiation from many laboratories support a
model (Figure 14.3) in which O-GlcNAcylation plays an important part in transcriptional
initiation. According to this model, O-GlcNAc residues function in the assembly of the
preinitiation complex. Subsequent to initiation, the O-GlcNAc moieties on the CTD are
removed, and extensive phosphorylation of the CTD allows elongation to occur. Further study is
required to evaluate the precise roles of O-GlcNAcylation in the regulation of transcriptional
initiation and elongation. However, it is clear that many (most?) of the proteins involved in
transcriptional regulation are dynamically O-GlcNAcylated and that the saccharide is directly
involved at one or more steps in the transcription process.
Figure 14.3. Model for the role of O-GlcNAc in transcription. The hypothesis is that O-GlcNAc
has a role in transcriptional initiation and is removed from the CTD of RNA polymerase prior to
elongation, allowing the extensive phosphorylation of the CTD. Recent studies have also shown
that the TATA-binding protein is modified by O-GlcNAc. GlcNAc is depicted as closed squares
according to the standard nomenclature used throughout this text. Casein kinase II, which is
predominantly a nuclear enzyme that has been implicated in regulating numerous cellular
processes including growth, proliferation, and differentiation, is multiply O-GlcNAcylated on
both its α and α subunits, but not on the β-subunit. Interestingly, one major site of O-
GlcNAcylation on casein kinase II occurs adjacent to a regulatory phosphorylation site used by
Cdc2/p34 kinase.
Nuclear Oncogene and Tumor Suppressor Proteins
c-Myc oncoprotein is a helix-loop-helix leucine zipper phosphoprotein that heterodimerizes with

the Max protein and regulates gene transcription. c-Myc was found to have O-GlcNAc both in
mammalian systems and in recombinant proteins overexpressed in baculovirus. Analyses of
deletion mutants localized the O-GlcNAcylation to within the trans-activation domain that
associates with the retinoblastoma gene product RB in vitro and is the mutation hot spot region
of c-Myc in Burkitt and AIDS-related lymphomas. Site analysis of recombinant c-Myc
demonstrated that the major site of O-GlcNAcylation is Thr-58. Figure 14.4 illustrates the major
steps used to map sites of O-GlcNAc on a protein such as c-Myc. Importantly, Thr-58 is also an
in vivo phosphorylation site within the trans-activation domain and is frequently mutated to non-
hydroxyl-containing amino acids in retroviral v-Myc proteins and in Myc proteins of human
Burkitt and AIDS-related lymphomas. Mutation of Thr-58 results in enhanced transforming
activity and increased tumor-inducing potential in the oncogene protein. Hierarchical
phosphorylation of Thr-58 and Ser-62 plays an important part in c-Myc regulation of the cell
cycle.
Figure 14.4. Outline of methods used to map O-GlcNAc attachment sites. The protein is purified
to homogeneity and cleaved with trypsin or another cleavage method, and the glycopeptides are
"tagged" with galactosyltransferase (GT)-mediated galactosylation. Galactose-labeled
glycopeptides are purified by several rounds of RP-HPLC, and purified peptides are analyzed by
gas phase microsequencing. Saccharides are localized by measuring at what cycle the labeled
sugar is removed during manual Edman degradation. Sites are confirmed by mass spectrometry.
These studies provide direct structural evidence that reciprocal glycosylation and
phosphorylation at this biologically significant site play an important part in mediating the
functions of the c-Myc transcription factor/oncogene protein. Clearly, an important message
from these findings is that caution must be used in the interpretation of site-directed mutagenesis
data in which a serine or threonine is mutated. It may be impossible from mutagenesis alone to
conclude whether the observed biological effect is due to lack of phosphorylation, lack of O-
GlcNAcylation, or lack of both modifications.
p53, a tumor suppressor and transcription factor that is mutated in about one half of all human
tumors, has also been shown to be O-GlcNAcylated. The high biological activity and high
affinity for DNA of EB-1 p53 are reported to be dependent on O-GlcNAc masking of a basic
region in the carboxyl terminus of p53, which otherwise represses DNA binding. Therefore, the
dynamic O-GlcNAcylation of this region of p53 appears to have an important role in modulating
the affinity of the protein for DNA.
SV40 T antigen also contains O-GlcNAc moieties, as does the nuclear oncogene, the v-Erb-a
oncoprotein. v-Erb-a is encoded by the avian erythroblastosis virus and is derived from a cellular
gene for a thyroid hormone (T4/T3 thyronine). It is likely that many of the ligand-responsive
transcription factors, such as steroid receptors, will also be found to be O-GlcNAcylated, as has
recently been reported for the estrogen receptors.
Cytoskeletal and Membrane O-GlcNAc Proteins (5, 11)

The first cytoskeletal protein shown to be O-GlcNAcylated was human erythrocyte Band 4.1,
which has an important role in maintaining erythrocyte cell shape. As early as 1988, clear
evidence was published for the presence of cytoplasmically O-GlcNAcylated transmembrane
proteins in both the ER and Golgi apparatus. More recently, a variety of cytoskeletal and a few
important membrane-associated proteins have been documented to bear O-GlcNAc moieties. Of
particular note is the finding of O-GlcNAc on the cytoplasmic domain of the β-amyloid
precursor protein. The similarity of O-GlcNAc attachment sites and PEST sequences that target
proteins for degradation has led to the postulate that O-GlcNAcylation might have a role in the
degradation of the β-amyloid precursor protein that produces the β-amyloid peptides associated
with lesions in Alzheimer's disease.
Cytoskeletal "Bridging" Proteins
It is noteworthy that several O-GlcNAcylated cytoskeletal proteins, such as Band 4.1, vinculin,
talin, and synapsin, are involved in the phosphorylation-dependent reversible bridging of the
cytoskeleton to the membrane or other structures (see Table 14.1). For example, O-GlcNAc has
been found on platelet vinculin and appears to be dynamically responsive to thrombin-mediated
platelet activation. Clathrin assembly protein (AP3) is both phosphorylated and O-GlcNAcylated
in its central 50-kD structural domain. Consistent with the saccharide's location in the
nonclathrin-binding domain, O-GlcNAc does not appear to be involved in AP3's interactions
with clathrin. Although the α crystallins are major components of the lens of eye, in other cell
types, such as the heart, they act as chaperones that modulate intermediate filament assembly.
The α crystallins in both lens and nonlens tissues are dynamically glycosylated with O-GlcNAc,
which turns over rapidly, cycling many times in the life of the polypeptide. Talin, which serves
to bridge integrins to the cytoskeleton via its interactions with vinculin, also contains O-GlcNAc.
It was suggested that the saccharide might have a role in the association of vinculin with talin.
Likewise, the O-GlcNAcylated protein synapsin I is thought to mediate the interactions of
synaptic vesicles with the cytoskeleton. Thus, although direct data are still lacking, it has been
proposed that O-GlcNAcylation has an important role in mediating protein-protein interactions
involved in a wide variety of cellular functions, including the organization of the cytoskeleton.
Intermediate Filament Proteins
Cytokeratins are a family of intermediate filament proteins comprising an essential component of

the cytoskeleton of epithelial cells. More than 21 different cytokeratins have thus far been
identified, which are expressed in a tissue-specific and differentiation-dependent manner.
Cytokeratin 13, one of the most abundant intermediate filament proteins of many internal
stratified epithelia, was shown to have O-GlcNAc by direct linkage and saccharide analyses.
WGA-blotting studies suggest that the O-GlcNAcylation of cytokeratin 13 is highly variable and
cell-type-dependent. Cytokeratins 8 and 18, derived from human HT29 colonic cells, were also
subsequently shown to have O-GlcNAc. Peptide mapping suggested multiple sites of O-
GlcNAcylation, and pulse-chase analysis indicates that the saccharide turns over much more
rapidly than the cytokeratin polypeptides, further supporting the dynamic nature of this form of
glycosylation. In HT29 cells arrested at the G2/M phase of the cell cycle by colcemid, both the
phosphorylation and O-GlcNAcylation of cytokeratins increased. However, cytokeratin 8
showed a preferential increase in phosphorylation, whereas cytokeratin 18 displayed a
preferential increase in O-GlcNAcylation during the G2/M block. In contrast, aphidicolin-
synchronized S-phase HT29 cells did not have cytokeratins with increased O-GlcNAcylation.
These studies suggest that the head and proximal rod domains of cytokeratin 18 are the primary
regions of both types of posttranslational modifications. Even though O-GlcNAcylation and
phosphorylation appeared to increase concomitantly at G2/M, they each appeared to occur on
different molecules. When overexpressed in baculovirus-infected insect cells, cytokeratins 8 and
18 were both glycosylated and phosphorylated, but each modification again occurred on different
molecules.
Neurofilaments
Site-mapping studies on neurofilament proteins indicate that the different types of

neurofilaments, H, M or L, are heavily O-GlcNAcylated. On the basis of prior site-directed
mutagenesis studies of these neurofilaments, the locations of the O-GlcNAc moieties suggest
that the saccharide is directly involved in mediating neurofilament assembly. In fact, most of the
O-GlcNAc residues on assembled neurofilaments are inaccessible to galactosyltransferase or
hexosaminidase treatments, unless the protein is first denatured or fragmented with protease
treatments. The O-GlcNAc moieties on α crystallins, which are also small heat shock proteins in
nonlens tissues, and those on the adenovirus fiber proteins, are similarly inaccessible on the
native molecules, whereas O-GlcNAc on undenatured nucleoporins appear to be largely on the
surface of the molecules.
Microtubule-associated Proteins
The normal microtubule-associated protein tau from bovine brain is heavily O-GlcNAcylated,
with apparently more than 12 O-GlcNAcylation sites and an average stoichiometry of
approximately four O-GlcNAc residues per mole of protein. In normal neurons, tau plays a key
part in organizing microtubules in the axons. However, in neurons from Alzheimer's disease
patients, tau is abnormally hyperphosphorylated, causing it to form abnormal filaments (PHF-
tau), which may be involved in neuronal death. As shown in Figure 14.5, these studies suggest a
possible model in which the abnormal hyperphosphorylation of PHF-tau in Alzheimer's disease
neurons may result from a defect in the normal O-GlcNAcylation of serine or threonine residues,
which allows abnormal phosphorylation to take place. A putative role for O-GlcNAcylation in
Alzheimer's disease is even more intriguing with the recent finding of O-GlcNAc on the β-
amyloid precursor protein. Likewise, a possible role for O-GlcNAc in generally mediating the
associations of microtubule-binding proteins must be considered in light of recent reports
describing the O-GlcNAcylation of the high-molecular-weight microtubule-associated proteins,
including MAP1, MAP2, and MAP4.
Figure 14.5. Possible model for a role of O-GlcNAc in Alzheimer's disease. The model proposes
that the MAP tau is normally O-GlcNAcylated. Indeed, so is the β-amyloid precursor protein at
its cytoplasmic domain. Hypoglycosylation could allow exposure of potential phosphorylation
sites on tau, which allow it to become hyperphosphorylated, leading to neurofibrillary tangles.
The Ser-262 site on the protein is illustrated. However, tau is both phosphorylated and O-
GlcNAcylated at many sites, some of which also have an important role in Alzheimer's disease.
O-GlcNAc on Viral and Parasite Proteins (5, 12)

O-GlcNAc modifications have been demonstrated on a number of viral proteins (see Table 14.1).
O-GlcNAc is found on the major tegument proteins (the region between the viral capsid and the
viral envelope) of human CMV and baculovirus. The function of the basic phosphoprotein
(UL32) of human CMV is presently unknown, but its location in the tegument region of the virus
suggests that it might act as a signal for final envelopment of the capsid. O-GlcNAc could be a
signal for oligomerization of the basic phosphoprotein or it could be the means of attachment to
the cellular compartment used for viral envelopment. The function of O-GlcNAc on any of these
proteins is not yet known; however, the fiber proteins of adenovirus are known to form mature
trimeric structures that are involved in virus attachment to the host-cell surface. Interestingly, the
O-GlcNAc moieties in the mature trimeric structures are inaccessible to labeling with
galactosyltransferase unless the fibers are denatured with detergents, indicating that the O-
GlcNAc moieties are buried in the trimeric structures. The demonstration that gp41 of
baculovirus contains O-GlcNAc made it clear that insect cells are fully capable of adding this
modification to proteins. Furthermore, this finding has opened the door to using the baculovirus
overexpression system to study the glycosylation of many low-abundance proteins that contain
O-GlcNAc, including transcription factors and oncogenes.
An approximately 92-kD cytoplasmic O-GlcNAcylated protein has been identified and cloned
from Leishmania major. A protective antigen of the protozoan parasite, Entamoeba histolytica,
the serine-rich E. histolytica protein (SREHP), has a high serine content (52/233 amino acids)
and is phosphorylated and O-GlcNAcylated. O-GlcNAc is a major protein modification in the
human blood fluke, Schistosoma mansoni, in which it accounts for more than 10% of the
metabolic incorporation of tritiated GlcNAc. O-GlcNAcylation also appears to be a major form
of protein glycosylation in malarial parasites.
O-GlcNAc as a Dynamic Regulatory Modification (7, 8,13 20)
Enzymes of O-GlcNAc Cycling
A UDP-GlcNAc:polypeptide O-GlcNAc transferase activity has been identified and purified to

apparent homogeneity from the cytosol of rat liver. Some of the properties of this enzyme
include the following: (1) cytosolic and nuclear localization of its active site; (2) UDP-GlcNAc
is the nucleotide sugar donor with a Km of about 545 nm; (3) the liver enzyme appears to contain
two subunits with an Mr of 110 kD (α-subunit) and 78 kD (β-subunit); (4) the holoenzyme is
about 340 kD, thus suggesting a heterotrimer of α2β configuration; and (5) photoaffinity labeling
studies indicate that the α-subunit contains the active site. Both subunits of the enzyme are
tyrosine-phosphorylated and O-GlcNAcylated.
The gene encoding the O-GlcNAc transferase has recently been cloned from both rat liver and
the nematode, C. elegans. The O-GlcNAc transferase protein is unlike any known
glycosyltransferase previously described. It is very highly conserved at the primary sequence
level from C. elegans to humans. One striking structural feature of this O-GlcNAc transferase is
the presence of 11 tetratricopeptide repeats. These repeat domains are thought to promote a
variety of both intra- and interprotein interactions. The enzyme's structure suggests that its
specificity or activity might be regulated by numerous tetratricopeptide-binding factors in a
manner analogous to the regulation of RNA polymerase II by transcription factors. The
availability of the cloned DNA encoding an O-GlcNAc transferase should allow rapid progress
on the evaluation of both the regulation of O-GlcNAcylation and the determinations of the
saccharide's functions in a wide variety of biological systems.
A neutral, O-GlcNAc-specific, β-d-N-acetylglucosaminidase activity from rat spleen cytosol has

also been identified and recently purified and characterized. The number of different O-
GlcNAcases is still unknown. The purified O-GlcNAcase activity was distinguished from
lysosomal hexosaminidases by its neutral pH optimum (pH 6.4) and by its insensitivity to
GalNAc or GalNAc analogs, either as inhibitors or as substrates. The enzyme activity can
specifically remove O-GlcNAc from peptide substrates that had been glycosylated with the O-
GlcNAc transferase with a sixfold higher relative activity than equal amounts of Diplococcus
pneumoniae hexosaminidase. As yet, almost nothing is known about the regulation of O-
GlcNAcases, but the cloning of the cDNA encoding the rat cytosolic enzyme should allow for
rapid progress.
Regulation of Protein Synthesis
A 67-kD multiply O-GlcNAc-modified glycoprotein (p67) has been described that can bind to
eIF-2 and protect it from phosphorylation by eIF-2 kinases, thereby maintaining protein
synthesis initiation. Several lines of evidence indicate that O-GlcNAcylated p67 protects the α-
subunit of eIF-2 from phosphorylation under normal conditions, but during starvation, p67 is
rapidly deglycosylated and then degraded, which allows eIF-2 kinases to phosphorylate the eIF-2
and prevent protein synthesis initiation. Reticulocyte lysates contain a "deglycosylase" (O-
GlcNAcase) that remains in a latent form in the presence of hemin. However, when hemin is
absent, the deglycosylase is activated to remove O-GlcNAc from p67 and to begin the cascade
toward inhibition of proteins synthesis. These data suggest that reversible O-GlcNAcylation has
a key role in protein synthesis. If correct, they have important implications with respect to the
overall significance of this saccharide modification.
Regulation of Glucose Homeostasis
Much of the pathophysiology of diabetes is clearly linked to hyperglycemia itself. Recently,

many studies have pointed to the conclusion that the hexosamine biosynthetic pathway serves as
a glucose sensor coupled to a negative feedback system that can limit the extent of glucose
uptake in response to hyperglycemic and hyperinsulinemic conditions. The connection between
glucosamine and insulin resistance, and the ubiquitous O-GlcNAcylation of transcription factors,
has led to the hypothesis that the abnormal glycosylation of transcription factors, perhaps due to
elevated UDP-GlcNAc levels (which can approach levels as high as ATP levels in some cells),
may be a major mechanism behind the pleiotropic effects of glucose toxicity and insulin
resistance. Glucosamine itself appears to be particularly important in the generation of "insulin
resistance." For example, in adipocytes, insulin, glucose, and glutamine in combination cause a
desensitization to insulin, suggesting the involvement of glutamine:Fru-6-P amidotransferase, a
key enzyme in glucosamine metabolism. Glucosamine is 40-fold more potent in inducing insulin
resistance than is glucose. Glutamine:Fru-6-P amidotransferase appears to play a key part in the
development of insulin resistance by directing the flow of incoming glucose into hexosamines,
which are postulated to serve as glucose sensors coupled to an "unknown" negative feedback
system. Glucosamine increases TGF-α mRNA by 6-fold and protein as well; 2 mm glucosamine
causes a 12-fold increase in TGF-α promoter activity. It has been postulated that elevated
glucosamine also contributes to glucose toxicity by stimulating the transcription of growth factor
genes giving rise to the vascular complications of diabetes. Thus, it is apparent that since O-
GlcNAc is a key posttranslational modification on many of the cell's regulatory proteins, it
follows that hyperglycemia-induced perturbation of O-GlcNAc metabolism, perhaps mediated in
part by elevation of UDP-GlcNAc pools, could play a major molecular part in the pathologies of
diabetes.
Future Directions
Even though Ser(Thr)-O-GlcNAcylation was only discovered about 14 years ago, pulse-chase
studies and comparative analyses of two-dimensional gels make clear that this modification is as
abundant and dynamic as phosphorylation on many eukaryotic nuclear and cytoplasmic proteins.
O-GlcNAcylation is characterized by three key features: (1) O-GlcNAc occurs at sites similar to
those used by many important kinases; (2) O-GlcNAcylation is reciprocal to phosphorylation on
well-studied proteins, and may have a "yin-yang" relationship with phosphorylation in terms of
its functions on many proteins; and (3) O-GlcNAcylation is highly dynamic with rapid cycling in
response to cellular signals or cellular stages, again exactly analogous to phosphorylation. This
chapter has highlighted the major paradigms that will likely lead to an understanding of the real
significance of O-GlcNAcylation. With the advances in methodologies, and the availability of
cDNAs and potent inhibitors of enzymes involved in O-GlcNAc cycling, there soon should be
rapid progress in evaluating the overall importance of O-GlcNAcylation.
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15. Sialic Acids
Diego).
THIS CHAPTER DESCRIBES THE DIVERSITY of the sialic acid family of monosaccharides
in nature, with respect to their biosynthesis, structure, and linkage to the underlying sugar chain.
Also mentioned are the general principles behind the different methods for the study of sialic
acids. The biological roles of sialic acids are briefly considered, especially concerning the roles
of sialic-acid-binding lectins.
More than 50 years ago, Blix, Klenk, and other investigators discovered N-acetylneuraminic acid
(Neu5Ac; see Figure 15.1) as a major product released by mild acid hydrolysis of brain
glycolipids or salivary mucins. The complete structure, chemistry, and biosynthesis of this
molecule were subsequently characterized in the 1950s and 1960s by several groups, including
those of Gottschalk, Roseman, Brossmer, Warren, Yamakawa, and Glick. Meanwhile, sialic
acids emerged as the targets for recognition by influenza viruses. Chargaff's group discovered
that the "receptor-destroying enzyme" activity of influenza viruses acts as a sialidase, releasing
sialic acids from macromolecules, and Meyer's group found a similar activity in bacterial
sources. Early on, it was apparent that this 9-carbon acidic sugar was actually a common member
of a whole family of compounds related to neuraminic acid. Partly because of their original
discovery in salivary mucins, this family was christened the "sialic acids." By the 1980s, more
than 30 types of sialic acids had been discovered by Schauer and other investigators, and many
were shown to be expressed in a cell-type-specific and developmentally regulated manner. More
recently, the discovery of KDN (which is not a neuraminic acid, see Figure 15.1) by Inoue and
colleagues has further expanded the family of sialic acids.
Figure 15.1. "Primary" sialic acids: 2-keto-5-acetamido-3,5-dideoxy-d-glycero-d-

galactononulosonic acid (Neu5Ac) and 2-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN).
The only difference is the substitution at the 5-carbon position. Neu5Ac is much more common
in most vertebrate cell types. All other sialic acids are thought to be metabolically derived from
these two.
Diversity in the Structure and Linkage of Sialic Acids (2,4, 7,9 13)
The sialic acids are typically found at the outermost ends of N-glycans, O-glycans, and
glycosphingolipids (and occasionally capping the side chains of GPI anchors). They are subject
to a wide variety of modifications (Figure 15.2). The carboxylate group at the 1-carbon position
is typically ionized at physiological pH, but it can also be occasionally found in lactone ester
with hydroxyl groups of adjacent saccharides. As shown in Figure 15.1, the 5-carbon position
commonly has an N-acetyl group (giving Neu5Ac) or a hydroxyl group (as in KDN). The 5-N-
acetyl group can also be hydroxylated, giving N-glycolylneuraminic acid (Neu5Gc).
Occasionally, the 5-N-acetyl group is de-N-acetylated, giving neuraminic acid (Neu). These four
molecules (Neu5Ac, Neu5Gc, KDN, and Neu) have the potential for additional substitutions at
the hydroxyl groups on the 4-, 7-, 8-, and 9-carbons (O-acetyl, O-methyl, O-sulfate, and
phosphate groups). Additional complexity arises from the fact that the O-acetyl esters can
migrate along the side chain (from the 7- to the 9-position) under physiological conditions.
Unsaturated and dehydro forms of sialic acids are also known to exist. Not all of the possible
combinations shown in Figure 15.2 have been reported in nature, but more than 40 are known to
date.
Figure 15.2. Diversity in the sialic acids. The 9-carbon backbone common to all known sialic
acids is shown. Natural substitutions that have been described to date (at R4, R5, R7, R8, and R9)
are indicated. Additional diversity can be generated by occurrence of lactones (from R1 to
hydroxyl groups on other saccharides), by dehydro forms (eliminating R2), by the internal
anhydro forms indicated, as well as by various types of glycosidic linkages (via R3, to the
underlying sugar chain). (Reprinted, with permission, from [13] Varki 1997.)
Further diversity in sialic acid presentation is generated by different α-linkages from the 2-
carbon to underlying sugar chains (Figure 15.2). Of these, the most common are to the 3- or 6-
position of Gal residues or to the 6-position of GalNAc residues. In some instances, sialic acids
can also occupy internal positions within glycans, the most common situation being another
sialic acid residue attached to the 8-position (see section on polysialic acids below).
Combinations of different glycosidic linkages with the various substitutions mentioned above
give hundreds of ways in which sialic acids can present themselves on the surface of
glycoconjugates. This structural diversity of sialic acids can determine and/or modify the
recognition by antibodies, as well as by a variety of sialic-acid-binding lectins of endogenous
and exogenous origin (see below).
Nomenclature and Abbreviations (4, 7, 12)
The complete chemical names of the different sialic acids are too cumbersome for routine use. A
uniform and simple nomenclature system is now available and is seeing increasing use. The root
abbreviation Neu denotes the core structure neuraminic acid, and KDN denotes the core 2-keto-
3-deoxy-nonulosonic acid. Various substitutions are then designated by letter codes (Ac = acetyl,
Gc = glycolyl, Me = methyl, Lt = lactyl, S = sulfate), and these are listed along with numbers
indicating their location relative to the 9-carbon positions. Thus, for example, N-glycolyl-
neuraminic acid is Neu5Gc, and 9-O-acetyl-8-O-methyl-N-acetyl-neuraminic acid is
Neu5,9Ac28Me, and 7, 8, 9-tri-O-acetyl-N-glycolyl-neuraminic acid is Neu5Gc7,8,9Ac3. When
not certain of the type of the sialic acid present at a particular location, the generic abbreviation
Sia should be used. If other partial information is available, this could be incorporated, e.g., a
sialic acid of otherwise unknown type with an acetyl substitution at the 9-position could be
written as Sia9Ac. If a substitution is present, but the type is unknown, it can be written with an
X, e.g., SiaX.
Oligosialic and Polysialic Acids (9,14 19)
Polysialic acid is a remarkable extended homopolymer of sialic acid found only on a few animal
glycoproteins, e.g., the N-CAM and fish egg glycoproteins, as well as in the capsular
polysaccharides of certain pathogenic bacteria (Figure 15.3). It has recently been recognized that
much shorter "oligosialic acids" consisting of two or three units can exist on many other
glycoconjugates (Figure 15.3). The polysialic acid polymer can be also subjected to O-
acetylation at the 7- or 9-position. Polysialic acid structures based on Neu5Gc, Neu5Ac, or KDN
have been reported. The linkages between the sialic acid units can also vary. A bacteriophage
that attacks polysialic-acid-containing bacteria produces a highly specific endosialidase that has
proven to be a powerful tool for studying the biology of polysialic acid. The expression of
polysialic acid on N-CAM decreases markedly with development and is thought to play a part in
maintaining developmental plasticity, apparently by regulating both homotypic and heterotypic
interactions involving neuronal cells. In keeping with this notion, increases in polysialic acid
expression are correlated with "neural plasticity," i.e., neurite sprouting and other situations
involving neuronal damage repair or axon migration. Polysialic acids are generally primed on an
initiating α2 3-linked sialic acid.
Figure 15.3. Terminal sialic acids, oligosialic acids, polysialic acids, and the enzymes that can
degrade them. The arrows indicate typical cleavage points for the action of the enzymes.
Tissue-specific and Molecule-specific Expression of Sialic Acid
Linkages and Modifications (2 4, 7, 9, 20)
In a variety of systems where they have been studied, the various linkages and modifications of
sialic acids show tissue-specific and developmentally regulated expression. Some are even
molecule-specific, i.e., found only on certain types of glycoconjugates in a given cell type. Even
within a particular group of glycoconjugates, a modification may be restricted to certain sialic
acid residues at specific positions on a glycan. Such findings suggest highly specific roles for
these modifications in tissue development and/or organization. They also indicate the occurrence
of specific enzymatic mechanisms for their generation and regulation (see below).
Sialic acids seem to have appeared late in evolution, and with rare reported exceptions that
remain controversial, they are not generally found in plants, prokaryotes, or most invertebrates.
However, sialic acid has been reported in Drosophila embryos, and certain strains of bacteria
contain large amounts of sialic acids in their capsular polysaccharides. Since many of these
bacteria are pathogenic, the possibility of gene transfer from host eukaryotes was suggested, but
proof for this has not been forthcoming. Instead, it seems that the enzymes involved in
synthesizing bacterial sialic acids may have evolved independently, deriving from the gene
products that normally synthesize KDO, an acidic bacterial sugar that has some structural
similarities to sialic acids. There have also been occasional reports of sialic acids in insects other
than Drosophila.
With these notable exceptions, sialic acids have been found only in animals of the deuterostome
lineage, which comprises the vertebrates and some "higher" invertebrates (such as Echinoderms)
that emerged at the Cambrian expansion, approximately 550 million years ago. Early studies
suggested that there was species specificity in the different types of modifications of sialic acids.
However, with improvements in techniques for detection and analysis, it is evident that these
modifications are not species-specific but are simply expressed at differing levels of
detectability.
Biosynthesis and Turnover of Sialic Acids (4, 7, 9, 12,21 28)
Neu5Ac and KDN are believed to be the metabolic precursors for all other sialic acids. They are
derived by the condensation of ManNAc-6-P (for Neu5Ac) or Man-6-P (for KDN) with activated
forms of pyruvate. Following dephosphorylation, the free sialic acid is activated into the
nucleotide donor CMP-Sia (evidence indicates that this particular reaction takes place in the
nucleus, for unknown reasons). The CMP-Sias from the cytosol are finally pumped into the
lumen of Golgi compartments by the action of a specific antiporter (see Figure 15.4) that has
recently been cloned (see Chapter 6). The transfer of sialic acids from CMP donors to newly
synthesized glycoconjugates in the Golgi is catalyzed by a family of linkage-specific
sialyltransferases, many of which have been recently cloned and characterized (for their
nomenclature and specificity, see Chapter 17). As with most other glycosyltransferases, these
enzymes are type-2 membrane proteins with Golgi localization signals. Shared amino acid
sequence motifs (called sialyl motifs) were found in the first sialyltransferases cloned and were
then used to clone new members of the family (see Chapter 17). Recent work suggests that these
conserved regions represent the sugar-nucleotide-recognition sites.
Figure 15.4. General life cycle of sialic acids. The general pathways for biosynthesis, activation,
transfer, and eventual recycling of the common sialic acid N-acetylneuraminic acid are indicated.
The asterisks indicate the pathways considered to be the major sources of sialic acids for CMP-
Sia synthesis.
Once attached to glycoconjugates, sialic acids can eventually be removed at some point in the
life cycle of the molecule (Figure 15.4). In vertebrate systems, this occurs mainly in the acidic
compartments of the endosomal and lysosomal systems by the action of specific sialidases.
However, considerable evidence also exists for cell surface and cytosolic sialidases, as well as
for resialylation reactions involving molecules returning to the Golgi apparatus. Cell surface
sialidases are thought to be involved in the abrupt shedding of cell surface sialic acids that occurs
upon activation of some cell types (e.g., leukocytes). The functions of the cytosolic sialidases
remain obscure, since there is as yet no evidence for sialylated glycoconjugates in the cytosol nor
on the cytosolic leaflet of cellular membranes. Many microorganisms also express sialidases,
several of which have been cloned and characterized. Whereas the viral sialidases represent two
distinct families, the bacterial, fungal, and invertebrate enzymes are evolutionarily related to the
mammalian families (in this instance, horizontal gene transfer from animals to bacteria seems to
be likely). Most sialidases share a set of common "Asp boxes" (Ser-X-Asp-X-Gly-X-Thr-Tyr) of
as yet uncertain function. The three-dimensional structures of several viral and bacterial
sialidases have been elucidated, some in a complex with their substrates or with transition-state
analogs. A different type of sialidase is the "trans-sialidase" expressed by certain pathogenic
protozoa (e.g., trypanosomes). These novel enzymes remove sialic acids from mammalian cell
surfaces and transfer the sugar directly onto the parasite's own cell surface acceptors, apparently
giving protection from the host immune system (Chapter 36).
Once a sialic acid is released into the lysosome of a vertebrate cell, it is transported back into the
cytosol by a specific exporter (see Chapter 6). This allows sialic acids to be either reutilized or
degraded (Figure 15.4). In many cells, it appears that the bulk of the released sialic acids are
reutilized for new synthesis of CMP-Sias. When degradation does occur, it is catalyzed by sialic-
acid-specific pyruvate lyases that essentially cleave the molecule back into a N-acylhexosamine
and pyruvate. Similar pyruvate lyases exist in a variety of microorganisms.
Biosynthesis and Turnover of Sialic Acid Modifications (4, 7, 9, 12,21

23,29 31)
The different modifications of sialic acids are added to the parent molecule in defined
topological compartments. Most, if not all, of the synthesis of Neu5Gc is accounted for by the
conversion of CMP-Neu5Ac to CMP-Neu5Gc in the cytosol (Figure 15.5). The novel
hydroxylase responsible for this reaction has been cloned and found to be a cytosolic-iron-
dependent enzyme that utilizes the common electron transport chain of cytochrome b5 and b5
reductase. Once a Neu5Ac molecule has been converted into a Neu5Gc residue, there is no
obvious way to reverse the reaction, perhaps accounting for the accumulation of Neu5Gc in cells
that do express it (for the cellular pathways involving Neu5Gc, see Figure 15.5).
Figure 15.5. Biosynthesis and turnover of the N-acyl group of sialic acids. The general pathways
for biosynthesis, activation, and transfer of N-acetylneuraminic acid are shown. The step at
which the N-acetyl group can be hydroxylated is indicated.
In contrast to the conversion to Neu5Gc, which takes place at the nucleotide sugar level, the
addition of O-acetyl esters and other hydroxyl group modifications seems to occur within the
lumen of the Golgi apparatus or in Golgi-related organelles, after the transfer of sialic acids to
glycoconjugates. Among the O-acetyltransferases, there is evidence for distinct activities
involved in the O-acetylation of specific positions on sialic acids (e.g., 4-position vs. 9-position),
as well as enzymes specific for sialic acids on different classes of glycoconjugates (e.g.,
gangliosides vs. N-glycans). Unfortunately, the purification and cloning of these extremely labile
O-acetyltransferases has proven to be an intractable problem. Other types of substitutions of the
hydroxyl groups arise from utilization of the appropriate donors (e.g., S-adenosylmethionine for
methylated sialic acids, 3 -phosphoadenosine 5 -phosphosulfate for sulfated molecules). In some
cases such as the 9-O-lactyl group, it is difficult even to predict what the donor might be.
Appropriate enzymes should also exist to permit the turnover of each of these substitutions.
It is important to note that (with the exception of Neu5Gc) the other modified sialic acids studied
so far do not appear to be good substrates for activation in the CMP form for direct retransfer.
Thus, O-acetyl esters need to be removed at some point in the life cycle of the parent molecule,
either for terminal degradation or as part of an acetylation/deacetylation cycle. The best
interpretation of current data is that there are at least two 9-O-acetylesterases in mammalian
systems. One is a cytosolic enzyme that may serve to "recycle" O-acetylated sialic acids that are
exported from lysosomes into the cytosol. The other 9-O-acetylesterase is a glycoprotein with N-
glycans that traverses the ER-Golgi pathway and is targeted to lysosomal and endosomal
compartments. The cDNA for the latter enzyme has been cloned from the mouse. However, it
has a relatively high Km value for its substrate, and unlike classic lysosomal enzymes, it has a
neutral pH optimum. At present, it is not possible to reconcile these properties with a specific
role for this enzyme in the lysosomal turnover of O-acetylated sialic acids. Enzymes with sialic-
acid-specific 9-O-acetylesterase activity have also been reported from bacterial and viral sources.
The esterases from influenza C and coronaviruses are better characterized and seem to act as
receptor-destroying activities that are incorporated into the hemagglutinin molecule of the virus.
Notably, all of these esterases are specific for esters at the 9-position and are incapable of
releasing O-acetyl esters at the 7-position. However, 7-O-acetyl groups can migrate to the 9-
position and thus become substrates for these enzymes.
Since the de-N-acetylated form of Neu5Ac (neuraminic acid, Neu) is very unstable in the free
state, it had been assumed that it did not exist in nature. However, the glycosidically bound form
of neuraminic acid is at least as stable as the N-acetylated compound. Several groups now have
indirect evidence to indicate that small amounts of de-N-acetylated sialic acids do exist in nature
(presumably from the action of a specific de-N-acetylase) and that these molecules can be later
re-N-acetylated. The search is under way for the appropriate enzymes that remove and add back
the N-acetyl groups.
Various dehydrated or unsaturated sialic acids also occur in nature and appear to arise during
enzymatic or chemical degradation processes. These include 2,7-anhydro sialic acids released
following cleavage of bound sialic acids by certain unusual sialidases, the 2,3-didehydro 2,6-
anhydro compounds resulting from mild alkali-catalyzed breakdown of CMP-Sias, and 4,8-
anhydro compounds formed during release or deacetylation of 4-O-acetylated compounds.
Although many of these compounds have been detected in free form in biological fluids, it is not
clear if they arise from enzymatically catalyzed reactions or from spontaneous chemical
processes occurring at a slow rate under physiological conditions. Their biological significance is
also not known.
Methods for Studying Sialic Acids: Diversity Can Be Missed (4,

7,32 34)
Prior to accurate analysis, sialic acids from biological sources must be completely released and
purified, with their modifications intact. Once released and purified, sialic acids can be analyzed
by colorimetry, TLC, GLC, and GLC/MS, NMR, or mass spectrometry. The technique of
derivatization with 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB) followed
by HPLC analysis with fluorescent detection has proven to be particularly sensitive, specific, and
applicable to most sialic acids. The adaptation of this technique to on-line mass spectrometry has
been a powerful enhancement. Several techniques have also been developed for the detailed
analysis of substitutions on metabolically labeled sialic acids. Monoclonal antibodies and lectins
have also been used to identify O-acetylated molecules. A recombinant soluble form of the 9-O-
acetyl-specific hemagglutinin of influenza C virus has been successfully used to probe for such
molecules on cells and tissues.
Many studies of sialoglycoconjugates fail to take into account sialic acid complexity. The
reasons are mainly technical. Some substitutions are labile and can alter the behavior of sialic
acids during release, purification, and analysis. In addition, substitutions can slow down or even
completely prevent release of sialic acids by commonly used sialidases or by acid hydrolysis. On
the other hand, when stronger acidic conditions are used, destruction of some substitutions
occurs. Furthermore, many methods used in the structural analysis of intact glycans cause
destruction of sialic acid modifications. Thus, conventional approaches to the study of sialic
acids from biological sources could easily miss a significant amount of such modifications.
These substitutions can affect the size, shape, hydrophilicity, net charge, and biological
properties of the molecule. Thus, a careful analysis for their presence is worthwhile in situations
where sialic acids are thought to have biological roles. With regard to the O-acetylation of the
side chain, chemical and enzymatic improvements now allow near-quantitative release and
purification of such molecules, without loss or migration of the ester groups. With rarer
molecules such as O-methylated or sulfated sialic acids, much less is known about their
susceptibility to sialidases or their optimal release with acid, and other methods for their direct
detection are not available. It is evident that much needs to be done to improve methods for the
release and purification of sialic acids from biological sources.
Sialic-Acid-binding Lectins (12 13,35 38)
Because of their terminal location and negative charge, sialic acids have the potential to inhibit
many intermolecular and intercellular interactions. As discussed above, such inhibition can be of
biological relevance, as in the case of the polysialic acid chains on N-CAM. In contrast to these
roles, sialic acids can also be critical components of ligands for recognition by specific lectins.
Table 15.1 lists examples of such proteins from a wide variety of animal, plant, and microbial
origins (see also Chapters 24, 26, 28, and 30). Some of these lectins were first discovered by
virtue of their ability to agglutinate red blood cells in vitro and by the loss of this
hemagglutination upon sialidase treatment of the cells. With others, the discovery occurred
during the investigation of cell-cell interaction phenomena and the finding that binding was
sensitive to sialidase treatments. In recent times, such lectins have been found purely by virtue of
their sequence homology with other known lectins. The three-dimensional structures of a few of
these lectins have also been elucidated, sometimes in a complex with a cognate sialylated
oligosaccharide.
In most instances studied, the negatively charged carboxylate group at the C-1 position has
proven to be critical for recognition. The role of divalent cations and the underlying
oligosaccharide can vary from being absolutely required to being nonessential. The linkage of
the sialic acid is recognized specifically by most of the lectins, sometimes in the context of the
underlying sugar chain (for some examples, see Figure 15.6 ). As the figure demonstrates, this
selectivity in recognition can provide a biological readout for the complex pathways of terminal
Golgi glycosylation that often terminate in sialylation. The structural diversity in the sialic acids
also affects recognition by these lectins. The role of various linkages and substitutions is highly
variable, ranging from being completely unimportant to being crucial for recognition. Various
combinations of treatments with sialidases, 9-O-acetylesterases, and mild periodate oxidation
can be used to explore this matter. Table 15.2 summarizes some examples where published
information is available.
Figure 15.6. Terminal oligosaccharide sequences recognized by some sialic-acid-binding lectins.
GlcNAc or GalNAc residues on glycoproteins and/or glycolipids can be extended by several
biosynthetic pathways, some examples of which are indicated. The sialylated sequences shown
as being recognized are based on published literature and/or reasonable predictions based on
known specificities. The sequences shown are the minimal structural motifs necessary for
binding: Natural high-affinity ligands may be more complex. Recognition can also be affected
by modifications of sialic acid (for examples, see Table 15.2 ). In the case of influenza A
hemagglutinin, relative preference for α2 3 and α2 6 linkages can vary with different viral
strains. Some of the key enzymes involved in the final biosynthetic steps are shown: ST3Gal-I =
Core 1: α2 3Sialyltransferase; ST3Gal-II = Gal: α2 3Sialyltransferase; ST3Gal-III = LacNAc:
α2 3Sialyltransferase; ST3Gal-IV = Gal: α2 3Sialyltransferase; ST6GalNAc-I = Core 1 α2
6Sialyltransferase; ST6Gal-I = LacNAc: α2 6Sialyltransferase; and OAcT = Sialate: O-
acetyltransferase. For other abbreviations, see Tables 15.1 and 15.2. (Reprinted, with permission,
from [13] Varki 1997.)
Table 15.1. Examples of naturally occurring sialic-acid-binding lectins
Vertebrate
C-type: Selectins (see Chapter 26)
I-type: Siglecs (see Chapter 24)
Unclassified: Complement factor H, laminin
Arthropod
Crab lectins: Limulin (American horseshoe crab, Limulus polyphemus)
Lobster and prawn lectins: l-agglutinin (lobster, Homarus americanus)
Scorpion lectins: Whip scorpion lectin (Masticoproctus giganteus)
Other insect lectins: Allo A-II (beetle lectin, Allomyrina dichotoma)
Mollusc
Slug and snail lectins: Limax flavus agglutinin (LFA, slug, Limax flavus)
Mussel and oyster lectins: Pacific oyster lectin (Crassostrea gigas)
Protozoal
Parasite lectins: Merozoite erythrocyte-binding antigen (Plasmodium falciparum)
Plant
SN agglutinin (elderberry bark lectin, Sambucus nigra), TJ agglutinin (Tricosanthes
japonicum), MA agglutinin (Maackia amurensis), wheat-germ agglutinin (Triticum vulgaris)
Bacterial
Bacterial adhesins: S-adhesin (Escherichia coli K99), adhesin I and adhesin II (Helicobacter
pylori)
Bacterial toxins: Cholera toxin (Vibrio cholerae), tetanus toxin (Clostridium tetani), botulinum
toxin (Clostridium botulinum), pertussis toxin (Bordetella pertussis)
Mycoplasma lectins: Mycoplasma pneumoniae hemagglutinin
Viral
Hemagglutinins: Influenza A and B viruses, Primate polyomaviruses, Rotaviruses
Hemagglutinin-neuraminidases: New Castle disease virus, Sendai virus, fowl plague virus
Hemagglutinin esterases: Influenza C viruses, human and bovine coronaviruses
Table 15.2. Structural requirements for recognition by some sialic-acid-
binding lectins
Preferences/requirements for recognition
Lectin name (see also Table sialic acid carboxylate 5-acyl side 9-O- 4-O-
15.1) linkage group chain acetyl acetyl
Selectins α2 3 yes no no
CD22 α2 6 yes Ac,Gc yes blocks
Sialoadhesin (Sn) α2 3 yes Ac yes blocks
Myelin-associated α2 3 yes Ac yes blocks
glycoprotein (MAG)
CD33 α2 3 yes yes
Complement factor H all? yes yes blocks
Limulus polyphemus lectin all? Ac=Gc no
(limulin)
Limax flavus agglutinin all yes Ac yes blocks accepts
(LFA)
Plasmodium falciparum α2 3 blocks
merozoite lectin
Sambucus nigra agglutinin α2 6 yes yes blocks
(SNA)
Tricosanthes japonicum α2 6 yes
agglutinin (TJA)
Maackia amurensis α2 3 Gc/Ac no
agglutinin (MAA)
E. coli K99 S-adhesin α2 3 Gc
Vibrio cholerae toxin (B α2 3
subunit)
Influenza A hemagglutinin varies yes blocks
(InfAHA)
Influenza C hemagglutinin all yes
esterase (InfCHE)
The data presented are based on published literature and some unpublished observations. In
some cases, the data represent reasonable assumptions based on prior precedent in very similar
situations. The preference for the underlying saccharide by some lectins is not shown here (for
examples, see Figure 15.6).
Functions and Uses of Sialic-Acid-binding Lectins (12 13,35 37,39 42)
The first mammalian sialic-acid-binding protein reported was the complement regulatory factor
H, a soluble serum factor that binds to surfaces via the intact exocyclic (C7-C8-C9) side chain of
sialic acids and restricts alternative pathway activation. The addition of a 9-O-acetyl group to the
side chain of cell surface sialic acids (or the oxidation of the unsubstituted side chain with mild
periodate) blocks the binding of factor H and abrogates its function as a negative regulator of the
alternative pathway. The biological roles of the other vertebrate sialic-acid-binding lectins are
discussed elsewhere, including the selectins (see Chapter 26) and the Siglec subset of I-type
lectins (see Chapter 24). Very recently, the important interaction between β-dystroglycan and
laminin in muscle has been suggested to involve a sialic-acid-binding site on the latter and a
novel sialylated O-Man-linked glycan on the former. The cell-type-specific expression of the
Siglecs and of the sialyltransferases that generate their cognate ligands has raised expectations
that they are involved in highly specific biological roles. Indeed, data to date indicate that CD22
may be involved in interactions with the tyrosine phosphatase CD45, sialoadhesin may mediate
macrophage interactions with developing myeloid precursors, and myelin-associated
glycoprotein may interact with specific neuronal gangliosides to maintain the integrity and
function of myelin. Analysis of these types of functions is complicated by the fact that the
cognate oligosaccharide sequences for some of the lectins are found on a wide variety of
glycoconjugates. Thus, these lectins must function by specifically recognizing a few high-
affinity ligands in the midst of a milieu of low-affinity inhibitors. Further confusion arises
because some of these lectins can become fully occupied by binding to sialylated ligands present
on the same cell surface as the lectin itself. Interestingly, activation of cells can result in
spontaneous exposure of these "masked" binding sites.
A large number of microbial-host interactions are dependent on recognition of sialylated ligands

(see Table 15.1 and Chapters 9 and 28). Examples of medical relevance include the recognition
of sialic acids by influenza viruses, the binding of Helicobacter pylori (the cause of peptic ulcer
disease) to gastric mucins and glycosphingolipids via at least two different sialic-acid-dependent
mechanisms, the interaction of various pathogenic microbial toxins to mammalian cells, and the
binding of the merozoite stage of the malarial parasite Plasmodium falciparum to erythrocytes.
The interactions of some microbial lectins with sialic acids can be abolished by substitutions
such as 9-O-acetylation, which can be found on mammalian mucosal surfaces. Thus, it has been
suggested that such modifications serve a specific protective role in this location. Indeed, it is
possible that some of the complexities of sialic acid diversification are the outcome of the
ongoing "arms race" between animals and microbial pathogens (see Chapter 3). On the other
hand, the modified sialic acids in some internal organs and tissues must have critical structural
roles and/or be required for recognition by endogenous lectins that are yet to be discovered.
Some sialic-acid-binding lectins are found in organisms that are not themselves known to
express sialic acids (lower invertebrates, plants, and insects). One explanation is that their
primary function is in defense against exogenous sialylated pathogens. In keeping with this,
limulin, which is found in the hemolymph of the horseshoe crab, can mediate foreign cell
hemolysis. Another possibility is that the sialic-acid-binding properties are serendipitous and that
the real ligands are other anionic carbohydrates that are yet to be identified. Regardless of what
their natural ligands are, some of these lectins have proven to be powerful tools for studying the
biology of sialic acids. For example, wheat-germ agglutinin and Limax flavus agglutinin have
been used as general tools to bind sialylated glycoconjugates, and combinations of Sambucus
nigra agglutinin, Tricosanthes japonicum agglutinin, and Maackia amurensis agglutinin can
distinguish between different types of sialic acid linkages on terminal lactosamines (recombinant
soluble forms of the Siglecs such as CD22 and sialoadhesin can also be used for this purpose). A
recombinant soluble form of the influenza C hemagglutinin esterase can specifically probe for 9-
O-acetylated sialic acids. Of course, in all situations in which a lectin is used as a detection tool,
the absence of binding does not necessarily imply the absence of the cognate glycan structure.
There are many other indirect clues to other biological roles of sialic acids. Certain classes of
lymphocytes have O-acetylated sialic acids, whereas others do not, and the T cells of patients
with various malignancies have been reported to acquire increased O-acetylation. The expression
of polysialylation and O-acetylation in neural gangliosides varies with developmental stage and
location, and differences in O-acetylation of brain gangliosides have been reported between cold-
blooded and warm-blooded species and between awake and hibernating animals. Developmental
up-regulation of O-acetylation in the gut mucosa may appear in response to microbial
colonization and has been suggested to have a role in protecting against certain microorganisms.
Likewise, O-acetylation of sialic acids on murine erythrocytes appears to confer resistance to the
binding of the malarial parasite. Expression of O-acetyl and N-glycolyl groups on cell surfaces
can also limit the action of bacterial sialidases and block the binding of some pathogenic viruses.
With regard to the 2,3-didehydro 2,6-anhydro sialic acids found in biological fluids, it has been
hypothesized that they provide protection by virtue of their powerful inhibition of microbial
sialidases. Overall, although the data are supportive, no conclusive proof yet exists that sialic
acids or their modifications provide crucial protection from pathogens.
Sialic Acid Modifications in Development and Malignancy (31, 43, 44)

Transformation and malignant progression are accompanied by striking changes in the quantity,
linkage, and types of sialic acids on tumor cell surfaces (see Chapter 35). In general, the amount
of sialic acid goes up and switches occur to different linkages (α2 6 linkages become
particularly prominent). O-acetylation at the 9-position can either disappear (as occurs in colon
carcinomas) or appear (as in 9-O-acetyl-GD3 which appears in melanomas). Interestingly, in both
cases, the change represents a reversal to the embryonic state.
Neu5Gc can also be an onco-fetal antigen, specifically in humans and chickens. Although it is
thought to be expressed in fetal human tissue and in certain human tumors and human tumor cell
lines, it is not found in normal adult human tissues. In fact, molecules containing large amounts
of Neu5Gc are immunogenic in humans. Thus, for example, upon exposure to horse serum, a
major epitope recognized in the resulting "serum sickness" reaction is Neu5Gc. Spontaneously
occurring Hanganutziu-Deicher antibodies to Neu5Gc also occur in patients with cancer and
with certain infectious diseases, as well as in chickens with Marek's disease, a malignant
herpesvirus infection. In humans, the explanation for these findings is an exon deletion in the
CMP-Sia hydroxylase that occurred after our last common ancestor with the African great apes.
Thus, the reexpression of this sialic acid reported in some disease states such as cancer may be
mediated by an alternate pathway or derived from food sources. The presence of this substitution
can certainly change recognition by a variety of lectins, including endogenous lectins such as the
Siglecs (CD22, myelin-associated glycoprotein, and sialoadhesin). It can also affect the binding
of microbes such as influenza and Escherichia coli K99. A particularly intriguing observation is
the suppression of expression of Neu5Gc in the brains of all animals studied, including those that
have high levels expressed in other tissues.
Future Directions
Cultured cell lines exist that are grossly deficient in sialic acid addition to glycans. Thus, the
more important biological roles of sialic acids may only be evident when studied in intact,
complex mammalian systems. Naturally occurring genetic defects in the export of sialic acids
from lysosomes and in the failure of feedback regulation of production of sialic acid have been
reported. However, apart from the human loss of Neu5Gc production, genetic defects in sialic
acid modification have not been discovered. Thus, to better understand the biological functions
of sialic acids, it may be necessary to create mutants in sialic acids and their modifications in
intact higher animals. In this regard, transgenic mice expressing the coat protein from influenza
C virus consistently arrested development at the two-cell stage, suggesting that O-acetylated
sialic acids might be involved in embryo segmentation. Late expression in specific organs caused
developmental abnormalities. The functions of sialic acids are also being elucidated by the
genetic ablation of sialyltransferases in the intact mouse (see Chapters 32 and 33). It is clear that
a great deal remains to be done in the study of the structure, biosynthesis, and regulation of sialic
acids and their modifications. Further improvements in analytical methods are needed. The many
clues to the biological roles of these molecules must be explored, and new ones must be actively
sought. The genetic manipulation of sialic acids and their modifications in intact animals is likely
to yield the most compelling data.
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16. Structures Common to Different Types of Glycans
Primary contributions to this chapter were made by J.B. Lowe (HHMI, University of Michigan
Medical School, Ann Arbor) and J.D. Marth (HHMI, University of California at San Diego).
THIS CHAPTER DESCRIBES GLYCANS that are found attached to the various core
components of N-glycans, O-glycans, glycolipids, and proteoglycans. Although these glycan
structures can be unique to various core subtypes, many are found on more than one class of
glycan. In postcore glycan biosynthesis, subterminal and terminal monosaccharide linkages
existing at "outer" positions can establish the function(s) of the glycoconjugate.
Background
Glycan chain modifications found at outer or terminal positions generally result from the actions
of one or more glycosyltransferases that modify glycan core acceptors, or precursors. Such
acceptors are the product of biosynthetic processes discussed in Chapters 7, 8, and 9 and
correspond to N-linked, O-linked, or lipid-linked structures shown in Figures 16.1 through 16.3.
Most precursors are typically expressed by all cell types in a mammalian organism. Such
structures include the multiantennary N-linked glycan precursor, the linear (unbranched) or
biantennary O-linked glycan precursors, and linear (unbranched) ceramide-linked precursors. As
noted for N-linked glycans in Chapter 7, but also for O-linked and lipid-linked glycans, other
more complicated precursors can result from tissue- or cell-type-specific biosynthetic processes
that yield, for example, tetra-antennary N-glycans (Figure 16.1). These more elaborate
precursors are generally susceptible to the modification processes discussed here and in Chapter
17, leading to structural diversification at the termini of glycan chains.
Figure 16.1. N-glycan synthesis leads to "core" structures (theoretical multi-antennary structures
in these examples; see Chapter 7 for details) that may be modified subsequently on specific
GlcNAc residues by glycosylation reactions that may be tissue-specific, developmentally
regulated, or even protein-specific.
Figure 16.2. O-glycan synthesis leads to "core" structures (a "Core 2" structure, in this example;
see Chapter 8 for details) that may be modified subsequently on specific residues by
glycosylation reactions that may be tissue-specific, developmentally regulated, or protein-
specific. In some cases, such modifications are catalyzed by the same enzymes that modify N-
linked glycans.
Figure 16.3. Glycolipid synthesis leads to "core" structures (a neolactosylceramide structure, in

this example; see Chapter 9 for details) that may be modified on specific GlcNAc residues by
tissue-specific or developmentally regulated glycosylation reactions. In some cases, such
modifications are catalyzed by the same enzymes that modify N-linked and O-linked glycans.
Regulated Glycosylation of Constitutively Expressed Precursors

to Terminal Chain Structures (1 7)
As implied by Figures 16.1 through 16.3, regulated biosynthetic processes determine the precise
nature of the terminal modifications of such precursors. In contrast to precursor chain synthesis,
these processes are generally regulated in a tissue- or cell-lineage-specific manner. Many
terminal glycosylation reactions are regulated during embryogenesis and in the postnatal period
as part of the normal developmental program (see Chapter 34). Changes in terminal glycan
structures have also been observed in association with malignant transformation (see Chapter
35). Functional correlates for a number of such changes in outer chain structures have been
identified and are discussed throughout this book. Most researchers in the field will agree that
the majority of regulated changes observed in terminal glycosylation do not yet have a defined
functional correlate. As discussed in Chapter 17, the tissue- and/or lineage-specific regulation of
outer chain biosynthesis is largely a function of careful regulation of expression of the
glycosyltransferases responsible for outer chain modifications. Generally speaking, the specific
portfolio of terminally acting glycosyltransferases expressed in any given cell type will
determine the nature of the outer chain structures expressed by that cell type.
Type-2 Chains (1 3,8 12)
The core precursors representative of N-linked, O-linked, and lipid-linked glycans are typically
modified with GlcNAc residues (see Figures 16.1 through 16.3). With a few exceptions to be
noted later in this chapter, virtually all subsequent modifications characterized as terminal first
require modification of one or more of these GlcNAc residues by the addition of a galactose
moiety in β1 4 linkage. The resulting structure has been termed a "type-2" chain; its component
disaccharide is the lactosamine unit (Figure 16.4). Expression of this structure and the
corresponding β1 4 galactosyltransferase(s) can therefore be considered to be rather
constitutive, in contrast to most other outer chain modifications discussed here. As also
illustrated in this chapter, lactosamine-terminated structures on N-linked, O-linked, and lipid-
linked glycans serve as acceptor substrates for a large variety of subsequent modifications to the
now subterminal GlcNAc, as well as to the terminal galactose moiety itself. An alternative to
type-2 lactosamine production occurs when the "lacdiNAc" glycan structure is generated by the
action of a β1 4 GalNAc transferase (GalNAcβ1 4GlcNAc; not shown, see discussion below).
Aside from its role as a precursor to subsequent outer chain glycosylations, the lactosamine unit
is known to be recognized by the S-type lectins (see Chapter 27). The function of such
interactions in the context of the intact mammalian organism remains open to conjecture. As
related in Chapter 33, efforts have been made to uncover the biological role(s) for type-2 chains
through the generation and analysis of mice deficient in a β1 4 galactosyltransferase locus
thought to be solely responsible for type-2 chain biosynthesis. However, it is now apparent that
several different β1 4 galactosyltransferase loci can contribute to β1 4 galactosylation and type-
2 chain expression, which may act to distinguish the functions of this structure among various
physiologic systems of whole animals.
Figure 16.4. Modification of exposed GlcNAc moieties by galactosylation. Modification by β1

4-linked galactose residues (top) is generally found in all mammalian tissues. This reaction is
catalyzed by β1 4 galactosyltransferases(s) and yields the Galβ1 4GlcNAc unit (lactosamine
unit) that defines type-2 chains. Modification of such precursors by β1 3-linked galactose
residues (bottom) is restricted to certain tissues (see text). This reaction is catalyzed by β1 3
galactosyltransferases(s) and yields the Galβ1 3GlcNAc unit (neolactosamine unit) that defines
type-1 chains. Substrates for these processes include some N-linked, O-linked, or glycolipid
glycan precursors (indicated by R) that display terminal GlcNAc residues. The GlcNAc residues
and the galactose residues, on both type-2 and type-1 chains, are subject to further modification
by subsequent glycosylation reactions.
Type-1 Chains (4, 13, 14)

The GlcNAc residues that terminate N-linked, O-linked, and lipid-linked glycan core precursors
may also be modified by galactose in β1 3 linkage (Figure 16.4). The resulting structure has
been termed a "type-1" chain; its component disaccharide is the neolactosamine unit. In humans,
expression of type-1 chains is mostly restricted to epithelia, in the gastrointestinal or
reproductive tracts, for example. Such chains represent precursors to a variety of terminal
structures, through modification by glycosyltransferases that can utilize structures with the
Galβ1 3GlcNAc linkage and that modify either the terminal galactose moiety, or the now
subterminal GlcNAc moiety. As discussed below, these include sialylated structures, and blood-
group-active structures generated by fucosylation and by modification with terminal galactose or
GlcNAc moieties. Expression of the type-1 chains, and the corresponding β1 3
galactosyltransferase, represents examples of structures and enzymes that are regulated in a
tissue-specific manner, as is the case for many outer chain modifications. Although the type-1
structure functions as a precursor to subsequent outer chain glycosylations, specific roles for this
disaccharide have not been clearly defined.
Polylactosamines (15 28)
Glycoproteins and glycolipids frequently bear glycans that include linear polymers of the type-2
lactosamine unit. These structures are termed polylactosamines. Polylactosamine biosynthesis is
directed by the alternative actions of one or more β1 4 galactosyltransferases and one or more
β1 3 GlcNAcT (Figure 16.5). They have been described on a variety of glycoproteins and may
be components of N-glycans, O-glycans, and glycolipids. It is clear that some glycoproteins or
glycolipids are preferentially modified by polylactosamine chains, relative to others. These
observations imply that the enzymatic machinery responsible for polylactosamine biosynthesis is
capable of discriminating between distinct glycoprotein or glycolipid molecules that are
otherwise identical with respect to the exposed GlcNAc and galactose residues that serve as
proximal modification points for polylactosamine chain biosynthesis. Polylactosamines have
been noted to preferentially reside on multiantennary N-glycans, especially the β1 6 branch,
whose synthesis is under control of GlcNAcT-V (Figure 16.6). Similarly, polylactosamine chains
on O-chains associated with mucin-type glycoproteins are often preferentially displayed on the
β1 6 branch whose synthesis is directed by a Core 2 β1 6 GlcNAcT (Figure 16.6).
Polylactosamine length is also under apparent regulatory control, with N-linked
polylactosamines generally found to be longer than O-linked chains. Chapter 17 discusses the
mechanisms that might account for these regulatory events. As will be noted, polylactosamines
serve as precursors for subsequent modifications, such as fucosylation and sialylation. The linear
nature of these chains, and the hydrophilic character of their component disaccharide unit,
predicts that they will maintain an extended linear conformation. This implies that
polylactosamines may serve as scaffolds for the presentation of specific terminal glycan
structures, including sialylated and/or fucosylated structures, whose functions require them to be
displayed at or above the surface of the cell's glycocalyx. Polylactosamines are recognized with
high affinity by mammalian S-type lectins (see Chapter 27), in a way that implies recognition of
internal structures, independent of the presence of terminal galactosyl moieties. Chapter 27
discussed the physiological relevance of interactions between polylactosamines and S-type
lectins.
Figure 16.5. Polylactosamine synthesis.
Figure 16.6. Polylactosamine chains.
β1 6GlcNAc Branching Structures (29 33)
Polylactosamines are subject to further glycosyltransferase-dependent arborizing modifications.

Such branching modifications include addition of N-acetylglucosamine to internal galactose
residues, in β1 6 linkage, through the actions of β1 6 GlcNAcTs (Figure 16.7). Branched and
nonbranched polylactosamines correspond to the antigens of the Ii-blood group system. The I-
and i-blood group systems were discovered through an investigation of a cold-dependent
agglutinating antibody (cold agglutinin) in a patient with acquired hemolytic anemia. The
relationship between cold agglutinin disease, anti-I or anti-i antibodies, and infectious agents is
discussed in more detail in Chapter 37. In the instance of the anti-I antibody relevant to the
discovery of the Ii-blood group system, the antibody reacted with the red cells of all but rare
potential red cell donors. Nonreactive donors were classified as having the i-blood group,
whereas reactive donors were assigned to the I-blood group. β1 6-branched polylactosamine
structures correspond to the I-blood group antigen, a precursor to the ABO blood group antigens
on erythrocytes, whereas linear polylactosamine chains correspond to the so-called i-blood group
antigen. Acceptor site specificity studies indicate that two distinct β1 6 GlcNAcTs yield
different types of β1 6-branched products, starting with linear polylactosamine precursors
(Figure 16.7).
Figure 16.7. Polylactosamine-derived β1 6 GlcNAc branching structures associated with I-
antigen expression. Linear polylactosamine chains, displayed by asparagine-linked,
threonine/serine-linked or lipid-linked moieties (R) (see Figure 16.6), may be modified by at
least two β1 6 N-acetylglucosaminyltransferases (β1 6 GlcNAcTs). One such β1 6 GlcNAcT
(often termed cIGnT6) adds N-acetylglucosamine in β1 6 linkage to internal galactose residues,
as shown in the figure. A second distinct β1 6 GlcNAcT (often termed dlGnT6) requires an
exposed terminal N-acetylglucosamine residue on the polylactosamine chain for activity
(reaction not shown). The newly added β1 6-linked N-acetylglucosamine residues may then
serve as substrates for additional polylactosamine chain biosynthesis (as in Figure 16.5;
alternatively modified by β1 4 galactosyltransferase and β1 3 N-acetylglucosaminyltransferase
activities), yielding branched polylactosamine chains with I-antigen reactivity.
Branching I- and i-reactive glycan chains are expressed by many human cells and tissues. The i-
antigen is abundantly expressed on the surface of red cells taken from the human embryo,
erythrocytes in cord blood, or during times of altered erythropoiesis. Such cells are relatively
deficient in expression of I-antigen. However, during the first 18 months of life, I-antigen
reactivity on red cells reaches adult levels, and i-antigen reactivity declines to very low levels.
This developmentally regulated pattern is presumed to be consequent to developmental
regulation of a cognate I β1 6 GlcNAcT locus. Rare individuals have been described who never
express the I-antigen on their red cells, but who maintain levels of red cell i-antigen expression.
It is not known if other tissues in such individuals are also deficient in I-antigen. Persons with
this phenotype (i-phenotype) are presumed to be homozygous for null alleles at the β1 6
GlcNAcT locus responsible for synthesis of the β1 6-branching structures associated with I-
antigen expression. However, the molecular basis for this rare phenotype and the identity of the
relevant β1 6 GlcNAcT, among those currently identified by molecular cloning studies, remain
to be defined. Individuals with the i-phenotype have no obvious pathophysiological phenotype
associated with absence of red cell I-antigen expression.
The A, B, and H Blood Group Structures (34 71)
In humans, linear polylactosamines and their β1 6-branched variants are subject to tissue-
specific modifications that form glycans of the ABO blood group system (Figures 16.8 to 16.11).
This system was discovered early in the 20th century by Landsteiner and colleagues. Although
they were not aware of the underlying glycan basis, their work revealed that humans could be
divided into different classes according to the presence or absence of serum constituents that
would agglutinate red cells isolated from other humans. We now know that these serum
constituents are antibodies and that their cognate antigens correspond to glycans whose
structures are genetically polymorphic. These glycan structural polymorphisms are determined
by allelic glycosyltransferases with different functional properties.
Figure 16.8. Type-1 A, B, and O(H) blood group structures.
Figure 16.9. Type-2 A, B, and H blood group structures.

A, B, and O(H) blood group antigens are oligosaccharide moieties based on the type-1 and type-
2 precursors described above and on so-called type-3 and type-4 glycan precursors (Figures 16.8
to 16.11). The H blood group structure, in its unmodified form, results in the O blood type
grouping (see below). The A, B, and H antigens are formed on these precursors by the sequential
action of distinct glycosyltransferases, encoded by three genetic loci (the ABO, H, and Secretor
loci) (Figure 16.12). The pathway of ABO blood group antigen synthesis begins with the
modification of precursor glycans by α1 2 fucosyltransferases. These enzymes form the blood
group H determinant, represented by the disaccharide unit Fucα1 2Galβ1-. The human genome
encodes two different α1 2 fucosyltransferases, corresponding to the products of the H and the
Secretor (Se) blood group loci. The H-α1 2 fucosyltransferase is expressed in erythrocyte
precursors and utilizes type-2 and type-4 precursors to form type-2 and type-4 H antigens on red
cells (Figures 16.9, 16.11, and 16.12). The Seα1 2 fucosyltransferase is expressed in epithelial
cells and utilizes type-1 and type-3 precursors to form type-1 and type-3 H determinants (Figures
16.8, 16.10, and 16.12) in epithelia lining the lumen of the gastrointestinal, respiratory, and
reproductive tracts and salivary glands, for example.
Figure 16.12. Summary of A, B, and O(H) blood group structures and their synthesis.
A or B blood group determinants are subsequently formed from type-1, -2, -3, or -4 H-active
glycans by codominant glycosyltransferases encoded by the ABO blood group locus. The blood
group A molecule is formed by an α1 3 GalNAcT corresponding to the A allele of the ABO
locus (Figure 16.12). The blood group B allele encodes an α1 3 galactosyltransferase that forms
the blood group B determinant (Figure 16.12). O alleles encode functionally inert polypeptides
that do not further modify H-active precursors and therefore represent null alleles at this locus.
Thus, individuals capable of constructing A molecules exclusively (blood group A) will have
genotypes AA or AO, whereas blood group B individuals will have the genotypes BB or BO.
Individuals capable of expressing both A and B antigens (AB blood group) maintain the
genotype AB. Blood group O individuals do not express either A or B antigen, leaving their H
antigens unmodified, and are homozygous for the (null) O allele (genotype OO).
The ABO antigens are expressed on the surfaces of red cells and many other tissues, including
the vascular endothelium, and by a variety of epithelia. In this form, ABO blood group
molecules are displayed by integral membrane proteins and membrane-associated glycolipids.
Some tissues also synthesize water-soluble forms of these molecules, as glycans on secreted
glycoproteins, on glycosphingolipids, and on free oligosaccharides. As discussed below, the
ability to express soluble ABH-active blood group molecules is a genetically determined trait
that is a function of an individual's alleles at the Se locus.
On each human red blood cell, approximately 1 2 million ABH determinants are displayed by
the anion transport protein, also known as Band 3. This represents approximately 80% of the
total complement of red cell ABH determinants. Another 5 × 105 ABH determinants localize to
the red cell glucose transport protein (Band 4.5). Both of these integral membrane proteins
display ABH antigens on a single N-linked, branched polylactosamine whose terminal branches
can display several ABH determinants. Small numbers of ABH antigens are also expressed by
other red cell glycoproteins. Each red cell also expresses approximately 5×105 glycolipid-based
ABH determinants. Many of these glycolipids correspond to A, B, and H antigen-modified
polylactosamines linked to ceramide and have been termed polyglycosylceramides or
macroglycolipids. A, B, and H determinants based on type-4 chains (Figure 16.11) are also
represented in human red cell glycolipids.
ABH determinants expressed by the epidermis are primarily constructed from type-2 precursor
chains. Mucins derived from the gastric mucosa and from ovarian cyst fluid express type-3 A, B,
and H antigens. As noted above, the epithelia lining the digestive, respiratory, urinary, and
reproductive tracts express type-1 oligosaccharides, as do the epithelia of some salivary and
other exocrine glands. These tissues are responsible for synthesis of soluble forms of the ABH
determinants, which are therefore largely represented by type-1 molecules. Expression of the A,
B, and H determinants in such secretory tissues is a function of the Se locus-encoded α1 2
fucosyltransferase, since the H locus-encoded α1 2 fucosyltransferase is not expressed in these
tissues. Humans homozygous for null alleles at the Se locus are incapable of synthesizing H
determinants in these tissues and do not elaborate soluble forms of the H (or A or B)
determinants in saliva or in other tissues, even though such tissues in these individuals continue
to synthesize the soluble precursors used by the Se locus-encoded α1 2 fucosyltransferase. The
term nonsecretor is used to describe the phenotype of such persons and refers to the fact that
soluble blood group H, A, and B substances cannot be detected in their saliva.
Serological procedures used in the context of characterizing red cells for use in transfusion have
identified variants of A and B blood group determinants. These are known as A or B subgroups.
The A and B antigens of these subgroups are close structural variants of the A and B antigens,
but they typically yield weak reactivity with serological typing reagents used to characterize A
and B antigens on red cells. For example, the lectin Dolichos biflorus will agglutinate the red
cells from most blood group A individuals (known as A1 individuals), but it will not agglutinate
red cells from the rather less common A2 subgroup individuals. A antibodies may also be
prepared that react with A1, but not A2, cells, for example.
The biochemical basis for the serological discrepancies between A1 and A2 cells is partly
accounted for by the greater number of A-active molecules on A1 cells, relative to A2 cells. The
molecular structures of A1 and A2 subgroup antigens are also distinct (Figure 16.13). The
structural differences between A1 and A2 cells are accounted for by differences in the catalytic
activities of the allelic blood group A transferases corresponding to A1 and A2 alleles, with the
A1 transferase exhibiting a higher specific activity in vitro. It is clear that the variant antigenic
reactivities of the other A and B subgroups can be accounted for by variant A and B transferase
alleles, although the structural basis for these differing reactivities is not yet known.
Figure 16.13. Structural differences between the A1 and A2 subgroup antigens. The type-2 A
structure representative of the A2 phenotype (left) may be synthesized by A1 or A2 subgroup
transferases. The type-2 A structure is then modified by a β1 3 galactosyltransferase and then by
the H locus-encoded α1 2 fucosyltransferase to form a type-3 H structure. The type-3 H
structure is efficiently utilized by the A1 transferase to form the repetitive A-reactive unit
proposed to be responsible for the strong serological reactivity of the A1 phenotype. The A2
transferase is unable to efficiently complete this last reaction. R represents the underlying
glycoprotein or glycolipid substructure. (Open boxes) A-reactive portions of the molecule.
The heritable red cell antigenic polymorphisms determined by the ABO locus have important
medical implications. Early in life, the immune system in an individual generates IgM class
antibodies directed against the ABO oligosaccharide antigens that are absent from that
individual's red cells. The antibodies likely represent an immune response to oligosaccharide
antigens synthesized by bacterial and fungal organisms in the environment, and whose structures
are similar or identical to those of the A and B blood group molecules. For instance, type-O
individuals do not make A and B determinants and therefore maintain relatively high titers of
circulating IgM antibodies (termed isoagglutinins) that react with A and B blood group
molecules. Similarly, blood group B individuals maintain circulating IgM class anti-A
isoagglutinins, but they do not make isoagglutinins against the blood group B determinant, which
is, in these individuals, a "self" antigen. Sera taken from individuals typed as blood group A
contain IgM class anti-B antibodies, but not anti-A antibodies. Finally, individuals with the AB
blood group do not make either anti-A or anti-B IgM class isoagglutinins. Anti-H antibodies are
not made in most individuals because a substantial fraction of the H structures is not converted to
A and/or B determinants, even in those with a functional A or B transferase allele.
These IgM isoagglutinins are able to efficiently trigger the complement cascade and circulate in
human plasma at titers sufficient to cause complement-dependent lysis of transfused erythrocytes
that display the corresponding antigen. Such acute antibody- and complement-dependent red cell
lysis is associated with the clinical manifestations of an immediate, or acute, transfusion
reaction, which can include hypotension, shock, acute renal failure, and death from circulatory
collapse. This problem is avoided by ensuring that the ABO type of the transfused red cells is
compatible with the patient's ABO type. Specifically, this means choosing red cells that are
deficient in the ABO antigens which are also lacking in the recipient (i.e., an A recipient may
receive red cells from another A person, or from an O person, but not from a person typed as B
or from a person typed as AB). Practically speaking, this is accomplished in blood banks through
procedures known as typing and cross-matching. In the typing procedure, units of red cell
products, typed previously for the A and B antigens, are chosen that match the patient's ABO
type. To ensure that these prospective red cell units are then truly "compatible" with the
recipient, the patient's serum is cross-matched with each of the prospective donor red cell units.
The cross-match is done by mixing an aliquot of the patient's serum with a small aliquot of each
prospective red cell unit and examining the mixture under low-power magnification. The red
cells of compatible units do not agglutinate with the patient's serum, whereas incompatibility is
indicated by agglutinated red cells (agglutinated by antibodies in the recipient's serum).
Although these procedures are used to ensure compatibility between the ABO phenotype of
transfused red cells and recipient plasma, they are also used to ensure compatibility between the
red cells circulating in a recipient who must be transfused with plasma. Similar ABO
compatibility concerns are important in heart, kidney, liver, and bone marrow transplantation
procedures. The "type and cross" procedures have virtually eliminated ABO blood group
incompatibility-dependent red cell transfusion reactions in the United States. In the very rare
instances where such a transfusion reaction has occurred, the cause is usually accounted for by
clerical errors introduced during the processing of the patient's serum or the transfused red cells.
The cross-matching procedures discussed above helped to identify a rare ABO blood group
phenotype termed the Bombay phenotype, so named because the first identified H-deficient
individual lived in that city. These individuals were found to maintain red cells that were
deficient in red cell H, A, and B antigens, whereas their sera contained IgM class antibodies that
reacted with the red cells from virtually all donors, including O red cells (H-antigen-positive, A-
and B-antigen-negative). Subsequent investigations indicate that these persons are homozygous
for null alleles at the Hα1 2 fucosyltransferase locus and are also nonsecretors (due to
homozygosity for null alleles at the Seα1 2 fucosyltransferase locus). These persons therefore
are incapable of synthesizing A, B, or H determinants in any tissue, maintain robust titers of
circulating IgM class anti-H, anti-A, and anti-B antibodies, and are therefore cross-match-
incompatible with the red cells of all except other Bombay (H-deficient) donors. A related
phenotype, termed the para-Bombay phenotype, corresponds to nullizygosity at the H locus in
individuals that maintain at least one functional Seα1 2 fucosyltransferase allele (secretor-
positive).
Functions for the ABO blood group oligosaccharides are not known, and the processes that have
led to the polymorphisms at the ABO locus are also mysterious. Indeed, the fact that Bombay
individuals do not exhibit any remarkable pathological phenotype argues that functions for the
A, B, and H antigens that may have existed earlier in evolution are no longer relevant. Although
it has been proposed that polymorphism at the ABO locus may have provided a selective
advantage for protection from certain infectious agents in prehistoric times, strong evidence to
support this contention is not available. Thus, the nature of the selective pressures that yielded
polymorphism at the ABO locus remains unclear.
In this context, however, it is worth noting that a variety of associations have been made between
ABO blood group phenotype and the relative risk for a spectrum of diseases. Two such
associations are discussed below; the remainder are not discussed because they generally
represent modest and imperfect correlations, some of which have not withstood repetitive
examination, and virtually all are without a clear mechanistic relationship.
The first possible association concerns the well-known but mechanistically mysterious
correlation between an individual's ABO phenotype and plasma levels of von Willebrand factor
(vWF), a glycoprotein involved in hemostasis. Recent studies in the mice indicate that the
circulating half-life of vWF (and thus its level in plasma) varies according to the nature of the
alleles at a specific glycosyltransferase locus, which encodes an enzyme that modifies vWF
glycans. Specifically, in a strain of mice with very low levels of plasma vWF, the resulting von
Willebrand's disease phenotype is a dominantly inherited trait that maps to a β1 4-linked
GalNAcT locus for the Sda blood group, as discussed in more detail below. Moreover, the
disease trait is associated with an allele of a β1 4 GalNAcT locus that directs expression of the
enzyme to vascular endothelial cells, a major site of vWF synthesis. Expression of the enzyme in
endothelial cells decorates the glycans on vWF with the corresponding β1 4GalNAc-linked
modification. Glycoforms of vWF characterized by this β1 4-linked GalNAc modification are
rapidly removed from the plasma by the hepatic asialoglycoprotein receptor. In contrast, mice
with normal vWF levels do not express this β1 4 GalNAcT in vascular endothelium, so their
vWF is not modified by β1 4-linked GalNAc moieties and is cleared less rapidly. Thus,
inheritance of this form of murine von Willebrand's disease is accounted for by allelism at a
glycosyltransferase locus. In humans, glycoform-dependent clearance mechanisms could also
account for the generally lower vWF levels in persons of the O phenotype, since vWF is
modified by the ABO blood group antigens in humans and since the A, B, and H blood group
structures may be recognized with different affinities by the asialoglycoprotein receptor and
other such lectins.
A second possible exceptional association is the proposed role for gastrointestinal ABO and
Lewis blood group antigens in the pathogenesis of the ulcerogenic spirochete Helicobacter
pylori (discussed in further detail below, in the context of the Lewis blood group antigens). Well-
known associations exist between the group-A phenotype and a modest increase in the relative
risk for stomach cancer (relative risk of 1.2) and between blood group O phenotype and a slight
increase in the relative risk of developing peptic ulcers (relative risk of ~1.3). Both of these
disorders are now clearly and causally associated with infection by H. pylori, but it remains to be
determined if there is a mechanistic relationship between ABO-dependent ulcer and cancer risk
associations and H. pylori infection (discussed again in the context of the Lewis blood group
antigens below).
The oligosaccharides synthesized through the actions of the H and Seα1 2 fucosyltransferases
are also without clear function. There are numerous reports of associations between secretor
status and various diseases; most are weak associations and without clear mechanistic
relationship. However, the increased relative risks for recurrent urinary tract infection in female
nonsecretors and for peptic ulceration in nonsecretors generally are worthy of brief discussion. It
has been implied that these associations may be partly accounted for by adhesive interactions
between blood group substances expressed by the relevant epithelia and bacterial pathogens that
use such substances to promote epithelial invasion. There is some support for this hypothesis in
the context of peptic ulceration, since α1 2-fucosylated glycans constructed through the action
of the Seα1 2 fucosyltransferase can support adhesion of H. pylori. However, as noted below in
the context of the α1 3-fucosylated glycans of the Lewis blood group antigen family, the
relationship between susceptibility to H. pylori infection and blood group status is substantially
imperfect.
Lewis Blood Group Structures (72 94)
The Lewis blood group antigens correspond to a structurally similar set of α1 3-fucosylated
glycan structures (Figure 16.14). The term Lewis refers to the family name of individuals
suffering from a red blood cell incompatibility problem that helped lead to the discovery of this
blood group.
Figure 16.14. Representative type-1 and type-2 Lewis structures. (Top) Type-2-based structures;
(bottom) type-1-based structures. Type-1 and type-2 structures differ in the linkage of the
outermost Gal (β1 3 and β1 4, respectively) and in the linkage of the fucose moiety to the
internal GlcNAc (α1 3 and α1 4, respectively). R represents N-linked, O-linked, or glycolipid-
based substructures.
The Lewis A antigen (Lea) is synthesized by an α1 3/1 4 fucosyltransferase encoded by the

Lewis (Le) blood group locus (Figures 16.15). The Lewis B antigen (Leb) is synthesized by the
concerted actions of the Lewis α1 3 fucosyltransferase and the α1 2 fucosyltransferase encoded
by the Se blood group locus (Figure 16.15). The nature of the alleles at an individual's Le and Se
loci determines the complement of Lewis-active oligosaccharide molecules that will be
constructed in that individual (Figure 16.15). Secretor-positive individuals convert type-1
oligosaccharide precursors to type-1 H molecules. The resulting type-1 H determinants may then
be used as precursors by the Lewis locus-encoded α1 3/1 4 fucosyltransferase to form the Leb
structure (Figure 16.15). Individuals that construct Leb exhibit the Le(a-b+) phenotype (Figure
16.15). Nonsecretors do not synthesize type-1 H determinants in secretory epithelia, but such
unsubstituted type-1 molecules can be converted to Lea-active oligosaccharides by the Lewis
α1 3/1 4 fucosyltransferase. These individuals exhibit the Le(a+b-) phenotype (Figure 16.15).
Individuals that are homozygous for null alleles at the Lewis locus can have two different
phenotypes. Such Lewis-negative individuals who are positive for secretor status construct type-
1 H determinants, but these structures remain unconverted to Leb determinants (Figure 16.15).
These persons exhibit the Le(a-b-) phenotype. In contrast, in individuals who are Lewis-negative
nonsecretors, type-1 precursors are not substituted by either the Lewis or the Secretor
fucosyltransferases, accounting for absence of Lea, Leb, or H structures, and their Le(a-b-)
phenotype.
Figure 16.15. Lewis blood group phenotypes. The structures of the Lewis blood-group-active
glycolipids are determined by the presence or absence of the Se locus α1 2 fucosyltransferase
and the Lewis locus α1 4 fucosyltransferase. R is lactosylceramide.
Expression of Lea and Leb molecules, and the Lewis α1 3/1 4 fucosyltransferase, is restricted
largely to the same epithelia that express the Secretor α1 2 fucosyltransferase. Again, these
epithelia elaborate soluble forms of these antigens that are released into secretions and body
fluids. Lea and Leb antigens are also detectable on red cells, at a level of approximately 4500 and
7300 Lea molecules per cell. However, red cell precursors do not synthesize these molecules.
Instead, Lewis antigens are acquired by the red cell membrane through passive adsorption of
Lewis-positive glycosphingolipid molecules that circulate in plasma as lipoprotein complexes
and aqueous dispersions.
Antibodies against the Lea antigens have been implicated in occasional instances of transfusion
reactions, through the same mechanisms noted above for ABO incompatibility in transfusion.
Anti-Leb antibodies are rarely, if ever, associated with clinical problems. The relatively benign
attributes of these antibodies are accounted for by the fact that anti-Lea and anti-Leb antibodies
are effectively neutralized by soluble Lea or Leb substances in transfused plasma. Moreover,
transfused Lewis antigen-positive erythrocytes rapidly revert to a Lewis antigen-negative
phenotype in Lewis-negative recipients after transfusion, owing to reversal of the absorptive
process by which the red cells had accumulated these antigens in the donor.
There are other members of the Lewis blood group family of glycan structures. These include the
Lex (SSEA-1) and Ley molecules and forms of the Lea and Lex determinants that are sialylated
and or sulfated (see Figure 16.14). These structures are formed through the actions of one or
more α1 3 fucosyltransferases in addition to the Lewis α1 3(4) fucosyltransferase.
As also noted for the ABO and Se loci, numerous associations have been made between the
Lewis blood group phenotype and susceptibility to various diseases. As with the ABO and H-
dependent associations, most of these are typically weak and generally are without clear
mechanistic relationship. However, some members of the Lewis blood group antigen family
have functional relevance in the context of selectin-dependent leukocyte and tumor cell adhesion
processes (see Chapter 26). The relevant members include especially the sialylated and/or
sulfated members represented by the sialyl Lex tetrasaccharide and its sulfated variants (Figure
16.14). These molecules provide essential contributions to the glycoproteins and glycolipids that
function as selectin counter-receptors on leukocytes and probably also on tumor cells (see
Chapters 26 and 35).
The Lewis blood group antigens have also been implicated in the pathogenesis of H. pylori. It is
clear that this organism is a causative agent in chronic active gastritis. Infection with this
organism is also associated with hypertrophic gastropathy, duodenal ulcer, gastric
adenocarcinoma, and gastrointestinal lymphoma. Colonization of the stomach by H. pylori
almost certainly requires adhesion to the gastric mucosal epithelium and to the mucus released
by these cells. This organism can adhere to the Leb blood group antigen, at least in the limited
number of strains that have been examined, implying that this oligosaccharide may function as a
receptor for H. pylori in vivo. However, clinical studies that have characterized the relationship,
in vivo, between gastric colonization by H. pylori and the ABO, Lewis, and Secretor status of the
human host are not necessarily consistent with this hypothesis. Such studies found no correlation
between H. pylori infection rate and host Leb phenotype, little or no correlation between H.
pylori infection rate and ABO and Secretor phenotype, and a weak association between
lymphocytic infiltration in H. pylori infection and ABO or Secretor status. The Lewis blood
group adhesion hypothesis is further complicated by the fact that some strains of H. pylori can
themselves elaborate expression of Leb antigen. Thus, the physiological relationship, if any,
between H. pylori pathobiology and the Lewis or ABO antigens is probably much more complex
than can be accounted for by simple blood group antigen-dependent adhesive processes.
P Blood Group Structures (95 106)
The P blood group antigens are glycan structures displayed by membrane-associated

glycosphingolipids on red cells and on other tissues, including the urothelium (Figure 16.16).
The glycans in the P blood group system are constructed by the sequential action of a series of
distinct glycosyltransferases. Little is known about the enzymes or genes that direct expression
of the P family of antigens, causing the nomenclature, biochemistry, and genetics of this system
to be somewhat complex.
Figure 16.16. Antigens of the P blood group system.
Synthesis of the P antigens involves two different pathways, each of which begins with
lactosylceramide as a common precursor (Figures 16.17 and 16.18). In one pathway, P antigen
biosynthesis begins with an α1 4 galactosyltransferase (Pk transferase) that synthesizes the Pk
antigen. The Pk antigen is then modified by a β1 3 GalNAcT, termed the P transferase, to form
the P antigen. In the second pathway, P1 antigen biosynthesis involves three sequential
glycosylation reactions, again starting with lactosylceramide. The first two enzyme reactions
lead to paragloboside synthesis. Paragloboside is then used as a substrate by the P1 transferase,
which forms the P1 molecule.
Figure 16.17. Pk and P antigen biosynthesis.
Figure 16.18. P1 antigen biosynthesis via paragloboside.
Expression of these antigens is polymorphic in humans. The most common P blood group
phenotype is termed P1. These individuals express full activity of each of the enzymes in these
pathways, and their red cells express both P and P1 antigens. P1 individuals also express small
amounts of Pk determinants because the P transferase does not completely convert all Pk
precursor determinants into P determinants. The other common blood group phenotype is P2.
These individuals are apparently nullizygous at the P1 transferase locus. Red cells from P2
individuals express normal levels of P and Pk antigens, but are deficient in P1 determinants.
Three rare P blood group phenotypes have been described. The first, termed the P1k phenotype, is
associated with deficiency of P transferase activity, and is presumed to be consequent to
nullizygosity for the P transferase locus. P1k individuals cannot convert the Pk structure into the P
structure and therefore express more than the normal numbers of Pk determinants. The parallel P1
synthesis pathway is intact in these persons, yielding normal numbers of P1 determinants. The
second, termed the P2k phenotype, is found in rare individuals, who are presumed to be
nullizygous at both the P transferase and the P1 transferase loci. Consequently, neither of the
parallel pathways is completed, resulting in deficiencies in both P and P1 antigen expression, but
increased numbers of Pk determinants. The third, termed the p phenotype, is characterized by a
deficiency of all three P antigens (P, P1, and Pk). Nullizygosity at the Pk transferase and P1
transferase loci can account for this phenotype. P antigen is not synthesized, regardless of P
transferase activity, because the P precursor (Pk) is not present. However, p phenotype red cells
express low levels of the P antigen reactivity because the normal P transferase activity in these
persons can form a glycan with P antigen reactivity, through the addition of an α 1 4-linked
galactose to paragloboside derived from the other pathway (Figures 16.17 and Figure 16.18).
These considerations assume that the P, P1, and Pk transferases represent the products of three
different loci. However, there are alternative genetic models that could explain the observed
human phenotypes. A definitive understanding of this system will await the isolation and
characterization of the genes corresponding to these enzymes. Antibodies directed against
various P blood group antigens have been implicated in transfusion reactions in individuals with
the p phenotype, who often maintain antibodies against P, P1, and Pk determinants. Complement-
fixing, cold-reactive anti-P antibodies known as "Donath-Landsteiner" antibodies have been
implicated in intravascular hemolysis observed in paroxysmal cold hemoglobinuria (see Chapter
37).
Physiological functions for the P blood group antigens are not known. However, these molecules
have been assigned roles in the pathophysiology of urinary tract infections and in parvovirus
infection. A role for P blood group antigens in the pathogenesis of urinary tract infections is
implied by the observation that various uropathogenic strains of Escherichia coli express
adhesins that bind to the Galα1 4Gal moiety of the Pk and P1 antigens. The P1 determinant is
expressed on the urothelium of P1 individuals and may facilitate bacterial infection by mediating
attachment of bacteria to the lining of the urinary tract. This hypothesis is supported by the
observation that P1 individuals have a higher risk, relative to P2 individuals, for urinary tract
infections and pyelonephritis. It is also supported by the observation that adhesion of a
pyelonephritic strain of E. coli to renal tissue is mediated by a bacterial adhesin specific for the
Galα1 4Gal structure and that deficiency of the adhesin severely attenuates the pyelonephritic
phenotype of the organism.
The P blood group antigens have also been assigned a role as a receptor for the human
parvovirus B19. This virus causes erythema infectiosum and leads to congenital anemia and
hydrops fetalis following infection in utero. It is also associated with transient aplastic crisis in
patients with hemolytic anemia and with cases of pure red cell aplasia and chronic anemia in
immunocompromised individuals. Parvovirus B19 replication is restricted to erythroid
progenitor cells; an adhesive interaction between the virion and P-antigen-active glycolipids is
involved in viral infection of erythroid progenitors. Individuals with the p blood group
phenotype are apparently resistant to parvovirus B19 infection.
The α1 3Gal Structure (107 116)
The Galα1 3Gal epitope is synthesized from type-2 glycolipid and glycoprotein precursors by a
specific α1 3 galactosyltransferase (Figure 16.19). This structure and the corresponding α1 3
galactosyltransferase are expressed by New World primates and many nonprimate mammals but
are largely absent from the cells and tissues of Old World primates which include Homo sapiens.
The molecular basis for the species-specific absence of this enzyme and its oligosaccharide
product involves the inactivation of the locus encoding the α1 3 galactosyltransferase in primate
taxa that do not express the epitope (see Chapter 33).
Figure 16.19. Structure and synthesis of the Galα1 3Gal antigen. The α1 3
galactosyltransferase uses unsubstituted type-2 precursors to form the Galα1 3Gal epitope. R
may be glycolipid- or glycoprotein-based glycan structures.
A physiological function for the Galα1 3Gal epitope has not been identified. There have been
reports that the Galα1 3Gal epitope is found in small amounts on human red cells and may
participate in red cell turnover, but these observations are at odds with the apparent absence of a
functional α1 3 galactosyltransferase locus in humans. Similar considerations apply to the
apparent expression of the Galα1 3Gal epitope on human thyroid cells or human cancer cells. A
proposed role for the Galα1 3Gal epitope as a sperm receptor on mouse oocytes is contradicted
by studies showing that mice homozygous for an induced null mutation in this locus are fertile
(see Chapter 33). These mice also have cataracts, but a mechanistic relationship between this
phenotype and the absence of Galα1 3Gal epitopes is not available. These mice have no other
recognized pathological phenotype as of this writing.
Species that lack expression of the Galα1 3Gal epitope, including especially humans, elaborate
naturally occurring anti-Galα1 3Gal antibodies, by mechanisms that involve immunization
through exposure to microbial antigens similar or identical to the Galα1 3Gal epitope. These
anti-Galα1 3Gal antibodies present a major barrier to the use of porcine and other nonprimate
organs for xenotransplantation in humans, since they bind to Galα1 3Gal epitopes on the
vascular endothelium in such xenotransplants and mediate hyperacute graft rejection through
complement-mediated endothelial cell cytotoxicity. Efforts are in progress to overcome this
barrier through the generation of animal organ donors that have been genetically modified to
express proteins that diminish complement-dependent cytotoxicity (see Chapter 33). Approaches
include transgenic expression of enzymes such as the H α1 2 fucosyltransferase that may
diminish Galα1 3Gal expression by diverting type-2 precursor substrates toward H antigen
synthesis and away from their utilization by the α1 3 galactosyltransferase. The creation of
induced null mutations in the gene encoding α1 3 GalT of such animal organ donors represents
one definitive solution to the problem of Galα1 3-Gal-dependent hyperacute rejection.
Unfortunately, the pluripotent embryonic stem cells required for this approach are not yet
available for pigs or other large animal organ donors.
Naturally occurring anti-Galα1 3Gal antibodies have also been shown to significantly diminish
the infective efficiency of recombinant retroviruses. This problem occurs because the packaging
cell lines used to propagate these viruses are derived from species that express the Galα1 3Gal
epitope and decorate the viral coat proteins. This problem has been solved through the generation
of packaging cell lines that are deficient in the cognate α1 3 galactosyltransferase. This
technical problem may reflect a natural mechanism that acts to restrict the interspecies spread of
retroviral genomes (see Chapter 3).
The Forssman Antigen (117 123)
The Forssman antigen, also known as globopentosylceramide, is a glycolipid structure formed by

the addition of GalNAc in α1 3 linkage to the terminal GalNAc residue of globoside (Figure
16.20). There is also evidence in the literature that Forssman-specific monoclonal antibodies can
detect Forssman-reactive glycoproteins, but the nature of these molecules is not known. The
Forssman antigen molecule is expressed during embryonic and adult life in rodents and other
mammals (see Chapter 34), but uncertainty exists about the ability of humans to express this
antigen. For example, there is evidence that humans maintain moderate titers of naturally
occurring anti-Forssman antibodies in plasma, suggesting that humans do not express the
Forssman antigen. In contrast, there is evidence that such antibodies are not consistently present
in humans and that when generated, may contribute to the pathogenesis of the Guillain-Barre
syndrome by binding to glycolipid components of peripheral nerve myelin. Similarly, evidence
exists that small amounts of Forssman reactivity may be found on human gastrointestinal
epithelium, by various human cultured cell lines, by pulmonary and gastointestinal tract
carcinomas. These conflicting observations may be a reflection of varied specificities of the anti-
Forssman monoclonal antibodies used by different investigators and by differences in epitope
reactivity achieved with immunohistochemical procedures versus thin-layer
chromatography/antibody overlay procedures. The function of this antigen is not known. The
ability of anti-Forssman antibodies to disrupt tight junction formation, apical-basal polarization,
and adhesion suggests that this molecule may participate in cell-cell adhesion and
communication processes. The mechanisms that account for these observations are not defined,
nor are there corollary studies that have confirmed these observations in a more physiological
context.
Figure 16.20. Forssman antigen biosynthesis. Globoside serves as the substrate for the Forssman
α1 3 N-acetylgalactosaminyltransferase (α1 3 GalNAcT) that forms globopentosylceramide,
also termed the Forssman glycolipid.
Sulfated Terminal β-linked GalNAc Structures on Pituitary
Glycoproteins (124 127)
Glycans with sulfated terminal β-linked GalNAc moieties have been studied extensively in the
context of pituitary glycoprotein hormones lutropin (LH), thyrotropin (TSH), and follicle-
stimulating hormone (FSH). These heterodimeric glycoproteins are composed of a common α-
subunit and a unique β-subunit. Each subunit is decorated with biantennary N-linked glycans.
The N-glycans on TSH and LH contain an unusual structure consisting of a GalNAc moiety
attached to one or both GlcNAc residues on the biantennary glycan, and additionally modified by
sulfation or by α2 6-linked sialic acid (Figure 16.21). This modification contrasts with the N-
glycans on FSH and on most N-glycans, where GlcNAc residues are modified by β1 4-linked
galactose moieties, which are often then modified by α2 3- or α2 6-linked sialic acid. The free
α-subunit common to LH, TSH, and FSH, and present as a synthetic intermediate in pituitary
cells, is also modified by this determinant, as are other proteins synthesized by the pituitary and
in other tissues. This structure has also been found on the O-glycans of pro-opiomelanocortin.
Figure 16.21. Structure and synthesis of N-glycans bearing terminal GalNAc and sulfate
moieties, including those associated with pituitary hormones LH and FSH.
Synthesis of this sulfated GalNAc glycan determinant is controlled by a β1 4 GalNAcT activity

that modifies the subterminal GlcNAc residues of the biantennary glycan chains (Figure 16.21).
Expression of this enzyme is restricted to only a few cell types, including the cells in the
pituitary that elaborate LH, TSH, and FSH. This β1 4-linked GalNAc moiety is then sulfated by
a sulfotransferase also expressed in pituitary cells. In some tissues, including the pituitary, the
β1 4-linked GalNAc moiety is subsequently modified by α2 6-linked sialic acid residues.
Subterminal GlcNAc residues beneath the β1 4-linked GalNAc moiety have also been observed
to be modified by α1 3-linked fucose (and see Chapter 26). In contrast, synthesis of β1 4-linked
galactose-terminated biantennary chains characteristic of FSH is directed by a β1 4
galactosyltransferase (Figure 16.21), an enzyme activity found in virtually all cell types,
including those of the pituitary gland.
Although a β1 4 GalNAcT and a β1 4 galactosyltransferase are both expressed in the pituitary

cell, the N-glycans on LH and TSH are decorated with the unusual β1 4-linked GalNAc moiety,
whereas the identical N-glycans on the nearly identical glycoprotein FSH are instead exclusively
decorated with the alternate and more common β1 4-linked galactose moiety. This protein-
specific glycosylation event is a consequence of interaction between the β1 4 GalNAcT and
specific protein sequence motifs present on the α and β subunits of LH and TSH. This
interaction causes this enzyme to increase the catalytic efficiency with which it modifies the
biantennary N-glycans on LH and TSH, at the expense of modification by the competing β1 4
galactosyltransferase, which does not recognize the peptide motif. The resulting terminal β1 4-
linked GalNAc moieties are subsequently susceptible to modification by sulfation or by α2 6
sialylation (Figure 16.21). In contrast, the peptide sequence motif recognized by the β1 4
GalNAcT is not present in the sequence of the β-subunit of FSH, and the recognition motif on
the α-subunit of FSH is not available to the enzyme. Consequently, the biantennary N-glycans
on FSH are not susceptible to modification by the β1 4 GalNAcT and are instead modified
exclusively by the competing β1 4 galactosyltransferase.
These differential glycosylation events have profound consequences for the ovulatory cycle
throughout vertebrate taxa. Maximal stimulation of the ovary by LH during the preovulatory
surge is associated with levels of circulating LH that rise and fall in a highly pulsatile manner.
The pulsatile characteristic of circulating LH levels assures maximal stimulation of the ovarian
LH receptor, since sustained high LH levels of this receptor would lead to LH receptor
desensitization. The pulsatile rise and fall in LH levels are due in part to pulsatile release of the
hormone by the pituitary. However, it is also a consequence of a rapid clearance of the hormone
from the circulation. Clearance of the hormone from the circulation is mediated by a receptor
specific for the sulfated GalNAcβ1 4 GlcNAc terminus. This receptor is expressed by hepatic
endothelial cells, and by Kupffer cells, and binds LH with an apparent Km of approximately 160
nm. Receptor binding is followed by internalization and lysosomal degradation. The protein
sequence of the receptor is identical to a previously characterized protein termed the macrophage
mannose receptor. The mechanisms that account for tissue-specific differences in this receptor's
ligand recognition characteristics remain to be defined.
Similar GalNAcβ1 4-GlcNAc (LacdiNAc) termini have also been reported on N-glycans from
other vertebrate sources, such as bovine milk, rat prolactin, and kidney epithelial cells, as well as
in invertebrates such as snails and parasitic worms. These residues generally do not become
sulfated as in pituitary hormones, but are frequently 2 6-sialylated in vertebrates. It is not clear
how the GalNAcTs responsible for producing these structures are related to the enzyme acting
specifically on pituitary hormones.
Sialylated Terminal β-linked GalNAc Structures (128 138)
Terminal GalNAc modifications of α2 3-sialylated structures are found on glycoproteins and on

glycolipids (Figure 16.22). The former correspond to the human Sda blood group structure, and a
murine oligosaccharide, termed the CT1 or CT2 antigen, first described on cytotoxic T
lymphocytes (CTLs). The glycolipid-based form of this structure is known as the ganglioside
GM2.
Figure 16.22. Production of the Sda or CT antigen and the glycolipid GM2.
The polypeptide-linked form of this structure was first identified as the pentasaccharide Siaα2
3(GalNAcβ1 4)Galβ1 4GlcNAcβ1 3Gal-, released from N-glycans present on Tamm-Horsfall
glycoprotein isolated from human urine. This structure is formed by addition of β1 4-linked
GalNAc to the galactose moiety of α2 3-sialylated type-2 chains (Figure 16.22). A β1 4
GalNAcT capable of catalyzing this reaction has been found in human kidney and urine,
intestine, colon, and blood plasma. Rare humans lacking the ability to construct this determinant
can form naturally occurring antibodies against it and are said to lack the corresponding (Sda)
blood group determinant. Absence of this antigen in these humans has no apparent associated
detrimental phenotype.
In the mouse, this structure corresponds to the antigen(s) recognized by a pair of IgM class
monoclonal antibodies termed CT1 and CT2, isolated for their ability to block lysis of cellular
targets by a murine CTL clone. These antibodies recognize similar but nonidentical antigens
expressed by activated CTLs, but not by naive T lymphocytes. Activation-dependent display of
the CT antigens correlates with inducible expression of the cognate enzyme activity. Both CT
antibodies also bind to intraepithelial lymphocytes, a type of constitutively activated T-
lymphocyte resident in the intestinal mucosa. The major proteins immunoprecipitated from CTL
cell lines by CT antibodies belong to isoforms of the CD45 cell surface transmembrane tyrosine
phosphatase required for T-cell proliferation in response to antigen. It is likely that other
lymphocyte cell surface proteins are also modified with this structure. As with human cells, this
glycan structure is constructed by a β1 4 GalNAcT that modifies β1 4-linked galactose
substituted with α2 3-linked sialic acid on O- and N-glycans. These human and mouse β1 4
GalNAcTs can transfer GalNAc to both N- and O-glycans present on glycoproteins but not on to
the glycolipid GM3 (Siaα2 3Galβ1 4Glc-Cer), even though both can efficiently use 3 -
sialyllactose (Siaα2 3Galβ1 4Glc) as a substrate in vitro.
The function of this structure in humans or mice is unknown. As noted earlier in this chapter, in
rare strains of mice with a dominantly inherited form of von Willebrand's disease, the
corresponding β1 4 GalNAcT is aberrantly expressed in vascular endothelium. Since the blood-
clotting protein vWF is also expressed by endothelial cells, expression of the enzyme at this
unusual location decorates the glycans on vWF in these mice. The resulting vWF glycoform is
rapidly cleared from the circulation, which accounts for the low levels of this protein in this
strain of mice. Rapid clearance is mediated by the asialoglycoprotein receptor, which exhibits
affinity for terminal GalNAc structures.
The glycolipid-linked form of this structure, termed GM2, is synthesized from the ganglioside
GM3 (Figure 16.22). GM2 synthesis is catalyzed by a β1 4 GalNAcT (GM2 synthase) that shares
primary sequence similarity with the β1 4 GalNAcT responsible for synthesis of the
glycoprotein form of this structure. However, this enzyme cannot effectively utilize glycoprotein
glycan precursors. The function of this structure is not known. However, it is widely expressed
in the CNS and PNS, and in the adrenal gland, for example, suggesting a role in these organ
systems. This possibility is supported by analysis of mice homozygous for an induced null
mutation at the GM2 synthase locus. These mice exhibit modest conduction defects in the PNS
and exhibit biochemical defects resulting in male sterility (and see Chapter 33). Studies are in
progress to determine how GM2 contributes to homeostasis in these systems and to characterize
the mechanisms responsible for these abnormal phenotypes.
α2 3-sialylated Structures (139 154)
Sialic acid is found in α2 3 linkage on many, and perhaps all, cells and tissues in vertebrates.
Members of a family of at least five different α2 3 sialyltransferases (ST3Gal-I, ST3Gal-II,
ST3Gal-III, ST3Gal-IV, and ST3Gal-V) are responsible for synthesis of these structures (Figure
16.23). Studies of the expression patterns of these genes indicate that STGal-III and STGal-IV
are expressed in most tissues and cells in adult mammals. These observations are consistent with
the identification of α2 3-sialylated glycans on many different cell types, including different
glycoproteins and glycolipids. In contrast, ST3Gal-I transcripts in humans and mice are abundant
in the spleen, liver, bone marrow, thymus, and salivary glands and are less abundant in other
tissues. Like ST3Gal-I, ST3Gal-II is also more restricted in its expression pattern, with
transcripts most abundant in the brain and much lower levels of expression in some other tissues.
The relatively robust expression of ST3Gal-II in the brain is consistent with the abundance of
α2 3-sialylated glycolipids in this organ and with the part played by this enzyme in glycolipid
(ganglioside) synthesis (Figure 16.23). The ST3Gal-V locus is also expressed in the brain,
skeletal muscle, testes, and the liver.
Figure 16.23. Synthesis of glycoproteins and glycolipids bearing terminal α2 3-linked sialic
acids by the ST3Gal series of sialyltransferases. Enzymes in parentheses contribute at relatively
low levels in vitro to the reactions indicated.
In vertebrates, α2 3 sialic acid linkages are found on terminal galactose moieties (Figure 16.23).
Such structures are substrates for further modification by α1 3 fucosylation (see Figure 16.14
and Chapter 26), β1 4 GalNAc linkage formation in limited circumstances (see Figure 16.21),
α2 6 sialylation (Figure 16.24), α2 8 sialylation (Figures 16.25 and 16.26), and sulfation
(Figure 16.27). In contrast, the α2 3-sialylated structures represented in Figure 16.23 are
generally not substrates for other enzymes, including α1 2 fucosyltransferases, α1 3
galactosyltransferases, GlcNAcTs, or GalNAcTs. The latter enzymes may compete for terminal
α2 3 sialylation as a glycan chain-terminating modification.
Figure 16.24. Synthesis of α2 6-sialylated termini on O-glycans, and glycolipids (and see
Chapters 8 and 9) by the ST6GalNAc series of sialyltransferases. Enzymes in parentheses
contribute at relatively low levels in vitro to the reactions indicated.
Figure 16.25. Structure and synthesis of polysialic acid on N-glycans.

Figure 16.26. Structure and synthesis of polysialic acid on glycolipids. Enzymes in parentheses
contribute at low levels in vitro to the reactions indicated.
Figure 16.27. Sulfation of α2 3-sialylated, α1 3 fucosylated glycans represented by the sialyl

Lex tetrasaccharide have been observed at the 6-hydroxyl positions of the galactose residue and
the GlcNAc residue. Both may contribute to L-selectin ligand activity. The biosynthesis of these
structures, the enzymes that participate in this process, and their functional attributes are
discussed further in Chapter 26.
Structures bearing α2 3 sialic acid linkages have been assigned a function in only a few
circumstances as of this writing. As discussed in detail in Chapter 26, the glycan ligands of the
selectin family of leukocyte adhesion molecules include α2 3-sialylated structures, modified by
α1 3 fucosylation or by α1 3 fucosylation and sulfation. The α2 3-linked sialic acid
component of such selectin ligands is likely essential for physiologic ligand formation involving
E-selectin, P-selectin, and L-selectin. Analyses of the tertiary structures of the CRD of E-selectin
derived from crystallographic studies and mutagenesis studies imply that specific positively
charged amino acid residues interact with the negatively charged sialic acid moiety on the
glycolipid and glycoprotein counterreceptors. Recognition is specific for sialic acid in α2 3-
linkage, since adhesive interactions are not supported by variants of selectin ligands in which
α2 3-linked sialic acid is substituted with α2 6-linked sialic acid.
Structures bearing α2 3 sialic acid linkages may contribute to the homeostatic maintenance of
circulating half-life of plasma glycoproteins, by virtue of "masking" terminal galactose residues
that would ordinarily contribute to the removal of such proteins from the circulation through the
asialoglycoprotein receptor (see Chapter 25). The physiological role for this process in uncertain,
however, at least with respect to the asialoglycoprotein receptor, since mice deficient in this
receptor do not have increased plasma levels of desialylated glycoproteins or lipoproteins in their
circulation. However, recent studies demonstrate that these mice accumulate endogenous, but as
yet unidentified, ligands for this receptor. The converse also seems to be true, in that α2 3 sialic-
acid-terminated glycans bearing a β1 4 GalNAc moiety (see Figure 16.21) undergo enhanced
asialoglycoprotein receptor-dependent clearance, at least in the case of one plasma protein.
In other studies, the role of ST3Gal-I in the production of the Siaα2 3Galβ1 3GalNAcα-
Ser/Thr glycan is important for the viability of peripheral CD8+ T cells. Mice lacking the
ST3Gal-I enzyme exhibit a decreased cytotoxic-T-cell response with an increase in the apoptotic
death of naive CD8+ T cells (see Chapter 33). The timing, specificity, and location of this
enhanced apoptotic death indicate that it is unlikely to be due to the liver asialoglycoprotein
receptor and may result from an endogenous lectin yet to be recognized that binds to Galβ1
3GalNAcα-Ser/Thr glycans.
The α2 3 sialic acid linkages that decorate some glycoproteins and glycolipids also contribute to
microbial pathogenesis. The most extensively studied system in this context concerns the role of
such structures as receptors for the influenza virus. Sialic acid in α2 3-linkage is recognized by
the influenza virus hemagglutinin commonly found in birds and pigs and prior to its
recombination, yielding binding to α2 6 sialic acid linkages as found frequently in isolates from
the human population. The viral hemagglutinin mediates attachment of the virus to cells that
express α2 3 sialic acid linkages, promoting viral neuraminidase-dependent release of sialic
acids with delivery of the viral genome to the interior of the cell (see Chapter 28).
Structures bearing α2 3 sialic acid linkages have also been implicated in contributing to
bacterial pathogenesis. For example, α2-3 sialic acid structures support adhesion of H. pylori,
the spirochete implicated in the pathogenesis of gastritis, gastric ulcers, and lymphoma of the
gastrointestinal tract mucosa. However, it remains to be determined if these in vitro observations
have a physiological correlate. In contrast, there is strong evidence for a physiological role for
the ganglioside GM1 (GM1a; Galβ1 3GalNAcβ1 4[Siaα2 3]Galβ1 4Glcβ1-Cer) as a receptor
for cholera toxin, produced by Vibrio cholerae, and heat-labile enterotoxin (LT-1), produced by
enterotoxigenic E. coli (see Chapter 28). Cholera toxin is responsible for the severe
enteropathogenicity that accompanies infection with V. cholerae. Heat-labile enterotoxin is the
causative agent of traveler's diarrhea and contributes to a substantial amount of childhood
mortality in the developing countries. Both toxins are heterohexamers composed of a single toxic
subunit with ADP-ribosylating activity (the A subunit), complexed with a pentamer composed of
five B subunits that bind GM1. The cytotoxic A subunit is delivered to enterocytes by binding of
the heterohexamer to the intestinal epithelial cells via the branched pentasaccharide of the
ganglioside GM1a. Binding of these toxins to GM1a is clearly of pathophysiological relevance;
glycan-based inhibitors of these interactions are currently under evaluation in humans for their
ability to diminish the symptoms and progression of cholera and traveler's diarrhea.
α2 6-sialylated Structures (139, 140, 149,155 157)
Sialic acid in α2 6 linkage is expressed by various vertebrate cells and tissue types and
displayed on a wide variety of glycoproteins and glycolipids. Synthesis of α2 6-linked sialic
moieties is directed by members of a family of at least five different α2 6 sialyltransferases (see
Figure 16.24). The genes of these have been defined by molecular cloning approaches (ST6Gal-
I, ST6GalNAc-I, ST6GalNAc-II, ST6GalNAc-III, and ST6GalNAc-IV). A gene corresponding
to a sixth activity, ST6GlcNAc-I, has not yet been genetically isolated. Surveys of tissues and
cells in a variety of mammals, and some lower vertebrates, indicate that α2 6-sialylated glycans
may be found in various (but not all) cell types, as they appear less ubiquitous than the α2 3-
linked sialic-acid-bearing glycans.
In vertebrates, α2 6 sialic acid linkages are found on terminal galactose moieties, on terminal or
subterminal moieties, or on an internal GalNAc moiety. Glycans modified by terminal α2 6
sialic acid are generally not modified further, except possibly by some members of the poly α2
8 sialyltransferase family, as discussed below. The products of ST6Gal-I are typically found on
N-glycans, although the in vitro acceptor substrate specificity of this enzyme indicates that the
β1 4-linked galactose of a terminal lactosamine repeat unit on O-glycans can be modified by
this enzyme. ST6Gal-I is expressed at a relatively high level in hepatocytes and lymphocytes and
is responsible for α2 6 sialylation of serum glycoproteins and glycoproteins of the antigen
receptor complex. In contrast, the α2 6-sialylated products and the ST6GalNAc-I and
ST6GalNAc-II enzymes are restricted to structures displayed on O-glycans. The ST6GalNAc-III
enzyme is responsible for substitution of core N-acetylgalactosamine moiety on O-lycans,
whereas the ST6GalNAc-IV enzyme appears to use glycosphingolipid precursors as the
preferred acceptor.
Few definitive functions have been assigned to α2 6-linked sialic acid modifications. As noted
above (and see Chapter 28), α2 6-linked sialic acid serves as a receptor for human infectious
strains of the influenza virus. The common occurrence of α2 6-linked terminal sialic acid
moieties on plasma glycoproteins may mask terminal galactose and N-acetylgalactosamine
moieties on the glycoproteins that would ordinarily lead to their clearance by the hepatic
asialoglycoprotein receptor. As also discussed previously, experiments using mice with an
induced mutation in the asialoglycoprotein receptor indicate that this notion is not as
straightforward as once thought, since such mice do not have obvious quantitative alterations in
plasma glycoprotein levels.
The clearest information concerning the function of some α2 6-linked sialic acids comes from a
characterization of a role for such structures by a genetically induced absence of ST6Gal-I and in
the context as ligands for CD22, a member of the I-type lectin family (see also Chapters 24 and
33). CD22 is restricted in its expression to the surfaces of B lymphocytes. The extracellular
domain of CD22 specifically recognizes Siaα2 6Galβ1 4GlcNAc-, where the sialylation is the
product of the ST6Gal-I enzyme. This glycan has been found on a variety of leukocytic
glycoproteins, including the protein tyrosine phosphatase CD45, with attendant possibilities for
CD22-dependent signal transduction processes mediated by interactions between CD22 on B
lymphocytes and α2 6-sialylated glycans on CD45. This structure is also displayed by CD22
itself, implying that CD22 may participate in homophilic interactions, both between cells and on
the same cells. Since CD22 has been associated with the antigen receptor on B-lymphocyte
proteins, and since its cytosolic domain is tyrosine-phosphorylated in some circumstances, it had
been proposed that homophilic and heterophilic interactions involving CD22 might also effect
biologically important signal transduction events in the immune system.
Mice lacking ST6Gal-I manifest an immunodeficiency characterized by a diminished antibody

response to T-lymphocyte-dependent and -independent antigens, by reduced B-lymphocyte
proliferation in response to cross-linking of the B-cell surface glycoproteins CD40 and surface
IgM, by reductions in the expression of B-cell surface IgM and CD22, and by an approximately
65% reduction in serum IgM levels. B-lymphocyte antigen receptor-dependent signal
transduction processes are also attenuated in these mice. These observations disclose an
important role for the ST6Gal-I in the immune system. Considered together, inconsistency exists
among the different reports on the phenotypes observed in mice with induced mutations in the
CD22 locus (discussed in Chapter 24), and these phenotypes are not necessarily completely
consistent with, or the biological converse of, the phenotype obtained with the CD22 ligand-
deficient (STGal-I null) mice. Some studies have implied that other lectins may exist that bind to
the ST6Gal-I-generated glycans. Additional experiments will be required to resolve these issues.
α2 8-sialylated Structures (139, 140,158 168)
Glycans modified by sialic acid in α2 8 linkage have been identified as developmentally

regulated antigens in the vertebrate CNS (Chapter 34); the biology of α2 8-sialylated
glycoconjugates has been studied most extensively in the context of neural development. These
α2 8-sialylated glycans are expressed in other, nonneuronal tissues during embryogenesis,
however, and in the adult vertebrate, and these glycans have been observed on transformed cells.
At least five different α2 8 sialyltransferases direct the synthesis of α2 8 sialylated structures
(see Figures 16.25 and 16.26). Two of these enzymes, termed ST8Sia-II (also termed STX) and
ST8Sia-IV (also termed PST-1 and PST), catalyze the synthesis of linear polymers of α2 8
sialic acid, termed polysialic acid, or PSA (see Figure 16.25). These polymers may be composed
of 100 or more sialic acid residues. N-glycans with terminal sialic acid moieties in α2 3 linkage
serve as the substrate for attachment of the initial α2 8-linked sialic acid residue, although there
is some evidence that α2 6-linked sialic acids may also support attachment of the first α2 8-
linked sialic acid residue. This so-called "initiase" reaction is followed by an "elongation"
reaction in which the α2 8 sialic acid moiety added in the preceding step serves as the
attachment site for the next α2 8-linked sialic acid. These two "activities" may be subsumed by
ST8Sia-II and ST8Sia-IV themselves or are the province of distinct sialyltransferases.
PSA is most extensively studied as a component of the posttranslational modifications of N-

CAM, a member of the immunoglobulin superfamily (and see Chapter 34). The α-subunit of the
voltage-gated sodium channel may also be modified with PSA. The polysialyltransferases
ST8Sia-II and ST8Sia-IV are each subject to autocatalytic polysialylation which can occur on N-
glycans that decorate each enzyme, although polysialylation is not a prerequisite for
polysialyltransferase activity. This process can yield membrane-associated, poly-α2 8-sialylated
forms of the enzymes, which may account for the observation that some cultured cell lines which
do not express N-CAM or the sodium channel can be induced to express surface-localized PSA
when transfected with ST8Sia-II or with ST8Sia-IV.
Modification of N-CAM by PSA is apparently directed by specific peptide segments composed

of NCAM's immunoglobulin domain 5 and its adjacent Ig4 and fibronectin type III repeats,
along with a membrane attachment segment. Regulated expression of PSA during development
is directed in part by tissue-specific regulation of expression of ST8Sia-II and ST8Sia-IV
(Chapter 34). ST8Sia-II is prominently expressed in fetal brain, but it declines markedly during
the postnatal period. In contrast, ST8Sia-IV continues to be expressed postnatally in the brain
and in nonneuronal tissues such as the heart, lung, and spleen. Posttranscriptional mechanisms
can also regulate PSA expression.
PSA negatively modulates the homotypic adhesive properties of N-CAM on opposing cells. This
property correlates with the observation that the embryonic form of N-CAM is extensively
modified by PSA and is less able to participate in homotypic adhesive interactions than is the
adult form of PSA, which is not highly modified by PSA. PSA can also diminish interactions
promoted by other adhesion molecules, including L1-dependent attachment to laminin or
collagen. Since PSA is highly negatively charged, is highly hydrated, and contributes up to one
third of the molecular mass of N-CAM, it is believed that PSA negatively modulates cell
adhesion processes by physical interference with apposition of the plasma membranes of
adjacent cells. PSA can also positively modulate N-CAM-dependent adhesive interactions with
other adhesion counterreceptors, however, and can modulate the adhesive activity of
glycoproteins such as L1 on the same cell. Mechanisms to account for these observations are the
subject of ongoing study. In general, however, PSA-dependent modulation of cell-cell and cell-
matrix interactions are believed to be important to targeting of growing axons to sites of
innervation, to the migration of neuronal cells during neural development, and to physiological
plasticity in the CNS (see Chapter 34). A confirmation of these hypotheses, and discovery of
other potential functions for PSA, will await the generation and analysis of mice that are
specifically deficient in PSA expression.
The second general type of α2 8 sialic acid occurs in certain members of the ganglioside family.
These are constructed by three α2 8 sialyltransferases that are distinct from the two responsible
for PSA synthesis. These are termed ST8Sia-I (also known as GD3 synthase), ST8Sia-III, and
ST8Sia-V (see Figure 16.26). They can construct single or oligomeric α2 8 sialic acid linkages
but not the polysialylated structures elaborated by ST8Sia-II or ST8Sia-IV. These three enzymes
are generally thought to act primarily on glycolipid substrates, but in vitro studies suggest that
ST8Sia-III can also utilize N-glycans. The precise nature of the products formed by these
enzymes, in vivo, is not clear at present. An overview of some of their in vitro substrate
specificities is presented in Figure 16.26. These enzymes are prominently expressed in the brain,
where each exhibits a distinct developmentally regulated expression pattern. ST8Sia-I is also
expressed in the kidney and thymus, where it presumably contributes to the synthesis of α2 8-
sialylated gangliosides. In vitro experiments imply that certain α2 8-sialylated gangliosides may
participate in signal transduction processes in neuronal cell types, yielding differentiative
phenotypes. In vivo correlates for these observations are not yet available, and other clear
functions for the α2 8-sialylated gangliosides remain to be discovered.
Sulfated Glycans: L-Selectin Ligands, HNK-1, and Keratan

Sulfate (169 182)
In principle, any free hydroxyl group on a monosaccharide component of a glycoconjugate is a
potential position for modification by sulfation. However, in vertebrates, glycan sulfation is
generally restricted to Gal, GlcNAc, GlcA, IdA, and GalNAc moieties. Sulfation can occur at
internal or terminal positions. Chapter 11 discusses the internally sulfated glycans represented by
heparin, heparan sulfate, and chondroitin sulfate proteoglycans that contribute to the ECM of
vertebrate organisms. Only sulfated terminal glycan structures are discussed in this chapter. The
most common and well-understood members of this group of glycans include the sulfated
structures that contribute to selectin counterreceptor activity, the HNK-1 epitope, and keratan
sulfate. This group also includes the sulfated structures found on some pituitary glycoprotein
hormones. This latter topic is discussed in some detail above and is not dealt with further here.
Certain sulfated glycans contribute to the process used by lymphocytes to home to peripheral
lymph nodes. These glycans have been identified as components of the glycocalyx on the
specialized endothelium that lines the postcapillary venules in peripheral lymph nodes and other
secondary lymphoid organs. These so-called high endothelial venules support adhesion of
lymphocytes via the C-type lectin L-selectin. L-selectin-dependent adhesion occurs through
recognition of mucin-type L-selectin counterreceptors elaborated by high endothelial venules.
Glycan structural analyses disclose that sulfated forms of α2 3-sialylated, α1 3-fucosylated
glycans represented by the sialyl Lex tetrasaccharide (Figures 16.14 and 16.27) provide an
essential contribution to the L-selectin counterreceptor activity of these glycoproteins. Sulfation
has been observed at the 6-hydroxyl positions of the galactose residue and the GlcNAc residue,
both of which may contribute to L-selectin ligand activity. The biosynthesis of these structures,
the enzymes that participate in this process, and their functional attributes are discussed further
in Chapter 26.
The HNK-1 antigen is a terminally sulfated glycan that was first described on human natural
killer cells and has the cluster designation CD57. The HNK-1 epitope is also expressed by a
variety of cell types in the vertebrate nervous system, where its cell-type-specific expression
patterns change during neural development (and see Chapter 34). The HNK-1 glycan is
composed of a 3-O-sulfated GlcA moiety, in which GlcA is attached in β1 3 linkage to a
terminal galactose (Figure 16.28). This structure is synthesized by specific
glucuronosyltransferases that act on terminal (poly)lactosamine units of N-linked glycans.
Glucuronylation is followed by 3-O-sulfation of the GlcA by one or more specific
sulfotransferases. The HNK-1 epitope has also been described on O-glycans of glycoproteins, on
proteoglycans, and on glycolipids, where similar enzymatic processes lead to its synthesis. There
is evidence that different glucuronosyltransferases may be involved in the synthesis of HNK-1
epitopes on glycoproteins and glycolipids.
Figure 16.28. Synthesis and structure of the HNK-1 epitope.
The HNK-1 structure has been described as a posttranslational modification of a variety of

neuronal cell proteins, including N-CAM-1, contactin, myelin-associated glycoprotein,
telencephalin, L1, contactin, and P0, the major glycoprotein of peripheral nerve myelin. This
sulfated glycan can function as a ligand for laminin, for L-selectin and P-selectin, and for a
cerebellar adhesion protein termed amphoterin. HNK-1 can also mediate homotypic adhesive
interactions involving P0. HNK-1-dependent adhesive interactions have been implicated in cell
migration processes involving cell-cell and cell-matrix interactions and may participate in
reinnervation of muscles by motor neurons.
Keratan sulfate represents a prominent posttranslational modification of several ECM proteins,

whose structures and functions are discussed in detail in Chapter 11. Keratan sulfate is a
polylactosamine attached to proteins via typical N-glycans (KS type I) or by a typical O-glycan
moiety (KS type II). It is believed that keratan sulfate is synthesized in a stepwise manner by
enzymes involved in polylactosamine synthesis, in coordination with a pair of 6-O-
sulfotransferase activities (Figure 16.29). A keratan-specific N-acetylglucosamine 6-O-
sulfotransferase activity utilizes unsubstituted (terminal) N-acetylglucosamine moieties but will
not effectively modify internal N-acetylglucosamine residues. This enzyme will not utilize the
GalNAc moieties in chondroitin-sulfate-type glycans, whose component is the GalNAc-GlcA
disaccharide repeat. In contrast, a second molecularly distinct 6-O-sulfotransferase activity has
been described that can apparently catalyze 6-O-sulfation of GlcNAc moieties in keratan sulfate
and GalNAc moieties in chondroitin sulfate. The relative quantitative contributions made by
each GlcNAc 6-O-sulfotransferase to keratan sulfate synthesis remain to be determined.
Figure 16.29. Synthesis of keratan sulfate by enzymes involved in polylactosamine synthesis
and with a pair of 6-O-sulfotransferase activities. Keratan sulfate is attached (R) to proteins via
an N-linked glycan (KS type I) or an O-linked glycan (KS type II).
The galactose 6-O-sulfotransferase efficiently sulfates galactose moieties within keratan sulfate
chains but is apparently more efficient in this sulfation reaction when the galactose to be
modified is adjacent to a 6-O-sulfated GlcNAc moiety. These observations imply that sulfation
of GlcNAc moieties can only occur when free GlcNAc moieties exist during synthesis and
elongation of polylactosamine chains, whereas sulfation of galactose moieties may occur at any
time during polylactosamine chain synthesis.
There are as yet few defined functions for keratan sulfate. Corneal keratan sulfate (KS I)
apparently maintains the proper spatial organization of type I collagen fibrils in the cornea that
promotes transparency of this structure. Alterations in the degree of sulfation of keratan sulfate
are associated with corneal opacity in a disease known as macular corneal dystrophy. Alterations
in the amount of sulfation of brain keratan sulfate have been found in the brains of Alzheimer's
disease patients. The pathophysiological relevance of the latter observation has not yet been
defined.
Future Directions
The presently large number of terminal glycan chain modifications continues to expand with
ongoing experimentation involving glycan isolation and increasingly sensitive structural
analyses. Where it has been explored, the careful regulation of terminal glycan structural
diversity in whole animals implies that these structures may function to transfer biological
signals between cells and their neighbors or between cells and the molecules in their
environment. There are also examples where specific terminal glycan moieties mediate receptor-
dependent interactions leading to glycoprotein clearance or to cell adhesion. Nevertheless, the
significant structural diversity displayed by glycans is not yet accompanied by a corresponding
functional diversity. As noted in Chapter 33, genetic modification of the glycosylation phenotype
in intact organisms represents a powerful approach to exploring and confirming postulated roles
for terminal glycan structures, as well as for discovering new functions for them. The large
number of glycosyltransferase loci that contribute to the terminal glycan structural diversity, the
overlap existing in some of their biosynthetic pathways, and the multiplicity of organs and
tissues where these structures may be found together imply that a correspondingly large number
of such experiments will be needed before a more complete understanding can be expected of the
functions generated by glycan diversification.
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17. Glycosyltransferases
Primary contributions to this chapter were made by J.B. Lowe (HHMI/University of Michigan
Medical School, Ann Arbor) and A. Varki (University of California at San Diego).
THIS CHAPTER OUTLINES GENERAL INFORMATION regarding the glycosyltransferases

involved in the biosynthesis of glycan chains, focusing mainly on enzymes involved in the
synthesis of various glycan classes in the Golgi apparatus. An outline is presented of their
general structural features, the intracellular processing events to which they are subjected, and
the regulation of their expression. Sequence relationships among families of functionally related
enzymes are briefly mentioned. The glycosidic linkages constructed by these enzymes and issues
of their biological significance are dealt with in other chapters.
Glycan Biosynthesis Is Primarily Mediated by

Glycosyltransferases (1 12)
A few of the enzymes required for glycan assembly are processing glycosidases, trimming
specific monosaccharides from precursors to form synthetic intermediates. These are not
discussed here. Most of the enzymes involved in glycan biosynthesis are glycosyltransferases,
which typically act by adding monosaccharides one at a time to specific positions on specific
precursors. As noted in Chapter 16, and elsewhere in this volume, the biosynthesis of glycans is
primarily determined by these sequentially acting enzymes, which assemble monosaccharides
into linear and branched sugar chains. Many, but not all, of these enzymes are found within the
ER-Golgi pathway for export of newly synthesized glycoconjugates. As might be expected from
the complex array of glycan structures found in nature, the glycosyltransferases are a very large
family of enzymes of ancient evolutionary origin. However, they share some common features,
which are discussed in this chapter.
With few exceptions, glycosyltransferases catalyze transglycosylation reactions where the

monosaccharide component of a high-energy nucleotide sugar donor (e.g., GDP-Fuc or CMP-
Sia; see Chapter 6) is transferred to a precursor termed an acceptor (Figure 17.1). Exceptions to
this generalization include the oligosaccharyltransferase, an enzyme that utilizes a large,
dolichol-linked oligosaccharide substrate (see Chapter 7), some of the mannosyltransferases and
glucosyltransferases involved with assembly of this dolichol-linked oligosaccharide, which
utilize dolichol-phosphate-linked mannose or glucose as high-energy donors, and related
transferases in bacteria that utilize undecaprenyl-phosphate-linked sugars. The acceptor
molecules for most glycosyltransferases are usually oligosaccharides themselves (see Chapter
16). However, glycoprotein synthesis can require an initiating attachment of a single
monosaccharide to a threonine or serine residue (see Chapters 8, 11, 12, and 14). In this instance,
a protein serves as the acceptor substrate. Likewise, the oligosaccharyltransferase utilizes a
protein as an acceptor in the initiation of N-glycosylation. Finally, glycolipid biosynthesis begins
with a galactosyltransferase or glucosyltransferase that utilizes a ceramide as an acceptor
molecule (see Chapter 9). There are, of course, other glycosyltransferases in nature that are not
involved in glycan biosynthesis, such as those involved in forming small sugar glycosides (e.g.,
in the detoxification of drugs by glucuronidation). These glycosyltransferase reactions are not
considered in this book.
Figure 17.1. A generic glycosylation reaction. A glycosyltransferase utilizes a glycosyl donor
and an acceptor substrate. Glycosyl donors can include nucleotide sugars and dolichol-
phosphate-linked mono- and oligosaccharides. Acceptors can take the form of oligosaccharides
(most commonly) or (rarely) can be monosaccharides. Proteins and ceramides are also acceptors
for the glycosyltransferases that initiate glycoprotein and glycolipid synthesis.
General Properties of Glycosyltransferase Reactions (1, 2, 4,5,7, 12)

Most glycosyltransferase-dependent transglycosylations involve a divalent cation as a cofactor
(typically Mg++ or Mn++), and the enzymes tend to be most active in the pH range of 5.0 to 7.0,
which reflects pH values found in various parts of the ER-Golgi-plasmalemma pathway.
Glycosyltransferases typically exhibit Michaelis-Menten constants (Kms) for nucleotide sugar
substrates in the low micromolar range, when assayed in vitro. These values are thought to
reflect the estimated concentrations of nucleotide sugars within the relevant intracellular
compartments, but the actual concentrations inside the Golgi have not been directly measured.
Generally speaking, Km values for acceptor substrates observed in vitro can vary dramatically for
different enzymes, ranging from low micromolar values to low millimolar values. However, in
vitro assays are not likely to faithfully recapitulate circumstances found in the subcellular
compartments of the secretory pathway where these enzymes function. Moreover, these assays
often utilize acceptor analogs composed of only a small portion of the physiological substrate.
For these reasons, in vitro acceptor Km data in the literature may not reflect actual affinities for
the natural substrates. Furthermore, the catalytic sites of most glycosyltransferases face the
lumen of various compartments in the Golgi apparatus, and the low-molecular-weight sugar
nucleotide-donors are made in the cytosol. Thus, for glycosylation to occur, these donors must be
specifically transported from the cytosol into the lumen of the Golgi compartment (see Chapter
6). Thus, several factors can regulate the action of each Golgi glycosyltransferase, including the
intraluminal concentration of the sugar nucleotide donor resulting from donor transport, the
presence of specific sugar nucleotide degrading enzymes, competition by other
glycosyltransferases for the same donors or acceptors, the intraluminal concentration of the
acceptors, the intraluminal pH, and the time taken for passage of the acceptor molecule through a
given Golgi compartment. In an intact cell, many or all of these factors may interact to determine
the final structures of the glycans synthesized on a given glycoconjugate that is passing through
the Golgi apparatus. For obvious reasons, many of these topological and kinetic considerations
do not apply to cytosolic glycosyltransferases, which generally have direct access to both the
donor and the acceptor (see Chapters 14 and 15).
Sequential Actions of Glycosyltransferases (1 5,7,12 15)
Generally speaking, glycosyltransferases act sequentially, such that the oligosaccharide product
of one enzyme yields a product that serves as a preferred acceptor substrate for the subsequent
action of other glycosyltransferases. The end result is a linear and/or branched polymer
composed of monosaccharides linked to one another. As implied by Chapter 16, and other
chapters in this book, the component monosaccharides are linked to one another with
extraordinary precision. This precision is a function of the very narrow acceptor substrate
specificity exhibited by most glycosyltransferases.
The human B blood group galactosyltransferases provide an excellent example of such

specificity. As detailed in Chapter 16, this enzyme catalyzes a transglycosylation in which
galactose is added in α anomeric linkage to carbon 3 of a subjacent galactosyl residue (Figure
17.2). This enzyme's inherent catalytic attributes dictate that it will utilize only UDP-Gal as a
donor, and catalyze the formation of an α anomeric linkage, but not a β anomeric linkage.
Furthermore, the enzyme will add the new residue only to the hydroxyl group of carbon 3 on the
galactose moiety of the acceptor substrate, but not to any other hydroxyl group on the galactose
moiety, nor to any other hydroxyl group on the acceptor molecule that could in principle allow
the formation of a glycosidic linkage. Importantly, the galactose moiety on the acceptor substrate
must have been modified first by the attachment of fucose in α1 2 linkage (see Chapter 16,
which describes individuals with the Bombay blood group who fail to add the fucose in α1 2
linkage, hence preventing the action of the A or B transferases). Furthermore, prior modification
of the galactose moiety by other monosaccharides, like an α2 6-linked sialic acid moiety (Figure
17.2), yields a glycan that is no longer utilized by the blood group B enzyme, even if this
modification leaves the galactose moiety's carbon 3-OH "available." Similar general
considerations apply to virtually all known vertebrate glycosyltransferases and led early on to the
concept that each glycosidic linkage is the product of a single enzyme.
Figure 17.2. Strict acceptor substrate requirement of the human B blood group α1 3
galactosyltransferase. The B transferase adds galactose in α1 3 linkage to the H antigen (top).
This enzyme requires the α1 2-linked fucose modification of the H antigen for activity, since
the B transferase does not add to an unmodified type-2 precursor (middle), nor to precursors
modified by sialyl residues (bottom) or other monosaccharides (not shown).
Exceptions to the One-Linkage-One-Enzyme Paradigm (12,16 19)
Several exceptions to the one-linkage-one-enzyme paradigm have emerged. First, it is becoming

increasingly clear that a specific glycosidic linkage may actually be the product of one of several
structurally and genetically related enzymes. Many such catalytically redundant
glycosyltransferase families are known, with members of the family of mammalian α2 8
sialyltransferases providing an excellent example (see Chapter 16). Second, it is clear that a few
glycosyltransferases (notably, the α1 3 fucosyltransferase, FucT-III) can synthesize two
different glycosidic linkages. Third, the acceptor specificity of an enzyme can be modified by
another protein (the classic example is α-lactalbumin, which switches the acceptor specificity of
β1 4 galactosyltransferase from GlcNAc to Glc, allowing lactose synthesis during milk
formation). Finally, some of the enzymes can catalyze two stepwise glycosyltransferase reactions
(e.g., the copolymerases that synthesize the backbones of glycosaminoglycan chains; see Chapter
11). In some cases, mutant cell lines (Chapter 31) or mice (Chapter 33) have also revealed
alternate pathways and/or the existence of new glycosyltransferases. Regardless of these added
complexities, the sum of the diverse yet extremely precise acceptor substrate specificities
exhibited by the repertoire of glycosyltransferases in a given cell type limits the combinatorial
possibilities for linking several monosaccharides together. Thus, it is reasonable to expect that
the set of glycan structures observed on any given cell type will generally reflect the set of
glycosyltransferases expressed by the cell. However, the situation is complicated by the many
other factors that affect the action of a given enzyme (see above) and the way in which
glycosyltransferases are organized in subcompartments of the cell (see below).
Effects of the Underlying Polypeptide on Glycosyltransferase

Action (20 25)
For most glycosyltransferases, specific recognition of the acceptor involves only one or a few
monosaccharide units or another residue, such as a lipid or an amino acid. However, in a few
cases, the underlying protein is known to specifically dictate the action of a particular
glycosyltransferase. As examples, the GlcNAc-phosphotransferase involved in lysosomal
enzyme targeting (see Chapter 23) recognizes specific lysine residues and other structural motifs
in the secondary structure of lysosomal enzymes and selectively phosphorylates the N-glycans
on these proteins; pituitary glycoprotein hormones have a Pro-Xaa-Arg/Lys sequence that
dictates the addition of GalNAc residues that subsequently undergo a biologically important 4-
O-sulfation (see Chapter 16), and features of the core protein dictate whether a proteoglycan
receives a heparan sulfate or chondroitin sulfate chain (see Chapter 11). However, for most
proteins, there is as yet no clear evidence for such recognition by glycosyltransferases. Despite
this, specific glycosylation sites of most proteins from normal tissues carry predictable and
specific glycan structures. To some degree, this may be because the underlying polypeptide
chain may serve to sterically limit the range of action of glycosyltransferases. However, direct in
vitro competition studies between glycosyltransferases suggest that additional as yet unknown
features allow glycoproteins to regulate their own glycosylation.
Molecular Cloning of Glycosyltransferase Genes (5 12,26 30)
It has been estimated that more than 100 distinct glycosidic linkages are present in the
glycoconjugate repertoire of any given vertebrate species. Since each linkage is usually the
product of a different glycosyltransferase, and each linkage is often found to be the product of
several distinct glycosyltransferases, it appears that there are at least several hundred different
glycosyltransferase loci in the genome of any given vertebrate. It is somewhat remarkable that
virtually nothing was known about the primary structures of these genetic loci and their cognate
enzymes until 1986, with the first report of the molecular cloning of a mammalian β1 4
galactosyltransferase. The delay in molecular cloning efforts relevant to glycosyltransferases can
be explained by the fact that these enzymes are often extraordinarily difficult to purify in
quantities sufficient to obtain protein sequence information or to generate specific antisera.
Although this approach has continued to be used successfully for the molecular cloning of some
glycosyltransferases, expression cloning approaches have also contributed substantially to the
repertoire of cloned glycosyltransferase sequences. Subsequently, it has become possible to
clone new glycosyltransferase loci using low-stringency cross-hybridization approaches, and
methods involving the PCR with PCR primers derived from conserved sequences among
members of a glycosyltransferase gene family. More recently, expressed sequence tag (EST)
databases have served as a rich source for finding new glycosyltransferases.
Primary Sequence Relationships among Glycosyltransferases (5

11,26 29,31 38)
As this repertoire of cloned glycosyltransferases has expanded, it has become clear that extensive
primary nucleotide sequence similarity exists within members of some enzyme families.
However, it is somewhat surprising that there is relatively limited homology between members
of different families. Thus, for example, sialyltransferases have no detectable homology with β-
galactosyltransferases. In contrast, there can be a high degree of conservation of the sequence of
a given enzyme between widely divergent vertebrate and invertebrate taxa. These observations
imply that the different glycosyltransferase families are evolutionarily very ancient and that there
have been strong selection pressures maintaining sequence similarities within families.
Analysis of sequence similarities within families has yielded a few shared amino acid sequence
motifs. The first of these recognized were the "sialyl-motifs" that are shared among different
sialyltransferases (Figure 17.3). Mutagenesis studies of the sialyl motifs suggest that these may
be involved in nucleotide sugar recognition. More recent comparisons among newly recognized
galactosyltransferases, fucosyltransferases, and N-acetylgalactosaminyltransferases suggest the
presence of other motifs unique to each of these families. It is likely that additional motifs will
emerge within each family as more members are cloned and sequenced. In this regard, an
intriguing report indicates that motifs common to glycosyltransferases involved in bacterial lipo-
oligosaccharide biosynthesis share some homology with Drosophila Fringe (FNG) and Brainiac
(BRN) proteins, which are involved in pattern formation during development. As crystal
structures of glycosyltransferases begin to emerge, a better understanding can be expected of the
three-dimensional organization and function of such sequences.
Figure 17.3. Domain structure of a typical sialyltransferase showing the sialyl motifs shared by
this family of enzymes. The sialyl L motif of 48 49 amino acids shares significant homology
among members and may be up to 65% identical in amino acid sequence. The sialyl S motif is
smaller (~23 amino acids) and diverges more among members of the family, with only two
identical amino acids. In both cases, identical residues are indicated and positions of homologous
residues are denoted by parentheses. The asterisk denotes the position of a highly conserved
sequence H-X(4)-E.
Golgi Glycosyltransferases Have a Shared Secondary Structure
(3,5, 6,39 44)
Despite the lack of sequence homology between different families of glycosyltransferase, almost
all of the Golgi enzymes do share some secondary features. Early studies of the cell biology and
biochemistry of vertebrate glycosyltransferases indicated that some glycosyltransferase activities
could be found in soluble form in secretions and body fluids; others were identified as
membrane-bound activities within cells. Some activities exhibited both properties. Cell
fractionation studies generally found cell-associated glycosyltransferase activities in membrane-
rich microsomal fractions, which could be liberated in soluble form with the aid of detergents.
These observations implied that some glycosyltransferases probably represent membrane-
spanning proteins, whereas others correspond to secreted proteins. It was only following the
initial molecular cloning efforts that defined the primary sequences of a β1 4
galactosyltransferase, an α2 6 sialyltransferase, and the blood group A α1 3 N-
acetylgalactosaminyltransferase, that it became clear that Golgi glycosyltransferases shared a
common secondary structure that could account for all of these previous findings. Indeed, all
Golgi glycosyltransferases described to date have a single transmembrane domain flanked by a
short amino-terminal domain and a longer carboxy-terminal domain. This structure is
characteristic of so-called type II transmembrane proteins, whose single amino-terminal
membrane-spanning domain functions as a signal-anchor sequence, placing the short amino-
terminal segment within the cytosol, while directing the larger carboxy-terminal domain to the
other side of the biological membrane into which the signal-anchor has been inserted (Figure
17.4). For plasma membrane-associated type II proteins, the "other side" means the extracellular
surface. For glycosyltransferases, the "other side" means the lumen of the membrane-delimited
compartments that constitute the ER-Golgi pathway. These include vesicles that transit from the
ER to the cis-cisterna of the Golgi, the cisternae of the Golgi apparatus itself, the vesico-tubular
network of the trans-Golgi network, and membrane-delimited structures distal to the trans-Golgi
network. This arrangement predicts that the large carboxy-terminal domain corresponds to the
catalytic domain of the glycosyltransferase, and this supposition has extensive experimental
support. The intraluminal location of this domain allows it to participate in the synthesis of the
growing glycans displayed by glycoproteins and glycolipids during their transit through the
secretory pathway. This type II transmembrane topology predicted by the sequences of
vertebrate glycosyltransferases has been widely confirmed experimentally. The topology may
also explain reports of the expression of glycosyltransferases at the surface of mammalian cells
(see Chapter 34). Interestingly, several other Golgi enzymes (processing glycosidases and
sulfotransferases) that have been cloned share a similar topological arrangement. However, two
clear exceptions are the UDP-GlcNAc:lysosomal-enzyme N-acetylglucosamine-1-
phosphotransferase (GlcNAc-phosphotransferase) and the GlcNAc-1-phosphodiester α-N-
acetylglucosaminidase, which are involved in the synthesis of the Man-6-P targeting signal of
newly synthesized lysosomal hydrolases (see Chapter 23). The former is a multisubunit complex,
and the latter is a type I membrane-spanning glycoprotein with its amino terminus in the lumen
of the Golgi apparatus.
Figure 17.4. Typical transmembrane topology and proteolytic processing of vertebrate
glycosyltransferases. Vertebrate glycosyltransferases generally have a single hydrophobic
segment that functions as a signal-anchor sequence. This segment spans the lipid bilayer of the
tubular and vesicular structures of the secretory pathway, including the membrane of the Golgi
apparatus. This topology places the catalytic domain of a glycosyltransferase within the lumen of
the Golgi apparatus, and other membrane-delimited structures of the secretory pathway. The
membrane-tethered form of a glycosyltransferase is susceptible to one or more proteolytic
cleavage events that transect the enzyme within its "stem" region. Proteolysis can liberate a
catalytically active, soluble form of the enzyme that may be released from the cell. With few
exceptions, vertebrate glycosyltransferases have one or more potential asparagine-linked
glycosylation sites (depicted by the forked symbols). Where examined experimentally, one or
more of these sites are utilized, indicating that most glycosyltransferases are glycoproteins.
All of the above considerations also do not apply to the glycosyltransferases involved in nuclear
and cytoplasmic glycan synthesis. For example, the GlcNAc transferase responsible for
synthesizing the O-linked GlcNAc of nuclear and cytoplasmic proteins (see Chapter 14 ) has no
detectable homology with the Golgi GlcNAc transferases. There are currently too few of these
nuclear and cytoplasmic glycosyltransferases cloned to determine whether they share any
primary or secondary structural features.
Proteolytic Cleavage and Secretion of Golgi

Glycosyltransferases (3, 6, 9,39 42, 45)
As mentioned above, many Golgi enzymes are secreted by cells, sometimes in large quantities.
The nature of the secreted forms of glycosyltransferases first became clear from an analysis of
amino-terminal peptide sequences of purified mammalian glycosyltransferases that had been
isolated as soluble forms without the aid of detergents. These studies demonstrate that the
soluble forms were actually derived from their membrane-associated forms by virtue of one or
more proteolytic cleavage events that occurred at a position a short distance away from the
membrane-spanning region, in the carboxy-terminal direction (Figure 17.4). These proteolytic
cleavage events release a catalytically active fragment of the glycosyltransferase from its
transmembrane tether and allow the cell to export this fragment to the extracellular milieu. The
cleavage of Golgi glycosyltransferases frequently occurs in the "stem region," which loosely
refers to the region between the membrane-spanning segment and the position(s) where
proteolytic cleavage occurs. The existence of catalytically active fragments of
glycosyltransferases that are also deficient in various portions of the stem region, together with
mutational analyses, implies that the stem region contributes little, if anything, to the catalytic
function of a glycosyltransferase. Nevertheless, some experimental analyses imply that peptide
sequences within the stem region can contribute to acceptor substrate utilization parameters. The
nature of the signals within the glycosyltransferase sequence that direct proteolysis is not
defined, but it appears that the proteolytic cleavages are relatively specific and are generated by
certain cathepsin-like proteases functioning in the trans regions of the Golgi apparatus and
beyond. The practical consequence is that many glycosyltransferases are also found in soluble
form in the circulation and in various body fluids. The production of these soluble enzymes from
cell types such as hepatocytes and endothelium can also be dramatically up-regulated under
certain inflammatory conditions. Since these circulating enzymes do not have access to adequate
concentrations of donor sugar nucleotides, which are primarily inside cells, they are functionally
incapable of carrying out a transfer reaction. The biological significance of these soluble
transferases therefore remains a mystery. Possibilities to consider include a lectin-like activity
recognizing their acceptor substrates, and a role in scavenging small amounts of circulating sugar
nucleotides that might otherwise be available to certain microbes, such as gonococci.
Glycosyltransferases Are Often Glycoproteins Themselves (3, 6,39

42, 46)
Many Golgi glycosyltransferases have consensus N-glycosylation sequences, as well as serine

and threonine residues that could be modified by glycosylation processes. Biochemical analyses
indicate that many mammalian glycosyltransferases are indeed posttranslationally modified by
glycosylation, especially N-glycosylation. Glycosylation is, in some instances, required for
proper folding and/or activity, and limited studies indicate that glycosyltransferases are also
subject to "autoglycosylation." There is also limited evidence that glycosyltransferases may be
modified by phosphorylation. The functional relevance of such posttranslational modifications
remains unknown.
Retention of Glycosyltransferases in Golgi Subcompartments (47

52)
Biochemical and ultrastructural studies indicate that glycosyltransferases partially segregate into
distinct compartments within the secretory pathway. Generally speaking, enzymes acting early in
glycan biosynthetic pathways have been localized to cis and medial compartments of the Golgi,
whereas enzymes acting later in the biosynthetic pathway tend to colocalize in the trans-Golgi
cisternae and the trans-Golgi network. These observations have prompted extensive exploration
of the mechanisms whereby glycosyltransferases achieve this compartmental segregation. An
effort was made to find Golgi-retention sequences, by analogy with the KDEL tetrapeptide
implicated in retention of ER-associated proteins. Although some general conclusions are
available from these studies, the reader is urged to consider the following caveats. First,
observations made with one enzyme are not necessarily applicable to others. Second, the Golgi
retention properties of any given glycosyltransferase may vary depending on the cell type in
which localization is examined. Third, variations in the level of expression of a
glycosyltransferase in an experimental system may have a major influence on
retention/localization properties. Fourth, many of these studies have used chimeric proteins
composed of segments of a glycosyltransferase fused to a reporter protein, but conclusions
derived from such experiments have not always been verified using intact glycosyltransferases or
chimeras composed of an additional reporter protein. Finally, in vitro studies using intact Golgi
compartments indicate some spatial and functional overlap between enzymes previously thought
to be segregated on the basis of other less sensitive techniques.
Most information relevant to retention of glycosyltransferases within specific Golgi

compartments derives from experiments done with α2 6 sialyltransferase (ST6Gal-I), a β1 4
galactosyltransferase (GalT-I), and an N-acetylglucosaminyltransferase I (GlcNAcT-I). The
former pair of enzymes tends to concentrate in the trans-Golgi compartments and the trans-
Golgi network, whereas GlcNAcT-I localizes mostly to the medial-Golgi compartment. With
respect to ST6Gal-I, the transmembrane domain and the sequences that flank it are sufficient to
direct heterologous proteins to the Golgi. However, it appears that the length of the
transmembrane segment (provided it is hydrophobic in nature) and not the precise sequence of
this domain may be another determinant of this enzyme's Golgi localization. Efficient Golgi
retention may be dictated by the nature of the amino acids that flank the transmembrane
segment. To complicate matters, some evidence suggests that the stem region of ST6Gal-I can
also have a profound influence on Golgi retention and can in some circumstances dominate over
Golgi retention properties conferred upon the protein by the transmembrane segment. In contrast,
an examination of the Golgi-retention determinants of GalT-I points mainly to an important role
for the transmembrane domain in this process. The sequences that flank the membrane-spanning
domain seem less important in Golgi retention for this enzyme than for ST6Gal-I. Retention of
GlcNAcT-I is also dictated largely by its transmembrane domain, although this enzyme's stem
region and residues flanking the transmembrane domain can also direct localization. Considered
together, the available experimental observations imply that retention of glycosyltransferases
within specific regions of the Golgi apparatus is probably not determined by simple primary
sequence motifs. Rather, this process is apparently determined by several different regions of
each enzyme. By analogy with the lysosomal enzymes that display a recognition motif for the
phosphotransferase that decorates them with the Man-6-P (see Chapter 23), it seems possible that
a localization or retention "signal" might consist of a group of specific amino acids brought
together to form a specific "shape" on the surface of a properly folded glycosyltransferase.
Two models have been proposed to account for retention of specific glycosyltransferases to
specific Golgi subcompartments. The first of these, termed the kin-recognition model, proposes
that members of a set of glycosyltransferases that have evolved to localize to a specific
compartment will aggregate with themselves in a multimeric complex, after arriving at the
proper Golgi compartment. This hypothetical process would involve homo- and hetero-
oligomerization through interactions between the stem regions and/or transmembrane segments
of the glycosyltransferases in the aggregate. This model also proposes that these oligomers will
be anchored to the Golgi structure through interactions between the cytosolic domains of the
enzymes in the aggregate and proteins in the Golgi matrix. Experimental support for this model
comes from the observation that some pairs of glycosyltransferases known to colocalize to a
specific Golgi compartment are coimmunoprecipitated from cells, even when one is directed to
the lumen of the ER. However, this type of association has not been demonstrated for most of the
enzymes.
A second model proposes that a cholesterol concentration gradient in the secretory pathway
yields lipid bilayers of increasing thickness in the direction of cis to trans across the Golgi
stacks. Thus, each glycosyltransferase sorts itself into the proper Golgi location by virtue of the
length of its transmembrane segment, which will retain the enzyme once it reaches the proper
compartment during the enzyme's transit through the secretory pathway. This model was
formulated largely on the basis of experiments involving ST6Gal-I, where the length of the
membrane-spanning domain appears to play an important part in Golgi retention. The general
applicability of this model is not apparent, however, since there is not a consistent relationship
between the length of the transmembrane segment and retention in a specific Golgi compartment
for a variety of glycosyltransferases. However, it is notable that Golgi enzymes in general tend to
have shorter membrane-spanning regions than plasma membrane proteins. This model may
therefore help to better explain the overall phenomenon of retention of glycosyltransferases in
the Golgi apparatus versus the delivery of other proteins to the plasma membrane.
Regulation of Glycosyltransferase Gene Expression (9, 12,53 57)
Apart from the Golgi factors that can regulate glycosyltransferase function, their actual
expression can be controlled at the level of RNA synthesis or turnover. Studies have indicated
that although the expression patterns of some glycosyltransferase mRNAs are highly regulated in
a tissue-specific and developmentally regulated manner, others have a widespread so-called
"housekeeping" type of distribution. Examples of both can be found within any given family of
enzymes. For the most part, it appears that differential regulation is due to the action of specific
promoter regions in the 5 region of the corresponding genes (see Chapter 34). In the case of
some genes (e.g., ST6Gal-I), there is evidence for multiple tissue-specific promoters that are
activated under different biological circumstances. An additional finding of interest is that
glycosyltransferase mRNAs in general tend to have long 5 -untranslated regions with extensive
secondary structure, which might result in differential message stability, especially during the
cell cycle.
Future Directions
A great many glycosyltransferases remain to be cloned and characterized. If past experience is
any guide, most new enzymes cloned will turn out to be "founder" members of a family of
related gene products. Thus, the glycosyltransferase superfamily may well turn out to represent
1% or more of the entire genetic complement of a given genome. Obviously, much work remains
to characterize the expression and regulation of these genes, and gene disruption experiments in
intact animals are likely to yield the most useful biological conclusions regarding
glycosyltransferase function in vivo. The availability of recombinant soluble enzymes will also
greatly enhance our ability to study the structure and function of these enzymes at the level of
atomic resolution. Such cloned well-characterized enzymes will also add to the armamentarium
of the chemist who synthesizes defined glycan structures for in vitro studies and in vivo
therapeutic use. Much also remains to be done to understand how the Golgi apparatus is able to
organize these numerous "workers" into an effective assembly line for the regulated production
of biologically important glycans, and how the cells modify their glycan repertoire in response to
external signals.
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18. Degradation and Turnover of Glycans
Jolla, California).
THIS CHAPTER EXPLORES THE DEGRADATION AND TURNOVER of glycoconjugates.

Most glycoconjugates are degraded in lysosomes, and a portion of the liberated monosaccharides
are reused for glycoconjugate synthesis (see Chapter 6). The degradation of glycans is ordered
and often highly specific. It involves both endo- and exoglycosidases that eventually liberate
monosaccharides, sometimes with the aid of noncatalytic proteins. Work on a series of rare
human genetic disorders called lysosomal storage diseases was critical to unraveling these
complex pathways (see Chapter 23). In each disease, a limited number of undegraded molecules
accumulate in the lysosomes. Clever experiments combining enzymology with carbohydrate
structural analysis revealed the steps of the pathways and also unlocked the mechanism of
lysosomal enzyme targeting as described in Chapter 23. The dismantling of selected glycans is
described, since each illustrates unique features.
The Lysosomal Enzymes (1 2)
Most of the endo- and exoglycosidases that degrade sugar chains and their modifications have
pH optima between 4.0 and 5.5, but exceptions exist. Some lysosomal enzymes have much
higher pH optima. Exoglycosidases cleave the glycosidic linkage of terminal sugars from the
nonreducing end of the chain. By convention, that means that the residue at the outermost end of
the molecule is at the extreme left end in a graphical depiction. The exoglycosidases recognize
only one (occasionally two) monosaccharide together with its anomeric linkage, but they are
much less particular about the structure of the molecule beyond the glycosidic linkage. This
feature allows these enzymes broad specificity. However, exoglycosidases do not usually act
unless all of the hydroxyl groups of the substrate sugar are unmodified. Substitutions such as O-
acetyl groups, sulfate, or phosphate esters usually must be removed before exoglycosidases can
work. Esterases cleave acetyl groups, and specific sulfatases cleave various sulfate groups from
glycosaminoglycans and from N- or O-linked sugar chains before exoglycosidase cleavage. The
endoglycosidases cleave internal glycosidic linkages of larger chains, yielding fragments that can
then be degraded by exoglycosidases. Endoglycosidases are often more tolerant of modifications
of the sugar chain residues, and in some cases, they even require them for optimal cleavage.
Even though the lysosomal glycosidases carry out similar reactions, they have only about 15
20% identical and 40 45% similar amino acids. There are no highly conserved "glycosidase"
catalytic domains. Lysosomal enzymes are all N-glycosylated and most are targeted to the
lysosome by the Man-6-P pathway discussed in Chapter 23. Therefore, they must share aspects
of the recognition marker for GlcNAc-phosphotransferase and variable affinities for the large
and small Man-6-P receptors. The concentration of enzymes within the lysosome is difficult to
determine, but proteinases such as cathepsins B, D, and L have been estimated at 1 mm.
Glycosidases are probably present at much lower concentrations.
Genetic Defects in Lysosomal Degradation of Glycans (1,3 5,9 10)
Loss of a lysosomal enzyme leads to accumulation of undegraded material in tissues and

secretion of fragments into the urine. Most of the human disorders listed in Table 18.1 have
animal models. Most of these affect glycoprotein degradation, except for the loss of β-
galactosidase and β-N-acetylhexosaminidase, which also affects glycolipids.
Table 18.1. Defects in glycoprotein degradation
Affects degradation of
Disorder Defect glycoprotein glycolipid Clinical symptoms
α-Mannosidosis types I α-mannosidase major none type I: infantile onset,

and II progressive mental
retardation, hepatomegaly,
death between 3 and 12
years
type II: juvenile/adult onset,
milder, slowly progressive
β-Mannosidosis β-mannosidase major none severe quadriplegia, death
by 15 months in most
severe; mild cases have
mental retardation,
angioker-atoma, facial
dysmorphism
Aspartylglucosaminuria aspartyl- major none progressive, coarse facies,
glucosaminidase mental retardation
Sialidosis sialidase major minor progressive, severe
(mucolipidosis I) mucopolysaccharidosis-like
features, mental retardation
Schindler types I and II α-N-acetyl yes ? type I: infantile onset,
galactosaminidase neuroaxonal dystrophy,
severe psychomotor and
mental retardation, cortical
blindness neurodegeneration
type II: mild intellectual
impairment, angiokeratoma
corpis diffusum
Galactosialidosis protective major minor coarse facies, skeletal
protein/cathepsin dysplasia, early death
A
Fucosidosis α-fucosidase major present spectrum of severities
includes psychomotor
retardation, coarse facies,
growth retardation
GM1 gangliosidosis β-galactosidase present major progressive neurologic
disease and skeletal
dysplasia in severe infantile
form
GM2 gangliosidosis β-hexosaminidase present major severe form:
neurodegenerative with
death by 4 years
less severe: slower onset of
symptoms and variable
symptoms, all relating to
various parts of the CNS
Tables 18.1, 18.2, and 18.3 also show some of the major clinical symptoms of diseases
associated with degradation of each type of glycoconjugate. In comparing all of the different
diseases in these three tables, two features stand out. First, many of the diseases share
overlapping symptoms, and yet each disease has unique features that rely on experienced
clinicians for specific diagnosis. Second, many of the diseases present with a range of severities.
Usually an infantile onset is the most severe, and the juvenile or adult onsets are milder. The
later-onset forms may even affect different systems than those affected by the early-onset forms.
It is not clear how accumulating different types of "undegraded material" in a lysosome leads to
the different symptoms in each disease. It may mean that accumulating material is not the cause
of different clinical manifestations, but it is an indicator of its severity. To the best of our
knowledge, the cell has no way of sensing the chemical nature of accumulated material. The
"lysosomal storage disease" label provides a convenient unifying theme. The range of clinical
severities and time of onset may be related to the amount of residual activity in the defective
enzyme and to the fine balance between synthesis and degradation of multiple glycans. Reducing
synthesis somewhat may help to retard accumulation of undegraded material. These aspects are
discussed later in this chapter.
Table 18.2. Mucopolysaccharidoses: Classification of defects and clinical

symptoms
Common Enzyme Glycosaminoglycan

Number name deficiency affected Clinical symptoms
MPS I Hurler α-l-iduronidase DS, HS corneal clouding,

Ha organomegaly, heart
disease, mental
retardation, death in
childhood
MPS II Hunter iduronate-2-sulfatase DS, HS severe: organomegaly, no
corneal clouding, mental
retardation, death before
15 years
less severe: normal
intelligence, short stature,
survival age to 20 60
MPS III Sanfilippo heparan N-sulfatase HS Profound mental
A A deterioration,
hyperactivity, relatively
mild somatic
manifestations
MPS III Sanfilippo α-N- HS similar to III A
B B acetylglucosaminidase
MPS III Sanfilippo acetyl CoA:α- HS similar to III A
C C glucosaminide
acetyltransferase
MPS III Sanfilippo N-acetylglucosamine 6- HS similar to III A
D D sulfatase
MPS IV Morquio A galactose-6-sulfatase KS, CS distinctive skeletal
A abnormalities, corneal
clouding, odontoid
hypoplasia, milder forms
known to exist
MPS IV Morquio B β-galactosidase KS same as IV A
B
MPS VI Maroteaux- N-acetylgalactosamine 4- DS corneal clouding, normal
Lamy sulfatase intelligence, survival to
teens in severe form;
milder forms known to
exist
MPS Sly β-glucuronidase DS, HS, chondroitin wide spectrum of
VII 4-, 6-sulfates severity, including
hydrops fetalis and
neonatal form
Adapted, with permission, from [11] Neufeld and Muenzer (1995).

a
MPS is mucopolysaccharidoses.
Table 18.3. Disorders in glycolipid degradation
Disease name Enzyme or protein deficiency Clinical symptoms
Tay-Sachs β-hexosaminidase A severest form: neurodegenerative with death

by 4 years
less severe: slower onset of symptoms and
variable symptoms all relating to parts of the
nervous system
Sandhoff β-hexosaminidases A and B same as Tay-Sachs
GM1 gangliosidosis β-galactosidase see Table 18.1
Sialidosis sialidase see Table 18.1
Fabry α-galactosidase severe pain, angiokeratoma, corneal
opacities, death from renal or
cerebrovascular disease
Gaucher β-glucosceramidase severe form: childhood or infancy onset,
hepatosplenomegaly, neurodegeneration
mild form: child/adult onset, no
neurodegenerative course
Krabbe β-galactoceramidase early onset with progression to severe
mental and motor deterioration
Metachromatic arylsulfatase A (cerebroside infantile, juvenile, and adult forms can
leukodystrophy sulfatase) SO4GalβCer include mental regression, peripheral
accumulates neuropathy, seizures, dementia
Multiple sulfatase cysteine formyl glycine hypotonia, retarded psychomotor
deficiency development, quadriplegia
Saposin deficiency saposin precursor similar to Tay-Sachs and Sandhoff
Glycoprotein Degradation
The great majority of N- and O-linked oligosaccharide chains reaching the lysosome contain
only six sugars β-GlcNAc, α/β-GalNAc, α/β-Gal, α/β-Man, α-Fuc, α-Sia and each should
theoretically require only one anomer-specific glycosidase, assuming that each enzyme cleaves
its substrate when bound to any other component. This number is actually quite close to the
known number of enzymes in the degradation pathways. However, some linkages require a
specific enzyme outside of this group, β-N-acetylhexosaminidase cleaves both β-GlcNAc and β-
GalNAc residues, and degradation of the GlcNAcβ-Asn and GalNAcα-Ser/Thr linkages also
requires specific enzymes.
Complex N-linked Chains (1,3 5)
Much of what we know of this pathway comes from analysis of products that accumulate in
patients missing one of the degradative enzymes (Table 18.1) or from structural analysis of
monosaccharide-labeled glycoproteins during degradation in perfused rat liver. By carrying out
the latter studies in the presence of inhibitors of different lysosomal enzymes, a picture emerges
of simultaneous and independent bidirectional degradation of the protein and the carbohydrate
chains (Figure 18.1). The relative degradation rates vary depending on structural and steric
factors of the protein and the sugar chains. The accumulation of GlcNAcβ1 4GlcNAcβ-Asn in
cells that cannot cleave the GlcNAcβ-Asn linkage clearly shows that degradation of the sugar
chain does not require prior cleavage from the protein. Much of the protein is probably degraded
before N-glycan catabolism begins. Removal of the core fucose (Fucα1 6GlcNAc) and probably
any peripheral fucose residues linked to the outer branches of the chain (Fucα1 3GlcNAc)
appears to be the first step in degradation, since patients lacking this enzyme still have intact N-
linked chains bound to asparagine. Glycosylasparaginase (aspartyl-N-acetyl-β-d-
glucosaminidase) then cleaves the GlcNAcβ-Asn bond, provided the α-amino group is not in
peptide linkage. In rodents and primates, chitobiase (an endo-β-N-acetylglucosaminidase)
removes the reducing GlcNAc, leaving the oligosaccharide with only one terminal GlcNAc. In
many other species, splitting of the chitobiose linkage (GlcNAcβ1 4GlcNAc) uses the β-N-
acetylhexosaminidase mentioned below as the last step in degradation. Because either pathway
appears to get the job done, the presence of this endo-β-N-acetylglucosaminidase (also called
chitobiase) in some species is unexplained. The oligosaccharide chain is then sequentially
degraded by sialidases and/or α-galactosidase and then β-galactosidase, β-N-
acetylhexosaminidase, and α-mannosidases. The remaining Manβ1 4GlcNAc is split by
mannosidase to mannose and GlcNAc or, in those species that do not have chitobiase, to
chitobiose, which is then degraded by β-N-acetylhexosaminidase.
Figure 18.1. Degradation of complex N-linked oligosaccharide chains in primates and rodents.
The lysosomal degradation pathway of glycoproteins carrying complex-type oligosaccharides
proceeds simultaneously on the protein and oligosaccharide moieties. The oligosaccharides are
sequentially degraded by the indicated exoglycosidase in a specific order as discussed in the text.
The asterisks indicate the residue targeted for hydrolysis in the next step.
Lysosomal sialidase (neuraminidase), β-galactosidase, and a serine carboxypeptidase called

protective protein/cathepsin A form a complex in the lysosome that is required for efficient
degradation of sialylated glycoconjugates. Cathepsin A protects β-galactosidase from rapid
degradation and also activates the sialidase precursor. Mutations in this protective protein lead to
galactosialidosis in which the simultaneous deficiencies in β-galactosidase and α-sialidase are
secondary effects.
Other oligosaccharides having GalNAcβ1 4GlcNAc or GlcAβ1 3Gal or GalαGal on the outer
branches must first have these residues removed by β-N-acetyl-hexosaminidase, β-
glucuronidase, and α-galactosidase prior to any further digestion of the underlying
oligosaccharide chain.
High-mannose-type Oligosaccharides (6 7)
High-mannose-type chains are hydrolyzed in the lysosome by an α-mannosidase to yield

Manα1-6Manβ1 4GlcNAc, a common intermediate derived from hybrid and complex chains, in
addition to the high-mannose type. A second α1,6-specific mannosidase can cleave this linkage
in humans and rats, but only on molecules that have a single core region GlcNAc, i.e., those
generated by chitobiase cleavage. Finally, β-mannosidase completes the job.
ER and Cytosolic Degradation of Oligosaccharides Derived from Dolichol

Precursors and Misfolded Glycoproteins (6 8)
High-mannose-type oligosaccharides are also partially degraded in the cytosol by a different set
of α-mannosidases with higher pH optima. These enzymes degrade free glycans liberated from
the dolichol-linked precursors or from newly synthesized but misfolded glycoproteins that are en
route to proteasome-mediated degradation. The degradation of the lipid-linked precursor begins
with a pyrophosphatase cleave of the nonglucosylated cytosolic-facing precursor, primarily
Man5. Normally, the next step in the biosynthetic pathway involves flipping the lipid-linked
oligosaccharide to the lumen of the ER for completion. Oligosaccharide precursors that fail to
flip may be degraded in this way. Once the phosphorylated Man5 chain is liberated from the
lipid, a phosphatase and a cytosolic endo-β-N-acetylglucosaminidase remove the phosphate and
reducing GlcNAc. Other dolichol-linked oligosaccharides may also be hydrolyzed within the
lumen of the ER, and transported into the cytosol, but the details of this pathway and its relation
to cleavage of oligosaccharides from proteins in the ER are less clear. Regardless of the
mechanism, the cytosolic endo-β-N-acetylglucosaminidase mentioned above (a chitobiase
distinct from the lysosomal one) clips a single GlcNAc. Several mannose residues are also
removed, producing the same Man5 structure mentioned above. This molecule is then imported
into the lysosome for its final degradation. Up to one third of the total lipid-linked
oligosaccharides can be recovered as free glycans. These cleavages may be important for
coordinating the level of oligosaccharide intermediates with protein synthetic rates.
Poorly folded glycoproteins were previously thought to be degraded in the ER, but more
recently, it has become clear that this degradation occurs in the cytosol as an ATP-dependent
process. The emerging view is that misfolded glycoproteins are reverse-translocated into the
cytoplasm through the Sec61 translocation channel in the ER. The release of the polypeptide into
the cytoplasm depends on ubiquitination, and final degradation is proteasome-dependent.
Depending on the protein, the N-glycan may require a small amount of mannose trimming prior
to ubiquitination, but complete degradation of the protein requires removal of all N-glycans
using a cytosolic peptide-N-glycoamidase (not endoglycosidase) activity. These liberated
oligosaccharides can then be cleaved by the cytosolic endo-β-N-acetylglucosaminidase and α-
mannosidases similar to the degradation of the chains liberated from lipid-linked
oligosaccharide.
Some evidence suggests that the removal of glycan chains from proteins might be
physiologically important. Proximal peptide-N-glycoamidases can release the entire
oligosaccharide chain, and in doing so, they convert an asparagine to an aspartic acid residue.
Amino acid sequencing of some proteins shows aspartic acid where the DNA sequence predicts
asparagine. The liberated glycans cannot be simply added to other glycoproteins in the ER, since
addition requires the pyrophosphate-linked dolichol donors and not just the sugar chain itself.
The liberated chains may need to be exported to prevent them from competing with ER lectins
such as calnexin, calreticulin, or ERGIC53, a mannose-binding lectin in the ER.
O-Linked Oligosaccharides (9)
Degradation of typical α-GalNAc-initiated O-glycans has not been systematically studied. Since
so many of the structures seen on N-linked chains are also found on O-glycans, they are probably
degraded by the same group of exoglycosidases discussed above. The exception to this of course
is the linkage region, GalNAcα-O-Ser/Thr. Patients with Schindler disease lack an α-N-
acetylgalactosaminidase. This enzyme is specific for α-GalNAc and will not cleave α-GlcNAc.
Patients lacking α-N-acetylgalactosaminidase accumulate GalNAc-containing glycopeptides in
their urine, but curiously they also accumulate more complex, extended glycopeptides containing
GlcNAc, galactose, and sialic acid. The structures are the same as those found on some native
glycoconjugates. Their production may result from a general slowdown in degradation of the
oligosaccharides, but this seems unlikely. Alternatively, the terminal GalNAcα-O-Ser/Thr
glycopeptide could be generated as a normal degradation product using normal glycosidases and
then rebuilt once again. How would this happen? There are several possibilities. One is that some
of the partially graded glycopeptides enter a compartment (Golgi?) that contains the appropriate
glycosyl transferases and sugar nucleotides. Monosaccharides are added sequentially before the
products exit the cell. In this way, the glycopeptides would be behaving like oligosaccharide
primers (see Chapter 40). However, another possibility is that the concentration of GalNAcα-O-
Ser/Thr is high enough in the lysosome that lysosomal glycosidases use them as acceptors in a
series of transglycosylation reactions. α-N-acetylgalactosaminidase has also been found to be
part of the sialidase/β-galactosidase/cathepsinA complex mentioned above. If lysosomal
glycosidases form complexes to degrade glycoconjugates more efficiently, partially degraded
substrates may be in a preferred location to be acceptors in transglycosylation reactions. The
same α-GalNAcase probably removes terminal α-GalNAc from blood-group-A-containing
glycans (GalNAcα1,3Gal) and some glycolipids such as the Forsmann antigen,
GalNAcα1,3GalNAcβ1,3Galα1,4Galβ1,4Glcβ-Cer.
Glycosaminoglycan Degradation (1,2,4,11 13)
The glycosaminoglycans, including heparan sulfate, chondroitin sulfate, dermatan sulfate,

keratan sulfate, and hyaluronan, are degraded in a highly ordered fashion. The first three are O-
xylose-linked to their core proteins, keratan sulfate can be both N- and O-linked depending on
the tissue source, and hyaluronan is made as a free chain (see Chapter 11). Some proteoglycans
are internalized from the cell surface, and the protein components are degraded. Then, the GAG
chains are partially cleaved by enzymes such as endo-β-glucuronidases or endohexosaminidases
that clip them at a few specific sites, probably depending on the sequence, generating
approximately 10-kD fragments. This is probably important, because simple exoglycosidase
digestion starting from one end would be very slow for such large chains. Endoglycosidase
cleavage creates multiple terminal residues that can all be degraded by unique or overlapping
sets of sulfatases and exoglycosidases. In general, an exoglycosidase will not degrade a
substituted sugar, so these must first be removed. Structural analysis of partially degraded
fragments in the lysosomes of cells from patients with a variety of genetic disorders called
mucopolysaccharidoses (see Table 18.2) was critical to solving these degradation pathways.
Again, note that there are a range of clinical severities and manifestations.
Hyaluronan
The most abundant GAG is HA, which is degraded in the lysosome by hyaluronidase (see Figure
18.2). The products are tetrasaccharides and larger fragments. Because HA is not synthesized on
a protein, proteolysis is not required, and it is not sulfated; therefore, only β-glucuronidase and
β-N-acetylhexosaminidase are needed for degradation, which occurs sequentially from the
nonreducing end. Pig liver hyaluronidase has a sequence identical to that of the heme-binding
serum protein hemopexin. Although serum hemopexin binds to HA, it does not appear to have
hyaluronidase activity. Expression of the pig hyaluronidase enzyme in baculovirus produces a
protein with hyaluronidase activity, showing that this primary sequence has the information
needed for enzymatic activity. The relationship between lysosomal hyaluronidase and
hemopexin is still unclear.
Figure 18.2. Degradation of hyaluronan. Hyaluronidase, an endoglycosidase, cleaves large

chains into smaller fragments, each of which is then sequentially degraded from the nonreducing
end.
Heparan Sulfate (14 15)
HS chains are first degraded by an endoglucuronidase followed by well-ordered sequential

degradation (see Figure 18.3). A terminal IdoA-2-sulfate must be desulfated by a specific IdoA-
2-sulfatase to make it a suitable substrate for α-iduronidase. If a GlcA-2-sulfate were at this
position, a GlcA-2-sulfatase would first remove the sulfate, followed by β-glucuronidase
cleavage. The terminal GlcNSO4 is the next sugar for cleavage, but this requires two steps. The
sulfate is removed by N-sulfatase forming glucosamine, but this residue cannot be cleaved by α-
N-acetylglucoaminidase. The amino group must be N-acetylated by an N-acetyl transferase
embedded in the lysosomal membrane. In the first step, acetyl-CoA donates the acetyl group to a
cytosolic-facing histidine residue at neutral pH. The acetyl group then becomes available on the
luminal side of the lysosomal membrane and transferred at low pH to the amino group of
glucosamine on the partially degraded HS chain. The terminal α-GlcNAc can now be cleaved. If
the glucuronate residue is 2-sulfated, the sulfate is first removed, followed by β-glucuronidase
cleavage. If the next α-GlcNAc is 6-O-sulfated, the sulfate is removed by a specific GlcNAc-6-
sulfatase. Figure 18.3 and Table 18.2 provide a listing of the enzymatic defects and the disorders
they cause.
Figure 18.3. Degradation of heparan sulfate. An endoglucuronidase first cleaves large chains
into smaller fragments, and then each monosaccharide is removed from the nonreducing end as
described in the text. N- and O-sulfate esters must first be removed before the exoglycosidases
can act. An unusual feature of HS degradation is that it also involves a synthetic step. After
removal of the N-sulfate residue on GlcNSO4, the nonacetylated GlcN must first be acetylated
using acetyl-CoA before α-N-acetylglucosaminidase can cleave this residue.
Dermatan Sulfate and Chondroitin Sulfate
A combination of endoglycosidases, sulfatases, and exoglycosidases degrade DS in the lysosome

(Figure 18.4). Iduronate-2-sulfatase is followed by α-iduronidase. The terminal GalNAc-4-SO4
can be removed by either of two pathways. In the first pathway, a GalNAc-4-sulfate sulfatase
acts, followed by β-N-acetylhexosaminidase A or B to remove GalNAc. In the second, β-N-
acetylhexosaminidase A removes the entire GalNAc-4-SO4 unit followed by sulfatase cleavage.
Absence of this sulfatase (MPS VI) is unique to the DS pathway. β-glucuronidase cleaves the β-
GlcA residue and the process is repeated on the rest of the molecule.
To degrade CS, GalNAc-6-SO4 sulfatase and GalNAc-4-SO4 sulfatase work in combination with
β-N-acetylhexosaminidase A or B and β-glucuronidase. Hyaluronidase may also degrade CS, but
no CS-specific endoglycosidases have been found.
Figure 18.4. Degradation of dermatan/chondroitin sulfate. Sequential degradation can proceed
by two different routes. One route uses GalNAc-4-SO4ase followed by cleavage with β-N-
acetylhexosaminidase A or B. The other route uses only β-N-acetylhexosaminidase A, since this
is one of the few exoglycosidases that can cleave sulfated amino sugars at low pH.
Keratan Sulfate
KS is a heavily sulfated polylactosamine chain. Although some organisms have an endo-β-

galactosidase that could cleave similar chains, mammalian cells do not have an endoglycosidase
to break KS down. Sequential action of sulfatases and exoglycosidases is needed. Galactose-6-
SO4 sulfatase is the same enzyme that desulfates GalNAc-6-sulfatase in CS degradation. This
hydrolysis is followed by β-galactosidase digestion, leaving a terminal GlcNAc-6-SO4.
Desulfation by the sulfatase followed by cleavage with β-N-acetylhexosaminidase A or B
eliminates GlcNAc-6-SO4. Alternatively, β-N-acetylhexosaminidase A can directly release
GlcNAc-6-SO4 followed by desulfation of the monosaccharide.
Degradation of the Linkage Regions of Proteoglycans
Degradation of the core region O-linked KS (skeletal type; type II) probably occurs by the same
route as the other typical O-linked chains (Figure 18.5). The familiar N-linked corneal-type KS
(type I) and the more recently discovered family of N-linked GAG chains are probably also
degraded by the same set of enzymes used for N-linked degradation. The more typical xylose-
linked GAG chains (DS, CS, HS) all share a common core tetrasaccharide. The finding of
GlcAβ1,3Galβ1,3Galβ1,4Xylβ-O-Ser and xylose- and galactose-terminated core fragments
suggests that endo-β-xylosidase and endo-β-galactosidase exist. An endo-β-xylosidase has been
detected in the rabbit liver. It is possible that similar enzymes cleave different types of GAGs,
but the details of how the linkage regions of GAGs are degraded remain to be established.
Figure 18.5. Stepwise lysosomal degradation of keratan sulfate. Sequential degradation of KS
occurs from the nonreducing end, and like the degradation of DS and CS, the terminal GlcNAc-
6-SO4 can be cleaved sequentially by a sulfatase and then by β-N-acetyl-hexosaminidase A or B,
or alternatively, β-N-acetyl-hexosaminidase A can cleave GlcNAc-6-SO4 directly at low pH.
Glycosphingolipid Degradation (1,16 18)
Glycosphingolipids (see Chapter 9) are degraded from the nonreducing ends by exoglycosidases
while they are still bound to the lipid moiety, ceramide. Since glycosphingolipids have many of
the same outer sugar sequences that are found in N- and O-glycans, many of the same
glycosidases are used for their degradation (Figure 18.6). However, specialized hydrolases are
needed for cleaving the glucose-ceramide and galactose-ceramide bonds and other linkages near
the membrane. Besides these specific enzymes, additional noncatalytic sphingolipid activator
proteins (some called saposins) help present the substrate to the enzyme for cleavage.
Endoglycoceramidases have been reported in leech and earthworm and may be present in higher
animals. This enzyme, like the peptide-N-glucoamidase, releases the entire glycan from the lipid.
Figure 18.6. Degradation of glycolipids. Required activator proteins are shown in parentheses.
Specialized Enzymes and Proteins for Glycosphingolipid Degradation
Some exoglycosidases for glycoprotein and GAG chain degradation also degrade glycolipids,
but others are unique to glycolipid degradation. The absence of these unique enzymes causes
glycolipid storage diseases listed in Table 18.3. Glucocerebrosidase, also called β-
glucoceramidase, is specific for the degradation of the Glcβ-Cer bond, and its loss causes
Gaucher's disease.
A specialized β-galactosidase, called β-galactoceramidase, hydrolyzes the bond between

galactose and ceramide, and it can also cleave the terminal galactose from lactosyl ceramide. Its
loss produces Krabbe disease. Galactosyl-ceramide is often found with a 3-sulfate ester
(sulfatide), and thus a specific sulfatase, arylsulfatase A, is needed for its removal prior to β-
galactosylceramidase action. Loss of this sulfatase causes metachromatic leukodystrophy and the
accumulation of sulfatide. Glycolipids terminated with α-galactosamine residues are degraded
by a specific α-galactosidase and its loss causes Fabry disease.
Activator Proteins
Two genes code for all of the known sphingolipid activator proteins. These proteins can also be
used to form complexes with more than one degradative enzyme for more efficient hydrolysis.
GM2 Activator
GM2 activator protein forms a complex with either GM2 or GA2 and presents them to β-N-
acetylhexosaminidase A for cleavage of terminal β-GalNAc. Sugars that lie too close to the lipid
bilayer apparently have limited access to the soluble hexosaminidase. The activator protein binds
a molecule of glycolipid, forming a soluble complex that can be cleaved by the hexosaminidase.
The resulting product is now inserted back into the membrane, and the activator presents the next
GM2 molecule, and so on. Genetic loss of this activator protein causes the accumulation of GM2
and GA2, resulting in the AB variant of GM2 gangliosidosis. The activator is probably targeted to
the lysosomes via the Man-6-P pathway.
Saposins
Saposins are derived from a 524-amino-acid precursor called prosaposin which is processed into
four homologous activator proteins, Sap-A, Sap-B, Sap-C, and Sap-D, each having about 80
amino acids and each having different properties. Sap-A and Sap-C both help β-glucosyl and β-
galactosyl ceramidase degradation. Sap-B assists arylsulfatase A, α-galactosidase, α-sialidase,
and β-galactosidase. The mechanism is thought to be similar to that of the GM2 activator by
partially lifting a glycolipid molecule out of the membrane to form a complex that is cleaved by
the soluble enzyme, followed by reinsertion of the substrate back into the membrane. Sap-D and
Sap-B also assist in sphingomyelin degradation by sphingomyelinase. Complete absence of
prosaposin is lethal in humans, and deficiencies in Sap-B and Sap-C lead to defects that
clinically resemble aryl sulfatase A deficiency (metachromatic leukodystrophy) and Gaucher's
disease. These symptoms might be predicted based on the enzyme that these saposins assist.
A very rare human disorder is the multiple sulfatase deficiency. All sulfatases are casualties of
this defect, since they all require a posttranslational conversion of an active site cysteine residue
to a 2-amino-3-oxopropionic acid for activity. In essence, the S group of cysteine is replaced by
a double-bonded oxygen atom that probably acts as an acceptor for cleaved sulfate groups. Loss
of the enzyme carrying out that reaction leads to inactive sulfatases.
Inhibition of Degradation (19 20)
There are several ways to prevent lysosomal degradation of glycoconjugates. One is by raising
the intralysosomal pH in cells. Another is to inactivate a particular glycosidase genetically or to
use a specific inhibitor. Although proteolysis inhibitors may also prevent efficient degradation,
they will not totally block it.
General Inhibitors
Since nearly all lysosomal enzymes have acid pH optima, degradation can be severely curtailed
by increasing the intralysosomal pH from 5 5.5 to 7, using agents such as ammonium chloride
and chloroquine. These agents lower the rate of degradation, but they may not completely block
it. Some lysosomal enzymes have relatively broad pH optima, and some of these enzymes
actually have pH optima higher than the lysosome. It has been suggested that lysosomes may not
always remain at low pH. Some "lysosomal" enzymes are actually present and active in early and
late endosomes that have a higher pH than lysosomes.
Specific Inhibitors
Glycosylation inhibitors are covered in Chapter 40; however, some of these also inhibit specific
lysosomal enzymes. For example, swainsonine blocks lysosomal α-mannosidase in addition to
blocking the α-mannosidase II involved in glycoprotein processing. Sheep and cattle become
neurologically deranged by eating food rich in swainsonine, which is also called locoweed. Since
there are no neurological effects seen in transgenic knockout mice lacking the processing α-
mannosidase II, it is likely that these temporary symptoms are induced because lysosomal α-
mannosidase is inhibited. Undegraded oligosaccharides probably accumulate in affected animals.
Although many inhibitors affect various enzymes when tested in in vitro enzymatic assays, they
may not be effective in cells or whole animals because they may not enter the lysosome, or if
they do, their concentration may not be sufficient.
Perspective on Synthesis and Degradation and Genetic Disorders
Genetic defects in lysosomal enzymes have provided the best insight into the catabolic pathways
and their importance. For example, cocultivation of fibroblasts from patients with different
storage diseases leads to the disappearance of stored material in both types of cells. Each
supplies the corrective factors (lysosomal enzyme) that the other lacks through uptake via the
Man-6-P receptors on the cell surface (for further details, see Chapter 23 on the discovery of
Man-6-P targeting of lysosomal enzymes). Cross-correction highlights the fact that only a
relatively small amount of enzyme may be needed to prevent accumulation of storage products
and the complications they cause. Some estimates suggest that only 10% of normal β-N-
acetylhexoaminidase activity may be sufficient to prevent pathological symptoms of Tay-Sachs
disease. Since accumulation of stored material depends on the relative rates of glycoconjugate
synthesis and degradation, the near absence of a glycosidase can be ameliorated by reducing the
synthetic rate of the stored compound. This was cleverly tested in a mouse model of Tay-Sachs
disease by using N-butyldeoxynojirimycin, an inhibitor of glucosylceramide synthase, the first
step in glycosphingolipid biosynthesis. When normal mice were treated with this compound, the
amount of glycosphingolipids fell 50 70% in all tissues without obvious pathological effects.
When this compound was given to Tay-Sachs mice, the accumulation of GM2 in the brain was
blocked and the amount of stored ganglioside was reduced. Thus, reducing the synthesis of the
primary precursor reduced the load of GM2 that could be degraded by the mice.
As is discussed in Chapter 6, it is assumed that most of the monosaccharides derived from

glycoconjugate breakdown are reutilized. The few studies exploring this issue suggest that
reutilization of glycosidase-liberated sugars may be quite substantial.
Degradation and Resynthesis (21 22)
Degradation of glycoconjugates is not always complete. Partially degraded or incomplete

glycans on glycoproteins and glycosphingolipids at the cell surface can be internalized and then
elongated within a functional Golgi compartment containing the sugar nucleotide and the
appropriate glycosyltransferase. In the case of glycosphingolipids, this pathway makes a
substantial contribution to the total cellular synthesis, whereas in the case of glycoproteins, it
probably makes a relatively small contribution. It is unknown whether these pathways are simply
salvage and repair mechanisms or whether they play an integral part in an as yet unidentified
physiological pathway.
Glycoprotein Reglycosylation
A variety of different experiments clearly show that the half-life of specific membrane proteins
is longer than the half-life of their glycans. The more terminal monosaccharides turn over faster
than those closer to the reducing end of the chain, suggesting that the monosaccharides are
sequentially removed by exoglycosidases. This may occur at the cell surface or when proteins
are endocytosed in the course of normal membrane recycling. Since the mildly acidic late
endosomes contain lysosomal enzymes, whose pH optima can be quite broad, terminal sugars
such as sialic acid can be cleaved. If the endocytosed proteins are not degraded in lysosomes,
they may encounter sialyltransferases in the Golgi, become resialylated, and reappear again on
the cell surface. A similar situation may occur if a glycoprotein has lost both sialic acid and
galactose residues from its glycans. Some membrane proteins synthesized in the presence of
oligosaccharide processing inhibitors can still reach the cell surface in an unprocessed form.
Subsequent incubation in the absence of inhibitors leads to normal processing over time. The
extent of processing and the kinetics depend on the protein and the cell line. Since Golgi
enzymes are not distributed identically in all cells, it is difficult to make a general statement
about how much "reprocessing" can occur. Most studies have monitored N-linked glycans, but
membrane proteins with O-glycans show a similar response. Newly arrived plasma membrane
proteins will acquire additional O-glycans at previously unoccupied glycosylation sites over
time. Initiation of O-glycans is thought to occur in the cis-Golgi, but it is difficult to determine
whether there is a small amount of O-GalNAc transferase that is active in a late (trans) Golgi
compartment or whether the acceptor protein briefly encounters a high concentration of the
glycosyltransferase in an early compartment.
Glycosphingolipid Recycling
The majority of the newly synthesized glucosyl ceramide arrives at the cell surface by a Golgi-
independent cytosolic pathway. Some of this material, as well as Glc-Cer generated by partial
degradation of more complex glycolipids, can recycle to the Golgi. There, like glycoproteins,
they can serve as acceptors for synthesis of longer chains. In the lysosome, sphingosine and
sphinganine are produced by complete degradation of glycosphingolipids, and reutilized.
Together, these pathways, especially the latter one, may actually account for the majority of
complex glycolipid synthesis in many cells. Depending on the physiological state of the cell and
synthetic demands, the de novo pathway starting from serine and palmitoyl CoA may account for
only 20 30% of the total synthesis. Shuttling the recycled components through and among the
various organelles appears to involve vimentin intermediate filaments. Mechanisms and details
of this process are presently lacking, but these studies offer a physiological function for
glycolipid transfer proteins that were purified from brain preparations many years ago. For
details of the biosynthetic pathway, see discussion in Chapter 9.
Future Directions
A recurring theme in this chapter is the fine balance between synthesis, degradation, and the
identification of salvage pathways. Investigation of monosaccharide salvage pathways will be an
area of future research and should reveal the importance of dietary contributions and recycling of
degraded molecules to glycoconjugate synthesis. Lysosomal storage diseases have been treated
by enzyme replacement therapy, such as in the case of Gaucher's disease, and gene replacement
therapy is also a possibility. However, as mentioned above, an interesting alternative is to
decrease the rate of synthesis of some glycans that contribute to the accumulation of stored
material. This may lead to some improvement in patients' conditions.
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19. Glycosylation in "Model" Organisms
Jolla, California).
THIS CHAPTER HIGHLIGHTS GLYCOSYLATION AND GLYCOBIOLOGY in several

biological model systems. It covers structure, biosynthesis, and newly emerging functional roles
of glycoconjugates. The advantages and limitations of each system are outlined. With the
exception of yeast, exploration of biosynthesis and structural analysis are much less developed in
these models than in mammalian cells. However, these more complex systems combine genetics,
embryonic development, and behavior and therefore offer great potential for understanding the
functional roles of glycans.
Background
Model organisms have had important roles for understanding biochemistry, physiology, and
behavior of more complex organisms. Each biological system brings its own strengths such as
rapid growth, availability of large quantities of material, the ability to carry out genetic
manipulations, and the ability to perform detailed analysis of cellular differentiation and
embryonic development. These biological advantages were not driving forces behind the early
studies in glycobiology. Instead, they focused on understanding the structure and biosynthesis of
glycoconjugates. The technical limitations of structural analysis required large quantities of
material. Milk, serum, and blood provided easily purified glycoproteins, cartilage yielded
proteoglycans, and brains were rich in glycolipids. The study of glycosylation and
glycoconjugate biosynthesis mostly was limited to microbes or cultured mammalian cells. The
idea of exploring the function of glycans in an entire multicellular organism was practically
nonexistent. Recently, this situation changed radically as the functional roles of glycosylation in
fundamental developmental processes have emerged from genetic studies.
The choice of model organisms depends to a large extent on the question being studied. Yeast
(Saccharomyces cerevisiae) has provided many insights into the biosynthesis of glycosylation
precursors and their assembly into oligosaccharides. Dictyostelium discoideum has the added
advantage of cellular differentiation, tissue proportioning, adhesion, and simple behavior. More
sophisticated organisms such as Caenorhabditis elegans, sea urchins, Drosophila melanogaster,
and Xenopus provide opportunities to study processes that are unique to multicellular organisms
such as fertilization, embryogenesis, organogenesis, and complex behaviors. These systems now
figure prominently in glycobiology research and provide a basis for understanding similar
processes in mammals.
Currently, homology searches for glycosyltransferases and the awesome power of functional
genetic analysis are being used in organisms such as Drosophila and C. elegans to make
quantum leaps in understanding the functional importance of glycans. The structural and
biosynthetic studies lag behind the functional studies. In the future, functional links will drive the
structural analysis of glycans in these model systems.
Yeast (1 4)
Nearly 14% of the dry weight of S. cerevisiae is mannose, mostly found in N- and O-glycans as
mannans located in the cell wall. Highly glycosylated enzymes such as invertase also typically
have large extended N-glycans. Clever genetic selection techniques and easy production of
grams of material have made yeast the ideal choice for dissection of the biosynthetic pathways,
enzymology, and structural analysis of glycans. More recently, the sequencing of the entire S.
cerevisiae genome was achieved. Homology searches promise to reveal additional genes
involved in glycosylation in both yeast and higher organisms. Mutants have been isolated in the
synthesis of biosynthetic precursors, sugar nucleotide transporters, and many
glycosyltransferases involved in the lipid-linked oligosaccharide biosynthesis and early
processing steps of N-glycans. In several cases, this information has led to cloning human genes
that proved to be mutated in at least three types of human carbohydrate-deficient glycoprotein
syndrome disorders (see Chapter 32).
N-Glycans
Yeast appears to be the most "primitive" eukaryote making the familiar N-glycans. The earliest
parts of the pathway have been well preserved in mammals. Yeast synthesize the typical
eukaryotic lipid-linked oligosaccharide precursor by the specific ordered addition of each N-
acetylglucosamine, mannose, and glucose residue to the chain (see Chapter 7). Mutants in most
of the lipid-linked oligosaccharide biosynthetic steps are available (Figure 19.1). The early
processing steps within the ER are similar to those seen in higher organisms: removal of all the
glucose residues and a single mannose residue prior to export. The endomannosidase that cleaves
the Glcα1 3Man from newly formed glycoproteins in mammals is not found in yeast. Another
difference is that yeast cleaves only a single mannose residue in the ER and then adds more
mannose residues in the Golgi, instead of removing many additional mannose residues. Each of
these mannose residues is added directly from GDP-Man, rather than Dol-P-Man. Examples of
some extensions are shown in Figure 19.2. For instance, in mannan synthesis, the core is
extended using an α1 6 mannosyltransferase to form a long repeating backbone. Each of the
α1,6 units is branched by adding α1 2 Man and α1 3 Man using linkage-specific
mannosyltransferases. There is recent evidence that the α1 6 and α1 2 transferases exist as part
of a physical complex in the yeast Golgi.
Figure 19.1. Lipid-linked oligosaccharide biosynthesis and mutants in yeast. Lipid-linked

oligosaccharide synthesis and transfer to protein in the ER is genetically defined by a wealth of
functional mutations that effect lipid-linked oligosaccharide structure and transfer to protein.
Some of the steps in the synthesis of the earliest precursors such as Man-6-P and Man-1-P have
also been detected but are not shown here. Defects in dolichol biosynthesis and recycling are
expected to also produce mutations that would reduce glycosylation efficiency. (Light gray)
Mannose residues that originate from GDP-Man; (dark gray) mannose residues from
dolichylphosphomannose. (Modified, with permission, from [3] Burda and Aebi 1999 [©
Elsevier Science].)
Figure 19.2. A portion of oligosaccharide processing pathway in yeast. Processing in yeast is

different from that seen in other eukaryotic systems in that usually only one mannose residue is
removed by the ER α-mannosidase to yield the structure (in the large box) derived from the
Glc3Man9GlcNAc2 oligosaccharide shown in Figure 19.1. A small portion of these molecules are
extended to give an inner core of 9 15 mannose as shown in some of the shaded residues. Other
oligosaccharides are elongated by extensive buildup of a linear sequence of α-6 Man units,
shown in the brackets. This core can be further modified by the addition of α1 2- and α1 3-
linked mannose residues to yield a complex structure (mannan) found in the cell walls of yeast.
In addition, the mannans are also phosphorylated by the addition of Man-1-P residues to the C-6
of an α1 2Man to form a phosphodiester. Other modifications are possible, and each of the
major steps is defined by mutant strains that produce oligosaccharides with altered structures. All
of the mannose residues are in α-linkage except for the β-linked mannose linked to N-
acetylglucosamine residue.
Another feature in mannan synthesis is the addition of Man-1-P to the 6-position of one of the
α1 3 Man units in the extended chain. Like the other mannosylation reactions, the donor for this
reaction is GDP-Man. Other species of yeast also synthesize similar phosphodiesters with Glc-1-
P (UDP-Glc donor) in place of Man-1-P. The formation of these phosphodiesters is reminiscent
of the first step in the Man-6-P biosynthetic pathway used for targeting lysosomal enzymes of
higher organisms (see Chapter 23). Knowing that phosphorylated mannan biosynthesis in yeast
generated a phosphodiester linkage provided a critical clue to understanding the pathway of
Man-6-P-containing oligosaccharides in mammalian cells. However, yeast do not use Man-6-P
for targeting lysosomal enzymes; rather, targeting relies on recognition of peptide motifs.
O-Glycans
Mannans also contain O-glycans that are initiated with mannose instead of the typical GalNAc-
based chains seen in mammals. Dol-P-Man is the donor for the first residue which is attached to
a Ser/Thr in the ER. Subsequent elongation of the chain by the addition of two to three more
mannose residues occurs in the Golgi and uses GDP-Man as the donor. As might be predicted,
the Golgi GDP-Man transporter is critical for synthesis of both the extended N- and O-glycans in
yeast. In fact, the coupling of sugar nucleotide transport to nucleotide diphosphate hydrolysis
was firmly established in yeast mutants. It is not yet known whether the abundant O-mannose-
based oligosaccharide chains found in the mammalian brain tissue (see Chapter 12) are related to
the O-mannose pathway in yeast.
Glycophospholipid Anchors
The basic carbohydrate structure of GPI anchors in yeast is similar to those seen in trypanosomes
and mammalian glycoproteins. One significant difference is that the yeast GPI anchors have a
ceramide in place of one of the acyl groups. The ceramide is resistant to base-catalyzed cleavage
(see Chapter 10).
Advantages and Limitations
As mentioned above, the yeast model has many advantages, including the ability to make large
quantities of glycans for structural analysis and the ability to generate mutants and a fully
sequenced genome. Yeast will undoubtedly continue to make substantial contributions for many
years to come, especially in identifying genes that may be defective in human glycosylation
diseases. This is expected to be a very fruitful area, but yeast also has limitations. First, there is
little N-glycan processing, which means that the trimming α-mannosidases, GlcNAc, galactose,
and sialic acid transferases are not found. There are no typical O-glycans, glycosaminoglycans,
or glycosphingolipids. Another aspect to consider is that the quantity of glycosylation in yeast is
about 100-fold greater than that in mammalian cells. Like most microbes, yeast are most
concerned with rapid and efficient growth. In multicellular organisms, the demand for
glycosylation is much less, and the emphasis is on regulation and fine tuning, rather than strictly
on aggressive growth.
Dictyostelium discoideum(5 7)
Life Cycle and Development
D. discoideum is a haploid amoeba that normally phagocytoses bacteria. When food is depleted,
it undergoes a complex 24-hour morphogenesis that generates a group of spores setting on top of
a strand of evacuolated stalk cells that resembles a cotton fiber (Figure 19.3). Development
begins when groups of 100,000 cells chemotax to cAMP and become mutually adhesive. Cells
form a mound that becomes covered with a cellulose and glycoprotein sheath, which seals an
elongating 1 2-mm-long migrating "slug." Cells at the anterior tip will generate the stalk and
those in the rear will generate the spores. When slug migration stops, the tip rears back, bringing
it into a vertical position. A small collar of cells in the center of the aggregate then synthesizes a
ring of cellulose that resembles a straw. Cells at the upper apex of the tube migrate into it and
stop, and each encases itself within a cellulose wall. The entire aggregate rises off the substratum
as more cells enter the stalk tube forming their own walls. The prespore cells climb the 2-mm
stalk and secrete a mixture of preformed polysaccharides and glycoproteins from vesicles unique
to prespore cells. Some glycoproteins form cellulose-binding complexes that seal the spore
against the environment. A raindrop, passing animal, or hungry predator distributes the resistant
spores from their perch to more fertile ground.
Figure 19.3. Development in D. discoideum. Scanning electron photomicrographs show
morphological stages in the development of D. discoideum. The flat mound in the center of the
picture is composed of cells that have just finished aggregating after being deprived of nutrients.
In the next stage a few hours later, the elevated mound is covered with a surface sheath primarily
made of protein. Depending on the environmental conditions, the mound can elongate into a
phototatic slug shown at the lower left as it migrates left. In the slug, the cells near the tip at the
left end of the slug will become stalk cells, whereas the remaining 80% will transform into
spores later on in development. A series of distinct morphological stages, including the
"Mexican hat" stage, occur during the next 7 8 hr that produce a cotton-fiber thin stalk
composed of evacuolated cells which support a group of cellulose-encased spore cells that
ascend the stalk, awaiting the 2-mm fall back onto fertile ground where the spores emerge to
feed on bacteria. (Courtesy of R.L. Blanton.)
N-Glycans
Like other eukaryotes, D. discoideum contains N-glycans and processes them, but those made
during vegetative growth undergo little processing. Most of the chains have seven to eight
mannose residues, and many contain a single GlcNAc located in the "bisecting" or "intersecting"
positions on mannose branches. This resembles some high-mannose and complex-type chains in
higher organisms modified by GlcNAc T-III (see Chapter 7), but typical biantennary glycans are
not found in Dictyostelium. Another common modification is the addition of an α1 6 Fuc to the
proximal GlcNAc in the core. This structure is also typically found in complex-type chains in
higher animals, where addition is thought to require the prior initiation of complex chains with
GlcNAc. This cannot be the case in D. discoideum, since it does not make these complex-type
glycans. A few high-mannose-type N-glycans with core fucosylation similar to that of
Dictyostelium have been found recently in mammalian cells, so the pathway may be more
diverse than previously appreciated.
Two oligosaccharide-processing α-mannosidases appear during development. On the basis of

inhibitor sensitivities and the structures formed, the first enzyme is typical of Golgi α-
mannosidase I generating Man5GlcNAc2 oligosaccharides and the second enzyme resembles α-
mannosidase II or (more likely) α-mannosidase III, since it generates Man3 containing chains.
Core Fucα1 6GlcNAc formation is found in vegetative cells but is very low during
development. On the other hand, Fucα1 3GlcNAc is found only during development in a
reciprocal relationship to the α1 6-linked core fucose. Core Fucα1 3GlcNAc is typically found
on many plant glycoproteins (see Chapter 20).
Man-6-P Biosynthesis
Dictyostelium lysosomal enzymes have N-glycans that carry Man-6-P. Biosynthesis is much like
that in mammalian cells. The first step is the transfer of GlcNAc-1-P to the 6-position of selected
mannose residues. The Dictyostelium transferase recognizes the sugar chain much like its
mammalian counterpart. Nearly identical structures can be made, but in contrast to the
mammalian GlcNAc phosphotransferase, the Dictyostelium enzyme does not interact with a
specific recognition marker on lysosomal enzymes. No Man-6-P receptor has been found in
Dictyostelium, but its lysosomal enzymes are recognized by the large Man-6-P/IGF-II receptor
and taken up into mammalian cells. The next biosynthetic step in mammals and in Dictyostelium
is the removal of the capping GlcNAc to generate Man-6-P, which is the final step in the
pathway of higher organisms. However, Dictyostelium has a Man-6-P-specific, S-
adenosylmethionine-dependent methyltransferase that converts the phosphatase-sensitive
phosphomonoester into a phosphodiester, Man-6-P-OCH3. The diester may protect Man-6-P
from degradation and enable it to participate in multiple rounds of cycling between various
cellular compartments without losing its Man-6-P. Although no Man-6-P-dependent targeting
has been proven, Dictyostelium does have two classes of lysosomes. Each contains proteins with
mutually exclusive sugar modifications, either Man-6-P or GlcNAc-1-P-Ser (see Chapter 12).
These groups of proteins seem to remain segregated despite repeated fusion of lysosomes with
cargo-containing vesicles. These results suggest that sugars may have a role in segregating the
proteins from each other.
Unusual Glycosylations
Several unusual types of O-linked sugars are found in Dictyostelium, as discussed in Chapter 12.
An α-GlcNAc directly linked to threonine residues is found on some proteins, and its loss results
in greater sensitivity of cell surface proteins to proteases. It is likely that additional
monosaccharides are added to the α-GlcNAc as well. Several different types of
phosphoglycosylations exist. GlcNAc-1-P-Ser was mentioned previously. Fuc-β-1-P-Ser is
found on several proteins that are used to construct the spore coat. Mutant strains without this
carbohydrate have more porous spore coats, and these spores would probably not survive well in
nature. The most elaborate form of cytosolic glycosylation known is found in the Dictyostelium
protein SKP1, which is involved in the ubiquitination of certain cell cycle proteins. A
hydroxyproline residue in some molecules of SKP1 contains a linear pentasaccharide composed
of GlcNAc, galactose, and fucose. The function of the oligosaccharide is unknown. For
additional discussion, see Chapter 13.
Polysaccharides and Cellulose
Dictyostelium does not have proteoglycans typical of mammalian cells, but it makes
polysaccharides of known composition. Cells that will become spores by virtue of their position
in the slug are called prespore cells. They have storage depots called prespore vesicles that
contain spore coat proteins and a polysaccharide called galuran composed of Gal, GalNAc, and
GalA. The entire contents of the vesicle are jettisoned at the time of final fruiting body
construction. They assemble into a multilayered spore coat containing at least eight proteins that
appear to self-assemble, with some of them becoming disulfide-cross-linked to each other. Some
of these proteins have high affinity for cellulose, and this interaction is probably involved in the
normal assembly of the spore coat. Cellulose fibrils are made by each individual spore cell, but
are not stored in the prespore vesicles. Cellulose is not found in vegetative cells, but it first
appears when the aggregating mass is sealed by the synthesis and secretion of the protein surface
sheath. Cellulose occurs in the sheath, and it also surrounds each of the stalk cells and is part of
the stalk tube found on the outside of the stalk.
Advantages and Limitations
Dictyostelium has an optional multicellular stage, and thus mutations effecting different parts of
the program can be studied independently. Like yeast, large amounts of vegetative and
synchronously developing cells are relatively easy to obtain. The Dictyostelium genome contains
about 34 Mb of DNA and an estimated 8000 genes and will soon be sequenced (scheduled for
completion in the year 2001). The precise morphological program allows random gene tagging
to disrupt development, and mutants are easily screened with a dissecting microscope.
Disruption of α-glucosidase and cellulose synthase leads to easily recognizable phenotypes due
to alterations in glycoprotein processing and cell wall assembly. Glycosylation has been
implicated in cell adhesion, chemotactic migration, and intracellular protein trafficking.
Dictyostelium has the most elaborate type of cytosolic glycosylation seen to date (see Chapter
13), but it has some limitations in that it does not make complex N-glycans, proteoglycans, or
glycosphingolipids. However, it does make ceramide-based glycophospholipid anchors much
like those seen in yeast. Dictyostelium has only very simple behaviors, such as phototaxis, and
development is limited to the differentiation of only a few cell types. Although many
carbohydrate-specific antibodies are available, most of the structures are not well characterized,
and expression cloning from cDNA libraries to complement mutant strains lacking these
antigens has not yet been successful.
Caenorhabditis elegans(8 17)
Overview, Advantages, and Limitations
The 97-Mb sequence of the nematode C. elegans is now completely sequenced, and with 19,099
genes, it is nearly eight times the size of the S. cerevisiae genome. Of the 20 most highly
populated gene families, 2 are involved in glycosylation or formation of carbohydrate-binding
proteins; 150 genes have C-type lectin domains and 70 genes appear to belong to the UDP-
glucuronosyltransferase and UDP-glucosyltransferase families. The latter group is as abundant as
the EGF-like domains common to many membrane proteins. Some of the gene families in C.
elegans are unique to multicellular organisms. The 959 cells of C. elegans hermaphrodite whose
origin and fate are known in detail include 302 cells in the nervous system. The relatively
simple, transparent body plan is amenable to easy visualization, especially with GFP-tagged
proteins, allowing exploration of spatial and temporal specific gene expression, pattern
formation, and cell fate studies. Functional knockouts can be done by injecting double-stranded
RNA matching the specific gene into the worm oocytes. This approach holds promise for
revealing the functions of glycosylation. In several instances, putative glycosyltransferase genes
have been implicated in developmental functions. Homology screening for glycosyltransferases
has been successful, but detection of functional roles for glycosylation based on homology are
often not accompanied by documentation of enzyme activity. This is important because
differences of only a few amino acids can change the specificity of glycosyltransferases and lead
to different products.
An additional problem is that the amount of C. elegans glycans available for structural analysis
has been quite limited. The discussion below describes the few known structures, genes, and
proven enzymatic activities.
General Screening
Histochemical lectin-binding studies first showed that mammalian-like glycosylations were

probably present in the nematode. Enzymatic assays of C. elegans crude extracts showed activity
of GlcNAcT-I, β1 3 GalT, Core 4 GlcNAcT, and GlcNAcT-V. Searches of the DNA database
confirmed that genes related to N-, O-glycosylation, GPI-linked glycoproteins, mucins, and
proteoglycans are all present as well. If the proteins coded by these genes were enzymatically
active, they would encompass much of the typical glycosylation pathways seen in mammals.
This would be a considerable leap in glycosylation complexity compared to either yeast or
Dictyostelium; however, there is no evidence for sialic acids in C. elegans.
Proven Structures
Specific structures of N-glycans in C. elegans have not been reported, but the N-glycans from
another nematode, Haemonchus contortus, have been established. It contains a series of unique
N-glycans, some with up to three core fucose residues. Specific antibodies have been used to
show that C. elegans also contains glycans with a lac-DiNAc structure (GalNAcβ1 4GlcNAc)
(see Chapter 36).
A few glycolipid structures of the arthro series have been identified: Glcβ1Cer, Manβ1
4Glcβ1Cer, and GlcNAcβ1 3Manβ1 4Glcβ1Cer (see Chapter 9). At this time, no specific
structures have been reported for O-glycans, glycosaminoglycans, or glycophospholipid anchors,
but they undoubtedly exist, as shown by the accumulating evidence for genes that code for the
critical glycosyltransferase.
Glycosyltransferase Genes
N-linked Pathway
Genes for GlcNAcT-I and GlcNAcT-II have been identified, and their proteins have been shown
to be enzymatically active. Three cDNAs homologous to GlcNAcT-I are designated gly-12, gly-
13, and gly-14 and presently one gene homologous to GlcNAcT-II (C03E10). gly-12 and gly-14
are highly active, but gly-13 appears to be inactive. Both the gly-12 and gly-13 promoters are
expressed throughout development in tissues, including intestine, muscle, hypodermal, epithelial
cells, and in ganglia of the head and tail region. The gly-14 promoter is expressed only in gut
cells throughout larval development and in the adult. The C. elegans GlcNAcT-II cDNA encodes
a protein with a typical type-2 membrane protein topology, but activity has not been
demonstrated.
The C. elegans GlcNAcT-V homolog is called gly-2. It has a typical single-membrane-spanning

segment and is 37% identical and 60% similar to the mammalian enzyme. A soluble construct
has GlcNAcT-V activity when assayed in vitro and is therefore comparable to the mammalian
gene. This point is reinforced by the ability of the C. elegans enzyme to complement a mutant
strain of CHO cells called Lec4A. This strain has a GlcNAcT-V that is mislocalized to the wrong
compartment as a result of a point mutation. The enzyme is still active when assayed in vitro, but
the mislocalization prevents synthesis of oligosaccharides that bind to the lectin L-PHA (see
Chapter 30). The normal C. elegans gene rescues the phenotype of Lec4A, but when a similar
point mutation is introduced into the gly-2 gene, it can no longer rescue Lec4A. Thus, the C.
elegans GlcNAcT-V is probably localized using the same mechanisms as mammalian cells. On
the basis of the functional interchangeability of the enzyme with the mammalian form, the worm
gene has the characteristics of an ortholog of GlcNAcT-V.
O-linked Pathway
C. elegans has mucin-type O-glycosylation, and 9 genes (gly-3 to gly-11) express 13 cDNAs that
have homology with O-α-GalNAc transferases. Each encodes a type II membrane protein that
has 60 80% amino acid sequence similarity with the catalytic domain of mammalian homologs.
Each has specific histidine and carboxylate side chains essential for activity and a DxH or "DxD-
like" motif found in many glycosyltransferases. Some cDNA clones show alternative message
processing, a reading frameshift, and premature termination codon in the carboxy-terminal
lectin-like domain. Five of the cDNAs are able to O-glycosylate a mammalian peptide substrate
containing multiple potential glycosylation sites, demonstrating that some have homologous
functions and similar amino acid sequence requirements for O-glycosylation. On the basis of
information derived from analysis of expressed mammalian genes, it is likely that different
transferases recognize different peptide sequences or require the prior action of another α-
GalNAcT with a different acceptor specificity.
Proteoglycans and Glycosaminoglycans
Glycosaminoglycans most likely exist in C. elegans, as described below. Homologs of glypicans,

the glycophospholipid-anchored heparan sulfates, have been identified (see Chapter 11).
Mutations in the unc-52 gene produce a disorganized muscle phenotype because it affects
attachment of the myofilament lattice to the muscle cell membrane. The unc-52 gene encodes a
nematode homolog of perlecan, a mammalian basement membrane heparan sulfate proteoglycan.
There are also close homologs to the mammalian core glucuronosyltransferase used for adding
the first GlcA residue in the core region of glycosaminoglycan chains, the GlcNAc N-
deacetylase/N-sulfotransferase and the GlcA C5 epimerase.
One of the more interesting and provocative findings in C. elegans is the possible involvement of
glycosylation in the development of the vulva. This tube is formed by an invagination of
specialized epithelial cells to form a contact with the uterine epithelium. The vulva is needed for
the hermaphrodite to lay eggs and receive sperm from males. Tube formation requires localized
changes in cell shape that are needed for making contacts with the ECM and with other cells.
Multiple mutations in eight different genes produce a similar "squashed vulva" (sqv) phenotype.
Three of the genes, sqv-3, sqv-7, and sqv-8, may be involved in oligosaccharide biosynthesis.
sqv-3 is predicted to encode a protein with similarity to vertebrate β1 4 GalTs and β1 4
GlcNAcTs. The predicted protein from sqv-8 resembles β1 3 GlcAT that adds GlcA to Gal β1
4 GlcNAc, and sqv-7 has features of the sugar nucleotide transporter family. Even though the
proteins have not been expressed or shown to have enzymatic activity, the results suggest that a
glycosylation pathway has a vital role in this part of normal morphogenesis. The homology of
the C. elegans genes with their mammalian counterparts is not sufficient to predict which
specific glycosylation pathway would be affected.
Sea Urchins (18 26)
A Model System for Fertilization and Early Development
The sea urchin has long been one of the premiere models for developmental biologists. These
echinoderms supply ample quantities of pure eggs and sperm on demand. The gametes are ready
to initiate a synchronous and well-defined developmental program with various morphological
stages and easily recognizable structures. The eggs are surrounded by a protective egg jelly that
shields the plasma membrane from the environment. The sperm must first attach to the jelly
before fertilization. The tip of the sperm head contains the acrosome, a specialized lysosome that
is loaded with degradative enzymes. These are released in a species-specific, ion-channel-
mediated event that allows the sperm to burrow through the egg jelly to reach the plasma
membrane prior to actual fertilization. Since fertilization occurs in seawater, egg and sperm of
the same species must specifically recognize their partners. And then, only when the sperm is
close enough to the egg, is the acrosome reaction initiated, releasing the enzymes at the target. A
premature acrosome reaction would dilute the enzymes in seawater, leaving the sperm unable to
penetrate the viscous egg jelly. A biochemical approach showed that much of the recognition
depends on protein interactions with unusual carbohydrates (see also Chapter 34).
The Acrosome Reaction
The acrosome reaction in sperm occurs when a fucose sulfate polymer, called fucan, in the egg
jelly binds to a protein receptor on the sperm. Two different fucan structures are found in the
eggs of most females of the species Strongylocentrotus perpuratus. They have either one or the
other and a small number have both. The backbone structures of repeating α1,3-linked fucose
have variable sulfation patterns. Both of the fucans are about equally active at inducing the
acrosome reaction. They bind to the receptor protein on the sperm, which has two different C-
type lectin domains. This 210-kD receptor protein has considerable homology with polycystin,
the PKD1 gene, which is the major protein defective in human polycystic kidney disease. In
humans, this protein is thought to be involved in ion transport, which would be consistent with
the ion-channel-mediated acrosome reaction.
Egg and Sperm Binding
After the sperm penetrates the egg jelly, it still must bind to the egg for fertilization. This binding
is also carbohydrate-mediated. A 350-kD glycoprotein, which is 70% carbohydrate, is the egg
receptor for sperm (ERS). It contains both N- and O-glycans, but only the O-glycans mediate
binding of acrosome-reacted sperm, possibly by stabilizing the interaction. The critical
component in binding appears to be the sulfate ester located at the nonreducing end of an
unusual disialic acid chain (Figure 19.4). The functional trisaccharide unit is composed of 9-O-
SO4-Sia2 5Sia-GalNAc-. Typically, most polysialylated molecules contain sialic acid in a α2 8
linkage, but in this case, the sialic acid, N-glycolylneuraminic acid (Neu5Gc), has an -OH group
at the 5-position that forms the glycosidic linkage (see Chapter 15).
Figure 19.4. Structure of sialic acid chain on the egg receptor for sperm (ERS). The structure of
the O-α-GalNAc-linked 9-O-sulfated disialic acid structure is linked through the glycolyl group
(5Gc) of this sialic acid.
Sperm-egg binding appears to be a two-step process. First, the sperm binds with low affinity to
the sulfated polysialic acid oligosaccharide and to a 247-amino-acid amino-terminal portion of
the ERS protein. The reaction does not have genus specificity. This is followed by a high-
affinity, genus-specific binding that involves a 32-amino-acid amino-terminal portion of the ERS
protein. Gamete fusion can then occur.
N-Glycosylation and Spicule Formation
N-glycosylation is required for gastrulation in sea urchins. Some of the early enzymes in the
pathway have low activities through the blastula stage and then increase during gastrulation. At
this time, three GPI-linked cell surface proteins of 130, 205, and 250 kD become involved in
calcium uptake and formation of the skeleton. A monoclonal antibody against this protein
inhibits calcium uptake and growth of the calcium carbonate spicules produced by embryonic
mesenchyme cells in vitro. The antibody recognizes an anionic N-glycan that may sequester
calcium ions for skeleton formation. Two other proteins were found to carry the epitope and may
be involved in spicule formation, but there is no other information on the oligosaccharide
structure.
Drosophila melanogaster(27 43)
Overview, Advantages, and Limitations
In 1910, T.H. Morgan published the first paper about the genetics of the fruit fly D.
melanogaster, which showed that white eye color was a sex-linked trait. Since then, this model
system has been the predominant organism for genetic analysis. Its advantages include an easily
studied developmental program, a relatively complex neural system, behavior, and the ability to
discern literally thousands of different phenotypes in morphology, development, and behavior.
The Drosophila genome will be sequenced within the next several years (estimated in the year
2001), providing all of the additional advantages of homology screening now available in yeast
and C. elegans. Drosophila is the most complex and versatile nonvertebrate model system
available. Unwary Drosophila geneticists in pursuit of genes that regulate and control pattern
formation and boundary demarcations ran head-on into glycobiology. The studies revealed genes
that ultimately control morphogenesis by establishing boundaries, regulating cell communication
and cell division via signal transduction pathways. These emerging areas hold great promise in
the near future for detecting new functional roles for glycoconjugates and unraveling their
mechanisms.
Even though Drosophila biology has aimed toward functional analysis, there is a paucity of data
on the structures of its glycoconjugates. Many of the exciting functional implications are based
on homology with genes that code for enzymes and lectins known in mammalian cells, but none
of the putative glycosyltransferase gene products have actually been shown to have the expected
activity. Sialic acids and extensive build-up of N-glycans have not been demonstrated, but the
existence of multiple GlcNAcTs in C. elegans indicates that similar processing will probably
occur in Drosophila.
Structures and Biosynthesis

N-linked Pathway
Drosophila has the typical biosynthetic enzymes needed for the lipid-linked oligosaccharide
precursor in the N-linked pathway. Processing includes the expected α-glucosidases, but the
endomannosidase that removes the Glcα1 3Man from newly synthesized chains is not found. α-
mannosidase-mediated processing occurs, and mutants lacking mannosidase I have only a mildly
altered wing and eye morphology, suggesting that another compensatory pathway may bypass
the defect. High-mannose-type glycans with a core Fucα1 6GlcNAc are found, and Man-6-P
has been reported on a lysosomal DNase. Honeybees make biantennary GlcNAc-terminated
chains in which the core GlcNAc is substituted by both α-3-linked and α-6-linked fucose
residues. It is likely that the fruit flies make them as well.
O-linked Pathway
Typical Galβ1 3GalNAcα-Thr/Ser (Core 1) structure has been identified in Drosophila. This
disaccharide is also seen in other insects and insect cell lines such as Sf9. One functional role for
Galβ1 3GalNAc appears to be in defending against microbial attack. A 19-amino-acid peptide
that is produced when insects are challenged by bacteria or trauma requires this disaccharide for
full activity. An identical in-vitro-synthesized peptide has five- to tenfold less activity. Insects
also use mucins as protective barriers in the intestine. For instance, a mucin-like protein, IIM, is
associated with the chitin-containing peritrophic membrane. Invading bacculovirus produces an
enhancin during invasion. This protein has mucin-degrading activity both in vitro and in vivo.
Enhancin-mediated degradation of IIM in vivo enhances viral infectivity, showing that viruses
have evolved an interesting mechanism for penetrating the critical intestinal mucinous barrier.
Mutations in the rotated abdomen locus (rt) cause a clockwise helical rotation of the abdomen.
Null alleles are viable and the rotation is expressed in the mesoderm and midgut. The rt gene
encodes a transmembrane protein that closely resembles the yeast O-mannosyltransferase genes,
PMT1 and PMT2, both of which use Dol-P-Man as a donor. Neither the enzymatic activity nor
the putative acceptor or the product has been established in the insect.
Glycolipids
The glycosphingolipids made by Drosophila have not been well characterized, but those from
other insects have structures that are probably similar (Figure 19.5). Note that the second
monosaccharide from the reducing end is mannose, not galactose, as seen in the glycolipids from
higher organisms. Additional monosaccharides are added that are similar to some of those seen
in higher organisms. However, some glycolipids have terminal GlcA and others have GlcNAc-6-
phosphoethanolamine (GlcNAc-6-P-Etn) residues making the molecule a zwitterion. Both GlcA
and GlcNAc-6-P-Etn can also occur on the same molecule. Drosophila also contains GPI-
anchored proteins.
Figure 19.5. Examples of glycolipids found in insects. Glycosphingolipids of insects (flies) have
unusual structures that include neutral, neutral zwitterionic, and acidic zwitterionic species. The
ethanolamine phosphate residues confer the zwitterionic properties and glucuronic acids provide
negative charge.
Other Types of Glycosylation?
Notch is a large, multimodular receptor protein involved in cell-fate determination. Members of

the Notch family of receptor proteins are widely distributed from C. elegans to humans. One
characteristic of Notch is the presence of epidermal EGF-like modules. As discussed in Chapter
12, the EGF-like domain modules in the human Notch homolog have been shown to contain two
unusual types of O-glycosylations, O-β-glucose and O-α-fucose which have been implicated in
Notch's interactions with its ligands. It is likely that such posttranslational modifications of the
EGF-like modules could influence binding and associated interactions. Although Drosophila
also has notch protein, it has not yet been shown to have these modifications.
Antibodies to Carbohydrate Structures
Proteins in the nervous system of many arthropods, including Drosophila, cross-react with
antibodies that recognize the β1 2-linked xylose and α1 3-linked fucose residues in the core
region of N-glycans. These oligosaccharide antigens are found in many plant glycoproteins such
as horseradish peroxidase. Neural cell bodies and axons of the central and peripheral nervous
systems in embryos and adults are reactive with polyclonal and monoclonal antibodies against
this epitope. Mutants that lack antibody binding have altered neural development and behavioral
deficits, suggesting that the tissue-specific neural cell interactions are mediated by these
oligosaccharides. Specific endogenous lectins in the Drosophila embryo that bind to the
horseradish peroxidase epitope have not been identified.
Frontiers of Drosophila Glycobiology

Lectins
Mutations in the furrowed (fw) gene reduce the size of the eyes and create furrows and crevices
in the retina. The ommatidia have an altered morphology and an abnormal number of cells. The
bristles also show an altered structure and can be duplicated or missing entirely. This phenotype
suggests that the fw gene is involved in events that affect cell determination in the sensory
organs. The gene has a C-type lectin-binding domain and a series of ten complement binding
domains. These features are reminiscent of the selectin architecture. The lectin-binding domain
was well matched in critical cysteine residues and hydrophobic clusters when compared to E-
and L-selectins, to the mannose-binding protein, to the lectin-binding domain from barnacle
lectin and another from the flesh fly. Since it is expressed in the larval imaginal discs, it may
mediate some carbohydrate protein interactions. However, the expressed protein has not yet been
shown to bind to any carbohydrate structure from insects or any other organism. It is important
to remember that relatively small changes in the primary structures of lectins can substantially
alter their carbohydrate-binding properties. Since sialylated oligosaccharides are not found in
Drosophila, the glycans that bind to the fw gene product are unlikely to be sialylated. Another C-
type lectin has been characterized from pupae and appears to specifically bind galactose and may
be involved in pathogen clearance.
Lectin-carbohydrate interactions are likely to mediate some cell-specific connections in the

developing nervous system. Screening of an embryonic cDNA expression library produced one
candidate lectin called "gliolectin." The predicted cDNA would code for a protein of
approximately 25 kD, but a monoclonal antibody against the expressed protein precipitates a 46-
kD molecule, suggesting that it has extensive posttranslational modifications. The molecule is
expressed in selected embryonic glial cells found at the midline of the developing ventral nerve
cord. The first axons to extend within the central nervous system of the Drosophila embryo
establish pathways that cross the midline to form axonal commissures. Spatial and temporal
expression of gliolectin is compatible with specific glia-glia or glia-neuron interactions.
Gliolectin binds specifically to a subset of neutral and zwitterionic (phosphorylethanolamine-
containing) glycolipids found in Drosophila embryos. Mutant lines lacking gliolectin show
defects in axonal pathfinding, suggesting that gliolectin is important for commissure formation.
Signaling Proteins
Fringe is a signaling protein that is able to induce wing margin formation in Drosophila. This
process involves specifying the fate of cells at specific boundaries. Homologs have been found in
the mouse where they are expressed at developmentally important boundaries in mesodermal,
neuroepithelial, epidermal, and hematopoietic tissues. All of the genes have significant
homology and secondary structural aspects that are similar to those of bacterial
glycosyltransferases. The fringe expression boundaries coincide with the notch-dependent
patterning and with the Notch ligand expression boundaries. The fringe protein has not been
shown to have any glycosyltransferase activity, but this may not be surprising, since activity is
dependent on selecting the correct substrate and identifying the correct donor. Neither of these
critical properties is necessarily predictable from their homology with a bacterial
glycosyltransferase.
Among P-element insertional studies in Drosophila was the surprising identification of two P-
element mutants that effect signaling pathways apparently by disrupting heparan sulfate
proteoglycan synthesis. In Drosophila, the wingless gene is part of the Wnt family of proteins
which are responsible for regulation of cell proliferation in embyonic development. wingless
signaling is required for proper patterning of entire segments of the Drosophila embryo. Defects
in two genes disrupt the wingless and FGF signaling pathways. Although the genes have been
variously christened by different labs, the names sugarless (sgl) and sulfateless (sfl) are the most
convenient. sgl encodes a protein that has homology with UDP-Glc dehydrogenase (see Chapter
6), and sfl encodes a protein that has homology with heparan sulfate N-deacetylase/N-
sulfotransferase. The first enzyme is essential for producing activated UDP-GlcA from UDP-Glc
and the second is essential for biosynthesis of heparan sulfate chains, showing that heparan
sulfate must have important functional roles (see Chapter 11). sgl and sfl were initially identified
as having a "wingless-like" phenotype since the cuticles in both sgl and sfl were virtually
identical to that seen in the wingless mutant. In some tissues, such as the stomatogastric nervous
system of the sgl and sfl mutants, Wingless signaling is reduced, but not completely blocked.
Wingless overexpression in the mutants bypasses the effects of these mutations and leads to a
normal Wingless signaling pathway. The most likely interpretation of these results is that
heparan sulfate facilitates the binding of the Wingless protein to its receptor. This would be
consistent with the well-known binding and presentation of growth factors by heparan sulfate.
In support of this is the finding that sgl or sfl mutant embryos have altered migration of
mesodermal cells resembling the severe effects of mutations in the genes Heartless (Htl) and
Breathless (Btl), two Drosophila FGF receptors that control the embryonic migration of
mesodermal and tracheal cells, respectively. In the htl mutant, MAP kinase is downstream from
the FGF receptor, and MAP kinase activation in mesodermal cell and tracheal pits is dependent
on the activities of Htl and Btl receptors, respectively. By staining with an antibody against the
active form of MAP kinase, sgl and sfl mutant embryos were found to be blocked in MAP kinase
activity in the critical areas of mesodermal cell and tracheal pits. Sgl and Sfl appear to be critical
for the signal transduction mediated by the Htl and Btl receptor.
A gene called dally (division abnormally delayed) was identified while screening for mutants
that affect development of the visual system. When dally is defective, there is a delay in
progression through the cell cycle, and therefore, cell divisions do not occur on schedule. This
causes defects in the morphogenesis of the antenna, wing, and genitalia. dally codes for a
glycophospholipid-anchored heparan sulfate proteoglycan of the glypican family, and it is
probably a coreceptor for signaling mediated by the Decapentaplegic (Dpp) pathway that
involves the TGF-β/BMP superfamily of growth factors.
Xenopus laevis(44 50)
Overview
The African clawed toad, Xenopus laevis, is a well-established model organism for studying
fertilization and embryonic development. Recent studies of developmentally important genes
illustrate the power and limitations of gene homology, and the importance of biochemical
analysis in dealing with molecules whose synthesis is poorly understood. A new type of lectin
has also been found in Xenopus.
Fertilization
The initial step in fertilization is likely to involve carbohydrate recognition (see Chapter 34).
Sperm binding to the egg vitelline envelope can be inhibited by two glycoproteins (gp69 and
gp64) purified from the envelope. Polyclonal antibodies against these proteins specifically block
sperm binding and fertilization, and glycopeptides made from gp69/64 glycoproteins also inhibit
sperm-egg binding. Sodium periodate treatment of the glycopeptides destroys their blocking
ability, showing that the intact sugar structure is required for inhibition of sperm-egg binding.
DG42, Hyaluronan, and Chitin Oligosaccharide Synthesis
The DG42 gene is expressed in endoderm cells briefly during the mid-late gastrulation stage and
disappears at the end of neurulation. It has homology with fungal chitin synthase and a
Rhizobium NodC gene, which makes short chitin oligomers (GlcNAcβ1 4GlcNAc)n. These
products and some of their complicated derivatives are required for Rhizobium to invade plant
root hairs and produce a nodule for nitrogen fixation. Expressing the DG42 gene product resulted
in chitin oligomers that were digestible by chitinase, and this was taken as evidence that
vertebrates, at least vertebrate embryos, could synthesize short chitin oligosaccharides. However,
DG42 also has considerable homology with the hasA gene from Streptococcus, which is
responsible for hyaluronan synthesis (GlcAβ1 3GlcNAcβ1 4)n (see Chapter 11). Expression of
DG42 in several cell lines and in yeast, which does not make hyaluronan, convincingly showed
that the expressed gene increased the synthesis of hyaluronan (hyaluronic acid) in those cells. In
fact, all mammalian hyaluronan synthase genes isolated and expressed to date have considerable
homology with DG42. Thus, by homology and initial biochemical characterization, there is
evidence for the synthesis of both types of glycans. The controversy is not completely settled at
this time.
The situation is complicated because hyaluronan biosynthesis in vertebrates is poorly understood

(Chapter 11). It is not made in the ER/Golgi pathway, but rather at or near the cell surface.
Furthermore, it is the only vertebrate polysaccharide that is not initiated on a protein. No
"acceptor" or primer has been identified for hyaluronan synthesis. In fact, the nature of the
reducing end of hyaluronan is not known. Favoring hyaluronan synthase activity for DG42 is the
finding that large (2 10 × 106 daltons) chains can be made in vitro by yeast expressing
recombinant DG42. Yeast does not normally synthesize hyaluronan and lacks a primer. Only
UDP-GlcA and UDP-GlcNAc were used to produce a high-molecular-weight hyaluronan, and
kinetic values were comparable to those obtained from membranes of vertebrate cells.
This controversy generated new insights into the importance of chitin oligosaccharides in
development. Xenopus, zebrafish, and carp embryos may all synthesize chitin oligosaccharides
only during late gastrula stages. In vitro synthesis can be inhibited by an antibody against DG42.
Injection of the DG42 antibody into fertilized developing eggs leads to severe defects in the
development of the trunk and tail. The same effects are seen if the embryos are injected with the
Bradyrhizobium NodZ enzyme that fucosylates chitin oligosaccharides. These observations
support a functional role for the chitin oligosaccharides in specific locations and times in
embryonic development.
A New Type of Lectin Discovered in Xenopus
X. laevis oocyte cortical granules contain a lectin (XL35), which is released at fertilization and
binds to mucin oligosaccharides in the egg jelly, and form a layer of the fertilization membrane.
XL35 binds to melibiose and requires calcium, although it does not have the typical sequence
motif for "C-type" lectins. Recombinant lectin is active in cell agglutination assays and is
inhibitable by EDTA and melibiose. The mRNA is expressed in oocytes and remains high
through late gastrula and is still detectable until tadpole stages. The protein is variably N-
glycosylated. This lectin is probably identical to that detected in localized regions of the
developing embryos, where it might be involved in regulating cell adhesion and migration.
Similar lectins have now been found in humans, although they appear to have different glycan-
binding specificities. The human homologs appear to be almost exclusively localized to
endothelial cells lining small blood vessels in selected tissues. The functions in humans are
unknown, but they likely involve intercellular adhesion. Homologs have also been discovered in
C. elegans.
Future Directions
Model systems to find genes that are important for cell adhesion, signal transduction, cellular
communications, and development have identified homologs of enzymes involved in glycan
biosynthesis. This is an important step, but the challenge in the future will be to show that the
suspected homologs actually code for proteins with the expected enzymatic activity or
carbohydrate-binding specificity. This requires in vitro assays, and in some cases, those assays
will have specific substrate requirements or restricted binding properties. This in turn means that
the structure and the biosynthetic pathways of the glycans will need to be understood in some
detail before the activities can be clearly assessed. This was not possible previously, but the
advent of highly sensitive analytical and structural techniques will pave the way. In addition, as
new model systems develop, exploration of their glycobiology will present greater opportunities.
A good example of this is the zebrafish system, where no explorations of glycobiology have
been reported.
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20. Glycobiology of Plant Cells
Primary contributions to this chapter were made by M.J. Chrispeels (University of California at
San Diego).
THE GLYCOBIOLOGY OF PLANTS is a huge area of research. This chapter therefore

addresses only the highlights of plant glycobiology, particularly those fields in which researchers
are making the most rapid progress. Plant scientists tend to focus their research on aspects of
biology that are unique to plants, although some investigations that run parallel to work being
done with animal cells also take place. Therefore, the focus of this chapter is also on problems
that are unique to plants. Plant cells have a flexible cell wall that should be thought of as an
extracellular matrix, rather than a box outside the cell. There are unique aspects to its
biosynthesis, its role in cellular architecture, and its role as a source of signals for patterning and
morphogenesis. The cell wall can also be a source of small glycans that signal pathogen
invasion. Plant cells have well-developed signaling cascades that turn on defense genes in
response to such oligosaccharide signals. In addition, glycolipid signals are used by bacteria to
establish species-specific symbiotic associations with plants. The complex N-glycans made by
plants differ from those made in mammals and resemble those made in insects and mollusks.
The Polysaccharide-rich Cell Wall Is the ECM of the Plant Cell

(1 4)
A discussion of plant glycobiology must start with a description of the structure and function of
the cell wall or extracellular matrix. Until the 1960s, the wall was viewed as a dead box that
surrounds the living protoplasm. In the 1970s, it came alive as cell wall proteins and enzymes
were discovered. In the 1980s, it was found to be a source of signals that plants use to detect
pathogens attempting to break down the walls of plant cells, and in the 1990s, the cell wall's
important signaling role in cell growth and differentiation has been recognized. No longer the
dead box, the ECM is very much part of the living cell. This brings us full circle, because in
1665 Robert Hooke discovered little boxes in cork, pith, and cortex tissues and called them
"cells." The plant cell wall remains an extremely active subject of research in plant biology.
We currently view the cell wall as a three-dimensional network of cellulose microfibrils and
cross-linking polysaccharides (hemicellulose) embedded in a gel matrix of acidic
polysaccharides rich in galacturonic acid (pectins) and held together by calcium bridges (Figure
20.1). The two major groups of flowering plants, the monocots and the dicots, differ in their
hemicellulosic polymers: Monocots have arabinoxylans and dicots have xyloglucans. This
network is enmeshed with a variety of structural proteins and glycoproteins and is partially
cross-linked by aromatic substances (monolignols). Enzymes needed for the biosynthesis or
modification of the network (e.g., peroxidase, xyloglucan endotransglycosylase, α-fucosidase,
and glucanase) or for the modification of metabolites (e.g., invertase, phosphatase, and ascorbic
acid oxidase) may be ionically or covalently bound to the hemicellulose network or the pectin
matrix.
Figure 20.1. Model of the primary wall (type I) found in most flowering plants. Three parallel
cellulose microfibrils are embedded in a xyloglucan network that shows bond breakage as a
result of cellular expansion. The vertically oriented pectin (polygalacturonic acid or PGA)
molecules form a hydrated gel. The orientation of the rod-like extensin molecules is unknown.
(Reprinted, with permission, from [2] Carpita and Gibeaut 1993.)
Assembly of the cell wall requires the coordination of the activities of large cellulose synthase
complexes in the plasma membrane with those of 20 25 different glycosyltransferases in the
Golgi that assemble the pectin and hemicellulosic polymers. These polymers are transported to
the plasma membrane in secretory vesicles, as are the cell wall proteins and glycoproteins, and
their cross-linking is catalyzed by cell wall enzymes. Despite this cross-linking, the walls of
young cells remain flexible and can extend through breaking and reforming of cross-links to
accommodate the enormous volume changes that characterize the growth of plant cells. In highly
differentiated cells such as xylem vessels (which carry water and nutrients up from the roots) and
fibers, the walls become thick, stiff, and incompressible after impregnation with lignin, a
polymer that is formed extracellularly by polymerization of secreted monolignol subunits.
Cell differentiation is accompanied by the synthesis and secretion of different components of the
ECM of plant cells, as visualized by interaction of monoclonal antibodies against cell wall
epitopes. This process has recently been studied in Zinnia cells. When exposed to the hormones
auxin and cytokinin, Zinnia mesophyll cells trans-differentiate into tracheary elements. This
trans-differentiation is accompanied by a new pattern of gene expression and changes in the
repertoire of proteins and polysaccharides at the cell surface.
ECM Glycoproteins Have Hydroxyproline Residues O-

glycosylated with Short Arabinosides (5 7)
The ECM of plant cells contains at least three types of structural proteins: glycine-rich proteins,
proline-rich proteins, and hydroxyproline-rich glycoproteins (HRGP). The best characterized
HRGP is carrot extensin, a protein with a molecular mass of 86 kD. The polypeptide has a
molecular mass of 30 kD and contains 25 repeats of the pentapeptide SerHypHypHypHyp
interspersed with TyrLysTyr units. Hydroxyproline residues are frequently O-glycosylated with
short arabinofuranosides that contain from one to four arabinosyl residues. The proximal
arabinose is linked 1 4 to hydroxyproline, the next two arabinoses are linked 1 2, and the
terminal arabinose is linked 1 3 in this short chain. The proportion of total hydroxyproline
residues in SerHyp4 pentapeptides varies between different HRGPs. Proline-rich proteins and
HRGPs may form a continuum rather than two distinct classes of proteins, because proteins that
are rich in proline and hydroxyproline have also been identified. The glycosylation of
hydroxyproline residues follows the contiguity rule, i.e., a hydroxyproline residue must be next
to another hydroxyproline to be glycosylated. The analysis of a Douglas fir HRGP showed that
IleProProHyp is never glycosylated, LysProHypValHyp is occasionally mono-arabinosylated at
Hyp-5, and LysProHypHypVal is always glycosylated with a tri-arabinoside at Hyp-3. In carrot
extensin, three of the four hydroxyproline residues in SerHyp4 pentapeptides are glycosylated
with tetra-arabinosides. Galactose residues are O-linked to the serine residues of extensin. The
carbohydrates serve to stabilize the protein into a rigid rod-like structure. Other HRGPs, called
arabinogalactan proteins, have larger glycans attached to hydroxyproline or serine residues (see
below). The TyrLysTyr repeated tripeptides serve a different structural role. The ECM-located
enzyme extensin peroxidase cross-links the tyrosine residues to form isodityrosine intra- and
intermolecular cross-links. This creates intermolecular as well as intramolecular cross-links.
Extracellular Arabinogalactan Proteins Are Markers for Cell

Fate and Can Induce Embryogenesis (8 9)
Arabinogalactan proteins (AGPs) are extracellular or plasma-membrane-anchored proteins that
are typically more than 90% carbohydrate. The polypeptide backbone has a domain that is rich in
alanine, serine, threonine, and hydroxyproline, and the carbohydrate moiety consists of short
arabinosides attached to hydroxyproline (as in carrot extensin, see above) and arabinogalactans
O-linked to hydroxyproline or serine. Both galactosyl-O-serine and galactosyl-O-hydroxyproline
linkages have been identified. The arabinogalactan side chains have 30 150 sugar residues, and
most have a pure β(1 3)-d galactan backbone with terminal l-arabinofuranosyl residues on
short side chains. The secreted AGPs generally have much more carbohydrate than the plasma-
membrane-anchored forms, and the relationship between the two is not known. AGPs are highly
heterogeneous, and each organ and cell type are associated with a characteristic subset of AGPs.
The cDNAs of several AGPs have now been cloned and have been classified as "classical" AGPs
(130 150 amino acids), consisting of a signal peptide, a hydroxyproline-, alanine-, serine-, and
threonine-rich domain, and a membrane-anchoring helix, and as "non-classical" AGPs, which are
larger (170 450 amino acids) and have a signal peptide and a hydroxyproline-, alanine-, serine-,
and threonine-rich domain followed by a large carboxy-terminal domain of variable length and
character (Figure 20.2). The variability found in AGP amino acid sequences, the degree of
glycosylation, and the size of the glycans are similar to those observed in polypeptide backbones
and glycosylation of the proteoglycans in the ECM of animal cells.
Figure 20.2. Schematic representation of the domain structures of three different AGPs from
Nicotiana alata (see reference 8).
AGPs are found abundantly in conditioned plant cell culture media and have recently been
shown to have an important role in cell fate determination and somatic embryogenesis. Newly
established suspension cultures of carrot are embryogenic: Upon the withdrawal of the hormone
auxin, some of the cells develop into somatic embryos. Such cultures contain three major
different cell types that persist as long as the culture keeps its embryogenic potential: spherical
cells that are either densely cytoplasmic or vacuolate and elongated cells that are vacuolate. Only
spherical cells give rise to embryos; elongated cells seldom do. A culture of carrot cells
maintains its embryonic potential as long as there are spherical cytoplasmic cells. The presence
of AGPs on these cell types can be detected with monoclonal antibody JIM8; only the vacuolate
spherical cells are JIM8(+). The densely cytoplasmic spherical cells, which are JIM8(-), only
develop into embryos if they are cultured with JIM8(+) cells or if AGPs are added to the culture
medium (Figure 20.3). Elongated cells do not develop into embryos even when AGPs are
present. This recent work confirms a number of older but less detailed observations that AGPs
can stimulate embryogenesis in cell culture.
Figure 20.3. Developmental fate of different cell types of carrot (Daucus carota) in suspension
culture. The "halo" around the cell indicates staining of AGPs by monoclonal antibody JIM8. B
cells can divide asymmetrically, and in the presence of AGPs, the spherical densely cytoplasmic
cell can give rise to an embryo.
Growth of the Plant Cell Requires Loosening of the Xyloglucan

Network, a Process Mediated by Proteins and Oligosaccharides
(10 14)
Young plant cells increase enormously in volume, a process that requires a loosening of the
hydrogen bonds that bind xyloglucans to cellulose microfibrils, the enzymatic cleavage of
xyloglucan chains, and an internal osmotic pressure that pushes the microfibrils apart. This is
accompanied by a laying down of layers of new cellulose microfibrils with their associated
hemicellulose polymers. The process of cell wall loosening is catalyzed by expansins, endo-β-
glucanases, and xyloglucan-endotransglycosylases (XET). Expansins are small (27 kD) cell-
wall-associated proteins that are capable of inducing cellular extension in vitro. They do so by
disrupting noncovalent steric or hydrogen bonding between cellulose and the polysaccharides of
the matrix. The derived amino acid sequence of expansin shows the presence of several domains:
a cysteine-rich domain with eight conserved cysteines, a basic domain, and a tryptophan domain.
The spacing of these domains is similar to that of the cellulose-binding domains of hydrolases
synthesized by cellulolytic bacteria. Recent evidence shows that expansin expression is up-
regulated whenever new leaf primordia are initiated in tomato meristems, suggesting that these
proteins have a major role in plant development.
The importance of the xyloglucan network and of the enzymes that hydrolyze covalent bonds in
this network is underscored by numerous observations showing that higher β-1 4-glucanase
activities are correlated with higher rates of cell expansion; furthermore, antibodies to these
enzymes inhibit cell expansion. The action of either endo-β-1 4-glucanase or XET on the
xyloglucan network results in the release of xyloglucan fragments, including nonasaccharides
called XXFG (Figure 20.4). XXFG oligosaccharides were first found to inhibit auxin-stimulated
cell growth at nanomolar concentrations and were later shown to be normal constituents of the
cell wall. How XXFG causes cell wall properties to be changed remains obscure. Removal of the
terminal fucose from XXFG by a cell-wall-localized α-fucosidase inactivates the
oligosaccharide, but substitution by galactose does not. Thus, the abundance of the active
oligosaccharide in the ECM may be regulated by the enzymes that cause its formation (endo-β-
1 4-glucanase or XET) and its inactivation (α-fucosidase).
Figure 20.4. Xyloglucan-derived nonasaccharide XXFG that inhibits auxin-induced growth in

stems.
The ECM Is a Source of Oligosaccharide Signals for Defense

against Pathogens (15 16)
When plants are invaded by pathogens, they mount elaborate defense responses. If the pathogen
is detected early on, necrotic spots develop at the site of infection, and this limits the spread of
the pathogen. If detection is too slow, the plant mounts a feeble response and the pathogen gains
the upper hand. The response consists of the activation of a number of genes that lead to cross-
linking of cell wall polymers, the production of additional structural cell wall components (e.g.,
monolignols and hydroxyproline-rich glycoproteins), phytoalexins (small molecules that kill the
invader), and enzymes such as β-glucanases and chitinases that degrade the cell wall of the
invading pathogens. In this interaction between the plant and the invading pathogen,
oligosaccharides coming from the cell wall of either partner can act as elicitors, triggering the
defense responses (Figure 20.5).
Figure 20.5. Oligosaccharides from fungal and plant cell walls that elicit plant defense
responses.
Oligosaccharide elicitors generally have between 4 and 20 sugar residues. Degradation of fungal
cell walls yields β-glucan, chitin, and chitosan oligosaccharide elicitors. Degradation of the plant
cell wall results in oligogalacturonides, pectin fragments with a degree of polymerization of 10
20, that act as potent elicitors. Little is known as yet about the mechanisms responsible for
perceiving these oligosaccharide signals and transducing this information to the nucleus, so that
defense genes can be turned on. Photoaffinity-labeling experiments identified a 70-kD soybean
plasma membrane protein that binds the β-glucan heptasaccharide from Phytophtera sojae, a
fungal pathogen of soybean. Whether this protein is a receptor and is responsible for signal
transduction remain to be demonstrated. The involvement of calcium, a universal intracellular
messenger, in signal transduction by the β-glucan heptasaccharide has been postulated on the
basis of experiments with calcium ionophores and calcium channel blockers.
Signaling by oligogalacturonides may involve systemic second messengers. Oligogalacturonides

are released from cell wall pectins at the site where pathogen attack occurs, but
oligogalacturonides are not mobile in the plant. In tomato (Lycopersicum esculentum), the
elicitation of a defense response at some distance from the site of infection is due to the
production of systemin, an 18-amino-acid peptide that is transported in the plant. Systemin
activates the same plant defense genes as oligogalacturonides but is a million times more active.
Whether elicitation of defense genes by systemin or a similar peptide occurs in other plant
species is not known.
Defense genes are also activated by jasmonate, which is synthesized from linolenic acid by the
octadecenoic pathway. Within minutes after an insect begins to feed on a plant, the jasmonate
content of the leaves rises, possibly as a result of the release of vacuolar lipases. Whether these
three signaling molecules, oligogalacturonides, systemin, and jasmonate, act in parallel or in
series in the activation of the defense response remains to be elucidated. One possible model for
the role of these three compounds in activating defense responses is shown in Figure 20.6.
Figure 20.6. Proposed model for the signaling leading to the activation of defense genes. (PK)
Protein kinase; (LOX) lipoxygenase; (JA) jasmonic acid.
Glycolipid Signals Have a Key Role in Establishing a Symbiotic

Relationship between Nitrogen-fixing Bacteria and Plants (17 22)
To establish a symbiotic relationship with legumes, soil-dwelling rhizobia must invade the roots
of the plants and induce the formation of nodules, specialized organs in which the bacteria
multiply and fix atmospheric nitrogen while deriving energy and carbon skeletons from the
photosynthetic products supplied by the plant. This complex process requires the expression of
numerous genes in both partners and involves signals from the plant and from the rhizobia.
Flavonoids released by plant roots induce bacterial nod genes that encode enzymes involved in
the synthesis of Nod factors, lipochitooligosaccharides (LCOs) consisting of a tetra- or
pentameric chitooligosaccharide backbone to which a long-chain unsaturated fatty acid is
attached at the nonreducing end (Figure 20.7). The recognition between the legume host and
Rhizobium symbiont is species-specific, and depending on the Rhizobium species, there is
variation in the structure of the fatty acids and in substitutions at the reducing and the
nonreducing ends of the glycan. LCOs elicit the formation of root nodules at extremely low
concentrations. The responses of the susceptible root zone near the root tip to the addition of
LCOs include root hair curling, induction of calcium pulses, induction of cell division by
activation of the cell cycle of cortex cells, and the activation of numerous genes, the products of
which play an important part in the early steps of symbiosis (ENOD or early nodulin genes).
Figure 20.7. Structures of LCOs produced by Rhizobium strains. The length of the
oligosaccharide backbone can vary between 3 and 5 sugar units. The lipid can, for example, be
C18:4 (R. leguminosarum bv. viciae), C16:2, or C16:3 (Rhizobium meliloti), or 18:1
(Bradyrhizobium japonicum). Additional substituents on the sugar backbone can be sulfate, 2-O-
methylfucose, an O-acetyl group, or an N-methyl group. The fatty acid shown is C18:4.
LCOs may have other roles in plant development. In the temperature-sensitive carrot cell line
mutant ts11, somatic embryogenesis does not proceed beyond the globular stage at the
nonpermissive temperature. This defect can be rescued by the addition of endochitinase the
mutant embryos fail to synthesize endochitinase or by the addition of GlcNAc-containing
glycans or by LCOs with an 18:4 fatty acid. The results suggest that GlcNAc-containing glycans
that resemble LCOs and are released from larger precursor molecules through the action of
extracellular endochitinases have an important role in this developmental process.
Complex N-Glycans on Plant Glycoproteins Differ from Those

on Mammalian and Yeast Glycoproteins and Can Be Highly
Immunogenic (23 29)
Like other eukaryotic organisms, plants have glycoproteins with N- and O-glycans. N-glycans
are attached to asparagine residues and O-linked glycans are attached to hydroxyproline, serine,
or threonine residues. Only a few plant proteins have been described with both types of glycans.
N-linked glycans are commonly found on secreted and vacuolar proteins and on proteins of the
endomembrane system. Two types of N-glycans are known: high-mannose glycans with the
composition Man5 9GlcNAc2 and complex glycans with fewer mannose residues and additional
residues of other sugars (Fuc, Xyl, GlcNAc, Gal) (Figure 20.8). The biosynthesis of high-
mannose glycans in plants is identical to that in yeast and mammals (see Chapter 7) and involves
the en bloc transfer of the Glc3Man9GlcNAc2 oligosaccharide from dolichylpyrophosphate to
specific asparagine residues in nascent polypeptide chains. Removal of glucose residues by
glucosidase I and II in the lumen of the ER (see Chapter 7) is followed by transport of the protein
to the Golgi and modification of certain high-mannose glycans (those that are accessible to the
modifying enzyme in the Golgi) to complex glycans. The initial modifications in the Golgi
resemble those found in mammalian cells: removal of four mannose residues by mannosidase I,
attachment of a peripheral GlcNAc by GlcNAc transferase I, removal of two more mannose
residues by mannosidase II, and attachment of a second peripheral GlcNAc residue. The first two
reactions always precede the activity of two glycosyl transferases that attach an α1 3 fucose
residue to the proximal GlcNAc of the chitobiose core and a β1 2 xylose residue to the proximal
mannose of the core. The fucose and xylose substituents are not present on all complex glycans.
Transport of such glycoproteins to the vacuole invariably results in the removal of the peripheral
GlcNAc residues, because vacuoles contain N-acetylglucosaminidases. Similarly, the presence
of α-mannosidase in vacuoles may result in trimming of the high-mannose glycans that escaped
the action of mannosidase I in the Golgi. Thus, many complex glycans on vacuolar proteins are
rather small and have the structure (Xyl)Man3GlcNAc(Fuc)GlcNAc. It is important to note that
mammalian glycoproteins have not been reported to contain α1 3 fucose or β1 2 xylose
residues. However, similar constituents and epitopes are reported on the glycoproteins of lower
animals (e.g., molluscs, insects, and spiders). Plant glycoproteins are unusually immunogenic
when injected into mammals, and the resulting sera broadly recognize all glycoproteins with
complex glycans found on plant glycoproteins. It is likely that the α-1,3 fucose and β-1,2 xylose
residues account for this immunogenicity in mammals, and the potential for inducing allergic
responses.
Figure 20.8. Structures of a high-mannose N-glycan and three complex N-glycans found in plant
glycoproteins.
Secreted glycoproteins retain their peripheral GlcNAc residues because the cell wall has little or
no N-acetylglucosaminidase activities. The secreted protein laccase is the only protein so far
identified that has larger complex glycans. The antennae of the complex glycans on laccase are
elongated by galactose and fucose residues and have the same structure as a Lewis A antigen
(Galβ1 3[Fucα 1 4]GlcNAc). N-glycans on plant glycoproteins stabilize them against
proteolytic degradation, but the function of complex glycans (as opposed to high-mannose
glycans) remains obscure. An Arabidopsis mutant (cgl) that lacks GlcNAc transferase I
(GlcNAcT-I) and is unable to convert high-mannose glycans to complex glycans has no visible
phenotype when grown under standard laboratory conditions. This is in striking contrast to the
effects of GlcNAcT-I deficiency in mice, which results in embryonic lethality at day 9.5 (see
Chapters 7 and 33). Interestingly, when the mutant cells are transformed with a mammalian
GlcNAcT-I, the transgenic cells synthesize glycoproteins with normal complex glycans,
suggesting that the mammalian enzyme is correctly targeted to and functions in the plant Golgi
apparatus.
Production of Mammalian Glycoproteins in Plants Requires the
Use of Mutant Plants That Synthesize Different N-linked
Glycans
One of the attractive uses of transgenic plants is for the production of recombinant
pharmaceuticals such as antibodies or lysosomal enzymes that are needed in large quantities
(molecular pharming). Projects are already under way to produce a number of mammalian
proteins in plants, with clinical trials set to begin in the near future. Nevertheless, if these
mammalian proteins are glycoproteins, they will carry the usual highly immunogenic and
potentially allergenic N-linked glycans of plants. Several approaches can be used to avoid these
potential problems. If mammalian proteins are produced in GlcNAcT-I-deficient mutant plants
similar to those described by von Schaewen et al., then all the glycans will be of the Man5
GlcNAc2 type, and such glycans are not immunogenic. However, the mannose-terminating
glycans could cause them to be rapidly cleared by the macrophage mannose receptor of
mammals. A second approach would be to eliminate (knock out) certain hydrolases (e.g.,
vacuolar N-acetylglucosaminidase) and glycosyltransferases (e.g., for xylose and fucose) and
add specific mammalian glycosyltransferases. Using plant cells transformed with mammalian
GlcNAcT-I, Gomez and Chrispeels showed that this mammalian Golgi enzyme can function
correctly in a plant cellular environment, and thus it should be possible to obtain plants that make
N-glycans without xylose and with α-1,6 fucose on the proximal GlcNAc. It is not clear whether
plants could readily synthesize sialylated glycoproteins, because this would require
transformation with a large number of genes, since plants are not known to synthesize sialic acid.
Thus, enzymes to make this sugar, to couple it to CMP, and the protein to transport CMP-Sia
across the Golgi cisternal membrane would have to be incorporated into the transgenic plants as
well as a sialyltransferase. An alternate approach is to produce the mammalian proteins with
carboxy-terminal signals (KDEL or HDEL) that cause the proteins to be retained in the ER.
Proteins that have been modified in this way have been shown to accumulate to quite a large
extent in the ER of leaves. Such proteins typically have high-mannose glycans similar to those of
other ER residents.
Lectins Are Involved in Recognition of Pathogens, Pests, and

Symbionts (30 36)
Lectins were first discovered in extracts of seeds as a result of their hemagglutinating properties
(see Chapter 30), and this activity was later shown to be caused by an interaction of the lectin
with cell surface glycans on red blood cells. Since then, seed lectins have been shown to interact
with the glycans of a variety of organisms, but a true role for seed lectins in the plant has never
been established. Lectins are often abundant proteins not only in the seeds of certain plant
species, but also in leaves, bark, or bulbs. In some cases, the same lectin gene is highly expressed
in one organ (e.g., the seed) but at a much lower level in another organ; in other cases,
homologous genes are expressed in different organs. Wherever lectins are abundant, they are
generally thought to be plant defense proteins.
Lectins are either sequestered in vacuoles or sometimes secreted in the ECM or intercellular
spaces. In plants, lectins are invariably synthesized as preproteins with a signal peptide that
directs the protein into the ER so that it can be either secreted or targeted to the vacuole.
Although plants contain lectins that interact with high-mannose glycans (e.g., ConA), lectins that
interact with the unusual complex glycans found on plant glycoproteins have not yet been found
in plants. Neither do plants contain receptors for their own lectins, except for the high-mannose
glycans present on many glycoproteins.
Phytohemagglutinin is among the most thoroughly studied of all the plant lectins. PHA makes up
4 8% of the protein in the seeds of the common bean (Phaseolus vulgaris) and is widely used in
mammalian cell culture because it is a mitogen. Extensive research by A. Pusztai and his
collaborators has shown that PHA is toxic to mammals. PHA is not readily degraded either by
digestive enzymes or by bacteria in the cecum and colon, and biologically active PHA survives
intestinal passage in rats. PHA recognizes and binds to the complex glycans present on brush
border membranes of the intestinal epithelium. Such binding has local as well as systemic
consequences: hyperplastic growth of the small intestine, increased turnover of brush border
cells, and atrophy of skeletal muscle and thymus. Some of these changes are due to PHA-
induced changes in hormone balance. An interesting consequence of PHA in the diet is an
increase in terminally mannosylated brush border glycoproteins characteristic of immature brush
border cells and a dramatic overgrowth of type-1 mannose-binding fimbriated Escherichia coli
in the intestinal lumen. Although more research has been done with PHA, other lectins such as
those of elderberry and black locust have similar toxic effects.
Lectins have also been shown to be toxic to insects. Wheat-germ agglutinin and Griffonia
simplicifolia lectin II, both GlcNAc-specific lectins, retard the growth of certain insect larvae.
Snow drop lectin (GNA), a mannose-specific lectin, is toxic to sucking insects such as aphids.
These lectins exert their activity by binding to glycoconjugates in the intestinal tract.
Recently, the common bean and the hyacinth bean (Dolichos lablab) have been shown to contain
a new mannose-specific lectin that preserves human cord blood progenitor cells in suspension
culture for up to 1 month without medium changes. This unusual property is yet another example
of the unexpected interactions between plant lectins and animal cells.
It has long been postulated that legume lectins may be involved in recognizing or at least
interacting with Rhizobium symbionts. It is now clear that they are not responsible for the
species-specific interaction between host and symbiont. That role is fulfilled by the LCOs (see
above). Lectins may have a role in "agglutinating" the bacteria onto the roots so that subsequent
interactions can proceed. The role of lectins in nodulation has been investigated in gain-of-
function mutants (overexpression in transgenic plants) and in loss-of- function mutants
(antisense transgenic plants and a soybean insertion mutant). Overexpression of pea lectin in the
roots of white and red clover leads to nodulation of these roots by Rhizobium leguminosarum bv.
viciae, which does not normally nodulate clover. However, this effect can be ascribed to a
greater sensitivity of the clover roots to the LCO produced by R. leguminosarum, rather than to
direct lectin action. A. Hirsch and coworkers characterized two alfalfa lectin genes, Mslec1 and
Mslec2, expressed in roots and flowers, respectively. Alfalfa plants with greatly decreased
expression of Mslec1 (antisense plants) showed normal nodulation by alfalfa rhizobia, although
the nodules quickly senesced. Their experiments show that lectins participate in the host-specific
interaction of rhizobia and plants and that lectins are necessary for establishing functional
nodules, but not sufficient for recognition. Antisense Mslec2 plants had normal nodules, but
various other developmental abnormalities, indicating that lectins have endogenous roles in
plants that remain to be explored.
Future Directions
Many key questions in plant glycobiology remain: How is the plant ECM synthesized and
assembled? How is the synthesis of the various components coordinated? How does the ECM
"grow" to permit changes in cell volume? What are the molecular events in the loosening of the
xyloglucan network? What are the molecules involved in the ECM-plasma membrane-
cytoskeleton continuum? How are signals from within the cell perceived to alter the architecture
of the ECM? What is the role of turgor pressure in this signaling? How do extracellular
macromolecules influence events in the cell and the nucleus? What is the role of complex N-
glycans? If mutant plants that cannot make them are normal, then when and for which proteins
are these glycans essential? Is this pathway only important when plants are under stress? Lectins
function in defense and symbiont recognition, but do they have other roles in physiology and
development and what are these roles? Presumably, plants have the same types of lectins for
cellular processes that have been found in animal cells, but no one is looking for them as yet
because this is not a plant-specific problem. Genome projects will make the cognate genes for
these lectins available. With respect to signaling pathways (glycolipids and glycan elicitors), the
receptors and the molecules that transduce the signals need to be identified, likely with studies of
response mutants.
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21. Bacterial Polysaccharides
Diego).
THIS CHAPTER DESCRIBES THE STRUCTURE AND ASSEMBLY of glycoconjugates and

glycans present in the bacterial cell wall: peptidoglycan, periplasmic glucans,
lipopolysaccharide, and the capsular polysaccharides. The biological significance of bacterial
glycosylation is discussed throughout the chapter, and the recent discovery of bacterial
glycoproteins is presented.
Introduction (1)
Bacteria produce a variety of glycoconjugates and polysaccharides as part of their cell walls, and
they have many "strange" sugars not found in vertebrate glycoconjugates, including KDO,
heptoses, and variously modified hexoses. The various glycans exhibit enormous structural
diversity and complexity, even in a "simple" bacterium such as Escherichia coli (Figure 21.1).
As in other gram-negative bacteria, the E. coli cell wall consists of an inner and outer membrane
separated by a periplasmic space. Peptidoglycan, a polysaccharide covalently linked to short
peptides, represents the major structural component of the periplasm. The periplasm in some
gram-negative bacteria also contains β-glucans, which play a part in osmoregulation. The outer
membrane has an unusual structure for a lipid bilayer since it contains mostly lipopolysaccharide
in the outer leaflet. Mucoid (slimy) strains contain a polysaccharide capsule that surrounds the
whole cell, which may have a role in virulence. Gram-positive bacteria have a similar cell wall
structure, except that they lack the outer membrane and have a much thicker peptidoglycan layer
with additional specialized polysaccharides known as teichoic acids.
Figure 21.1. Structure of the cell wall of E. coli. The cell wall of gram-negative bacteria consists
of several layers of various polysaccharides. The periplasm contains peptidoglycan, a copolymer
of polysaccharide and short peptides, and a class of β-glucans known as MDOs. The outer leaflet
of the outer membrane is rich in LPS. In mucoid strains, a capsular polysaccharide covers the
entire cell (not shown). (Adapted, with permission, from [8] Raetz 1993.)
As discussed below, the polysaccharide components have important structural and functional
roles in the life of a bacterial cell. The capsular polysaccharides and LPS represent the first line
of defense against complement and bacteriophages. They also contain the major antigenic
determinants that distinguish various serotypes of bacteria, which are sometimes correlated with
disease. These components can also have profound effects on the mammalian host. LPS contains
lipid A, also known as endotoxin, which causes various secondary complications of infections,
such as septic shock, multiple organ failure, and mortality. Thus, considerable interest exists in
developing agents to block these deleterious endotoxin-stimulated effects.
Peptidoglycan (1 5)
Peptidoglycan (also known as murein) makes up about 10% of the dry weight of the cell wall in
gram-negative bacteria. Its cross-linked structure confers mechanical strength and shape to the
cell and provides a barrier to withstand internal osmotic pressure. Nevertheless, it has sufficient
plasticity to allow cell growth (elongation) and division (septation).
Peptidoglycan consists of parallel strands of polysaccharide composed of GlcNAc and N-

acetylmuramic acid (MurNAc; Figure 21.2) in β1,4 linkages, cross-linked by covalently bound
short peptides composed of l-Ala, unusual d-amino acids (d-Glu and d-Ala), and l-
diaminopimelic acid (l-DAP). The strands are thought to be arranged as a monolayer or possibly
a bilayer oriented parallel to the circumference of the bacterium (Figure 21.1). The peptide cross-
links occur frequently along the chains, but the structure is highly dynamic since about 50% of
the peptidoglycan turns over each generation (~30 minutes).
Figure 21.2. Structure of the disaccharide-peptide (muropeptide) subunit. Peptidoglycan consists

of a backbone of repeating disaccharide-peptides with interstrand cross-links. (DAP)
Diaminopimelic acid.
Assembly of peptidoglycan takes place according to the following scheme (Figure 21.3):
Figure 21.3. Assembly of the peptidoglycan. Assembly of the peptidoglycan occurs by transfer
of the disaccharide pentapeptide to nascent peptidoglycan. Undecaprenol phosphate (P) has a
catalytic role in the process.
• UDP-MurNAc (formed by condensation of phosphoenolpyruvate with UDP-GlcNAc

followed by reduction) is modified by the sequential addition of amino acids to form a
UDP-MurNAc-pentapeptide derivative. The addition of each amino acid requires a
specific amino acid ligase, and each reaction is driven by the hydrolysis of ATP. The
final two amino acids (d-Ala d-Ala) are added as a dipeptide unit. All of these reactions
are catalyzed by cytosolic enzymes.
• The MurNAc-pentapeptide is then transferred by a membrane translocase to
undecaprenyl (C55) phosphate, also known as bactoprenyl phosphate. This lipid
resembles the dolichol carrier in eukaryotes used for the formation of the precursor
oligosaccharide of N-glycans found on glycoproteins. The final product contains a
pyrophosphate linkage.
• A second enzyme (transferase) on the inner surface of the inner membrane transfers
GlcNAc from UDP-GlcNAc to the undecaprenyl-PP-MurNAc-pentapeptide to form the
disaccharide pentapeptide linked to the lipid carrier. This precursor, called the
muropeptide, constitutes the subunit from which peptidoglycan will assemble.
• The undecaprenol acts as a carrier to move the subunits from their site of assembly in the
cytosol across the inner membrane, but little is known about the transport mechanism.
The subunit then transfers en bloc to existing peptidoglycan by a transglycosylation
reaction. Two mechanisms have been proposed: growth from the reducing end (the 4-OH
group of the nonreducing GlcNAc residue of a muropeptide attacks the MurNAc-P
linkage of a nascent [lipid-bound] peptidoglycan chain displacing undecaprenyl-PP) or
growth from the nonreducing end (the nonreducing end GlcNAc of the nascent
peptidoglycan attacks MurNAc-P linkage in a subunit, again with liberation of
undecaprenyl-PP). A typical chain contains on average 15 subunits, but the mechanism
controlling chain length is unknown. The final release of the peptidoglycan chain is
coupled to the formation of 1,6 anhydroMurNAc on the "reducing end" of the chain (not
shown).
• The undecaprenyl-PP undergoes cleavage of one phosphate group, which then makes it
available for another round of transfer.
• The final reaction involves formation of interchain cross-links by transpeptidation, in
which cleavage of the terminal d-Ala residue results in transfer of the liberated carboxyl
group of the new terminal d-Ala residue to the amino group of a DAP unit of a
neighboring strand. Thus, the final structure contains tetrapeptide cross-links located on
average every other subunit (Figure 21.4).
Figure 21.4. Cross-linked structure of peptidoglycan. Peptidoglycan consists of layers of

MurNAc-GlcNAc polysaccharide cross-linked by peptides. (A) Ala; (G) Glu; (D) DAP;
(hexagon) MurNAc; (closed box) GlcNAc.
Many well known antibiotics act on the ligases and the transpeptidase involved in the formation
of the peptidoglycan layer. Bacitracin blocks the dephosphorylation and recycling of bactoprenol
pyrophosphate. Both gram-positive and gram-negative bacteria contain penicillin-binding
proteins that participate in the transglycosylation and transpeptidation reactions. As their name
implies, penicillin and other β-lactam antibiotics bind these proteins and inhibit the insertion of
the peptidoglycan subunits. Vancomycin binds to the d-Ala d-Ala dipeptide in the muropeptide.
Interestingly, vancomycin-resistant strains have a mutation in the ligase that allows the enzyme
to form d-Ala d-lactate instead of d-Ala d-Ala.
Peptidoglycan is highly dynamic, with about 50% turnover per generation catalyzed by
autolysins (glycosidases, peptidases, and amidases). The continuous turnover of the
peptidoglycan in the presence of antibiotics results in loss of peptidoglycan, loss of cell wall
integrity, and lysis due to osmotic swelling.
Lysozyme will cleave the peptidoglycan between the GlcNAc and MurNAc disaccharides. In
fact, this is the basis for breaking open gram-negative bacteria in the laboratory: Lysozyme-
treated cells are osmotically sensitive and easily disrupted by shearing. Homogenization
followed by density gradient centrifugation separates cytosol and the inner and outer membranes.
A host defense mechanism in vertebrates involves creating holes in the outer membrane with
complement, which allows lysozyme secreted by leukocytes to penetrate and disrupt the murein
layer.
Gram-positive bacteria lack the outer membrane of gram-negative bacteria and have a thicker
peptidoglycan layer, representing as much as 20 25% of the dry weight. In gram-positive
bacteria, the polysaccharide backbone typically contains 100 disaccharides, and every tenth unit
on average contains a teichoic acid. Bacillus strains contain teichoic acids composed of
polyribitolphosphate or polyglycerophosphate, but many other types of teichoic acids exist.
Some teichoic acids contain a reducing terminal phosphatidic acid and go by the name of
lipoteichoic acids. All teichoic acids bound to peptidoglycan appear to be linked by a conserved
linkage unit consisting of ManNAc-GlcNAc-1-P linked to C6 of a MurNAc residue. The teichoic
acid polymer composed of polyribitolphosphate and polyglycerophosphate forms on the linkage
unit while still attached to core disaccharide precursor ([ribitolphosphate]n-[glycerophosphate]2-
3-ManNAc-GlcNAc-PP-undecaprenol), and the entire unit is thought to be transferred en bloc to
the peptidoglycan. The outer ribose and glycerol units can then be decorated with d-Ala, sugars,
and other substituents. The function of teichoic acids is unknown, but they may be necessary for
viability, since growth is slowed by daptomycin, which blocks teichoic acid formation.
Membrane-derived Oligosaccharides (6)

Approximately 1 5% of the dry weight of E. coli (and about 0.1% of gram-positive bacteria)
consists of small β-glucans present in the periplasmic space. Historically, the β-glucans were
discovered in studies of phospholipid turnover, which showed that the polar head groups of
phosphatidylglycerol and phosphatidylethanolamine were transferred to a group of low-
molecular-weight water-soluble oligosaccharides, which came to be known as the membrane-
derived oligosaccharides (MDO). Although the precise structure of MDO varies, it generally
consists of 6 12 glucose units, mostly in β1-2 linkages with β1-6 branches (Figure 21.5). In
addition, the oligosaccharides contain phosphoethanolamine, phosphoglycerol, and succinyl
groups, which confer a high net negative charge. E. coli, Pseudomonas, Rhizobia, and
Agrobacteria produce MDO, and in some cases, cyclic structures have been found. The
assembly of MDO requires UDP-Glc and undecaprenyl-PP-Glc as a primer and carrier for
transport across the inner membrane.
Figure 21.5. . Partial structure of MDO in E. coli. MDOs consists of small β-glucans and various
charged substituents thought to confer osmotic resistance.
Bacteria encounter extreme differences in osmolarity in the environment (like a bicycle tire, up
to 6 atmospheres of turgor pressure!), and therefore they have devised both physical and
chemical mechanisms to resist disruption. Peptidoglycan provides a structural barrier to osmotic
swelling. The high charge of MDO together with the charge layer from the murein
polysaccharide creates an osmotic buffer in the periplasm, which protects the inner membrane.
Interestingly, low osmotic conditions induce MDO biosynthesis.
Lipopolysaccharide and Endotoxin (7 13)
The outer membrane of gram-negative bacteria consists of a lipid bilayer, but unlike other cell
membranes composed of a bilayer of phospholipids, the outer leaflet of the outer membrane
contains mostly LPS. Each bacterial cell contains about 106 molecules of LPS (vs. 107
phospholipids). Its structure is stabilized by divalent cations; chelators that bind divalent cations
(e.g., EDTA) make the outer membrane permeable, even to large proteins such as lysozyme,
which can cleave the peptidoglycan.
LPS was first discovered more than 100 years ago as heat-stabile toxin associated with bacteria
(in contrast to heat-labile exotoxins discussed in Chapter 28). Its structure is complex, consisting
of three distinct regions (Figure 21.6), composed of both common and unusual sugars (Figure
21.7). The partial structure of the lipid A core was described in the 1950s but remained uncertain
until 1983. The complete synthesis of lipid A in 1985 confirmed its identity as the heat-stable
endotoxin associated with gram-negative sepsis. Lipid A also serves to anchor LPS in the outer
membrane and as the scaffold for assembly of the inner core region and the outer O-antigen
oligosaccharides.
Figure 21.6. Schematic structure of LPS in E. coli. LPS can be divided into three domains. The
lipid A domain is the same as endotoxin and contains two acylated GlcNAc-P residues (G). The
core domain consists of KDO (K), heptoses (H), and neutral sugars such as galactose. The outer
O-antigen consists of units of two to eight sugars repeated many times. (Open hexagons) An
unspecified monosaccharide. (Adapted, with permission, from [10] Holst et al. 1996 [© Elsevier
Science].)
Figure 21.7. Structure of heptose and KDO. LPS contains several unusual sugars not found in
vertebrates.
The basic structure of lipid A consists of two glucosamine residues in β1-6 linkage. The
reducing terminal sugar contains phosphate at C1 and two units of β-hydroxymyristic acid in
ester linkage at C3 and in amide linkage at C2 (Figure 21.8). The stereochemistry at these chiral
centers is identical to that found in the glycerol backbone of glycerophospholipids, and the acyl
amide is reminiscent of the one found in mammalian sphingolipids (see Chapter 9). The second
glucosamine residue also contains two β-hydroxymyristic acids with additional lauroyl groups
attached to the β-hydroxyls (which resemble waxes). All lipid A molecules contain from 1 to 4
units of KDO (Figure 21.7), which together with the phosphate groups at C1 and C4 on the core
disaccharide represent the binding sites for the divalent cations that stabilize the outer
membrane. The extent of acylation, phosphorylation, and the types of fatty acids vary
considerably in different gram-negative bacteria.
Figure 21.8. Structure of KDO2 lipid A in E. coli. (Reprinted, with permission, from [11] Raetz
1996.)
Lipid A biosynthesis initiates by acylation of UDP-GlcNAc at C3, followed by N-deacetylation,

N-acylation, and cleavage of the pyrophosphate linkage to form 2,3 diacylglucosamine-1-
phosphate. This can condense with another molecule of UDP-diacylglucosamine to form the
tetraacyl disaccharide core. A 4 kinase phosphorylates the nonreducing sugar and then KDO
transferases initiate the formation of the core region. Interestingly, the other two β-hydroxyl-
linked fatty acids are added only after the KDO units are added. The formation of KDO2-lipid A
is essential for survival of E. coli, but this might not be true for all gram-negative bacteria. Deep
rough mutants of E. coli and Salmonella lack the core and outer chains but contain a KDO-
bearing lipid A. The assembly process occurs in the inner membrane, but very little is known
about the translocation of lipid A to the outer leaflet of the outer membrane.
Lipid A has powerful biological effects in mammals, causing fever, septic shock, and a variety of
deleterious physiological effects. When released into the circulation, LPS binds to CD14 on
monocytes and macrophages, which triggers secretion of various cytokines. At low levels,
endotoxin serves as an adjuvant causing polyclonal B-cell expansion; however, at high levels, it
causes morbidity and mortality. Because of its significance, antagonists are being sought based
on lipid A analogs and inhibitors of LPS synthesis, by targeting the enzymes involved in its
formation.
After assembly of lipid A, the inner core regions of heptoses, hexoses, and phosphate groups are
added (Figure 21.7). A large number of genes encoding various transferases (clustered in the rfa
locus) participate in this process. The biosynthesis of the outer O-antigens occurs independently
of lipid A and the inner core and utilizes activated nucleotide sugars and undecaprenyl-
phosphate. Apparently, the chain grows from the reducing terminus while attached to the carrier.
When outer chain synthesis is completed, it is transferred en bloc to the core region of LPS. The
assembly process occurs by membrane-bound enzymes facing the cytosol, but the ligation to the
inner core is thought to occur in the periplasm.
The O-antigens consist of two to eight sugars, repeated 50 times. A broad range of sugars are
present, including free and amidated uronic acids, amino sugars, methylated and deoxygenated
derivatives, acetylated sugars, and others containing covalently bound amino acids and
phosphate. Three examples are provided in Table 21.1. The O-antigens, as their name implies,
define various serotypes distinguished by their reactivity with human antisera. More than 170
serotypes of E. coli are known. No strict correlation exists between serotypes and disease,
although some infections are more typical of certain serotypes. From the bacterium's point of
view, the O-antigen probably provides a hydrophilic barrier to hydrophobic antibiotics (natural
fungal and bacterial metabolites), bile acids (in enterobacteria), and complement.
Table 21.1. Examples of O-antigens
Capsular Polysaccharides (14 18)
Many bacteria in the wild produce polysaccharide capsules, which are called the K-antigens
(Kapsel antigen) to distinguish them from the O-antigens of LPS and the F-antigens of fimbrae
and flagella. Capsules represent the outermost layer and impart a mucoid appearance to colonies
grown on soft agar. More than 80 different capsule types are known in E. coli, and several are
shared by other bacteria (e.g., E. coli K1 and Meningitidis B both produce sialic-acid-containing
capsules). The capsular polysaccharides exhibit extraordinary diversity in structure, but the
presence of certain common sugars forms the basis for different groups (Table 21.2). Group Ia
capsules contain hexuronic acids and neutral sugars, group Ib capsules contain hexuronic acids
and N-acetylated hexosamine, and group II capsules contain hexuronic acids, KDO, or sialic
acids in combination with neutral or amino sugars.
Table 21.2. Examples of capsular polysaccharides
The assembly of capsules involves genes that are clustered in the chromosome in three
contiguous regions (I III). Region II genes, also known as the serotype region, encode enzymes
involved in nucleotide sugar formation and capsule-specific transferases. Group I capsule
polysaccharide assembly takes place by polymerization of the oligosaccharide repeat units linked
to undecaprenol pyrophosphate. Bacitracin, which blocks the recycling of undecaprenyl-P,
inhibits the formation of these types of capsules. However, other capsular polysaccharides do not
require undecaprenol and are resistant to the action of bacitracin. Some of these capsules contain
phosphatidic acid KDO conjugates or lipid A at the reducing termini of the oligosaccharides,
suggesting that different types of primers may exist. Well-studied examples include the assembly
of K1 capsules composed of α2-8-linked polysialic acid. Biosynthesis occurs from the reducing
end in most likely a processive mechanism. However, the mechanism of initiation is unclear. The
formation of hyaluronic acid capsules in Streptococcus A strains occurs in a similar way, but in
this case, the enzyme contains both GlcNAc and GlcA transferase activities (cf. Chapter 11). The
formation of other copolymeric capsules may involve similar dual functional enzymes (e.g., K5).
Additional work is needed to clarify the assembly process in vivo.
Regions I and III in the gene cluster contain transport activities required for movement of the
polysaccharides across the inner membrane and periplasm, but genes required for transport
across the outer membrane have not yet been identified. Transport occurs independently of
serotype. The arrangement of the loci in clusters allows a simple mechanism for changing
capsule types by merely swapping different serotype cassettes. In fact, the serotype locus can be
transferred by plasmids between compatible bacteria, resulting in switching of the capsule
composition.
Capsules have multiple functions. They can act as adhesion receptors during colonization of
tissues (see Chapter 28). Because they attract water, they act as a deterrent to desiccation. Their
varied structure may confer resistance to various phages and to complement (alternate pathway)
in the vertebrate host, which means that some capsules are virulence factors. Some capsules tend
not to be immunogenic due to the presence of structures identical to those found in the host. For
example, the hyaluronic acid capsules of Streptococcus A are identical to the hyaluronic acid
polymers generated by mammalian cells (see Chapter 11). Molecular mimicry like this also
occurs in bacteria with K1 capsules (polysialic acid is found in the brain) or K5 capsules (N-
acetylheparosan is the backbone of heparan sulfate), and group IIIB Streptococcus
(Neu5Acα3Galβ4Glc, which resembles ganglioside GM3).
Bacterial Glycoproteins (19 22)
Recently, certain species of Archaea and Bacteria were shown to glycosylate proteins. These
bacterial glycoproteins include surface layer (S layer) proteins, flagellar proteins, and
polysaccharide-degrading enzymes secreted from cells. Many different oligosaccharides have
been described so far, which vary in core structure to a much greater extent than seen in
eukaryotes. For example, a novel type of glycoconjugate was observed in the cell envelope of the
extremely halophilic archaeon, Natronococcus occultus. The conjugate consists of a glycosylated
polyglutamyl polymer.
Bacteria also appear to be able to make glycoconjugates closely related to those found in
mammals. For example, Chlamydia trachomatis makes a high-mannose-type N-glycan on
glycoprotein, similar to the type assembled on eukaryotic glycoproteins. They also produce a
heparin-like molecule important in infection of the eukaryotic host. Neisseria meningitidis
produces adhesive fimbriae that contain an O-glycan in the pilin structural subunit (see Chapter
28). The assembly of eukaryotic-like glycans by bacteria, especially pathogens, may be a form of
molecular mimicry for bacteria, presumably to avoid or compromise the immune system of the
host. This new area of research may reflect horizontal gene transfer in which the bacteria has
acquired genes of eukaryotic origin. Almost certainly, it will provide novel insights into the
evolution of glycosylation and new targets for therapeutic intervention.
Future Directions
As should be evident from the preceding discussion, bacteria have evolved a variety of
glycoconjugates that serve both structural and functional roles in cell growth and division.
Recently, the entire genomes of several bacteria have become available, and a survey of open
reading frames indicates that proteins involved in carbohydrate metabolism, carbohydrate-
binding proteins, and cell wall assembly constitute major families of expressed genes. These
genes are good candidates for the development of new antimicrobial agents. Especially appealing
is the idea of targeting the formation of essential intermediates, such as lipid A or the unusual
sugars unique to bacteria (e.g., KDO and heptoses). This is a problem of paramount importance,
since clinical isolates of bacteria resistant to all known antibiotics have now appeared.
Although much is known about the structure of bacterial glycans and the pathways for their
assembly, very little information is available about the topology of assembly. All of the
precursors appear to be made in the cytoplasm from water-soluble nucleotides and various
sugars. But the assembly process for membrane glycans takes place in the hydrophobic milieu of
the inner and outer membranes, and peptidoglycan biogenesis takes place in the periplasmic
space. How the precursors access these different compartments remains for the most part a
mysterious and fertile area for future work.
Acknowledgment
Many thanks to Christian R.H. Raetz for helpful comments on this chapter.
References
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F. Beckmann, U. Seydel, K. Brandenburg, A.J. Ulmer, T. Mattern, H. Heine, J. Schletter, H.
Loppnow, U. Schonbeck, H.D. Flad, S. Hauschildt, U.F. Schade, F. Di Padova, S. Kusumoto,
and R.R. Schumann. 1996. Bacterial endotoxin: Chemical constitution, biological recognition,
host response, and immunological detoxification Curr. Top. Microbiol. Immunol. 216: 39-81.
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Proteins That Recognize
Glycans
22. Discovery and Classification of Animal Lectins
Diego).
THIS CHAPTER PRESENTS SOME HISTORICAL BACKGROUND regarding the discovery

of animal lectins and a general introduction to the topic and then discusses the current
classification of these molecules, based on sequence homologies and probable evolutionary
relatedness. Some general principles regarding the structure and function of animal lectins are
also considered, as well as some aspects of their carbohydrate-binding properties. Further
information regarding each of the major classes of animal lectins is covered in Chapters 23 27.
For details regarding the analysis of carbohydrate-protein interactions and the physical principles
involved, see Chapter 4.
Historical Background of the Discovery of Animal Lectins (1 31)
Lectins are carbohydrate-binding proteins. Although they were first discovered more than 100
years ago in plants (see Chapter 30), they are now known to be present throughout nature
(including the microbial world, wherein they tend to be called by other names, such as
hemagglutinins, adhesins, and toxins; see Chapter 28). Interactions between animal cells
involving carbohydrates were first discovered by observing the phenomenon of reaggregation of
dissociated marine sponge cells, a form of species-specific recognition. There was also evidence
for the presence of hemagglutinins (red cell agglutinating activity) in the body fluids of various
crustaceans and arachnids. However, until the 1960s, such interactions had not been discovered
in complex multicellular animals such as vertebrates. The first inkling that there might be lectins
endogenous to a vertebrate system came from the studies of Ginsburg and colleagues in which
blood leukocytes from the rat were treated with bacterial glycosidases and then injected back
into the circulation. The treatments resulted in changes in the homing of cells to different internal
organs. Given the nature of the tools available at that time, it was difficult to ascertain whether
these phenomena were mediated by an endogenous lectin. In retrospect, these studies were
probably demonstrating the generation of ligands for a hepatic galactose receptor (due to
sialidase action) and/or possibly the selectins (due to fucosidase action). The first direct evidence
for a mammalian lectin arose serendipitously during work by Ashwell and colleagues, who were
studying the mechanisms that controlled the turnover of glycoproteins in the blood circulation.
Looking for an improved way to introduce a radioactive tracer into purified glycoproteins before
reinjection, they attempted to transfer the tritium label from tritiated borohydride into the sugar
chains of these proteins, which contain the terminal sequence Sia-Gal-GlcNAc. This was
successfully done either following mild periodate oxidation of sialic acid side chains (which left
the rest of the sialic acid molecule intact) or following oxidation of the 6-position of galactose
residues (which required removing the outer sialic acid residues). To their surprise, there was a
dramatic difference between the circulating half-lives of these two preparations, which
terminated either in sialic acid or galactose residues. The molecules that retained labeled sialic
acids remained in the circulation for many days, whereas those that had lost them (and now
terminated in galactose residues) disappeared within minutes. The importance of these terminal
β-linked galactose residues was confirmed by in vitro resialylation or by β-galactosidase
treatment, both of which partially restored stability in circulation. The site of accumulation of the
desialylated glycoproteins was found to be primarily the liver. This led to the discovery of the
"asialoglycoprotein receptor," a hepatocyte membrane protein complex that specifically
recognizes terminal β-linked galactose or GalNAc residues on circulating glycoproteins or cells.
It was subsequently found that this lectin activity was markedly inhibited by plasma from birds
and reptiles. This, in turn, led to the discovery of a chicken hepatic lectin, which preferentially
recognizes terminal GlcNAc residues. Thus, chickens have a GlcNAc-specific hepatic receptor
instead of a galactose-specific one, permitting the circulation of asialoglycoproteins, but not
glycoproteins that also have their galactose residues removed.
Affinity columns of immobilized asialoglycoproteins were then used to purify the hepatic
asialoglycoprotein receptor. The same approach was applied by investigators working in other
systems, uncovering a variety of galactose-binding lectins in cell types, ranging from the slime
mold Dictyostelium discoideum to mammalian tissues such as the heart. Most of these lectins
eventually proved to be quite different from the original asialoglycoprotein receptor, being water
soluble and of relatively low molecular weight (now known as the galectins, Chapter 27).
Meanwhile, Hill and colleagues reported yet another type of hepatic uptake system that seemed
to involve recognition of fucose moieties.
In the early 1970s, Neufeld and colleagues reported a carbohydrate-dependent system mediating
the uptake of lysosomal enzymes by fibroblasts. In 1977, Sly and colleagues demonstrated
specific blockade of this uptake by the monosaccharide Man-6-P. Further work by the groups of
Kornfeld, Jourdian, Von Figura, and others led to the discovery of Man-6-P receptors, which
recognize phosphorylated high-mannose-type oligosaccharides that are selectively expressed on
lysosomal enzymes. Encouraged by the prospect of using this uptake system to correct lysosomal
enzyme deficiency diseases in humans, other investigators infused intact labeled lysosomal
enzymes into intact animals and followed their fate. As it turned out, mature lysosomal enzymes
were not actually rich in Man-6-P (the phosphate esters are mostly removed after initial targeting
to lysosomes; see Chapter 23); instead, the clearance predominantly involved recognition of
terminal mannose and GlcNAc residues. This led to the discovery of the macrophage mannose
receptor. Thus, by the beginning of the 1980s, the concept of vertebrate lectins that could
recognize specific endogenous ligands had become firmly established.
Meanwhile, several circulating soluble lectins were discovered in the blood plasma of various
species, with varied carbohydrate-binding specificities. Initially, it had been thought that sialic
acids, while serving as ligands for exogenous microbial pathogens, generally acted as "masks"
within vertebrates, preventing binding by endogenous lectins that recognized underlying
saccharides. The discovery of some arachnid and crustacean lectins that could recognize sialic
acids in vitro did not change this impression, since these organisms did not themselves express
endogenous sialic acids. The first indication that sialic acids might serve as endogenous ligands
within vertebrates came from Fearon and Austen, who showed that the binding of the
complement regulatory H protein to "self" cell surfaces was dependent on sialic acid residues.
Rosen and colleagues then showed that sialidase treatment of the rat lymph node sections
abolished binding of lymphocytes to high endothelial venules; this was the first demonstration of
the involvement of carbohydrates in recognition by what turned out to be the selectin family of
vascular receptors (see Chapter 26). Meanwhile, the discoveries that the anticoagulant effects of
heparin chains were mediated by a structural sequence specific for antithrombin III and that the
cartilage link protein could bind to hyaluronan established the concept that protein interactions
with polyanionic glycosaminoglycan chains could also be based on highly specific recognition
(see Chapter 29). In the late 1980s, it became evident that the primary amino acid sequence of a
protein could be used to predict carbohydrate recognition properties of proteins (see Current
Classification of Animal Lectins below). This led to the recognition of the hyaluronan-binding
properties of CD44 and the correct prediction that selectins would recognize carbohydrates. In
the current decade, the discovery of sialic-acid-dependent binding by the B-cell molecule CD22
and the cloning of the macrophage receptor sialoadhesin led to the definition of another new
family of lectins (the Siglecs) belonging to the immunoglobulin superfamily (see Chapter 24). A
specific clearance system was also discovered that recognized the sulfated GalNAc residues on
pituitary glycoprotein hormones. Most recently, several lectins have been discovered within the
ER-Golgi pathway itself, where sugar chain biosynthesis occurs.
Current Classification of Animal Lectins (7, 15, 19, 23, 26,31 40)
For a while after their discovery, animal lectins were classified according to the carbohydrate
sequences to which they bound best (e.g., β-galactoside-binding lectins). Only with the advent of
molecular cloning did a more consistent classification emerge, based on amino acid sequence
homology and evolutionary relatedness of these lectins (for classification, see Table 22.1, and for
schematic examples, see Figure 22.1). The first such classification was proposed by Drickamer,
based on some highly conserved amino acid sequence motifs in the carbohydrate recognition
domains of two groups of lectins: The first group required calcium for recognition and was
therefore called C-type lectins, and the other group required "free" thiols for stability and was
termed S-type lectins. Meanwhile, the two lectins that recognized Man-6-P were sequenced and
found to be homologous, but distinct from all the others, justifying their recognition as P-type
lectins. Although some classes of lectins such as the P-type and S-type seemed to recognize a
single class of sugars (Man-6-P and β-galactosides, respectively), others like the C-type
encompassed a variety of molecules that shared in common only a lectin protein module. A
major breakthrough occurred when the independent cloning of three homologous vascular
adhesin receptors revealed a common amino-terminal C-type lectin motif; these three molecules
eventually turned out to be the selectins. This was the first time that carbohydrate recognition
had been predicted on the basis of the primary amino acid sequence of a cloned protein,
validating the concept of classification based on sequence homology. The cloning of a variety of
circulating soluble lectins also led to the recognition of a subset of C-type lectins designated as
the "collectin family." In addition, two calcium-binding lectins (calnexin and calcireticulin) are
unrelated to the C-type lectins (not all calcium-requiring lectins are C-type lectins) and
specifically recognize glucose residues on newly synthesized glycoproteins. Studies in the 1990s
have also revealed that immunoglobulin superfamily members can recognize carbohydrates,
leading to a new group of I-type lectins (see Chapter 24). A subgroup of these molecules that
specifically recognize sialic acids have been recently designated the "Siglecs" (for sialic
acid/immunoglobulin superfamily/lectins). Another class of evolutionarily very ancient
circulating soluble lectins called the pentraxins is recognized not so much by primary sequence
homologies, but by a consistent pentameric structural organization and a probable role in the
primary host immune response.
Table 22.1. Animal lectin classes and families
Lectin class Number Defining features in Calcium Known carbohydrate

of protein sequence dependence recognition
members
Sequence homologies
known
C-type (includes <20 C-type lectin sequence yes (most) highly variable
selectin, collectins, motif
etc.)
S-type (galectins) >8 S-type lectin no β-galactosides and
sequence motif poly-
N-acetyl-lactosamines
P-type (M6PRs) 2 unique repeating motif variable Man-6-P on high-
mannose-type-N-
glycans
I-type (includes >5 immunoglobulin-like no sialic acids (Siglecs),
Siglec family) domains others
calnexin, 2 homology with each yes glucosylated high-
calcireticulin, calmegin mannose-type N-
glycans in the ER
hyaluronan-binding >5 sequence homology in no hyaluronan chains
proteins (CD44, link binding region
protein, verscan,
aggrecan)
frog egg lectins unknown sequence homology yes galactose, sialic acids,
heparins
Sequence homologies
not known
Pentraxins >5 pentameric subunit yes (most) variable
arrangement
glycosaminoglycan- >20 basic amino acid no glycosaminoglycans
binding proteins clusters (variable)
ERGIC-53 and VIP- unknown similarity to plant lectin glycans in the ER-
36 sequences Golgi pathway?
S4GGnM receptor unknown cysteine-rich domain in no sulfated GaINAc
mannose-binding in residues on pituitary
mannose-binding glycoprotein hormones
protein
ganglioside-binding unknown no sequence no sialylated glycolipids
proteins information known
sulfoglucoronosyl unknown no sequenceinformation yes sulfoglucoronosyl
lipid-binding protein known glycolipids
insect hemolyph unknown no sequence sialic acids
lectins information known
interleukins I and II unknown no high-mannose
oligosaccharides
Figure 22.1. Schematic examples of major groupings of animal lectins, based on protein
structure. Examples of some of the major families are shown. The emphasis is on the
extracellular domain structure and topology. The following are the defined carbohydrate-binding
domains: (CL) C-type lectin CRD; (SL) S-type lectin CRD; (MP) P-type lectin CRD; (IL) I-type
lectin CRD. Other domains follow the recommendations of the International Workshop on
Sequence, Structure, Function, and Evolution of Extracellular Protein Modules: (EG), EGF-like;
(IG [I2]) Immunoglobulin C-set; (TM) transmembrane region; (C3) complement regulatory
repeat.
These general groupings are based primarily on sequence homologies and probable evolutionary
relatedness and include the majority of known animal lectins. However, many others do not
show any obvious sequence homologies or evolutionary relationships (Table 22.1). Another
large group that defies easy classification based on sequence data or general structure are the
proteins that bind glycosaminoglycans such as heparin (GAG-binding proteins; see Chapter 29).
Unlike other animal lectins that tend to recognize specific terminal aspects of sugar chains by
fitting them into shallow but relatively well-defined binding pockets, GAG-protein interactions
seem to involve surface clusters of positively charged amino acids that line up against internal
regions of the anionic GAG chains. Thus, despite the fact that the GAG structural motifs
recognized can be quite specific, and many involve the general motifs XBBXBX, XBBBXXBX,
or TXXBXXTBXXXTBB (where B is a basic residue and X is a hydropathic residue), most
GAG-binding proteins do not seem to be evolutionarily related to each other. Partly for these
reasons, the term "lectin" is not even commonly applied to GAG-binding proteins.
For further details regarding many of these different classes of animal lectins, see Chapters 23
27, and for details regarding the principles of carbohydrate:protein interactions, see Chapter 4.
The rest of this chapter provides an overview of general principles regarding the biosynthesis,
physical structure, binding properties, and regulation of animal lectins.
Biosynthesis, Trafficking, and Regulation of Animal Lectins (12 13,

15, 21, 23,25 26, 30, 34,37, 41)
Some lectin genes are expressed constitutively, whereas others are induced by gene activation
under specific biological circumstances. All membrane-bound and many soluble lectins are
synthesized on ER-bound ribosomes and delivered to their eventual destinations via the ER-
Golgi pathway. Thus, the lectins themselves are often glycoproteins. However, a significant
subset of soluble lectins (galectins, heparin-binding growth factors, and some cytokines) are
synthesized on free ribosomes and delivered directly to the exterior of the cell by an as yet
poorly understood mechanism involving extrusion through the plasma membrane. This makes
some teleological sense, since several of these lectins can recognize biosynthetic intermediates
that occur in the Golgi-ER pathway (e.g., galactosides and high-mannose oligosaccharides). By
circumventing the conventional pathway of secretion, these molecules can avoid unwanted
premature interactions with potential ligands that are synthesized within the same cell. In
addition, some of these lectins (such as the galectins) are sensitive to the redox state of the
environment and can remain active only in the reducing environment of the cytosol. Upon
entering the oxidizing environment of the extracellular space, they must therefore immediately
bind to ligands or become progressively inactivated. Another form of regulation occurs when the
lectin binds to cognate sugar chains present on the same molecule or the same cell surface and
hence becomes functionally inactive (e.g., the Siglecs, where sialic-acid-bearing ligands from the
same cell surface must be removed before the lectin can be active). Some membrane-bound
lectins are internalized upon binding to ligands, with delivery to internal acidic compartments
(endosomes). There the cargo is released, and some of the receptors can recycle back to their
original location.
Soluble and Membrane-bound Forms of Animal Lectins (42 43)
From a functional point of view, it is worth considering these carbohydrate-binding proteins in

two physical classes: soluble and membrane-bound. Cell-membrane-bound lectins are more
likely to be involved in endocytosis or cell adhesion and to stay confined to the cell type of their
original synthesis. On the other hand, soluble lectins are capable of diffusing locally in tissues
and/or entering the blood circulation. Although useful in functional terms, this type of physical
classification is confounded by two issues: First, lectins that start out life as membrane-bound
proteins can be proteolytically shed into the extracellular fluid, and second, soluble multivalent
lectins can become attached to cell surfaces via their carbohydrate-binding sites. Figure 22.2
indicates some examples of these relationships and the nature of the potential interactions with
natural ligands, which, in turn, can also be soluble or membrane-bound.
Figure 22.2. Possible mechanisms of regulation of an animal lectin by cognate ligands. Potential
ligands can be on the cell surface and/or on soluble glycoproteins (including sugar chains
attached to the lectin itself). As discussed in the text, the "lectin-binding structure" or "ligand"
depicted in this cartoon can be a very complex motif that includes more than one
monosaccharide and/or additional components. Direct cell-cell interactions could occur among
lectin-positive cells (A), or between lectin-positive cells and other cell types bearing cognate
ligands (B). Soluble glycoprotein ligands could interact directly with lectin-positive cells (C),
bridge between two such cells (D), or inhibit cell-cell interactions involving the lectin (E).
Expression of ligands on lectin-positive cells could inactivate the lectin function by either inter-
or intramolecular interactions (F).
Nature of Lectin-Ligand Interactions (42 43)
Several crystal structures of animal lectins with their cognate ligands have been elucidated,
allowing an understanding of these interactions at the level of atomic resolution. These can be
divided into two general groups: those involving GAG chains (mostly mediated by ordered
arrays of surface charge contacts; Chapter 29) and those involving N- and O-glycans. The
principles that have emerged about the latter are as follows: First, the binding sites are of
relatively low affinity and are found in shallow indentations on the surface of the proteins.
Second, selectivity is mostly achieved via a combination of hydrogen bonds (involving the
hydroxyl groups of the sugars) and by van der Waals' packing of the hydrophobic face of
monosaccharide rings against aromatic amino acid side chains. Third, further selectivity can be
achieved by additional contacts between the saccharide and the protein, sometimes involving
bridging water molecules or divalent cations. Finally, the actual region of contact between the
saccharide and the polypeptide typically involves only one to three monosaccharide residues. As
a consequence of all of the above, these lectin-binding sites tend to be of relatively low affinity,
but of high specificity. The ability of such low-affinity sites to mediate biologically relevant
interactions in the intact system thus appears to require multivalency.
Animal Lectins Are Generally Multivalent (14, 21, 26, 31, 34,44 46)
Until the 1990s, all of the animal lectins discovered were found to be naturally multivalent,
either because of their defined multisubunit structure or by virtue of having multiple
carbohydrate-binding sites within a single polypeptide. Indeed, high avidity generated by
multivalent binding of low-affinity single sites appears to be a common mechanism for
optimizing lectin function in nature, and a traditional definition for a lectin was "a multivalent
carbohydrate-binding protein that is not an antibody." The first exception to this general rule
appeared to be the selectins, which have only a single CRD site within their extracellular
polypeptide domains (see Chapter 26). The same situation applies to the Siglecs (see Chapter
24). However, in both these instances, evidence is appearing that the molecules become
functionally multimeric either by noncovalent association or by clustering on cell surfaces. It
remains to be seen whether biologically significant binding by any animal lectin can arise from a
strictly monovalent interaction. It is also of note that a single lectin can carry multiple binding
sites for multiple ligands, e.g., the macrophage mannose receptor is now known to bind not only
to mannans, but also via a distinct CRD to the 4-O-sulfated GalNAc residues of pituitary
glycoprotein hormones.
Nature of the Ligands for Animal Lectins (6, 12,14 15,17 18, 21, 23, 27, 29, 31, 34,
41,43 45,47 48)
Details about the natural ligands for animal lectins can be found in other chapters in this volume.
In the few instances where crystal structures of animal lectins with their cognate ligands are
available, it is evident that the contact regions between the two usually occupy only one to three
monosaccharide units (see Chapter 4). On the other hand, despite their stereospecificity,
monosaccharides or small oligosaccharide units tend to be weak inhibitors of lectin interactions.
The natural ligands for most lectins are typically complex glycoconjugates that carry clustered
arrays of the cognate carbohydrate, thus cooperating with clustered lectin-binding sites to
generate high-avidity binding, which is further enhanced by mass transport effects (high local
concentrations of ligands). In some instances (e.g., the selectins), the nature of this clustering is
not easily defined, and cooperation with other aspects of the underlying polypeptide may be
necessary to generate optimal binding. Partly for this reason, it is common to see the names of
the underlying polypeptide backbone used to define the nature of a ligand, e.g., PSGL-1 is the
ligand for P-selectin. However, it should be recognized that unless it is correctly glycosylated
and/or otherwise modified (e.g., sulfated), the polypeptide is not itself the ligand. Typically,
these polypeptides are simply carriers of the true ligands for lectins, which are made up of
combinations of glycan units. In addition, recombinant lectins that are often used to identify
potential biological ligands are usually multimeric in structure and/or are presented in
multivalent clustered arrays in soluble complexes or on solid supports. Thus, although a variety
of molecules may be found to bind to a given recombinant lectin in a glycosylation-dependent
manner, only a few of these "ligands" may be actually involved in mediating biologically
significant interactions. The challenge then is to tell the difference between what can bind to a
recombinant lectin in an in vitro experiment, and what actually does bind in vivo to the native
lectin in a biologically relevant manner. Indeed, the term ligand should probably be reserved for
the latter type of biologically relevant structures. It should also be kept in mind that the natural
ligands of some animal lectins may be present primarily on foreign invaders (e.g., the circulating
soluble mannan-binding protein may serve to bind and opsonize microorganisms bearing high
densities of mannose, such as yeasts and other fungi).
Types of Functions Mediated by Animal Lectins (6,11 13, 15,20 21,23 25,28
29, 31,37,49 51)
Animal lectins provide one functional explanation for the enormous diversity of glycan
structures found on animal cells (i.e., recognition of endogenous ligands). Details about the
function of various animal lectins can be found in later chapters. Table 22.2 lists some examples
of specific known or putative functions. It can be seen that nature has capitalized on the
versatility and diversity of carbohydrate binding to generate a wide variety of functional
outcomes. In some of these instances, the biological significance of the interactions is evident
from the consequences of natural or experimental genetic mutations in the expression of the
lectin or its ligand. Of course, there are still several other instances wherein the lectin and its
specificities are well defined, but the biological functions of the interaction remain elusive. In
these cases (e.g., the Siglecs), the highly restricted cell-type-specific expression of the lectins
makes it reasonable to predict that they do serve highly specific biological functions.
Table 22.2. Some examples of biological functions of animal lectins
Lectin name Class Expression Natural Proposed Functions

(family) location ligand(s) biological confirmed by
functions genetic mutations
in intact animals?
Cation- P-type most cell phosphorylated trafficking of ligands (I-disease)

high-
types mannose-type enzymes to and receptors
independent
Man-6-P glycans of prelysosomal (mouse mutants)
receptor lysosomal compartment
enzymes
P-selectin C-type platelets and PSGL-1 tissue homing ligands (human
carrying
sialyl Lewis of leukocytes; LAD-type II
endothelial X
cells and tyrosine interactions of and FucT-VII
sulfate platelets with mouse mutant)
residues
monocytes and receptor
(mouse mutant)
CD22 I-type mature B α2-6-linked interactions of ligands and
receptors
(Siglec-2) cells sialic acid CD22 with (CD22 and
ST6Gal I
residues on B-cell and/or mouse mutants)
unknown T-cell
carriers
glycoproteins
Antithrombin heparin- plasma heparin-like inhibits receptor (human
binding protein sequences coagulation AT deficiency)
proteins (on by enhancing
endothelial
cells?) antithrombin
inactivation of
IIa and Xa
Calnexin grouped ER newly sensor for no
synthesized
with glycoproteins incomplete
with α- folding of
calcireticulin glucosylated
N-glycans glycoproteins;
facilitates
chaperone
functions?
Galectin-I S-type many cell poly-N-acetyl- induction of no
(galectins) types lactosamine apoptotic
units
on developing responses
thymocytes
and on
migrating
leukocytes
Future Directions
The discovery of new animal lectins will likely continue as it has in the past, i.e., arising from
serendipitous observations, unexpected sequence homologies of newly cloned molecules to
known lectins, or directed discovery attempts using defined carbohydrate probes as affinity
ligands. The ultimate goal must be not only the identification and structural characterization of
all of their natural ligands, and the elucidation of the nature of the relevant binding interactions at
atomic resolution, but also a full understanding of the biological roles of these molecules. The
latter must eventually be confirmed by natural or genetically manipulated mutations in the
expression of the lectins and/or their ligands in intact animals.
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23. P-type Lectins
Diego).
LYSOSOMES ARE INTRACELLULAR ORGANELLES that carry out the final degradation of
most cellular macromolecules. This is achieved primarily by the action of a number of lysosomal
enzymes (often called acid hydrolases because of the low internal pH characteristic of
lysosomes). These enzymes are synthesized on membrane-bound ribosomes in the ER, and
traverse the ER-Golgi pathway along with other newly synthesized proteins. Eventually, they are
segregrated away from all other glycoproteins and selectively delivered to the lysosomes. In
most higher animal cells, this specialized trafficking is achieved primarily by a selective
carbohydrate marker that is recognized by specific receptors. This chapter describes the
discovery and characterization of this Man-6-P pathway for the trafficking of lysosomal
enzymes, the biosynthetic steps involved in the generation of the marker, and the structure and
function of the Man-6-P receptors ("P-type" lectins) and discusses the known human and murine
genetic defects in this pathway. It is noteworthy that the discovery and elucidation of this
pathway were the first demonstrated link between glycoprotein biosynthesis and human disease.
I-Cell Disease and the "Common Recognition Marker" of

Lysosomal Enzymes (1 4)
During the 1960s, studies of human genetic "storage disorders" indicated a failure of intracellular
degradation of cellular components, which therefore accumulated in the lysosomes (Chapter 18).
Neufeld and coworkers demonstrated that cultured fibroblasts from some of these patients
accumulated "mucopolysaccharides" (now called glycosaminoglycans), which could be
metabolically labeled by [35S]sulfate. This [35S]sulfate accumulation was corrected by
cocultivating the cells with normal fibroblasts (or with cells from patients with a different
clinical phenotype). Soluble "corrective factors" were found to be responsible for this result.
Upon purification, these factors turned out to be different lysosomal enzymes that were deficient
in the patients with different diseases and were being secreted in small quantities by the normal
cells (or by cells from patients with a different defect) (Figure 23.1). These enzymes were found
to exist in two forms: a high-uptake form that could correct deficient cells and a low-uptake form
that was inactive. Direct binding studies showed saturable, high-affinity receptors for the high-
uptake molecules. Meanwhile, fibroblasts from an unusual human genetic disease with very
prominent inclusion bodies in cultured cells (therefore termed I-cell disease) were found to lack
not one, but almost all lysosomal enzymes. In I-cells, all of the enzymes were actually being
made, but they were almost completely secreted into the medium. Hickman and Neufeld made
the seminal observation that although I-cells could take up the high-uptake enzymes secreted by
normal cells, the enzymes secreted from I-cells were not taken up by other cells. They proposed
that I-cell disease resulted from a failure to add a common recognition marker present on all
lysosomal enzymes, which they assumed was responsible for retention in the cell and normal
trafficking to the lysosomes. Since the high-uptake property could be destroyed by periodate
treatment, they predicted that this marker contained carbohydrate.
Figure 23.1. "Cross-correction" of lysosomal enzyme deficiencies in cultured cells. Low levels
of high-uptake lysosomal enzymes secreted by fibroblasts can be taken up by other cells and can
correct an intrinsic genetic lack of a single enzyme. In contrast, I-cell disease fibroblasts secrete
large amounts of all lysosomal enzymes of the low-uptake variety; i.e., they cannot correct
deficiencies in other cells. I-cells retain the capability to accept high-uptake enzymes secreted by
other cells.
Discovery of the Phosphomannosyl Recognition Marker (5 9)
The next major breakthrough occurred when Kaplan et al. found that the uptake of high-uptake
lysosomal enzymes was specifically blocked by the sugar Man-6-P and its stereoisomer Fru-1-P.
Although millimolar concentrations were required, similar concentrations of other sugars and
sugar phosphates had no comparable effect. By this time, the general pathway for processing of
N-glycans had been worked out (see Chapter 7). Since mannose residues were known to occur
on high-mannose-type N-glycans, it was predicted that these might be phosphorylated
specifically on lysosomal enzymes. This was confirmed by alkaline phosphatase treatment,
which abolished high-uptake activity, and by tunicamycin treatment, which blocked N-
glycosylation and caused secretion of lysosomal enzymes from cells. Soon thereafter, Man-6-P
was shown to be present in high-uptake forms of lysosomal enzymes and on endo-β-N-
acetylglucosaminidase H (Endo H)-sensitive oligosaccharides from these enzymes. A surprising
result occurred when the groups of Kornfeld and Von Figura isolated the Endo-H-sensitive N-
glycans from newly synthesized lysosomal enzymes. Although the molecules contained the
predicted phosphate esters on mannose residues, most of these were found to be "blocked" by
outer α-linked GlcNAc residues, which could be removed by cleaving the phosphodiesters with
mild acid, generating the phosphomonoesters of Man-6-P.
Enzymatic Mechanism for Generation of the Recognition

Marker (7,10 12)
Further studies of the N-glycans showed that each molecule carried one or two phosphate
residues on various different mannose residues. Comparison of glycans with phosphodiesters and
phosphomonoesters predicted that the former must be the metabolic precursors. This led to the
prediction that phosphorylation was mediated not by an ATP-dependent kinase, but by a UDP-
GlcNAc-dependent GlcNAc-1-phosphotransferase. This was proven using a double-labeled
substrate:
The next step was the discovery of a Golgi enzyme that could remove the outer GlcNAc residues
and "uncover" the phosphomonoesters. Pulse-chase studies confirmed the order of events,
indicating that more than one oligosaccharide on a given enzyme could be phosphorylated and
that removal of outer mannose residues by the processing Golgi mannosidases was also required
(Figure 23.2). In most cell types, the phosphomonoesters are eventually lost, presumably upon
exposure to an acid phosphatase in the lysosomes. However, during passage through the Golgi,
the phosphate residues block the complete action of the processing mannosidases, maintaining
the glycans in a high-mannose form (Figure 23.2). The lack of phosphorylation in I-cell disease
(see below) likely explains why the secreted lysosomal enzymes in these patients carry more
sialylated complex-type N-linked oligosaccharides.
Figure 23.2. Pathways for biosynthesis of N-glycans bearing the phosphomannosyl recognition
marker. Following early processing (see Chapter 7), a single GlcNAc phosphodiester is added to
the N-glycans of lysosomal enzymes, on one of three mannose residues on the side with the α1
6-linked mannose to the β-linked mannose (structure A). A second phosphodiester can then be
added to the other side of the glycan (structure B). Removal of the outer GlcNAc residues and
further processing of mannose residues gives structures C and D. Further mannose removal is
restricted by the phosphate esters. The asterisks indicate alternate locations of these esters. Thus,
C and D represent only two of several possible structures bearing one or two
phosphomonoesters. Glycans that are not phosphorylated become typical complex-type or
hybrid-type oligosaccharides. Some hybrid phosphorylated molecules are also found (structure
E). Binding studies of these glycans with purified MPRs have shown the relative affinities
indicated in the figure.
After many years of unsuccessful effort by many laboratories, purification and cloning of the two
primary enzymes involved in the pathway have recently occurred. The UDP-N-
acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-P-T)
was partially purified and then used to generate a specific monoclonal antibody capable of
immunoprecipitating the activity. This antibody was then used to immunopurify the enzyme
about 480,000-fold to apparent homogeneity. The purified enzyme is a 540-kD complex
composed of disulfide-linked homodimers of 166-kD and 51-kD subunits and two identical,
noncovalently associated 56-kD subunits. The 166-kD subunit was identified as the catalytic
subunit by photoaffinity labeling with azido- [β-32P]UDPGlc. A similar monoclonal-antibody-
based approach has recently yielded the purification of the second enzyme, α-N-
acetylglucosaminyl-l-phosphodiester glycosidase, which is a 272-kD complex of four identical
68-kD subunits arranged as two disulfide-linked homodimers. It appears that unlike other Golgi
enzymes, this is a type I membrane-spanning glycoprotein with its amino terminus in the lumen
of the Golgi. Cloning of genes encoding both enzymes is nearly complete (W. Canfield, pers.
comm.).
Enzymatic Basis for I-Cell Disease and Pseudo-Hurler

Polydystrophy (13 15)
Soon after the discovery of the GlcNAc-P-T enzyme, analysis of fibroblasts from patients with I-
cell disease (also called mucolipidosis-II, ML-II) revealed defects in this activity. A milder
variant called pseudo-Hurler polydystrophy (mucolipidosis-III, ML-III)showed a less severe
enzyme deficiency. Metabolic labeling of fibroblasts corroborated the failure of mannose
phosphorylation of lysosomal hydrolases in these diseases, and obligate heterozygotes showed a
partial deficiency in this phosphotransferase, with slightly elevated levels of serum lysosomal
enzymes, but no phenotype. With the cloning of the gene for the GlcNAc-P-T enzyme, final
proof is expected soon that GlcNAc-P-T deficiency is the primary genetic disorder in these
diseases. Regarding the phosphodiester glycosidase, only one family with a partial deficiency
has been reported, associated with an elevated level of lysosomal enzymes. This presumably
represents a heterozygous state for enzyme deficiency.
Variants of I-Cell Disease and Pseudo-Hurler Polydystrophy

(14,16 17)
The simplest in vitro substrate for the GlcNAc-P-T is α-methylmannoside. However, it is a

poorer substrate than a high-mannose-type N-glycan, which in turn is much poorer than a native
lysosomal enzyme. Other glycoproteins bearing high-mannose-type N-glycans are also poor
substrates. These data indicated that the GlcNAc-P-T must specifically recognize lysosomal
enzymes in preference over other N-glycan-bearing glycoproteins, via a second recognition site
(Figure 23.3). In most cases of ML-II and ML-III assays with the monosaccharide and lysosomal
acceptors gave congruent reductions in activity. However, one pair of siblings with ML-III
showed normal activity with the α-methylmannoside acceptor, but markedly decreased activity
with lysosomal enzyme acceptors. The GlcNAc-P-T in these patients is presumed to be present
in normal catalytic amounts, but it fails to recognize lysosomal enzymes as special acceptors for
phosphorylation. This provided genetic evidence for specific recognition of lysosomal enzymes
by the GlcNAc-P-T. The molecular basis of this variant enzyme has very recently been
determined (W. Canfield, pers. comm.).
Figure 23.3. Selective recognition of lysosomal enzymes by the GlcNAc-P-T. The normal
GlcNAc-P-T has three independent binding sites, one for the oligosaccharide substrate, one for
the UDP-GlcNAc donor, and one for the selective recognition of lysosomal enzymes. The last
site is a critical determinant of the selective phosphorylation of lysosomal enzymes; this site is
presumed to be mutated in some variant cases of pseudo-Hurler polydystrophy.
As for most sporadic inherited disorders, these diseases are genetically very heterogeneous.
Several complementation groups have been identified among fibroblasts from these patients,
including a heat-labile form of the enzyme. Stabilization of such enzymes may explain the
correction of GlcNAc-P-T activity and lysosomal sorting in some of these cell lines when grown
in 88 mm sucrose. Another novel variant is a case of moderately severe I-cell disease in which
the patient's tissues consist of a mosaic of cells that were either homozygous or heterozygous for
deficiency of the GlcNAc-P-T. Although the genetic basis for this mosaicism remains
unexplained, this experiment of nature does show that complete "cross-correction" of lysosomal
enzymes between all normal and deficient cells cannot occur, even when both cell types are
growing together in the same multicellular organism.
Recognition of Lysosomal Enzymes by the Phosphotransferase

(18 23)
Since the high-mannose-type N-glycans of lysosomal enzymes are identical to those of many
other glycoproteins passing through the ER-Golgi pathway, the specific recognition of the
former by the GlcNAc-P-T is crucial to achieve their selective trafficking. This recognition is not
explained by any similarities in the primary polypeptide sequences of lysosomal enzymes.
Indeed, denatured lysosomal enzymes lost their specialized acceptor activity, indicating that
features of secondary or tertiary structure are crucial. Kornfeld and colleagues carried out
systematic "swapping" of primary sequences between cathepsin D and pepsinogen, two
homologous enzymes that are or are not recognized by the GlcNAc-P-T. The results showed that
scattered basic residues and an adjacent peptide loop explain GlcNAc-P-T recognition. A novel
combinatorial mutagenesis strategy (shuffle mutagenesis) was then used to show that two
regions of the cathepsin D amino lobe are involved in proper folding, surface expression, and
selective phosphorylation of the carboxyl lobe N-glycan. These regions appear to cooperate with
a recognition element in the carboxyl lobe to allow efficient phosphorylation of both the amino
and carboxyl lobe oligosaccharides.
Studies on some other lysosomal enzymes have generalized these findings. With human
lysosomal aspartylglucosaminidase, three lysines and one tyrosine residing in three spatially
distinct regions of the aspartylglucosaminidase polypeptide were found to be necessary for N-
glycan phosphorylation. Two of the lysines are especially important for the lysosomal targeting
efficiency of aspartylglucosaminidase, which seems to be mostly dictated by the degree of
phosphorylation of the "α" subunit oligosaccharide. Phosphorylation of DNase I (a secreted
protein that carries low levels of Man-6-P) decreased from about 13% to about 2% when Lys-50,
Lys-124, and Arg-27 were mutated to alanines. Mutations of lysines at other positions did not
impair GlcNAc-P-T, demonstrating the selectivity of this process. Interestingly, when Arg-27
and/or Asn-74 in DNase I were replaced with a lysine, phosphorylation actually increased,
showing that GlcNAc-P-T prefers lysine residues to arginine residues. Moreover, the Arg Lys-
27 and Arg Lys-74 mutations caused selective phosphorylation of the neighboring asparagine-
linked oligosaccharide. Various models for extending the catalytic reach of the GlcNAc-P-T to
widely spaced oligosaccharides on the surface of a lysosomal hydrolase target have been
proposed. With the recent cloning of the genes for phosphorylating enzyme, these models can
now be directly tested in vitro.
Man-6-P Receptors (6 9, 18,24 29)
The first candidate receptor for the phosphomannosyl recognition marker was isolated from
bovine liver by Sahagian and Jourdian, using affinity chromatography on immobilized yeast
phosphomannan. Shortly thereafter, the same receptor was isolated from other sources by several
other investigators, using a variety of methods. This molecule (m.w. ~275,000) bound Man-6-P
in the absence of cations. The observation that certain cells deficient in this receptor still showed
Man-6-P-inhibitable binding of lysosomal enzymes led to the discovery of a second Man-6-P
receptor of about 45 kD, which required divalent cations for optimal binding. The larger cation-
independent Man-6-P receptor (CI-MPR) bound with highest affinity in a 1:1 stoichiometry to
oligosaccharides carrying two phosphomonoesters (structure C, Figure 23.2) and poorly to
molecules bearing GlcNAc-P-Man phosphodiesters (structures A and B). Binding to molecules
carrying one phosphomonoester was intermediate in affinity (structure D). The smaller cation-
dependent Man-6-P receptor (CD-MPR) seemed to have only a binding site for a single
phosphomonoester. In vitro removal of the "blocking" GlcNAc residues from molecules carrying
two phosphodiesters caused improved binding to the receptors, but treatment with an α-
mannosidase enhanced this further, confirming the predictions made from the structural and
pulse-chase studies that removal of outer mannose residues by the processing Golgi
mannosidases was a requirement. These findings with isolated chains were also confirmed and
extended by studying direct uptake of phosphorylated glycans into cells.
Genes encoding both of the Man-6-P receptors have since been cloned and extensively
characterized from several species (see Table 23.1 and Figure 23.4). Both receptors are type-I
membrane glycoproteins with large extracytoplasmic domains, single transmembrane
hydrophobic regions, and relatively small carboxy-terminal intracytoplasmic domains. The larger
CI-MPR has 15 unique contiguous repetitive units of about 145 amino acids each that have
partial identity to one another. The small cation-dependent receptor CD-MPR has a single
extracellular domain that has homology with some of the repeating domains of the large
receptor. Together with the conservation of certain intron-exon boundaries, this homology
suggests that the two genes evolved from a common ancestor. On the basis of their sequence
relationships and unique carbohydrate-binding properties, the two MPRs have been formally
classified as P-type lectins. There are as yet no other members in this family.
Table 23.1. Comparison of the two mammalian MPRs (P-type lectins)
Cation-independent Cation-dependent
Feature (large) receptor (small) receptor
Topology type I membrane type I membrane glycoprotein

glycoprotein
Subunit molecular mass (SDS- 300 250 kD 45 kD
PAGE)
Core polypeptide molecular mass 275 kD 28 kD
Optimal pH for binding 6.0 7.0 6.0 6.5
Cation dependence for binding no Mn++>Mg++ = Ca++
Domain structure 15 homologous repeating single 155-amino-acid unit
units of ~145 amino acids homologous to repeating units
each of large receptor
Native oligomeric state monomer dimer or tetramer
Stoichiometry of binding to Man- two per monomer one per monomer
6-P
Kd for glycan with two Man-6-P 2 × 10-9 m 2 × 10-7 m
units
Binding of other ligands yes no
methylphosphomannose
IGF-II yes (no in chicken/Xenopus) no
retinoic acid yes ?
urokinase-type plasminogen yes no
activator receptor (uPAR)
Role in biosynthetic pathway yes yes
Role in endocytotic pathway yes no (?except at high density)
Figure 23.4. Schematic diagram of the domain structure and topology of the two known
vertebrate MPRs. The CD-MPR is shown in the most common dimeric form. Two possible
modes of recognition of phosphorylated glycans on lysosomal enzymes are shown. These are not
necessarily mutually exclusive to the individual receptors. Enzymes with only a single mannose
phosphate residue do not bind with high affinity.
The CD-MPR exists mainly as a dimer, with each monomer component binding one residue of
Man-6-P. However, monomeric and tetrameric forms are also found, and the equilibrium
between the forms is affected somewhat by temperature, pH, and the presence of ligands. The
CI-MPR seems to be primarily in a monomeric state. Somewhat surprisingly, this much larger
molecule binds only two residues of Man-6-P, utilizing just 2 of its 15 repeating units.
Mutagenesis studies have identified specific residues of these receptors involved in Man-6-P
binding, and the crystal structure of a single extracytoplasmic domain of the CI-MPR has
recently been obtained, in complex with Man-6-P. This domain crystallized as a dimer, and each
monomer was found to fold into a nine-stranded flattened β-barrel, which has a striking
resemblance to avidin (Figure 23.5). The distance between the two ligand-binding sites of the
dimer provides a good explanation for the differences in binding affinity shown by the CD-MPR
toward various lysosomal enzymes.
Figure 23.5. Ribbon diagram of the bovine cation-dependent CD-MPR. The two monomers
(magenta ribbon and cyan ribbon) of the dimer as well as the ligand, Man-6-P (gold ball-and-
stick model), are shown. (Modified, with permission, from [29] Roberts et al. 1998 [© Cell
Press].)
The term phosphomannosyl recognition marker thus encompasses a family of Man-6-P-bearing

glycans, with varying degrees of affinity for the receptors, based on the position of the phosphate
groups and the structure of the underlying oligosaccharide (Figure 23.2). The number and
distribution of such glycans on each acid hydrolase could further alter the effective binding to the
two receptors. Thus, whereas both MPRs have a preference for enzymes containing glycans with
two phosphomonoesters, it appears that two appropriately spaced monophosphorylated
oligosaccharides can together provide a high-affinity ligand. A cohort of newly synthesized
lysosomal enzymes therefore presents a spectrum of affinities. Taken together with factors such
as the number, compartmental localization, and availability of the receptor molecules, the
differences in the properties of the two receptors, and the concentration of cations, there is
clearly much flexibility in this trafficking mechanism. Indeed, different cell types target different
Man-6-P-containing proteins to their lysosomes at different rates, with varying proportions being
secreted.
The Cation-independent Receptor Binds to Other Ligands (27

28,30 37)
IGF-II was previously known to bind to two receptors, one identical to the IGF-I receptor and
another independent receptor of reported size of about 220 250 kD. Molecular cloning of the
latter revealed the surprising fact that it is identical to the CI-MPR. A number of studies have
since explored the potential interactions between these seemingly disparate ligands. Although it
is clear that the two ligands bind to distinct sites on the receptor, there are some conflicting
reports regarding potential interactions between the two systems. In various studies, the two
ligands were found to have either synergistic or antagonistic actions. It has also been suggested
that the redistribution of the receptor upon insulin stimulation could explain some of the known
metabolic effects of this hormone on protein degradation, by altering the trafficking of lysosomal
enzymes. However, the CI-MPRs of the chicken and Xenopus do not bind IGF-II, although their
cells can respond to IGF-II. This finding makes it less likely that the overlap in binding
specificity is of vital importance to animal cells in general. Rather, it appears that the CI-MPR
acts primarily as a general "sink" for excess IGF-II in the extracellular fluid, carrying it away to
the lysosome for degradation and reducing the amount available to bind to the IGF-I receptor.
It was also recently found that the CI-MPR binds retinoic acid with high affinity at a site that is
distinct from those for Man-6-P and IGF-II. The binding of retinoic acid to the Man-6-P/IGF-II
receptor seems to enhance the primary functions of this receptor, and the biological consequence
appears to be the suppression of cell proliferation and/or induction of apoptosis. The significance
of this unexpected observation is still being explored. There are also some unexplained changes
in CI-MPR expression in relation to malignancy. Loss of heterozygosity at the CI-MPR locus
occurred in more than 50 dysplastic liver lesions and hepatocellular carcinomas associated with
the high-risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining
allele were detected in about 50% of these tumors, which also seemed to frequently develop
from clonal expansions of phenotypically normal, CI-MPR-mutated hepatocytes. Thus, the CI-
MPR functions as a liver tumor-suppressor gene.
Consequences of Natural and Induced Genetic Defects in the

MPRs (28,38 45)
Targeted disruption of the CD-MPR gene in mice is associated with normal or only slightly
elevated levels of lysosomal enzymes in the circulation and an otherwise normal phenotype.
However, in thymocytes from homozygous null mice or in primary cultures of fibroblasts from
such animals, there is a clear increase in the amount of phosphorylated lysosomal enzymes
secreted into the extracellular medium. Thus, there must be mechanisms that compensate for the
deficiency in vivo. Injection of inhibitors of other glycan-specific endocytotic receptors (the
mannose-specific receptor of macrophages and the asiaoglycoprotein receptor of hepatocytes)
gave a marked increase of lysosomal enzymes in the serum of the deficient mice, indicating that
such receptors are part of these in vivo compensatory mechanisms.
Meanwhile, other workers had shown that the mouse CI-MPR is part of the naturally occurring
Tme locus, a maternally imprinted region of chromosome 17 (i.e., expressed only from the
maternal chromosome). Mice that inherit a deletion of the Tme locus from their mother die at day
15 of gestation. That this lethality is due to the lack of the CI-MPR was directly proven by
genetic disruption of this gene. Maternal inheritance of a null allele or homozygosity for the
inactive allele was generally lethal at birth, and mutants are about 30% larger in size. The
phenotype is probably caused by an excess of IGF-II, because the introduction of an IGF-II null
allele rescued the mutant mice. The mutant mice also have organ and skeletal abnormalities.
Cell lines lacking either or both MPRs were also obtained by mating CD-MPR-deficient mice
with the mice heterozygous for a CI-MPR-deleted allele. Fibroblasts prepared from embryos that
lacked both receptors showed a massive missorting of multiple lysosomal enzymes and
accumulated undigested material in their endocytotic compartments. Like fibroblasts that lacked
only the CD-MPR, fibroblasts that lacked only the CI-MPR had only partial impairment in
sorting. This demonstrates that both receptors are required for efficient intracellular targeting of
lysosomal enzymes. Comparison of the phosphorylated proteins secreted by the different cell
types indicates that the two receptors may interact preferentially with different subgroups of
hydrolases. Thus, the structural heterogeneity of the phosphomannosyl recognition marker
within a single lysosomal enzyme molecule and among different enzymes is a rational
explanation for the evolution of two MPRs with complementary binding properties, to ensure an
efficient but varied targeting of lysosomal proteins in different cell types or tissues.
Relative Roles of the Two MPRs in Lysosomal Enzyme
Trafficking (6 9, 18,27 28,43 47)
The biosynthesis, intracellular distribution, and trafficking of both MPRs have been extensively
studied. Each is synthesized as an N-glycosylated glycoprotein that traverses the ER-Golgi
pathway to end up in cell surface and internal pools that are constantly mixing with one another
(Figure 23.6). Most of the MPRs are found in the trans portion of the Golgi apparatus and in
adjacent late-endosomal compartments. Thus, most newly synthesized lysosomal enzymes
traverse the entire Golgi pathway and are diverted from the trans-Golgi into endosomal
compartments by binding to one or the other of the MPRs that are collected into clathrin-coated
pits. In many cells, a minority of the newly synthesized lysosomal enzymes escape sorting and
are secreted into the medium carrying Man-6-P residues. These secreted molecules are subject to
recapture by the same cell or presumably by adjacent cells bearing similar receptors. Regardless
of the source, the enzymes that bind to cell surface MPRs are endocytosed via clathrin-coated
pits and vesicles, eventually arriving in the same late endosomal compartments where newly
synthesized molecules have arrived from the Golgi. Here, the acidic environment causes
dissociation of both populations of enzymes from the receptors, which then recycle to the cell
surface or the Golgi apparatus. There is general agreement that MPRs are not found in mature
lysosomes, which seem to obtain their cargo from late endosomes by other mechanisms. The
"secretion-recapture" pathway from the cell surface is a minor pathway in most cells, with
potential importance in some situations. For example, some activated macrophages secrete a
large portion of their lysosomal enzymes directly into the medium. It is possible that under
inflammatory situations, it is useful for such secreted enzymes to be returned to lysosomes from
the extracellular fluid via the Man-6-P pathway.
Figure 23.6. Subcellular pathways followed by glycoproteins, including lysosomal enzymes and
the MPRs. Newly synthesized glycoproteins originating from the rough ER pass through the
Golgi stacks and are then sorted to various destinations as indicated. Along this route, lysosomal
enzymes are modified as indicated (the proposed locations where these modifications are
believed to occur are noted). Lysosomal enzymes re-entering the cell via the endocytotic
pathway can intersect with those following the biosynthetic route, as depicted. Open arrowheads
indicate pathways general to many glycoproteins; closed arrowheads indicate specific itineraries
of lysosomal enzymes. Beyond the trans-Golgi, the latter are primarily mediated by the MPRs.
The fuzzy border indicates a clathrin-coated pit or vesicle.
As indicated above, cells genetically deficient in both receptors secrete most of their enzymes,
much like cells from patients with I-cell disease. It appears that only the CI-MPR is responsible
for endocytosis from the cell surface going to the lysosomes via endosomes. However, when the
CD-MPR is strongly overexpressed, it is capable of mediating uptake from the surface. These
differences may be due to the very narrow binding pH optimum of the CD-MPR and/or its
variable oligomeric state. Taken together, the results indicate that although the CI-MPR is a
major determinant of trafficking in the biosynthetic pathway, the CD-MPR also contributes
significantly. However, when the CD-MPR is overexpressed in cells containing the CI-MPR,
increased secretion of lysosomal enzymes can result. Thus, the CD-MPR may modulate the
pathway in the direction of either retention or secretion, perhaps based on other factors such as
its oligomeric state, expression level, subcompartmental pH values, divalent cation availability,
amounts of the other receptor present, or differences in precise affinities for multivalent ligands
(Table 23.1). In the final analysis, different combinations of amounts and locations of the two
receptors, together with the spectrum of phosphomannosyl recognition marker structures on
various molecules, could explain the highly variable physiology of lysosomal enzyme trafficking
in different cell types.
Mechanisms of Subcellular Trafficking of the Two MPRs (6 9, 18,27

28,47 54)
There are many elegant studies of the molecular determinants of subcellular trafficking of the
MPRs, which mainly involve determinants in the cytosolic tails of the molecules. These
interesting studies deserve a detailed and separate review, but they can only be summarized
briefly here. Certain amino acid motifs in the cytoplasmic tail of the receptors can bind
specifically to "adaptor" proteins, which in turn are known to interact with clathrin. Both
endocytosis from the plasma membrane and the budding of transport vesicles from the trans-
Golgi network involve the interaction of the receptor with the clathrin-coated vesicle-associated
protein complexes AP1 and AP2. Interestingly, the two adaptors AP1 and AP2 show selective
distribution to the Golgi complex and plasma membrane, respectively, the two sites where
trafficking decisions of the MPRs must be determined. Using mouse fibroblasts lacking both
MPRs or reexpressing physiological levels of either MPR, it was found that the amount of AP1
bound to membranes and associated with clathrin-coated vesicles depends on the expression
level of the MPRs and on the integrity of their cytoplasmic domains. Thus, the concentration of
the MPRs, i.e., the major transmembrane proteins sorted toward the endosomes in the trans-
Golgi network, determines the total number of clathrin-coated vesicles formed in this region.
Endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino-
acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also
present in the cytoplasmic tail of other receptors known to be internalized from the plasma
membrane, this feature may prove to be a general determinant for endosomal sorting. Transport
from the trans-Golgi network to the endosomal pathway by the CI-MPR is inhibited by the
phosphoinositide-3-kinase inhibitor wortmannin, consistent with the established role of this
kinase in the equivalent transport process in yeast. In contrast, CI-MPR endocytosis and
transport to the trans-Golgi network are not inhibited by wortmannin. The two cysteine residues
(Cys-30 and Cys-34) in the cytoplasmic tail of the CD-MPR are palmitoylated via thioesters,
which are turned over rapidly. The data suggest that they are involved in the anchoring of this
region of the cytoplasmic tail to the lipid bilayer. Anchoring via Cys-34 is essential for the
normal trafficking and lysosomal enzyme-sorting function of the receptor. The cytoplasmic tail
of the CI-MPR also contains a signal that prevents the receptor from entering lysosomes where it
would be degraded.
Evolutionary Origins of the MPR System (27 28, 55)
Although the MPR clearly has a major role in lysosomal enzyme trafficking in vertebrate cells,
its role in invertebrate systems is not prominent. Lysosomal enzymes are successfully targeted in
lower eukaryotes such as Saccharomyces, Trypanosoma, and Dictyostelium, without the aid of
identifiable MPRs. The slime mold Dictyostelium discoideum produces a novel
methylphosphomannose sequence on some of its lysosomal enzymes that can be recognized in
vitro by the mammalian CI-MPR (not the CD-MPR). However, despite the presence of a
GlcNAc-P-T that recognizes α1 2-linked mannose residues, no receptor for the phosphorylated
mannose residues has been found in these organisms. Notably, although this phosphotransferase
does not show the specific recognition of lysosomal hydrolases seen with the mammalian
enzyme, it produces another transferase that selectively adds GlcNAc-1-P to serine residues (see
Chapter 12). In contrast to this situation, the protozoan Acanthameba produces a GlcNAc-P-T
that does show specific recognition of lysosomal enzymes. Although some of these organisms
show evidence for an "uncovering" enzyme, no definable MPR has yet been found. The
evolutionary divergence point at which the complete MPR system came into being has yet to be
identified.
Alternate Pathways for the Trafficking of Lysosomal Enzymes (9,

56)
Although the phosphomannosyl recognition marker has a crucial role in the trafficking of newly
synthesized lysosomal enzymes to lysosomes in vertebrate cells, several lines of evidence
indicate that alternate mechanisms must exist in some cell types. Even in I-cell disease, some
cells and tissues (e.g., liver and circulating granulocytes) have essentially normal levels of
enzymes. B-lymphoblast lines derived from these patients also do not show the complete
phenotype of enzyme deficiency seen in fibroblasts. One interpretation is that the Man-6-P
pathway for trafficking of lysosomal enzymes is a specialized form of targeting, superimposed
on some other basic mechanisms that remain undefined. Two lysosomal enzymes, acid
phosphatase and β-glucocerebrosidase, are not at all affected in their distribution, even in I-cell
disease fibroblasts. With acid phosphatase, the enzyme is synthesized initially as a membrane-
bound protein, and once in the lysosome, it is proteolytically cleaved to generate the mature
soluble form. Glucocerebrosidase is also membrane-associated, does not show phosphorylation
of its glycans, and is targeted to lysosomes independent of this pathway. Likewise, integral
membrane proteins of the lysosome such as the LAMP/lgp proteins do not require the
phosphomannosyl recognition marker pathway for trafficking to lysosomes. Rather, they seem to
utilize motifs in their cytosolic tails similar to those of the MPRs.
Significance of Man-6-P on Nonlysosomal Proteins (57 63)
Man-6-P esters have been reported on a variety of nonlysosomal proteins. Some are hydrolytic
enzymes that seem to take a predominantly secretory route, e.g., uteroferrin and DNase I. In the
first case, the failure of removal of the blocking GlcNAc residues may be the cause for secretion.
In the case of DNase I, the native level of phosphorylation appears to be very low. These appear
to be variations on the basic MPR pathway.
Man-6-P has also been found on the TGF-β precursor and the phosphate is then lost in the
mature form. It appears that Man-6-P may serve to target the precursor to an acidic compartment
for activation in the intact cell. Other nonlysosomal proteins reported to carry Man-6-P are
proliferin, leukemia inhibitory factor, and thyroglobulin. In the latter case, it is suggested that the
Man-6-P-containing chains are used to target the protein for degradation and release of thyroid
hormone. The varicella virus glycoprotein has been shown to contain Man-6-P on its complex-
type N-glycans, and it has been suggested that this form of Man-6-P originates from an entirely
distinct pathway. The significance of finding Man-6-P on these nonlysosomal proteins is unclear
in most cases. One should not necessarily assume that the phosphomannose residues on all of
these proteins are involved in intracellular trafficking. Just as phosphorylation of serine residues
has diverse biological roles, Man-6-P might be utilized for more than one purpose in a complex
multicellular organism. Further investigation of each situation is therefore needed, with an open
mind to all of the possibilities.
Future Directions
The trafficking of mannose-6-phosphorylated lysosomal enzymes by the P-type lectins can be
considered as one of the most clear-cut examples of specific biological roles of the glycans
involving recognition by an endogenous lectin. The next logical steps are the further
characterization of the recently cloned biosynthetic enzymes and the understanding of all the
molecular interactions in the system at the level of atomic resolution. The multifunctional nature
of the CI-MPR and its aberrations in malignancy also deserve further investigation. The more
detailed characterization of the genetic defects in the GlcNAc-P-T and the search for mutations
in the phosphodiester glycosidase are also worthwhile.
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24. I-type Lectins
Diego).
I-TYPE LECTINS ARE A FAMILY of carbohydrate-binding proteins within the

immunoglobulin superfamily. Included are a group of sialic-acid-binding lectins (the Siglecs)
and several non-sialic-acid-binding lectins. Details of the discovery, biochemical
characterization, binding properties, and biology of the Siglecs are provided here, along with a
discussion of the functional implications of these proteins within mammalian biology.
Primary contributions to this chapter were made by L. Powell (University of California at San
Diego).
Historical Background and Discovery(1 12)
The immunoglobulin superfamily is a large family of proteins, and the majority are involved in
protein-protein-binding interactions as either receptors, cell adhesion molecules, or antibodies.
Of late, a subset of this family has been identified that functions as carbohydrate-binding
proteins (lectins; see Table 24.1). The defining features of this subset, termed the I
(immunoglobulin)-type lectins, include both their function (as carbohydrate-binding proteins)
and their IgSF-like amino acid sequences. Of note, only one member of this family
(sialoadhesin) was initially isolated and characterized on the basis of its carbohydrate-binding
properties. All of the other I-type lectins were isolated in the course of other studies, and their
lectin function was subsequently established, as discussed below.
Table 24.1. Established and putative members of the I-type lectin family
Lectin Alternate Tissue/Cell type distribution Domain Minimal carbohydrate

name structure structure(s) recognized
Siglecs
Siglec sialoadhesin macrophages in spleen, lymph (V)1-(C2)16 Siaα2 3Galβ1
1 nodes, and bone marrow 3(4)GlcNAc-
Siaα2 3Galβ1 3 GlcNAc-
Siglec CD22 B cells (V)1-(C2)6 Siaα2 6Galβ1 4GlcNAc-
2
Siglec CD33 myeloid cell lineage (V)1-(C2)1 Siaα2 3Galβ1
3 3(4)GlcNAc-
Siaα2 3Galβ1 3GlcNAc-
Siglec MAG PNS (V)1-(C2)4 Siaα2 3Galβ1 3GalNAc
4a
Siglec Schwann cell Schwann cells in quail (V)1-(C2)4 Siaα2 3Gal-
4b
Siglec myelin granulocytes and monocytes (V)1-(C2)3 Siaα2 3Gal- and Siaα2 6-
5 protein Gal-
Non-Siglecs
platelets heparin
PECAM
P0 PNS (V)1 SO3GlUAβ1 3Galβ1-R
(HNK1 epitope)a
N- PNS and CNS (C2)5 high-mannose
CAM oligosaccharidea
ICAM- blood cells, endothelium, etc. (C2)5 hyaluronan (GlcNAcβ1
1 3GlUAβ1 4)n
leukosialin (sialylated
mucin)a
CD48 activated B cells (V)1-(C2)1 heparin and heparan sulfate
a
In these cases, the evidence for specific binding involving carbohydrates is indirect.
Sialoadhesin was initially described in 1986 as a murine macrophage receptor responsible for
mediating sialic-acid-dependent binding of nonopsinized sheep erythrocytes. Because of its
considerable size, it was not sequenced until 1994, which established its membership in the IgSF.
MAG (myelin-associated glycoprotein) was initially described in 1972 and sequenced in 1987.
CD33 and CD22 were initially identified by monoclonal antibodies as myeloid and B-cell-
restricted cell surface glycoproteins, respectively, and subsequently cloned and sequenced in
1988 and 1990. Schwann cell myelin protein was identified and sequenced from avian cells in
1992. In 1993, the lectin function of CD22 was characterized. The considerable sequence
homology of CD22 and sialoadhesin with CD33, Schwann cell myelin protein, and MAG led to
the correct prediction that these three latter glycoproteins function as sialic-acid-dependent
lectins as well. By searching DNA databases for cDNAs that have homology with CD33, an
additional protein (now termed Siglec-5) was identified that on subsequent testing, functioned in
vitro as a sialic-acid-binding protein analogous to that of the other Siglecs. A few other IgSF
member proteins have been identified that recognize nonsialylated carbohydrate structures.
Included here are N-CAM, P0, PECAM (platelet-endothelial cell adhesion molecule), CD48, and
ICAM-1 (intracellular adhesion molecule) (Table 24.1). Consequently, the entire family of
lectins is termed the I-type lectins, whereas the subset recognizing sialylated structures is termed
the Siglecs (sialic acid/Immunoglobulin/lectin) and has been numbered sequentially by common
agreement. This chapter reviews structural and functional data about this group of lectins,
concentrating on the Siglecs, about which the most is known.
Common Features of Siglecs

Domain Structure and Genetics(13 21)
The primary amino acid sequence of the I-type lectins indicates that they are members of the
IgSF, containing an amino-terminal V-set domain followed by a variable number of C2-set
domains (Vl-C2n) (see Figure 24.1). The C2-set domains are a variation of the more common
C1-set domains and are believed to represent a primordial motif predating the evolution of
antibody genes. A significant degree of amino acid sequence relatedness can be found between
the Siglecs (Figure 24.1). On the basis of their similarities with IgSF proteins and the crystal
structure of the first domain of sialoadhesin, the first domains are predicted to form a structure
consisting of a β-sandwich made of two β sheets termed ABED and GFCC , using the standard
immunoglobulin nomenclature for labeling the individual β strands. The location of these strands
is indicated in Figure 24.1. Notable features in the V domain include the placement of the second
cysteine residue in the E strand rather than the F strand (the typical location in immunoglobulin
domains), leading to the prediction of an intra-β-sheet disulfide bond rather than an inter-β-sheet
disulfide bond. The fact that the cysteine in the F strand is occupied by a hydrophobic residue
suggests that hydrophobic interactions unite the β-sandwich of the domain. The X-ray crystal
structure of sialoadhesin has verified this prediction. Another common feature is the presence of
an odd number of cysteine residues in the first and second domains, suggesting an interdomain
disulfide linkage. This was not seen in the crystal structure of sialoadhesin but has been
demonstrated biochemically for MAG. Several conserved arginine residues are also seen in the F
and G strands of the first domain (Figure 24.1). Site-directed mutagenesis indicated that these
residues are essential for the binding of sialylated glycoconjugates by sialoadhesin and CD22 by
interaction with the carboxyl group on sialic acid. The crystallographic structure of sialoadhesin
bound to sialyllactose further demonstrates the presence of hydrophobic interactions between
sialic acid and two conserved aromatic residues in the A and G strands of the first domain (see
Figures 24.1 and 24.2).
Figure 24.1. Sequence alignments of the first two domains of the murine forms of CD22,
sialoadhesin, MAG, and CD33 are shown, with regions of identity included in boxes and regions
of similarity in yellow. Above the sequences are the regions of predicted and confirmed (for
sialoadhesin) β strands. Asterisks indicate the residues known to be involved in binding of
sialyllactose in sialoadhesin and are identified in Figure 24.2. The leader peptides have been
deleted from each sequence.
Figure 24.2. Defined interactions of specific amino acid residues in sialoadhesin with 3
sialyllactose. Solvent molecules are depicted as ovals and 3 sialyllactose is shown in thick black
lines. Note that the seven-amino-acid residues involved in interactions with the glycan ligand
correspond to highly conserved residues within the Siglec family (indicated by asterisks in
Figure 24.1). (Reprinted, with permission, from [15] May et al. 1998.)
Chromosomal localization studies indicate that CD22, CD33, Siglec-5, and MAG are clustered
on human chromosome 19q and the syntenic murine chromosome 7. Twenty related genes of the
IgSF, including members of the carcinoembryonic antigen and pregnancy-specific glycoprotein
families, are also found in close proximity, suggesting their evolutionary origin by gene
duplication. Sialoadhesin maps to murine chromosome 2 and human chromosome 20
(determined using the murine sialoadhesin as a probe), and thus is unlinked to the other Siglecs
in both species. The presence of other Siglecs in proximity to the sialoadhesin gene has yet to be
explored.
Specificity for Recognition of Sialylated Structures (9 11, 17,22 26)
Another very significant feature of the Siglecs is their recognition of sialic-acid-containing

structures in a linkage-specific manner. CD22 is absolutely specific for α2 6-linked sialic acid
residues and MAG, Schwann cell myelin protein, and CD33, for α2 3-linked sialic acid
residues; sialoadhesin can recognize α2 3-linked and α2 8-linked residues. The initial analysis
of Siglec-5 using synthetic sialosides indicates that it is capable of binding to α2 3- and α2 6-
linked sialic acid residues.
Some preference for sialylated glycoconjugates formed by the α2 3 sialyltransferases can also
be seen in some assay systems (Table 24.2). These observations are of significance, given the
large and ever expanding family of sialyltransferases that have marked differences in linkages
formed, acceptor glycoconjugate structures recognized, and cellular expression patterns (Table
24.2; see Chapters 16 and 17). Thus, the Siglecs are one of a small number of mammalian
proteins capable of "reading" the information content reflected in the differential expression of
the multiple sialyltransferase genes.
Table 24.2. Sialyltransferases relevant to forming ligands for the Siglecs
Sialyltransferase Acronym Acceptor Sialylated Potential Siglec

disaccharide structure formed ligands
O-glycan Core 1 α2 3 ST3Gal-I Galβ1 Siaα2 3Galβ1 SMP, MAG,

sialyltransferase 3GalNAc 3GalNAc sialoadhesin, CD33
O-glycan α2 3 ST3Gal-II Galβ1 Siaα2 3Galβ1 SMP, MAG,
3GalNAc 3GalNAc
sailytransferase ST3Gal- Galβ1 Siaα2 3Galβ1 sialoadhesin, CD33
III 3GlcNAc 3GlcNAc
Galβ1 Siaα2 3Galβ1 sialoadhesin, CD33 >
4GlcNAc 4GlcNAc MAG
LacNAc: α2 3 ST3Gal- Galβ1 Siaα2 3Galβ1 SMP, MAG,
sialyltransferase IV 3GalNAc 3GalNAc
sailytransferase Galβ1 Siaα2 3Galβ1 sialoadhesin, CD33
3GlcNAc 3GlcNAc
N/O α2 3 sialyltransferase Galβ1 Siaα2 3Galβ1
4GlcNAc 4GlcNAc
Galβ1 4 GlcNAc α2 6 STL6Gal- Galβ1 Siaα2 6Galβ1 CD22
sialyltransferase I 4GlcNAc 4GlcNAc
In addition to their preference for specific sialic acid linkages, the Siglecs also are sensitive to
modifications of the sialic acid residue itself. As presented in Chapter 15, the sialic acids are a
family of more than 40 different molecules based on the parent molecules, neuraminic acid, and
keto-deoxynonulosonic acid. The two most common forms found in vertebrates are Neu5Ac and
Neu5Gc (for structures and nomenclature, see Chapter 15). Murine sialoadhesin and murine
MAG show a marked reduction in binding to Neu5Gc-containing structures, whereas murine
CD22 shows a marked preference for Neu5Gc-containing structures. In contrast, human CD22
binds equally well to both Neu5Gc and Neu5Ac. These observations are of interest, considering
that NeuGc is not found in human tissues but in primates and nonprimate species, where the ratio
of NeuGc to NeuAc varies between tissues within the species. Modifications of the C9 hydroxyl
group on the side chain of sialic acid also negatively impacts the binding of the Siglecs, whether
it be by 9-O-acetylation or by its truncation by chemical oxidation. As 9-O-acetylation is a
naturally occurring and dynamic modification, it may have a meaningful role in the regulation of
Siglec-dependent functions.
Site-directed mutagenesis of CD22 and sialoadhesin, along with the crystallographic structure of
sialoadhesin, explain the roles of several conserved amino acid residues (Figures 24.1 and 24.2).
The sialylated structure binds to a site formed on the face of the GFCC C β-sheet. The invariant
arginine residue in the F strand (Arg-97 in sialoadhesin) forms a salt linkage with the carboxylate
moiety of sialic acid. The conserved basic residue (Arg, Gln, or Lys) in the G strand (Arg-105 in
sialoadhesin) forms a hydrogen bond with the acetamido hydrogen. The conserved hydrophobic
residues (Tyr, Trp, Phe) in the A and G strands (Trp-2 and Trp-106 of sialoadhesin) form
hydrophobic bonds with the acetamido methyl group and glycerol side chain carbon residues,
respectively. Several hydrogen bonds are formed with the hydroxyl groups on the glycerol side
chain of sialic acid, explaining the sensitivity of Siglec binding to both periodate oxidation of the
side chain and 9-O-acetylation of the side chain. Furthermore, the crystal structure of
sialoadhesin with α2 3 sialyllactose suggests that the 7-O- or 8-O-acetylated derivatives of sialic
acid will not bind.
Lectin Functions of the Siglecs

Siglec-1 (Sialoadhesin) (10, 22,27 33)
Sialoadhesin was initially identified as the receptor on murine macrophages responsible for their
sialic-acid-dependent binding of nonopsonized sheep erythrocytes. Initially, it was recognized
that this binding was due to receptors distinct from those responsible for binding desialylated or
opsinized erythrocytes. The extracellular portion of this lectin consists of 17 immunoglobulin
domains, in a (V)1-(C2)16 arrangement, with the lectin function localized to the first domain. The
internal domains 4 17 are composed of seven homologous pairs of domains, which suggests that
they arose by repeated gene duplication events. The only function identified for these multiple
domains is to position the amino-terminal lectin domain away from the sialic-acid-rich
glycocalyx that surrounds the cell, which otherwise would block its lectin site. This hypothesis is
supported by three observations, including a linear structure for sialoadhesin in electron
micrographs, a reduction in adhesion efficiency with the deletion of 7 of the 17 extracellular
domains, and the conservation of sialoadhesin's length between mice and humans.
Compared to the other Siglecs, sialoadhesin binds to the widest array of sialylated structures.
Early studies demonstrated binding to α2 3 sialic acid residues, including Siaα2 3Galβ1
3GalNAc and Siaα2 3Galβ1 3(4)GlcNAc. These structures are the products of several different
α2 3-specific sialyltransferases and can be found on glycolipids and N-linked and O-linked
chains on glycoproteins (Table 24.2). More recently, sialoadhesin has been demonstrated to bind
to gangliosides carrying terminal α2 8 sialic acid residues such as GD3 and GD1b. Thus, potential
ligands for sialoadhesin are likely to be found on virtually all cell types. However, sialoadhesin
(expressed on COS cells, macrophages, or as an Sn-Rg [sialoadhesion-receptor globulin]
construct) shows marked preference for binding to granulocytes and erythroblasts over other cell
types. For example, in comparison with MAG, both erythrocytes and thymocytes bind higher
levels of MAG than sialoadhesin, a result that would not have been predicted by the binding
preferences of MAG and sialoadhesin to purified gangliosides. Thus, there appears to be
additional structural information involved in lectin-ligand binding beyond what is contained in
the sialylated oligosaccharide structure itself. Whether this information has to do with structural
features in the oligosaccharide itself, in the scaffold (glycoprotein or glycolipid) that expresses
the oligosaccharide, or with its density/presentation on the cell surface has yet to be determined.
Sialoadhesin has an apparent Kd of approximately 0.5 mm for α2 3 sialyllactose. How does such
a low-affinity lectin function in biological systems? The apparent affinity of a receptor can be
increased by factors such as density of the lectin on macrophages and its target ligand. Given the
preference of a soluble Sn-Rg protein to bind to different cells independent of α2 3 sialic acid
levels, it is likely that the formation of high-affinity interactions is dependent on structural
features of the scaffold protein or glycolipid carrying the sialylated oligosaccharide structures. Its
apparently poor Kd for α2 3 sialyllactose would explain why total serum glycoproteins, which
contain approximately 1 2 mmα2 3 sialic acid residues, are not able to block sialoadhesin-
dependent binding between different cell types. The identification of the naturally occurring
macromolecular ligand for sialoadhesin, and its subsequent characterization, will certainly help
resolve these issues.
Sialoadhesin is not constitutively expressed on all macrophages, but only on specific subsets of
tissue-phase or activated macrophage populations. In this regard, it is important to consider the
diversity of macrophage morphology and function in different organs. Resting macrophages
isolated from lung, liver, thymus, heart, brain, peritoneum, and skin have low levels of
sialoadhesin expression. Levels are up-regulated in acute and chronic inflammatory states and
for peritoneal macrophages, by exposure to factors contained in mouse serum. Only in bone
marrow, spleen, and lymph nodes do macrophages constitutively express high levels of
sialoadhesin. In human spleen, strongly sialoadhesin-positive macrophages are selectively
localized in sheaths around capillaries found in the perifollicular zone, a position that may put
them in intimate contact with lymphocytes or other cells entering or exiting the developing
follicles. In mouse bone marrow samples, sialoadhesin-positive macrophages are found in cell
clusters with developing erythroblasts, where electron microscopy showed that sialoadhesin is
clustered at the points of cell-cell contact. Sialoadhesin also binds to myeloid cells within the
bone marrow. These observations all support the hypothesis that this lectin functions as a cell
adhesion molecule, but with potentially different roles on different macrophage populations in
different tissues. The cytoplasmic tail of sialoadhesin consists of only 28 amino acid residues,
and it is not phosphorylated (unlike CD22, CD33, and MAG). However, it may still play a part
in intracellular signaling through noncovalent association with cytoplasmic proteins. A potential
role(s) for sialoadhesion includes the facilitation of granulocyte development and clearance,
macrophage margination and activation, antigen-specific lymphocyte activation, and/or homing
of activated lymphocytes to lymphoid follicles.
Siglec-2 (CD22) (4 5, 7 8, 17 18, 20, 25, 34 48)
CD22 was the first Siglec identified both as a member of the IgSF and as a lectin. Two human
isoforms of CD22 have been isolated, differing in the presence or absence of the third and fourth
immunoglobulin domains. The oligosaccharide-binding properties of these two isoforms have
not been directly compared, but given the data indicating that the lectin site is contained in the
first two immunoglobulin domains, it is likely that both would have identical sugar-binding
properties. No short isoform of CD22 from the mouse has been isolated, but two CD22 alleles
exist, differing in six deletions and eight substitutions in the amino acid sequence, clustering in
the C and C β strands of domains 1 and 2, respectively. None of the residues replaced
correspond to the amino acid residues essential for sialic acid binding, as determined for
sialoadhesin and CD22, although the fine binding specificities of these different alleles to
potential glycoprotein ligands have not been explored.
CD22 was initially identified in 1986 as a developmentally regulated cell surface glycoprotein on
B cells. It is expressed at approximately the time of immunoglobulin gene rearrangement and is
expressed preferentially on B cells in the lymph node follicular mantle zone and splenic marginal
zone areas. Cloned and sequenced in 1990, CD22 was demonstrated to function as an adhesion
molecule by its ability to cause CD22-transfected COS cells to rosette monocytes, erythrocytes,
and certain populations of lymphocytes. CD22-dependent adhesion could be blocked by anti-
CD45 monoclonal antibodies, indicating that antigen was one major ligand for CD22. One of the
blocking antibodies was UCHL-1, a monoclonal antibody known to recognize a sialic-acid-
dependent epitope on CD45RO. With this observation as a clue, the binding of CD22 itself was
demonstrated to be abrogated by pretreatment of target cells with sialidase, indicating it
recognized a sialic-acid-dependent epitope. Direct binding studies between isolated glycans and
CD22 established its lectin function, with no evidence for binding requiring protein-protein
interactions. The minimal carbohydrate structure recognized is Neu5Acα2 6Galβ-R, with a Kd
of approximately 30 µm measured for α2 6 sialyllactose and α2 6 sialyllactosamine structures.
These structures are predominantly found on N-glycans (the product of the Golgi enzyme
ST6Gal-I; see Table 14.2), yet can also be found in the terminal position of lactosamine units
found on some mucin and glycolipid structures. Both α2 6-sialylated GlcNAc and GalNAc
structures can be bound by CD22. Although some of these different α2 6-sialylated structures
bind to CD22 with an apparent affinity 10 30-fold less than that observed for α2 6 sialyllactose
(which would predict a Kd in the 0.5 1.0 mm range), these low anticipated affinities do not
exclude these interactions from potential biological relevance. For example, the approximate
affinity of sialoadhesin to α2 3 sialyllactose is estimated to be 0.5 mm, and the affinities of the
selectins for simple sialylated and fucosylated glycans are also in this range (see Chapter 26).
CD22 exists as a noncovalent multimer on the surface of cells, as demonstrated by cross-linking

studies. Furthermore, inhibition studies performed with mono- and bi- α2 6-sialylated
biantennary N-glycans indicate that the two lectin sites on this noncovalent dimer are in close
proximity. The existence of this noncovalent dimer suggests a role for the internal
immunoglobulin domains of CD22 in forming this structure. In lymphocytes, CD22 shows
preference in binding to only a subset of α2 6-sialylated glycoproteins, including CD45.
However, several other as yet unidentified glycoproteins are also bound. In serum, the major
binding glycoproteins are haptoglobin and pentameric IgM. Of note, both on lymphoid cells and
in serum glycoproteins, many of the nonbinding proteins contain significant amounts of α2 6-
linked sialic acid residues. Why these glycoproteins fail to bind to CD22 is not clear and may
reflect contributing factors, such as the density of sialic acid epitopes and/or other structural
features in the peptide backbone. In fact, in serum, the concentration of α2 6 sialic acid residues
is approximately 0.5 mm, and serum concentrations of more than 4% block the binding of all
glycoproteins to CD22. Therefore, these other glycoproteins, although not of sufficiently high
affinity to be identified as ligands for CD22, can function as ligand antagonists. These
observations present the unanswered questions: How CD22 can effectively function as a lectin in
an apparent "sea" of α2 6 sialic-acid-containing structures?
Using a soluble CD22-immunoglobulin fusion protein as an immunohistochemical reagent,

murine lymphohematopoietic tissues have been stained for the expression of potential ligands.
Ligand expression appears to be highly regulated, as only discrete populations of lymphocytes in
the lymph node and spleen expressed potential ligands. Furthermore, potential ligands were
masked by O-acetyl residues, as de-O-acetylation increased the staining patterns in all tissues
examined. One would expect that staining would reflect the expression of ST6Gal-I, but as
discussed in the preceding paragraph, not all proteins containing α2 6-linked sialic acid residues
bind to CD22. Consequently, the tissue-specific and cell-specific staining patterns observed in
these studies likely reflect the expression of both ST6Gal-I and specific glycoproteins.
Although the role of ST6Gal-I in regulating expression of potential ligands on target cells is
evident, ST6Gal-I can also mask CD22's;s lectin function. This is demonstrated experimentally
by the binding of an appropriately sialylated probe to cells transfected to express CD22, but not
to cells transfected to express both CD22 and ST6Gal-I. It is important to realize that the first
demonstration of the ability of CD22 to function as an adhesion molecule was done with CD22
transfected into COS cells, which do not express ST6Gal-I. CHO cells, which are also frequently
employed in these adhesion assays, also do not express ST6Gal-I. However, B cells do express
this sialyltransferase, and this results in the masking of CD22's lectin function on native B-cell
populations, at least as measured either by cell adhesion assays or by probing with α2 6 sialic-
acid-containing glycoproteins or acrylamide polymers. A specific role has been proposed for the
first N-glycosylation site of CD22 in masking its lectin function. However, other cell surface
glycoproteins may also be involved. Upon cellular activation, some of the previously masked
CD22-binding sites become unmasked, by as yet unknown mechanisms.
How CD22's lectin property relates to CD22's immunoregulatory role has yet to be established.
A minority of cell surface CD22 is found noncovalently associated with the B-cell IgR complex.
Its cytoplasmic tail rapidly becomes tyrosine-phosphorylated following B-cell immunoglobulin
receptor stimulation by the lyn kinase, a modification that serves to recruit the protein tyrosine
phosphatase SHP-1. Given the association of CD22 with the IgR complex, the recruitment of this
phosphatase may function as a negative regulatory signal. In keeping with this hypothesis,
CD22-null mice have evidence of immune disregulation including hyper- or hyporesponsiveness
of B-cell responses following IgR-dependent triggering, along with increased levels of serum
autoantibody and expanded numbers of peritoneal B cells. The specific roles of its lectin function
and the ligands involved in these humoral immune processes remain unclear. However, the
presence of more severe immunological abnormalities in mice with a null mutation in ST6Gal-I
(see Chapters 16 and 33) indicates a clear role for α2 6 sialic-acid-containing structures in the
regulation of B-cell functions. Whether CD22-dependent functions are due to CD22 interaction
with sialylated ligands coexpressed on the CD22+ cells, neighboring cells, or a soluble
glycoprotein is not known.
Siglec-3 (CD33) (3, 9,49 50)
CD33 was initially identified as a marker of early myeloid precursors in 1983 and was cloned in
1988. It is the smallest of the Siglecs, having a two-domain extracellular portion and an
approximately 80-amino-acid cytoplasmic domain. Its mass by SDS-PAGE is about 70 kD,
depending on the particular cell expressing CD33, indicating N-linked carbohydrate content of
approximately 40%. The lectin function of CD33 was initially demonstrated on CD33-
expressing COS cells. Demonstration of its lectin function is absolutely dependent on
pretreatment of CD33-expressing cells with sialidase, as is the case with Siglec-5. Of interest, the
rosetting activity of sialidase-treated CD33+ COS cells can be readily demonstrated, but naturally
occurring CD33-positive cell lines such as HL60 and U937 cells will not rosette erythrocytes,
even after sialidase treatment. Forced differentiation of these cells with agents such as phorbol
esters, which induce monocytic features, yields adherent cells capable of rosetting erythrocytes
in a sialic-acid-dependent manner; but due to a lectin function that is not CD33 (based on
antibody blocking studies), this may be due to the newly described Siglec-5. A CD33-
immunoglobulin construct also bound to erythrocytes in a sialic-acid-specific manner, showing a
preference for α2 3 sialic acid residues (added to asialoerythrocytes by specific ST3 GalTs).
Although a specific binding affinity has not been determined, it appears to be the weakest of all
the Siglecs identified to date. A murine homolog of CD33 has been isolated that has a high
degree of sequence homology over the two immunoglobulin domains (61% amino acid identity),
yet with a cytoplasmic domain showing considerably less homology with the human isoform. In
contrast, the cytoplasmic domains of CD22 are reasonably well conserved between humans and
mice. Long and short cytoplasmic domains of murine CD33 were initially isolated, but the
longer isoform may be a splicing intermediate.
CD33 is expressed on immature myeloid and erythroid cells, maturing myeloid cells, and at low
levels on mature monocytes, granulocytes, and some dendritic cell populations. It is a reliable
marker of myeloid leukemias. It does appear on some biphenotypic lymphoblastic leukemias and
on T cells after long-term in vitro expansion. Monoclonal antibodies directed against CD33 have
no effect on the proliferation of myeloid cell lines. CD33 is rapidly phosphorylated in a number
of different cell lines as a downstream consequence of protein kinase C activation on serine
residues. Preliminary data indicate that phosphorylation down-regulates its lectin activity and,
likewise, that occupancy of the lectin site inhibits its ability to be phosphorylated. The role of
CD33 in myeloid cell biology remains undetermined at present.
Siglec 4a (MAG) and Siglec 4b (SMP) (2, 6, 10, 16, 24, 26,51 55)
MAG was initially identified in 1972 as a glycoprotein in central and peripheral nervous system
myelin and was cloned in 1987. MAG was known to bind structures expressed on the surface of
neurons, although its specific ligands were not known. SMP (Schwann cell myelin protein)
cloned from quail Schwann cells in 1992 has 43% sequence homology with rat MAG. Although
SMP may approximate the avian homolog of MAG, it differs from MAG in some respects,
including some cell expression patterns, and thus may not have the same function(s) as MAG.
However, it will be considered here with MAG because of the overall similarities of the two
proteins.
Both proteins have five extracellular immunoglobulin domains, and detailed studies with MAG
support the existence of a cysteine disulfide bridge between the unpaired cysteine residues of the
first two domains. MAG contains five N-glycosylation sites and its molecular mass (100 kD, by
SDS-PAGE) is approximately one-third carbohydrate. Two isoforms of MAG (L-MAG and S-
MAG) differ in the presence of an early stop codon in the cytoplasmic domain, yielding domains
of 74- or 105-amino-acid residues in length. Several potential Ser/Thr phosphokinase substrate
sites are identifiable, and isolated L-MAG is a substrate for protein kinase C. L-MAG is
expressed during the phase of myelination during embryogenesis and juvenile development,
whereas S-MAG is found predominantly in adult animals.
MAG's lectin function was initially identified by the rosetting of selectively resialylated
erythrocytes to COS cells transfected to express MAG. These studies indicated that binding was
specific for α2 3 sialic acid residues. A preference for erythrocytes resialylated with ST3Gal-I
over ST3Gal-III (suggesting a preference for Siaα2 3Galβ1 3GalβNAc over Siaα2 3Galβ1
3(4) GlcNAc-containing structures) was initially demonstrated. This preference has been
confirmed with synthetic sialylated glycolipids (gangliosides). In examining the binding of
detached MAG+ or SMP+ COS cells to surfaces adsorbed with different ganglioside species,
binding is demonstrable only with structures containing a terminal α2 3 sialic acid linkage. GM1,
which contains just an internal α2 3 sialic acid residue, was not a ligand for SMP+ or MAG+
COS cells, but it was a ligand for Sn+ COS cells. These observations are consistent with the
proposal that MAG's natural ligands might include brain gangliosides.
Immunohistochemical studies showed that MAG is expressed on myelin-forming cells, including

oligodendrocytes in the CNS and Schwann cells in the PNS. In fully developed axons, it is
located in the periaxonal region but not in the myelinated areas. This localization suggested a
role in the process of myelination. Somewhat surprisingly, MAG-/- mice develop normally with a
normal myelin ultrastructure. However, these mice have an inability to maintain the normal
myelination of axons throughout adulthood, suggesting a role in the preservation rather than
development of myelin integrity. Alternatively, MAG may also function as a regulator of neurite
outgrowth. Myelin contains substances that inhibit neurite outgrowth and repair after injury, and
both soluble MAG and MAG expressed on CHO cells can function in vitro to inhibit neurite
outgrowth from adult dorsal root ganglion neurons. In contrast, MAG promotes neurite
outgrowth from newborn dorsal root ganglion neurons. In contrast to MAG's distribution, SMP
is found on myelinating and nonmyelinating cells as well, which suggests that SMP may not be
the functional avian homolog of MAG. Potential functional roles of SMP have yet to be
described.
Given the abundance of gangliosides in myelin, these studies certainly imply that they may
function as natural ligands for these two lectins. However, other lines of evidence suggest that
glycoproteins are involved as well. The binding of MAG to some populations of neurites is
trypsin-sensitive. A soluble form of MAG isolated from rat brain was able to bind to collagen,
although binding was not seen with liposomally incorporated MAG. Recent data further indicate
the complexity of MAG-dependent interactions and the challenge of identifying natural ligands.
As expected from the mutational data on sialoadhesin, the sialic-acid-binding function of a three-
domain MAG-Rg construct was eliminated by mutating Arg-118 (which corresponds to the
essential Arg-97 in sialoadhesin) to alanine. However, full-length MAG, with the same mutation,
when expressed on COS cells retained its ability to inhibit neurite outgrowth. Furthermore,
neurite outgrowth was inhibited by a five-domain, but not three-domain, MAG-Rg construct.
These data imply a role of the two membrane-proximal extracellular domains in neurite growth
regulation and, furthermore, that its sialic acid lectin property is probably not essential in these in
vitro assays.
Siglec-5 (11, 56)
Sequence homology searches of cDNA and expressed sequence tag databases identified three
protein sequences that have significant homologies with the Siglecs, termed OB-BP1, OB-BP2,
and CD33-L. OB-BP1 and OB-BP2 have been deposited in Genbank as "leptin-binding
proteins," but no published data are available as yet. CD33-L was cloned from human placenta,
and its sequence is almost identical to OB-BP1. Using human CD33 sequence as a probe, the
expressed sequence tag databases were searched and a clone was identified that on subsequent
isolation and sequencing proved to be 99.6% identical to OB-BP2. Termed Siglec-5, this protein
is expressed on human monocytes and granulocytes and is able to function as a sialic-acid-
dependent lectin when examined in the erythrocyte rosetting assay with COS cells transiently
transfected to express Siglec-5. Of note, Siglec-5 was able to bind equally well to synthetic
polymers containing either α2 3-linked or α2 6-linked sialic acid residues. The cytoplasmic
domain of Siglec-5 (89 amino acid residues) contains notable sequence homology with CD33
and OB-BP1, including an ITIM motif. ITIM motifs, when phosphorylated on tyrosine residues,
form a docking site for the phosphatases SHP-1 and SHIP. The sialic-acid-binding properties of
OB-BP1 or CD33-L have not been described, although the existence of a sialic-acid-binding
lectin in human placenta has been demonstrated. These observations suggest the existence of a
larger family of Siglecs.
I-type Lectins Binding to Nonsialylated Structures (57 61)
Within the broad grouping of I-type lectins are several proteins that are not Siglecs (Table 24.1).
Limited sequence homologies are found between these lectins and the Siglecs, although the
homologies are not as strong as those among the Siglecs themselves. The fourth domain of N-
CAM appears to interact with high-mannose-type N-glycans on other proteins, a binding
property that is apparently independent of the homotypic protein-based interactions of this
molecule. ICAM-1 can act as a receptor for the anionic oligosaccharide hyaluronan and possibly
for the heavily sialylated glycoprotein leukosialin (CD43), although the dependence of this
binding on leukosialin's sialylation has not been explored. In addition, indirect evidence suggests
that the single anionic complex-type N-glycan present on the small IgSF member P0 may be
involved in modulating some of its binding functions. At least two other IgSF members are
known to recognize heparin (PECAM and CD48). The biological significance of most of these
carbohydrate-binding phenomena remains relatively unexplored and mostly unexplained.
Future Directions
The activity of the I-type lectins and lectin functions, especially the Siglecs, have been
established largely by in vitro experimentation. However, it is highly probable that this lectin
feature is an essential property of the biological function(s) of these molecules. One of the
challenges in understanding these molecules will be to develop an understanding of biological
processes dependent on weak interactions occurring on the cell surface, with a lectin exposed not
to a unique high-affinity ligand, but to a mixed population of high- and low-affinity binding
molecules in what conceptually could be envisioned as a two-dimensional environment.
Biophysical parameters of such a system would likely include features of ligand proximity,
receptor and/or ligand clustering, and the simultaneous exposure of the lectin receptor to mixed
populations of receptor agonists and antagonists. The regulation of lectin function might occur at
several levels including expression of the lectin itself and the expression of glycosyltransferases
(either cis or trans) and other modifying enzymes (e.g., acetylation reactions). The complexity of
these systems may lend them to a level of fine tuning essential for certain biological processes.
Using the experimental methodologies that have been successful in identifying the I-type lectins
already characterized, additional I-type lectins will likely be discovered in the future.
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25. C-type Lectins
Primary contributions to this chapter were made by R.D. Cummings (University of Oklahoma at
Oklahoma City).
THE C-TYPE LECTINS REPRESENT A LARGE FAMILY of Ca++-dependent lectins that

share primary structural homology in their carbohydrate-recognition domains. This very large
family, which includes many endocytic receptors, many proteoglycans, and all known collectins
and selectins, is found throughout the animal kingdom. Most of the members of this family
differ, however, with respect to the types of carbohydrate structures that they recognize with
high affinity. The C-type lectins are involved in many immune-system functions, such as
inflammation and immunity to tumor and virally infected cells, whereas the collectins are
involved in innate immunity. This chapter emphasizes the diversity of the C-type lectin family
and presents the current state of knowledge about the overall structures of these proteins and the
breadth of their biological distribution and functions.
Historical Background and Discovery of C-type Lectins (1 4)
The first lectin identified in animals was the C-type lectin known as the hepatic
asialoglycoprotein receptor or the hepatic Gal/GalNAc receptor. It was identified by Ashwell and
Morell and their colleagues, who were examining the structure and function of the sialylated
serum glycoprotein ceruloplasmin. On the basis of observations that most serum glycoproteins
contained terminal sialic acid residues, these authors hypothesized that sialylation was essential
for the proper lifetime of glycoproteins in serum. In a key experiment, they enzymatically
desialylated a radioactively labeled glycoprotein and injected it into rabbits; they discovered that
the desialylated glycoprotein was removed much more rapidly from the circulation than the
parent glycoprotein. In addition, the penultimate galactose residues exposed upon desialylation
were critical for clearance, since treatment with galactose oxidase, which oxidizes the C-6
hydroxyl of galactose residues, prolonged the serum survival time. Resialylation of the
desialylated glycoproteins by a preparation of sialyltransferase and CMPNeuAc restored normal
circulatory lifetime. The asialoglycoproteins removed from the circulation were found to be
sequestered in the liver and principally in lysosomes.
Using radioactively labeled asialoglycoproteins, a specific Ca++-dependent receptor for

asialoglycoproteins was identified in hepatocyte plasma membrane fractions, and the hepatic
receptor was purified by affinity chromatography on columns of asialo-orosomucoid-Sepharose.
The receptor consisted of a major subunit of approximately 48 kD and a minor subunit of about
40 kD. The purified rabbit hepatocyte receptor was able to agglutinate desialylated human and
rabbit erythrocytes and to induce mitogenesis in desialylated peripheral lymphocytes. Notably,
this was the first demonstration that an animal lectin could have such profound effects on cellular
metabolism. The rabbit hepatic lectin was inhibited in its binding by both GalNAc and galactose,
with the former being more potent than the latter, and it was able to bind to glycoproteins
containing either nonreducing terminal GalNAc or galactose. A similar lectin was identified in
rats and termed the rat hepatic lectin. It also contained an unusual trimeric structure composed of
two subunits, which is termed rat hepatic lectin R2/3. A lectin was also identified in chicken
hepatocytes, but the avian lectin was different in that it only recognized glycoproteins containing
terminal GlcNAc residues. Thus, only glycoproteins that were enzymatically both desialylated
and degalactosylated were rapidly cleared by the avian liver.
Definition of the C-type Lectins and Sequence Motifs (5 7)
The sequencing of several mammalian Ca++-dependent lectins, notably the chicken hepatic
lectin, the rat hepatic lectin, and the hepatic mannose receptor, led to the finding that all of these
proteins shared a common structural motif. The sequence homology was noted in the CRD, and
proteins with this motif were classified as members of the C-type (Ca++-dependent) lectin family
by Drickamer. The domain of the C-type lectins responsible for binding carbohydrate was
originally identified by analyzing proteolytic fragments, and it was found that the CRD is
contained in these fragments. The CRD of the C-type lectin family is an approximately 115 130-
amino-acid segment containing a recognizable consensus sequence shown in Figure 25.1. Of
particular significance is the presence and spacing of cysteine residues that are disulfide-bonded,
as shown, to contribute to the folded lectin domain.
Figure 25.1. (A) Conserved primary structures within the CRD in C-type lectins. Invariant
cysteine residues are underlined and highly conserved residues are indicated in the parentheses.
The amino acid spacing between residues is indicated by Xs within parentheses. (B) Disulfide
bonding within the C-type lectin CRD; (C) comparison of the rat MBP C and murine L-selectin
within the CRD. Identical residues between the two sequences are boxed, and those residues
within the canonical C-type lectin CRD are in boldfaced letters.
Different Subfamilies of C-type Lectins (7)

The discovery of the CRD for the C-type lectins opened the way to characterize other related
proteins that also displayed Ca++-dependent binding to carbohydrate ligands (Table 25.1 and
Figure 25.2). To date, more than 20 different proteins containing a C-type lectin CRD have been
identified in humans and corresponding homologs have also been found in many other higher
animals. In addition, C-type lectins occur in many other vertebrates, including reptiles, and in
invertebrates. From the genomic sequencing of Caenorhabditis elegans, approximately 150 C-
type lectin genes have been identified. These many C-type lectins in higher animals are classified
into subfamilies, based on their function or unique localization.
Table 25.1. Different subfamilies of C-type lectins
Endocytic receptors
• rat kupffer cell receptor
• human macrophage mannose receptor
• rat asialoglycoprotein receptor R2/3 (hepatic lectin 2/3)
• human asialoglycoprotein receptor (hepatic lectin H1) chicken hepatic lectin
• dendritic cell and thymic epithelial cells DEC-205 (homolog of macrophage mannose-
binding receptor)
• murine macrophage asialoglycoprotein-binding protein (macrophage Gal/GalNAc-
specific lectin [MMGL])
• bovine 180-kD secretory phospholipase A2 receptor
• DEC 205 receptor
Collectins
• bovine collectin-43
• bovine conglutinin
• rat mannose-binding proteins A and C
• human mannose-binding protein
• human pulmonary surfactant-associated protein A (SP-A)
• human pulmonary surfactant-associated protein D (SP-D)
• human tetranectin (plasminogen-kringle-4-binding protein)
Selectins
• L-selectin
• E-selectin
• P-selectin
Lymphocyte lectins
• human early activation antigen (CD69)
• human B-cell differentation antigen (CD72)
• murine T-cell surface glycoprotein YE1/48 (LY49A,B,D,E,F,G,H antigen)
• human NK antigen (CD94)
• human NKG2A,B,C,D,E
• human mast-cell-function-associated antigen (MCFA)
• activation-induced C-type lectin (AICL)
• human eosinophil granule major basic protein
• human low-affinity immunoglobulin epsilon Fc receptor (CD23)
• P47 pr LSLCL (lymphocytic secreted long form of C-type lectin)
Proteoglycans
• human versican core protein (large fibroblast proteoglycan-CS proteoglycan core protein-
2-glial hyaluronate-binding protein)
• human aggregan core protein (cartilage-specific proteoglycan core protein-CSPCP-CS
proteoglycan core protein-1)
• rat brevican core protein (brain-enriched HA-binding protein)
• rat neurocan core protein (245-kD early postnatal core glycoprotein)
Others
• human integral membrane protein DGCR2/IDD
• human lithostathine 1 α-precursor (pancreatic stone protein, PSP)
• human polycystin
• human endothelial cell scavenger receptor
• human pancreatitis-associated protein 1 (PAP or HIP)
• human pancreatic β-cell growth factor (INGAP)
Invertebrate lectins
• human clotting factor (hemolymph of horseshoe crab Tachypleus tridentatus)
• lectin BRA-2 (coelomic fluid of acorn barnacle Megabalanus rosa)
• newt lectin (oviduct of Iberian ribbed newt Pleurideles waltii)
• inducible flesh fly lectin (Sarcophaga peregrina)
• tunicate lectin (Polyandrocarpa misakiensis)
• integral spicule matrix lectin in sea urchin (Strongylocentrotus purpuratus)
• sea urchin echinoidin (Anthocidaris crassispina)
• cockroach lectin (hemolymph of Periplaneta americana)
• antifreeze protein (AFP) from the sea raven (Hemitripterus americanus)
Viral lectins
• hepatic lectin homolog in fowl poxvirus
• gp22 24 in vaccinia virus
Snakes and venoms

• alboaggregin A subunit 1 (white-lipped pit viper Trimeresurus albolabris)
• phospholipase A2 inhibitor subunit B (Trimeresurus flavoviridis)
• echicetin α-subunit (saw-scaled viper Echis carinatus)
• coagulation factor IX/factor X-binding protein A (IX/X-BP) (Trimeresurus flavoviridis)
• galactose-specific lectin (Crotalus atrox)
• botrocetin, α chain (platelet coagglutinin) (Bothrops jararaca)
Figure 25.2. Organization of the CRD in different members of the C-type lectin family.
(Adapted, with permission, from [7] Drickamer and Taylor 1993 [© Annual Reviews].)
The hepatic lectins discussed above represent a class of C-type lectins capable of mediating
endocytosis of bound ligands (Table 25.1 and Figure 25.2). The endocytic pathway involves
lectin recognition of ligands at the cell surface, internalization via coated pits, and delivery of the
complex to endosomal compartments where the low pH induces dissociation of ligand and lectin.
The lectins recycle to the cell surface and repeat the process. The mammalian asialoglycoprotein
receptor is trimeric and has two different polypeptides encoded by different genes. Although one
polypeptide is usually dominant, both polypeptides are essential for functional assembly. In
contrast, the chicken hepatic lectin is a trimer containing a single polypeptide species. This lectin
occurs in trimeric and hexameric forms that promote high affinity for specific glycoconjugate
ligands. Clustering of the CRDs may determine both the specificity and affinity of the lectins,
since each individual CRD can act independently to bind sugar. The asialoglycoprotein receptor
and most endocytic receptors are type II transmembrane proteins, whereas the macrophage
mannose receptor is a type I protein. The hepatic lectins have a single C-type CRD, and the
macrophage mannose receptor has eight CRDs and is the only known protein with multiple
independent CRDs in a single polypeptide. The adjacent CRDs in the mannose receptor may
help to direct its binding to specific multivalent, mannose-containing glycans. The macrophage
mannose receptor can internalize lysosomal enzymes containing high-mannose-type N-glycans,
as well as participate in the phagocytosis of several pathogens, such as yeast, Pneumocystis
carinii, and Leishmania.
Although the function of the hepatic asialoglycoprotein was predicted to contribute to

homeostasis and maintenance of serum glycoprotein levels, mice containing a null mutation in
the MHL-2 subunit were viable and fertile without obvious phenotypic abnormalities and
without excessive accumulation of endogenous serum-containing glycoproteins. When
challenged with exogenous asialoglycoproteins, however, the mutant mice are defective in
hepatic uptake and clearance. These results raise the possibilities that the hepatic receptors may
be required to regulate serum glycoprotein levels in periods of induced stress, which is known to
cause elevation of serum glycoprotein levels, and/or to be involved in specific interactions with
glycoconjugates from pathogenic organisms. For example, the hepatic asialoglycoprotein
receptor promotes endocytosis-dependent entry of hepatitis B virus particles into liver cells. It is
interesting that autoimmune hepatitis is associated with autoantibodies to the human hepatic
ASGPR, and there is evidence that hepatitis virus invasion can augment this response.
It is also possible that other domains of the C-type lectins outside the canonical CRD may also
have receptor activity. For example, the cysteine-rich domain of the "mannose" receptor
mediates GalNAc-4-SO4 binding and may be involved in internalization and removal from the
serum of circulating pituitary hormones containing this determinant. This ability to bind multiple
ligands is also exemplified by the Ca++-independent Man-6-P receptor (a P-type lectin), which
binds Man-6-P-containing ligands via interactions of a few of its 15 repeating domains and binds
IGF-II and the urokinase-type plasminogen activator receptor through interactions with other
domains.
The Collectins (14 17)
The collectins are a class of C-type lectins containing a collagen-like domain that usually
assemble in large oligomeric complexes containing 9 27 subunits (Table 25.1 and Figure 25.3).
Some collectins, such as mannose-binding protein A and human surfactant SP-A, organize into a
"bouquet" form, whereas others, such as bovine conglutinin and surfactant SP-D, organize into a
"cruciform" shape. One of the best studied serum collectins is MBP. Bovine collectin-43 (CL-
43) is structurally one of the simplest collectins and consists of only three polypeptides, each of
which contains a terminal C-type lectin domain. Rats possess two serum MBPs designated A and
C, sometimes called mannan-binding proteins. Humans appear to have only a single MBP
corresponding to the rat MBP-A.
Figure 25.3. Subunit arrangements in the collectin family of C-type lectins. (Adapted, with
permission, from [7] Drickamer and Taylor 1993 [© Annual Reviews].)
The collectins contribute to "innate" immunity and act before induction of an antibody-mediated
response. Collectins can stimulate in vitro phagocytosis by recognizing surface glycans on
pathogens, chemotaxis, and production of reactive oxygen, and regulate cytokine release by
immune cells. Lung surfactant lipids have the ability to suppress a number of immune cell
functions such as proliferation, and this is augmented by SP-A.
MBP forms a trimeric helical structure via interactions in the collagenous tails that is stabilized
by disulfide bonds in the cysteine-rich amino-terminal region. These trimers can aggregate to
generate three or six trimers in a type of "bouquet" organization. Each CRD in the trimer is
separated by about 53 Å, which is critical to the function of the lectin; this is because each
individual CRD has a relatively low affinity and relatively low specificity. The spacing between
CRDs provides an interesting degree of regulation, since the far spacing lessens potential
interactions of the MBL with host glycoconjugates and enhances the potential interactions with
extended mannan-containing glycoconjugates, especially those on bacteria, yeast, and parasites.
Binding of MBL and other collectins to a target cell can directly activate complement via the
classical pathway. For human MBP, this activation appears to result from a novel type of C1s-
like serine protease that complexes with MBP and initiates the complement cascade in vivo. It is
interesting that some individuals have mutations in the Gly-X-Y repeat, encoded within exon 1
of the MBP gene, and display MBP deficiency syndrome. Mutations within exon 1, which are
highly variable among human populations, inhibit subunit interactions, leading to increased risk
of the individuals to microbial infections.
An interesting member of the collectin family is tetranectin, which is a plasminogen-binding

protein that recognizes the kringle 4 region in plasminogen. Tetranectin may be involved in the
packaging of molecules destined for exocytosis. Human tetranectin is a homotrimer forming a
triple α-helical coiled coil, and each monomer consists of a CRD connected to a long α-helix.
Tetranectin could be classified in a distinct subfamily of the C-type lectin superfamily, but the
protein has the most structural similarity to collectins.
The Selectins (18 20)
The selectins are a class of type I membrane-bound C-type lectins expressed in vascular
endothelium and in circulating leukocytes. They are involved in selective cell adhesion. So far,
only three selectins have been identified: L-selectin, expressed on all leukocytes; E-selectin,
expressed by cytokine-activated endothelial cells; and P-selectin, expressed constitutively in
granules of platelets and Weibel-Palade bodies of endothelial cells and on the surface of
activated platelets or cells. For a more complete discussion of selectins, see Chapter 26. The
selectins are involved in leukocyte-leukocyte interactions (L-selectin) and leukocyte-endothelial
cell interactions (L-, E-, and P-selectin).
Each selectin has a C-type CRD at the amino terminus, followed by a consensus EGF-like
domain and a number of complement regulatory domain repeats. The proteins have a single
transmembrane domain and a large cytoplasmic domain. Each domain of the protein is probably
important for functional interactions with cells under vascular fluid flow. All of the selectins
display modest specificity and affinity for the sialylated, fucosylated structure known as the
sialyl Lewis X antigen NeuAcα2 3Galβ1 4(Fucα1 3)GlcNAcβ-1-R. However, each of the
selectins has much higher-affinity interactions with specific macromolecular ligands, and in most
cases, these ligands are mucins containing sialylated, fucosylated O-glycans. In addition, ligands
for L-selectin (GlyCAM-1) contain Gal-6-sulfated and/or GlcNAc-6-sulfated sialyl Lewis X
antigens, and the major ligand for P-selectin, termed P-selectin glycoprotein ligand (PSGL-1),
has sulfated tyrosine residues adjacent to O-glycans containing sialyl Lewis X antigen.
The Lymphocyte Lectins (21)

A growing list of proteins containing the C-type CRD has been identified on human and rodent
lymphocytes (Table 25.1). For the most part, the functions of these proteins are poorly
understood and their ability to bind carbohydrate has not been demonstrated. Most of the
lymphocyte C-type lectins are type II membrane proteins with a single C-type CRD. The
immunoglobulin-like receptors and C-type lectin receptors are two families of MHC class-I-
specific receptors found on NK cells. In mice, the C-type lectins on NK cells are represented by
the Ly49 family of receptors. Human NK cells express a distantly related molecule designated
CD94. In human NK cells, CD94 forms MHC class-I-specific disulfide-linked heterodimers with
NKG2 family members, which also contain a C-type lectin CRD.
The NK gene complex occurs on mouse chromosome 6 and its human homolog occurs on
chromosome 12. These complexes encode type II transmembrane proteins with a C-type lectin
domain that can trigger or inhibit target cell lysis by NK cells (e.g., NKR-P1, Ly49, NKG2, and
CD94) or can activate various hematopoietic cells (CD69). CD69 is an early activation antigen
expressed on the surfaces of activated T cells, B cells, NK cells, neutrophils, eosinophils,
epidermal Langerhans cells, and platelets and can be detected within hours following mitogenic
stimulation. The B-cell differentiation antigen (CD72) is expressed on human pre-B cells and B
cells, but it is absent from terminally differentiated plasma cells and is associated with CD5.
Another C-type lectin on lymphocytes is termed the activation-induced C-type lectin (AICL),
whose expression is rapidly increased following lymphocyte stimulation. AICL is encoded by a
gene that also closely maps to the human NK gene complex and proximal to the CD69 gene. The
mast-cell-function-associated antigen (MCFA) is a C-type lectin, whose clustering by antigen
inhibits mast cell secretory responses stimulated via Fc epsilon RI. The lymphocyte low-affinity
IgE Fc receptor (CD23) binds in a Ca++-dependent fashion via its CRD to IgE, but this binding
may be carbohydrate-independent, again indicating the potential of CRDs to function in protein-
protein interactions rather than carbohydrate-protein interactions.
The Proteoglycans (22 23)
The C-type CRD has also been identified in several classic proteoglycans, recently termed
lecticans, that lack transmembrane domains and occur in the ECM. These include aggregan,
brevican, versican, and neurocan (Table 25.1 and Figure 25.2). Like the selectins, each of these
core proteins contains a C-type CRD, an EGF-like domain, and a complement-regulatory domain
repeat, but their ordering is different and they are located in the carboxyl terminus of the protein.
A large region containing attachment sites for chondroitin sulfate and keratan sulfate is proximal
to the lectin domain. For a more complete discussion of the proteoglycans, see Chapter 11. The
exact functions of the lectin domain in these proteins are not known. The CRD of aggregan binds
to supports containing a high density of sugar, but it displays a broad specificity, with the best
ligands being fucose and galactose. Some recent studies suggest that the CRD may not be
directly important for carbohydrate binding in these proteins, but it may be a structural domain
promoting protein-protein interactions with other ECM molecules. The C-type lectin domain of
versican can bind to tenascin-R, an ECM protein specifically expressed in the nervous system,
presumably by carbohydrate-protein interaction. However, there is also evidence that the C-type
lectin domain of all the lecticans, including brevican, can bind tenascin-R in a carbohydrate-
independent fashion. Thus, the C-type CRD may be a structural feature of proteins capable of
promoting specific protein-protein interactions.
Other Types of C-type Lectins (24 25)
A number of proteins with C-type CRDs have been identified in the pancreas and kidney, but the
importance of the CRD and carbohydrate binding to their functions is unclear (Table 25.1).
Autosomal dominant polycystic kidney disease (ADPKD) is a commonly occurring hereditary
disease accounting for approximately 10% of end-stage renal disease. PKD1, one of two recently
isolated ADPKD gene products, has been implicated in cell-cell and cell-matrix interactions. The
PKD1 gene encodes a novel protein named polycystin that has multiple cell-recognition domains
including a single C-type CRD at its amino-terminal region. The function of PKD1 and effect of
mutations on its possible activity are unclear. Interestingly, some C-type lectins can be extremely
small, as exhibited by HIP and PSP. They are virtually free C-type CRDs that are preceded by a
signal sequence.
Lower vertebrates, invertebrates, and some viruses also synthesize proteins with a C-type CRD,
and some of these proteins have been shown to bind carbohydrate. For example, the galactose-
specific lectin from the snake Crotalus atrox binds a variety of galactose-containing glycolipids
in a Ca++-dependent fashion. A number of related venom proteins are known to inhibit platelet
function and/or the coagulation cascade. Alboaggregin A from Trimeresurus albolabris (the
white-lipped pit viper) contains subunits 1 4, and subunit 1 contains a single C-type CRD in its
amino-terminal domain. The protein binds to the platelet GPIb/IX receptor and stimulates
agglutination, but the potential role of carbohydrate recognition in this process is presently
unknown.
Tertiary/Quaternary Structures of C-type Lectins (26 30)
The crystal structure of the trimeric rat MBP-A is shown in Figure 25.4. Each CRD has a
continuous segment of polypeptide folded into a series of four loops stabilized by two disulfide
bonds and each contains two binding sites for Ca++. This interesting structure identified the role
of the C-type CRDs, since many of the acidic residues help to coordinate Ca++, and the common
glycine and proline residues are found at critical turns. In addition, the carboxyl and amino
termini of the CRD are near each other. This helps explain how the C-type CRD can be similar
in both type I transmembrane proteins, such as selectins, and in type II transmembrane proteins,
such as the hepatic asialoglycoprotein receptor. The CRD binds to sugar ligands in a shallow
surface pocket, which classifies the C-type CRD as a type-2 carbohydrate-binding domain, as
opposed to a deep-pocket type-1-binding domain, which are typically found in monosaccharide
transporters and bacterial toxins such as cholera toxin (see Chapter 4). Interaction of the CRD
with mannose in the rat serum MBP occurs through equatorial hydroxyl groups 3 and 4 of
terminal mannose coordinated to Ca++. This complex is stabilized by H bonds to each hydroxyl
group. The loss of affinity of C-type CRD in the endocytotic receptors upon lowering the pH is
probably due to loss of coordinated Ca++.
Figure 25.4. Crystal structure of a trimeric rat mannose-binding protein A (MBP-A) complexed
with mannose at 1.8 Å resolution (see reference 27). Mannose is indicated in the stick figure and
Ca++ and Cl are indicated by the green and blue balls, respectively. Note that Ca++ is closely
coordinated with the bound sugar.
Future Directions
The discovery of the C-type lectins represents the exciting fulfillment of predictions made years
earlier by many investigators in the field that the carbohydrate moieties of soluble and
membrane-bound glycoconjugates would be important in cell-cell and cell-matrix and other such
cellular interactions. Future studies are likely to concentrate on identifying more C-type lectins
and defining their roles by genetic mutations in animal model systems and searching for more
naturally occurring mutations affecting their expression and localization. In addition, the
multitude of C-type lectins in animal cells, with their vast differences in carbohydrate-binding
specificity for macromolecular glycoconjugates, suggests that many carbohydrate structures in
the vast animal repertoire may have specific binding partners. This notion is becoming more
compelling as a host of other carbohydrate-binding proteins in addition to the C-type family have
also been identified in animals.
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26. Selectins
Primary contributions to this chapter were made by R.D. Cummings (University of Oklahoma
Health Sciences Center) and J.B. Lowe (HHMI, University of Michigan Medical School).
THIS CHAPTER SUMMARIZES THE CURRENT KNOWLEDGE concerning the structure,

function, and expression of the family of C-type lectins termed selectins and the corresponding
glycan structures that decorate the ligands for these molecules and are required for ligand
activity. The three members of this family, termed L-selectin, E-selectin, and P-selectin, have
been given the cluster designations CD62L, CD62E, and CD62P. These molecules share overall
structure and primary sequences and exhibit similarities in the cognate glycans that contribute to
their adhesion function, and each participates in mediating interactions among blood cells and
endothelial cells. In most cases, these interactions are characterized by relatively rapid on/off
kinetics of binding and are most relevant in the context of vascular shear flow.
Background (1 13)
The discovery of the selectins arose from efforts to define the molecules and mechanisms that
facilitate the recirculation of lymphoid cells from the intravascular compartment to the secondary
lymphoid organs, including lymph nodes and Peyer's patches, from which these cells then return
to the circulation through the lymphatic system. This recirculation process provides lymphocytes
the opportunity to encounter foreign antigens displayed by antigen-presenting cells within
secondary lymphoid organs. Early studies indicated that blood lymphocytes enter lymph nodes in
postcapillary venules within these organs. These postcapillary venules are lined with specialized
endothelium and are termed high endothelial venules, or HEVs. These endothelial cells are rather
cuboidal in shape and their surfaces are decorated with certain glycoproteins required for
adhesion of lymphocytes to the HEVs, leading to subsequent transmigration.
Early studies of this adhesion process involved the generation of a monoclonal antibody called
MEL-14 that blocked both in vitro adhesion of lymphocytes to HEVs and in vivo homing of
lymphocytes to peripheral lymph nodes. Contemporaneous studies provided indirect evidence
that oligosaccharide components of the HEV glycocalyx contributed to lymphocyte-HEV
adhesive events. For instance, formaldehyde fixation of HEVs exposed on frozen sections did
not disrupt lymphocyte HEV adhesion (the Stamper-Woodruff assay), implying that the fixation-
resistant glycan components of the HEV surface are responsible for adhesion. Furthermore,
assay of lymphocyte-HEV interactions using this approach showed that various specific mono-
and polysaccharides could inhibit lymphocyte-HEV interactions. The most potent
polysaccharides were fucoidan (a sulfated high-molecular-weight polysaccharide from brown
seaweed containing α2- and α3-linked fucose residues) and PPME (a yeast-derived mannan-
based polysaccharide containing an abundance of Man-6-P groups). Both inhibited lymphocyte
adhesion at concentrations in the low nanomolar range. Moreover, treatment of lymphocytes
with PPME inhibited their adhesion to HEVs, whereas pretreatment of the HEV did not inhibit
adhesion. These observations and others showing direct binding of PPME to lymphocytes
implied that PPME was recognized by a carbohydrate-binding protein or lectin expressed on the
surface of lymphocytes and that it was involved in adhesion to HEVs. Subsequent studies
demonstrated that PPME interacts with the lymphocyte cell surface protein recognized by the
MEL-14 antibody. These observations implied that the MEL-14 antigen corresponded to a
carbohydrate-binding protein, now known as L-selectin (CD62L), that mediates lymphocyte
adhesion to HEV through recognition of specific carbohydrate-based ligands on this
endothelium.
This hypothesis was further substantiated by studies demonstrating a requirement for sialic acids
in this interaction. Specifically, sialidase treatment of paraformaldehyde-fixed HEVs or
intravascular injection of bacterial sialidases yielded, respectively, decreased lymphocyte
adhesion to HEV in vitro and diminished short-term homing of lymphocytes to peripheral lymph
nodes in vivo. These observations indicated that sialic acid is an important contributor to the
carbohydrate-based molecules on the HEVs that mediated lymphocyte trafficking.
Meanwhile, the molecule now known as E-selectin (CD62E) was identified as endothelial
leukocyte adhesion molecule I (ELAM-1). This arose from studies that sought to identify
leukocyte adhesion molecules expressed by vascular endothelium in the context of inflammation.
Monoclonal antibodies generated against endothelial cell surface molecules induced following
treatment with IL1-β or TNF-α were screened for their ability to block adhesion of neutrophils to
this activated endothelium. These antibodies were later used to clone cDNAs encoding E-
selectin.
In parallel with this work, P-selectin was discovered as an antigen expressed on the surfaces of
activated platelets; it was originally termed granule membrane protein 140 (GMP-140) or
platelet activation-dependent granule to external membrane (PADGEM) protein, because of its
localization to platelet α granules and its release following platelet activation. These terms are no
longer in general use. However, P-selectin (CD62P) is also expressed in megakaryocytes, and in
the Weibel-Palade bodies of vascular endothelial cells. Within minutes following activation of
either platelets or endothelial cells, P-selectin is expressed on the cell surface as a result of fusion
of the intracellular storage compartments with the plasma membrane. Storage granule
mobilization and fusion are observed following treatment of endothelial cells with histamine,
protein kinase C activators, fragments of complement, and thrombin, for example.
In 1989, the nearly simultaneous cloning of these three proteins resulted in the realization that
they belonged to a single unique family. The presence of the amino-terminal C-type lectin
domain in all three cDNAs and the previous evidence for carbohydrate involvement in L-selectin
binding triggered work in many laboratories that eventually led to the recognition of their
carbohydrate ligands and their functions in vivo.
L-Selectin Structure and Expression (3, 4, 8, 13)

Molecular cloning studies subsequently isolated cDNAs corresponding to the MEL-14 antigen
and its human homolog. This protein is termed L-selectin and has been previously referred to as
LEC-CAM, Ly-22, Leu8, TQ1, DREG-56, and LAM-1 by different groups, although these terms
are no longer in general use. The polypeptide sequence derived from the cloned cDNAs
predicted a molecule corresponding to a type I transmembrane glycoprotein with a major amino-
terminal extracellular segment (Figure. 26.1). Analysis of this polypeptide sequence defined a
series of discrete domains, beginning with an amino-terminal signal peptide sequence. The
amino-terminal segment of the mature protein shows primary sequence similarity to other
members of the C-type (Ca++-dependent) lectin family (see Chapter 25). The amino-terminal 116
residues of mature L-selectin protein share approximately 25 30% amino acid sequence identity
with other C-type lectins, such as the CRDs of the mammalian hepatocyte asialoglycoprotein
receptor, the Kupffer cell fucose-binding protein, and several mannose-binding proteins. Four
cysteine residues conserved among the C-type lectins are similarly maintained in L-selectin.
Mouse and human L-selectin carbohydrate recognition domains share 83% amino acid sequence
identity, which is consistent with observations implying that mouse and human L-selectin share
ligand recognition properties.
Figure 26.1. Domain structures of L-selectin, E-selectin, and P-selectin.
The CRD in L-selectin is followed by a domain with primary sequence similarity to the EGF
precursor. The EGF-like domain corresponds to a protein sequence motif found in many
proteins, including the Notch protein in Drosophila, coagulation factor X, and the LDL receptor.
The carboxyl terminus in the EGF-like domain is adjacent to a tandemly repeated pair of 62-
amino-acid-long domains. Each of these domains shares primary sequence similarity to a
sequence motif in decay accelerating factor, complement receptor I, and the receptor for the
complement component C3d, among others. This motif is called the CR repeat (complement-
regulatory or consensus repeat) because of its frequent occurrence in proteins that regulate the
complement pathway. The CRD, EGF-like, and CR domains of L-selectin are in turn attached to
a transmembrane segment linked in turn to a short cytoplasmic domain. Analyses of the genomic
structures of the human and mouse L-selectin loci indicate that the signal peptide sequence, the
CRD, the EGF domain, each CR repeat, and the transmembrane segment are encoded by discrete
exons. The cytosolic domain is encoded by two exons. The human L-selectin locus resides on
chromosome 1 between bands q22 and q25, at a substantial distance from the genes encoding
several other CR repeat-containing proteins that localize to band 1q32. As noted below, the E-
selectin and P-selectin loci are organized similarly to the L-selectin locus and are closely linked
to this locus.
The functional contributions of each domain within L-selectin have been examined using
antibody blocking approaches and recombinant L-selectin chimeras. In general, these studies
confirm that the CRD in L-selectin is directly involved in adhesion interactions with glycan-
based ligands, including especially amino acids within the carboxy-terminal portion of the CRD.
Such studies also indicate that the EGF-like domain plays an important part in helping to
maintain the CRD in its proper configuration for ligand recognition. The two CR repeat
segments within L-selectin are required to maintain optimal recognition of its ligands.
L-selectin is expressed on blood monocytes, blood neutrophils, subsets of natural killer cells, and
T and B lymphocytes, including virtually all lymphocytes of the "naive" phenotype, both in
blood and in lymphoid organs. L-selectin is generally not expressed by lymphocytes that exhibit
the "memory" phenotype. L-selectin is also expressed in both early and mature hematopoietic
cells in the bone marrow, except that cells of the B-lymphocyte lineage seem to express this
molecule only near the latter stages of the B-lineage developmental program.
L-selectin is a glycoprotein that varies in size according to the type of leukocyte in which it is
produced. These observations imply that lineage-specific glycosylation processes regulate glycan
structures associated with L-selectin. L-selectin is shed from the surface of leukocytes when they
are activated, and this shedding involves proteolytic cleavage immediately adjacent to the outer
surface of the cell membrane, between residues 283 and 284 of the mature protein. L-selectin
shedding is catalyzed by one or more metalloproteinases (secretases), including the tumor
necrosis-α converting enzyme. There is evidence that calmodulin regulates the protease-
dependent shedding process. It has been proposed that L-selectin shedding serves to facilitate
release of adhesion of cells from the endothelium during the transmigration process, but
evidence also exists to the contrary, leaving this issue unresolved. The soluble form of L-selectin
ocurs in serum from normal and diseased individuals. The functional significance of the
circulating form of this molecule is not clear.
Adhesion mediated by L-selectin is characterized by interactions of apparently low affinity (0.1

mm range), exhibiting relatively rapid "on-rate" and "off-rate" characteristics. These adhesive
interactions operate predominantly under conditions of vascular shear flow. Similar
considerations apply to the cell adhesion processes that involve E-selectin and P-selectin,
although L-selectin exhibits an interesting "shear-dependent" cell adhesion. Selectin-dependent
cell adhesion in vivo is characterized by "rolling" of leukocytes along the surface of the
endothelial cell monolayer. This process is mediated by torque exerted on the leukocyte as a
consequence of a force differential between the more rapidly flowing stream near the center of
the vessel (and on the luminal surface of the rolling leukocyte) and the relatively slow flow near
the endothelial cell surface (and the adjacent abluminal side of the rolling leukocyte). These
forces, together with the relatively rapid on and off rates of selectin-selectin ligand adhesive
interactions occurring at the leukocyte-endothelial cell interface, cause the leukocyte to roll
along the endothelial lining of the vessel.
L-selectin-dependent lymphocyte rolling on HEVs and the corresponding process that leads to
lymphocyte homing in vivo require localization of L-selectin to the tips of the microvilli
characteristic of the surfaces of lymphocytes and other leukocytes. Microvillus localization
seems especially important for L-selectin-dependent tethering events involved in the initiation of
rolling. L-selectin-dependent rolling activity is also dependent on an association between the
cytosolic domain of L-selectin and the actin cytoskeleton, although this is apparently not related
to ligand recognition or to microvillus-sorting processes. The cytosolic domain of L-selectin is
also apparently involved in signal transduction processes, since cross-linking of L-selectin, or
ligation of L-selectin by multivalent ligand analogs, can lead to intracellular phosphorylation
events implicated in signal transduction processes in other systems. The relevance of these
observations to leukocyte trafficking and effector cell function remains to be discovered.
L-Selectin Ligands on High Endothelial Venules (13 31)
Glycan-based, HEV-borne molecules recognized by lymphocyte L-selectin have been

characterized using a variety of biochemical and cell biological approaches, including
purification of such ligand molecules using L-selectin immunoglobulin chimeras, followed by
characterization of their polypeptide components through molecular cloning, and analysis of
their glycan components through oligosaccharide structural analyses and monoclonal-antibody-
staining approaches. Early efforts demonstrated that the molecules elaborated by HEVs, and
precipitable by L-selectin-Ig chimeras, represented glycoproteins whose component glycan
moieties were most likely serine/threonine-linked and were modified by sulfate, fucose, and
sialic acid. Several apparently distinct, L-selectin-reactive polypeptides were seen in these
studies, implying the existence of multiple glycoprotein ligands for L-selectin.
Biochemical and molecular cloning approaches have now defined four distinct, HEV-borne
glycoproteins that are recognized by L-selectin. The first of these to be isolated, termed
GlyCAM-1, for glycosylation-dependent cell adhesion molecule, is a protein of 132 amino acids
containing numerous serine and threonine residues. The predicated molecular mass of this
protein, 14,154 daltons, is significantly less than the size observed with electrophoretic methods
with the glycoprotein (50 kD). GlyCAM-1 corresponds to the L-selectin-Ig-precipitable
molecule termed Sgp50. GlyCAM-1 is elaborated as a soluble molecule by HEVs in organ
cultures, and immunoelectron microscopy studies do not identify significant amounts of
GlyCAM-1 at the HEV cell surface. These observations implied that O-glycans contribute
substantially to the apparent molecular mass of GlyCAM-1, and further imply that the molecule
is a mucin-type glycoprotein. These observations are consistent with the fact that the sequence of
GlyCAM-1 does not predict a transmembrane segment, suggesting that any association between
GlyCAM-1 and the HEV cell surface must be mediated by virtue of a 21-residue amphipathic
helix at its carboxyl terminus, or more likely by interactions with one or more other membrane-
tethered molecules. GlyCAM-1 is detectable in serum and has been proposed to regulate the
adhesive and/or activation state of L-selectin-bearing leukocytes through interactions with L-
selectin on such cells.
A second L-selectin ligand expressed by HEV is CD34, which corresponds to the L-selectin-Ig-
precipitable molecule termed Sgp90. CD34 is a sialomucin containing several mucin-like
domains with numerous serine and threonine residues predicted to be heavily O-glycosylated.
Since CD34 is a transmembrane glycoprotein, this L-selectin ligand is positioned to contribute
substantially to the process of tethering and adhesion of circulating lymphocytes to lymph node
HEVs. However, CD34 is clearly not the sole contributor of L-selectin ligand activity in HEVs,
since CD34-null mice maintain virtually normal L-selectin-dependent lymphocyte homing
activity. CD34 is expressed elsewhere, including on vascular endothelial cells, but does not
function as an L-selectin ligand at these locations because it is not appropriately
posttranslationally modified.
A third molecule identified as an L-selectin ligand is termed MAdCAM-1, for mucosal addressin
cell adhesion molecule. MAdCAM-1 had been studied previously as the ligand for the integrin-
type lymphocyte homing receptor α4β7, a molecule responsible for the bulk of lymphocyte
homing to Peyer's patches. Biochemical experiments indicate that MAdCAM-1 expressed in
mesenteric lymph nodes is decorated with O-glycans that can support L-selectin-dependent
leukocyte adhesion. MAdCAM-1 is therefore predicted to maintain ligand activity for both L-
selectin and the α4β7 integrin.
A fourth HEV-expressed L-selectin ligand is the transmembrane sialomucin podocalyxin-like

protein or PCLP. Like CD34, PCLP is expressed on some vascular endothelia, but it is also
expressed on the foot processes of glomerular podocytes. HEV-derived PCLP interacts with
recombinant L-selectin-Ig chimera and supports L-selectin-dependent lymphocyte adhesion
under physiological conditions.
As noted above, early biochemical studies provided indirect evidence that L-selectin ligands
required sulfate and sialic acid moieties for proper function. For instance, broad-spectrum
sialidases inactivate L-selectin ligand activity in a variety of in vitro and in vivo assays, and the
use of a sialidase specific for α2 3-linked sialic acid residues (Newcastle disease virus
neuraminidase) inactivates ligand activity displayed by GlyCAM-1. These studies implied that
sialic acid in α2 3 linkage is required for L-selectin ligand activity. A requirement for sulfation
was inferred by studies in which chlorate was used to inhibit sulfation in organ cultures of
peripheral lymph nodes, from which GlyCAM-1 was then purified. The resulting nonsulfated
GlyCAM-1 was not bound by an L-selectin-Ig chimera. (Chlorate competitively blocks synthesis
of PAPS, the donor for all known sulfotransferase reactions in cells.)
Direct biochemical analysis of the O-glycans on GlyCAM-1 yielded a series of candidate O-

glycan "capping" structures implicated in L-selectin recognition. The proposed structures
correspond to Core-2-type O-glycans with the terminal structures 6-sulfosialyl Lex, 6 -sulfosialyl
Lex, and 6,6 -bis sulfosialyl Lex (Figure 26.2).
Figure 26.2. Candidate "capping" structures for the glycans of HEV-borne L-selectin ligands.
Evidence supporting the hypothesis that one or more of these molecules are indeed essential, or
at least important, contributors to L-selectin ligand activity comes from studies involving
inhibition of L-selectin-dependent adhesion using these structures or their variants. Supporting
evidence also derives from studies using monoclonal antibodies that recognize these structures
and mice lacking specific glycosyltransferases (and see below). In vitro studies that assess the
relative ability of these structures, and related ones, to block L-selectin-dependent recognition of
its ligand(s) have yielded conflicting results over the relative inhibitory "potencies" of 6 -
sulfosialyl Lex, 6-sulfosialyl Lex, and some structural analogs of the 6 ,6-sulfosialyl Lex moiety.
However, studies using monoclonal antibodies directed against these structures, and their
isomers, indicate that 6-sulfosialyl Lex is abundantly expressed on human peripheral node HEVs
and imply that the 6 -sulfosialyl Lex and the 6 ,6-sulfosialyl Lex structures are not necessarily
present in substantial amounts on HEVs.
Biochemical analyses and genetic studies have provided evidence for an ordered biosynthetic
scheme in the elaboration of these sulfated, sialylated, fucosylated structures (Figure 26.3). Some
of the glycosyltransferases participating in this biosynthesis have been defined, but most others
remain to be identified (and see further below). This scheme begins with Core 2 O-glycan branch
formation, followed by lactosamine unit addition. There is evidence that 6-O sulfation of the
subterminal GlcNAc residue precedes addition of β1 4-linked galactose. Addition of 6-O-linked
sulfate to the terminal galactose residue and addition of α2 3-linked sialic acid to this galactose
moiety are presumed to occur subsequently, but the relative order of their addition is not known.
It is generally accepted that the addition of fucose in α1 3 linkage represents a terminal step in
the pathway. The relative contributions of each of these three proposed structures to
physiological L-selectin-dependent adhesion events in HEVs are not yet clear. Since the HEV-
borne L-selectin ligands each displays multiple O-glycans, it is possible that each of these three
structures exists on all four ligands and may substitute the component glycans at multiple sites,
possibly at positions that are idiosyncratic to the individual structure, with the attendant
possibility for functional importance. Detailed structural analyses and corresponding functional
correlations are needed to clarify these issues.
Figure 26.3. Proposed biosynthetic scheme for "capping" structures shown in Figure 26.2. A
Core-2-type O-glycan (R), containing a terminal N-acetylglucosamine residue (I), serves as the
synthetic precursor for both capping structures. The pathway leading to synthesis of 6-sulfosialyl
Lex initiates with sulfation of the terminal GlcNAc moiety in I by a 6-O N-
acetylglucosaminylsulfotransferase (6 sulfoT), forming the synthetic intermediate II. This
intermediate is then modified sequentially by β1 4 galactosyltransferase (β1 4 GalT; forming
III), by an α2 3 sialyltransferase (ST3Gal; forming IV), and finally by the α1 3
fucosyltransferase FucT-VII. The pathway leading to synthesis of 6 -sulfosialyl Lex is proposed
to initiate with modification of I by β1 4 galactosyltransferase (β1 4 GalT; forming V),
followed by sialylation by an α2 3 sialyltransferase (ST3Gal; forming VI). This intermediate is
subsequently fucosylated by FucT-VII and sulfated by one or more 6-O
galactosylsulfotransferases (6 sulfoT). The order of action of these two enzymes is not known.
L-Selectin-dependent Leukocyte Adhesion to Microvascular

Endothelium (32 42)
L-selectin also has a role in adhesion of neutrophils, eosinophils, and monocytes to nonlymphoid
vascular endothelium (Figure 26.4). Much of the evidence for this process stems from in vitro
and in vivo observations showing that anti-L-selectin antibodies can block flow-dependent
rolling of leukocytes on activated vascular endothelium. At least three different mechanisms
have been proposed to account for this process. The first mechanism proposes that neutrophil-
borne L-selectin displays glycan ligands for E-selectin, which is expressed by the activated, pro-
adhesive endothelial cell. As discussed later in this chapter, E-selectin mediates adhesion of
neutrophils to activated vascular endothelium by recognition of sialylated, fucosylated
glycoconjugates expressed by neutrophils. L-selectin expressed by human neutrophils is
decorated with sialylated, fucosylated glycans within the family of such molecules implicated in
E-selectin ligand activity. These considerations imply that a component of the L-selectin-
dependent rolling processes observed in vitro, and in vivo, may be accounted for by
"presentation" of E-selectin ligands by neutrophil L-selectin.
Figure 26.4. Rolling adhesion of circulating leukocytes to activated endothelium via interactions
between selectins and their ligands. The example shows a neutrophil expressing PSGL-1 and L-
selectin on its microvilli and interacting by random contact with membrane P-selectin on
endothelial cells. E-selectin may also participate in these interactions by binding to PSGL-1 or
other ligands on these cells. Following interactions, cells are tethered and roll along the
endothelium. Leukocyte-leukocyte interactions involving L-selectin and PSGL-1 are depicted,
but L-selectin may also have other ligands expressed by the activated endothelium. Adherent
cells become activated by regionally presented chemokines or lipid autacoids. The activated
leukocytes express integrins that interact with Ig-like counterreceptors on endothelial cells,
strengthen the adhesion, and promote the emigration or extravasation (diapidesis) of cells into
the underlying tissues in response to chemotactic gradients.
Evidence also exists that the endothelial cell displays molecules that directly mediate L-selectin-
dependent adhesion. Biochemical analyses disclose that a substantial proportion of such L-
selectin ligand activity is Ca++dependent and is contributed by heparan sulfate proteoglycans.
These molecules may contribute to L-selectin-dependent adhesion of monocytes and neutrophils,
and possibly other leukocytes. The structure of such molecules and the regulation of their
expression are not yet well defined.
The third mechanism through which L-selectin can mediate adhesion of neutrophils and other
leukocytes to nonlymphoid vascular endothelium involves leukocyte-leukocyte interactions.
Observations using an in vitro flow chamber disclosed that adherent neutrophils can support L-
selectin-dependent rolling of other leukocytes. These initial studies implied that the adherent
leukocytes represent a stationary surface that displays L-selectin ligand activity. Such activity
was sialidase-sensitive, temporally transient, and operative in a unidirectional manner
characterized by adhesion between L-selectin on the rolling leukocyte and the ligand on the
adherent leukocyte. These observations implied that neutrophils display both L-selectin and L-
selectin ligands and would seem to predict L-selectin-dependent leukocyte aggregation in vivo.
However, L-selectin-dependent adhesion is only evident under certain conditions of shear flow,
accounting for the general absence of L-selectin-dependent leukocyte agglutination in vivo. The
apparently unidirectional nature of the L-selectin-dependent leukocyte-leukocyte rolling
phenomenon noted in the initial in vitro observations may be accounted for by the relative
absence of L-selectin on the adherent neutrophils, consequent to activation-dependent shedding
of L-selectin from those cells. However, it is now clear that L-selectin, displayed as a chimeric
molecule or by adherent cell monolayers, can initiate tethering and capture of incoming
leukocytes under shear, indicating that L-selectin-dependent tethering and capture of neutrophils
by other neutrophils can be a bidirectional process. An important component of neutrophil
ligands for L-selectin is the molecule PSGL-1 (P-selectin glycoprotein ligand-1), a mucin-type
sialylated, fucosylated glycoprotein that is discussed subsequently in this chapter. There is also
evidence that leukocytes express other mucin-type ligands for L-selectin that are distinct from
PSGL-1. Although it is clear that sialic acid residues are clearly important for such L-selectin
ligand activities, important contributions by other glycan components may also exist, but they
have not yet been defined.
Functions for L-selectin in vivo have been explored through the generation and analysis of mice
that are homozygous for null alleles at the L-selectin locus. These mice have a modest defect in
neutrophil trafficking through extravascular sites in the context of inflammation, which is
consistent with roles proposed for neutrophil L-selectin in adhesion to vascular endothelium and
to adherent neutrophils. These mice are also deficient in the homing of naive, L-selectin-positive
T lymphocytes to peripheral and mesenteric lymph nodes. The lymph nodes in these mice are
consequently small and relatively deficient in naive T lymphocytes. This deficit leads to an
inability of these mice to mount a delayed contact hypersensitivity response.
E-Selectin Structure and Regulation (5, 8, 13,43 44)
The sequence of the E-selectin cDNA predicts a type-1 transmembrane glycoprotein with a
CRD, EGF-like domain, and four CR repeat domains (see Figure 26.1). A recombinant fragment
of E-selectin composed of the CRD and EGF-like domain has been crystallized and its structure
solved. The E-selectin CRD superimposes substantially on the CRD of the mannose-binding
protein but differs from the latter molecule in that the E-selectin CRD coordinates a single
calcium ion, whereas the mannose-binding protein and other crystallized C-type lectins
coordinate two calcium ions. Furthermore, the segment of the E-selectin CRD that coordinates
calcium differs from that found in the mannose-binding protein. Mutagenesis of the E-selectin
CRD and the CRDs of L-selectin and P-selectin localizes shared and distinct amino acids
associated with binding of small carbohydrate ligands. These residues typically map within the
vicinity of the calcium-coordinating site of the protein. Positively charged residues invariant
among the three selectins have also been disclosed by such mutagenesis analyses and are
proposed to interact with sialic acid or fucose, which make essential contributions to selectin
ligand affinity, as discussed below.
As noted above for L-selectin, the E-selectin CRD, EGF-like, CR repeat, and transmembrane
domains are each encoded by separate exons, whereas the E-selectin cytosolic domain derives
from two exons. The E-selectin locus flanks the L-selectin locus on chromosome 1.
In primates and rodents, there is little, if any, constitutive expression of E-selectin by the
vascular endothelium. Cytokine-dependent transcriptional processes lead to an inducible
expression of E-selectin at the surface of the endothelium. Inducible transcription of the E-
selectin locus is mediated by TNF-α, IL1-β, and lipopolysaccharide, for example, and is
mediated at least in part through NF-κB-dependent events. Cytokine-dependent regulation of the
E-selectin locus yields E-selectin expression beginning approximately 2 hours after cytokine
treatment, with maximal expression at approximately 4 hours. In vitro, E-selectin expression
declines to initial levels within 12 hours, but it may be expressed chronically at sites of
inflammation in vivo. Decline of E-selectin expression is associated with decreased transcription
of the E-selectin locus, degradation of the E-selectin transcripts, and internalization and
degradation of E-selectin molecules by the endothelium. Soluble forms of E-selectin are
detectable in the serum, and increased levels of circulating E-selectin have been associated with
acute and chronic inflammatory states. The functional relevance of soluble E-selectin in the
blood is unknown. Acute and chronic inflammatory conditions associated with E-selectin
expression include, for example, sepsis, kidney inflammation, rheumatoid arthritis, and organ
transplantation.
E-Selectin Ligands (43 46)
E-selectin ligands are expressed by neutrophils, monocytes, eosinophils, memory-effector T-like

lymphocytes, and natural killer cells. Each of these cell types is found in acute and chronic
inflammatory sites in association with expression of E-selectin, thus implicating E-selectin in the
recruitment of these cells to such inflammatory sites. Efforts to assign a causal relationship to
this association have included the generation and analysis of mice homozygous for null alleles at
the E-selectin locus. These mice maintain virtually normal leukocyte recruitment in a peritoneal
exudate model and in a delayed-type hypersensitivity model, both of which measure recruitment
of neutrophils and other leukocytes in the context of acute inflammation. However, a leukocyte-
trafficking defect in these mice is unmasked by administration of a blocking monoclonal
antibody against P-selectin, a treatment that does not yield a leukocyte-trafficking defect in wild-
type animals. These observations imply that both E- and P-selectin maintain an apparent
functional redundancy, at least in mice, in the limited circumstances where inflammation has
been carefully examined. Nevertheless, other animal studies using blocking anti-E-selectin
antibodies have shown that E-selectin can provide an essential, nonredundant contribution to
leukocyte trafficking in acute inflammation.
The presence of a CRD in E-selectin suggested that it recognizes glycoconjugate-based

leukocyte ligands. This possibility was supported by the close structural similarity between E-
selectin and L-selectin and by studies implying that L-selectin recognizes carbohydrate-
dependent ligands. Clues to the identity of E-selectin ligands could be found among the glycans
expressed by the types of leukocytes recognized by E-selectin (e.g., neutrophils), but not on
blood cells that do not interact with E-selectin (e.g., red blood cells). Glycan structural analyses
on myeloid lineage cells and erythroid lineage cells had previously identified a group of
fucosylated, sialylated oligosaccharides absent from erythroid cells, but abundant on myeloid
cells. These glycans could be isolated from the glycoprotein and glycolipid components of the
myeloid cell membranes and included polylactosamine chains modified by terminal or
subterminal substitution with α2 3-linked or α2 6-linked sialic acid residues. Myeloid lineage
specificity on such structures was conferred by the presence of α1 3-linked fucose residues
absent from red cell glycans. The prototype for these myeloid-specific structures is the sialyl Lex
tetrasaccharide (Figure 26.5). This tetrasaccharide is representative of a family of α2 3-
sialylated α1 3-fucosylated glycans that exist on N-glycans, on lipid-linked glycoconjugates,
and on O-glycans. The latter set includes Core-2-based lactosamine and polylactosamine chains.
The polylactosamine chains representative of these molecules may be fucosylated variably, at
one or more GlcNAc residues in the polymer.
Figure 26.5. Structures of all fucosylated, sialylated oligosaccharides that have been shown to
mediate E-selectin-dependent cell adhesion. These include sLex, difucosylated sLex, sLea, 3 sulfo
Lex, 3 sulfo Lea, "GalNAc-Lex (also termed the fucosylated lacdiNAc or LDNF) and VIM-2.
Evidence supporting a role for such molecules in E-selectin-dependent leukocyte adhesion is

derived from studies in which soluble forms were tested in vitro for their ability to inhibit such
adhesion. Similarly, gene transfer approaches have been used to force the expression of these
glycan structures on various cultured cell lines that point to the sialyl Lex structure and its
variants as important components of E-selectin ligands. Considered together, this rather large
body of data indicates that the sialyl Lex and sialyl Lea structure and some (but not all) sulfated
forms of these molecules can function in vitro as E-selectin ligands (Figure 26.5). These studies
also indicate that the unsialylated Lex or Lea structures do not support E-selectin-dependent cell
adhesion, nor does the Lex structure artificially substituted with α2 6-linked sialic acid. Type-2
lactosamine chains with a terminal α2 3-linked sialic acid residue singly substituted with α1 3-
linked fucose on an internal GlcNAc residue (VIM-2; Figure 26.5) do support the E-selectin-
dependent cell adhesion under some in vitro circumstances.
Assignment of the sialyl Lex tetrasaccharide and/or its structural variants as "ligands" for E-
selectin is not as straightforward as originally envisioned. Specifically, consideration must be
given to discriminating between structures that can support E-selectin-dependent cell adhesion in
vitro and those that actually do support this process in vivo. Clearly, this issue depends also on
the nature of the in vitro assays used to characterize putative E-selectin ligands. In this regard, it
is important to point out that the sialyl Lea and sulfated forms of sialyl Lex and sialyl Lea are not
known to be found on leukocytes, implying that they do not participate in E-selectin-dependent
leukocyte adhesion events, even though they can clearly support E-selectin binding in the
laboratory. It is also important to note that although monoclonal antibodies directed against such
structures have played an important part in establishing correlations between structures and E-
selectin ligand "activity," such antibodies generally only identify surrogate markers for E-
selectin ligand activity. For example, the monoclonal antibodies CSLEX-1 and HECA-452
recognize epitopes on leukocytes that are strongly correlated with E-selectin activity. However,
HECA-452 clearly reacts with the sialyl Lea structure, whereas CSLEX-1 does not. Similarly,
skin-homing memory T cells that are known to express E-selectin ligands do not react with the
CSLEX-1 antibody, but they do bind the HECA-452 antibody. Thus, it is important to try to
purify physiological E-selectin ligands from leukocytes, to characterize the composition and
structure of their glycan components, and to evaluate the contribution of each of the glycan
components to E-selectin adhesion activity.
Such studies have assigned E-selectin ligand function to at least three different glycoproteins.
The first of these, PSGL-1, which was discussed above as an L-selectin ligand and is clearly also
a ligand for P-selectin, may serve as a "scaffold" to present α2 3-sialylated, α1 3-fucosylated
glycans to E-selectin. Importantly, PSGL-1 will not support E-selectin-dependent binding
interactions if it is not modified by α2 3 sialylation or α1 3 fucosylation. Furthermore, PSGL-1
is clearly not required for E-selectin ligand activity in some leukocyte cell lines, although it is
possible that this mucin-like molecule may contribute to E-selectin ligand activity in leukocytes
in vivo.
A second candidate E-selectin ligand is a glycoprotein purified from bovine γ/δ T lymphocytes.
This protein has an unreduced molecular mass of 250 kD, and may also function as a ligand for
P-selectin. Although this molecule is apparently not the bovine homolog of PSGL-1, its structure
and other functional properties remain to be explored.
A third E-selectin ligand candidate, termed ESL-1, has been purified from murine myeloid
lineage cells. ESL-1 corresponds to a splice variant of a murine homolog of a previously studied
chicken FGF-binding protein. ESL-1 displays N-glycans with E-selectin ligand activity, and O-
glycans are not involved. This molecule has been found both in the Golgi and on microvilli of
the cell surface.
Glycolipids have also been assigned E-selectin ligand activity. Experiments in which myeloid
cell glycolipids have been purified and fractionated identify α2 3-sialylated α1 3-fucosylated
gangliosides that support E-selectin-dependent cell adhesion. These structures are characterized
by α1 3 fucosylation at internal GlcNAc residues, in the absence of α1 3 fucosylation of the
most terminal GlcNAc residue, i.e., VIM-2 structures. In contrast, glycolipid molecules whose
terminal GlcNAc residue is α1 3-fucosylated do not exhibit E-selectin ligand activity. As with
the studies involving candidate glycoprotein ligands for E-selectin, it is important to note that
assay of these glycolipid structures for E-selectin ligand activity involves in vitro biochemical
procedures that do not necessarily reflect physiological circumstances. In vivo studies that
support a role for these glycolipids and selectin ligand activity are not yet available.
P-Selectin Structure and Expression (6 8,47 55)
Molecular cloning studies have defined the amino acid sequence of P-selectin and the genomic
organization of the P-selectin locus. P-selectin shares the domain structure described for E-
selectin and L-selectin, consisting of a single peptide, a CRD, a single EGF-like motif, nine CR
repeats in human P-selectin, a single transmembrane segment, and a short cytosolic domain (see
Figure 26.1). Each of these domains corresponds to a single exon at the P-selectin locus,
excepting the cytosolic domain, which is derived from two different exons. This organization
mirrors those defined for the E-selectin and L-selectin molecules. The P-selectin locus is
adjacent to the L-selectin locus on human chromosome 1.
P-selectin is found within the Weibel-Palade bodies of endothelial cells and in α-granules of
platelets. Sequences within the cytoplasmic domain of P-selectin apparently mediate sorting to
the granule, through interactions with molecules that direct this process. Cell surface expression
of P-selectin is generally transient and is a consequence of rapid endocytotic events mediated
through the protein's cytoplasmic tail. Splice variants of P-selectin transcripts yield forms of P-
selectin that are without a transmembrane domain, accounting for the fact that some P-selectin is
released as a soluble form.
The cytosolic tail of P-selectin undergoes phosphorylation at specific threonine, tyrosine, and
histidine residues. The P-selectin cytosolic tail is also myristoylated at a single cysteine in this
domain. The functional relevance of these posttranslational modifications has not yet been
defined. P-selectin expression is also under transcriptional control, with induction by TNF-α,
IL1-β, or LPS. The time course of expression of P-selectin in these contexts is similar to that
observed for E-selectin.
P-selectin contributes to leukocyte recruitment in a variety of acute and chronic inflammatory

contexts. Its role in leukocyte recruitment and inflammation is a function of both acute and
chronic expression of P-selectin by endothelium and by platelet activation-dependent P-selectin
expression. The role of this molecule in leukocyte recruitment in the context of acute
inflammation is well illustrated by analysis of mice that are homozygous for null P-selectin
alleles. These mice exhibit a substantial delay in neutrophil recruitment in the context of acute
inflammation, relative to wild-type mice, indicating that P-selectin plays an important part in the
early phases of leukocyte recruitment. P-selectin can also contribute to chronic inflammation,
presumably through transcriptional up-regulation of its expression during the latter stages of an
inflammatory response. For instance, P-selectin-deficient mice exhibit delayed trafficking of
monocytes to an inflamed peritoneal space during the most acute phase of this inflammatory
model. These observations imply that transcription-dependent expression of P-selectin at later
times normally contributes to monocyte recruitment. Similarly, T lymphocytes exhibit a
requirement for P-selectin when trafficking to skin in the context of chronic inflammation.
Platelet-derived P-selectin also contributes to leukocyte trafficking, as well as to wound healing

and blood clotting. For example, activated platelets adhere to neutrophils, monocytes, natural
killer cells, and some subsets of T lymphocytes. This adhesion is mediated through P-selectin, in
part, and represents a mechanism to augment the recruitment of leukocytes and platelets to sites
of vascular compromise. Platelet-derived P-selectin may also contribute to hemostatic processes
by its ability to stimulate monocytes to express tissue factor and by facilitating fibrin deposition
during clot formation. The inferred role for platelet-derived P-selectin in hemostatic processes is
not completely consistent with the generally mild hemostatic defect observed in P-selectin null
mice, and this issue remains to be explored further.
P-Selectin Ligands (56 86)
P-selectin binding to cells is inhibited by many different anionic compounds, including sulfated
glycoconjugates (e.g., heparin and sulfatide) and phosphorylated compounds (e.g., Man-1-P).
These early observations led to the belief that P-selectin might be highly undiscriminating in the
types of ligands it recognizes. Remarkably, leukocytes express only one glycoprotein that binds
with high affinity to P-selectin, and that is PSGL-1, which is a relatively minor mucin on
leukocyte surfaces (Figure 26.6A). The identification and characterization of a single specific
receptor for P-selectin were especially surprising, given the view up to recent times that most
lectins only recognize carbohydrate determinants (or modifications of the carbohydrate) and the
concept that typical carbohydrate determinants in a cell are expressed on a multitude of
glycoproteins.
Figure 26.6. (A) Predicted disulfide-bonded dimeric form of PSGL-1 in leukocytes. The multiple
O-glycan and three potential N-glycans on the molecule are indicated. (B) Two major fucose-
containing Core 2 O-glycans identified in PSGL-1. The sialyl Lex determinant in each glycan is
boxed. (C) Predicted interaction of the tyrosine sulfate residues and sialyl Lex Core 2 O-glycans
at the extreme amino terminus of PSGL-1 with the C-type lectin domain of P-selectin.
The binding of P-selectin to neutrophils, however, is abolished upon sialidase treatment, which
suggested originally that sialic acid may be a critical determinant required for P-selectin
recognition. The possible importance of the sialyl Lex moiety to P-selectin recognition was
derived from experiments showing that (1) P-selectin binds to a mutant CHO cell line expressing
the sialyl Lex moiety, but not to cells lacking the epitope; (2) antibodies to the sialyl Lex epitope,
but not the Lex epitope, can block binding of cells to P-selectin; and (3) glycans containing the
sialyl Lex moiety inhibit P-selectin-mediated adhesion, and P-selectin binds directly to
immobilized glycans containing the sialyl Lex epitope. However, the binding of P-selectin to
neutrophils displays much higher affinity interactions than to nonmyeloid cells, even those
expressing sialyl Lex, originally suggesting that myeloid cells possess a unique ligand(s) for P-
selectin. Using 125I-labeled P-selectin blotting and affinity chromatography on immobilized
human P-selectin, a glycoprotein ligand for P-selectin was identified and purified, starting with
total membrane glycoproteins extracted from human neutrophils and the human promyelocytic
cell line HL60. The purified ligand, now known as PSGL-1, behaves as a disulfide-bonded
approximately 250-kD protein in nonreducing SDS-PAGE and approximately 120-kD in
reducing SDS-PAGE. In contrast to other selectin ligands, PSGL-1 binding to P-selectin is
characterized by high-affinity binding (Kd in the range of 100 nm) and very fast association and
dissociation rates.
The cDNA encoding PSGL-1 was expression-cloned in COS cells transfected to coexpress an
α1 3(4)-fucosyltransferase (human FucT-III), which allows both sialyl Lex and sialyl Lea
synthesis in these cells. The sequence of PSGL-1 predicts a protein of 412 amino acids with an
18-amino-acid signal sequence and a tetrapeptide consensus cleavage site for a paired basic
amino-acid-converting enzyme in leukocytes at residues 38 41 (-R-D-R-R). Thus, the amino
terminus of the mature protein begins at residue 42. There are 16 decapeptide repeating units
with the consensus sequence -A-T/M-E-A-Q-T-T-X-P/L-A/T- spanning residues 118 277 in the
long form of the protein, and the short form is missing the residues 132 141. A single cysteine
residue occurs at position 320 in the extracellular domain preceding the predicted single
transmembrane-domain-spanning residues 321 341 and the cytoplasmic domain of residues
342 412. The coding region for human PSGL-1 is contained entirely in exon 2 of the gene,
which maps to chromosome 12q24. The cDNA for the murine PSGL-1 encodes a predicted 397-
amino-acid protein that has recognizable homology with the human sequence. The murine
protein contains a predicted signal sequence and propeptide identical in size to that of human
PSGL-1; the mature murine PSGL-1 is also predicted to begin at residue 42. However, the
mouse homolog has only ten decameric repeats with the consensus sequence -E-T-S-Q/K-P-A-P-
T/M-E-A- that are obviously different in sequence from that of the human PSGL-1. The highest
homology between the human and murine PSGL-1 occurs in the transmembrane (83%) and
cytoplasmic domains (76%).
Each subunit of human PSGL-1 contains 70 serine and threonine residues in the extracellular
domain that are potential sites for O-glycosylation and three potential sites for N-glycosylation.
The murine PSGL-1 also contains numerous extracellular serine and threonine residues and two
potential sites for N-glycosylation. The murine PSGL-1 also contains a single unpaired
extracellular cysteine at residue 307 that preceeds the predicted transmembrane domain.
Interestingly, human PSGL-1 contains three predicted tyrosine sulfation sites at residues 46, 48,
and 51 that fall in the consensus sequence in which tyrosine residues are flanked by acidic
residues. The murine PSGL-1 contains two predicted tyrosine sulfation sites at residues 54 and
56.
The large size and extensive glycosylation of PSGL-1 present a daunting challenge to
understanding how it is specifically recognized by P-selectin. It was anticipated at first that
PSGL-1 might be a high-affinity and unique ligand for P-selectin by virtue of its mucin-like
nature and the presumption that the ligand contained large amounts of the sialyl Lex antigen,
thereby enhancing its avidity for P-selectin. However, as discussed below, this prediction was
not correct.
Treatment of purified PSGL-1 with sialidase abolishes its binding to P-selectin, but treatment of
neutrophil-derived PSGL-1 with peptide N-glycosidase F, which removes most, if not all, N-
glycans on the molecule, does not affect its recognition by P-selectin. Such results suggested that
sialylated O-glycans, rather than N-glycans, are important determinants for P-selectin
recognition. The involvement of O-glycans was supported by the observation that treatment of
either neutrophils or purified PSGL-1 with the O-sialoglycoprotease from Pasteurella
hemolytica, an enzyme that degrades sialylated mucins, blocks binding of the cells or ligand,
respectively, to P-selectin. In addition, when HL60 cells are treated with benzyl-α-GalNAc,
which inhibits extension of O-glycans, the cells bind less to P-selectin. Other studies
demonstrated that treatment of isolated PSGL-1 with endo-β-galactosidase, a bacterial
endoglycosidase capable of degrading type-2 polylactosamine repeats [-3Galβ1 4GlcNAcβ1-]n,
significantly reduces binding to P-selectin, thus indicating that polylactosamine, presumably on
O-glycans, may also be important for binding.
The structures of the O-glycans of native PSGL-1 purified from human HL60 cells reveal that
most contain a simple Core 2 structure with one or two sialic acid residues and generally lack
fucose residues. Fucose is present in only two relatively minor O-glycans, termed glycan 1 and
glycan 2 (Figure 26.6B). Both glycans 1 and 2 contain the sialyl Lex antigen; however, glycan 1,
but not glycan 2, contains a polylactosamine on the Core 2 structure with multiple fucose
residues. The possible synthesis of glycan 1 is discussed below (Figure 26.7). Thus, the O-
glycans of PSGL-1 generally lack fucose and only a few O-glycans display the sialyl Lex
antigen, presumed to be important in P-selectin binding. The fact that glycan 2 is predicted to
occur in substoichiometric quantities led to the expectation that glycan 1 may be the more
important O-glycan for P-selectin recognition, but this is not yet clear. The glycosyltransferases
participating in P-selectin ligand formation are discussed in the next section.
Figure 26.7. Synthetic scheme for synthesis of sialyl Lex, difucosylated sialyl Lex, Lex, and
VIM-2. A polylactosamine chain (I) displayed by O-linked, N-linked, or lipid-linked
glycoconjugates (R) serves as the precursor for the pathways. One important example of such
glycoconjugates includes the polylactosamine moiety on a Core 2 O-glycan displayed by PSGL-
1. This precursor may be modified on galactose terminal moiety by α2 3-linked sialic acid,
through the action of one or more α2 3 sialyltransferases (ST3Gal), forming the synthetic
intermediate II. The subterminal GlcNAc moiety of II may be modified with α1 3-linked
fucose, through the action of the α1 3 fucosyltransferase FucT-VII, to form compound III, a
sialyl Lex-active, monofucosylated α2 3-sialylated lactosamine. In contrast, the α1 3
fucosyltransferase FucT-IV is generally not capable of efficiently completing this reaction.
Alternatively, synthetic intermediate II can be used by FucT-IV to form compound IV, whose
internal GlcNAc moieties are α1 3-fucosylated. This structure exhibits reactivity with the VIM-
2 antibody, but not with CSLEX-1 or other sialyl Lex-reactive antibodies. Structures III and IV
can serve as synthetic precursors for V, through α1 3 fucosylation, catalyzed by FucT-IV or by
FucT-VII, respectively. Product V is therefore thought to be the product of the combined actions
of FucT-IV and FucT-VII, operating sequentially, or simultaneously, in vivo, on synthetic
intermediate II. Product IV, a multiply fucosylated neutral polylactosamine structure, may be
formed by direct fucosylation of synthetic intermediate I by the α1 3 fucosyltransferase FucT-
IV. FucT-VII does not complete this reaction. Compound VI cannot serve as a precursor for
synthesis of α2 3-sialylated structures such as V, since α2 3 sialyltransferases performing this
reaction have not been identified. The double-strike indicates an enzyme that cannot catalyze the
reaction indicated. An X indicates a reaction pathway that does not occur.
Although proper glycosylation of PSGL-1 is required for its binding to P-selectin, other studies
suggested the sulfation is also important. Treatment of purified PSGL-1 with a bacterial aryl
sulfatase, which removes sulfate from tyrosine residues in PSGL-1, abrogates binding of the
molecule to P-selectin. Furthermore, recombinant forms of PSGL-1 in which the three tyrosine
residues, which occur in a putative tyrosine sulfate motif at the extreme amino terminus of the
molecule, have been changed to phenylalanine also fail to bind P-selectin. In addition, treatment
of cells with sodium chlorate also blocks expression of a functional PSGL-1 molecule.
The critical binding domain appears to reside in the extreme amino terminus of PSGL-1. Several
different lines of evidence are consistent with this possibility: (1) A blocking monoclonal
antibody (PL1) was developed that recognized a peptide epitope overlapping the tyrosine sulfate
consensus sites, whereas nonblocking monoclonal antibodies (e.g., PL2) mapped to epitopes
outside this region. The PL1 monoclonal antibody prevents neutrophil, monocyte, eosinophil,
and lymphocyte adhesion and rolling on P- and L-selectin. (2) Chimeric glycoproteins containing
the extreme amino-terminal domain of PSGL-1, when produced in cells expressing appropriate
glycosyltransferases, are recognized by P-selectin. (3) Treatment of neutrophils with
mocarhagin, a cobra venom metalloproteinase, removes the extreme amino-terminal ten amino
acid residues from PSGL-1 and abrogates its binding to P-selectin. Such data point to a model in
which the combination of tyrosine sulfate residues and oligosaccharides on the protein are
required for high-affinity binding to P-selectin (Figure 26.6C).
Another approach to explore the fine structure of the PSGL-1 amino-terminal domain required
for P-selectin recognition is selective mutagenesis of amino acids in that domain and assessment
of the binding of recombinant PSGL-1 to P-selectin. Replacement of all three amino-terminal
tyrosine residues with phenylalanine abolishes binding of the recombinant PSGL-1 to P-selectin,
but not E-selectin. Interestingly, only one of the three potential tyrosine sulfate residues is
absolutely necessary for binding to P-selectin. Even more surprising is the finding that
replacement of threonine at position 57 with alanine blocks binding of the recombinant molecule
to P-selectin. These data support the model in Figure 26.6C, where the C-type lectin domain of
PSGL-1 may contact tyrosine sulfate residues and the sialyl Lex structure on a Core 2 O-glycan
at Thr-57. However, it must be stressed that this model is only a prediction, and there is no direct
evidence that such an O-glycan, as depicted in Figure 26.6C, even occurs at Thr-57 in PSGL-1,
nor is there yet any direct proof that there is coordinate binding of such an O-glycan and one or
more tyrosine sulfates.
The specific in vivo functions of PSGL-1 have been explored using blocking antibodies to the
protein and recombinant forms. A blocking monoclonal antibody to PSGL-1 (PL1) and its F(ab)
fragments dramatically reduced rolling of human polymorphonuclear neutrophils and HL60 cells
in venules of acutely exteriorized rat mesentery, indicating that PSGL-1 is important in vivo for
rolling of myeloic cells in mesenteric venules at physiologic shear stress. Another approach to
study PSGL-1 function in vivo during inflammation is to explore its role in ischemia/reperfusion
injury models, in which blood flow is blocked, thereby stimulating P-selectin expression by
endothelial cells. In a rat model of ischemia/reperfusion injury, treatment of animals with
recombinant PSGL-1 significantly enhanced rat survival and liver function and recovery. PSGL-
1 may also be important for lymphocyte recruitment to sites of inflammation in vivo, since
blocking antibodies to mouse PSGL-1 blocks entry of T-helper-1 cells into inflamed areas of the
skin during a cutaneous delayed-type hypersensitivity reaction model. There is also evidence that
P- and possibly E-selectin are important contributing factors to development of atherosclerotic
lesions. Although such studies are beginning to define a role for PSGL-1 in leukocyte functions
in vivo, many more studies are needed to understand the involvement of PSGL-1, as opposed to
or in concert with other selectin ligands, in the overall response to inflammation.
Glycosyltransferases Involved in Selectin Ligand Biosynthesis

(87 100)
Much remains to be learned concerning the structure of the physiological leukocyte ligands for
the selectin, but the nature of the genetic loci responsible for synthesis of these structures has
become more apparent in recent years. For instance, the identities of the leukocyte α1 3
fucosyltransferases that decorate leukocyte selectin ligands and contribute to selectin ligand
activity have been identified from analysis of cells transfected with candidate fucosyltransferase
genes and from an analysis of animals in which these genes have been deleted. Similar
approaches have identified the role of a Core 2 β1 6 N-acteylglucosaminyltransferase in selectin
ligand biosynthesis (see below).
The human genome maintains at least five distinct α1 3 fucosyltransferase loci. Three of these,
FucT-III, FucT-V, and FucT-VI, are not apparently expressed in leukocytes or their progenitors,
and there is no obvious defect in selectin ligand synthesis in individuals who are homozygous for
null alleles at the FucT-III locus or at the FucT-VI locus. In contrast, the α1 3
fucosyltransferases FucT-IV and FucT-VII are expressed in the leukocyte lineages and have
represented strong candidates for participation in the fucosylation of E-selectin ligands.
Published evidence for a role for FucT-IV in this process is conflicting, since this gene is able to
determine expression of E-selectin ligand activity, i.e., sialyl Lex, can in some culture cell lines,
but not others. Biochemical analyses indicate that FucT-IV can generate the Lex structure and
can generate internally fucosylated products using α2 3-sialylated polylactosamine precursors
(Figure 26.7). However, this enzyme is rather ineffective in the formation of the sialyl Lex
tetrasaccharide from α2 3-sialylated lactosamine chain precursors, except in the context of its
expression in some cultured cell lines.
FucT-IV and FucT-VII may make mutually exclusive contributions to fucosylation of the α2 3-
sialylated lactosamine precursors to glycans with selectin ligand activity. These observations
further suggest that such distinct fucosylation events may make correspondingly distinct
contributions to selectin ligand activity. These ideas have yet to be confirmed in experiments in
which selectin ligands bearing glycan structures determined by FucT-IV, or by FucT-VII, or by
the two operating together, have been studied with respect to their relative selectin ligand
"activity." From the possible biosynthetic pathway shown in Figure 26.7, FucT-VII is predicted
to act cooperatively with FucT-IV to generate a high-affinity selectin ligand, particularly with
regard to PSGL-1.
Evidence supporting a central role for the fucose residue in providing L-selectin ligand structures
comes from an analysis of mice made deficient in the α1 3 fucosyltransferase, FucT-VII, which
is expressed in peripheral node HEVs. Mice homozygous for a FucT-VII null allele are deficient
in L-selectin ligand formation in the HEVs. They also exhibit a marked leukocytosis i.e.,
elevated leukocyte levels, with a deficiency in lymphocyte colonization of the lymph nodes.
Moreover, mice lacking FucT-VII are impaired in experimentally induced inflammatory
responses by assessing neutrophil recruitment to inflamed peritoneum. HEV-borne L-selectin
ligand activity is reduced more than 90% when assessed by immunohistochemical procedures or
by in vivo lymphocyte-homing studies, confirming an essential role for α3-linked fucose
residues in L-selectin ligand activity. In vitro, FucT-VII effectively utilizes the nonfucosylated 6-
sulfated, sialylated precursor to form the 6-sulfosialyl Lex structure. In contrast, the 6 -sulfated,
sialylated precursor is not utilized by FucT-VII to form the 6 -sulfosialyl Lex structure. No
information is yet available about the ability of FucT-VII to use the bis-sulfated precursor.
There is similarly strong evidence for an essential role for FucT-VII in E- and P-selectin ligand
biosynthesis. For example, FucT-VII consistently generates α1 3-fucosylated, α2 3-sialylated
epitopes recognized by the CSLEX-1 and HECA-452 antibodies, e.g., when expressed in
cultured cell lines with glycosylation phenotypes capable of supporting this synthetic pathway.
FucT-VII maintains a rather limited acceptor substrate specificity, in comparison to the other
α1 3 fucosyltransferases, since it is unable to utilize neutral precursors to form the Lex antigen,
nor is it able to utilize type I precursors to form the Lea or sialyl Lea structure. There is also
biochemical evidence that FucT-VII operates exclusively on N-acetylglucosamine residues at the
terminus of an α2 3-sialylated polylactosamines, to the exclusion of activity on N-
acetylglucosamine residues at more proximal positions.
The recombinant form of PSGL-1 when expressed in CHO cells fails to bind P-selectin.
However, if the cells are transfected to coexpress α1 3 fucosyltransferases, which can support
sialyl Lex synthesis, and the Core 2 GlcNAcT participating in Core 2 O-glycan formation, the
recombinant PSGL-1 efficiently binds to P-selectin. Such results suggested that expression of
both the sialyl Lex antigen and Core 2 GlcNAcT is required for PSGL-1 binding to P-selectin.
The vast majority of leukocyte binding to P-selectin is lost in mice rendered genetically deficient
in FucT-VII.
The relevance of the Core 2 O-glycan structure to selectin ligand formation has been explored in
mice that are homozygous for null alleles at the Core 2 GlcNAcT locus. The L-selectin ligand
activity of the peripheral lymph node HEVs in these mice is only partially reduced as assessed
by immunohistochemical procedures but not functionally affected as judged by normal
lymphocyte abundance in the nodes, and in vivo lymphocyte homing assays. The apparent
absence of a physiologic contribution to L-selectin ligand activity by the Core 2 GlcNAc linkage
may be explained by compensatory processes in the mice and/or by N-linked or lipid-linked (O-
sialyloglycoproteinase-resistant) L-selectin ligands that have been described on peripheral lymph
node HEVs.
In contrast, neutrophils from Core 2 GlcNAcT-null mice bind poorly to P-selectin, and especially
to E-selectin and L-selectin. Although the detailed structures of O- and N-glycans on murine
leukocytes have not yet been defined, these studies strongly implicate the Core 2 O-glycans
containing the sialyl Lex antigen, as observed on human PSGL-1, as critical determinants for
selectin recognition. The inflammation response in Core 2 GlcNAcT-null mice is severely
affected, similar to the response deficit in FucT-VII-null mice. A moderate leukocytosis occurs
in mice lacking Core 2 GlcNAcT, which is almost completely due to increased levels of
neutrophils. The myeloid specificity and nature of the phenotype indicate that selectin ligand
function, in either lymphocyte homing or myeloid inflammation, appears segregated by Core 2
O-glycan biosynthesis.
Much remains to be understood regarding the biosynthesis of physiologic selectin ligands. A

glycan structural correlate for loss of selectin binding with FucT-VII deficiency in leukocytes of
null mice is not yet available. The cDNAs encoding two human tyrosylprotein sulfotransferases
capable of sulfating Tyr residues in PSGL-1 have been identified, and a cDNA encoding a
GlcNAc 6-sulfotransferase capable of synthesizing 6-sulfosialyl Lex has been identified.
However, the nature of other sulfotransferases relevant to selectin ligand biosynthesis is under
intense study. Interestingly, the precise nature of the α2 3 sialyltransferase(s) relevant to the
biosynthetic scheme in HEVs has not been defined as of this writing.
Future Directions
The identification of selectins as carbohydrate-binding proteins important in regulating leukocyte
trafficking and adhesion marked a milestone in the development of the field of glycobiology.
Selectins serve as a premier example of the structure-function relationships between
glycosyltransferases and their cognate carbohydrate structural products as recognition molecules.
In addition, a better understanding of selectin biology is leading to the development of specific
pharmaceuticals capable of blocking selectin-mediated adhesive events and represents a major
effort in the budding field of glycobiotechnology. It is anticipated that further understanding of
the regulated expression of selectins and their ligands and the detailed molecular nature of their
interactions will provide new paradigms into how carbohydrate-binding proteins interact with
specific glycans to regulate adhesive interactions among blood cells.
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27. S-type Lectins (Galectins)
Primary contributions to this chapter were made by R.D. Cummings (The University of
Oklahoma Health Sciences Center, Oklahoma City).
THE S-TYPE LECTINS, MORE RECENTLY TERMED GALECTINS, represent a group of

proteins that bind β-galactosyl-containing glycoconjugates and share primary structural
homology in their carbohydrate recognition domains (CRDs). They are completely different
from the C-type lectins in both structure and lack of requirement for divalent cations. Galectins
are widely distributed throughout the animal kingdom. Most galectins are soluble proteins that
are secreted by an unusual pathway and display a requirement for reducing conditions to
maintain activity in the absence of ligands. Certain members of the galectin family can promote
cell-cell adhesion, whereas some have potent biological activities, such as the ability to induce
apoptosis, or programmed cell death, and to induce metabolic changes, such as cellular
activation and mitosis. This chapter describes the diversity of the galectin family and presents
what is known about their biosynthesis, secretion, and biological activities.
Historical Background and Discovery of Galectins (1 11)
Following the discovery of agglutinins in plants and of lectins (Discoidin I) in Dictyostelium

discoideum in the early 1970s by Barondes and Rosen and colleagues, many investigators began
to search for lectins in animal tissues. As noted in Chapter 25, the first reported lectin in animal
cells was the hepatic asialoglycoprotein receptor, a C-type lectin. The next reported lectin in
animals was the protein now recognized as the first S-type lectin. It was originally described by
Teichberg and colleagues during studies on the possible presence of lectins in electric organs of
the electric eel. The protein, termed electrolectin, had hemagglutinating activity inhibitable by β-
galactosides and could be isolated by affinity chromatography on β-galactoside supports.
Notably, this activity required the inclusion of β-mercaptoethanol in the isolation buffers to
maintain the activity, suggesting the presence of one or more free cysteine residues. Electrolectin
is approximately 15 kD in size and occurs as a noncovalently linked homodimer.
The first S-type lectin found in mammals, now termed galectin-1, was isolated in 1976 by
Kornfeld and colleagues from extracts of calf heart and lung. Interestingly, lactose was required
to efficiently extract the protein from macromolecular glycoconjugates in the extracts. The lectin
was isolated by affinity chromatography on asialofetuin-Sepharose and also required reducing
conditions to maintain activity. The calf heart/lung galectin-1 is approximately 15 kD in size and
occurs as a noncovalent dimer. In the same year, Barondes and colleagues also identified an S-
type lectin in chick muscle extracts. In the early 1980s, Wang and colleagues identified a 35-kD
carbohydrate-binding protein, now known as galectin-3, from human and mouse fibroblasts that
also bound to β-galactosides. All of these proteins demonstrated hemagglutinin activity, but the
choice of erythrocytes was crucial. Rabbit erythrocytes, which display more terminal galactose
residues than human erythrocytes, are readily agglutinated by most galectins, whereas human
erythrocytes require treatment with neuraminidase to enhance their agglutinability. The soluble
nature of these proteins, their sequence homologies, and their requirement for reducing
conditions led Drickamer to term these lectins S-type to denote their sulfhydryl dependency and
solubility, in much the same way the C-type denotes the Ca++ dependency for that class of
lectins. The nomenclature for galectins was systematized in 1994. The first galectin found
(electrolectin, β-galactoside-binding lectin, galaptin, L-14, etc., depending on its source) was
termed galectin-1. Its nearest homolog was termed galectin-2. The 35-kD carbohydrate-binding
lectin was termed galectin-3, and other members of this family were then numbered
consecutively by order of discovery.
Definition of the Galectins and Sequence Motifs (12)
The canonical CRD of galectins has approximately 130 amino acids, although only a small
number of residues that make up the carbohydrate-binding site directly contact carbohydrate
ligands. A comparison of the sequences of approximately 30 galectins from many different
sources reveals that eight residues, which have been shown to be involved in carbohydrate
binding by X-ray crystallographic analyses, are invariant. In addition, another dozen residues
appear to be highly conserved. The common sequence motif used to identify galectins is shown
in Figure 27.1, along with a comparison of the human and chicken galectin-1 sequences.
Although all galectins share a high degree of homology in their CRDs, two general subgroups of
galectins can be distinguished, based on sequence homologies, and these are the galectin-1
subfamily, which includes galectins 1 and 2, and the galectin-3 subfamily, which includes all
others. In comparison to human galectin-1, the mushroom galectin from Coprinus cinereus is
overall about 20% identical and the 14-kD and 16-kD galactins in chickens are both about 60%
identical.
Figure 27.1. (Top) Conserved primary structures within the CRD in galectin family members.
Italicized letters indicate those highly conserved residues known to make contacts with
carbohydrate ligands. Nonconserved amino acids are indicated by -x-. (Bottom) A comparison of
the human and chick galectin-1 sequences within the CRD. Identical residues between the two
sequences are boxed and those residues within the canonical galectin CRD are in boldface
letters.
Different Subfamilies of Galectins (6)

A large number of galectins have now been identified in animals based on the conserved galectin
CRD, and these all appear to recognize simple β-galactosides, although relatively weakly in most
cases. A list of the mammalian proteins with a galectin CRD is shown in Table 27.1 and Figure
27.2. Some galectins are monomers and some are noncovalent dimers. In addition, some of the
proteins, such as galectins 4, 6, 8, and 9, are monomers that contain a protease-sensitive
intersubunit bridging peptide connecting two CRDs. In addition to this list, galectins are found in
many other animals. Birds contain 14-kD, 16-kD, and 30-kD forms of galectins that are probably
the avian homologs of galectins 1 and 3. Galectins have also been found in the skin (16 kD) and
oocytes (15 kD) of the amphibians Xenopus laevis and Bufo arenarum, respectively. As noted,
the electrolectin from electric eel was the first galectin to be investigated, but the conger eel also
has a 16-kD dimeric galectin in its skin mucus. Galectin-1 and -4 homologs have been found in
the free-living nematode Caenorhabditis elegans and in sponges and fungi. Thus, galectins
probably occur in all animals and multicellular organisms.
Table 27.1. The galectin family and its distribution in mammals
Type Source Distribution Molecular Oligomeric Remarks

mass (kD) structure
Galectin- human, rat, muscle, heart, lung, 14.5 dimer most common and
1 mouse, liver, lymph node, abundant galectin
hamster, thymus, prostate,
monkey, colon
bovine, pig
Galectin- human, small intestine 14.5 dimer minor level compared
2 mouse to galectin-1
Galectin- human, rat, macrophage, colon 29 35 monomer amino-terminal domain
3 mouse, dog, is collagen-like
hamster
Galectin- human, rat, alimentary tract 36 monomer linkage of two CRDs is
4 pig, mouse protease-sensitive
Galectin- rat erythrocytes 17 18 monomer 85% identical to
5 carboxy-terminal CRD
of galectin-9
Galectin- mouse gastrointestinal 34 ? 85% identical to
6 galectin-4
Galectin- human, rat skin 14.5 ? marker of stratified
7 epithelia
Galectin- human, rat liver, kidney, lung 34 monomer
8
Galectin- human, rat, thymus, kidney, 35 ? carboxy-terminal
9 mouse Hodgkin's lymphoma domain is 85%
identical to galectin-5
Galectin- human oesinophil, basophil 17 dimer Charcot-Leyden crystal
10 protein,
lysophospholipase
Figure 27.2. Schematic illustrations of the structures of galectins. The CRDs are indicated in
gray.
Carbohydrate Ligands for Galectins (13 15)
All members of the galectin family tested so far bind simple β-galactosides, but the affinity is
relatively low, in the millimolar range. Surprisingly, the detailed glycan specificities for most
galectins are not clear, and each galectin may differ in its overall specificity. All galectins appear
to bind terminal β-galactosides, but some galectins differ significantly in their recognition of
galactosyl residues within oligosaccharides. For example, both galectins 1 and 3 bind simple
lactosaminyl units as well as polylactosamine. However, galectin-3 binding to oligosaccharides
is enhanced if the penultimate galactosyl residues are substituted with Galα1 3, GalNAcα1 3,
or Fucα1 2 residues. In contrast, such substitutions dramatically decrease binding by galectin-1.
Cocrystallization of galectins with simple β-galactoside-containing disaccharides has revealed a
tight recognition of the C4 and C6 hydroxyl of galactose and the C3 hydroxyl of GlcNAc.
Several studies on galectin-1 have revealed that it displays much higher affinity for larger
glycans containing repeating galactosyl residues, as occur in type-2 polylactosamine sequences
represented by the structure: -3Galβ1 4GlcNAcβ1 3Galβ1 4GlcNAcβ1 3Galβ1 4GlcNAcβ1-
R. Interestingly, at least for galectin-1, its interaction of the polylactosamine is not dependent on
terminal galactose residues, but it does require at least two linear repeating disaccharide units. It
is possible, as discussed below in the section on galectin structure, that the interaction of
galectin-1 with polylactosamine and other extended glycans may be due to contributions of
secondary binding sites on the protein.
The potential endogenous glycoconjugate ligands have been investigated for only a few
members of the galectin family. Potential ligands for galectins 1 and 3 include basement
membrane proteins, such as laminin and fibronectin; membrane receptors, such as integrin α7β1,
CD43, and CD45; lysosome-associated membrane proteins (LAMPs); and even certain
gangliosides. However, the precise carbohydrate structures on these macromolecules that are
recognized by galectins are not well defined. It is possible that each galectin differs somewhat in
both oligosaccharide binding specificity and affinity for macromolecular ligands. The fact that
galectin-1, for example, binds to a limited set of glycoconjugates suggests that the mere presence
of galactose residues in glycoconjugates is not sufficient to promote their high-affinity binding to
this lectin.
Biosynthesis and Secretion of Galectins (16 22)
All members of the galectin family lack classical signal sequences or membrane-anchoring
domains and appear to be synthesized on free polysomes in the cytoplasm and accumulate there
prior to secretion. One rather common modification of galectins is blockage of the amino
terminus, although galectin-3 has been shown to also have phosphorylated serine. Newly
synthesized galectins isolated directly from the cytosol of cells are functional in binding β-
galactosides. The complexity of galectin biosynthesis, secretion, and oligomerization is
illustrated schematically for galectin-1 in Figure 27.3. Curiously, the export of galectins from
cells does not involve direct movement through the secretory apparatus. The mysterious process
by which these proteins are exported has been explored in several ways, but the basic mechanism
is still unknown. Several other relatively small-sized growth factors and ECM molecules are also
secreted by a nonclassical pathway, but whether their secretory pathway converges with that of
galectins is not known.
Figure 27.3. Possible biosynthetic routes for galectin-1 in animal cells. The protein is translated
on free polysomes in the cytoplasm and the newly synthesized protein there is capable of binding
carbohydrate ligands or other proteins within the cell. After secretion or export by an undefined
mechanism, the newly synthesized galectin is unstable, but it associates with carbohydrate
ligands that stabilize its structure. The stable monomeric protein may dimerize and interact with
ligands on the cell surface and in the extracellular matrix.
In exploring a possible mechanism for export, recombinant galectin-1 expressed in yeast was
exported by a transmembrane protein, but so far, this transporter appears to be novel and limited
to yeast. Whether different transporters occur in animal cells is unknown. It is also possible that
export involves membranous structures. For example, galectin-1 appears to exit from myoblasts
via evaginations of the plasma membrane, which pinch off to form lectin-enriched vesicles.
Similarly, galectin-3 assembles into patches that eventually appear to underlie the plasma
membrane as a prelude to deposition in the extracellular space. Chimeric forms of galectin-3,
containing introduced sites for myristolylation and palmitoylation, are rapidly transported to
plasma membrane domains and appear to be released from cells in vesicles that can be isolated
from the culture media. The spontaneous rupture of these vesicles may allow release of the
protein. However, the nature of these vesicles and how galectins are sequestered within them are
not known.
Although the mechanism of export is unclear, most galectins rapidly lose activity in a
nonreducing environment, as exists outside cells. However, studies on the biosynthesis of
galectin-1 found that the newly exported protein is extremely unstable in the absence of
carbohydrate ligands, but when high-affinity ligands are available, the protein is stable. Thus, the
regulated secretion and availability of ligands may regulate activity and stability of galectins.
Structure: Tertiary/Quaternary (13,23 26)
The crystal structures of several galectins have now been reported, including the structure of
bovine galectin-1 complexed with either N-acetyllactosamine or diantennary N-glycans; human
galectins 2, 3, and 7 in complexes with monosaccharides or disaccharides, such as lactose; and
toad galectin-1 complexed with a disaccharide. All of the structures show that the CRD of
galectin subunits is composed of five- and six-stranded antiparallel β sheets arranged in a β-
sandwich or jelly-roll configuration that completely lacks the α-helix (Figure 27.4). In the
dimeric proteins, such as galectins 1, 2, and 7, the subunits are related by a twofold rotational
axis perpendicular to the plane of the β sheets. The carbohydrate-binding sites in the CRD are
located at opposite ends of the dimer. The compactly arranged structure of the CRD partly
explains the protease resistance of the galectin CRD and the high degree of conservation and
requirement for the 130 amino acids in the CRD. Interestingly, the tertiary structure of galectins,
but not their primary structure, is similar to the jelly-roll configuration found in many
leguminous plant lectins (see Chapter 30).
Figure 27.4. Proposed structure of human galectin-2, based on the X-ray crystallographic
analyses of the protein complexed with lactose, shown in space-filling mode. The subunit
interface is based on interactions between the carboxy- and amino-terminal domains of each
subunit. (See Reference 23.)
As mentioned above, the galectin CRD displays highly specific interactions with galactose and
GlcNAc residues, but the open-ended structure of the carbohydrate-binding site might allow
access to extended galactose-containing glycans, such as the polylactosamines. There are several
subsites on the protein near the carbohydrate-binding site that could serve to enhance affinity for
more extended glycans. The crystal structure of bovine galectin-1 was also derived for bovine
galectin-1 complexed with biantennary N-glycans containing two terminal β-galactoside
residues. In this extended crystal structure, the N-glycan is bridged between two galectin dimers,
thus effectively creating a crystal latticework.
Proposed Functions of Galectins (15,27 37)
Because of their variety and the multiple subcellular localizations of galectins, many different
functions have been proposed for these proteins (Figure 27.5). Most of the galectins are
multivalent or oligomeric and in vitro are capable of agglutinating cells and cross-linking
ligands. The expression of galectins 1 and 3 in mice is developmentally regulated. The proposed
biological functions of galectins include roles in cell-cell adhesion, cell-matrix adhesion, direct
effects on cell growth and viability, and potential intracellular functions in regulating me
tabolism. Their expression in many types of tumor cells has led to the hypothesis that galectins
may also be involved in tumorigenesis and metastasis.
Figure 27.5. Proposed functions of galectins.
Cell-Matrix Interactions
The discovery that galectin-1 binds with high affinity to polylactosamine-containing ligands,
such as laminin, and the colocalization of galectin-1 with laminin in ECMs suggested that a
major function of the lectin could be to promote cell adhesion to glycoconjugates. Indeed,
galectin-1 can directly promote adhesion of cells to laminin in some systems, but it can interfere
with cell attachment to laminin in other systems. Galectin-1 interferes with integrin α7β1 binding
to laminin but not fibronectin, via carbohydrate-dependent interactions with the integrin. The
biological significance of this cross-linking ability of galectins has been recently more directly
demonstrated in sponges, where the sponge galectin was shown to be a linking protein between
the sponge cells and a glycosylated aggregation factor.
Apoptosis
Thymocyte maturation in the thymus is accompanied by changes in the sialylation of the cells;
i.e., immature cells are less sialylated than mature cells, and this correlates with expression of
ST3Gal-I sialyltransferase (Chapters 8, 33, and 34). These observations led to studies regarding
the possibility that maturing thymocytes interact with endogenous thymic lectins. Subsequent
experiments in vitro demonstrated that galectin-1, which is expressed by thymic epithelium, can
bind to both activated and resting thymocytes and that its binding to activated T cells and T-cell
leukemic cell lines induces apoptosis, or programmed cell death. Apoptosis is a recognized form
of biologically controlled cell death that is controlled by specific surface receptors capable of
oligomerizing and signaling an intracellular protease (caspase) cascade. The thymocyte receptors
for galectin-1 appear to be CD45 and CD43, both of which are highly glycosylated membrane
glycoproteins. Interestingly, galectin-9, which is also highly enriched in thymus, can also induce
apoptosis of human thymocytes. Thus, both galectins 1 and 9 may contribute to regulating T-cell
development.
In contrast, other studies have now shown that galectin-3 may have an anti-apoptotic effect in T
cells infected with human T-cell leukemia virus type I, compared to uninfected cells, perhaps
through the ability of galectin-3 to interact with the apoptosis-inhibiting protein Bcl-2. Similarly,
when galectin-3 is overexpressed in human breast carcinoma cells, it inhibits drug-induced
apoptosis. Thus, the galectins may have a general function of regulating apoptosis in a variety of
cell types. This regulation may involve both cell surface receptors and cytoplasmic interactions
with other proteins that also participate in apoptosis-regulated pathways.
Intracellular Ligands for Galectins
Galectin-3 occurs both in the nucleus and cytoplasm of many cells and on the cell surface. On
tissue macrophages, galectin-3 is a major surface antigen, known as the Mac-2 antigen. The
multiple locations of this galectin-3 have suggested that it may have multiple functions. For
example, galectin-3 is located in the nucleus of some cells and may be functionally important in
RNA synthesis by being a component of the splicing complex with pre-mRNA. Galectin-3 can
also bind cytokeratin, a cytoskeletal protein. Unexpectedly, cytokeratin was recently shown to
contain a unique modification with GalNAcα1 3Gal(NAc)β1 R. Galectin-3 binds with high
affinity and in a carbohydrate-dependent fashion to cytokeratin, but the biological significance of
this interaction is not clear.
Genetic Mutations in Galectins
Because galectin expression is highly developmentally regulated, it was proposed that these
proteins are important in the developmental processes. To explore this possibility, null mutations
for galectins 1 and 3 have been generated in mice. Unexpectedly, neither null mutations for
either lectin nor combined null mutations for both lectins appear to result in obvious
developmental defects in the animals. It is possible that the redundancy of galectin family
members may contribute to survival of these null mutants or that these particular galectins are
involved in post-developmental processes, such as immune regulation. Consistent with this
possibility, recent investigations into the inflammatory response in galectin-3 null mice have
demonstrated a measurable decline in infiltrating neutrophils, suggesting that this galectin may
be important in neutrophil functions. It will be interesting to observe the phenotypes of these
combined null mutants when provided with a variety of environmental, pathogenic, and
antigenic challenges. Obviously, much remains to be learned about this large family of lectins
and their biological roles.
Future Directions
The exciting new discoveries of the wide variety of galectins and their potential functions in cell
adhesion, apoptosis, and gene expression suggest that these proteins have fundamental roles in
animal development and cellular differentiation and physiology. We still know little, however,
about the macromolecular ligands for the galectins, how they are secreted from cells, why
galectins all galectins appear to bind β-galactosides, and why there are so many different
galectins.
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28. Microbial Carbohydrate-binding Proteins
Diego).
MANY PATHOGENIC MICROORGANISMS exploit host cell-surface glycoconjugates as

receptors for cell attachment, tissue colonization, and invasion. This chapter provides examples
of proteins on the surface of microorganisms (adhesins or hemagglutinins) and their
carbohydrate-binding partners on mammalian cell surfaces (receptors). Examples include viruses
binding to sialic acids and glycosaminoglycans, and bacteria and toxins binding to
glycosphingolipids.
Introduction (1 8)
Many microbial interactions with animal hosts involve attachment to epithelial cells lining the
respiratory tract or the gastrointestinal tract, which are exposed to the environment. For infection
to occur, bacteria, viruses, and parasites must pass through the glycocalyx that surrounds the
cells, bind to cell surfaces or exposed ECM, and colonize the tissue. The first step involves
adhesion mediated through specific proteins on the surface of the microorganism, called either
bacterial adhesins or viral hemagglutinins, and ligands on the surface of the mammalian cells,
called receptors. (Note that the term receptor in this case is equivalent to "ligand" for animal cell
lectins.) During the last 15 years, many adhesins and hemagglutinins have been described,
cloned, and characterized. In some cases, binding involves protein ligands, but many bind to
complex carbohydrates found on glycoproteins, glycolipids, and proteoglycans. Binding can lead
to cell invasion, tissue destruction, and systemic infection.
Many adhesins are lectins and may contain carbohydrate recognition domains that bind to the
same carbohydrates as endogenous mammalian lectins (see Chapter 22). The adhesin present on
influenza A virus, the hemagglutinin, is by far the best-studied system to date, in part because of
its importance in public health. Wiley and his associates crystallized the viral hemagglutinin,
determined its structure in 1981, and later solved the structure of cocrystals prepared with
sialyllactose. Since then, the crystal structures for several other viral hemagglutinins and
bacterial toxins have been determined as well. Like animal cell lectins, some microbial adhesins
bind to terminal sugar residues, whereas others bind to internal sequences found in linear or
branched oligosaccharide chains (see Chapter 29). The detailed maps of the carbohydrate-
recognition domains have provided much insight into binding mechanisms and have led to the
development of ligands with even greater affinity. These synthetic ligands form the basis for
therapeutic agents to treat infection.
It should be kept in mind that colonization of tissues by microorganisms is not always

pathogenic. For example, the normal flora of the gastrointestinal tract is determined by
appropriate and desirable colonization by beneficial bacteria. Selective colonization of tissues by
microorganisms defines the tropism of the infectious agent. To a large extent, tropism is
determined by the composition and structure of the carbohydrate receptors expressed by cells in
the target tissue.
Methods for Studying Microbial Binding and Adhesion (7,9 12)
To study microbial adhesion in vitro, adherence assays have been developed in which
microorganisms are challenged to adhere to cultured cells, tissue sections, thin-layer
chromatography plates, blots prepared from SDS-PAGE gels, or plastic surfaces coated with
oligosaccharides or glycoconjugates. Many intact viruses cause hemagglutination if the red
blood cells contain the cognate carbohydrate receptor.
In a typical overlay procedure, an immobilized glycoconjugate (separated by electrophoreses or

chromatography) is incubated with a suspension of bacteria, virus, or toxin, and the extent of
binding is determined by autoradiography, antibody staining, or direct visualization of the bound
microorganism. These techniques have great sensitivity, especially when the putative receptors
have been concentrated into bands (e.g., on a thin-layer plate; see Figure 28.1), which is thought
to mimic the high density of ligands found on cell surfaces. To learn more about the ligand-
receptor interaction, adherence or binding can be measured in the presence of a competitive
ligand, such as another carbohydrate, or at different pH values and salt concentrations, or in the
presence of divalent cations.
Figure 28.1. One way to find carbohydrate receptors is through an overlay technique. In the
depicted thin-layer plate, labeled bacteria bind to a relatively minor glycolipid band.
In addition to direct binding experiments, one can examine how removal of a suspected
carbohydrate receptor from target cells affects adhesion. For example, treatment of red blood
cells with sialidase ablates hemagglutination by influenza virus. Resialylation with a specific
sialyltransferase and CMP-sialic acid restores defined types of linkages (α2 3 or α2 6), thus
allowing measurement of the linkage specificity of binding. The glycoconjugate composition can
also be modified metabolically (see Chapters 6 and 40), by mutation of genes encoding the
biosynthetic enzymes (see Chapters 31 and 32), or by transfection of cells with
glycosyltransferases.
Many glycoreceptors and microbial adhesins have been identified in the manner described
above. As adhesin cDNAs become available through molecular cloning, refined binding assays
can be carried out using various types of protein-carbohydrate assays (cf. Chapter 29). With the
development of transgenic and gene-targeted mice with altered glycosylation (see Chapter 33), it
should be possible to correlate the results of binding studies performed in vitro with microbial
pathogenicity in vivo.
Microbial Adhesins and Cell Surface Glycoconjugate Receptors

(8,13 15)
Microorganisms have evolved adhesins that interact with glycoproteins, proteoglycans, and
glycolipids. On bacteria, many of the adhesins are protein subunits of pili (hairs), also known as
fimbrae (threads). These structures typically have a diameter of 5 7 nm and can extend 100 200
nm in length, or about one-tenth the diameter of a bacterial cell. Thus, pili extend well beyond
the glycocalyx formed from lipopolysaccharide and capsular polysaccharides (see Chapter 21),
which can actually interfere with adhesin activity. The carbohydrate recognition domain of the
adhesin is typically at the tip of the pilus.
Some adhesins are monomeric or oligomeric membrane proteins. Most bacteria (and perhaps
other microorganisms) have multiple adhesins with different carbohydrate specificities, which
help define the range of susceptible tissues (i.e., the microbe's ecological niche). Binding is
generally of low affinity, but because the adhesins and the receptors often cluster in the plane of
the membrane, the resulting strength of the interaction (avidity) can be quite strong. In fact,
adhesion can require several hundred times the force of gravity to dissociate. Perhaps an
appropriate analogy for adhesin-receptor binding is the interaction of the two faces of VelcroTM
strips.
Adhesin-receptor interactions can result in signal transduction events critical for colonization and
infection. Many microbes must fuse with the cell surface (e.g., herpes simplex virus) or with
endosomal membranes after internalization (e.g., influenza). In some cases, the microbe survives
and replicates within a phagolysosome (e.g., Chlamydia and Leishmania), which implies that the
microbe can subvert normal processing pathways inside the cell. In other cases, binding results
in a defense reaction (e.g., binding might cause epithelial cells to secrete interleukins, which
results in a mucosal immune response). The relationship between microbes and the host can be
quite complex. For example, colonization of germ-free mice with Bacteroides thetaiotaomicron,
a normal resident microbe of the small intestine, induces an α1 2 fucosyltransferase in the
mucosal epithelial cells. The bacteria bind to l-fucose residues and also use it as a carbon source.
Rotaviruses, the major killer of children worldwide, can only bind to the intestinal epithelium of
newborn infants during a period that appears to correlate with the expression of specific types
and arrangements of sialic acids on glycoproteins. Thus, the intestine is operationally a
functioning micro-ecosystem in which glycosylation plays an important part.
Adhesins That Bind to Glycolipids (1, 5, 8,16 20)
Many bacteria, bacterial toxins, and parasites bind to glycolipids, and a large number of adhesins
target Galβ4Glc-containing oligosaccharides. Sometimes the adhesin binds to terminal Galβ4Glc
in lactosylceramide, but the core oligosaccharide is often capped by other sugars (e.g., blood
group antigens). Some bacteria secrete glycosidases that expose Galβ4Glc-Cer determinants,
whereas others bind to the internal sequence Galα4Gal. Other binding specificities have been
described as well (Table 28.1).
Table 28.1. Examples of interactions of bacterial adhesins with glycans
Microorganism Target tissue Adhesin Proposed specificity
Escherichia coli urinary P-fimbrae Galα4Galβ-

Escherichia coli intestine S-fimbrae Neu5Acα3Galβ4GlcβCer
GalNAcβ4(Neu5Acα3)Galβ4GlcβCer
Escherichia coli type-1 mannose, mannans, glycoproteins
fimbrae
Propionobacterium skin, ? Galβ4Glcβ-Cer
intestine
Streptococcus pneumoniae respiratory ? GlcNAcβ3Gal-
Staphylococcus urinary ? Galβ4GlcNAc-
saprophyticus
Actinomyces naeslundii mouth ? Galβ3GalNAcβ-
Pseudomonas aeruginosa respiratory ? GalNAcβ4Gal
Neisseria gonorrhoeae genital ? Galβ4Glcβ-Cer,
NeuAcα3Galβ4GlcNAc-
Helicobacter pylori stomach BabA [Fucα2]Galβ3[Fucα4]GlcNAc (Lewis
B)-
Bordetella pertussis respiratory FHA sulfated glycolipids, heparin
Mycoplasma respiratory ? Neu5Acα3Galβ4GlcNAcβ-
The specificity of binding can explain the tissue tropism of the organism. The columnar
epithelium that lines the large intestine expresses Galβ4Glc-Cer, whereas the cells lining the
small intestine do not. Thus, Bacterioides, Clostridium, Escherichia coli, and Lactobacillus only
colonize the large intestine under normal conditions. Several uropathogenic E. coli strains
recognize Galα4Gal-containing glycolipids as either internal or terminal structures on bladder
epithelia, consistent with the correlation of urinary tract infections with the P1 blood group
phenotype (see Chapter 16). The presence or absence of these antigens may be a factor
influencing the adherence of bacteria to the uroepithelial cells.
In addition to the organisms listed in Table 28.1, a variety of secreted bacterial toxins also bind
to glycolipid determinants (Table 28.2). The best-studied example is the toxin from Vibrio
cholera (cholera toxin), which consists of A and B subunits, in the ratio AB5. Its crystal structure
shows that the B subunits bind to GM1 ganglioside receptors through carbohydrate-recognition
domains located on the base of the subunits (Figure 28.2 and Figure 4.2). The A subunit (toxin)
is loosely held above the plane of the B subunits, with a single α-helix penetrating through a
central core created by the pentameric B subunits. Upon binding to membrane glycolipids
through the B subunits, the A subunit is delivered to the interior of the cell by an unknown
mechanism. The structures of related toxins from Shigella dysenteria, Bordetella pertussis, and
E. coli have also been solved.
Table 28.2. Examples of glycosphingolipid receptors for bacterial toxins
Microorganism Toxin Target Proposed receptor sequence

tissue
Vibrio cholera cholera small Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer (GM1)

toxin intestine
Escherichia coli heat-labile intestine Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer (GM1)
toxin
Clostridium tetanus nerve G1b gangliosides, with GT1b as the most efficient
tetani toxin membrane
Clostridium botulinum nerve (±NeuAcα8)NeuAcα3Galβ3GalNAcβ4
botulinum toxins (A- membrane (NeuAcα8NeuAcα3)Galβ4GlcβCer
E)
Clostridium toxin a large GalNAcβ3Galβ4GlcNAcβ3Galβ4GlcβCer
difficile intestine
Shigella shiga toxin large Galα4GalβCerGalα4Galβ4GlcβCerGlcNAcβ4GlcNAc
dysenteriae intestine
Figure 28.2. Crystal structure of cholera toxin B subunit pentamer with bound GM1
pentasaccharide. (A) Bottom view; (B) side view. (Reprinted, with permission, from [17] Merritt
et al. 1994 [© Cambridge University Press].)
Adhesins That Bind to Glycoproteins (5,21 34)
Compared to glycolipids, fewer interactions are known to occur between microorganisms and
glycoproteins. This apparent preference for glycolipids may be related to the juxtaposition of
glycolipid glycans to the membrane surface compared to the more distal location of glycoprotein
glycans. Binding of a toxin or bacterium to a glycolipid might provide a higher likelihood of
further interactions with the membrane (e.g., binding to another receptor or membrane
intercalation). In fact, Shiga toxin will bind to Galα4Gal determinants on both glycolipids and
glycoproteins, but only the binding to glycolipids results in cell death. Alternatively, the apparent
preference for glycolipids may often reflect the better methodology available for analyzing
glycolipids as receptors (e.g., overlay methods). In any case, several viruses and parasites have
infection strategies based on binding to glycoproteins (Table 28.3). For example, Entamoeba
histolytica expresses a 260-kD heterodimeric adhesin that binds to terminal Gal/GalNAc residues
on glycoproteins and glycolipids. Binding may have a role in attachment, invasion, and cytolysis
of intestinal epithelium, and it may function in binding bacteria as a food source.
Table 28.3. Examples of glycoprotein carbohydrate receptors for
microorganisms
Microorganism Target tissue Proposed receptor sequence
Entamoeba histolytica small intestinal mucosa terminal Gal/GalNAc residues

Plasmodium red blood cells sialic-acid-containing glycans (Neu5Acα2
falciparum 3Galβ-)
Trypanosoma cruzi blood sialic-acid-containing glycans (?)
Cryptosporidium intestinal epithelium terminal Gal-GalNAc
parum
Giardia lambia duodenum and small mannose-terminated oligosaccharides
intestine
Influenza upper respiratory tract sialic-acid-containing glycans
hemagglutinin mucosa
By far, the best studied example of a glycoprotein adhesin is the influenza hemagglutinin, which
binds to sialic-acid-containing glycans. Influenza A hemagglutinin associates into trimeric
oligomers that enhance the overall binding to multivalent surfaces. The specificity of this
interaction for A and B subtypes of influenza varies considerably, with human influenza viruses
binding only to cells containing Siaα6Gal and other animal influenza viruses binding to
Siaα3Gal linkages. This linkage preference is due to a single amino-acid change in the
hemagglutinin. Influenza C, in contrast, binds preferentially to glycoproteins containing 9-O-
acetylated sialic acids. Having the crystal structure available has made it possible to design better
synthetic ligands. For example, the crystal structure revealed a hydrophobic pocket near the
carbohydrate-binding site, which predicted that sialosides containing a hydrophobic aglycone
would bind with greater affinity.
In addition to the hemagglutinin, influenza A and B virions express a sialidase (traditionally

called neuraminidase) that cleaves sialic acid from glycoproteins. Its function may include
prevention of viral aggregation by removal of sialic acid residues from virion envelope
glycoproteins, dissociation of virions as they bud from the cell surface, or desialylation of
soluble mucin from sites of infection in order to improve access to membrane-bound sialic acids.
Interestingly, the sialidase specificity with respect to sialic acid linkage tends to evolve in
parallel to the hemagglutinin, suggesting an important trophic function as well. In influenza C
virus, a single glycoprotein contains both the hemagglutinin activity and the receptor destroying
activity, which in this case is an esterase that cleaves the 9-O-acetyl group from acetylated sialic
acid receptors. Powerful inhibitors have been designed based on the crystal structure of the
sialidase from influenza A. Some of these inhibit enzyme activity at nanomolar concentrations
and are in clinical studies to determine their utility as antiviral agents (see Chapter 41). Many
other viruses (e.g., reovirus, rotavirus, Sendai, and polyomavirus) also appear to use sialic acids
for infection.
The interaction of Plasmodium falciparum (malaria) merozoites with red blood cells also
depends on sialic acids present on the host cell. In this organism, attachment is mediated by a
specific sialic-acid-binding adhesin on merozoites called EBA-175. The adhesin binds to sialic
acids present on the major erythrocyte membrane protein, glycophorin, and prefers Neu5Ac
rather than 9-O-acetyl Neu5Ac or Neu5Gc. Soluble Neu5Ac and Neu5Acα6Gal-containing
oligosaccharides do not competitively inhibit the binding of EBA-175 to erythrocytes, but
Neu5Acα3Gal-containing oligosaccharides are effective inhibitors, indicating that the adhesin is
sensitive to the underlying oligosaccharide structure. Binding to erythrocytes leads to invasion
and eventual production of additional merozoites. Other organisms expressing sialic acid
adhesins also can bind to erythrocytes (e.g., influenza), but these interactions cannot lead to
productive infections in these nonnucleated cells. Thus, in these circumstances, the erythrocyte
might be considered a clearance mechanism for these agents (see Chapter 3). Plasmodium-
infected erythrocytes also express glycosaminoglycan-binding proteins that are thought to
facilitate adherence of the infected cells to tissues. As described below, in certain types of
malaria, another developmental form of the parasite, the circumsporozoite, selectively invades
hepatocytes by way of a heparan-sulfate-binding adhesin called the circumsporozoite protein.
Adhesins That Bind to Glycosaminoglycans (7,35 36)
Many bacteria, parasites, and viruses use proteoglycans as adhesion receptors (Table 28.4). Most
microorganisms bind to heparan sulfate rather than chondroitin sulfate, possibly due to its greater
prevalence on cell surfaces (see Chapter 29). Unlike adhesins that interact with glycolipids and
glycoproteins, the GAG-binding adhesins presumably pick out binding sites within the
polysaccharide chains as opposed to binding to terminal sugars. To date, the precise structure of
the carbohydrate recognition domain of a microbial glycosaminoglycan adhesin has not yet been
determined. Dengue flavivirus, the causative agent of dengue hemorrhagic fever, binds to
heparan sulfate. Modeling the primary sequence of the viral envelope protein on the crystal
structure of a related virion envelope protein suggests that the heparan-sulfate-binding site may
lie along a groove in the protein lined by positively charged amino acids (Figure 28.3). Thus, the
GAG-binding adhesins may have a more open structure, consistent with the binding sites of
other heparin-binding proteins (see Chapter 29).
Table 28.4. Examples of microorganisms that bind proteoglycan receptors on
eukaryotic cells
Microbe Target tissue
Bordetella pertussis ciliated epithelium in respiratory tract

Chlamydia trachomatis eyes, genital tract, lymphoid tissues
Haemophilus influenzae respiratory epithelium
Borrelia burgdorferi endothelium, epithelium, extracellular matrix
Neisseria gonorrhoeae genital tract
Staphylococcus aureus connective tissues, endothelial cells
Streptococcus pyogenes cardiac and kidney tissues
Mycobacterium tuberculosis respiratory epithelium
Plasmodium falciparum hepatocytes, placenta
(circumsporozoites)
Leishmania amazonensi macrophages, fibroblasts, epithelium
(amastigotes)
Trypanosoma cruzi heart, GI tract, nervous system, extracellular matrix
Herpes simplex virus (HSV) mucosal surfaces of mouth, eyes, genital tract, respiratory
tract; latent in nerve ganglia
Dengue flavivirus macrophages?
Cytomegalovirus neutrophils, monocytes
HIV-1 T lymphocytes
Figure 28.3. Putative structure of the heparin-binding site in dengue virus envelope protein.
Note the alignment of positively charged amino acids along an opening on the face of the
protein. (Reprinted, with permission, from [35] Chen et al. 1997.)
The different tissue tropism of glycosaminoglycan-binding microbes may reflect variation in the
fine structure of the heparan sulfate chains. Herpes simplex virus glycoproteins gpB and gpC
bind to heparin and have different requirements for sulfate groups along the chains (Table 28.5).
The differential expression of sequences rich in 2-O-sulfated uronic acids on various cells
therefore could partly explain the different tissue tropism of HSV-1 and HSV-2 subtypes. P.
falciparum sporozoites (malaria) also bind to heparin and heparan sulfate in a tissue-specific
manner, with preferred binding to the basolateral surface of hepatocytes and the basement
membrane of kidney tubules. The circumsporozoite protein that covers the sporozoite cell
surface mediates binding. The carboxyl terminus of the protein contains positively charged
residues. Clustering of the circumsporozoite protein on the surface of the organism may generate
a high concentration of positively charged residues that facilitate binding. How this would
achieve selective binding to hepatocyte proteoglycans is unclear.
Table 28.5. Microbial heparin-binding proteins
Microorganism Protein Binding domains
Plasmodium circumsporozoite carboxy-terminal region II+

falciparum protein PCSVTCGNGIQVRIK
HIV gp120 V3 loop NNTRKSIRIQRGPGRAFVTIGKIG
HSV gpC region SP-1, RxxxRCFRxxxR
Bordetella pertussis filamentous carboxy-terminal region
hemagglutinin
In many cases, the proteoglycans may be part of a coreceptor system in which the
microorganisms make initial contact with a cell surface proteoglycan, and later with another
receptor. For example, HSV binds to heparan sulfate on the cell surface, but infection requires
additional nonproteoglycan receptors. The coreceptor role of proteoglycan is reminiscent of the
formation of ternary complexes required for antithrombin inhibition of thrombin and basic FGF
signaling (see Chapters 29 and 34). Although it is clear that cell surface proteoglycans act as
adhesion receptors, their role in invasion and pathogenesis is unclear. The HSV glycoprotein
gpB binds heparan sulfate and promotes adherence, as well as virus-cell fusion and syncytium
formation. The mechanism by which heparan sulfate facilitates membrane fusion is unknown,
but perhaps it acts like a template facilitating the association of fusogenic membrane proteins (cf.
Chapter 29).
Microbial Carbohydrate Ligands for Animal Cell Lectins (3,37 44)
Some microorganisms mimic carbohydrate receptors on mammalian cell surfaces. For example,
Chlamydia has a complex mode of adhesion, in which heparan sulfate is thought to act as a
bridge, binding both host-cell protein receptors and Chlamydia receptors. A minimal
decasaccharide is needed, and on the basis of competition studies with chemically modified
heparin, the binding sequences for host and microbial receptors may differ. Interestingly,
Chlamydia produces its own sulfated heparin-like molecule, which may provide the opportunity
to infect cells with low levels of endogenous heparan sulfate or with heparan sulfate that lacks
appropriate binding sequences. Leishmania appears to utilize heparan sulfate in a similar way.
This is an open area of research since the heparin-binding proteins have not yet been described in
any detail, nor has the biosynthesis of a heparan-sulfate-like chain been studied to any extent in
these microbes.
Trypanosoma cruzi has developed an interesting strategy of molecular camouflage in which a

parasite-encoded trans-sialidase transfers sialic acid from serum glycoproteins in the host to
membrane proteins on its own surface. Although the primary function of this reaction is most
likely to cover surface glycans as a way of preventing host immune reactivity, the sialylated
glycans might be recognized by sialic-acid-binding lectins on cells (see Chapters 22 27). The
trans-sialidase may also act as an adhesin. Neisseria gonorrhoeae uses low levels of tissue CMP-
sialic acid to cover itself with sialic acid residues, making it resistant to complement.
Schistosomes, a parasitic filarial worm, contain the Lewis X antigen that is also found on human
leukocytes. Since Lewis X is recognized by selectins, the presence of these carbohydrates may
provide a mechanism for attachment or transcellular migration. (However, these glycans also
generate a massive anti-Lewis X antibody response in the host.) In a similar way, the capsules
surrounding bacteria, lipopolysaccharide, and yeast cell walls contain oligosaccharide sequences
that may be recognized by mammalian cell lectins. For example, yeast mannans are recognized
by both soluble and macrophage mannose-binding protein, which has an important role during
the preimmune phase in infants. The structure and biology of these types of carbohydrates and
carbohydrate-binding proteins are discussed in Chapters 19, 21, and 36.
Future Directions (5,45 48)
The interactions described above suggest a correlation between adhesin-receptor interactions and
microbial pathogenesis. Examination of virulent and nonvirulent isolates has revealed a
dependence on carbohydrate interactions in some cases, but much additional work is needed in
this area. The use of mouse models with genetic defects in specific steps in glycosylation (see
Chapters 32 and 33) will undoubtedly prove to be useful in this regard. In the end, however, it is
the ability to interfere with these processes that will establish whether a causal relationship exists
and whether the interaction is a suitable target for drug intervention.
Adhesin molecules represent potential targets for generating antibodies for vaccination.
However, the presence of multiple adhesins on cells may frustrate this strategy for controlling
infection. Another idea suggested by in vitro binding studies is to administer oligosaccharides
known to interact with an adhesin and to measure microbial distribution, tissue colonization, and
host survival in a suitable animal model. The ability of exogenous heparin and related
polysaccharides to inhibit viral replication suggests that this approach might lead to
polysaccharide-based antiviral pharmaceutical agents. Multivalent ligands should prove even
more potent, but their use may be limited to the respiratory and gastrointestinal tracts because of
difficulties in their delivery.
As more crystal structures become available, the ability to custom design small-molecule
inhibitors that fit into the carbohydrate recognition domains and the active sites of adhesins
should improve. Already, the structure of the influenza hemagglutinin and neuraminidase has
suggested numerous ways to modify sialic acid to better fit the active sites. Some of these
compounds are already being tested in human trials (see Chapters 40 and 41).
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29. Glycosaminoglycan-binding Proteins
Diego).
THE GLYCOSAMINOGLYCANS FOUND ON PROTEOGLYCANS are bound by a variety of

proteins, some of which have characteristic arrangements of positively charged amino acids.
This chapter focuses on examples of glycosaminoglycan-binding proteins for which specific
biological roles are known and where the carbohydrate-binding sequences are known. Methods
for measuring GAG-protein binding are also discussed. Information about three-dimensional
structures of glycosaminoglycans and glycosaminoglycan-binding proteins is presented.
Introduction (1)
Glycosaminoglycans interact with a variety of proteins. In fact, the binding of heparin to
antithrombin was one of the first examples of a specific, physiologically relevant interaction of a
protein with a complex carbohydrate (see Chapter 22). The elucidation of the substructures of
heparin that bind to antithrombin involved detailed chemical analysis of heparin fragments,
chemical synthesis of oligosaccharides, peptide mapping and mutational studies of antithrombin,
and ultimately the crystallization of antithrombin-heparin complexes. The results show that high-
affinity binding depends on a relatively rare pentasaccharide sequence in heparin interacting with
positively charged amino acids in a cleft formed by the apposition of two α helices. These
findings serve as a paradigm for GAG-protein interactions in other systems. In the last 5 years,
several other GAG-protein systems have been elucidated and some of the rules for thinking
about these interactions have emerged.
Many Glycosaminoglycan-binding Proteins Occur in Nature (1 9)
More than 100 GAG-binding proteins have been described in the literature, a few of which are
presented in Table 29.1. The interaction between GAGs and proteins can have profound
physiological effects on hemostasis, lipid transport and absorption, cell growth and migration,
and development. Binding to GAGs can result in immobilization of proteins at their sites of
production and in the matrix for future mobilization, regulation of enzyme activity, binding of
ligands to their receptors, and protection of proteins against degradation. In some cases, the
interaction has been shown to depend on a minor but very specific sequence of modified sugars
in the GAG chain. Most of the GAG-binding proteins that have been described interact with
heparin/heparan sulfate or hyaluronic acid; relatively few are known to interact with chondroitin
sulfate or keratan sulfate with comparable avidity and affinity. The reasons for this selectivity are
not known.
Table 29.1. Some examples of GAG-binding proteins and their biological
activity
Protein GAG Physiological effect of binding
Antithrombin heparin/HS systemic anticoagulation

Heparin cofactor II DS and heparin localized anticogulation
t-Plasminogen activator heparin/HS clot dissolution
Fibroblast growth factors heparin/HS mitogenesis
Hepatocyte growth factor heparin/HS mitogenesis
Chemokines IL-8/MIP-1b heparin/HS inflammation
L and P selectins heparin/HS inflammation
Extracellular superoxide dismutase heparin/HS host defense
Lipoprotein lipase heparin/HS localized lipolysis/turnover
apoE heparin/HS lipoprotein clearance
Fibronectin heparin/HS cell adhesion
Laminin heparin/HS cell adhesion
Type V collagen heparin/HS cell adhesion
Thrombospondin heparin/HS/CS cell adhesion/growth
CD44 hyaluronan cell adhesion/motility
RHAMM hyaluronan cell adhesion/motility
Aggrecan hyaluronan cartilage formation
Methods for Measuring Gag-Protein Binding (10 13)
Numerous methods are available for analyzing GAG-protein interactions, and some provide
direct measurement of Kd values. The most common method involves affinity fractionation of
proteins on Sepharose columns containing covalently linked GAG chains, usually heparin. The
bound proteins are eluted with different concentrations of NaCl, and the concentration required
for elution is generally proportional to the Kd. High-affinity interactions require at least 1 m
NaCl to displace bound ligand, which translates into Kd values of 10-7 to 10-9m (determined
under physiological salt concentrations by equilibrium binding). Proteins with low affinity (10-4
to 10-6m ) either do not bind under normal conditions (0.15 m NaCl) or require only 0.3 0.5 m
NaCl to elute. Typically, this method provides an assessment of relative affinity compared to
other proteins. By immobilizing the protein and passing GAGs over the column, the technique
also makes possible the purification of subsets of glycans with different affinities. In a few cases,
the glycans may bind in a divalent-cation-dependent manner (i.e., displaceable by chelation of
cations).
An electrophoretic separation technique called affinity coelectrophoresis allows simultaneous

measurement of affinity and selectivity. In this technique, radioactive GAGs are electrophoresed
through acrylamide gels containing a binding protein. Association of the GAGs with the protein
retards the mobility of the chains, which can be visualized by autoradiography. By varying the
concentration of protein in the gel, an apparent Kd value can be measured. This method also
allows separation of subpopulations of GAGs that bind from those that do not bind. Using this
technique, it is possible to show that only a subfraction of heparin binds to antithrombin (30% of
a typical preparation).
Another technique is a filter-binding assay in which specific proteins are incubated in solution
with radioactive GAGs, followed by separation of free and protein-bound carbohydrate on a
nitrocellulose filter (free carbohydrate chains do not bind to the filters). The advantage of this
technique is that the measurements are done in solution, and true equilibrium values for
association can be measured. By adding competing GAG chains to the assay, competition for the
protein ligand can be measured between a labeled GAG chain and unlabeled, size-fractionated
oligosaccharides. Various conditions can also be tested, e.g., different concentrations of salt,
divalent cations, pH, and temperature.
Regardless of the technique used, it must be kept in mind that binding to immobilized GAG
chains on a column or a solid support is not likely to be the same as binding to proteoglycans on
the cell surface or in the ECM. To determine the physiological relevance of the interaction,
binding may be measured under conditions that can lead to a biological response. For example,
one can measure binding to cells with altered GAG composition (see Chapter 31) or after
treatment with specific lyases to remove GAG chains from the cell surface (see Chapter 11), and
then measure whether the response is the same as that in the presence of GAG chains. The
interaction can then be studied more intensively using the in vitro assays described above.
Conformational and Sequence Considerations (14 15)
As mentioned above, most GAG-binding proteins interact with HS or heparin. It is unclear why
this should be the case, since HS, CS, KS, and hyaluronan are widely distributed in tissues. At
first glance, this might be explained on the basis of the greater heterogeneity of modified sugar
residues in HS, but several hyaluronan-binding proteins have been described even though
hyaluronan lacks any sugar modifications. Conrad has suggested that the difference may lie in
the conformational options found in heparin and HS afforded by the unusual conformation of
iduronic acid, as described below. Although this hypothesis is difficult to prove, it is instructive
to understand the influence of IdoA on the secondary structure of GAGs.
GAGs are linear helical structures, consisting of alternating residues of GlcNAc or GalNAc with
GlcA (except for KS, which consists of alternating GlcNAc and Gal residues; see Chapter 11).
Inspection of heparin oligosaccharides containing highly modified domains ([GlcNS6S-
IdoA2S]n) shows that the N-sulfate and 6-O-sulfate groups of each disaccharide repeat lie on
opposite sides of the helix from the 2-O-sulfate and carboxyl groups (Figure 29.1). Because of
rotational restrictions about the anomeric linkages from the N-acetyl and carboxyl groups, the
chains are relatively rigid, with limited end-to-end bending possible compared to polypeptides.
Thus, the GAG chains present a relatively fixed orientation of substituents.
Figure 29.1. Structure of heparin. A space-filling model of a heparin oligosaccharide (14-mer)
deduced by NMR is shown in panel A and the same structure in stick representation is shown in
panel B. Both renderings were made with RASMOL using data from the Molecular Modeling
Database (MMDB Id: 3448) at the NCBI. (Red) Oxygen; (gold) sulfur; (blue) nitrogen; (gray)
carbon; (white) hydrogen.
Analysis of the conformation of individual sugars shows that GlcNAc and GlcA residues assume
a preferred conformation in solution, designated 4C1 (indicating that carbon-4 is above the plane
defined by carbons 2, 3, and 5 and the ring oxygen and that carbon-1 is below the plane). In
contrast, IdoA assumes the 1C4 or the 2S0 conformation (Figure 29.2), which reorients the
position of the sulfate substituents and therefore creates a different orientation of charged groups.
When a protein binds to an HS chain, it can potentially induce a change in conformation in
regions rich in IdoA, which may result in a better fit and enhanced binding. IdoA residues are
always found in domains rich in N-sulfated glucosamine residues and O-sulfates (for
biosynthetic reasons, see Chapter 11), which is also where proteins usually bind. Thus, the
greater degree of conformational flexibility in these modified regions may explain why so many
more proteins bind with high affinity to heparin, HS, and DS than to other GAGs. Obvious
exceptions exist, which may imply evolution of proteins that more closely approximate the
structure of the other GAGs. Another possibility is that binding to other GAGs might require
longer oligosaccharides interacting over larger domains of the protein.
Figure 29.2. Conformations of individual sugars. GlcA exists in the 4C1 conformation, whereas
IdoA exists in equally energetic conformations designated 1C4 and 2S0.
Do Consensus Sequences Exist in Gag-binding Proteins? (4, 8,16 19)
The discovery of multiple GAG-binding proteins led a number of investigators to examine

whether a consensus sequence for GAG binding exists. In retrospect, this strategy was overly
simplistic, since it assumed that all GAG-binding proteins would recognize the same
oligosaccharide sequence within heparin, or at least sequences that would share many common
features. We now know that GAG-binding proteins interact with different oligosaccharide
sequences (Table 29.2). The binding sites in the protein always contain basic amino acids (lysine
and arginine) whose positive charges presumably interact with the negatively charged sulfates
and carboxylates of the GAG chains. However, the arrangement of these amino acids is quite
variable, consistent with the variation in fine structure of the GAG partner. A preference for
amino acids other than lysine or arginine does not appear to exist.
Table 29.2. Oligosaccharides recognized by GAG-binding proteins
Most proteins are formed from α helices, β strands, and loops. Therefore, to engage a linear
GAG chain, it would be predicted that the positively charged amino acid residues would have to
line up along the same side of the protein segment. α helices have periodicities of 3.4 residues
per turn, which would require the basic residues to occur every third or fourth position along the
helix in order to align with an oligosaccharide. In β strands, the side chains alternate sides every
other residue. Thus, positively charged residues in a GAG-binding protein should be located very
differently if the peptide chain folds into a β-strand.
On the basis of the structures of several heparin-binding proteins that were available in 1991,
Cardin and Weintraub proposed that typical heparin-binding sites had the sequence XBBXBX or
XBBBXXBX, where B is lysine or arginine and X is any other amino acid. From the structural
arguments provided above, only some of the basic residues in these sequences could participate
in GAG binding, the actual number being determined by whether the peptide sequence exists as
an α-helix or as β-sheet. It is now known that the presence of these sequences in a protein merely
suggests a possible interaction with heparin (or another GAG chain), but it does not prove that
the interaction occurs under normal conditions. In fact, the predicted binding sites for heparin in
bFGF turned out to be incorrect once the crystal structure was determined. It is likely that
binding involves multiple protein segments that juxtapose positively charged residues into a
three-dimensional recognition site. The specific arrangement of residues should vary according
to the type and fine structure of oligosaccharides involved in binding.
In host plant and animal lectins and in antibodies that recognize carbohydrates, the carbohydrate
recognition domains are typically shallow pockets that engage the terminal sugars of the
oligosaccharide chain (see Chapters 4 and 22). In GAG-binding proteins, the protein binds to
sugar residues that lie within the chain instead of at the terminus. Therefore, the binding sites in
GAG-binding proteins consist of clefts or sets of juxtaposed surface residues rather than pockets.
Given that GAGs exist in a helical conformation, only those residues on the face toward the
protein interact with amino acid residues; those on the other side of the helix might be free to
interact with a second ligand. Alternatively, residues in a binding cleft could interact with both
sides of the helix. Finally, it should be kept in mind that the oligosaccharides that bind represent
only a small segment of the GAG chain, and therefore a single glycan can bind multiple protein
ligands.
In contrast to HS, one might expect that a better consensus would exist for HA-binding proteins
due to the uniformity of the repeats in HA ([GlcAβ1 3GlcNAcβ1 4]n). Indeed, a consensus
sequence for HA binding was deduced by comparing a number of HA-binding proteins: BX7B,
where B is arginine or lysine (Table 29.3). However, close inspection of these motifs indicates
that they contain additional basic residues in the intervening seven amino acids. Note that the
actual location of the basic residues would allow their alignment if the peptide segment assumed
an α-helical conformation in the native protein.
Table 29.3. Proteins known to interact with HA
Protein Peptide sequence involved in binding
RHAMM 401KQKIKHVVKLK411
Link protein 316RYPISRPRKR325
CD44 38KNGRYSISR46
Hyaluronidase 96RGTRSGSTR104
Aggrecan 71RIKWSRVSK79
2109KRTMRPTRR2117
Antithrombin-Heparin: A Paradigm for Studying GAG-binding

Proteins (1,3, 16,20 22)
The best-studied example of protein-GAG interaction is the binding of antithrombin to heparin
and HS. This interaction is of great physiological importance in hemostasis, since heparin is used
clinically as an anticoagulant. Antithrombin is a member of the serpin family of protease
inhibitors, many of which bind to heparin. Binding has a twofold effect: First, it causes a
conformational change in the protein and activation of the protease-inhibiting action, resulting in
a 1000-fold enhancement in the rate at which it inactivates thrombin and Factor Xa. Second,
heparin acts as a template enhancing the physical approximation of thrombin and antithrombin.
Thus, both the protease (thrombin) and the inhibitor have GAG-binding sites.
Heparin acts as a catalyst in these reactions, by enhancing the rate of the reaction through
approximation of substrates and conformational change. After the irreversible inactivation of
thrombin by antithrombin occurs, the complex loses affinity for heparin and dissociates. The
heparin is then available to participate in another activation/inactivation cycle.
Early studies using affinity fractionation schemes showed that only about one third of the chains
in a heparin preparation actually will bind with high affinity to antithrombin. Comparing the
sequence of the bound glycans to those that did not bind failed to reveal any gross differences in
structure, consistent with the later discovery that the binding site consists of only five sugar
residues (the average heparin chain is about 50 sugar residues). This observation can be extended
to virtually all GAG-binding proteins, inferring that the binding sites represent a very small
segment of the chain.
Cleavage of heparin into smaller oligosaccharides using heparinases and chemical

depolymerization methods revealed that a pentasaccharide is the smallest oligosaccharide that
binds to antithrombin with high affinity. Analysis of the structure of the pentasaccharide
revealed an unusual modification in which a sulfate group is attached to the 3-OH position of an
internal N-sulfated/6-O-sulfated glucosamine residue (Table 29.2). Furthermore, the preceding
residue (toward the nonreducing side) is always GlcA, whereas the next residue is always IdoA.
The 3-O-sulfated glucosamine residue is a relatively minor component in heparin and HSs. 3-O-
sulfation can occur in other sequence contexts, possibly catalyzed by sulfotransferases different
from those involved in forming the antithrombin-binding site (see Chapter 11).
Recently, crystals of antithrombin were prepared and analyzed by X-ray diffraction to 2.6 Å
resolution (Figure 29.3). The docking site for the heparin pentasaccharide is formed by the
apposition of helices A and D, both of which contain critical arginine and lysine residues at the
interface. The sequence in the D helix (124AKLNCRLYRKANKSSKLVSANR145) places many
of the positively charged residues on one face of the helix, in proximity to the arginine residues
in the A helix (41PEATNRRVW49).
Figure 29.3. Antithrombin structure. (Red) D-helix; (green) A-helix; (blue) heparin-binding
residues. All of the positively charged residues point toward the heparin-binding site. (Reprinted,
with permission, from [22] Skinner et al. 1997.)
The pentasaccharide is sufficient to activate antithrombin binding toward Factor Xa, but it will
not facilitate the inactivation of thrombin. For this to occur, a larger oligosaccharide of at least
18 residues is needed. As mentioned above, thrombin also contains a heparin-binding site, and
the larger heparin oligosaccharide is thought to act as a template for the formation of a ternary
complex with thrombin and antithrombin. In contrast to antithrombin, thrombin exhibits little
oligosaccharide specificity. As might be expected, adding high concentrations of heparin actually
inhibits the reaction, since the formation of binary complexes of heparin and thrombin or heparin
and antithrombin predominate. This important principle of "activation at low concentrations and
inhibition at high concentration" also occurs in other systems where ternary complexes form (see
Chapter 28).
Heparin is solely the product of mast cells, and although it has proven to be of great therapeutic
use, the natural oligosaccharide that participates in the control of thrombin is most likely HS
present on cell surface proteoglycans of endothelial cells. A small percentage of endothelial cell
HS contains antithrombin-binding sequence, unlike HSs from other tissues and cells contain
none. The location of these binding sequences does not appear to be restricted to specific
proteoglycans.
FGF-Heparin Interactions Enhance Stimulation of FGF-

Receptor Signal Transduction (23 30)
A large number of growth factors can be purified on the basis of their affinity for heparin. The
heparin-binding family of growth factors has grown to more than nine members and includes the
prototype, FGF-2, otherwise known as basic fibroblast growth factor. FGF-2 has a very high
affinity for heparin (Kd ~10-9m) and requires 1.5 2 m NaCl to elute from heparin-Sepharose.
FGF-2 has potent mitogenic activity in cells that express one of the FGF signaling receptors
(four are known). Cell surface HS is thought to bind to both FGF-2 and its receptor, facilitating
the formation of a ternary complex. Both binding and the mitogenic response are greatly
stimulated by heparin or HS, possibly by dimerizing the ligand (Figure 29.4).
Figure 29.4. Formation of complexes between an HS proteoglycan, bFGF, and a bFGF receptor.
The binding of heparin or HS to bFGF lowers the concentration required for receptor activation.
The costimulatory role of HS (and heparin) in this system is reminiscent of the
heparin/antithrombin/thrombin story. Indeed, the minimal binding sequence for FGF-2 also
consists of a pentasaccharide (Table 29.2). However, the pentasaccharide is not sufficient to
trigger a biological response (mitogenesis). For this to occur, a longer oligosaccharide (12-mer)
containing the minimal sequence and a flanking set of residues rich in 6-O-sulfated, N-sulfated
glucosamine units are needed, presumably because these bind to the FGF receptor. The
pentasaccharide that binds to FGF-2 is prevalent in HS and heparin. The requirement for an
adjacent sequence differing in structure that binds to the receptor reduces the probability of
finding this particular arrangement in naturally occurring HS. Thus, some preparations of HS are
inactive, and those containing only one half of the bipartite binding sequence are actually
inhibitory.
The structure of FGF-2 cocrystallized with a heparin hexasaccharide has been obtained (Figure
29.5). The heparin fragment ([GlcNS6Sα1 4IdoA2Sα1 4]3) was helical and bound to a region
of the bFGF surface containing residues Asn-28, Arg-121, Lys-126, and Gln-135 and an
additional binding site formed by Lys-27, Asn-102, and Lys-136. The linear segment consisting
of the sequence 120KRTGQYKLGSKTGPGQK136 does not conform to the Cardin and Weintraub
prediction. Only one N-sulfate and the 2-O-sulfate from the adjacent IdoA are bound to the
growth factor in the first binding domain, and the next GlcNS residue is bound to the second site,
consistent with the minimal binding sequence determined with oligosaccharide fragments (Table
29.2). No significant conformational change in FGF-2 occurred upon heparin oligosaccharide
binding, consistent with the idea that heparin primarily serves to dimerize FGF-2 and juxtapose
components of the FGF signal transduction pathway. The crystal structure of FGF-1 has also
been solved and shows similar sequences on its surface.
Figure 29.5. Stereoview of the crystal structure of FGF-2 with a heparin hexasaccharide (shown
at the top of the figure; yellow balls indicate sulfur atoms). The stereo rendering was made with
RASMOL using data from the Molecular Modeling Database (MMDB Id: 4322) at the NCBI.
Hyaluronan-Protein Interactions (31 36)
As indicated above, most reported GAG-protein interactions involve HS/heparin. However,

several HA-binding proteins have also been described that have important roles in ECM
formation, cell adhesion, and motility. In cartilage, the large CS containing proteoglycan
(aggrecan) binds to HA (Table 29.2). Link protein stabilizes the aggregate by binding to both HA
and aggrecan with 1:1 stoichiometry (see Chapter 11 and Figure 11.1). These aggregates
constitute the major space-filling components of cartilage. Defects in their formation result in
abnormal cartilage and disproportionate dwarfisms in both mice and humans (Chapter 32).
HA has long been implicated in cell adhesion and locomotion, since it is abundantly expressed
by many cell types, especially by highly motile tumor cells, and is expressed during
morphogenesis. A search for cell surface receptors revealed two major HA-binding membrane
proteins, CD44 and RHAMM (receptor for hyaluronan and motility). CD44 is abundantly
expressed by many cell types and varies markedly in glycosylation, oligomerization, and protein
sequence due to differential mRNA splicing. All of these factors are thought to influence its
affinity for HA. CD44/HA interactions can mediate leukocyte rolling and extravasation in some
tissues. Changes in CD44 expression are associated with a wide variety of tumors and metastatic
spread of cancer, although as with other tumor-associated factors, a strict correlation does not
exist. Many cells also express RHAMM protein receptors, which are involved in cell motility
and cell transformation as well, possibly through a tyrosine kinase signal transduction pathway
that targets the formation of focal cell adhesions. The RHAMM pathway is thought to induce
focal adhesions to signal the cytoskeletal changes required for elevated cell motility seen in
tumor progression, invasion, and metastasis. Like CD44, RHAMM occurs as different splice
variants, some of which may be intracellular. Both RHAMM and CD44 contain HA-binding
motifs that match the BX7B consensus (Table 29.3).
Other Gag-Protein Interactions (7,37 40)
Heparin cofactor II, another thrombin inhibitor, will bind to dermatan sulfates as well as heparin.
The structure of heparin cofactor II is very similar to that of antithrombin and has a
complementary binding site for GAG. However, unlike antithrombin, heparin cofactor II is the
only serpin known to associate with DS. Since heparin cofactor II will not interact with HSs, its
function as an anticoagulant appears to be restricted to damaged tissue where DS proteoglycans
in the matrix become exposed. As shown in Table 29.2, the DS that binds consists of a repeating
structure rich in IdoA.
L and P selectins have been shown to bind to a subfraction of HSs and heparin in a divalent-
cation-dependent manner. The selectins are calcium-dependent C-type lectins that bind certain
sialylated, fucosylated, sulfated glycoprotein ligands (see Chapter 25). However, L and P
selectins also recognize endothelial proteoglycans in a calcium-dependent manner, by way of HS
chains enriched in unsubstituted glucosamine units. Porcine intestinal heparin shows similar
properties. Thus, endothelium-derived HS chains and mast-cell-derived heparin could have a role
in modulating the biology of selectins in vivo. Although the physiological relevance of these
interactions remains to be established, the observation raises the possibility that other types of
cation-dependent receptors for GAG chains may exist.
Viruses, bacteria, and parasites use cell-surface GAGs as adhesion receptors, which facilitates
colonization and possibly invasion of cells. Many of these interactions have been traced to
specific proteins on the microbial surface that bind to plasma membrane proteoglycans. This
topic is discussed in greater detail in Chapter 29.
Future Directions
As described above, a considerable body of molecular information is now available for various
GAG-binding proteins and their carbohydrate ligands. An excellent book by Conrad on heparin-
binding proteins recently appeared and should be a mainstay for any library on the subject of
GAG-protein interactions. Unlike other carbohydrate-binding proteins, such as plant and animal
lectins (see Chapters 22 27 and 30), GAG-binding proteins form a distinct group that cannot be
categorized according to primary sequence motifs. Thus, it will be difficult to discover additional
GAG-binding proteins by comparative sequence analysis. Instead, new members will have to be
found by direct binding measurements using soluble protein preparations and immobilized GAG
chains as affinity matrices. Fortunately, GAGs are available in large quantity, making the
generation of affinity columns relatively easy compared to making comparable matrices
composed of defined N-and O-glycans. As more crystal structures are solved, and with
improvements in synthetic approaches for generating defined GAG oligosaccharides, a better
understanding of GAG-protein interactions should emerge.
The availability of crystal structures of protein-GAG complexes should make it possible to

devise model compounds as GAG agonists and antagonists. Such agents could prove useful as
drugs in situations where GAG-protein interactions are important. Thus, we can anticipate the
development of synthetic compounds for treating infectious disease (see Chapter 28), to enhance
wound healing, and perhaps to alter tumor growth and metastasis (see Chapter 35). Future
studies should focus on the isolation and characterization of the various GAG-binding proteins
involved in these and related processes.
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30. Plant Lectins
Primary contributions to this chapter were made by R.D. Cummings (The University of
Oklahoma Health Sciences Center, Oklahoma City).
MOST PLANTS CONTAIN ONE OR MORE carbohydrate-binding proteins termed plant

lectins. Although the functions of these proteins in plants are just beginning to be understood,
lectins have been enormously important to the development of the field of glycobiology. Most
lectins are multivalent and capable of agglutinating cells. In addition, since lectins differ in the
types of carbohydrate structures they recognize with high affinity, they are also useful in the
characterization of glycoconjugates. This chapter describes the diversity of the plant lectins, how
they are isolated and characterized, and what is known about their structures and the
carbohydrates they recognize. In addition, the many ways in which plant lectins can be useful in
studying glycoconjugates are described.
Historical Background on Plant Lectins (1)

The presence of proteins in plant seeds that are capable of binding to and agglutinating cells was
identified during the last century; the proteins were designated hemagglutinins for their ability to
agglutinate erythrocytes. However, it was not then known whether the proteins recognized
carbohydrates. The great biochemist J.B. Sumner crystallized the protein now known as
concanavalin A from the common jackbean Canavalia ensiformis in 1919, well before the
crystallization of the first enzyme, urease, in 1926. Sumner later discovered in 1936 that ConA is
able to bind and precipitate some polysaccharides, such as glycogen and starch. However, the
term lectin (Latin: lectus, meaning to gather or select) was not generally adopted until 1954. In
general, lectins are defined as a class of proteins of nonimmune origin that bind carbohydrates
without modifying them. Originally, the term lectin was restricted to soluble, multivalent
proteins capable of agglutination and historically was limited to proteins of plant origin.
However, today the term lectin is used in a broad sense to denote all types of carbohydrate-
binding proteins that do not catalyze reactions with their ligands.
Since this early beginning, hundreds of different plant lectins have been identified and
characterized. The availability of these proteins and their exquisite specificity for complex
carbohydrates helped to catapult the field of glycobiology into the modern era. A short history of
the field of lectin research in Table 30.1 illustrates the enormous contribution plant lectin
research has had on modern biochemistry. The lessons learned about plant lectin isolation and
characterization and assays for their binding activity directly contributed to modern
breakthroughs on the discovery of the C-type and S-type (galectin) lectins.
Table 30.1. Historical aspects of plant lectins
Date Investigators Discovery
1888 H. Stillmark Ricinus communis plant extract has hemagglutinating

properties
1890 P. Ehrlich lectins used as antigens in early immunological studies
1908 K. Lansteiner and H. different hemagglutinating properties in different plant
Raubitsheck seeds
1919 J. Sumner crystallization of ConA
1936 J. Sumner lectins found to bind sugar; ConA precipitates glycogen
1940 W. Boyd, R. Reguera, lectins found to be specific for some human blood group
and K.O. Renkonen antigens
1952 W. Watkins and W. use of lectins and glycosidases to prove that blood group
Morgan antigens are sugars and to deduce the structures of the
antigens
1954 W. Boyd and E. the name lectin is proposed to replace hemagglutinin
Shyleigh
1960 P.C. Nowell red kidney bean lectin P. vulgaris found to be mitogenic for
resting lymphocytes
1960s and J.C. Aub, M. Burgern, lectins preferentially agglutinate some animal tumor cells
1970s and G. Nicolson
Classification and Sequence of Plant Lectins (2 6)
The best-characterized family of plant lectins is the Leguminosae (Table 30.2). This family
includes lectins such as ConA, soybean agglutinin, and lentil lectin. Two other smaller families
of plants whose lectins have been characterized are the Gramineae (cereals, such as wheat germ)
and Solanaceae (potatoes and tomatoes). Because of the tremendous diversity of carbohydrate-
binding specificities among the plant lectins, some researchers classify them according to the
small carbohydrate haptens they recognize, e.g., galactose-binding lectins or GlcNAc-binding
lectins. The leguminous and cereal lectins differ considerably in terms of
primary/secondary/tertiary structure and in the posttranslational modifications and requirements
for metals for carbohydrate-binding activity.
Table 30.2. Classification of plant lectins
Class Monosaccharide Subunit Subunits Binding Glycosylation -S-S- Metals

specificity m.w. (kD) sites per bonds
subunit
Legumes diverse 25 30 2 or 4 1 variable no Ca++,

Mn++
Cereals primarily amino ~18 2 2 variable yes no
sugars
(GlcNac/NeuAc)
Most leguminous lectins are metalloproteins with tightly bound Ca++ and Mn++, which are
essential for carbohydrate-binding activity. The metals can be chelated with EDTA to reversibly
inactivate the proteins. The leguminous lectins have shared consensus sequences that readily
allow their identification. These shared sequences are illustrated in Figure 30.1 for two typical
lectins, the lima bean lectin and soybean agglutinin. The amino termini of these lectins contain
two highly conserved phenylalanine residues. The carboxy-terminal domain contains a
consensus sequence with invariant valine and glycine residues. The domain responsible for
binding Mn++ and Ca++ contains invariant valine and aspartic acid residues. Leguminous lectins
are synthesized as precursor peptides of approximately 30 kD within the secretory organelles of
the plant cells and usually oligomerize to form homodimers and homotetramers.
Figure 30.1. (A) Comparison of the amino termini of two examples of leguminous lectins,
mature lima bean lectin and soybean agglutinin. (B) A conserved motif in the carboxy-terminal
domains of leguminous lectins. Invariant residues are indicated by boldfaced letters and
conserved residues are shown in parentheses. Nonconserved amino acids are indicated by -x-.
The sequences of lima bean lectin and soybean agglutinin are compared. Identical residues are
boxed. (C) Consensus pattern of the metal-binding domain in the carboxyl terminus of
leguminous lectins.
During biosynthesis, some of the leguminous lectins are proteolytically cleaved to generate a β-
chain, corresponding to the amino terminus, and an α-chain, corresponding to the carboxyl
terminus. For example, jacalin lectin, from the jackfruit Artocarpus heterophyllus, is a tetrameric
two-chain lectin (65 kD) (molecular mass 65 kD) with an α-chain of 133 amino acid residues
and a β-chain of 20 21 amino acid residues. An exceptional situation occurs with the well-
known lectin ConA from jack beans. ConA is also proteolytically processed, but the two chains
are transposed and rejoined with the formation of a new peptide bond to generate the intact
protein. Thus, with regard to other lectins, the mature amino terminus of ConA corresponds to an
α-chain and the carboxyl terminus corresponds to a β-chain. In sequence alignments with other
lectins, ConA exhibits what is called "circular" homology.
Many leguminous plant lectins are glycoproteins containing N-glycans that are synthesized by
the typical pathways found in animal cells. Two common lectins, ConA and wheat germ
agglutinin, are not glycosylated, but their precursor proteins contain carbohydrates that are lost
when propeptides are proteolytically removed. The lectins in the Solanaceae family, such as
potato lectin and D. stramonium agglutinin, are also glycoproteins, but they contain high
amounts of unusual sugars, such as tri- and tetra-l-arabinofuranosides in β-linkage to
hydroxyproline residues and galactose residues in α-linkage to serine residues. For some
leguminous lectins, the N-glycans may not be required for activity, since recombinant proteins
expressed in Escherichia coli can bind carbohydrates with a specificity similar to that of the
native lectins.
The oligomeric nature of most plant lectins generates multivalency and high affinity, since in
most cases, a single subunit has a single carbohydrate-binding site and displays relatively low
affinity. In some seeds, there are multiple lectin species and these can reversibly associate to
make heteromeric complexes. For example, in Phaseolus vulgaris, the red kidney bean, two
lectins, termed E4-PHA and L4-PHA, associate to make all possible heterotetramers E1L3-PHA,
E2L2-PHA, and E3L1-PHA. In most oligomeric plant lectins, the subunits are held together
relatively tightly, but noncovalently.
Toxicity of Plant Lectins (7 10)
Many, but not all, plant lectins are toxic to animal cells. Lectins consumed in the diet may be
innocuous, since they are usually denatured by cooking and proteolytically digested upon
consumption. However, raw lectins can have deleterious effects, since uncooked lectins are
extremely stable to proteases. For example, consumption of raw navy beans alters the intestinal
microflora and results in gastrointestinal dysfunction. Raw soybean lectin and wheat-germ
agglutinin induce release of cholecystokinin, suggesting that these types of lectins may have
direct effects on gastrointestinal function and growth.
Some plant lectins, more properly classified as toxins, however, are among the most poisonous
proteins on our planet and can readily result in death not only to cells in culture, but also to
animals. Such highly toxic plant lectins are usually heterodimeric and contain one subunit for
binding to carbohydrate ligands and a second subunit that has no carbohydrate-binding activity
but is instead an enzyme. For example, ricin from Ricinus communis, the castor bean plant,
occurs as a heterodimeric protein containing an A chain and a B chain. Other lectins like ricin
include modeccin and abrin and the mistletoe lectin I (ML-I) from Viscum album. The B chain is
the carbohydrate-binding subunit and the A chain is an enzyme with adenosine-N-glycosidase
activity that can catalytically inactivate ribosomes; such enzymes are termed ribosome-
inactivating proteins or RIPs. A single molecule of A subunit entering a cell by endocytosis can
completely block protein synthesis. The mechanism by which the A subunit enters the cytosol
from endocytic compartments is not clear. These toxic plant lectin subunits conjugated to
specific antibodies and other targeting ligands are now being tested as treatments for cancer and
other disorders of cellular proliferation.
Isolation of Plant Lectins (4,11)

Many seeds contain a considerable amount of lectin. For example, soybean agglutinin constitutes
10 15% of the total protein content of the seed. However, not all seeds contain lectins. For
example, tomato lectin occurs in a soluble form in the locular fluid within the tomato fruit and
not in tomato seeds. For the most part, plant lectins are relatively soluble and can be easily
extracted. In typical cases, a saline extract is taken from a ground seed preparation, filtered, and
centrifuged at high speed. Interestingly, most seeds do not contain significant quantities of
endogenous glycoconjugate ligands for lectins, and the lectins can usually be recovered
quantitatively in the supernatant following centrifugation. However, for those plant lectins from
stems and bark, the extraction procedures are more laborious. Once solubilized, lectins can be
purified to homogeneity on appropriate immobilized carbohydrate matrices and eluted by proper
haptens. The lectin activity is usually measured by an agglutination assay that uses erythrocytes,
leukocytes, and so forth. However, some lectins, such as ricin, are not agglutinins, since they
have only a single carbohydrate-binding subunit. In those cases, it is necessary to establish other
biological assays, such as toxicity measurements, for lectin activity.
Structure of Plant Lectins (12 20, 36)
Most of the leguminous plant lectins contain at least one N-glycan, but some lectins, such as
ConA, do not contain any covalently associated carbohydrate. The ease of purification and
availability have made plant lectins prime subjects of study for protein crystallography, and the
structures of many plant lectins have now been deduced. For the most part, leguminous lectins
assemble into a compact β-barrel configuration devoid of α helices and dominated by two
antiparallel pleated sheets (Figure 30.2). Interestingly, the organization of the antiparallel β
sheets and the overall tertiary structure of the leguminous plant lectins are very similar to that
seen for animal galectins, despite the fact that leguminous lectins and galectins share no
sequence homology and galectins do not require metals for activity (Figure 30.3). In leguminous
lectins, the metal-binding sites are located on a single long loop. For ConA, the monomers come
together to form a type of ellipsoidal dimer approximately 80 Å in length, and two such dimers
then come together to form a tetrahedral-shaped structure that is stabilized by salt links, H bonds,
and hydrophilic interactions.
Figure 30.2. Structure of tetrameric ConA at 2.35 Å. The trimannoside ligand is indicated in
space-filling mode and the coordinated Ca++ and Mn++ are shown as the large green balls and
small pink balls, respectively. The crystal structure was originally reported as a complex of
ConA and a trimannoside ligand by Naismith and Field (see reference 18). (Modified, with
permission, from Loris et al. 1996.)
Figure 30.3. Comparison of the subunit structures of soybean agglutinin (SBA) (left), complexed
with a pentasaccharide containing Galβ1,4-GlcNAc-R (see [37] Olsen et al. 1997), and human
galectin-2 (right), complexed with lactose (see [38] Lobsanov et al. 1993). Both lectins display a
related β-barrel configuration.
The binding site for carbohydrate in most leguminous lectins involves a combination of H bonds,
hydrophobic interactions, and van der Waals contacts. Nearby subsites on the proteins assist in
binding oligosaccharides and contribute to hydrophobic interactions with aglycon moieties. The
metals near the binding site do not make direct contact with the sugar but help stabilize amino
acid side chains required for binding. Even in cocrystals between a lectin and a more complex
glycan ligand, the lectin makes contact primarily with a single monosaccharide substituent. The
plant lectins appear to acquire high affinity via the multivalency they display in dimeric and
tetrameric forms. The quaternary structure of the lectins may also contribute to glycan
recognition. The ability of each subunit to individually bind sugars can lead to unique cross-
linked lattice structures, as have been observed exquisitely for soybean agglutinin in its
interactions with four isomeric biantennary N-glycans and for wheat-germ agglutinin. Wheat-
germ agglutinin, cocrystallized with a tryptic sialoglycopeptide from glycophorin A, adopts a
conformation in which there is an association between wheat-germ agglutinin dimers, composed
of two identical four-domain monomers. Interestingly, two independent modes of sugar binding
are observed: a dominant mode in which there is cross-linking of the sialylated O-glycans
between two lectin subunits, and a minor mode in which a single α-2,6-linked sialic acid residue
is bound to an aromatic-residue-rich binding site. This ability to form lattice structures allows
lectins to form complex interactions with cell surfaces and matrix glycoconjugates containing
multiple binding sites, which probably contributes to the biological activities of plant lectins.
Uses of Plant Lectins (21 29, 37, 38)
Although the natural functions of plant lectins are just now being understood, investigators over
the years have found dozens of uses for plant lectins (Table 30.3). Because of their ability to
distinguish carbohydrate determinants in human blood cells, lectins have historically been used
for blood typing. Lectins are useful in immunological studies, because at low concentrations, the
lectins, such as ConA, pokeweed mitogen, and L-PHA, are mitogenic to peripheral blood
lymphocytes. However, some lectins, such as ConA and L-PHA, are extremely cytotoxic to cells
at higher concentrations and have been used to select for cell lines mutated in glycosylation
pathways. Plant lectins are also useful in histochemical studies and in lectin-blotting assays
(Western-type blots) on electrophoretically separated glycoproteins. However, some lectins also
display nonspecific hybrophic binding to nonglycosylated molecules. Thus, it is important to
make sure that lectin binding is carbohydrate-specific and inhibitable by appropriate haptens.
Table 30.3. Some uses for plant lectins
Agglutination of cells and blood typing

Cell separation and analysis
Bacterial typing
Identification and selection of mutated cells with altered glycosylation
Toxic conjugates for tumor cell killing
Cytochemical characterization/staining of cells and tissues
Mitogenesis of cells
Mapping neuronal pathways
Purification of glycoconjugates
Assays of glycosyltransferases and glycosidases
Defining glycosylation status of target glycoconjugates
Lectins are also used extensively to characterize the structures of animal cell glycoconjugates, to
which their carbohydrate-binding specificities have been best defined. Figure 30.4 lists a large
number of plant lectins, the types of glycans they bind to with high affinity, and appropriate
haptens capable of specifically blocking the interactions. Plant lectins are routinely characterized
as binding to monosaccharides, but this simplification can be misleading. For example, as shown
in Figure 30.4, most plant lectins bind with high affinity to complex determinants, and
recognition often involves specific anomeric configuration and adjacent sugar residues. In
addition, the affinity of lectins for complex carbohydrates is often in the range of 1 10 µmKd,
whereas the affinity for monosaccharides is in the range of 1 10 mmKd. In addition, the haptenic
sugars used to inhibit a lectin may not reflect the structural nature of the glycans recognized with
high affinity. For example, the most effective monosaccharide hapten for L-PHA is GalNAc.
However, L-PHA binds to tri- and tetra-antennary complex-type N-glycans containing α1 6
mannose residues substituted at C-2 and C-6 with lactosaminyl disaccharides and binds weakly if
at all to glycoconjugates containing terminal GalNAc residues (Figure 30.4).
Figure 30.4. Complex-carbohydrate-binding specificities of plant lectins. Indicated are the
minimal glycan structures required for high-affinity binding to a variety of plant lectins. For
some lectins, such as ConA and LCA, a boxed area within an N-glycan that is recognized by the
lectin is shown. The types of hapten sugars used for dissociating lectin-glycan complexes are
indicated.
Given the impressive specificity of plant lectins for N- and O-glycans of animal cell
glycoconjugates, it is not surprising that immobilized forms of these lectins are used in affinity
purification of glycoproteins, glycopeptides, free glycans, and even glycolipids. Many of the
different terminal modifications of complex glycans, such as sialylation and fucosylation, can be
specifically recognized by plant lectins. In addition, the core structures of N- and O-glycans are
distinguishable by specific lectins. The most commonly used lectin in this way is ConA, which
binds very tightly to high-mannose-type N-glycans, and with weaker affinity to hybrid-type and
biantennary complex-type N-glycans. However, ConA does not bind detectably to more highly
branched complex-type N-glycans and O-glycans. When intact glycoproteins are analyzed for
their interactions with plant lectins, the interpretation of data may be complicated by the
multivalency of the glycoprotein. For example, glycoproteins containing multiple high-mannose-
type N-glycans bind so tightly to immobilized ConA that it is difficult to elute the bound
glycoprotein, even with extremely high concentrations of hapten under harsh conditions.
However, the interactions of free glycans with lectins is much better understood and complexes
are more easily dissociated with hapten sugars. A combined use of many immobilized lectins is
termed serial lectin affinity chromatography. It is especially useful in sorting complex mixtures
of glycans that may differ in structure but have rather similar size and charge characteristics. An
example of serial lectin affinity chromatography using a hypothetical mixture of glycans is
shown in Figure 30.5. At each step in the purification, haptenic sugars are added to a column to
force the reverse equilibrium and allow the bound glycans to be eluted from the lectin. The
combined use of lectin affinity chromatography and HPLC can provide highly purified
glycoconjugates for structural analysis.
Figure 30.5. Example of the use of different immobilized plant lectins in the serial
chromatography of complex mixtures of free glycans. In this example, a mixture of glycans is
applied to a column of immobilized ConA, and the bound glycans are eluted with increasing
concentration of hapten sugars (arrows) as shown in Figure 30.4. The recovered glycans are then
applied to a second set of columns containing immobilized LCA, and bound glycans are eluted
with hapten sugars. This process is repeated over other lectins, such as L-PHA and SNA. In
some cases, as for L-PHA, the glycans are retarded in their elution, and haptenic sugars may not
be necessary for elution.
Functions of Plant Lectins (30 35)
Despite the enormous interest in plant lectins, little is known about their functions in the plant.
Many leguminous lectins are found in seeds, but the same lectin and homologs are also found in
other parts of the plant, such as the bark, stem, and leaves (see also Chapter 20). As shown in
Table 30.4, lectins probably have many different and important functions in plants. There is a
high level of research interest in the possibility that leguminous lectins may be involved in
rhizobial attachment and root nodulation. For example, transgenic expression of the soybean
agglutinin gene in Lotus corniculatus, which is normally nodulated by Rhizobia loti, changes the
nodulation pattern of the transgenic plant to Bradyrhizobium japonicum, which normally
nodulates soybeans. Some of the nodulation factors released by the bacteria are glycoconjugates,
and it is intriguing to consider that some of the members of the plant lectin group may be
involved in this important plant signaling pathway. Plant lectins are toxic to many plant
pathogens, and this may be a major role within seeds and other peripheral tissues of the plants.
For example, the snowdrop lectin Galanthus nivalis agglutinin is toxic toward the sap-sucking
insect called the rice brown plant hopper. Transgenic expression of the gene encoding G. nivalis
agglutinin in rice plants decreases survival and fecundity of insects attacking the transgenic
plants. Although we usually think of plant lectins as only having an ability to bind carbohydrate,
evidence exists that these proteins may have additional activities. For example, some lectins,
such as Dolichus biflorus, bind adenine residues with high affinity and specificity in regions of
the protein outside the common carbohydrate-binding site.
Table 30.4. Some possible natural functions of plant lectins
Seed storage proteins

Aid in maintaining seed dormancy
Defense against fungal, viral, and bacterial pathogens
Defense against animal predators
Symbiosis in legumes
Transport of carbohydrates
Mitogenic stimulation of embryonic plant cells
Elongation of cell walls
Recognition of pollen
Future Directions
Plant lectins have made an enormous contribution to the development of interest in animal cell
glycobiology. They have provided important clues to the vast repertoire of carbohydrate
structures in animal cells and the possibilities that animal cells might also contain carbohydrate-
binding proteins. Undoubtedly, plant lectins will continue to have an important role as reagents
in their own right. The increased understanding of the carbohydrate-binding specificity of lectins
and their three-dimensional structures allows the possibility in the future of engineering lectins to
recognize specific glycoconjugates. In addition, the increased attention of biologists worldwide
to the phenomenal part carbohydrates play in cellular interactions and regulation may finally
contribute to a better understanding of the natural functions of the interesting plant proteins.
Genetically altering plants to allow expression of different lectins may afford protection to plants
from infectious diseases and may also enhance the nodulation and nitrogen-fixing ability of the
plants.
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Glycans in Genetic Disorders
and Disease
31. Genetic Disorders of Glycosylation in Cultured Cells
Diego).
MUCH OF WHAT WE KNOW about the biosynthesis and function of vertebrate glycosylation
originates from studies of induced somatic cell mutants of cultured cell lines. This chapter
reviews general methods used to isolate glycosylation mutants, the diversity of mutants currently
available, and the some of the uses of mutants in glycobiology research.
Introduction (1 6)
Somatic cell genetics grew out of a desire to understand the molecular basis of human genetic
disorders and metabolism by applying the principles of microbial genetics to mammalian
systems. The field began in the early 1950s, with pioneering studies of cultured fibroblasts and
cultured cell lines from CHO cells. These early efforts showed that stable cell lines could be
isolated from animal tissues and propagated in vitro under reasonably defined growth conditions.
Furthermore, mutants could be isolated at a respectable frequency, and their phenotypes
remained stable over many generations. Studying various processes in cultured cell mutants
circumvented the long generation times inherent in genetic studies of whole organisms and
allowed systematic control of environmental factors, such as nutrients, pH, and temperature.
The ability to isolate mutants in culture has made it possible to unravel metabolic pathways, to
enumerate structural and regulatory genes, and to map these genes on human chromosomes.
Mutants often accumulate intermediates upstream of the defective enzyme in a metabolic
pathway, which can reveal the chemical transformations that take place at each step in a
pathway. Sequencing mutant alleles can reveal how changes in specific amino acids affect an
enzyme activity. Finally, using cDNA transfection to introduce foreign glycosyltransferases, one
can revert the phenotype of mutants or convert the cell to produce different glycotypes. These
techniques were applied early in the development of the field of glycobiology, yielding
numerous mutants in glycoprotein biosynthesis and later in proteoglycan, GPI anchor, and
glycolipid synthesis. In most cases, the mutations depress the activity of an important enzyme in
a pathway, but in other cases, the mutations can activate a latent glycosyltransferase or elevate
the expression of an existing enzyme (Figure 31.1). In both situations, the mutations lead to
altered glycans on the cell surface and corresponding changes in biological responses dependent
on glycosylation.
Figure 31.1. Alteration of cell surface glycosylation by recessive and dominant

Induction and Isolation of Mutants (7 12)
In cell culture, mutations occur randomly at a low rate ( 10-7 mutations per generation), and thus
the likelihood of finding mutants in glycosyltransferase genes is quite low. To increase the
probability of finding desirable strains, cells can be treated with chemical (e.g., alkylating
agents), physical (e.g., ionizing radiation), or biological mutagens (e.g., a virus), which raises the
incidence of mutants in the population several orders of magnitude. Obviously, the DNA ploidy
state of the cell can affect the frequency of observing the mutant phenotype if the mutation is
recessive, as expected for loss-of-function defects in glycosyltransferases. However, the
frequency of finding recessive mutants is actually much higher than predicted, possibly because
of gene conversion effects. In CHO cells, many loci are functionally hemizygous, which means
that single hits are adequate to observe a recessive phenotype. Dominant mutations also have
been found, which in most cases infers a gain of function caused by the activation of a latent
transferase.
Even with mutagenesis, the incidence of mutants with defects specifically in glycosyltransferase
genes is relatively low, since genes involved in glycosylation represent only about 1% of the
genome. Thus, some form of selection or enrichment is also needed to find rare mutants bearing
the desired phenotypic alteration in glycosylation (Table 31.1).
Table 31.1. Classes of glycosylation mutants according to screening method
Selection/enrichment scheme Types of mutants isolated
Lectin resistance N-glycosylation

dolichol assembly
nucleotide sugar formation and transport
gain-of-function mutants
Radiation suicide N-glycosylation
nucleotide conversion
Cell sorting Antibody/complement-mediated lysis GPI anchors
Replica-plating GAG biosynthesis
N-glycosylation
nucleotide sugar formation
Direct selection schemes based on resistance to cytotoxic plant lectins have proven especially
useful for identifying mutants altered in N-glycosylation. Dozens of lectins are available with
different selectivities for sugars in various arrangements (see Chapter 30). Some of the plant
lectins, such as ricin and abrin, have cytotoxic activity due to their ability to inactivate
ribosomes. Thus, cells can become resistant to lectins by reducing the expression of the cognate
glycan ligand on the cell surface (Figure 31.1). Other lectins that do not have cytotoxic activity
are also useful for selecting mutants, since lectin binding may interfere with cell adhesion. Thus,
normal cells do not adhere in the presence of a lectin that binds to the cell surface, whereas
mutant cells lacking the carbohydrate determinant recognized by the lectin continue to adhere.
Mutations that affect both terminal and internal glycosylation reactions have been detected in
these ways. Mutations in the generation and transport of nucleotide sugar precursors have also
been found.
Virtually any agent that recognizes cell surface carbohydrates can be used to detect glycosylation
defects in mutagenized cells. Other approaches consist of using fluorescent tags, such as a
conjugated antibody or lectin, that bind to a cell surface carbohydrate or to a protein whose
expression on the cell surface depends on glycosylation (e.g., GPI-anchored proteins). Deficient
cells can be detected by fluorescence-activated cell sorting. Panning is a related technique that is
based on adhesion of cells to plastic or glass surfaces coated with a carbohydrate-binding agent.
For example, coating a plate with bFGF allows detection of mutant cells that fail to produce
heparan sulfate proteoglycans, since mutant cells fail to adhere to the coated plate (see Chapter
29). Antibodies to carbohydrate antigens have also been used in this way.
Radiation suicide provides a direct selection method for obtaining mutants. This procedure takes
advantage of the pathways used by cells to salvage free monosaccharides and other precursors
for macromolecular synthesis (e.g., mannose, fucose, and sulfate). Incubation of cells with a
radioactive sugar or sulfate at high radiospecific activity results in its incorporation and
concentration into glycoproteins, glycolipids, or proteoglycans. As the radioisotope decays, it
causes radiation damage to cells expressing the labeled glycoconjugates.
Animal cells also can be replica-plated, much like microbial colonies. In this procedure, animal
cells are plated at low density to allow colony formation to occur from single cells. The cells are
overlaid with a porous cloth made of polyester or nylon and a layer of glass beads that weighs
the disc against the bottom of the plate. As colonies form, some cells in each colony remain
firmly attached to the plate, whereas others grow into the disc, thus producing replicas of the
colony pattern on the dish and the disc. The colonies on the disc can then be used to measure
incorporation of macromolecular precursors (e.g., radioactive sugars or sulfate) or mutants that
fail to bind to a lectin or anti-carbohydrate antibody (Figure 31.2). An adaptation of this
technique allows detection of mutants by direct enzymatic assay in colony homogenates
generated on the discs. This latter technique provides much greater screening specificity than the
other procedures.
Figure 31.2. Screening for mutants using animal cell replica-plating. Animal cell colonies
transferred to discs can be screened for defects in incorporation of radioactive precursors,
binding to lectins and antibodies, or direct enzymatic assay.
Regardless of the technique used to detect mutants, the resulting strains must be carefully
characterized. Additional genetic tests include cell hybridization studies for dominant/recessive
testing and for sorting the mutants into different complementation groups. Biochemical analysis
involves the characterization of glycan structures produced by mutant cells (see Chapter 38), the
quantitation and analysis of intermediates, and assays for enzyme activities thought to be
missing, based on the properties of the mutant.
Mutants in N-Glycosylation (4, 6, 8,13 16)
Selection schemes based on resistance to cytotoxic plant lectins and other inhibitors have yielded
a large number of glycosylation mutants. Table 31.2 lists examples of strains altered in
nucleotide sugar formation or transport. As might be expected, some of the defects are
pleiotropic; i.e., they affect O-glycan formation and GAG synthesis as well as N-glycosylation
(e.g., UDP-Glc/Gal epimerase). The CHO-ldlD mutant is particularly interesting in this regard
since it lacks the enzyme responsible for converting UDP-Glc to UDP-Gal and UDP-GlcNAc to
UDP-GalNAc. Since salvage pathways for Gal and GalNAc exist (see Chapter 6), the
composition of oligosaccharides can be controlled in ldlD cells by nutritional supplementation
with Gal and/or GalNAc. Most of the mutants in the table lack a particular activity. SAP mutants
of CHO and D33W25-1 are examples of dominant mutations that activate a latent enzyme, in
this case a hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc.
Table 31.2. Examples of defects in nucleotide sugar formation and transport
Strain Biochemical defect Glycosylation phenotype
Lec32 (CHO) CMP-Neu5Ac sialic acid

synthetase
Lec2 (CHO) CMP-Neu5Ac reduced in Neu5Ac; N- and O-glycans terminate in
transporter Gal
Lec8 (CHO) UDP-Gal transporter reduced in Gal; N-glycans terminate in GalNAc
Lec13 (CHO) GDP-Man-2,4- reduced in Fuc residues
dehydratase
ldlD (CHO) UDP-Gal/UDP- N-glycans reduced in Gal and terminate in GlcNAc
GlcNAc-4-epimerase
O-glycans and chondroitin sulfate not present in the
absence of added GalNAc; GAG deficient in the
absence of Gal
D33W25-1 activation of CMP- terminate in Neu5Gc
(MDAY-D2) SAP Neu5Ac hydroxylase
(CHO)
Some of lectin-resistant mutants affect the formation of Dol-P oligosaccharides or the processing
enzymes that remove glucose and mannose residues after transfer of the glycan from dolichol
intermediates to glycoproteins (see Chapter 7). These latter mutants demonstrate the importance
of α-mannosidases in the formation of N-glycans in cultured cell lines, but subsequent studies in
which the α-mannosidase II was ablated in mice revealed that another enzyme exists with
overlapping activity. This finding emphasizes a limitation of somatic mutants: The investigation
is restricted to the cell line in which the gene is mutated. Since many glycosyltransferases appear
to be developmentally regulated in a tissue-specific manner, alternate pathways might be missed
by studying mutants of a single cell type.
Other examples of defects in N-glycan synthesis are given in Table 31.3. Note that some
mutations can affect the kinetic properties of enzymes (e.g., Lec1A) or their subcellular
localization (e.g., Lec4A). Sequencing the mutant allele provides leads for further in vitro
mutagenesis of the gene in order to define important functional domains of the protein required
for catalysis or sorting.
Table 31.3. Examples of mutants altered in late N-glycosylation reactions
Lec1 (CHO) GlcNAc-TI Man5GlcNAc2Asn

Clone 15B
(CHO)
Lec1A GlcNAc-TI (Km defect) complex mixture of glycans and
(CHO) Man5GlcNAc2Asn
RicR21 GlcNAc-TII Man3GlcNAc2Asn
(BHK)
Lec4A GlcNAc-TV located in the complex N-glycans lack β(1,6)GlcNAc
(CHO) incorrect compartment branch from αMan(1,6)
Sil (KB) α2,3sialyl-T reduced Neu5Ac on O-glycans
Another class of lectin-resistant mutants have dominant phenotypes, which correspond to a gain
of function due to expression of a glycosyltransferase that is normally suppressed or expressed at
very low levels. The activation of these glycosyltransferases may reflect a mutation in a
regulatory region of the gene or in a trans-acting suppressive factor. The generation of gain-of-
function mutants provides the opportunity to detect and analyze the effect of novel genes, which
in many cases were not previously known to exist. For example, LEC14 and LEC18 mutations
activate transferases that add branching GlcNAc residues to the core chitobiose of N-linked
chains, a previously undescribed modification (Table 31.4).
Table 31.4. Examples of dominant mutants altered in glycosylation reactions
LEC11 (CHO) (1,3)Fuc-T terminal Lewis X, SA-Lewis X, and VIM-2

LEC14 (CHO) GlcNAc-TVII additional GlcNAc in core
LEC18 (CHO) GlcNAc-TVIII additional GlcNAc in core
LEC10 (CHO) GlcNAc-TIII complex chains have the bisected GlcNAc residue
Mutants in GPI Anchor Biosynthesis (9, 17)

Lectins that selectively bind to GPI anchors have not been described. Thus, to isolate mutants
altered in GPI synthesis, a different approach was developed that took advantage of antibodies to
a GPI-anchored protein (Thy-1 on T-cell lymphoma) and cell sorting or complement-mediated
lysis. Cells expressing Thy-1 on their surface were incubated with an antibody to Thy-1 and
complement, which lysed cells expressing the antigen. Loss of GPI-anchor synthesis reduced the
expression of Thy-1 on the surface and conferred resistance to the cytolytic effect. Other mutants
were found by sorting cells with a fluorescent antibody assay. The mutants fall into eight
complementation groups, with different lesions in GPI-anchor biosynthesis (see Chapter 10;
Table 31.5). These mutants demonstrate the complexity of GPI-anchor synthesis: Three gene
products are involved in forming the GlcNAc linkage to phosphatidylinositol, the first committed
intermediate in the pathway. The mutants also established that Dol-P-Man was involved in the
linkage of mannose within the GPI anchor and that multiple enzymes are involved in the
attachment of ethanolamine phosphate residues (three in all). On the basis of the structure of
mammalian GPI anchors, other enzymes in the pathway must exist, but mutants in these genes
have not yet been identified. The available strains demonstrate the importance of mutagenesis
studies for identifying genes that might not be obvious from merely examining the enzyme
reactions in vitro.
Table 31.5. Mutants defective in GPI-anchor biosynthesis
A,C,H GlcNAc to PI transferase formation of GlcNAc-PI

J GlcNAc N-deacetylase accumulates GlcNAc-PI
E Dol-P-Man synthase additional GlcNAc in core
B addition of α1,2-linked mannose Man2GlcN-PI
F,K ethanolamine-phosphate addition reactions Man3(Eth-P)1 2GlcN-PI
Mutants in Proteoglycan Assembly (3)

A large collection of mutants have been obtained that are defective in proteoglycan biosynthesis
(Table 31.6). These mutants were obtained by replica-plating methods using sulfate
incorporation to monitor glycosaminoglycan biosynthesis in colonies (Figure 31.2). Mutants in
the early steps of glycosaminoglycan biosynthesis (complementation groups A, B, and G) lack
both chondroitin sulfate and heparan sulfate chains, and enzymatic assays showed that they were
lacking enzymes responsible for the assembly of the core protein-linkage tetrasaccharide shared
by both glycosaminoglycans (Chapter 11). Another class of mutants (group D) are defective in
heparan sulfate biosynthesis. This mutation defines a bifunctional enzyme that catalyzes the
alternating addition of GlcNAc and GlcA residues to growing chains. Some of the mutant alleles
depress both enzyme activities, whereas others only affect the GlcA transfer activity. Thus, the
mutants define different functional domains of the protein, which can be mapped by sequencing
various mutant alleles. Mutants in the GlcNAc N-deacetylase/N-sulfotransferase (another
bifunctional enzyme) showed only a partial deficiency in N-sulfation of the chains. Further
analysis of the mutant showed that more than one isozyme is present in CHO cells and that the
defect affected only one locus. Thus, the mutants revealed that the assembly of heparan sulfate is
much more complex than appreciated, based on structure of the glycans, enzymatic reactions
measured in cell extracts, and pulse-labeling experiments.
Table 31.6. Animal cell mutants defective in proteoglycan assembly
Strain Biochemical defect Phenotype
pgsA (CHO) xylosyltransferase defective formation of both HS and CS

pgsB (CHO) galactosyltransferase I defective formation of both HS and CS
pgsG (CHO) glucuronosyltransferase I defective formation of both HS and CS
pgsD (CHO) GlcA and GlcNAc transferase HS-deficient and accumulates CS
ldlD (CHO) UDP-Gal/UDP-GlcNAc-4- CS not present in the absence of added
epimerase GalNAc
GAG deficient in the absence of Gal
pgsC (CHO) sulfate transporter normal glycosaminoglycan biosynthesis
CM-15 (COS) N-sulfotransferase undersulfated HS
pgsF (CHO) heparan sulfate 2-O- defective 2-O-sulfation of HS; defective bFGF
sulfotransferase binding
Mouse LTA 3-O-sulfotransferase defective antithrombin binding
cells
Mutants Defective in Glycolipid and Mucin Assembly (18)

In contrast to the systems described above, very few mutants have been described that are
selectively blocked in glycolipid or mucin biosynthesis (Table 31.7). The absence of mutants in
these systems most likely reflects a lack of effort rather than anything fundamentally different
about the mutability of these pathways.
Table 31.7. Mutants altered in glycolipid and mucin biosynthesis
Strain Biochemical defect Phenotype
GM-95 (B16 glucosylceramide no glycosphingolipids

Melanoma)
ldlD (CHO) UDP-Gal/UDP- GlcNAc-4- O-glycans not present in the absence of
epimerase added GalNAc
Uses of Somatic Cell Glycosylation Mutants (3, 4, 6,15,19 22)
As illustrated above, mutants have played an important part in defining the pathways of
glycosylation in cells. Glycosylation mutants also provide an opportunity to study the function of
glycosylation in the context of a living cell. Examples include viral, bacterial, and parasite
adhesion and infection; leukocyte cell adhesion and motility; protein folding; intracellular sorting
and secretion of proteins; growth factor binding and activation; enzyme immobilization; and
receptor function. For additional details, see Chapters 5, 28, and 29, and cited review articles at
the end of this chapter.
Perhaps one of the most surprising results emerging from these studies is that many steps in
glycosylation may be dispensable in cultured cells. Thus, the N-glycans can undergo large
variation in structure without affecting cell growth. Similarly, proteoglycan formation and
glycolipid biosynthesis are unimportant for maintaining viability. However, it should be kept in
mind that some mutants have been difficult to obtain using very powerful selection methods that
in theory should have yielded the missing strains. An example of this is the β1 4
galactosyltransferase involved in glycoprotein formation. The failure to obtain mutants in this
step may reflect the essential nature of this enzyme, possibly in pathways that have not yet been
defined. Alternatively, the lack of mutants might indicate the presence of multiple isozymes.
Although many steps in glycosylation may be dispensable in cultured cells, this is probably not
true in vivo. Studies of glycosylation mutants showed that in some cases, glycosylation is
essential for tumor growth and metastasis. More importantly, gene ablation studies in mice have
shown that intact glycosylation is essential for embryogenesis. Examples include mutants in
GlcNAc-TI that exhibit embryonic lethality and a recent study of 2-O-sulfation of heparan
sulfate in mice. The corresponding mutations in cultured CHO cells do not cause a growth
phenotype (Lec1 and pgsF, respectively). Thus, glycosylation is important in the context of a
multicellular organism. This topic is discussed in greater detail in Chapters 32 34.
Future Studies
Somatic cell genetics arose from the need to manipulate the phenotype of cultured cells in vitro.
Today, the emphasis in genetics is shifting toward the generation of organismal mutants using
the newly developed techniques of transgenesis and homologous recombination. However, the
study of somatic cell mutants still plays an important part in glycobiology research, since it
provides a less expensive and faster method for studying the effect of deleting or expressing
particular gene products in metabolism. Furthermore, cell-based genetics makes it possible to
discover new genes by screening for phenotypic changes directly related to glycosylation, which
is more difficult (although not impossible) to do in animals. Thus, somatic cell mutants provide
access to new genes involved in glycosylation, which in turn is the material needed for
sophisticated gene ablation experiments in animals. By combining the two approaches, the
function of a particular glycosyltransferase or glycoconjugate in cell biology and
pathophysiology can be defined. Gene ablation in animals also makes it possible to derive cell
lines from a variety of tissues with desired defects in glycosylation. Thus, genetics appears to be
coming full circle as mutants in cells and animals provide mutually useful reagents for further
analysis of glycosylation.
Acknowledgment
Many thanks to Pamela Stanley (Albert Einstein School of Medicine) for providing information
for Tables 2 4.
References
1. T.T. Puck and F.-T. Kao. 1982. Somatic cell genetics and its application to medicine Annu.
Rev. Genet. 16: 225-274. (PubMed)
2. A. Takatsuki and G. Tamura. 1971. Effect of tunicamycin on the synthesis of macromolecules

in cultures of chick embryo fibroblasts infected with Newcastle disease virus J. Antibiot. 24:
785-794. (PubMed)
4. A. Takatsuki and G. Tamura. 1971. Tunicamycin, a new antibiotic. II. Some biological
properties of the antiviral activity of tunicamycin J. Antibiot. 24: 224-231. (PubMed)

7. R.M. Baker, C.B. Hirschberg, W.A. O'Brien, T.E. Awerbuch, and D. Watson. 1982. Isolation
of somatic cell glycoprotein mutants Methods Enzymol. 83: 444-458. (PubMed)
8. P. Stanley. 1983. Selection of lectin-resistant mutants of animal cells Methods Enzymol. 96:
157-184. (PubMed)
9. A.D. Elbein. 1991. The role of N-linked oligosaccharides in glycoprotein function Trends
Biotechnol. 9: 346-352. (PubMed)
10. J.D. Esko. 1989. Replica plating of animal cells Methods Cell Biol. 32: 387-422. (PubMed)
11. Schwarz R.T. 1991. Manipulation of the biosynthesis of protein-modifying glycoconjugates

by the use of specific inhibitors. Behring Inst. Mitt. , pp. 198 208.
12. S.C. Hubbard, L. Walls, H.E. Ruley, and E.A. Muchmore. 1994. Generation of Chinese
hamster ovary cell glycosylation mutants by retroviral insertional mutagenesis. Integration into a
discrete locus generates mutants expressing high levels of N-glycolylneuraminic acid J. Biol.
Chem. 269: 3717-3724. (PubMed)
13. A. Takatsuki, K. Arima, and G. Tamura. 1971. Tunicamycin, a new antibiotic. I. Isolation
and characterization of tunicamycin J. Antibiot. 24: 215-223. (PubMed)
14. P. Stanley. 1984. Glycosylation mutants of animal cells Annu. Rev. Genet. 18: 525-552.
(PubMed)
15. P. Stanley. 1987. Glycosylation mutants and the functions of mammalian carbohydrates
Trends Genet. 3: 77-81.
16. P. Stanley, T.S. Raju, and M. Bhaumik. 1996. CHO cells provide access to novel N-glycans
and developmentally regulated glycosyltransferases Glycobiology 6: 695-699. (PubMed)
17. R.P. Mohney, J.J. Knez, L. Ravi, D. Sevlever, T.L. Rosenberry, S. Hirose, and M.E. Medoff.
1994. Glycoinositol phospholipid anchor-defective K562 mutants with biochemical lesions
distinct from those in Thy-1-murine lymphoma mutants J .Biol. Chem. 269: 6536-6542.
(PubMed)
18. S. Ichikawa, N. Nakajo, H. Sakiyama, and Y. Hirabayashi. 1994. A mouse B16 melanoma
mutant deficient in glycolipids Proc. Natl. Acad. Sci. 91: 2703-2707. (PubMed) (Full Text in
PMC)
20. R.M. Mortensen and J.G. Seidman. 1994. Inactivation of G-protein genes: Double knockout
in cell lines Methods Enzymol. 237: 356-366. (PubMed)
21. A. Varki and J. Marth. 1995. Oligosaccharides in vertebrate development Dev. Biol. 6: 127-
138.
22. K.S. Rostand and J.D. Esko. 1997. Microbial adherence to and invasion through
proteoglycans Infect. Immun. 65: 1-8. (PubMed) (Full Text in PMC)
32. Naturally Occurring Genetic Disorders of Glycosylation
Primary contributions to this chapter were made by J.B. Lowe (HHMI/University of Michigan,
Ann Arbor) and H. Freeze (The Burnham Institute, La Jolla, California).
THIS CHAPTER DEALS WITH INHERITED DISEASES in glycan biosynthesis. It focuses on

an emerging group of carbohydrate-deficient glycoprotein syndromes (CDGS), leukocyte
adhesion deficiency syndrome II (LADII), congenital dyserythropoietic anemia type II
(HEMPAS), galactosemia, and abnormalities in proteoglycan synthesis. For a discussion of
inherited deficiencies in the blood group glycosyltransferases, see Chapters 16 and 17. Chapter
18 discusses lysosomal storage diseases resulting from defects in glycan degradation, and
Chapter 23 discusses disorders of lysosomal enzyme phosphorylation. Diseases such as cystic
fibrosis where altered glycosylation is a secondary effect are covered in Chapter 37.
Introduction
Inherited disorders of glycosylation are biochemically and clinically heterogeneous. Individuals
with CDGS, LADII, HEMPAS, and galactosemia typically suffer from malfunction of multiple
organ systems. Depending on the specific biosynthetic lesion, the structures and function of
many glycoproteins, glycolipids, and glycophosphotidylinositol-linked proteins may be affected.
Recent remarkable progress in this area has led to the identification of several primary genetic
defects and to successful therapies based on clear biochemical rationales. In addition, these
studies imply that many of the diseases are probably more genetically heterogeneous than once
expected and are probably underdiagnosed. Consequently, a new awareness of this group of
disorders by the clinical community should foster new insights into their pathogenesis and
treatment.
Spontaneous Mutations in Humans and Animal Models

Most of this chapter focuses on human glycosylation disorders; however, animals also show
spontaneous mutations in glycosylation. The defects in animals primarily affect proteoglycan
biosynthesis, since these defects produce dysmorphic features that are easy to recognize. Table
32.1 lists disorders in animals and humans (see similar tables in Chapters 7 and 33). In addition,
Chapter 23 covers mutations in several animal species for many of the lysosomal storage
disorders.
Table 32.1. Naturally occurring disorders in glycosylation pathways in animals
Disorder Defect Species
I-cell disease and pseudo-Hurler deficiency of UDP-GlcNac: lysosomal human

polydystrophy (mucolipidosis II and III) enzyme GlcNAc-1-phosphotransferase
Carbohydrate-deficient glycoprotein several genetic defects in the assembly, human
syndromes (CDGS) transfer, and early processing of N-
glycans (for details, see text)
Leukocyte adhesion deficiency type II decreased conversion of GDP-Man to human
primary defect unknown GDP-Fuc;
Galactosemia defects in forming UDP-Gal, e.g., Gal-1- human
phosphate uridyltransferase
Macular corneal dystrophy type I defective keratan sulfate biosynthesis human
Ehlers-Danlos syndrome with progeroid partial deficiency of xylosylprotein β4 human
syndrome galactosyltransferase (GalT-I)
Diastrophic dysplasia, achondrogenesis diastrophic dysplasia sulfate transporter; human
type Ib, atelosteogenesis type II defect in sulfate availability
Brachymorphism (bm) adenosine-phosphosulfate kinase mouse
deficiency (SK2); defect in synthesis of
sulfate donor (PAPS)
Cartilage matrix deficiency (cmd) aggrecan proteoglycan gene (frameshift) mouse
Nanomelia aggrecan proteoglycan gene (nonsense chicken
mutation)
Simpson-Golabi-Behmel overgrowth glypican-3 proteoglycan core protein human
syndrome deficiency
mouse
Hereditary erythrocytic multinuclearity partial deficiency of UDP-GlcNAc:α- human
with positive acidified serum test mannosidase GlcNAc transferase II or
(HEMPAS); also called congenital processing α-mannosidase II; primary
dyserythropoietic anemia type II (CDA genetic defect unclear
type II)
N-glycolylneuraminic acid deficiency exon deletion in CMP-Sia hydroxylase all humans
gene
α1 3 Gal epitope deficiency nonsense mutations in the UDP- all Old
Gal:LacNAc α1 3 galactosyltrasferase World
gene primates
Tn syndrome deficiency in β1 3 galactosyltransferase human
that synthesizes Core 1 O-glycans;
affects erythrocytes
IgA nephropathy same as for Tn syndrome, except that human
IgA is affected
Clinical and Laboratory Features of the CDGSs (1 10)
The CDGSs are an emerging group of clinically heterogeneous autosomal recessive

glycosylation disorders. Four types of CDGSs (types I through IV) have been defined on the
basis of their serum transferrin isoelectric focusing profile (see below). Systematic CDGS
nomenclature is still being refined and will probably be revised in the near future. CDGS type I
is the most common, with more than 200 patients described in the medical literature. In contrast,
only a few patients with CDGS types II, III, and IV have been reported. CDGS type I has now
been subdivided into three distinct forms, termed CDGS type Ia, CDGS type Ib, and CDGS type
Ic (also called type V), according to the nature of the clinical symptoms and molecular defects.
Types II, III, and IV are single distinct clinical entities at this time.
The diagnosis of CDGS is based on clinical signs and symptoms, together with isoelectric
focusing or chromatofocusing analysis of serum transferrin. These methods provide a very
sensitive indicator of the glycosylation state of serum transferrin. Other serum glycoproteins
have altered glycosylation, but transferrin is the most reliable, sensitive, and simplest indicator.
In normal humans, serum transferrin molecules have two glycosylation sites that are occupied
primarily (~80%) by disialylated, biantennary N-glycans. Most molecules contain four sialic acid
residues, and they exhibit a characteristic electrophoretic migration position (Figure 32.1). This
tetrasialylated transferrin glycoform is termed S4. Approximately 10 15% of normal human
serum transferrin molecules contain at least one trisialylated, triantennary N-glycan providing S5
and S6 glycoforms. These forms increase during an acute-phase response but are not important
for CDGS diagnostics. In CDGS patients, the S4 glycoform is either substantially diminished
(CDGS type I) or absent (CDGS type II), with compensatory increases in two other glycoforms,
termed S2 and S0. Structural analyses of the S2 and S0 glycoforms isolated from CDGS type I
patients show that each S2 transferrin molecule contains only a single disialylated biantennary
N-glycan (at one of the two normal attachment sites), whereas each S0 molecule is completely
deficient in N-glycan modification (Figure 32.1). At present, mutations in three different genetic
loci account for CDGS underglycosylation. In CDGS type II, the glycans are efficiently added,
but they are processed abnormally. In type IV, structurally altered glycans are added from the
lipid precursor and are also abnormally processed.
Figure 32.1. Schematic diagram of the glycan structures found on serum transferrin in normal
individuals and on serum transferrin in CDGS types I and II. The approximate fraction of total
serum transferrin that exhibits a particular glycoform is indicated at the right. The symbols
represent biantennary N-glycans. Absence of the GnT-II-dependent branch is illustrated by an
absence of one of the two solid diamonds in the serum transferrin glycoform that predominates
in CDGS type II.
CDGS Type Ia (11 25)
CDGS type Ia is the most common form of CDGS, representing approximately 70% of all cases
of CDGS type I. More than 180 patients with this disease have been reported in the literature
since its discovery in 1980. These individuals typically present at birth with hypotonia,
dysmorphic features, failure to thrive, liver dysfunction, and a pronounced susceptibility to
infection. Approximately 20% of these patients die within the first few years of life. Other
clinical features of CDGS type Ia include hypoplasia of the cerebrum, cerebellum, and
brainstem, and a significant delay in motor and language development. CDGS patients also
exhibit an unusual distribution of body fat, and suffer from polyneuropathy, ataxia, hypotonia,
skeletal deformities, retinitis pigmentosa, and oculomotor abnormalities. Liver function
abnormalities are characterized by elevated serum transaminases, decreased serum albumin, and
clinically significant decrements in the blood-clotting proteins antithrombin III, protein C,
protein S, heparin cofactor, and factor XI. CDGS type Ia patients also exhibit abnormalities in
endocrine homeostasis and typically display hypogonadism.
Biochemical and molecular genetic analyses disclose that the glycosylation defect in CDGS type
Ia is caused by defects in the phosphomannomutase 2 locus (PMM2). This gene is on human
chromosome 16p13, where linkage studies have localized the CDGS type I locus in some
families. The coding region of the PMM2 locus shares approximately 65% DNA sequence
identity with the PMM1 locus. PMM1 is found on human chromosome 22q13, which excludes
the involvement of this gene in the classical form of CDGS type Ia. A processed PMM2
pseudogene, termed PMM2psi, localizes to human chromosome 18p.
The PMM2 locus encodes an enzyme that catalyzes an essential step in the biosynthesis of GDP-
Man and Dol-P-Man (Figure 32.2). These two compounds are essential substrates for the
mannosyltransferases required for the synthesis of the lipid-linked precursor for N-glycosylation
(Figure 32.2). In CDGS type Ia, a deficiency of phosphomannomutase activity reduces the levels
of GDP-Man and Dol-P-Man. The reduced levels of these compounds reduce synthesis of
Glc3Man9GlcNAc2-PP-Dol, the physiological substrate for oligosaccharyltransferase. Reductions
in GDP-Man and Dol-P-Man also yield truncated forms of the Dol-PP-oligosaccharide.
However, these truncated forms, and especially those that are not glucosylated, are poor
substrates for oligosaccharyltransferase. Oligosaccharyltransferase preferentially uses the
physiological precursor substrate Glc3Man9GlcNAc2-PP-Dol, but its low level is rate-limiting,
causing the enzyme to leave some asparagine-linked sites unmodified. Nevertheless, a
substantial proportion of sites receive a normal Glc3Man9GlcNAc2 glycan that is trimmed and
remodeled normally, to give a disialylated biantennary oligosaccharide.
Figure 32.2. Synthetic reactions in N-glycan biosynthesis that are affected in CDGS. The
reactions affected in CDGS types Ia, Ib, and Ic, and type IV are indicated by the parenthetic
annotation below the relevant synthetic step.
It is important to point out that GDP-Man is also required for the synthesis of the four mannose
residues of glycophosphotidylinositol anchors (see Chapter 10) and is the major source of GDP-
Fuc required for terminal fucosylation (see Leukocyte Adhesion Deficiency II Syndrome below).
The diminished synthesis of GDP-Man in PMM-deficient CDGS type Ia is therefore likely to
cause deficits in the structure and function of molecules that depend on these processes. For
example, it is possible that the frequent and severe infectious complications suffered by CDGS
type Ia patients may be due to defective synthesis of leukocyte selectin ligands, which require
terminal α1 3-fucosylation for proper function (see Chapter 26, and Leukocyte Adhesion
Deficiency II Syndrome below).
The sequence of the PMM2 locus has been examined in more than 50 CDGS patients with
phosphomannomutase deficiency. A large fraction of these patients are compound heterozygotes
for missense mutations in the PMM2 locus. Less commonly, CDGS type Ia patients are
homozygous for missense mutations. Some PMM-deficient patients do not have mutations in the
coding region of the PMM2 gene. These latter observations suggest that PMM deficiency can
also be caused by mutations that affect the regulation of this gene or the splicing and stability of
its transcripts. Nevertheless, existing and emerging information about the nature of PMM
mutations in PMM deficiency will help advance encouraging early efforts to establish prenatal
diagnosis in CDGS type Ia.
Therapeutic approaches to CDGS type Ia remain problematic. In vitro studies demonstrate that
addition of exogenous mannose to CDGS type Ia fibroblasts can diminish the synthesis of short
lipid-linked oligosaccharides observed in these cells and is associated with an increase in the
incorporation of radioactive mannose into glycoproteins. These results suggested the possibility
that mannose supplementation might ameliorate the defect in CDGS type Ia patients. However,
the biochemical basis for these interesting and encouraging in vitro results is not yet clear, and
mannose therapy in CDGS type Ia patients has not been effective at the biochemical level, nor
does it seem to improve the clinical status of CDGS type Ia patients.
CDGS Type Ib (26 29)
CDGS type Ib is a recently described form of CDGS. There are about ten known patients, but
several have died before diagnosis. Patients with CDGS type Ib do not present with psychomotor
or developmental abnormalities. Instead, these individuals present in the first year of life with
hypoglycemia, severe vomiting and diarrhea, protein-losing enteropathy, and hepatic fibrosis.
CDGS type Ib patients are also susceptible to recurrent thromboses, presumably due to their low
levels of antithrombin III. The isoelectric-focusing pattern of serum transferrin in CDGS type Ib
is identical to that observed in type Ia. Phosphomannomutase activity is normal in CDGS type
Ib; however, phosphomannose isomerase activity is deficient in these patients (Figure 32.2).
Molecular analyses identified missense mutations in the phosphomannose isomerase (PMI) locus
of CDGS type Ib patients. As in CDGS type Ia, PMI deficiency (Figure 32.2) is expected to lead
to decreased levels of GDP-Man, Dol-P-Man, and Glc3Man9GlcNAc2-PP-Dol and elaboration of
truncated forms of this precursor. Again, this circumstance leads to the synthesis of
glycoproteins that are incompletely glycosylated with structurally correct N-glycans. In
principle, these patients may also have defects in the synthesis of other fucosylated glycans and
some glycophosphotidylinositol linkages.
A therapeutic approach to bypass PMI deficiency in CDGS type Ib was suggested by the position
of PMI within the metabolic pathway leading to GDP-Man and Dol-P-Man (Figure 32.2). Under
normal circumstances, Man-6-P can be derived both from Fru-6-P, via PMI, and from free
mannose, via hexokinase. This latter pathway remains intact in CDGS type Ib patients. Mannose
in this pathway presumably comes from glycoconjugates degraded in the lysosome and from the
blood where it is delivered by one or more mannose-specific transporters. Circulating mannose
presumably derives from dietary sources, including mannose-rich glycans in plant and animal
foodstuffs. Mannose in these glycans is liberated from the complex carbohydrates in food
through α-mannosidases that are located in the brush border of intestinal enterocytes.
Apparently, the amounts of mannose derived from a normal diet, and from glycoprotein
catabolism, are insufficient to circumvent the PMI defect in CDGS type Ib via the hexokinase-
dependent pathway. However, it is conceivable that the flux of mannose through this pathway
could yield enough Man-6-P, and its downstream products, to spare CDGS type Ib patients from
the more severe clinical signs and symptoms characteristic of CDGS type Ia.
These considerations prompted efforts to circumvent the PMI defect by providing a

supraphysiological flux of mannose through the hexokinase route to Man-6-P synthesis. In vitro
studies with PMI-deficient cells confirm that extracellular mannose can be used by CDGS type
Ib cells to fully compensate for PMI deficiency. In addition, in vivo studies demonstrate that oral
administration of mannose elevates serum mannose levels well above the Kuptake of the mannose
transporter. These observations provided the scientific rationale for initiating chronic oral
mannose therapy for several PMI-deficient CDGS type Ib patients. This approach was quite
successful and corrected hypoglycemia, protein-losing enteropathy, and intermittent
gastrointestinal problems. It increased plasma antithrombin III levels into the normal range, and
corrected the isoelectric-focusing pattern of serum transferrin and other serum glycoproteins.
Since orally administered mannose is well-tolerated, this approach is clearly a satisfyingly
effective, if not curative, therapy for this life-threatening condition.
CDGS Type Ic (30, 31)
CDGS type Ic (also called type V) has a transferrin glycoform phenotype identical to that
observed in CDGS types Ia and Ib, but PMM and PMI are both normal. CDGS type Ic was
identified quite recently. Two families have been reported in the literature, but nine more have
also been identified. The patients described in published reports have a mild CDGS type Ia
phenotype, which includes psychomotor abnormalities, involving diminished mentation capacity,
and deficiency of motor development. The patients also suffer from recurrent infections and have
decreased levels of several blood-clotting factors.
Biochemical analyses of type Ic fibroblasts show deficient synthesis of Glc3Man9GlcNAc2-PP-

Dol, and an accumulation of Man9GlcNAc2-PP-Dol, which is a poor substrate for
oligosaccharyltransferase (Figure 32.2). Dol-P-Glc and UDP-Glc syntheses are normal, but the
glucosyltransferase activity that adds the first glucosyl residue to the Man9GlcNAc2-PP-Dol
precursor is virtually undetectable in these fibroblasts by in vitro assays. The marked reduction
in glucosyltransferase activity results from mutations in the glucosyltransferase-coding sequence.
Unfortunately, understanding the biochemical lesion in these patients does not provide an
obvious therapeutic approach. Palliative treatment is all that can be offered at this time to these
patients.
It is important to point out that the defect in CDGS type Ic is confined to the N-glycosylation
pathway, since cells remain competent to synthesize normal levels of Man-6-P, GDP-Man, and
Dol-P-Man. This may account for the relatively milder clinical presentation compared to that of
CDGS type Ia patients, where defective synthesis of Man-6-P could affect N-glycosylation, GPI
anchor synthesis, and fucosylation, since GDP-Man is converted to GDP-Fuc.
CDGS Type II (32 36)
Two families have been described with CDGS type II. The patients have dysmorphic features
and psychomotor retardation that is even more severe than that typically seen in CDGS type Ia.
In addition, CDGS type II patients have a ventricular septal defect, but they do not have
peripheral neuropathy or cerebellar atrophy. They are relatively deficient in the blood-clotting
factors XI, antithrombin III, protein C, protein S, and heparin cofactor, as are other CDGS
patients, but they are also deficient in factors IX and XII. CDGS type II is easily distinguished
from types Ia, Ib, and Ic by transferrin isofocusing (see Figure 32.1). In CDGS type II, the
tetrasialylated glycoform S4 is virtually absent and is replaced by the disialylated glycoform S2.
Both glycosylation sites are occupied by truncated asparagine-linked glycans (Figure 32.3)
missing the "second" antenna which is normally initiated by the enzyme N-
acetylglucosaminyltransferase II (GlcNAcT-II; see Chapter 7). Biochemical analyses
demonstrated approximately 98% reduction in GlcNAcT-II activity in CDGS type II fibroblasts.
Molecular analyses show that the patients are homozygous for enzyme-inactivating missense
mutations in the GlcNAcT-II locus. There is no effective therapy for this disorder at present.
Figure 32.3. N-glycans that accumulate in CDGS type II and in HEMPAS. (Normal) N-glycans
that are present on red cell bands 3 and 4.5; "n" may be from 1 to several, and represents the
number of lactosamine units within the polylactosamine chain. CDGSII glycans are not modified
on the α1 6-linked mannose residue, whereas the glycan structure on the α1 3-linked branch is
normal. In HEMPAS patients, N-glycans on red cell bands 3 and 4.5 are deficient in
polylactosamine chains. Furthermore, there is often evidence for deficient trimming of the two
mannose residues that are normally removed by α-mannosidase II (indicated by the +/-
annotation). In some cases, these residues are removed, but the α1 6-branch is not further
modified, as if GlcNAcT-II is not able to function.
CDGS Types III and IV (37 41)
CDGS type III has been described in two families. This disorder is characterized by severe
stationary psychomotor retardation with tetraparesis, cerebral and optic atrophy, and dysmorphic
features. The clinical symptoms differ from CDGS type I in that polyneuropathy, retinal
pigmentation, and cerebellar hypoplasia are absent. Serum transferrin glycoform analysis shows
a predominance of the tetrasialylated isoform S4 but a somewhat increased level of all the
hyposialylated glycoforms.
CDGS type IV has been reported in two families. This form of CDGS is characterized by
psychomotor retardation associated with microcephaly and epileptic seizures and by an apparent
absence of liver disease. Serum transferrin glycoform analysis shows a relative excess of the
disialylated transferrin glycoform. One patient with this transferrin pattern and these clinical
features is defective in Dol-P-Man synthase activity. The patient primarily makes a truncated
lipid-linked oligosaccharide with only five mannose residues, compared to the normal individual
with nine residues. Although this oligosaccharide is transferred to proteins normally, subsequent
oligosaccharide processing in fibroblasts is abnormal. Hybrid chains with only one sialic acid
occur more frequently. This alteration probably accounts for the increase in S2 transferrin seen in
this patient. Addition of mannose to the culture medium of fibroblasts corrects the truncated size
of the precursor oligosaccharide and altered processing. The reason for the correction is
uncertain, but the Km for Dol-P-Man synthase is about fivefold higher than normal in the patient.
Presumably, increasing exogenous mannose increases the local concentration of GDP-Man pool
and drives the reactions. It is important to stress that a deficiency in Dol-P-Man will also affect
synthesis of glycophospholipid anchors, C-mannosylated proteins, and possibly the O-mannose-
based glycans seen in brain.
There are other recent reports of infants with neonatal psychomotor abnormalities, and other
clinical abnormalities, associated with aberrant serum transferrin glycoforms. Nearly all of these
patients were identified through transferrin analysis because they shared a few symptoms with
patients having known forms of CDGS. Transferrin is a powerful diagnostic tool because it can
theoretically detect the consequences of mutations in at least 30 glycosylation-related genes. It
remains to be seen whether such alterations actually have clinical consequences. Transferrin is a
useful marker, but it cannot be used to detect other altered glycosylations such as
polylactosamines or fucosylation, since the N-glycans of transferrin do not contain these
structures.
Leukocyte Adhesion Deficiency II Syndrome (42 53)
The term leukocyte adhesion deficiency (LAD) was originally chosen to describe a syndrome
characterized by frequent, nonpurulent bacterial infections, a leukocyte adhesion defect, and
peripheral blood leukocytosis. There now appear to be two types of these deficiencies, termed
LADI and LADII. The molecular basis of LADI has been shown to be due to diminished or
absent expression of the integrin-type leukocyte adhesion molecules LFA-1 (CD11a/CD18;
αLβ2), Mac-1 (CD11b/CD18; αMβ2), and p150/95 (CD11c/CD18; αXβ2), or normal expression of
dysfunctional forms of these molecules. Mutations in the gene encoding the β-subunit (CD18)
common to these heterodimeric β2 integrins account for the pathogenesis of this disease. The
clinical and laboratory findings are all compatible with the integrin-dependent leukocyte
adhesion defect.
More recently, a LAD-like syndrome has been described in two unrelated boys with normal
levels and function of CD18. Like LADI, there are recurrent, nonpyogenic infection and
leukocytosis. However, in addition to the CD18-dependent symptoms of LADI, these patients
also present with decreased growth rate, severe mental retardation and other neurological
manifestations, and various morphological and skeletal abnormalities not observed in LADI
patients. Furthermore, both patients exhibit the rare Bombay blood group phenotype,
characterized by a deficiency in red cell H blood group structures (see Chapter 16). Both patients
are also deficient in the Lewis blood group antigens and are non-Secretors of the blood group
antigens. Because the H and Lewis antigens correspond to fucosylated oligosaccharide
structures, and because the non-Secretor trait is also associated with an absence of expression of
fucosylated blood group substance (see Chapter 16), these observations suggested that these
patients might have a general defect in fucose metabolism. Since fucosylated leukocyte antigens,
such as sialyl Lewis X, contribute to selectin-dependent leukocyte adhesion (discussed in
Chapter 28), defective fucose metabolism could also account for the LAD-like phenotype in
these patients. In fact, their leukocytes do not express sialyl Lewis X. These observations
prompted Etzioni and colleagues to call β2-integrin-dependent disease LAD type I (LADI) and
the sialyl Lewis X deficiency as LAD type II (LADII).
Subsequent studies showed that LADII neutrophils lack ligands for E-selectin, P-selectin, and L-
selectin. This is implied by flow cytometry studies showing the absence of binding the
monoclonal antibody CSLEX-1, a surrogate marker for E- and P-selectin ligand activity. LADII
neutrophils do not adhere to E-selectin or P-selectin in vitro, nor do such cells participate in L-
selectin-dependent homotypic aggregation events in vitro. In vivo, intravital microscopy studies
demonstrate that LADII neutrophils do not roll on venular endothelium under circumstances
where endothelial E-selectin is expressed and mediates leukocyte rolling under shear flow.
Finally, skin window and skin chamber tests in a LADII patient document a profound reduction
in migration of neutrophils and monocytes into inflamed cutaneous sites. Considered together,
these observations demonstrate that LADII leukocytes do not maintain normal trafficking due to
a deficiency in selectin ligands. These observations, and the leukocytosis characteristic of the
LADII syndrome, are consistent with the defective leukocyte-trafficking phenotypes. They are
also consistent with the leukocytosis observed in P-selectin and/or E-selectin null mice and in
mice deficient in FucT-VII, the α1 3 FucT that makes an essential contribution to leukocyte
selectin ligand activity.
As noted above, LADII patients are deficient in three different fucosylated blood group antigens
(the H/ABO, Secretor, and Lewis systems), and in the fucosylated leukocyte antigen recognized
by the CSLEX-1 antibody. In principle, this constellation of deficiencies could be accounted for
by homozygous null alleles at each of the four fucosyltransferase loci making these antigens (the
H, Se, Lewis, and FucT-VII loci). However, it is now clear that the defect instead lies in the
pathway involving the synthesis of GDP-Fuc (Figure 32.4).
Figure 32.4. Synthesis of GDP-Fuc. The cytosolic de novo pathway begins with GDP-Man and
is catalyzed by two enzymes (a 4,6-dehydratase, and the FX protein) that yield GDP-Fuc.
Alternatively, the "salvage" pathway begins with cytosolic fucose derived from extracellular
sources or from intracellular catabolic sources (not shown). Two enzymes (fucose kinase and a
pyrophosphorylase) yield GDP-Fuc. Cytosolic GDP-Fuc is imported into the Golgi lumen by a
specific transporter.
De novo biosynthesis of GDP-Fuc involves three separate reactions that occur in the cytosol of
mammalian cells (see Chapter 6). This pathway begins with the conversion of GDP-Man to
GDP-4-keto-6-deoxymannose by the enzyme GDP-Man-4,6-dehydratase. GDP-4-keto-6-
deoxymannose is then converted to GDP-Fuc via a two-step reaction involving a 3,5-epimerase-
dependent conversion to GDP-4-keto-6-deoxy-l-Gal, and a subsequent 4-reductase-dependent
formation of GDP-Fuc (Figure 32.4). In mammalian cells, a single polypeptide catalyzes the
epimerization and reduction reactions. This epimerase-reductase polypeptide is also known as
the FX protein. The FX protein was first described as a "tumor-specific" antigen and was also
discovered as an abundant red cell protein with an unknown function. Molecular cloning studies
revealed that the FX protein shares primary sequence similarity with prokaryotic epimerase-
reductase enzymes involved in GDP-Fuc synthesis. Biochemical studies have confirmed that the
FX protein functions as the GDP-4-keto-6-deoxymannose 3,5-epimerase-4-reductase.
Mammalian cells can also synthesize GDP-Fuc using the salvage or "scavenger" pathway.
Extracellular fucose can be transported into the cytosolic compartment via fucose-specific
plasma membrane transporters. Alternatively, fucose cleaved from endocytosed glycoproteins
can enter the cytosol. Fucose kinase forms Fuc-1-P, and GDP-Fuc pyrophosphorylase (GDP-Fuc
"synthase") makes GDP-Fuc from Fuc-1-P. Cell culture experiments suggest that the scavenger
pathway makes a relatively minor contribution to the GDP-Fuc pools.
Following its synthesis in the cytosol, GDP-Fuc is subsequently transported into the lumen of the
Golgi apparatus (see Chapter 6). Luminal-localized GDP-Fuc serves as a substrate for Golgi-
localized fucosyltransferases that fucosylate membrane-associated and soluble glycoconjugates.
Studies exploring GDP-Fuc metabolism in cultured cells derived from LADII patients indicate
that the defect in this disorder lies in the de novo pathway of GDP-Fuc biosynthesis. Under
normal culture conditions, Epstein-Barr-virus-transformed B lymphocyte cell lines prepared
from LADII patients do not bind the fucose-specific lectin Lotus tetragonobolus agglutinin,
confirming that LADII cells do not express fucosylated cell surface glycans. However, growth of
the LADII cells in fucose-containing media restores lectin binding. These observations
demonstrate that the salvage pathway for GDP-Fuc synthesis is intact in LADII cells and imply
that transport of GDP-Fuc into the Golgi lumen is not impaired (Figure 32.4). In contrast, in vitro
studies reveal that the conversion of GDP-Man to the GDP-4-keto-6-deoxymannose intermediate
is defective, whereas the epimerase-reductase reaction is apparently unaffected by the LADII
lesion. However, 4,6-dehydratase activity exists in an inactive form in LADII cytosol but
becomes active after a preincubation step. Furthermore, although the 4,6-dehydratase reaction is
impaired in LADII cells, the coding sequence of the LADII dehydratase locus is intact, and the
4,6-dehydratase protein is expressed at normal levels in LADII cells. These observations imply
that the defect in LADII involves an unknown factor that may control the de novo GDP-Fuc
synthetic pathway, via an interaction with the GDP-Man4,6-dehydratase.
A recent report indicates that the immunological impairment in LADII may diminish during
maturation, since there seems to be a reduction in the incidence of infectious episodes in these
patients as they grow older. Since the fucosylation defect in LADII cells can be corrected via the
salvage pathway, a fucose-supplemented diet might lead to clinical improvement, since it might
partially restore defective leukocyte selectin ligand activity. This potential therapy is not without
risk, since it might also restore H blood group determinant expression in red cells, as well as A
and/or B determinants, depending on the patient's ABO genotype. Because LADII patients, like
most individuals with the Bombay phenotype, maintain high titers of the IgM class, complement
fixing anti-A, anti-B, and anti-H antibodies, robust restoration of these red cell antigens by oral
fucose therapy could precipitate an episode of acute, autoimmune hemolytic anemia.
Despite the possible complications, fucose supplementation was cautiously tested on the most
recently diagnosed LADII patient. This 2-year-old boy presented with all of the typical LADII
symptoms. The primary defect was not identified, but no mutations were found in the coding
sequences of either the dehydratase or the reductase-epimerase, and the respective mRNA levels
were normal. Fibroblasts from the patient were defective in fucosylation, but it was restored by
providing exogenous fucose. On the basis of this finding, the patient was given multiple daily
doses of fucose, which were efficiently absorbed into the blood. Soon after beginning therapy,
some fucosylated selectin ligands appeared on neutrophils and core fucosylation of serum
glycoproteins returned. During 5 months of treatment, infections and fever disappeared, elevated
neutrophil counts returned to normal, and, surprisingly, psychomotor capabilities even improved
based on standardized tests. Fortunately, H-antigen did not appear on the red cell surface and
there was no hemolytic anemia. These results underscore the ability of the salvage pathway to
contribute directly to fucosylation. The variable effectiveness of fucose therapy in restoring
fucosylation suggests that phenotypic correction may be cell-type-specific and glycoconjugate-
selective. Very little is known about fucose utilization or how it is regulated in higher animals
(see Chapter 6)
Congenital Dyserythropoietic Anemia Type II (54 64)
Hereditary erythroblastic multinuclearity with a positive acidified-serum lysis test (HEMPAS) is

also known as congenital dyserythropoietic anemia type II. It appears to be an autosomal
recessive disorder, but it is not clear if the primary defect is in glycosylation (see Chapter 37).
The acronym HEMPAS refers to the observation that serum from some normal individuals
contains an antibody that binds to red cells from HEMPAS patients and will lyse these cells
when the serum is acidified. The nature of the antigen recognized by this antibody in not known,
nor is it understood why this antibody is present in some but not all normal human sera. In
HEMPAS patients, the red cells and their marrow precursors are fragile and susceptible to lysis,
which accounts at least in part for the ineffective production of red cells by the marrow.
Hyperplasia of the erythroid precursors in the marrow is observed in HEMPAS patients and is
accompanied by the presence of multinucleated erythroblasts. Ultrastructural analyses disclose
abnormal membrane structure in cells of the erythroid lineage. Patients with HEMPAS suffer
from the consequences of ineffective erythropoiesis, including anemia, marrow hyperplasia,
enlarged spleen, gallstones, and liver disease, with excessive accumulation of iron in the liver.
The disease primarily affects the marrow red cell progenitors (erythroblasts) and the red cells
themselves, but the biochemical defects discussed below have sometimes been found in
leukocytes and other tissues in some individuals.
Analysis of red cell glycans in HEMPAS discloses a structural defect in N-glycans borne by a
pair of red cell membrane proteins termed band 3 (the anion exchange protein) and band 4.5 (the
glucose transporter protein). These proteins normally bear biantennary complex N-glycans that
are decorated with long polylactosamine on both antennae. However, in HEMPAS patients, red
cell bands 3 and 4.5 are virtually devoid of polylactosamine. In contrast, these cells contain
relatively large amounts of polylactosamine on glycolipids (i-antigen), which are relatively
minor components in normal red cells.
Structural analyses of the band-3 and band-4.5 glycans indicate that these vary to some
significant extent among different individuals with HEMPAS. In some such patients, the
abnormal glycans feature a truncation of the antenna attached to the α1 6-linked mannose
residue of the trimannosyl core structure in the N-glycan (Figure 32.3). This structure is the
immediate synthetic precursor to GlcNAcT-II, and its excessive accumulation has implied a
defect in the expression or activity of this enzyme in some HEMPAS patients (Figure 32.4).
Although it has been reported that the cells in some HEMPAS patients exhibit 70 90% reduction
in GlcNAcT-II activity, the pathophysiological relevance of these observations is not clear, since
the inherited GlcNAcT-II deficiency that causes CDGS type II is clearly associated with a much
more severe disease and involves multiple organ systems. It has been suggested that a red-cell-
specific decrement in GlcNAcT-II could account for the erythroid-specific manifestations in
HEMPAS, versus the pan-lineage deficit of this enzyme characteristic of CDGS type II.
However, it is not clear how an erythroid-specific deficiency of GlcNAcT-II could cause the
virtually complete deficit of band-3 polylactosamine observed in HEMPAS red cells, especially
since these glycans are only reduced by approximately 50% in CDGS type II. These
considerations make it unlikely that simple defects in the GlcNAcT-II locus will be found to
account for HEMPAS.
In other HEMPAS patients, hybrid-type N-glycans predominate and are characterized by the
retention of the α1 3- and α1 6-linked mannose residues attached, in turn, to the α1 6-linked
mannose of the trimannosyl core (Figure 32.3). This structure is the synthetic substrate for α-
mannosidase II and must be processed by this latter enzyme before GlcNAcT-II may act. The
excessive accumulation of this partially processed glycan in the red cells of some HEMPAS
patients has implied a defect in expression or activity of α-mannosidase II in these individuals
(Figure 32.3). In one such patient, α-mannosidase II activity is virtually absent in some cells, and
decreased accumulation of the α-mannosidase II RNA transcript seems to account for the
enzyme deficiency in this family. However, the molecular defect is unknown, but it may lie in
another locus that regulates its expression.
Why is the apparent α-mannosidase II deficiency in these HEMPAS cases limited to the
erythroid lineage? From studies of mice rendered deficient in α-mannosidase II, the best
explanation is the presence of another α-mannosidase II-like activity (termed α-mannosidase III)
in extra-erythroid tissues. Additionally, recent molecular cloning studies have identified a related
α-mannosidase locus, termed α-mannosidase IIx. The gene that encodes α-mannosidase III
activity has not yet been identified.
In summary, the molecular basis for HEMPAS is not clearly understood. Genetic linkage studies
in some HEMPAS pedigrees have excluded defects in the α-mannosidase II locus, the α-
mannosidase IIx locus, and the GlcNAcT-II locus. Furthermore, the biochemical basis for
diversion of erythroid polylactosamine biosynthesis from N-glycans to glycolipids is not at all
clear. These observations, together with evidence for significant genetic heterogeneity implied
by glycan structural analyses, indicate that significant challenges lie ahead in understanding the
pathophysiology of this disorder.
Galactosemia (65 70)
Galactosemia refers to a group of diseases caused by inherited defects in the genes encoding
three enzymes in galactose metabolism. One of these disorders, termed classical galactosemia, is
caused by a deficiency of Gal-1-P uridyl transferase (GALT; Figure 32.5). This disease may
decrease synthesis and availability of UDP-Gal. Defects in UDP-Gal 4 epimerase or in
galactokinase are rare, do not apparently impact on UDP-Gal synthesis or accumulation, and are
not discussed further.
Figure 32.5. UDP-Gal synthesis and galactosemia. The most common form of galactosemia is
due to a deficiency of Gal-1-P uridyltransferase (GALT). This enzyme normally utilizes Gal-1-P
derived from dietary galactose. In the absence of GALT, Gal-1-P accumulates, along with
excessive galactose and its oxidative and reductive products galactitol and galactonate (not
shown). UDP-Gal synthesis may also be impaired in the absence of GALT, but not completely
so, since UDP-Gal 4 epimerase (GALE) can form UDP-Gal from UDP-Glc, and can supply the
galactosyltransferases required for normal glycoconjugate biosynthesis.
GALT-deficient individuals present in infancy with a failure to thrive, enlarged liver, jaundice,
and cataracts. Institution of a lactose-free diet ameliorates most of the acute symptoms of the
disorder. This treatment reduces the amount of galactose entering the galactose metabolic
pathway and thereby diminishes the accumulation of excessive amounts of galactose and Gal-1-
P that are thought to contribute to the symptoms of the disease. The reduction in galactose
accumulation also helps to inhibit the formation of galactitol and galactonate, which are
produced via reductive or oxidative metabolism of galactose, respectively. Galactitol is not
metabolized further and has osmotic properties that can make a dominant contribution to cataract
formation. Unfortunately, a galactose-free diet apparently does not prevent the appearance of
cognitive disability, ataxia, growth retardation, and ovarian dysfunction characteristic of this
disease. It has been suggested that these long-term complications in treated GALT-deficient
individuals may be due to small amounts of toxic metabolites that continue to accumulate in
these patients (via small amounts of dietary galactose and via de novo synthesis from Glc-1-P;
Figure 32.5). It has also been suggested that GALT deficiency leads to a relative deficit of UDP-
Gal, the nucleotide sugar substrate used by galactosyltransferases, with a consequent deficiency
of galactosylated glycans that may contribute to the pathogenesis of this disease.
Hypogalactosylation of glycoproteins and glycolipids has been observed in some GALT-
deficient individuals and seems to support this hypothesis. In addition, patients not adhering to
galactose-free diets synthesize abnormal transferrin glycoforms typical of CDGS types 1a and
1b. The pattern returns to normal when they return to galactose-free diets. However, it is not
clear whether GALT-deficient patients have a physiologically relevant deficit of cellular UDP-
Gal, especially since significant amounts of this compound are formed from UDP-Glc by UDP-
Gal 4 epimerase (Figure 32.5). It is possible that accumulation of hypogalactosylated glycans is
secondary to a general metabolic abnormality in these patients. A relationship between the
accumulation of these abnormal structures and the neurological deficits in these patients remains
to be demonstrated.
Defects in Proteoglycan Synthesis (71 79)
Proteoglycans and their glycosaminoglycan GAG chains are critical components in extracellular
matricies. For a discussion of their biosynthesis, core proteins, and function, see Chapter 11.
Ehlers-Danlos syndrome (progeroid type) is a connective tissue disorder characterized by failure

to thrive, loose skin, skeletal abnormalities, hypotonia, and hypermobile joints, along with
delayed motor development and delayed speech. The molecular basis of the disorder in one
patient appears to be in the synthesis of the core region common to xylose-based GAG chains.
Decorin, a dermatan sulfate proteoglycan that binds to collagen fibrils, was partially deficient,
and some molecules were made without an extended GAG chain. The activity of galactosyl
transferase I, the enzyme that adds galactose to xylosylserine, was only 5% of normal in this
individual, whereas the parents had 50% of normal activity. The patient s enzyme was also
thermolabile. In addition, galactosyl transferase II, the enzyme responsible for adding the second
galactose residue to the GAG chain core, had only 20% of normal activity, and both parents
showed reduced activity. Further analysis will be needed to resolve the specific defect, but one
possible explanation is that the primary mutation affects the formation or stability of a
biosynthetic complex involving several GAG-chain biosynthetic enzymes. The selective effect
seen on decorin may reflect substrate preferences.
Three autosomal recessive disorders, diastrophic dystrophy (DTD), atelosteogenesis type II
(AOII), and achondrogenesis type IB (ACG-IB), all result from defective cartilage proteoglycan
sulfation. These forms of osteochondrodysplasia have various outcomes. AOII and ACG-IB are
parinatally lethal due to respiratory insufficiency, whereas DTD patients have symptoms only in
the cartilage and bone, including cleft palate, clubbed feet, and other skeletal abnormalities.
Those DTD patients surviving infancy often live a nearly normal life span. All of these disorders
result from different mutations in the DTD gene that encodes a sulfate transporter. Unlike
monosaccharides, sulfate released from degraded macromolecules in the lysosome does not seem
to be salvaged well. The heavy demand for sulfate in bone and cartilage proteoglycan synthesis
probably explains why the symptoms are most evident in these locations.
Keratan sulfate in the cornea is an N-glycan with polylactosamine repeats (GlcNAcβ1 3Galβ1
4) variably sulfated at the 6-position. Another autosomal recessive disease, macular corneal
dystrophy (MCD), causes the cornea to become opaque and corneal lesions develop. Two types
of MCD have been described. MCD I appears to be a deficiency in sulfating the repeating units.
Both galactose and GlcNAc are sulfated in keratan sulfate, but the enzyme that sulfates galactose
in keratan sulfate and GalNAc in chondroitin sulfate is normal in patients. This leaves the
GlcNAc 6-sulfotransferase as a more likely candidate for the defect, but this has not yet proven.
MCD II differs from MCD I in that the defect in MCD II is not proven, but it may be an allelic
form of MCD I.
Future Directions (80 83)
It is now clear that a number of syndromes characterized by neonatal presentation of severe

neurological and metabolic dysfunction are caused by defects in glycosylation typified by
CDGS. It seems likely that an increasing awareness of this constellation of diseases by
neonatologists and pediatricians and the availability of a relatively straightforward diagnostic test
for these disorders will mean that CDGS will be diagnosed with increasing frequency. To date,
only five specific genetic lesions have been identified to account for this disease. However, it
seems likely that defects in other genetic loci known to be required for N-glycan synthesis may
be found to cause other forms of CDGS. Most of these genes have now been cloned and
characterized, and it is likely that the remaining genes will soon be, based in part on successful
efforts to isolate these loci in yeast and other organisms, and on the emerging wealth of human
DNA sequence information in the public domain EST (expressed sequence tag) databases. Such
sequence information will help to define precisely the defect in patients with aberrant transferrin
glycoforms suggestive of CDGS and should facilitate prenatal diagnostic efforts. Importantly, as
best illustrated by CDGS type Ib and one case of LADII, the ability to discover effective
treatments for these devastating disorders will be aided by a clearer understanding of the
biochemical pathways involved and through identification of the genetic lesions that account for
defects specific to different forms of CDGS. It also seems appropriate to continue to develop
models of these diseases via induced mutation approaches in the mouse. These animals provide
an opportunity to uncover as yet unknown components of the mammalian glycan synthetic
pathways. Moreover, they offer the potential to test therapeutic approaches to the extent that
murine deficiencies in glycan synthesis can be shown to accurately reflect the corresponding
human pathophysiology.
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33. Determining Glycan Function Using Genetically
Modified Mice
at San Diego).
MANY CELL LINES THAT LACK GLYCOSYLTRANSFERASES or glycosidases do not

exhibit obvious phenotypes, whereas identical mutations in intact organisms yield pathologies
and in some cases embryonic lethality. Several approaches to determine oligosaccharide function
in vivo include expressing glycosidases, masking glycosyltransferases, competing
glycosyltransferases, and overexpressing endogenous glycosyltransferases (Figure 33.1). This
chapter details approaches that are useful in defining the function of glycans, especially by
transgenesis and gene targeting using the mouse as a model vertebrate. The specific techniques
used are reviewed. A significant amount of information on glycan function has been gained in
recent years using these approaches, and this information is also presented.
Figure 33.1. Approaches to altering glycosylation in studies of oligosaccharide function.

(Modified, with permission, from [66] Varki and Marth 1995.)
Background
Following the development of molecular genetic techniques to isolate and alter DNA structure in
vitro, methods to alter the germ-line DNA of intact animals were devised. In the early 1980s, a
procedure was successfully developed in the mouse, referred to as transgenesis, that introduces
exogenous DNA into the germ line. The term transgenesis refers to the production of a
transgenic organism, e.g., an organism in which DNA from another organism has been
transferred, so that the host acquires the genetic traits encoded by the transferred genes in its
chromosomal composition. The mouse was considered the best organism in which to initially
develop transgenesis, since all mammals have similar physiologies and mice can be maintained
easily in relatively little space. Additionally, mouse generation time is short, approximately 8
weeks, which is rapid enough for practical use. Why transgenic animals? Gene transfer is
valuable in diploid organisms with long life cycles where classic methods (breeding) are
impractical. In addition, studies of some systems (the immune system, for example), or of
ontogeny itself, require a physiologic "body."
In developing transgenesis, the earliest studies used blastocyst-stage mouse embryos for
injection of SV40 into the blastocoel cavity. After implantation in foster mothers, live-born
offspring were found to contain the SV40 genome in various tissues. However, the intact SV40
DNA did not integrate or colonize the germ line, and thus, germ-line transformation did not
occur. Although retroviral transduction of blastocyst embryos did achieve germ-line
modification, expression was poor, due to the now recognized presence of cellular factors that
inhibit retroviral expression. Subsequently, the development of pronuclear microinjection
techniques using purified DNA resulted in germ-line transformation and transgene expression.
Transgene expression can be regulated by the promoter-enhancer elements chosen in
constructing the transgene vector. Transgenesis by pronuclear injection results in an animal in
which the transgene is most frequently integrated in all cells of the body. This procedure is
effective in providing for dominant genetic studies in vivo.
An approach to generating specific recessive genetic lesions in vivo in intact vertebrates has long
been sought. Understanding the physiologic function of a gene may be best gained by studying
an organism rendered deficient in the function of that particular gene. In the 1980s, a
combination of techniques and reagents was developed that allowed the production of recessive
genetic lesions in intact mice. One development was the invention of PCR. Separately, the
derivation of pluripotent embryonic stem cell lines allowed the production of chimeric mice with
modified germ lines. Recessive genetic models are now commonly produced by a method often
referred to as gene targeting. A refinement to this approach has also been gained by development
of site-specific DNA recombination during and subsequent to gene targeting. Genetic lesions can
now be generated in specific cell types of intact mice and at specific times.
Transgenesis (1 8)
To produce transgenic animals requires some knowledge of the organism's embryonic

development, as well as competence in microinjection techniques and various surgical
procedures. In addition, to assess the validity of results achieved by transgenesis requires an
understanding of how transgenes affect endogenous genomic structure and how they become
expressed. Overall, the technique is rather complex, expensive, and inefficient. The production
of a transgene vector that incorporates the desired elements is relatively easy. Transgene DNA is
microinjected into a pronucleus of a fertilized egg (zygote), prior to the fusion of male and
female pronuclei (Figure 33.2). The next day, zygotes that have survived the injection and have
become two-cell-stage embryos are implanted into the oviduct of a female mouse that has been
prepared to accept an embryo by prior mating with a vasectomized male. It can require
approximately 100 zygotes to generate one transgenic mouse. On average, 50% of zygotes
survive DNA injection and divide to the two-cell stage. Of those, 50% may implant successfully
and develop to term. Anywhere from 5% to 25% (1 6 mice from 100 zygotes) may be transgenic
as usually determined by genotyping tail DNA.
Figure 33.2. Pronuclear injection of DNA into a mouse zygote during the production of
transgenic mice
The mechanism of DNA integration likely involves breaks in chromosomal DNA, since linear
DNA is fivefold more efficient at integration than is circular DNA. Integration is rapid,
occurring more than 90% of the time while embryos are at the one-cell stage. If integration
occurs at the two-cell stage, the transgenic mouse will be chimeric, because not all cells in the
body will have acquired the transgene. Transgenes integrate predominantly as head-to-tail arrays
of anywhere between one and several hundred copies. Approximately 5% of transgene
integrations occur within an endogenous gene and thus generate a break, and potentially a
mutation of a functional cellular gene. Insertional mutation events producing an inactivating
(null) mutation may lead to observable phenotypes that are not due to transgene expression. This
is the reason that multiple transgenic lines established by unique founders (the "founding"
transgenic mouse is the original born from a specific pronuclear injection) are considered
essential to control for the effect of the transgene on the endogenous genome.
Transgene expression is dependent on a number of factors, and early experiments have given
significant insights into chromosomal structure and function. Perhaps only 10 50% of transgenic
mice express the transgene, which was originally much lower before it was recognized that the
presence of plasmid sequences in transgenes is detrimental. It appears that host factors are able
to extinguish expression of transgenes incorporating bacterial and bacteriophage DNA
sequences. Most, but not all, transgenes examined are appropriately expressed, considering their
use of specific promoter and enhancer elements. Since transgene expression can be higher than
that of an endogenous homolog, optimal gene expression does not depend on normal
chromosomal context. However, chromatin structure at the site of integration can influence
transgene expression. Levels of expression vary with distinct lines of equal copy number and
different integration site, inferring that chromosomal position can influence expression. This may
be due to the varied accessibility to host transcription factors. In addition, a few transgenic lines
may not express at all, perhaps due to transgene integration into regions of heterochromatin.
Because multiple transgene copies are present in the arrays, there is no way to determine the
number of transcriptionally active transgenes present. However, the poor correlation of transgene
expression with transgene copy number implies that only a few of the transgenes are expressed
and that the array is sensitive to chromosomal position.
Tissue-specific transgene expression can be routinely achieved. In general, if a transgene is

expressed at all, it is usually appropriately expressed, despite its integration at different
chromosomal positions. Therefore, trans-acting proteins involved in establishing host-tissue-
specific gene expression can find their target sequences and activate transcription at most
chromosomal positions. This appears to be true even among species divergent by millions of
years of evolution. Therefore, many signals involved in tissue-specific gene regulation are
evolutionarily conserved. Occasionally, sequence effects in transgene vectors comprising
elements from divergent sources result in expression patterns not observed with either the gene
or promoter/enhancer element separately. Promoter and/or enhancer elements normally
expressed in many cell types are often the most sensitive, in transgenes, to the influence of
chromosomal position on expression. With a tissue-specific enhancer, the chromosomal position
can only influence expression levels in one or a few cell types. However, with a
promoter/enhancer that functions in a variety of cell types, the chromosomal position effect may
vary in different cell types and from one founder animal to another, leading to an apparently
random success rate in establishing transgene expression. Some transgenes have never been
successfully expressed, which may be due to embryonic lethality with transgene expression.
Gene "silencers" have also been reported, including the 3 region of the v-src-coding sequence
and a CD4 gene intron that controls T-cell lineage commitment.
Transgene expression in offspring is generally identical to that of founder parents. However,

alterations have been observed as a result of genomic imprinting. Genomic imprinting breaks the
Mendelian rule of haploid equivalency and was first recognized in studies of balanced
Robertsonian chromosomal translocations, where some alleles must be inherited from both male
and female genomes for embryo viability. The mechanism of genomic imprinting includes
changes in DNA methylation. Transgenes can also undergo imprinting, leading to variations in
expression from one generation of offspring to the next, depending on the sex of the parent
providing the transgene.
Modifying Glycosylation in Vivo by Transgenesis (9 17)
In the first transgenic experiment devised to alter glycosylation in vivo, an influenza virus sialic
acid O-acetylesterase was found to interfere with embryogenesis, as its expression resulted in a
developmental block at the two-cell stage (Figure 33.3). This viral enzyme removes the O-acetyl
group linked to many sialic acids contained on Golgi and cell surface oligosaccharides by an
endogenous sialic acid O-acetyltransferase activity (see Chapter 15). When the transgenic O-
acetylesterase was expressed preferentially in somatic compartments, alterations in tissue
morphogenesis were observed in the retina and adrenal gland. These results imply that the O-
acetylation of sialic acids is an oligosaccharide modification required for early preimplantation
embryogenesis and in organogenesis.
Figure 33.3. Developmental block at the two-cell embryo stage induced by the influenza sialic
acid O-acetylesterase following pronuclear injection. (Reprinted, with permission, from [16]
Varki et al. 1991 [© Cell Press].)
The above example demonstrates the use of an esterase to remove an endogenous

oligosaccharide modification (Figure 33.1). This is distinct from an approach to overexpress an
endogenous glycosyltransferase in an effort to notably enhance endogenous function. In studies
with a β1 4 galactosyltransferase transgene, overexpression led to reduced sperm-egg binding
and inhibited the development and lactation response of the mammary gland. This β1 4
galactosyltransferase is expressed in the Golgi of most cells normally, but it can also be found on
the sperm cell surface where it has been implicated in regulating sperm-egg binding during
fertilization. Ectopic expression of either α1 3 galactosyltransferase (Gal3T) or α1-3/4
fucosyltransferase (FucT-III) had no effect on development, although the expected
galactosylated or fucosylated glycoconjugates were found in higher abundance in adult tissues.
With overexpression of Gal3T, mice were reported with lower body weights, hair growth
alterations, and increased mortality. With increased α1-3/4 fucose linkages engineered in the
stomach of a transgenic mouse, the desired increase in Lewis b antigen was observed and may
provide a mouse model for studies of infection with Helicobacter pylori, a bacterium known to
cause stomach ulcers.
Masking the action of a "competing" glycosyltransferase may be effected by overexpression of a

glycosyltransferase or glycosidase. Formation of the α1 3Gal terminus on oligosaccharides by
Gal3T generates the major xenotransplantation antigen of human relevance. Old World primates
have specifically lost the ability to generate this structure from acquired germ-line mutations in
the Gal3T gene. As a result, human serum is rich in immunoglobulins that bind to this epitope
and initiate the immunologic rejection of organs transplanted from other species, such as pigs. A
means to reduce or eliminate cell surface α1 3Gal residues was desired, and one means to
achieve this involved overexpression of an α1 2 fucosyltransferase (EC 2.4.1.691) that normally
generates the H blood group locus and that may compete for the same substrate as the Gal3T.
The reduction reported in cell surface α1 3Gal residues was further enhanced when α1 2
fucosyltransferase transgenic mice were bred to be cotransgenic with a transgene encoding an α-
galactosidase that cleaves α1 3Gal linkages.
Transgenesis by pronuclear injection is a dominant genetic approach on a genomic background

already containing a complete set of genes, as well as loci possibly mutagenized by transgene
integration. In general, dominant transgenic approaches require reproducible, specific, and
relatively high expression levels of the transgene, sometimes in a long-term manner, and this is
not easily achieved. Although relevant physiologic information is still to be gained, a dominant
genetic approach can complicate the assignment of structure-function relationships, and the
random nature of transgene integration adds further complications.
Gene Targeting Using Embryonic Stem Cells (18 28)
The development of recessive genetic techniques in the mouse has provided a needed approach
to understand the molecular genetic basis of physiological systems. Gene-targeting techniques
are now routinely used to alter germ-line DNA by homologous recombination and therefore in a
highly specific and experimentally defined manner. Homologous recombination between
exogenous DNA and chromosomal DNA occurs at high efficiency in yeast and has been used
with great success to produce recessive genetic lesions and thereby reveal the function of
endogenous yeast genes. However, homologous recombination is a low-frequency event in the
mammalian genome. Detection of this event using PCR has enabled the clonal selection of cells
that have undergone homologous recombination in vitro following gene transfer. Coupling a
PCR detection system with use of ES cells provided a major breakthrough in the late 1980s with
the insertion of an experimentally altered allele into the mouse germ line.
ES cells are derived from the inner cell mass of blastocyst-stage embryos and can be maintained
in vitro indefinitely in the pluripotent state with appropriate medium. This medium must be
enriched in a cytokine originally termed leukemia inhibitory factor and now known by various
trade names. Gene transfer into ES cells by electroporation is easily accomplished with stably
transfected clones usually bearing single integration events that incorporate the neomycin
phosphotransferase gene. Stable integrants are thus often selected by resistance to the antibiotic
G418. The targeting vector is designed such that homology with genomic DNA is placed
flanking the mutation to be generated (Figure 33.4). PCR screening and clone isolation follow,
and subsequent studies at the genomic level following clone outgrowth can confirm the altered
allelic structure. The frequency of homologous recombination, which may range from 0.1% to
sometimes 10%, has been found to increase dramatically with the use of isogenic DNA, i.e.,
DNA from the same ES cell genome or strain of mice. Most ES cells have been derived from the
129 strain of mice, although ES cells from other strains, such as C57BL/6, have now been
isolated.
Figure 33.4. Classic gene-targeting strategy used to inactivate an endogenous allele. The
neomycin phosphotransferase gene (NEO) acts both as a selectable marker and as the insertional
mutation that disrupts a coding exon of the gene.
Following the isolation and characterization of correctly targeted ES cells, they are microinjected
into the "host" blastocyst-stage embryo (Figure 33.5). Approximately eight to ten ES cells are
injected, and these integrate into the inner call mass, thereby colonizing various tissues in the
developing embryo. If their colonization includes germ cells, the resulting mouse will transmit
the altered allele to its offspring. The genotype of the host embryo is often C57BL/6 because this
allows a coat color analysis, indicating the presence of chimeric mice born from 129-strain-
derived ES cells (Figure 33.6). The 129 ES cell genome is homozygous for the Agouti locus and
yields mice with brown coat color, whereas the C57BL/6 genome yields mice with black hair.
However, the Agouti locus is dominant, and the amount of Agouti (brown hair) present on the
chimeric mice coat tends to be an indicator of the likelihood that the ES cell genome has also
colonized the germ cells. Breeding chimeric mice with C57BL/6 mice is a test for this and can
generate mice heterozygous for the mutation. Pure Agouti offspring produced by this mating
indicates that the ES cell genome has colonized the germ line. Of Agouti offspring, 50% will be
heterozygous for a mutation that is "unlinked" in meiotic recombination to the Agouti locus.
Subsequently crossing mice heterozygous for the mutation produces offspring that are
homozygous for the mutant gene (25% on average), unless the mutation is lethal in
embryogenesis.
Figure 33.5. Microinjection of gene-targeted ES cells into a blastocyst-stage embryo in

producing chimeric mice.
Figure 33.6. Chimeric mice generated by injection of ES cells into a host (C57BL/6) blastocyst-
stage embryo. Different amounts of chimericism (Agouti coat color) are apparent.
In this manner, hundreds of specific gene mutations have now been engineered in the mouse
germ line in studies that are deciphering the molecular genetic basis of physiology and disease.
A significant proportion of these mutations are found to be highly disadvantageous to the
embryo or offspring born, often yielding serious developmental defects and lethality.
Investigating Gene Function in Vivo by Conditional

Mutagenesis (29 40)
The goal of developing conditional mutagenesis approaches in vivo demanded the use of a site-
specific DNA recombination system. The Cre and Flp recombination systems are intrinsic to
bacteriophage and yeast, respectively, and thus have been used for this purpose. Both Cre and
Flp are members of the integrase family of recombinases and can function without the use of
ATP or cellular cofactors. They bind to 34-bp DNA sequences termed loxP for Cre or frt for Flp.
Their mechanism of action is termed conservative and requires a Holliday intermediate structure
with specific nucleotide base pairing at the recombination synapse. Cre recombinase appears to
function better than Flp in mammalian cells, although development of more effective forms of
Flp recombinase has recently yielded promising results. Cre can act in an efficient, heritable,
tissue- and site-specific manner to excise DNA specifically flanked by direct repeats of loxP at
distinct chromosomal locations. Following recombination, excised DNA is degraded, as it does
not appear integrated elsewhere in the genome.
A novel mutagenesis strategy has been devised in which loxP sites are used in acquiring gene-
targeted ES cell clones. Homologous recombination results in the incorporation of loxP sites
flanking the gene element to be deleted (often a crucial exon) and the selectable markers, such as
Neo and HSV-TK. Following transient Cre expression in these parental gene-targeted ES cells,
subclones bearing either type I or type II recombination are selected for by the absence of the
HSV-TK gene using gancyclovir (Figure 33.7). The type I recombinant (one loxP site left) is
used to produce the systemic (classic) congenital deficiency. An advantage is the lack of
prokaryotic Neo and HSV-TK genes, which can influence the expression of neighboring genetic
loci and male fertility. The type II recombinant (two loxP sites remain) bears the conditional
mutation, with loxP sites flanking the gene in what is expected to be a nondeleterious manner,
from ES cell clones containing the conditional mutation. Following the production of chimeric
and heterozygous mice, a breeding strategy to achieve conditional gene mutation in vivo is
employed using transgenic mice bearing the Cre transgene (Figure 33.8). Mice homozygous for
the floxed allele and transgenic for Cre will have tissues that undergo gene mutation only where
Cre is expressed. Cre is provided as a transgene under the control of specific promoter-enhancer
elements to generate a restricted expression pattern.
Figure 33.7. Strategy for achieving systemic and conditional gene mutagenesis in ES cells using
the Cre-loxP recombination system.
Figure 33.8. Site-directed recombination and conditional gene mutagenesis in vivo. Mice
bearing a tissue-specific Cre recombinase transgene expression profile in somatic cells (circled)
are mated in generation 1 (G1) with mice heterozygous for a gene exon (boxed) innocuously
flanked by loxP sites (red arrowheads) in intronic regions following homologous recombination
in ES cells. Heterozygous (G2) and homozygous (G3) mutations occur specifically in cells
expressing the Cre recombinase. (Reprinted, with permission, from [36] Marth 1996 [©
American Society for Clinical Investigation].)
In practice, Cre recombination has been successfully demonstrated in vivo among virtually all
somatic cell types, including postmitotic cells in the brain and liver. The efficacy is high in
systems that produce high levels of nuclear-localized Cre protein, and chimeric tissues in which
some cells have not undergone recombination also occur. The use of inducible promoters such as
the tetracycline system and modification to the Cre sequence that allows hormonal activation are
additional refinements that link Cre recombination to exogenous and experimental stimulation.
Conditional mutagenesis in vivo obviously provides a more defined physiologic context in which
to study gene function and may in some cases be necessary when systemic gene ablation leads to
early lethality, thereby precluding the investigation of gene function in adult systems. The use of
conditional mutagenesis by site-directed recombination is likely to be especially applicable in
glycobiology where in vivo studies of glycans can require a focus on cell-cell interactions among
relatively disparate cell types in an intact organism.
Functions of Glycans Revealed by Recessive Genetic Lesions

(31,41 61)
To determine the function of glycans in vivo, enzymes participating in glycan diversification are
now routinely inactivated in the mouse germ line. Using this recessive genetic approach, such
mutations effectively restrict the formation of specific oligosaccharide linkages. When this
approach is coupled to a defined biosynthetic and diversification pathway, like that of N-glycans,
it is possible to define specific structure-function relationships of glycans in the intact animal.
Like mutations generated in other obviously hierarchical biological systems, such as in protein
phosphorylation, the inactivation of an enzyme can affect the structure of many "downstream"
molecules. Very few to more than a dozen glycoproteins or phosphoproteins can be altered in
systems deficient in a single enzyme. Nevertheless, such experiments are considered to represent
the best approach currently available to establish the functions of genes, especially those that are
conserved in phylogeny. As additional data are accumulated, it remains worth considering that
perhaps not all posttranscriptional modifications are physiologically relevant. Overlapping
functions have been found to exist, however, and some enzyme families and physiologic systems
may thus have the ability to compensate for single gene dysfunction during ontogeny.
More than 20 genes controlling glycan biosynthesis and diversification have been inactivated in
the mouse germ line as of this writing (Table 33.1). Studies have uncovered various and
unexpected physiologic functions controlled by endogenous oligosaccharide structures. The
phenotypes include embryonic and postnatal lethalities, widespread systemic disease with
models of human glycosylation deficiencies, cell death by apoptosis, immune dysfunction, and
defects in organogenesis. The lethalities observed thus far segregate to early steps in N-glycan
biosynthesis, GPI-anchor formation, hyaluronan biosynthesis, and proteoglycan sulfation. In 26
studies that produced an inherited systemic glycan deficiency by gene targeting, only 5 (19%)
caused embryonic lethality.
Table 33.1. Induced congenital oligosaccharide deficiencies in the mouse and
their biological effects
Enzyme Phenotype/Disease
GlcNAc-1-phosphotransferase embryonic lethality (E4.5)

GlcNAcT-I embryonic lethality (E9.5) with defects in vascularization,
heart loop asymmetry, and neural tube formation
α-Mannosidase II dyserythropoiesis and glomerulonephritis
GlcNAcT-II CDGS type-II-like syndrome with frequent postnatal lethality
GlcNAcT-III viable/under study
GlcNAcT-V immune dysfunction and intestinal hyperplasia
ST6Gal-I immunodeficiency with attenuated B-cell function
Polypeptide GalNAcT-1 viable/under study
Polypeptide GalNAcT-8 viable/under study
Core 2 GlcNAcT myeloid leukocytosis and defect in inflammation response
β1 4 GalT multiple defects including epithelial and endocrine
FucT-IV partial inflammation response deficit; collaboration with
FucT-VII
FucT-VII general leukocytosis; lymphoid homing defect; and
α1 3 GalT (Gal3T) cataracts
ST3Gal-I cytotoxic-T-cell deficiency by apoptosis
ST3Gal-II viable/under study
ST3Gal-III viable/under study
ST3Gal-IV viable/under study
ST8Sia-II (STX) viable/under study
Heparan sulfate 2- renal agenesis with neonatal lethality
sulfotransferase (hs2st)
N-deactylase/N- postnatal lethality
sulfotransferase-1 (NDST-1)
N-deactylase/N- mast cell defect
sulfotransferase-1 (NDST-2)
Hyaluronan synthase-2 (has2) embryonic lethality (E9.5)
Ceramide galactosyltransferase myelin abnormalities; paralysis; and postnatal death
GM2/GD2 synthase slight reduction in conduction velocity of some nerves; male
sterility
GPI synthesis (X-linked Pig-a) lethality if mutation is acquired in germ line or widespread in
somatic cells
Most effects of glycan deficiencies thus far produced are evident in specific physiological
systems and do not grossly alter morphogenesis or create systemic pathologies. This may reflect
the experimental focus and assays performed. Cells commonly affected include those of the
hematopoietic and nervous systems in processes that modulate hematopoiesis, immune function,
receptor-ligand activation, myeloid inflammation, lymphoid apoptosis, neural axon migration,
neuromuscular activity, endocrine function, and innervation. Additionally, lethal genetic
deficiencies in early N-glycan biosynthesis and proteoglycan sulfation were detrimental to the
development of the heart, kidney, neural tube, and vasculature. The modeling and study of
vertebrate disease states such as the carbohydrate-deficient glycoprotein syndrome type II and
the chronic glomerulonephritis induced by α-mannosidase II deficiency should provide
information on the etiology of these glycan-based maladies.
In general, the more proximal the defect in glycan biosynthesis, the more widespread and severe
the phenotypic outcome. This makes intuitive sense as the earliest steps in glycan diversification
are common to most glycans and occur widely in many cell types. However, several early
biosynthetic steps previously believed to be performed by a single enzyme have been found to
involve the presence of numerous isozymes in vivo. In the case of α-mannosidase II deficiency,
its role in early N-glycan biosynthesis remains as previously defined; however, its in vivo
function was essential only to the erythroid lineage in the mouse. A distinct α-mannosidase
activity was noted in Golgi extracts that could provide an efficient alternate pathway in complex
N-glycan biosynthesis among most cell types (Chapter 7). A second Core 2 GlcNAcT isozyme
has also been discovered (Chapter 8). Additional biosynthetic pathways operating in specific cell
types may be more frequent than presently defined and may be further discovered from studies
of in vivo recessive lesions among glycosyltransferase and glycosidase genes.
Several branchpoints exist in both N- and O-glycan biosynthesis (Chapters 7 and 8).
Glycosyltransferases and glycosidases operating at these branchpoints control the formation of
various structures in subsequent biosynthesis (see Chapter 16). Some of these structures are
found on multiple branches, whereas others are preferentially localized to one branch. These
branches may exist in part to create a multivalent ligand for the appropriate lectin. In studies
ablating enzymes controlling branch formation, results so far generally indicate that N- and O-
glycan branching provides unique functions that can be especially crucial for specific tissues or
cell types (Figure 33.9). Studies inactivating other glycan classes in the mouse, including the
biosynthesis of hyaluronan, GPI anchors, and glycosaminoglycan sulfation, have also provided a
first glimpse of structure-function relationships involving these classes (Figure 33.10).
Figure 33.9. Branch specificity is observed in N- and O-glycan structure-function relationships.
Circled structures represent those rendered deficient from induced germ-line mutations in the
mouse involving indicated genes/enzymes that control N- or O-glycan diversification.
Figure 33.10. Structure-function relationships in glycosaminoglycan, hyaluronan, and GPI-
anchor biosynthesis. Circled structures represent those rendered deficient by induced germ-line
mutations in the indicated genes/enzymes of the mouse.
In theory, studying glycan function should also be possible by in vivo mutagenesis of the genes
encoding specific lectin activities. Few such studies have been reported, and of those
accomplished, the results obtained may be complicated by the potentially distinct role of the
peptide component, as well as by the possible functional overlap with other lectins of similar
binding activities. Nevertheless, physiological connections between the functions of
glycosyltransferases and glycosidases and those of endogenous lectins are expected to contribute
significantly to our understanding of how glycans modulate embryonic and adult physiology.
Future Directions
Studies thus far indicate that extracellular glycans perform roles in regulating cell-cell adhesion
and the activation of receptor complexes through endogenous lectin-ligand interactions. These
activities suggest that many glycan functions may be described as nature's biological modifiers
or rheostat. Glycans can modulate the development and function of physiologic systems, as
exemplified by the role of sialyltransferases in lymphoid apoptosis and antigen receptor
signaling, and by the essential requirement for proteoglycans in fibroblast growth factor receptor
activation. Endogenous lectins may therefore represent the "other arm" of glycan-based
modulation systems and may be found to be more frequent and diverse among cells than
currently recognized. In other situations, glycans may act primarily through steric and
conformational influences. The finding of physiologic functions for genes controlling glycan
formation provides a route to defining the mechanisms of glycan function. It will be of
importance to develop new approaches to structural studies of glycans and their carriers as
derived from whole tissues and primary cells of transgenic and gene-targeted mice. In
understanding the physiologic processes that glycans modulate, experiments with various
multicellular organisms will also be informative, and in some cases, breeding together multiple
mutant genetic loci may uncover the presence of overlapping roles as well as yet to be
discovered functions.
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34. Glycosylation Changes in Ontogeny and Cell Activation
at San Diego).
THE ONTOGENY OF MULTICELLULAR ORGANISMS and the activation of various cell

types are associated with distinct changes in glycan structures. A vast amount of literature exists
on this topic. This chapter provides examples of specific glycosylation changes occurring during
development and physiologic responses with an emphasis on vertebrate systems, approaches
used to detect these changes, and their potential functions.
Background
Glycosylation changes occurring on cell surfaces during mammalian embryogenesis and cell
activation have been recognized for decades. Alterations in glycosylation have been proposed to
participate in cell adhesion, receptor activation, cell differentiation, and tissue morphogenesis.
Because the early embryo is composed of cells that must adhere to other embryonic cells, it
seemed likely that factors modulating this adherence would have important roles in early
development. Recognition that lectins promote various cell-cell adhesion events led to the
speculation that cell surface glycans may be involved in vertebrate embryonic development.
Indeed, studies with glycosylation inhibitors and exogenous glycans have resulted in substantial
alterations in the adherence of embryonic cells with detrimental effects on embryonic
development. Efforts to classify and determine the identities of these embryonic glycans began
with the derivation of monoclonal antibodies against what were originally defined as "stage-
specific" antigens. These antigenic structures were found to fluctuate at different times in
embryogenesis. A surprising finding was that most of these monoclonal antibodies were
carbohydrate-specific and recognized distinct glycan structures that wax and wane during
vertebrate ontogeny. Since then, both lectins and anticarbohydrate antibodies have been used to
derive information on vertebrate cell surface glycosylation in embryogenesis and cell activation.
The biological relevance of these glycan modifications has remained mostly unresolved.
However, in some cases, a direct role for certain glycans in vertebrate embryogenesis and cell
activation has been obtained.
Fertilization (1 9)
The process of oocyte fertilization appears to require glycan recognition by lectins and has been
explored in studies especially of the sea urchin, frog, and mouse. The sperm receptor on the sea
urchin egg surface is composed of sulfated O-glycans which when exogenously added can
inhibit fertilization. Additionally, sulfated glycans from sea urchin eggs can induce the sperm
acrosome reaction needed for sperm fusion with the egg membrane. Species-specific
glycosylation patterns are commonly found on vertebrate eggs. Sperm-egg binding in the frog
Xenopus laevis may require glycan structures on the gp69/64 glycoproteins of the egg vitelline
envelope. In the mouse, chemical removal of O-glycans has been shown to ablate sperm-egg
binding, whereas removal of N-glycans had no effect. Addition of UDP-Gal and UDP-Fuc
polymers also blocked sperm-egg binding. Although studies including the use of glycosidases
indicated that the α1 3Gal glycan terminus was probably involved, α1 3Gal-deficient mice do
not suffer from a deficiency of egg-sperm interaction or reduced fertility. Studies with β1 4
galactosyltransferase-deficient mice have suggested a function for this galactose linkage in the
oocyte acrosome reaction that is induced by sperm binding during fertilization. More recent
studies have suggested that α1 3-fucosylated O-glycans may be obligatory for high-affinity
binding sperm. Additional studies will be needed to define which endogenous glycan structures
are involved in mammalian fertilization.
Preimplantation Development (10 22)
Following sperm fertilization of an egg, the resulting one-cell embryo (or zygote) divides several
times with no increase in size until the early embryo has completed travel through the oviduct
(Fallopian tube) and entered the uterus, where it implants on the uterine epithelial wall.
Vertebrate embryogenesis is often denoted by various stages of development. From fertilization
until implantation is referred to as the preimplantation stage of development (Figure 34.1). This
is when the first differentiative events occur, as cell division and embryo size constraints
together produce daughter cells that have lost totipotency concurrent with cell-cell contacts that
are now unique from parental cells. This is the morula stage of embryogenesis. Such embryos
have approximately 16 cells, at which time they undergo the process of compaction, which is
also the first morphologic event in embryonic development. Subsequently, the last stage of
preimplantation development yields the blastocyst with approximately 64 cells. Embryos of this
stage include an inner cell mass that will become the embryo itself, whereas the outer layer of
trophoblast cells will ultimately form the placenta after implantation.
Figure 34.1. Preimplantation development in the mouse. (Reprinted, with permission, from [95]
Hogan et al. 1994.)
It is during the preimplantation phase that the embryonic genome becomes active and produces
the first embryonically derived proteins. This process actually begins early for some genes with
transcriptional activation at the two-cell embryo stage. In studies referring to the role of genes
and proteins during preimplantation development, it is necessary to consider the possibility that
maternally (oocyte)-derived mRNAs and proteins may aid in development prior to induction of
the embryonic genome. Such maternal sources may persist in preimplantation, but they are
eventually lost by degradation and dilution.
Production of monoclonal antibodies generated against preimplantation embryonic cells yielded
the first defined stage-specific embryonic antigens (SSEAs). SSEAs are glycan structures that
may exist on glycoproteins or glycolipids. Their expression changes significantly in early
embryogenesis (Figure 34.2). SSEA-1 is another term for what is also called the Lewis X
oligosaccharide (Chapter 16) and is transiently found on 8-cell embryos but is lost by the 32 64-
cell developmental stage. Absence of SSEA-1 correlates with the onset of compaction. These
glycans may be functionally important, since synthetic multivalent Lewis-X-bearing haptens
(attached to primary amino groups of lysyl-lysine) can decompact embryos (Figure 34.3). This
was not observed with haptens bearing the Lewis A oligosaccharide or free Lewis X
oligosaccharide added with free lysyl-lysine. In another study, glycans bearing fucose-linked
α1 3 and α1 4 to GalNAc also caused decompaction. When mouse preimplantation embryos
are further examined using antibodies that react with glycolipids, it is apparent that most
glycolipids are of the globo series (containing Galα1 4Galβ1 4Glcβ1-ceramide) which includes
SSEA-3, SSEA-4, and the Forssman antigen. SSEA-3 and SSEA-4 are found on membranes of
early oocytes and zygotes, indicating that they are synthesized by maternal sources. Their
expression is lost by the blastocyst stage. The Forssman antigen is found by the 8-cell stage and
is maintained on cells of the inner cell mass of blastocysts. The function of these carbohydrate
structures in embryogenesis is not known.
Figure 34.2. Carbohydrate variation in mouse preimplantation embryogenesis. (Modified, with

permission, from [14] Fenderson et al. 1990.)
Figure 34.3. Adhesion of preimplantation fibroblasts regulated by multivalent oligosaccharides.
(A) Compacted embryos treated with multivalent Lewis A (B) or multivalent SSEA-1/Lewis X
(C) oligosaccharide. (D) Untreated embryos compared with those treated with fibroblast
glycopeptides (E) or high-molecular-weight (F) polylactosaminoglycans from F9
teratocarcinoma cells. (Reprinted, with permission, from [14] Fenderson et al. 1990, originally
adapted from [97] Fenderson et al. 1984.)
Compaction (10 22)
The process of compaction can also be regulated by embryonic glycoconjugates bearing

polylactosamines, sometimes termed embryoglycans. Natural human antibodies and distinct
structural features define two types of polylactosamines: Those of the i type are unbranched and
found to be widespread in expression from the zygote stage onward. In contrast, the branched I-
type polylactosamines are absent from or expressed at low levels in preimplantation embryos
(and see Chapter 16). Enzymatic removal of polylactosamines from embryos results in a greatly
reduced rate of compaction, whereas addition of embryoglycans derived from F9 cells, but not
from adult fibroblasts, hyperagglutinates 8 16-cell embryos (Figure 34.3). The antibody ECMA-
2 is known to recognize a terminal α-linked galactose residue on polylactosamines of
embryoglycans. ECMA-2 expression is widespread in preimplantation but progressively
disappears by embryonic day (E)9 10 to become restricted to only a few subsets of adult tissues.
The function of the ECMA-2 epitope in embryogenesis is not known.
Various glycosylation inhibitors have dramatic effects on preimplantation development.

Tunicamycin blocks compaction and blastocyst formation but does not affect early cleavages.
Continuous embryo exposure to tunicamycin kills trophoblast cells, but apparently not cells of
the inner cell mass (Figure 34.4). This effect is similar to results obtained in embryos lacking the
gene encoding GlcNAc-1-phosphotransferase, which is essential for the production of the
dolichol oligosaccharide precursor needed in N-glycan biosynthesis; its absence ablates all
embryonic N-glycosylation (see Chapter 7). In another study of N-glycosylation during
preimplantation, compactin (an inhibitor of polyisoprenoid synthesis) was found to cause
developmental arrest; however, this is not reversed by dolichol supplementation.
Microenzymatic assays of enzymes involved in N-glycan biosynthesis, including Dol-P-Man
synthetase, Dol-P-Glc synthetase, and Dol-P-P-chitobiosyl synthetase, indicate that these
steadily decrease in activity from fertilization onward until they are induced at the blastocyst
stage.
Postimplantation Embryogenesis (10, 12, 14, 16, 18, 20,23 26)
Following implantation of the blastocyst-stage embryo on receptive uterine epithelium, various

morphogenic processes occur in further development. In implantation and subsequently, the
trophoblast cells act to interface the embryo with the maternal uterine epithelium and will
ultimately form the placenta. Increased levels of polylactosamines are associated with
implantation among cells involved in adhesion at the maternal-fetal interface, as well as with
metastatic activity in various cancers (see Chapter 35). Gastrulation results in the formation of
the mesoderm, thereby yielding the presence of all three embryonic germ layers: the endoderm,
mesoderm, and ectoderm. This occurs during E7 in the mouse. Various glycan expression
changes have been observed in early and late postimplantation embryos using lectins and
anticarbohydrate antibodies (Figure 34.5). As in preimplantation, i-antigen expression remains
widespread in the endoderm, ectoderm, and mesoderm. Sialylation of i-antigen structures,
yielding sialyl-i, is also widespread. However, I-type polylactosamines are induced from
gastrulation on and are more restricted in expression, with high levels found in the endoderm and
mesoderm but very little expression noted in the ectoderm. Sialyl-I structures are present in the
extraembryonic ectoderm. SSEA-3 and SSEA-4 reappear on the endoderm in early
postimplantation development from E5 to E7, especially on the extraembryonic endoderm,
which is involved in absorption, secretion, and formation of the placenta. The Forssman antigen
is found on the primitive ectoderm from E5 to E10. The primitive ectoderm bears pluripotent
stem cells that are derived from the inner cell mass.
Figure 34.5. Various changes in glycan structure throughout mouse gestation. (Reprinted, with
permission, from [16] Muramatsu 1988 [© Wiley-Liss, Inc.].)
Most cell surface N-glycans on adult cells are of the complex subtype and therefore require the
glycosyltransferase GlcNAcT-I. Embryonic induction of Mgat1-gene-encoded GlcNAcT-I has
been shown to occur between E7 and E9 (Figure 34.6). Mice engineered to be deficient in
GlcNAcT-I by inactivation of the Mgat1 gene die during E9, suggesting that embryonic Mgat1
gene induction occurs approximately at the time the embryo requires GlcNAcT-I function in
various cell types. In the absence of GlcNAcT-I, morphogenic processes are affected including
aberrant heart development (situs inversus), abnormal neural tube formation, and impaired
vascularization. Prior to E9, it appears that the Mgat1-null embryo can acquire complex N-
glycans from multiple parental sources including maternal RNA and glycoproteins with complex
N-glycans, the latter of which are transported through the extraembryonic endoderm (the
primitive placenta). These maternal sources may moderate the Mgat1-null phenotype and permit
development to E9. Use of ConA and E-PHA to visualize N-glycans in embryos indicates no
obvious distinctions among cell types of the three germ layers at E7 in the mouse, as all appear
to react with these lectins (Figure 34.6). However, a subtype of complex N-glycans defined by
L-PHA reactivity, and which requires GlcNAcT-V, is absent from E7 mesoderm and ectoderm
(Figure 34.7).
Figure 34.6. Induction of GlcNAc-TI-encoding Mgat1 RNA, necessary for hybrid and complex
N-glycan biosynthesis, in early postimplantation development of the mouse. Expression is shown
in embryos at day 7 (a,b,c) or 9.5 (d,e) of development. A control experiment (f) using sense
Mgat1 RNA is shown. (Reprinted, with permission, from [23] Campbell et al.1995 [© Oxford
University Press].)
Figure 34.7. E-PHA (A), L-PHA (B), and ConA (C)-binding profiles on the three embryonic
germ layers from E7 mouse embryos. Distinct absence of GlcNAc-TV branched N-glycans from
the mesoderm (m), amnion (am), and neural ectoderm (ne) is noted. Embryonic endoderm (ee)
and parietal endoderm (pe) contain these structures. (Reprinted, with permission, from [24]
Granovsky et al. 1995 [© Oxford University Press].)
Cell movements during gastrulation and neural crest migration (see below) correlate with spatial
changes in the composition of the ECM. Throughout the embryo, various pathways exist along
which these molecules accumulate. The ECM is rich in hyaluronan and various proteoglycans,
and these molecules may act to promote trafficking and differentiative events in this cytokine-
rich milieu. Proteoglycans have been shown to accumulate at areas of major morphogenic
developments, including organ and limb bud formation, and they can regulate the development
of cell lineages and organs (see below). Relatively few reagents are presently available to detect
changes in glycoaminoglycan structure in situ. However, proteoglycan sulfation detected with
antibodies appears to be regulated in various spatial-temporal patterns including epithelial-
mesenchymal cell boundaries and in regions of the developing brain.
Neurogenesis (27 51)
The finding that some monoclonal antibodies were specific for glycans restricted to neural cell
subtypes or regions of the CNS suggested that carbohydrates might control some processes in
neurogenesis. The SSEA-1 epitope expressed in early embryogenesis is reexpressed at later
embryonic stages in a subpopulation of astrocytes within the cerebellum and spinal sensory
neurons. Various lectins also detect changes in glycans in the developing nervous system. Early
studies reported a ventral-to-dorsal gradient of GM2 ganglioside binding in the retinal-tectum
system of vertebrates. Enzymatic activity that converts GM2 to GM1 by addition of a terminal
galactose residue was found in a ventral-to-dorsal gradient, consistent with the above
observation. Adhesion of neural cells to immobilized gangliosides was reported in vitro. Protease
treatment of cells ablated this binding, suggesting the presence of cell surface lectins. In fact, an
early study reported that chick retinal cell surfaces actually express an N-
acetylgalactosaminyltransferase. This was a surprising finding since sugar donors needed for
glycosylation are generally thought to be absent or at very low concentrations in the extracellular
environment. Nevertheless, numerous observations of glycosyltransferases and glycosidases at
the cell surface have led to the hypothesis that these enzymes may act as lectins in some
situations. In the retino-tectum system, treatment of cells with N-acetylhexosaminidase or
sialidase also decreased cell adhesion in the ventral region. Sialylation may thus also have a role.
ST6Gal-I RNA has been reported in the rat retina with high expression in ganglion cells and
various expression patterns among photoreceptor cells.
The JONES antigen is recognized by a monoclonal antibody specific for a 9-O-acetylated sialic
acid on the ganglioside GD3. O-acetylated GD3 is observed on some neuroblasts and axons and is
enriched on glial processes in regions through which axons migrate. In the retinal system,
expression of O-acetylated GD3 is also found in a dorsal-to-ventral gradient and is thus opposite
to the GM2 ganglioside binding described above. This is also distinct from the expression of GD3
itself, which does not show regional specificity in the retino-tectum system. Reactivity to the
JONES epitope is highest in neural regions at times of maximal cell migration. Other studies of
the developing retina revealed that removal of highly sulfated proteoglycans altered retinal
histogenesis. These changes imply the possibility that glycan structures may modulate cell and
axon guidance during neurogenesis (and see below).
A sulfated glycan structure termed HNK-1 (which refers to the monoclonal antibody) is found on
neural crest migratory cells of the neural epithelium and other cells of neuroectodermal origin
during postimplantation development. It is carried on both glycolipids and glycoproteins
including N-CAM, myelin-associated glycoprotein, and tenascin. The biological role of this
modification is unclear, although antibodies against HNK-1 have been reported to inhibit
neuron-astrocyte, astrocyte-astrocyte, neuron-oligodendrocyte, and oligodendrocyte-
oligodendrocyte cell adhesion.
Glycan involvement in the development of the synaptic junction has been proposed. The
neuromuscular junction binds selectively to the lectin DBA, indicating the presence of terminal
GalNAc residues. Other lectins specific for different saccharides bind synaptic and extrasynaptic
regions specifically. The molecules bearing terminal GalNAc moieties have been reported to
include the enzyme acetylcholinesterase and a glycolipid recognized by anti-SSEA-3 antibodies.
The muscular sodium channel is found in the vertebrate neuromuscular junction. Sialic acid
linkages and their abundance on various ion channels have been studied for more than a decade.
Sialidase treatment has indicated that ion channel function assessed by current flow and the
voltage dependence of gating are modulated by the presence of sialic acids. Ion channels in the
myocardium, neuromuscular junction, and various neural cell types may all be regulated by
electrostatic or steric mechanisms dependent on the number and positioning of sialic acid
linkages. Some of these channels have been reported to contain oligosialic or polysialic acid.
Perhaps the best studied of glycan structures regulated in the vertebrate nervous system is that of
PSA. PSA is a polymer of α2 8-linked sialic acid with chains commonly extending 60 100
residues (see Chapter 15). N-CAM carries the majority of PSA found in embryonic vertebrate
tissues. PSA expression is highly restricted and prominent in the developing CNS (Figure 34.8).
Most PSA is found on N-glycans of N-CAM. The expression of PSA is highly regulated and is
found specifically on the embryonic form of N-CAM and correlates with absence of homotypic
N-CAM adhesion. A role for PSA in inhibiting homotypic cell-cell adhesion is indicated.
Enzymatic removal of PSA using a phage endoneuraminidase specific for α2 8-linked sialic
acid yields evidence that PSA functions in neurite fasciculation (Figure 34.9). PSA has been
proposed to regulate axon fasciculation by large steric and negative charge properties. Removal
of PSA by neuraminidase injection into chick eyes perturbs genesis of the neural epithelium with
a resulting ectopic optic nerve axon fiber layer in the retina. Interestingly, mice engineered with
mutations in N-CAM by gene-targeting approaches led to a decreased size of the olfactory bulb
with axon accumulation in the ventricular zone and deficits in spatial learning (Figure 34.9).
Sialyltransferases involved in the production of PSA have been tentatively identified by in vitro
cotransfection studies also using N-CAM cDNAs. At least five sialyltransferases may generate
α2 8 sialic acid linkages; however, the STX sialyltransferase (ST8Sia-II) and PST (ST8Sia-IV)
are likely responsible for PSA production on N-CAM, and their expression is correlated with the
presence of this modification.
Figure 34.8. Expression of PSA is enriched in neural tissue during postimplantation
development in the mouse. (Adapted, with permission, from [40] Ong et al. 1998 [© Oxford
University Press].)
Figure 34.9. Neuronal fasciculation and olfactory bulb innervation are affected by loss of PSA.
Neuronal trajectory of motor axons through hindlimb in sham-treated (A) and treated with α2 8
linked sialic-acid-specific neuraminidase (B). In comparison to wild-type mice (C), N-CAM
mutant mice (D) exhibit reduced cell migration to the olfactory bulb and neuronal precursor
accumulation in the subventricular zone. (Reprinted, with permission, from [38] Landmesser et
al. 1990 and [44] Rutishauser 1996.)
Organogenesis (52 65)
Organogenesis reflects major morphogenic events in tissue pattern formation and limb
development. Alterations of these events by modulators of glycan formation can yield clues to
glycan roles in embryogenesis. Many such studies have been reported in vertebrate systems,
including the mouse. In general, among vertebrates studied, a correlation exists between
organogenesis and the increased production of ECM components, including hyaluronan and
glycosaminoglycans. Hyaluronan production is high in the developing mouse limb buds, heart,
and somites at critical stages in morphogenesis. This ECM component is bound by the
hyaluronan receptor CD44, which exists as numerous isoforms from alternate RNA splicing.
Chitin oligosaccharide synthetase activity has been discovered in zebrafish embryos during late
gastrulation. Injection of antibodies against the DG42 hyaluronan synthase controlling
production of this glycan leads to defects in morphogenesis involving trunk and limb
development. The biosynthesis of proteoglycans in the developing mouse cornea undergoes a
switch from heparan sulfate to keratan sulfate, the latter becoming a major consitutent of the
cornea, where it may have a role in corneal transparency. In addition, sulfation patterns on
proteoglycans have been found to change during vertebrate organogenesis and cartilage
formation. Experiments with lectins, including wheat-germ agglutinin, when applied to the
developing chick tail bud, resulted in a range of defects. During organogenesis, PSA has also
been observed on N-CAM in a variety of mesodermal and endodermal derivatives. In general,
PSA N-CAM expression is prominent on epithelial cells during epithelium formation and
decreases afterward.
The sulfated GalNAc found on a restricted subset of N-glycans controls the half-life of
glycoprotein hormones (see Chapter 16). Production of this unique N-glycan appears to
modulate endocrine functions and requires a GalNAc transferase specific for glycoprotein
hormones and a sulfotransferase specific for terminal-β-linked GalNAc. These two enzymes and
the resulting product are coordinately expressed in a subset of organs and their derivatives,
including pituitary, submaxillary, and parotid glands and the kidney. In other examples,
development of the rat pancreas in embryogenesis has been found to occur with specific changes
in lectin reactivity regarding the cytodifferentiation of the three cell types (acinar, endocrine, and
centroacinar). The plant lectins RCA II (Ricinus communis agglutinin) and SBA do not bind
endocrine or centroacinar cells at any developmental stage, whereas RCA I and WGA bind to all
three cell types. This lack of binding appears to be due to masking by sialic acid, as
neuraminidase treatment results in exposure of binding epitopes to these two lectins. PSA N-
CAM has also been reported on cells of the developing pancreas. Myocytes in the developing
heart preferentially bind the anti-GD antibody specific for Siaα2 3Galβ1 4GlcNAc. During
cardiac innervation and morphogenesis, PSA-containing N-CAM is found on various axons and
cells contributing to the development of the ventricular septal wall.
Many different glycan changes have been described in the developing lung. These include the
increased biosynthesis of proteoglycans and the presence of a sulfated N-glycans. Lewis antigens
change dramatically during lung development. In the earliest phase of lung bud development, the
Ley antigen is expressed, but Lewis X and sialyl Lewis X are absent. Subsequently, epithelial
cells of forming future bronchioles preferentially express Lewis X. Later, sialyl Lewis X is found
to be primarily expressed on terminal bud cells, but Lewis X and Ley are absent. Ciliation of
epithelial cells occurred with the reappearance of Lewis X and Ley expression. In adult lung,
neither of the Lewis antigens is found at appreciable levels on respiratory cells of the lung.
Kidney development is associated with various changes in glycan structure. Two examples
include fucosylated polylactosamine and PSA N-CAM. Antibodies against the fucosylated
polylactosamine bind to cells of the ampullae of the ingrowing ureteric branches but not the
remainder of the ureteric bud. This glycan is absent following fusion of the ureteric bud with the
S-shaped tubule but persists on segments of the convoluted tubule. A correlation is therefore
suggested between expression of this fucosylated polylactosamine and cells involved in renal
tubulogenesis. PSA expression is also reported in the developing kidney. At early stages, PSA
N-CAM was localized to the ureteric bud and the metanephrogenic mesenchyme. Expression in
the ureteric bud declined and was absent prior to birth. PSA N-CAM antibody reactivity was
highest during mesenchymal transformation into epithelium. A correlation is thereby indicated
between PSA N-CAM expression and changes in cell adhesion during kidney organogenesis.
Hematopoiesis and the Immune System (66 74)
Cells of the hematopoietic lineage are relatively well defined with regard to their differentiation
into various lineages (Figure 34.10). In ontogeny, hematopoietic stem cells first appear in the
embryonic yolk sac at E7 in the mouse and consist only of nucleated red blood cells and
macrophages. They are subsequently found in the developing liver of the mammalian embryo
beginning at about E10. Liver-based hematopoiesis produces mature anuclear erythrocytes and
continues to birth when the majority of hematopoietic stem cells have migrated to the bone
marrow, where they then reside throughout adult life.
Figure 34.10. Cell lineage production during hematopoiesis. (CFU-GEMM CFU-mix) Colony-
forming unit granulocyte, erythrocyte, megakaryocyte, monocyte (multiple progenitor cells);
(BFU-E) burst-forming unit erythrocyte (early erythroid progenitor cell); (CFU-E) colony-
forming unit erythrocyte (late erythroid progenitor cell); (CFU-GM) colony-forming unit
granulocyte/monocyte; (CFU-Eo) colony forming unit eosinophil; (CFU-Ba) colony-forming
unit basophil; (CFU-Meg) colony-forming unit megakaryocyte; (CFU-G) colony-forming unit
granulocyte; (CFU-M) colony-forming unit monocyte. (Reprinted, with permission, from [76]
Miltenyi Biotec 1998.)
Developmental changes in glycan structures have been described on the erythroid, myeloid, and
lymphoid lineages. A striking example involves the production of erythroid polylactosamine. In
immature and fetal red blood cells of mouse and humans, the majority of polylactosamine is of
the unbranched i type and is mostly present on glycolipids. This type of polylactosamine is also
observed in periods of hemodynamic stress associated with a high production and turnover of red
blood cells. In postnatal development, i antigen decreases significantly, and levels of branched I-
type polylactosamines become the predominant species. The human adult erythroid I antigen is
found at high levels on the Band-3 anion transporter, although mouse Band-3 may not carry this
structure. Alterations of erythroid N-glycans are directly indicated in the etiology of
dyserythropoiesis, with resulting high levels of i antigen on glycolipids. In comparison,
granulocyte polylactosamines are normally and predominantly of the i type and may be
fucosylated to generate the Lewis X antigen. This fucosylation occurs after the myeloblast stage
of differentiation, and increased levels of Lewis X are noted with further maturation. The ABH
blood group antigens are not found on human myeloid cells at any stage of differentiation. In
general, erythroid cells are enriched in blood group antigens, lymphoid cells in Siaα2 6 termini
on N-glycans, and myeloid cells in polylactosamines and sialyl Lewis X. The sialyl Lewis X
oligosaccharide is detectable on immature and developing avian B and T lymphocytes, however,
but is then absent from differentiated cells. Sialyl Lewis X reappears on mature neutrophils and
monocytes, consistent with its role in leukocyte cell adhesion and trafficking (see Chapter 26).
Lymphocytes and accessory cell types exhibit specific cell surface glycans. Chondroitin sulfate
makes up the majority of glycosaminoglycan structures on proteoglycans of immature B cells,
whereas more mature B cells commonly synthesize heparan sulfate. Chondroitin proteoglycan
sulfation on B lymphocytes is mostly at the 6-position, with much less 4-O-sulfation. T
lymphocytes undergo a series of specific changes in glycosylation during their development in
the thymus. This development requires interactions with thymic epithelial and dendritic cells.
These accessory cells provide stimuli that regulate positive and negative selection necessary
events to acquire a mature peripheral T-cell repertoire that is both immune-competent and self-
tolerant. Hyaluronic acid is produced at high levels by thymic epithelium, whereas thymocytes
produce much less in comparison. The ratio is similar to the proportion of sulfated
glycosaminoglycans produced by thymic epithelium and thymocytes. Among sulfated
glycosaminoglycans, both thymic epithelium and thymocytes predominantly synthesize
chondroitin sulfates and not heparan sulfates. Chondroitin sulfates of glycosaminoglycans on T
lymphocytes are predominantly modified with 4-O-sulfate and not with 6-O-sulfate. Changes in
thymocyte O-glycans have also been reported. Core 2 GlcNAcT expression is found in
CD4+CD8+ cortical thymocytes but is absent from the single positive medullary thymocytes,
indicating that most O-glycans in immature thymocytes are of the Core 1 subtype.
Sialylation of developing thymocytes is developmentally restricted. Binding of the plant lectin

SNA, which requires ST6Gal-I function, is found primarily on mature medullary thymocytes,
even though RNA in situ studies have shown ST6Gal-I expression is widespread and includes
immature cortical thymocytes. One of the most documented changes in thymocyte
oligosaccharide variation involves the lectin peanut agglutinin and the ST3Gal-I
sialyltransferase. Immature cortical thymocytes highly express the peanut-agglutinin-binding
structure Galβ1 3GalNAcα-Ser/Thr. This correlates with high levels of ST3Gal-I RNA in this
immature T-cell population. Absence of peanut-agglutinin reactivity is noted in mature
medullary thymic T cells, and this change is abrogated in the genetic absence of ST3Gal-I
function (Figure 34.11; see Chapter 23).
Figure 34.11. PNA lectin reactivity (Galβ1 3GalNAcα-Ser/Thr) defines the cortical-medullary
boundary in wild-type thymus tissue. PNA binding is high on immature cortical thymocytes and
absent from mature medullary thymic T cells. This difference is due to ST3Gal-I, as the
medullary thymocytes from mice lacking this enzyme are deficient in Siaα2 3Galβ1
3GalNAcα-Ser/Thr and retain PNA-binding epitopes.
Cell Activation and Signal Transduction (75 97)
Investigating glycan function in cell activation and signal transduction is an active research area.
The observed changes in glycan production in ontogeny might be expected to occur in part
through regulated variations in glycosyltransferase and glycosidase gene expression. Relatively
little is known about the mechanisms by which glycosyltransferase and glycosidase genes are
transcriptionally regulated. However, genetic loci encoding GlcNAcT-II and GlcNAcT-V are
regulated by promoter sequences bearing sites for various transcription factors and growth-
responsive elements, some of which either are defined as proto-oncogenes or are regulated by
proto-oncogenes. This includes the ets-responsive elements in the GlcNAcT-V promoter,
activated by the cellular src oncoprotein. In addition, liver transcription factors including HNF-1,
DBP, and LAP have been found to trans-activate the ST6Gal-I promoter. Control of glycan
alterations may be found to occur in a cell-cycle-specific manner. Production of the Lewis X
determinant by fucosylation was found to be induced upon postmitotic intestinal villus-
associated enterocytes that have reentered the cell cycle from exposure to the SV40 large T
antigen. Glycosylation variants of human thyroid-stimulating hormone have unique
physiological properties that have been shown to selectively activate signal transduction through
either the cAMP or inositol phospholipid hydroylsis pathways.
The majority of studies involving glycans in cell activation and signaling have focused on
hematopoietic cells because they are easily obtained, can generally be maintained in culture, and
can be activated in vitro. Many examples exist where lymphocyte immune activation can be
modified by changes in cell surface glycans and can be found in association with changes in
endogenous oligosaccharide production. Evidence that lymphocyte signal transduction can be
modulated by glycans was gathered in experiments that involved desialylation by sialidase,
followed by activation of antigen receptors by cross-linking or by a mixed lymphocyte reaction.
A significant increase in lymphocyte proliferation was obtained, suggesting that sialic acids may
normally act to dampen immunologic responsiveness. T-cell activation has also been reported to
occur with changes in O-glycosylation involving an increase in Core 2 GlcNAcT and Core 2 O-
glycans. Additionally, lymphocyte proliferation can be modified by exogenously added
proteoglycans, and lymphocyte stimulation itself induces a significant increase in proteoglycan
production, especially involving 6-O-sulfated chondroitin. In some systems, tumor cell
recognition by T cells leading to T-cell activation appeared to require sialic acids, perhaps of the
α2 6-linked variety found mostly on N-glycans, since inhibitors of N-glycan biosynthesis had
the same effect. Similar addition of deoxymannojirimycin or swainsonine to inhibit N-glycan
formation in B lymphocytes was found to reduce levels of immunoglobulin production upon
stimulation with the polyclonal activator pokeweed mitogen.
The sialyl Lewis X oligosaccharide has been studied for effects on leukocytes following binding
to the L-selectin. Following treatment of leukocytes with the L-selectin ligand GlyCAM-1, or
antibody-mediated cross-linked L-selectin, integrin activation has been observed with increased
cell binding to fibronectin. Studies indicate that L-selectin binding to GlyCAM-1 promotes
integrin activation, which leads to enhanced recruitment of leukocytes to peripheral lymph
nodes. The nature of this integrin-dependent activation mechanism is not fully established, but it
appears to include intracellular protein phosphorylation cascades.
Not all effects on vertebrate cell activation appear to be associated with changes among N- or O-
glycans. Alterations in cell differentiation have been found to occur with exposure to specific
glycolipids. For example, addition of GM3 ganglioside to cultures of the promonocytic HL60 cell
line induced differentiation into monocytes, whereas a ganglioside mixture derived from
neutrophils induced granulocytic differentiation. In another in vitro differentiation system, the
GQ1b ganglioside has been shown to induce neurite outgrowth in some human neuroblastomas.
Other glycolipids are able to modulate the activities of receptors for PDEF and EGF, although
the mechanism is not understood. Apoptosis is an important event in the development and
maintenance of the immune system in vertebrates. Gangliosides may be involved in apoptosis, as
overexpression of the GD3 synthase gene induces apoptosis by mediating CD95 (Fas) activation
in hematopoietic cells.
A mechanism by which glycans can control the activation and signal transduction of a cell
surface receptor has been suggested in a case involving the glycosaminoglycan chains of
proteoglycans. Invertebrate models of glycosaminoglycan sulfation deficiency yield severe
developmental defects that involve the dysfunction of growth factor receptor signal transduction
systems (see Chapter 11). It has long been known that many growth factors are sequestered by
glycosaminoglycans following their biosynthesis and secretion. For example, heparan sulfate
proteoglycans normally bind a variety of factors including the FGFs. Binding of FGF to
glycosaminoglycans has been shown to be necessary for efficient FGF receptor dimerization,
activation, and subsequently cell proliferation (Figure 34.12; see Chapter 29). FGF receptor
signaling is known to be essential for the development of vertebrate embryos. During ontogeny,
a switch in proteoglycan-binding specificity for FGF-1 over FGF-2 has been found to parallel the
induction of FGF-1 RNA. Although most receptor tyrosine kinases require dimerization to
activate their cytoplasmic tyrosine kinase catalytic domains, the mechanisms by which receptor
dimerization occurs are generally unresolved but may involve glycan structure.
Figure 34.12. Glycosaminoglycan dependence on the activation of the FGF receptor.

Glycosaminoglycan chains on proteoglycans bind to the FGF and the FGF receptor and are
essential for efficient receptor activator with autophosphorylation (P).
Signal transduction events controlled by lectin-ligand interactions may occur between distinct
cell surfaces or between molecules on the same cell surface. The CD22 lectin is found
exclusively on B lymphocytes and specifically binds to the Siaα2 6Galβ1 4GlcNAc
trisaccharide terminus of some N-glycans. It appears that lymphocytes in the mouse are
especially enriched for this terminal trisaccharide moiety, including B lymphocytes themselves.
CD22-binding activity has been shown to occur not only on the same cell surfaces, but also to
CD22 itself as it also carries Siaα2 6Galβ1 4GlcNAc-R. The function of CD22 involves
modulating B-lymphocyte immune activation, and thus modification of its lectin activity has
been proposed as a mechanism of action. The ST6Gal-I sialyltransferase is required for Siaα2
6Galβ1 4GlcNAc-R production and is also expressed in a tissue-specific manner with high
levels in lymphocytes. ST6Gal-I-deficient mice have been generated and are reported to lack the
Siaα2 6Galβ1 4GlcNAc-R (see Chapter 33). Although otherwise seemingly unaffected, they
exhibit an immunodeficient phenotype with reduced circulating IgM levels and impaired B-
lymphocyte function. B lymphocytes from ST6Gal-I-deficient mice are deficient in tyrosine
phosphorylation following antigen receptor cross-linking. In B lymphocytes, antigen receptor-
induced tyrosine phosphorylation is crucial for the recruitment and activation of various kinases
and phosphatases that subsequently become active and transmit signals downstream which
promote B-cell immune function.
Future Directions
The large degree of variation in glycan structures found to be associated with ontogeny and cell
activation has engendered many hypotheses purporting the presence of glycan-dependent
regulatory mechanisms. In some cases, the use of exogenous glycosylation inhibitors or
glycosidases has provided compelling evidence that indeed indicates a modulatory role for
glycans in cell-cell interactions, cell-migration patterns during development, and receptor
activation responses. From research on glycosyltransferase- and glycosidase-deficient cell lines,
and the emergence of genetic studies of intact animals, it has become apparent that glycans are
important components in a variety of developmental and postnatal processes. Future research in
this area will further discriminate between the provocative changes in glycans observed in vivo
and those changes that are physiologically significant to the intact organism. It should be of
interest and perhaps foretelling to compare variations in glycosylation changes among species, as
well as those glycan expression profiles that are most closely conserved in evolution.
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35. Glycosylation Changes in Cancer
Diego, California).
ALTERED GLYCOSYLATION IS A UNIVERSAL FEATURE of cancer cells, and certain

types of glycan structures are well-known markers for tumor progression. This chapter discusses
some specific enzymatic pathways that are frequently altered in cancer cells, the correlational
patterns between altered glycosylation and clinical prognosis, the genetic basis of some of these
changes, and the in vitro assays and in vivo studies that indicate the importance of these
pathways in malignancy.
Like normal cells during embryogenesis, tumor cells undergo activation and rapid growth,
adhere to a variety of other cell types and cell matrices, and invade tissues. As described in
Chapter 34, embryonic development and cellular activation in vertebrates are typically
accompanied by changes in cellular glycosylation profiles. Thus, it is not surprising that
glycosylation changes are also a universal feature of malignant transformation and tumor
progression. The earliest evidence came from observing the enhanced binding of plant lectins
(such as ConA and WGA) to animal tumor cells. Next, it was found that in vitro transformation
of cells was frequently accompanied by a general increase in the size of metabolically labeled
glycopeptides. With the advent of monoclonal antibody technology in the late 1970s,
investigators in search of a "magic bullet" against cancer cells found that many of their "tumor-
specific" antibodies were directed against carbohydrate epitopes, especially against those borne
on glycosphingolipids. In most cases, further studies showed that these epitopes were "onco-fetal
antigens," i.e., they were also expressed in embryonic tissues, in tumor cells, and in a few cell
types in the normal adult. Significant correlations between certain types of altered glycosylation
and the actual prognosis of tumor-bearing animals or patients increased interest in these changes.
In some instances, in vitro cellular assays have further supported the notion that these changes
may indeed be critical to aspects of tumor cell behavior. However, in most situations, definitive
proof of the mechanisms by which these glycosylation changes alter tumor behavior is still
lacking. This chapter outlines the changes that have been described to date and their potential
relevance, particularly in relation to glycan recognition by endogenous vertebrate lectins.
Ways in Which Glycosylation Can Be Altered in Malignant

Cells (4 7)
The changes that occur in malignant cells can take a variety of forms. Examples have been found
of either loss of expression or excessive expression of certain structures, the persistence of
incomplete or truncated structures, the accumulation of precursors, and, less commonly, the
appearance of novel structures. Changes in early branchpoints in the normal pathways of
biosynthesis can markedly affect the relative amount of one class of structures while causing the
dominance of another. However, this is not simply the random consequence of disordered
biology in tumor cells. As more data have become available, it is evident that only a limited
subset of biosynthetic pathways are frequently correlated with malignant transformation and
tumor progression. The commonest of these changes in glycosylation are discussed further
below, including a discussion of the biosynthetic mechanisms likely to be involved, and the
possible biological consequences of each.
Altered Branching of N-Glycans (5 14)
The classic reports of increased size of tumor cell glycopeptides have now been convincingly
explained by an increase in β1 6 branching of N-glycans (Figure 35.1), which results from an
increased expression of GlcNAc transferase V (GlcNAcT-V; see Chapter 7). This change in
enzyme expression seems to occur primarily at the transcriptional level and can be induced by a
variety of mechanisms, including viral and chemical carcinogenesis. Recent studies link such
responses to specific features of the 5 -promoter region of GlcNAcT-V. Cell lines with increased
GlcNAcT-V expression show an increased frequency of metastasis, and spontaneous revertants
for loss of enzyme activity also lose this metastatic phenotype. Clinical specimens of some
human tumors show an increase in staining with the plant lectin L-PHA, which is known to
preferentially recognize N-glycans bearing the GlcNAcT-V product. Most convincingly,
transfection of GlcNAcT-V cDNA into cultured cells causes a visually obvious transformed
phenotype associated with colony formation in soft agar, increased cell spreading, enhanced
invasiveness through membranes, and tumorigenic behavior by previously nontumorigenic cells.
By conventional criteria, such a response is typical of a true oncogene. Conversely, metabolic
inhibition of N-glycan processing by swainsonine (which blocks α-mannosidase II, preventing
complete processing of N-glycans and thereby abrogating addition of the β1 6 branch)
demonstrates reversal of tumorigenic behavior.
Figure 35.1. The increased size of N-glycans occurring upon transformation can be explained by
an increased activity of GlcNAcT-V, resulting in β1 6 branching of N-glycans,. This, in turn,
leads to enhanced expression of poly-N-acetylactosamines, which can also be sialylated and
fucosylated. These structures are potentially recognized by galectins and selectins, respectively.
The consequences of increased expression of GlcNAcT-III are also shown.
Taken together, all of these data indicate that GlcNAcT-V plays a very important part in the
biology of cancer. What remains to be shown is the precise mechanism(s) by which this
biochemical and structural change results in the biological outcomes observed. Possibilities
(Figure 35.1) include an increase of polylactosamine chains (potentially recognized by galectins)
that are preferentially found on this branch, increased outer-chain polyfucosylation and sialyl
Lewis X production (potentially recognized by the selectins), and a general biophysical effect of
the branching itself on membrane protein structure. In the last instance, it is suggested that the
β1 6 branch has a conformation very different from that of the other outer antennae of N-
glycans, tending to exist in a "broken wing" conformation, perhaps directly associating the
glycan chain with the nearby polypeptide surface. In this manner, β1 6 branching may affect the
physical properties and functional behavior of glycosylated cell surface adhesion molecules,
such as the integrins.
Enhanced expression of another GlcNAc transferase affecting N-glycan structure (GlcNAcT-III,

which catalyzes the addition of the bisecting GlcNAc branch) has been reported in certain
tumors, such as rat hepatomas (Figure 35.1). However, deliberate overexpression of GlcNAcT-
III in other tumor cell types caused suppressed function of growth factor receptors and altered
cellular morphology and actually reduced the rate of tumor metastasis. A recent study shows that
mice genetically deficient in GlcNAcT-III show a reduced rate of chemical hepatocellular
carcinogenesis, despite the fact that (unlike the situation in rats) GlcNAcT-III is not up-regulated
in mouse hepatomas. The data suggest that an independent glycoprotein factor with bisecting
GlcNAc residues facilitates tumor progression in the mouse liver. Overall, although GlcNAcT-
III expression appears to affect the biology of tumors, the results are not as clear-cut and
consistent as those seen with GlcNAcT-V.
Changes in the Amount, Linkage, and Acetylation of Sialic

Acids (15 21)
The classic observation of increased wheat-germ agglutinin binding to animal tumor cells is most
likely explained by an overall increase in cell surface sialic acid content, which in turn reduces
attachment of metastatic tumor cells to collagen type IV and fibronectin. This increase in
sialylation is often manifested as specific increases in α2 6-linked sialic acids attached to outer
lactosamine (Galβ1 4GlcNAc units) or to inner GalNAc-O-Ser/Thr units on O-glycans. The
latter epitope Siaα-2 6GalNAcα1-0 Ser/Thr (called sialyl-Tn) is currently a target for attempts
at immunotherapy in some cancers (see below). Apart from the amount and linkage of sialic
acids, there can also be significant changes in their modifications. Sialic acid 9-O-acetylation
either can be up-regulated (e.g., the ganglioside epitope 9-O-acetylated GD3 [Figure 35.2] is
increased in melanoma cells from a wide variety of species, ranging from humans to fish) or may
be decreased (e.g., on the O-glycans of colon carcinomas). Some tumor cell types have also been
reported to express small amounts of de-N-acetyl gangliosides (Figure 35.2), wherein the N-acyl
group typical of sialic acids is removed, exposing a free amino group. Again, the pathological
significance of these molecules is uncertain; some studies have suggested that they act by
stimulating tyrosine phosphorylation by the EGF receptor.
Figure 35.2. Pathways for accumulation of gangliosides in human neuroectodermal tumors. The
heavy arrows indicate pathways that are up-regulated. The dashed arrows indicate pathways that
have not yet been formally proven. O-acetylation of the terminal sialic acid can occur at the 7- or
9-position, with the former migrating to the latter position under physiological conditions.
Recent work has uncovered a subfamily of I-type lectins called Siglecs, which specifically
recognize many structural features of sialic acids, including the different linkages (see Chapter
24). Since some of these lectins (CD22, CD33, sialoadhesin, and Siglec-5) are exposed to the
bloodstream, the question may be asked whether alterations in sialylation of tumor cells affect
interactions with any of these lectins. Of particular interest is the up-regulation of α2 6-linked
sialic acids in some tumors, which could be recognized by CD22 on B cells or Siglec-5 on
monocytes and neutrophils. How such an interaction might be beneficial to the tumor cell is
unclear.
Expression of N-Glycolylneuraminic Acid in Human Tumors (22,

23)
A particularly interesting phenomenon is the aberrant expression of Neu5Gc in human tumor

cells. This sialic acid differs from the common sialic acid N-acetylneuraminic acid (Neu5Ac) by
the addition of a single oxygen atom (Figure 35.3). Adult humans do not express significant
levels of Neu5Gc on their normal cells, and they mount an immune response to this epitope
when infused with Neu5Gc-containing animal serum. Recent evidence indicates that this is
because the hydroxylase responsible for creating Neu5Gc is mutated in humans. In view of this,
earlier reports of aberrant Neu5Gc expression in human tumors must be explained by an
alternate pathway for the synthesis of Neu5Gc that becomes amplified in certain cancerous
lesions (or by its uptake from dietary sources). Regardless of the mechanism involved, this
reexpression of Neu5Gc may explain why some patients with cancer spontaneously develop so-
called "Hanganutziu-Deicher" antibodies that are directed against Neu5Gc-containing
gangliosides.
Figure 35.3. α-N-glycolyl-neuraminic acid (Neu5Gc). A negatively charged carboxylate occurs

at the 1-position, and linkages to the underlying sugar chain occur from the 2-position. The
arrow points to the additional oxygen atom that distinguishes Neu5Gc from its precursor, N-
acetyl-neuraminic acid (Neu5Ac).
Alterations in Glycosaminoglycan Expression (24, 25)

Significant changes in content of proteoglycans have been reported in the stroma surrounding
tumors, and it is suggested that these alterations can support tumor growth as well as progression
and invasion. The levels of decorin, a leucine-rich proteoglycan capable of regulating matrix
assembly and cell proliferation, are markedly elevated in the stroma of colon carcinomas. Other
reported changes in malignancy include a decrease in the level of sulfation of heparan sulfate and
an increased expression of hyaluronan. In the latter case, the high levels of this anionic polymer
are thought to loosen the matrix surrounding tumor cells and thus facilitate invasion.
CD44 is a major cell surface receptor for hyaluronan and certain chondroitin-sulfate-containing
proteoglycans. The hyaluronan-binding activity of CD44 is affected by its own glycosylation,
including N-linked, O-linked, and glycosaminoglycan chain additions, as well as by the
expression of various isoforms. Changes in CD44 expression (including alterations in
glycoforms and isoforms) are associated with a wide variety of cancers, and in some cases, these
changes correlate directly with metastatic behavior. Whether this is mediated through changes in
hyaluronan recognition is not yet clear.
Pathological Roles of Mucins with Altered Glycosylation (26 38)
Mucins are large glycoproteins with a "rod-like" conformation caused by the presence of many
clustered glycosylated serines and threonines in tandem repeat regions (see Chapter 8). Most
epithelial mucin polypeptides belong to the MUC family. In the normal polarized epithelium,
mucins are expressed exclusively on the apical domain, toward the lumen of the organ. Likewise,
soluble mucins are secreted exclusively into the lumen. However, the loss of correct topology in
malignant epithelial cells (Figure 35.4) allows mucins to be expressed on all aspects of the cells,
and soluble mucins can then enter the extracellular space and body fluids such as the blood
plasma.
Figure 35.4. Loss of normal topology and polarization of epithelial cells in cancer results in
secretion of mucins into the bloodstream. The tumor cells invading the tissues and bloodstream
also present such mucins on their cell surfaces.
Overexpression of mucins in carcinomas has been described for many years, starting with the
classic studies of "Episialin" on mouse tumor cells by Codington, Jeanloz, Bhavanandan, and
colleagues. The simultaneous expression of both membrane-bound and secreted forms of mucins
by many carcinoma cells confounds any consistent discussion of their pathophysiological roles,
since the two forms of mucins could actually have opposing effects. Regardless of this issue, the
secreted mucins often appear in the bloodstream of patients with cancer and can be detected by
monoclonal antibodies. In many instances, mucins appear to be the major carriers of altered
glycosylation in carcinomas. A critical pathophysiological role of mucins in malignancy is
suggested by the metastatic potential of various carcinoma lines in athymic mice and by
inhibitory effects of preincubation with benzyl-α-GalNAc, which blocks O-glycosylation (see
Chapter 40). Apart from specific interactions of the O-glycans with endogenous lectins (see
below), the rod-like structures of the mucins and their negative charge are thought to repel
intercellular interactions and sterically prevent other adhesion molecules such as cadherins and
integrins from carrying out their functions. Thus, in some instances, mucins may act as "anti-
adhesins" that can also promote displacement of a cell from the primary tumor during the
initiation of metastates. Some evidence suggests that they might also block adhesion between
blood-borne carcinoma cells and the host cytolytic cells such as natural killer cells. In addition,
mucins may mask expression of antigenic peptides by MHC molecules.
Another abnormal feature of carcinoma mucins is incomplete glycosylation. One common

consequence is the expression of Tn and T antigens (Figure 35.5; see Chapter 8). Since such
structures occur infrequently under normal circumstances, it is thought that they may provoke an
immune response in the patient. Indeed, there is a correlation between the expression of the T
and Tn antigens, the spontaneous expression of antibodies directed against them, and the
prognosis of patients with carcinomas. The most extreme form of underglycosylation results in
the expression of "naked" mucin polypeptides. Clinical trials are under way to deliberately
provoke or enhance these immune responses by injecting patients with synthetic peptide
antigens, sometimes bearing Tn or sialyl-Tn structures. It is of note that the best immune
responses to the glycosylated antigens are seen when they are presented in arrays, exactly as seen
on the mucins. This is presumably because of multivalent binding of the antigen to the surface
immunoglobulin of B cells, giving maximum cellular activation.
Figure 35.5. Incomplete glycosylation in the O-linked pathway results in expression of the Tn
antigen, the sialylated Tn antigen (a "dead-end" structure), or the T antigen (Thomsen-
Friedenreich antigen, or unmodified Core 1 structure). Multiple copies of these structures may
occur in a closely spaced array on the polypeptide. Some regions of the mucin backbone may
also be "naked," i.e., completely unglycosylated.
Sialylated Lewis Structures and Selectin Ligands on Cancer

Cells (26, 36,39 43)
Immunohistochemical studies on tumor specimens show that Lewis X/A structures are
frequently overexpressed in carcinomas, being carried on glycosphingolipids as well as on N-
and O-glycans. Indeed, sialyl Lewis X and sialyl Lewis A were first identified as tumor antigens.
The expression of these antigens by epithelial carcinomas consistently correlates with tumor
progression, metastatic spread, and poor prognosis in humans and with metastatic potential in
mice. These sialylated fucosylated structures also form critical components of most natural
ligands for the selectins. Thus, it is reasonable to postulate that such tumor cells gain a selective
advantage by presenting pathological selectin ligands that mediate interactions with endogenous
selectins. Indeed, calcium-dependent selectin ligands on carcinoma cells have been demonstrated
by several investigators, and mucin-like tumor antigens binding to E-selectin were directly
demonstrated in the blood of colon carcinoma patients. Likewise, overexpression of E-selectin in
the transgenic mouse liver induced redirection of the metastatic patterns of syngeneic carcinomas
that normally colonize the lung. Tumor metastasis is also attenuated in mice with a P-selectin
deficiency. These studies indicate that interactions between mucins and selectin molecules play a
part in the metastatic cascade of some carcinoma cells (Figure 35.6). This may tie in with the
classic observation that cancer cells entering the bloodstream tend to form complex
thromboemboli with platelets and leukocytes, which are thought to facilitate transport to ectopic
sites and help in evasion of the immune system.
Figure 35.6. Potential interactions that could occur between tumor cells and endogenous lectins.
Most of these have not yet been formally proven to be important in tumor biology. See text for
discussion. (Modified, with permission, from [7] Kim and Varki 1997 [© Kluwer].)
Several animal studies have shown that a lowering of circulating platelet counts can lower the
rates of tumor metastasis. Recent data suggest that this phenomenon may be explained by
interactions involving platelet P-selectin with carcinoma mucins. Thus, human colon carcinoma
cells show a reduced metastatic rate in P-selectin-deficient mice, which may be explained by a
lack of P-selectin-dependent rosetting of platelets on the tumor cells. In this regard, it is
interesting that cancer patients sometimes develop thromboemboli and hypercoagulable states
that may also be associated with cancer cell-platelet interactions. The potential for interactions
between L-selectin on normal leukocytes and the mucins on carcinoma cells is less well studied.
Altered Expression and Shedding of Glycosphingolipids (44 49)
Many of the "tumor-specific" monoclonal antibodies raised against cancer cells seem to be
reactive with the carbohydrate portion of glycosphingolipids. Some of these structures are highly
enriched in specific types of tumors (e.g., Gb3/CD77 in Burkitt's lymphoma and GD3 in
melanomas). Several types of tumors (particularly those of neuroectodermal origin, such as
melanoma and neuroblastoma) are characterized by synthesis of very high levels of sialylated
glycosphingolipids (gangliosides, see Chapter 9). Some of these, e.g., GD2 and GM2, are not
normally found at high levels in extraneural gangliosides. As such, they are currently targets for
both passive immunotherapy (monoclonal antibody infusion) and active immunotherapy
(immunization with purified glycolipid preparations). In some cases, gangliosides are also the
major carriers of modified sialic acids (see above). Many in vitro studies suggest that some
gangliosides have effects upon growth control, and it is suggested (but not proven) therefore that
this may be the case in vivo. Some investigators have also noted strong immunosuppressive
effects of the large quantities of gangliosides "shed" from the cell surface by some tumors. It is
not known whether this type of immunosuppression is a purely pathological phenomenon with
no natural counterpart. Regardless, the amount of free ganglioside that can be found in the body
fluids of some patients with these tumors is such that immunosuppression is likely to be
medically relevant.
Galectins and Polylactosamine Expression (50 53)
Increased expression of galectins (especially galectin-3) has also been associated with tumor
progression. The molecular significance of this correlation is proposed to be the interactions of
galectins with polylactosamines on matrix proteins such as laminin, aiding cellular invasion.
Since polylactosamines are also expressed on cancer mucins and enriched on the β1 6-branched
glycans of tumor N-glycans (see above and Figure 35.1), this molecular interaction could
mediate homotypic adhesion of carcinoma cells as well (Figure 35.6). Galectin recognition may
also explain how adding cell surface galactose to tumor mutants lacking the Golgi UDP-Gal
transporter enhances metastasis. Overall, it remains to be elucidated exactly how galectin-
polylactosamine interactions alter the biology of cancer.
Altered Expression of the ABH(O) Blood-Group-related

Structures (54 56)
The loss of normal AB blood group expression (accompanied by increased expression of H and
Ley structures) is associated with a poorer clinical prognosis of carcinomas in several studies.
The reason for this correlation remains unknown. There are also rare instances in which a tumor
may present an "illegal" blood group structure (i.e., expression of a B blood group antigen in an
A-positive patient; Chapter 16 details the ABO blood group antigens). The genetic basis for such
a change remains unexplained, since the "B" transferase would theoretically require four
independent amino acid changes to convert it into an A transferase (see Chapter 16). Regardless
of the underlying mechanism, tumor regression has been noted in a few such cases, presumably
mediated by the naturally occurring endogenous antibodies directed against the illegal structure.
Loss of Glycophospholipid Anchor Expression (57, 58)

A complete loss of expression of GPI-anchored proteins is seen in some cases of malignant and
premalignant states involving the hematopoietic system. This results from acquired somatic
mutations of hematopoietic stem cells in the PIG-A gene (required for an early step in the
biosynthesis of the GPI anchor precursor; see Chapter 10). The consequence is a marked or
complete loss of cell surface expression of several GPI-anchored proteins on the progeny of a
single hematopoietic stem cell clone. Since this mutation affects only a single stem cell, one
might imagine that its progeny should not be easily detected. However, it is commonly found
that this clone gradually replaces all of the others, giving rise to a syndrome called paroxysmal
nocturnal hemoglobinuria (see Chapter 10). Although the mutation itself does not confer a
malignant phenotype, the affected clone is prone to become malignant, i.e., giving rise to a
leukemia. The mechanism(s) predisposing to the subsequent transforming event is unknown.
Practical Significance of Altered Glycosylation in Tumor Cells
Although many tumor-specific monoclonal antibodies have been generated by injection of tumor
glycolipids, the appearance of the corresponding antigens in the bloodstream is probably caused
by the spillover of mucin glycoproteins (Figure 35.5) bearing similar terminal glycan structures.
The latter are of well-established value for detecting and monitoring the growth status of tumors
(e.g., assays for CA125 in ovarian carcinoma allow the physician to monitor the amount of
tumor remaining in a patient after surgery or chemotherapy). However, these circulating mucins
also pose a serious practical obstacle against the therapeutic use of monoclonal antibodies
directed against these antigens, since they represent a "sink" that would absorb any infused
antibody, preventing it from reaching the tumor. As mentioned above, attempts are also being
made at specific passive and active immunotherapy against certain tumor-associated
gangliosides and incompletely glycosylated mucins. The most promising results to date have
been seen with a humanized anti-GD2 antibody that has shown apparent activity against
childhood neuroblastomas in phase-1 trials. With regard to the increased β1 6 branching of N-
glycans, early clinical studies of the metabolic inhibitor swainsonine, which inhibits N-glycan
processing, have shown some promising results. In most other instances, there is insufficient
understanding of the underlying mechanisms to suggest a specific approach to diagnosis or
prognosis.
Future Directions
There is general agreement that the early events in the evolution of neoplasia involve alterations
in oncogenes, tumor suppressor genes, mutator genes, and apoptosis-related pathways.
Subsequent tumor growth, invasion, and metastasis involve the survival of the fittest cells, and it
is therefore likely that the highly selective changes seen in tumor cell glycosylation have the
greatest functional consequences in these later stages. Unfortunately, the intrinsic genetic
instability and cellular heterogeneity of advanced tumors have previously made it difficult to
determine the functional consequences of specific glycosylation changes (for the same reason,
exceptions to every suggested hypothesis are also likely to be found). However, in most cancers,
it is invasion and metastasis that finally kill the patient. Thus, it seems worthwhile to pursue this
difficult area of research. The most specific consequences of aberrant glycosylation are likely to
involve endogenous lectins, and these should be the focus of further attention. One approach to
dissecting these complex situations is to take advantage of mice with induced mutations in
specific endogenous lectins and in the glycosyltransferases that generate their cognate ligands.
For example, the effects of genetic ablation of the enzymes required to express selectin ligands
on tumor biology could be explored. Comparing the biology of tumors in these animals to that
occurring in their wild-type or heterozygous littermates may help to delineate specific roles for
the lectins and their carbohydrate ligands. As precise molecular explanations for the functional
effects of these aberrant glycans become clear, inhibitors such as free oligosaccharides or
synthetic mimics may become worth pursuing.
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36. Glycobiology of Protozoal and Helminthic Parasites
Primary contributions to this chapter were made by R.D. Cummings (University of Oklahoma
Health Science Center).
MANY PROTOZOAL AND HELMINTHIC PARASITES synthesize unusual carbohydrate

structures, which are often antigenic and required for invasion of their hosts. Parasites also
synthesize carbohydrate-binding proteins for attachment and invasion of host cells. The major
protozoal parasites include Plasmodium species (causing malaria), Entamoeba histolytica
(causing amebiasis), Leishmania species (causing leishmaniasis), and Trypanosoma species
(causing sleeping sickness and Chagas disease). The major helminthic parasites include
nematodes, such as Ascaris lumbricoides; trematodes, such as Schistosoma mansoni (causing
schistosomiasis); and cestodes or tapeworms, such as Taenia solium (causing taeniiasis). This
chapter focuses on what is known about the structures of the glycoconjugates and lectins
synthesized by these parasites and the roles of glycoconjugates in host-parasite interactions.
Background on Parasite Glycobiology (1)

Parasitism may be defined as a condition in which one organism (the parasite) either harms its
host or survives in some way at the expense of the host. Parasites affect millions of people
worldwide and cause tremendous suffering and death, especially in less developed countries
some sobering statistics are listed in Table 36.1 and thus the importance of research into
parasite glycobiology and biochemistry. In addition, the study of such organisms that have
evolved with great success to deceive and compromise the immune systems of infected animals
may provide important new insights into molecular pathology. However, parasite glycobiology
can be frustrating because of the difficulty in both obtaining sufficient amounts of material for
study and carrying out in vitro experimentation. In addition, many parasites have specific
primary and intermediate hosts, making it difficult to study all stages of the life cycle.
Table 36.1. Worldwide distribution of some major parasitic diseases
Type of disease Estimated human infections Estimated deaths per year
All helminths 4.5 billion ?

Ascaris 1 billion 20 thousand
Hookworms 900 million 50 60 thousand
Trichuris 750 million ?
Filarial worms 657 million 20 50 thousand
Schistosomes 200 million 0.5 1.0 million
Malaria 489 million 1 2 million
Adapted, with permission, from [1] Schmidt and Roberts (1996) (The McGraw-Hill Companies).
The figures show the total number of infections worldwide (some individuals may have more
than one infection) and the number of infection-related deaths per year.
Despite these obstacles, exciting new results demonstrate that glycoconjugates are very
important in the life cycles and pathology of most major parasites. It is becoming clear that some
of the protozoal and helminthic parasites rely on carbohydrate-binding proteins in the host to
promote their parasitism (also see Chapter 28), and they have elaborated intriguing strategies to
defeat the anticarbohydrate immunity of the host. Some of the major parasitic diseases infecting
humans and animals are listed as two categories: the protozoal (single-celled) parasites (Table
36.2) and helminths (Table 36.3).
Table 36.2. Some of the major protozoal parasites of humans
Parasite Comments
Amoeba infecting humans

Entamoeba histolytica causes amoebic dysentery; can cause liver abscesses
Intestinal and genital flaggellates
Giardia lamblia causes diarrhea; one of the most common parasites in North
America
Trichomonas vaginalis causes inflammation of reproductive organs; very common
Haemoflagellates
Leshmania donovani causes visceral leshmaniasis (kala-azal), hepatosplenomegaly
L. mexicana causes fulminating, cutaneous ulcers
L. major causes cutaneous ulcers
Trypanosoma brucei sp. causes sleeping sickness in humans and cattle (African
trypanosomiasis)
T. cruzi causes Chagas disease (South American trypanosomiasis)
Gregarines, coccidia, and related organisms
Plasmodium falciparum major cause of human malaria
P. vivax, P. ovale, P. also causes human malaria
malariae
Causes of opportunistic infections in immunodeficiency states
Toxoplasma gondii causes muscle and intracellular pain
Pneumocystis carinii causes interstitial cell pneumonia
Cryptosporidium parvum intracellular parasites of intestinal cells resulting in diarrhea
Table 36.3. Some of the major helminthic parasites of mammals
Parasite Comments
Trematodes
Blood flukes
Schistosoma mansoni causes human schistosomiasis (affects mesenteric veins draining large
intestine)
S. haematobium causes human schistosomiasis (affects urinary bladder plexus)
S. japonicum causes human schistosomiasis (affects mesentery veins in the small
intestine)
Liver flukes
Fasciola hepatica primarily infects ruminants and occasionally humans; worms live in
biliary tract
Clonorchis sinensis most prevalent liver fluke in humans; can be acquired by eating raw fish
Cestodes
Taenia solium long human tapeworm acquired by eating undercooked pork
Echinococcus short, human tapeworm acquired by eating undercooked lamb; parasitic
granulosus cysts(hydatids) occur in liver and elsewhere
Taeniaarhynchus long, human tapeworm acquired by eating undercooked beef
saginatus
Nematodes
Ascaris lumbricoides most common intestinal roundworm in humans
Trichurus trichuiura intestinal whipworm in humans
Enterobius tiny intestinal roundworm; causes perianal night itch in children
vermicularis
Necator americanus intestinal hookworm of humans; causes anemia
Ancylostoma intestinal hookworm of humans; causes anemia
duodenale
Strongyloides intestinal parasite; causes autoinfection
stercoralis
Haemonchus contortus intestinal parasite of sheep and goats
Trichinella spiralis smallest nematode parasite of humans (trichinosis) residing in muscle
fibers; parasite acquired from uncooked pork
Onchocerca volvulus filarial parasite; causes river blindness
Wuchereria babcrofti filaria live in lymph nodes causing elephantiasis
Brugia malayi filaria live in lymph nodes causing elephantiasis
Dirofilaria immitis dog heartworm
Malaria (2 10)
Malaria is caused by Plasmodium species, and several major species infect humans, with P.
falciparum being the most virulent. The life cycle of P. falciparum is complex (Table 36.4), and
there are sexual and asexual stages in the human and insect hosts. Cell-cell interactions between
parasite and host are critical for the successful completion of each step in the life cycle. The
interaction of sporozoites with hepatocytes requires a protein expressed by the malarial
circumsporozoites that recognizes hepatocyte heparan sulfate, possibly in association with the
low-density lipoprotein receptor-related protein (LRP) (Table 36.5; also see Chapter 28).
Sulfated glycoconjugates can inhibit sporozoite interactions with cells in vitro, and malarial
sporozoites and the circumsporozoite protein do not bind to Chinese hamster cell mutants
defective in glycosaminoglycan synthesis (see Chapter 31).
Table 36.4. Life cycle of Plasmodium falciparum that causes malaria
Sporozoites released from salivary gland of mosquito during blood meal; attach via heparan
sulfate and enter liver cells of host
Development into the pre-erythrocytic stage
Cells rupture, releasing merozoites
Merozoites attach and invade red blood cells via sialic acid recognition and enter within a
vacuole
Merozoites in the vacuole transform into trophozoites that digest hemoglobin to form the
malarial pigment haemozoin
Trophozoites on maturation undergo schizogony and form daughter merozoites
Merozoites after several schizogonic cycles develop into sexually differentiated cells (male and
female gametocytes)
Gametocytes ingested by mosquito during blood meal
Gametocytes in midgut of mosquito become male microgametes and female macrogametes
Union of microgametes and macrogametes leads to zygote
Zygote is transformed into an ookinete that penetrates the intestinal wall and is transformed into
a circular oocyst
Inside the oocyst, the sporozoites develop from germinal cells known as sporoblasts
Sporozoites emerge from the oocysts and migrate to salivary gland
Table 36.5. Some major parasites and their carbohydrate-binding proteins
Parasite Stage Protein Specificity
Plasmodium falciparum merozoite EBA-175 Neu5Acα2 3Gal

merozoite MSA-1 Neu5Ac?
sporozoite circumsporozoite protein heparan sulfate
Trypanosoma cruzi trypomastigote trans-sialidase Neu5Acα2 3Gal
penetrin heparan sulfate
Entamoeba histolytica trophozoite Gal/GalNAc lectin Gal/GalNAc
220-kD lectin chitotriose
80 kD hyaluronan
Giardia lamblia trophozoite taglin Man-6-P
Cryptosporidium parvum sporozoite Gal/GalNAc lectin Gal/GalNAc
Acanthamoeba keratitis
136-kD mannose-binding protein mannose
In addition, sialic acid on the erythrocytes is critical for binding and invasion of those cells by
the merozoite stage. Desialylation of erythrocytes blocks interactions, and individuals lacking
glycophorin A (Ena-) or glycophorin B (MkMk) are resistant to invasion. The glycophorins are
the major sialic-acid-containing glycoproteins on erythrocytes. Merozoites probably contain
several sialic-acid-binding proteins; two have been identified so far and named EBA-175 and
MSA-1, which are immunologically distinct and may have different functions. Hapten inhibition
experiments indicate that both of these proteins bind sialic acid specifically and that the sialic
acid linkage is important.
Glycoconjugates synthesized by the parasite may also be important in the invasion process.
Plasmodium synthesizes both free and protein-bound GPI lipids, which are structurally different
and whose expression is developmentally regulated (see Chapter 10). The free GPI lipids from
Plasmodium are bioactive and can elevate expression of host adhesion molecules, such as I-
CAM-1, V-CAM-1, and E-selectin in human umbilical vein endothelial cells. This activity
occurs via a tyrosine phosphorylation cascade that is blocked by a specific antagonist of tyrosine
kinases, but the specific mechanism of action is unclear.
Trypanosomiasis (11 18)
Trypanosoma cruzi is the cause of Chagas disease, a debilitating disease in South America
affecting multiple organ systems; T. brucei gambiense and T.b. rhodesiense are responsible for
African sleeping sickness in humans. Still another subspecies, T.b. brucei, does not infect
humans, but it causes a disease called nagana in native antelopes and other African ruminants. T.
brucei lives free in the host blood and lymph, where it is constantly exposed to host immunity,
whereas T. cruzi is sequestered in host cells, where it can partly evade the immune system. The
complex life cycle of T. cruzi is shown in Figure 36.1. To evade host immunity, T. brucei
expresses high levels of a GPI-anchored variable surface glycoprotein on its plasma membrane
(~10 million copies per cell) (see Chapter 10). The primary structure of the VSG changes
periodically during infection due to expression of a different VSG gene. The organism can
express up to 1000 different VSGs, but only one is expressed at a time. The new variants are not
recognized by preexisting antibodies, thus allowing a temporary evasion of immunity. When
immunity arises to these variants resulting in their destruction, the numbers of other variants that
are not recognized by the immune system expand. This cycle continues until the hosts dies.
Other trypanosomes also express GPI anchors, like all eukaryotes, but the core structures differ
in terms of their modifications (Figure 36.2). GPI anchors in T. cruzi are required for effective
parasitism. Induction of a GPI deficiency in T. cruzi by the heterologous expression of T. brucei
GPI-phospholipase C depresses surface glycoprotein expression and decreases virulence in
infected animals.
Figure 36.1. Life cycle of Trypanosoma cruzi. (Adapted, with permission, from [11] Adam et al.
1971.)
Figure 36.2. GPI anchors of Trypanosoma sp. and Leishmania sp. In T. brucei VSG: R1, R2, and
R24 = OH, R3 = αGal0 5, and the lipid is a diacyl-glycerol. In T. cruzi 1G7: R1 = αMan, R2, R3,
and R4 = OH, and the lipid is undefined. In L. major PSP: R1, R2, R3, and R4 = OH and the
As discussed for Plasmodium, sialic acid residues are also critical for succcessful attachment of
circulating T. cruzi trypomastigotes to host cells. The interesting adhesion molecule for this
attachment was identified as a GPI-anchored trans-sialidase on the surface of the protozoa. The
enzyme can act as a conventional sialidase, but it also is capable of removing sialic acid from
host glycoconjugates and transferring it to its own glycoconjugates. These observations explain
the anomaly that the parasites are sialylated but lack a conventional sialyltransferase. This
activity is specific for sialic acid linkage (α2,3) and for attachment to terminal galactose
residues. The sialic acids appear to protect the organism from complement activation and
antibody formation. Antibodies induced to the trans-sialidase by immunization with cDNA for
the enzyme provide protective immunity to challenged animals. Trypomastigotes also contain a
surface heparin-binding protein of approximately 60 kD termed penetrin that interacts with
heparan sulfate molecules of host cells. Mannose-containing glycoproteins on amastigotes may
also be ligands for human macrophage mannose-binding proteins (see Chapter 28).
In addition to the obvious value of studying trypanosomes for insights into their biology, studies
of N-glycan biosynthesis in trypanosomes have been particularly fruitful in defining the general
functions of glycoconjugates in glycoprotein biosynthesis. Trypanosomes are unable to
synthesize Dol-P-Glc and therefore synthesize a lipid-linked oligosaccharide donor that is
Man6,7,or9GlcNAc2-P-P-Dol, depending on the species. The oligosaccharyltransferase of
trypanosomes, unlike the mammalian enzyme, efficiently transfers these nonglucosylated donor
oligosaccharides to asparagine residues in newly synthesized glycoproteins. However,
trypanosomes have a glucosidase-II-like activity, which was shown to be required for
deglucosylation of N-glycans glucosylated by the UDP-Glc:glycoprotein glucosyltransferase, an
activity that was first detected in those parasites. Glucosylation of newly synthesized
glycoproteins is probably an important aspect of chaperone-assisted (calnexin/calreticulin)
glycoprotein folding and exit from the ER in all eukaryotes (see Chapter 7).
Leishmaniasis (19 24)
Leishmania donovani is a protozoal parasite that causes visceral leishmaniasis; other species
cause mucocutaneous leishmaniasis (see Figure 36.3). The surface of the promastigote is covered
with a macromolecular glycoconjugate, a phosphoglycan, containing high amounts of mannose.
In L. donovani, this is present in the lipophosphoglycan, shown in Figure 36.4, that is not
attached to protein. In contrast, the phosphoglycan of L. mexicana is linked to serine residues in
proteins, such as secreted acid phosphatase (sAP); in L. major, the phosphoglycan is serine
linked to a proteophosphoglycan. The LPG of L. donovani is synthesized by sequential addition
of Man-1-P from GDP-Man and galactose from UDP-Gal. The LPG of L. donovani is a critical
ligand for recognition by the macrophage mannose receptor and entry of the parasite into
macrophages. In addition, the L. donovani LPG can inhibit phagosome-endosome fusion and
prevent lysis of the endocytosed parasite. The LPG of L. donovani is generally required for the
parasite's survival; mutagenized Leishmania promastigotes that cannot synthesize LPG grow
well in vitro, but they cannot grow within the sand fly nor establish infections in macrophages.
Although the bioactivity of the LPG is unclear, it inhibits protein kinase C in macrophages,
which is normally required for oxidative burst in these cells. Thus, the phosphoglycans may all
function to allow survival and reproduction of parasites within vacuoles of infected cells. One of
the mutations in the Leishmania affecting mannose incorporation into LPG was identified to be
within a gene encoding a protein that has predicted homology with membrane transporters and is
predicted to encode a GDP-Man transporter (see Chapter 6).
Figure 36.3. Life cycle of Leishmania donovani. (Adapted from [57] Zaman and Keong 1989.)
Figure 36.4. Phosphoglycans of Leishmania sp. (A) L. donovani LPG. The procyclic LPG
contains approximately 15 repeating units; (B) L. mexicana secreted acid phosphatase (sAP); (C)
L. major proteophosphoglycan (PPG). The phosphoglycans, represented by the wavy lines, are
attached via serine residues within the sAP and PPG.
The LPG of L. donovani also has other interesting biological activities. For example, it can
activate HIV-1 replication in monocytoid cells and directly up-regulate HIV-1 transcription in T
cells, via activation of transcription factors that recognize NF-κB-binding sites. These results
support the possibility that L. donovani may be a putative cofactor in HIV-1 pathogenesis.
Altogether, the phosphoglycans expressed by Leishmania sp. may be among the most potent
weapons of defense presented by protozoal parasites, since these organisms are able to infect and
thrive in host macrophages.
Schistosomiasis (25 45)
Schistosomiasis is caused by a parasitic trematode, and three major species exist that infect
humans worldwide (Figure 36.5). The parasite is unique in that the male/female pair live in the
blood vessels of its host and lay eggs that adhere to the endothelium. The eggs may become
lodged in the host tissues; many eventually pass into the stool and continue the cycle through
intermediate snail hosts, which differ for each Schistosoma species.
Figure 36.5. Life cycle of Schistosoma mansoni and its movement within its vertebrate host.
Schistosomes generate huge quantities of membrane-bound and circulating glycoproteins

containing fucosylated antigens. Three of the notable antigenic carbohydrate structures found in
schistosome glycans include the Lewis X antigen, LDN, LDNF, and several others (Figure 36.6).
Overall, fucosylation is a common theme for most schistosome glycoconjugates. Interestingly,
other helminths, such as Echinococcus granulosus, Dirofilaria immitis, and Haemonchus
contortus, also synthesize glycoproteins containing LDN and LDNF, in addition to other
fucosylated and xylosylated glycans (Figure 36.7). In general, schistosomes appear to be
especially rich in carbohydrate structures and contain an impressive array of glycosphingolipids
and O- and N-glycans on a multitude of glycoproteins. However, among all helminths tested so
far, only schistosomes appear to synthesize the Lewis X antigen.
Figure 36.6. Some fucosylated schistosome glycoconjugates.

Figure 36.7. Other types of glycoconjugates from schistosomes and Haemonchus contortus.
Another theme in schistosomes and other helminths studied so far is the absence of sialic acid in
glycoconjugates synthesized by the worms and the absence of sialyltransferase activities.
Schistosomes synthesize many interesting glycoconjugates in their so-called cercarial glycocalyx
and in eggs. Glycoproteins derived from the tegument, gut, and eggs of the parasite are highly
antigenic and occur in the circulation of the infected animal. The expression of many of these
glycan structures is developmentally regulated and stage-specific, but their fundamental roles in
parasite development and host pathogenesis are not clear. It is also likely that the different
schistosome species differ in several ways in terms of their glycoconjugate structures. For
example, the S. mansoni glycosphingolipids have an extended difucosylated oligosaccharide, but
the terminal difucosylated GalNAc is absent from glycosphingolipids of S. japonicum (Figure
36.7).
Individuals infected with Schistosoma species develop an autoimmunity to the Lewis X antigen.
This is surprising in many ways, since the Lewis X antigen is a common mammalian leukocyte
marker (CD15) (see Chapter 16). The adult worms have a rough tegumental syncytial membrane
that is highly regenerative, even in response to complement attack. This is an interesting
phenomenon, because schistosomiasis is characterized by intense humoral immunity to
carbohydrate antigens, but immune attack is ineffective in destroying the mature worms, except
when the curative drug praziquantel is administerd, which requires humoral immunity for
effectiveness. Evidence now exists that expression of the Lewis X antigen by the parasite may be
important in compromising host cellular immunity. During chronic schistosome infection, Th2
immune responses (promoting humoral immunity) predominate over Th1 responses (promoting
cellular immunity). In response to glycans containing the Lewis X antigen, murine B-1 cells
secrete in vitro large amounts of IL-10. Because IL-10 can depress Th1 responses in animals, the
Lewis X antigen may partly contribute to Th2 dominance in early stages of schistomiasis.
Glycans synthesized by schistosome eggs may also be involved in adhesion to the endothelium
and may be ligands for selectins, such as soluble forms of L-selectin, a member of the C-type
family of carbohydrate-binding proteins expressed in the vascular bed (see Chapter 26).
Surprisingly, the schistosomes themselves are reported to express selectin-like molecules that
interact with host cells expressing fucosylated glycans. The identification of many different
antigenic glycoconjugates from schistosomes is helping design new diagnostic procedures for
schistosomiasis. In addition, characterization of the glycosyltransferases in schistosomes and
responsible for antigenic glycan biosynthesis and of enzymes in their intermediate snail hosts
may help identify new drug targets and develop carbohydrate-based vaccines.
Glycobiology of Other Parasites (46 57)
Glycoconjugates and carbohydrate-binding proteins, as discussed above, play an impressive part

in host infection by many different parasites. Several additional examples are shown in Table
36.4. Many parasitic protozoans appear to use carbohydrate-binding proteins as a major
mechanism for host-cell attachment and invasion (see Chapter 28). For example, one of the
major glycoproteins expressed by Entamoeba histolytica, a major cause of amoebic dysentery, is
a lectin that recognizes Gal/GalNAc residues, resulting in adherence of trophozoites to host cells.
Adhesion is followed by contact-dependent cytolysis of host cells. Acanthamoeba keratitis,
which causes severe eye infections involving the corneal epithelium, also adheres via a lectin
interaction. This lectin-mediated adhesion of A. keratitis to host cells is a prerequisite for the
amoeba-induced cytolysis of target cells. The adhesion occurs via a mannose-binding protein
that is highly inhibited by Manα1 3Man disaccharides. Interestingly, mannose and Man-6-P can
inhibit adhesion of Giardia lamblia trophozoites. One of the proteins from the parasite that may
bind mannose and Man-6-P is termed taglin. The cDNAs of several galectin family members,
identified initially as antigenic proteins, have been cloned from parasitic nematodes, such as
Teladorsagia circumcincta. Sporozoites from Cryptosporidium parvum, an opportunistic
protozoan infecting individuals with compromised immunity, have hemagglutinating activity,
and a lectin on the parasite surface may have a crucial role in host-cell attachment.
In addition to these ongoing studies on carbohydrate-binding proteins in parasites, the

carbohydrate antigens of many parasites are being characterized in the hope that the information
may lead to the development of vaccines and new diagnostics for these diseases. For example,
the major antigenic glycoconjugates synthesized by larvae of the parasitic nematodes Toxocara
canis and T. cati are O-methylated trisaccharides containing 2-O-methyl fucosyl and galactosyl
residues. The intestinal nematode Trichinella spiralis synthesizes several highly immunogenic
glycoproteins that contain the unusual sugar tyvelose (3,6-dideoxy-d-arabino-hexose). Tyvelose
is found in complex-type N-glycans of larvae from the T. spiralis and is a critical antigenic
determinant recognized by antibodies in infected animals. Strong immunity to these glycans
provide protective immunity, which causes expulsion of the invading larvae from the intestine.
Protective immunity is also provided by antibodies to uncharacterized carbohydrate antigens
from Haemonchus contortus, an intestinal parasitic nematode of ruminants.
Future Directions
Although much more work remains to identify and fully characterize the roles that parasite-
derived lectins and glycans have in parasitic infections, the information gathered so far suggests
that glycoconjugates are immensely important to many types of protozoal and helminthic
parasites. Future studies in this area will probably aim at developing new drugs that block unique
parasite glycoconjugate biosynthesis or interfere with parasite recognition by host receptors.
Information about the important carbohydrate-binding proteins of the parasites and the unique
glycoconjugates expressed by the parasites might promote development of new types of
diagnostic assays. In addition, because many parasites express specific lectins required for cell
adhesion, vaccines based on these lectins could be particularly effective in limiting infection.
Vaccines could also be developed against specific parasite glycoconjugates identified as being
uniquely antigenic and important for parasite invasion.
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37. Acquired Glycosylation Changes in Human Disease
Diego).
SEVERAL HUMAN DISEASE STATES are known to involve acquired (noninherited) changes
in glycosylation and/or in the recognition of glycans. This chapter discusses some examples of
these situations and considers the mechanisms of the changes seen, as well as the
pathophysiological roles of glycans. Wherever it is relevant, the potential therapeutic
significance of the information is mentioned. Details regarding some of these situations are
covered elsewhere (e.g., glycosylation changes in cancer are covered in Chapter 35). Human
glycopathologies resulting from inherited genetic disorders are discussed separately in Chapter
32.
Cardiovascular Medicine
Role of Selectins in Reperfusion Injury (1)
A variety of common cardiovascular disorders (e.g., stroke, myocardial infarction, and

hypovolemic shock) are characterized by a period of decreased or absent blood flow, followed
by a state of reperfusion, which occurs either by natural mechanisms or because flow has been
restored by medical intervention. Despite rescue of the tissue from permanent anoxemic
necrosis, the entry of leukocytes into the reperfused area can initiate a cascade of events that
ultimately results in substantial tissue damage. P-selectin on the activated endothelium in the
reperfused area and L-selectin on the leukocytes have a vital role in mediating the initial steps of
this cascade (see Chapter 26). Substantial data in animal model systems indicate that blockade of
this initial selectin-based recognition can markedly ameliorate the subsequent tissue damage. A
major goal of some pharmaceutical and biotechnology companies is to make small-molecule
inhibitors that can be used to achieve this blockade in human patients (see Chapters 26 and 40
regarding the synthesis of small glycan molecules designed to be selectin inhibitors).
Role of Selectins, Glycosaminoglycans, and Sialic Acids in Atherosclerosis (1

7)
High levels of low-density lipoprotein cholesterol and low levels of high-density lipoprotein
cholesterol are associated with an increase in the risk of atherosclerotic lesions of the large
arteries, which are the major cause of heart attacks, strokes, and other serious diseases. The very
earliest phase of the development of atherosclerotic lesions (the fatty streak) involves the entry
of monocytes into the subendothelial regions of the blood vessels. There is evidence that this
process involves the expression of P-selectin on the endothelium, which recognizes PSGL-1 on
circulating monocytes. Indeed, lesions in LDL receptor-deficient mice showed delayed
progression in a P-selectin-deficient background, and even slower progression occurs in a
combined P- and E-selectin-deficient state. The induction of endothelial P-selectin expression
may result from oxidized lipids that are present in LDL particles and/or the inflammatory
process that occurs in the early atheromatous plaque. Whether or not it is feasible to intervene in
this process remains to be seen, since the early lesions probably develop very slowly and
relatively early in life.
The subendothelial retention of LDLs in the early plaque is thought to occur at least partly via
their interactions with proteoglycans. The interaction is thought to cause irreversible structural
alterations of LDL, potentiating oxidation and uptake by macrophages and smooth muscle cells.
At the molecular level, clusters of basic amino acids present in Apo-B (the protein moiety of
LDL) are thought to bind the negatively charged glycosaminoglycans of proteoglycans. Several
reports also indicate a lowered sialylation of LDL in patients with coronary artery disease. The
pathophysiological significance of this finding and the mechanism(s) involved remain unclear.
One hypothesis is that the desialylated LDL is more prone to be taken up and incorporated into
atheromatous plaques. However, there are also some contradictory data in the literature
concerning this issue.
Dermatology: Role of Selectins in Inflammatory Skin Diseases (8

12)
Several inflammatory skin diseases (e.g., atopic dermatitis and contact dermatitis) are
characterized by the entry of leukocytes into the dermis, which have a pathogenic role in
recruiting other types of cells and in mediating tissue damage. These types of skin lesions are
sometimes associated with the chronic persistent expression of E-selectin on the endothelial
cells. Independent evidence indicates that E-selectin can recruit circulating lymphocytes carrying
the cutaneous lymphocyte antigen (detected by the antibody HECA452), which appears to be a
specific E-selectin ligand epitope carried on a subset of PSGL-1 molecules (see Chapter 26).
There is also evidence that some T-helper-1 lymphocytes can be recruited into the skin by virtue
of their expression of the PSGL-1 ligand for P-selectin. Many of these observations have been
made only in experimental models. The potential for therapeutic intervention in these selectin-
mediated processes has not yet been pursued.
Endocrinology and Metabolism: Altered Glycosylation and the

Complications of Diabetes Mellitus (13 20)
Diabetes mellitus is a disease of dysregulated glucose metabolism resulting from relative or
absolute lack of insulin action. It is accompanied by characteristic long-term vascular and
neurologic complications. The high levels of glucose in body fluids cause an acceleration of a
well-known normal process in which the open-chain (aldehyde) form of the glucose reacts
randomly with lysine residues on various proteins, resulting in reversible Schiff bases. With
time, some of these adducts can undergo the irreversible Amadori rearrangement. These then
undergo a series of "browning" (Maillard) reactions, which eventually progress to advanced
glycation end products. The resulting protein cross-links can damage cellular functions, and the
adducts can be recognized by some receptors (e.g., the macrophage scavenger receptor, perhaps
participating in the process of atherogenesis). The theory is that this is a normal process of aging,
which is accelerated in the setting of the chronic persistent hyperglycemia of uncontrolled
diabetes mellitus. It is important to differentiate mechanistically and semantically between this
nonenzymatic glycation (or glucosylation) process and the enzymatic glycosylation process that
normally takes place in the Golgi apparatus utilizing glycosyltransferases and sugar nucleotides.
Another metabolic change of interest in diabetes mellitus is the increased production of UDP-
GlcNAc caused by the conversion of excess glucose via the glucosamine:fructose
aminotransferase (GFAT pathway). A current hypothesis is that this increase in cytosolic UDP-
GlcNAc may give a secondary increase of O-GlcNAc levels on nuclear and cytosolic
glycoproteins (see Chapter 13).
Nephropathy is a diabetic complication associated with high mortality. It begins with low levels
of albumin excretion, or microalbuminuria, which progresses to frank macroalbuminuria.
Ultimately, the nephrotic syndrome and the decrease in glomerular filtration rate progress to end-
stage renal disease. The proteinuria has been correlated with a reduction in the heparan sulfate
proteoglycan content of the glomerular basement membrane. The underlying mechanism may
involve a reduction in the heparan sulfate synthesis by glomerular epithelial cells that may be, in
turn, caused by the high glucose in the environment. There is evidence that the N-deacetylase:N-
sulfotransferase which plays a key part in heparan sulfate biosynthesis may have reduced activity
in poorly regulated diabetic animals. The resulting decrease in anionic change and loss of
heparan sulfate proteoglycan are thought to affect the porosity of the glomerular basement
membrane.
Gastroenterology
Role of Gut Epithelial Glycans in Gastrointestinal Infections (21 26)
A large number of gastrointestinal pathogens interact with the gut mucosa via recognition of
glycan structures (see Chapter 28). Prominent examples include cholera toxin (which binds GM1
ganglioside) and Helicobacter pylori, the causative agent of peptic ulcer disease and gastritis,
which binds a variety of glycans in the stomach. Consideration is now being given to using
orally administered soluble glycan inhibitors to impede the progress of such pathogens in the gut.
In this regard, it is interesting that a time-honored treatment for peptic ulcer disease was a
combination of antacids and milk (which contains large amounts of free sialyloligosaccharides).
Changes in Sialic Acid O-acetylation in Ulcerative Colitis (27)
Ulcerative colitis is an inflammatory disease typically affecting the superficial epithelial layer of
the rectum and the distal colon. The cause of the disease is unknown, and remissions and
exacerbations are common. The sialic acids of the colonic mucosa, which are normally heavily
O-acetylated, lose this modification in ulcerative colitis. Whether or not this is of pathogenic
significance is uncertain, but these modifications do render the sialic acids more resistant to
bacterial sialidases.
Hematology
Acquired Anticoagulation Due to Circulating Heparan Sulfate (28)
Occasionally, patients with diseases like cirrhosis and hepatocellular carcinoma spontaneously
secrete a circulating anticoagulant and an unusual coagulation test profile that makes it appear
that the patient is heparinized. The anticoagulant activity can be purified from the plasma and
identified as a heparan sulfate GAG. The precise source of secretion has not been defined, and
therapy is often difficult unless the underlying disease can be corrected.
Abnormal Glycosylation of Plasma Fibrinogen in Hepatoma and Liver

Disorders (29 32)
Plasma fibrinogen is heavily sialylated, and the sialic acids are involved in binding calcium.
Certain genetic disorders of fibrinogen are known to be associated with altered glycosylation of
the N-glycans, which causes altered function in clotting. Patients with hepatomas and other liver
disorders can also sometimes manifest increased branching and/or number of N-glycans,
resulting in an overall increase in sialic acid content. This can present clinically as a bleeding
disorder associated with a prolonged thrombin time.
Paroxysmal Nocturnal Hemoglobinuria (33, 34)

This unusual form of acquired hemolytic anemia usually appears in adults. The defect arises
through a somatic mutation in bone marrow stem cells that causes the production of one or more
abnormal clones. The defect is an inactivation of the single active copy of the PIG-A gene and
X-linked locus involved in the first stages of biosynthesis of GPI anchors (for details on GPI
anchor biosynthesis, see Chapter 10). Although several blood cell types show abnormalities, the
red cell defect is the most prominent, being characterized by an abnormal susceptibility to the
action of complement. This is now known to be due to the lack of expression of certain GPI-
anchored proteins, such as decay accelerating factor, that normally down-regulate complement
activation on "self" surfaces.
Paroxysmal Cold Hemoglobinuria (35)
Patients with this rare disorder have cold-induced intravascular destruction of red cells
(hemolysis), which appears to be caused by the presence of a circulating IgG antibody directed
against the red cell P blood group system. The pathogenesis of this disorder is unknown, but it
tends to occur in the setting of some viral infections and in syphilis. The IgG antibody is
demonstrated by the so-called "Donath-Landsteiner test," where the patient's serum is mixed
either with the patient's own red cells or with those from a normal person, and chilled to 4°C.
Hemolysis occurs after warming to 37°C.
Cold Agglutinin Disease (36 41)
This disease is caused by autoimmune IgM antibodies directed against glycans on red blood
cells. High titers of IgM agglutinins are present in serum and are maximally active at 4°C.
Presumably, this IgM reacts with erythrocytes that are circulating in the cooled blood of
peripheral regions of the body. The antibody fixes complement, which then destroys the cells
when they reach warmer areas of the body. There are several variants of the disease. One affects
young adults and follows infection with Mycoplasma pneumoniae or Epstein-Barr virus
(infectious mononucleosis). This antibody is typically directed against the so-called i antigen
(polylactosamine), is polyclonal, and is generally short-lived. Since M. pneumoniae is itself
known to have a receptor that recognizes polylactosamine, it is hypothesized that the antibody
results from a mirror-image anti-idiotypic reaction to the initial antibody directed against the
mycoplasma's binding site. A more common idiopathic variant of cold agglutinin disease affects
older individuals, involves a monoclonal IgM, and can be a prodrome or an accompaniment to a
lymphoproliferative disease like Waldenström's macroglobulinemia, chronic lymphocytic
leukemia, or other lymphomas. These antibodies are typically directed against the I antigen (β1
6-branched polylactosamine). Some less common variants of cold agglutinin disease express
antibodies directed against sialylated lactosamines. In some patients on chronic hemodialysis, the
syndrome is due to the formation of antibody directed against the sialylated blood group antigen
N.
Tn Polyagglutinin Syndrome (42 44)
Tn polyagglutinability syndrome is an acquired condition where the blood cells made by the
bone marrow express the Tn antigen (O-linked N-acetylgalactosamine, GalNAcα1-O-Ser/Thr)
and sialosyl-Tn (Siaα2 6GalNAcα1-O-Ser/Thr), thus becoming susceptible to hemagglutination
by the naturally occurring anti-Tn antibodies that are present in most normal human sera. The
defect tends to be incomplete, with some circulating cells expressing the more complete
sialylated tri- and tetrasaccharide O-glycans as well. These observations are best explained by
the finding of an acquired stem-cell-based loss of expression of the O-glycan Core 1 β1 3
galactosyltransferase activity. Recently, two cases of this syndrome have been studied in more
detail and have been shown to have repression of a functional allele of this enzyme. Thus,
treatment of cultured lymphoblasts with 5-azacytidine or butyrate resulted in derepresson of
enzyme expression (the recent discovery of at least five distinct gene loci encoding this activity
complicates interpretation of this experiment). Patients with this syndrome show a wide range of
symptoms. Some are picked up simply because the polyagglutinability of their red blood cells is
detected when blood typing is done for a possible transfusion. Others have varying degrees of
hemolytic anemia and/or decreases in other blood cell types. Some of these patients can
subsequently progress into frank leukemia. It is unclear how the primary syndrome predisposes
to the development of the malignancy.
Hereditary Erythrocytic Multinuclearity with Positive Acidified Serum Test

(45 50)
HEMPAS syndrome is a form of congenital dyserythropoietic anemia (CDA type II) diagnosed
by the presence of erythroblastic multinuclearity in the bone marrow and lysis of the blood cells
by acidified serum (Ham's test). Patients generally live a normal life span, although
complications may develop with age, including an enlarged liver, jaundice, gallstones, or
diabetes. In more severe cases, removal of the spleen has been effective in alleviating the
anemia. Erythroid cells in HEMPAS exhibit altered glycosylation, involving loss of complex N-
glycans and an increase in hybrid forms. Additionally, the presence of polylactosamines on
glycosphingolipids is greatly increased. Although the syndrome appears to be genetic in origin, it
is not clear if it is a primary inherited disorder of glycosylation (see also Chapter 32). Reduced
expression of α-mannosidase II and/or GlcNAcT-II enzyme activity has been observed in many,
but not all, cases of HEMPAS. A decrease in α-mannosidase II RNA was previously reported in
one case, but genetic mutation in α-mannosidase II or GlcNAcT-II has not yet been reported.
The mouse model of α-mannosidase II deficiency (see Chapters 7 and 33) does manifest some
aspects of the HEMPAS phenotype and suggests an erythroid-specific function for α-
mannosidase II. However, only a subset of the features of human HEMPAS are observed. In a
survey of six families, each with two or more cases of HEMPAS among the children, linkage
analysis excluded α-mannosidase II, α-mannosidase IIx/III, or GlcNAcT-II (see Chapter 7) as
candidate genes and suggested a primary genetic lesion on chromosome 20q11. An alteration of
a transcriptional factor or mechanism regulating expression of all three enzymes has been
suggested.
Hemolytic Transfusion Reactions
The unnatural practice of blood transfusion uncovered the existence of the ABO blood group
system, which is dictated by different alleles of a lactosamine-specific αGal(NAc) transferase
(for details, see Chapter 16). These and other less prominent carbohydrate antigens are
responsible for many of the hemolytic transfusion reactions that can occur when errors are made
in blood typing.
Immunology and Rheumatology

Changes in IgG Glycosylation in Rheumatoid Arthritis (51 54)
The IgG class of circulating immunoglobulins carry N-glycans. Of these, those in the constant
(CH2 or Fc) region of human IgG are reported to have several unusual properties. First, the
glycans are buried between the folds of the two constant regions. Second, they are often
sufficiently immobilized by carbohydrate-protein interactions that they can be seen in the crystal
structure of the protein (most glycans are not visible in crystal structures). Third, although they
become processed into biantennary complex-type N-glycans, they are hardly ever completed into
fully sialylated molecules. Instead, most of the molecules remain with one or more terminal β-
linked galactose residue (so-called G1 and G2 molecules). It was previously noted that in the
patients with a chronic systemic disease called rheumatoid arthritis, a major fraction of the serum
IgG molecules has decreased galactosylation of N-glycans, some carrying no galactose at all (so-
called G0 molecules). The severity of the disease tends to correlate with the extent of the
glycosylation change, and the spontaneous improvement that occurs during pregnancy is
correlated with an increase in galactosylation of IgG.
One function attributed to the Fc N-glycans is to maintain the conformation of the Fc domains as
well as the hinge regions. These structural features are necessary for effector functions such as
complement binding and Fc-dependent cytotoxicity. NMR studies have shown that the G0 N-
glycans have an increased mobility resulting from the loss of interactions between the glycan and
the Fc protein surface. Thus, it is thought that regions of the protein surface that are normally
covered by the glycan are exposed in rheumatoid arthritis. In addition, some studies suggest that
the more mobile G0 N-glycan may be recognized by the circulating mannose-binding protein,
which can activate complement directly. Rheumatoid arthritis is also characterized by circulating
immune complexes consisting of antibody molecules (called rheumatoid factor) that seem to be
directed against the Fc region of other IgG molecules. With regard to the mechanism for the
underglycosylation, some groups have reported lowered activities of the enzyme β-
galactosyltransferase in lymphocytes from patients with rheumatoid arthritis. It remains an open
question whether the altered glycosylation of IgG has a primary pathogenic role in rheumatoid
arthritis, since the appearance of G0 molecules is a general feature of other unrelated chronic
granulomatous diseases (e.g., Crohn's disease and tuberculosis). Furthermore, the glycan change
is also seen in osteoarthritis, a form of chronic degenerative arthritis with a different
pathogenesis. Overall, the change in IgG glycosylation in rheumatoid arthritis remains an
interesting phenomenon whose precise significance and pathogenic role have yet to be
confirmed.
Secondary Changes in the O-glycans of CD43 in Wiskott-Aldrich Syndrome

(55, 56)
This inherited genetic disease is characterized by skin eczema, altered cellular immune
responses, and low platelet counts appearing in childhood. Early studies had suggested an
absence of CD43 (leukosialin, sialophorin), the major O-glycosylated protein of lymphocytes.
However, in retrospect, it is clear that the polypeptide is still expressed normally but had
changed gel mobility because of a markedly increased branching of O-glycans. Recent data
indicate that the primary defect in this syndrome is not in glycosylation but in a transcription
factor. However, the glycan changes seen in resting T cells of these patients are exactly the same
as those that can be induced upon activation of normal T cells. Thus, it remains possible that
some aspects of the immune disorders in this disease are due to a secondary change in
glycosylation.
Infectious Disease
Recognition of Glycans by Bacterial Adhesins, Toxins, and Viral
Hemagglutinins
A wide variety of pathogens initiate infection by specifically recognizing cell surface glycans. In
some instances, the differences in infection rate between individuals can be attributed to
variations in the expression of the cognate structure. For example, adhesion of certain pathogenic
strains of Escherichia coli to cells in the urinary tract can be mediated by P fimbriae, involving a
specific glycan receptor on the P blood group antigens. Infections do not occur in individuals
who are P negative. P fimbriae also appear to be important in determining the propensity for
bacterial bloodstream invasion from the kidney. For a discussion of this topic, see Chapter 28.
Desialylation of Blood Cells by Circulating Microbial Sialidases during

Infections (57, 58)
Several microorganisms produce sialidases (classically called neuraminidases) that are involved
in the pathogenesis of the diseases which they cause. In most instances, this enzyme remains
localized to the site of infection. However, in some severe infections, e.g., Clostridium
perfringens-mediated gas gangrene, a sufficient amount of the sialidase is produced so that it can
appear in the plasma. In this situation, the surface of circulating blood cells can become
desialylated, resulting in enhanced clearance and anemia. The detection of the circulating
sialidase has been proposed to have diagnostic and prognostic significance. Whether inhibiting
the sialidase with appropriate inhibitors will have a therapeutic value has not been investigated.
Nephrology
Loss of Glomerular Sialic Acids in Nephrotic Syndromes (59 61)
Nephrotic syndrome occurs when the kidney glomerulus fails to retain serum proteins during the
initial filtration of plasma; these proteins then leak into the urine. The epithelial/endothelial
mucin called podocalyxin present on the foot processes (pedicles) of glomerular podocytes is
thought to have a role in maintaining pore integrity and in excluding large molecules, such as
proteins from the glomerular filtrate. The sialic acid residues of podocalyxin molecules are
believed to be critical in this process. Loss of glomerular sialic acid is seen in the spontaneous
so-called "minimal change renal disease" in children and in the nephrotic syndrome that follows
some bacterial infections. Several animal models seem to mimic this situation. Proteinuria and
renal failure developed in a dose-dependent manner after a single inoculation of Vibrio cholerae
sialidase, and this was correlated with removal of α2 6-linked sialic acids from the glomerulus.
This was also accompanied by the effacement of foot processes and the apparent formation of
tight junctions between podocytes. The anionic charge returned to endothelial and epithelial sites
within 2 days of sialidase inoculation, but the foot process loss remained. Another model is
termed aminonucleoside nephrosis, induced in rats by injection of puromycin. Defective
sialylation of a podocalyxin and glomerular glycosphingolipids has also been detected in this
model.
Changes in the O-glycans in IgA Nephropathy (62, 63)
Aggregation of the IgA1 molecule is thought to be involved in a form of nephrotic syndrome

called IgA nephropathy. Studies of the O-glycans on serum IgA showed a loss of sialylation of
Galβ1 3GalNAc in the IgA nephropathy group compared with a negative control group and a
decrease in galactosylation. One of the functions of the carbohydrate side chains is thought to be
to stabilize the three-dimensional structure of the molecule. Studies of heat-induced aggregation
support the notion that the altered glycosylation on the hinge region of IgA1 results in a loss of
conformational stiffness, perhaps explaining the aggregation phenomenon. Removal of glycans
from the IgA1 molecule also resulted in noncovalent self-aggregation and a significant increase
in adhesion to the ECM proteins. It is therefore suggested that the underglycosylation of the
IgA1 molecule found in IgA nephropathy is involved in the nonimmunologic glomerular
accumulation of IgA1. The primary mechanism of underglycosylation remains unknown.
Neurology and Psychiatry
Pathogenic Autoimmune Antibodies Directed against Neuronal Glycans (64
70)
A variety of diseases are associated with circulating antibodies directed against certain glycan
molecules that are highly enriched in the nervous system. As a consequence, these patients suffer
from symptoms related to autoimmune nerve damage. The antibodies can arise via at least three
distinct pathogenic mechanisms. In the first situation, patients with benign or malignant B-cell
neoplasms (e.g., benign monoclonal gammopathy of unknown significance [MGUS],
Waldenstrom's macroglobulinemia, and plasma cell myeloma) secrete monoclonal IgM or IgA
antibodies that are highly specific for either ganglio-series gangliosides or more commonly,
sulfated glucuronosyl glycans (the so-called HNK-1 epitope). These antibodies react with the
glycolipid bearing this epitope 3-O-SO3-GlAβ1 4Galβ1 4GlcNAcβ1 3Galβ1 4Glc-Cer (3
sulfoglucuronosylparagloboside) and against the N-glycans on a variety of CNS glycoproteins
(MAG, P0, L1, N-CAM) that bear the same terminal sequence (3-O-SO3-GlAβ1 4Galβ1
4GlcNAcβ1-). The resulting peripheral demyelinating neuropathy can sometimes be more
damaging than the primary disease itself. Therapy consists of attempts to treat the primary
disease with chemotherapy or to remove the immunoglobulin by plasmapheresis. Both
approaches are usually unsuccessful at lowering the immunoglobulin to a level sufficient to
diminish the symptoms. The second situation appears to be an immune reaction to the molecular
mimicry of ganglioside structures by the lipo-oligosaccharides of bacteria such as
Campylobacter jejuni. Following an intestinal infection with such organisms, circulating
antibodies against gangliosides like GM1 and GQ1b appear in the plasma. These are typically
associated with the onset of symptoms of a demyelinating neuropathy involving the peripheral
and central nervous systems: the Guillain-Barre and Miller-Fisher syndromes, respectively. The
third situation seems to be a man-made disease arising from recent attempts to treat patients with
disease such as stroke using intravenous injections of mixed brain gangliosides. Although some
evidence exists that this treatment may be beneficial for the primary disease, several cases of
Guillain-Barre syndrome have been reported as a likely side effect.
Role of Glycans in the Histopathology of Alzheimer's Disease (71 75)
Alzheimer's disease is a common primary degenerative dementia of humans having an insidious

onset and a progressive course. The ultimate diagnosis can only be made by histological
examination of brain tissue, which shows characteristic amyloid plaques with neurofibrillary
tangles that are associated with neuronal death. Two types of glycans have been implicated in the
histopathogenesis of the lesions: O-GlcNAc and heparan sulfate glycosaminoglycans.
Paired helical filaments are the major component of the neurofibrillary tangle. These are
primarily composed of microtubule-associated protein tau, which is present in a
hyperphosphorylated state. This abnormally hyperphosphorylated tau no longer binds
microtubules and self-assembles to form the paired helical filaments that may contribute to
neuronal death. Normal brain tau is known to be multiply modified by Ser(Thr)-O-linked
GlcNAc, a dynamic and abundant posttranslational modification that is often reciprocal to
Ser(Thr) phosphorylation (see Chapter 13). The hypothesis currently being investigated is that
site-specific or stoichiometric changes in O-GlcNAc addition may modulate tau function and
may also play a part in the formation of paired helical filaments, by allowing excessive
phosphorylation.
The hyperphosphorylated tau in Alzheimer's disease brain is found in association with heparan
sulfate proteoglycans. Nonphosphorylated tau isoforms with three microtubule-binding repeats
form paired helical-like filaments under physiological conditions in vitro when incubated with
heparan sulfate. Heparin prevents tau from binding to microtubules and promotes microtubule
disassembly. These findings, together with previous evidence that heparin stimulates tau
phosphorylation by protein kinases, have been used to argue that sulfated glycosaminoglycans
may be a critical factor in the formation of the neurofibrillary tangles. However, no significant
difference was noted between the detailed structure of heparan sulfate obtained from control
brains and from Alzheimer's disease. Furthermore, the topological separation of tau (in the
cytosol) from glycosaminoglycans (extracellular) indicates that this physical association can only
occur after cell death. Heparan sulfate proteoglycans may also have an important role in amyloid
plaque deposition. Investigators have demonstrated high-affinity binding between heparan
sulfate proteoglycans and the amyloid precursor, as well as with the A4 peptide derived from the
precursor. In addition, a specific vascular heparan sulfate proteoglycan found in senile plaques
bound with high affinity to two amyloid protein precursors. Overall, the data indicate that
heparan sulfate chains may play an important part in the pathogenesis of the histological lesions.
Oncology: Altered Glycosylation in Cancer

Altered glycosylation is a universal feature of cancer cells, but only certain specific glycan
changes are frequently associated with tumors. These include increased β1 6GlcNAc branching
of N-glycans; changes in the amount, linkage, and acetylation of sialic acids; reexpression of N-
glycolylneuraminic acid; expression of sialylated Lewis structures and selectin ligands; altered
expression and enhanced shedding of glycosphingolipids; increased expression of galectins and
polylactosamines; altered expression of the ABH(O) blood-group-related structures; alterations
in sulfation of GAGs; increased expression of hyaluronan; or loss of expression of
glycophospholipid anchors. Some of these changes, e.g., increased β1 6GlcNAc branching of
N-glycans and expression of selectin ligands, have been shown to have pathophysiological
significance in model tumor systems and some are also targets for diagnostic and therapeutic
approaches to cancer. For details of these topics, see Chapter 35.
Pulmonary Medicine
Role of Selectins in Bronchial Asthma (76 78)
Asthma is a disease characterized by a hyperresponsiveness of the tracheobronchial tree to

various stimuli, resulting in widespread narrowing of the airways that changes in severity, either
spontaneously or as a result of therapy. The two dominant pathological features of asthma are
airway wall inflammation and luminal obstruction of airways by inflammatory exudate. At least
some cases are due to the presence of antigen-specific IgE antibodies, which then become fixed
to mast cells as well as to basophils and certain other cell types. Subsequently, antigen can cross-
link adjacent IgE molecules, triggering an explosive release of vasoactive, bronchoactive, and
chemotactic agents from mast cell granules into the extracellular milieu. Eosinophils may
contribute to the pathogenesis of asthma in several ways, by synthesizing leukotriene C4,
stimulating histamine release from mast cells and basophils, providing a positive feedback loop,
and releasing major basic protein, a granule-derived protein that has toxic effects on the
respiratory epithelium. Recent evidence indicates that the selectins are intimately involved in the
recruitment of eosinophils and basophils into the lung, raising the hope that small-molecule
inhibitors of selectin function can be used to treat the early stages of an asthmatic attack.
Role of Selectins in the Acute Respiratory Distress Syndrome (1,79 83)

This serious pathophysiological process is the final common pathway of lung injury arising from
a variety of events (e.g., shock, trauma, and sepsis). It is characterized by diffuse pulmonary
endothelial injury, progressing to pulmonary edema, resulting from a marked increase in
capillary permeability. Selectins and integrins help circulating neutrophils to adhere to the
endothelium and release injurious oxidants, proteolytic enzymes, and arachidonic acid
metabolites, resulting in endothelial cell dysfunction and destruction. The presence of many
neutrophils and secretory products in bronchoalveolar lavage liquid emphasizes the critical role
of the underlying inflammatory response. Again, the hope is that small-molecule selectin
inhibitors can be used in the early stages of this syndrome, before it progresses to extensive lung
damage and respiratory failure.
Altered Glycosylation of Epithelial Glycoproteins in Cystic Fibrosis (84 94)
Cystic fibrosis is a very common genetic disorder caused by a mutation in the cystic fibrosis
transmembrane conductance regulator. This causes defective chloride conduction across the
apical membrane of involved epithelial cells. Cystic fibrosis is associated with increased
production of viscous mucins in the gut and lungs, which leads to many of the symptoms. There
are known to be widespread reductions in sialylation of secreted proteins and increases in the
sulfation and fucosylation of mucus glycoproteins. Data have indicated that the cystic fibrosis
conductance regulator defect is partly responsible for effective acidification of the Golgi
apparatus and that a higher Golgi pH in cystic fibrosis results in the abnormalities in
glycosylation (there is currently some controversy about this conclusion). The major cause of
morbidity in the disease is the colonization of respiratory epithelium by an alginate-producing
form of Pseudomonas. Certain glycolipids have been suggested to be the Pseudomonas receptors
that help to maintain the colonization. The defects in epithelial Golgi sialylation as well as
production of a sialidase by the bacteria may help to enhance the production of potential binding
targets for organ colonization.
Future Directions
It is evident from the examples presented in this chapter that acquired changes in glycans and/or
their recognition may have a significant role in a variety of human diseases. In some cases, the
evidence remains circumstantial, and further work is needed to define whether the glycan
changes have a primary role. In many of the situations, detailed knowledge of the nature of
glycan/receptor interactions could result in improved diagnostic or therapeutic approaches.
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Methods and Applications
38. Principles of Structural Analysis and Sequencing of
Glycans
Primary contributions to this chapter were made by A.E. Manzi (Nextran Corporation, San
Diego, California) and H. van Halbeek (University of California at San Diego).
THIS CHAPTER SURVEYS VARIOUS APPROACHES for analyzing the composition and
primary structure of biologically relevant glycans. An overview of different analytical strategies
is provided, leading from detection methods for native and derivatized ("tagged") glycans, via
metabolic labeling with radioactive sugars, the use of endo- and exoglycosidases, to quantitative
monosaccharide composition analyses, various forms of glycan release and chromatographic
separations, chemical methods for linkage position analysis, and ultimately to mass
spectrometric and NMR spectroscopic methods for complete sequence determination.
Introduction
Fully understanding the biology of glycoconjugates requires in-depth knowledge of the primary
sequence and three-dimensional structure of carbohydrate chains. The primary structure of a
carbohydrate is defined by the parameters listed in Table 38.1 (also see Chapter 2). Additional
parameters required to describe the conformation of a carbohydrate of known primary structure
are listed in Table 38.2. For most carbohydrates, the secondary and higher-order structures in
solution are not readily defined, due to their inherent flexibility. Characterization of carbohydrate
dynamics by experimental and theoretical methods remains an area of active research.
Conformational analysis of carbohydrates is not discussed in further detail in this chapter.
Instead, the analytical methods used to determine composition and sequence are described.
Table 38.1. Primary structural features of a complex carbohydrate
General description Specific examplesa
Qualitative and quantitative composition, i.e., nature and number Gal, GlcNAc, Man, Fuc in
of constituting monosaccharides, including ratio 2:4:3:1
absolute configuration (d or l) and d-Gal, d-GlcNAc, d-Man, l-
Fuc
ring size (pyranose [p] or furanose [f]) Manp, Galf
Configurations of glycosidic linkages α, β
Positions of glycosidic linkages (1 2), (1 3), (1 4), (1 6)
Sequence of monosaccharides, including occurrence of Manα(1 6)[Manα(1
branchpoints (double or triple substitution of a monosaccharide) 3)]Manβ(1 4)GlcNAc
Nature, number, and location of appended noncarbohydrate IdoA2SO3, Neu5Ac9OAc
groups: phosphate, sulfate, acetate (peptide, lipid)
a
For a definition of these parameters, see Chapter 2.
Table 38.2. Secondary structural features of a complex carbohydrate
Conformational aspect Defined bya
Precise ring conformation of each complete set of H-C-C -H dihedral angles

monosaccharide
Orientation of monosaccharides with torsional angles φ, ψ (ω) around glycosidic bonds
respect to each other and/or interatomic distances
Flexibility of the spatial structure dynamics parameters (rotational correlation times,
order parameters)
a
For a definition of these parameters, see Chapter 2 and Reference 17.
The structural characterization of the glycans of a typical glycoprotein (bovine fetuin) is included
here to illustrate actual strategies. Guidance is provided on how to make the appropriate choices
to obtain the data required, given the type and quantity of sample available. General indications
are given for primary structural analysis of other major types of vertebrate carbohydrates.
Additional information can be found in References 1 16.
General Considerations for Analyzing the Primary Structure of

a Carbohydrate
The structural diversity of naturally occurring glycans is great, making it difficult to define a
universal protocol for their analysis. The building blocks of complex carbohydrates (i.e., the
monosaccharides) exhibit very similar structures; thus, their differentiation is more involved than
that of amino acids. The structural analysis of carbohydrates requires a flexible approach, which
all but precludes full automation of the process. Only within a particular subclass of
carbohydrate structures (e.g., for N- and O-glycans commonly found in glycoproteins) can some
general techniques be applied universally. At any given juncture in the procedure, the choice of
methodology is dictated by the amount and purity of carbohydrate material available. If pure
carbohydrate material is abundantly available, the goals for structural characterization can be set
as high as possible: Investigators should be able to delineate the complete primary structure. In
situations where purity and/or amounts are limiting factors, partial characterization may still be
possible.
Detection of Carbohydrates
The approaches taken to detect carbohydrates in glycoconjugates include chemical reactions and
radioactive labeling, as well as detection with specific lectins or antibodies. Detection methods
based on the susceptibility of sugars to periodate oxidation have been traditionally used to
identify glycoproteins in gels with the periodic acid-Schiff (PAS) reaction. Commercially
available kits now allow detection of 5 10 ng of glycoprotein, using the same periodate reaction
with subsequent amplification by means of biotin-hydrazide/streptavidin-alkaline phosphatase.
Lectin overlays of blots can also be used to detect the presence of carbohydrates with
comparable sensitivity.
Alternatively, proteins metabolically labeled with radioactive sugar precursors can be detected
directly by autoradiography. Cells incubated with 3H- or 14C-labeled mannose, glucosamine,
galactose, or fucose will incorporate the label into the glycan chains of glycoproteins,
glycolipids, and proteoglycans. Such molecules can be detected as radioactively labeled bands
when a mixture of proteins is analyzed by SDS-PAGE, or the lipid extract is analyzed by TLC.
The ability to label proteins with specific radioactive precursors (e.g., myo-inositol,
ethanolamine) may suggest that they are GPI-anchored. Proteoglycans containing radioactively
labeled GAGs can be detected using polysaccharide lyases. Another approach is to transfer a
label from radioactive sugar nucleotides to endogenous glycans using purified
glycosyltransferases (e.g., O-linked GlcNAc residues can be detected by using
galactosyltransferase and UDP-[3H]Gal). Glycosaminoglycan chains on proteoglycans can be
liberated with a nonspecific protease or by alkaline β-elimination. GAGs are then isolated by
anion-exchange chromatography or cetyl pyridinium chloride/ethanol precipitation. In
radioactively labeled samples (e.g., with 35SO4 or [3H]GlcNH2), GAGs are quantitated by direct
scintillation counting, whereas nonradioactive GAGs are usually quantified by measuring the
content of uronic acid.
When chemical radioactive labeling reactions are applied, glycoconjugates will incorporate
radioactivity if specific structural features are present (e.g., mild periodate oxidation of terminal
sialic acid side chains followed by reduction of the resulting aldehyde with sodium
[3H]borohydride). These radioactive labeling approaches have the advantage of being easy to
perform and to monitor. Radioactively labeled sugar chains can also be isolated following the
same methodology used for nonlabeled sugars with the advantage that radiometric purity is much
easier to achieve and sufficient for further analysis. However, the information obtained from
such analyses is limited, and a complete structural identification requires the isolation of non-
labeled material.
The following subsections deal with more specific issues for detecting carbohydrates, either as
such or as constituents of different classes of glycoconjugates.
Glycoproteins and Proteoglycans
A glycosylated protein typically presents one or more diffuse bands during gel electrophoresis,
resulting from heterogeneity in the carbohydrate moiety. Visualized by protein staining reagents,
this phenomenon is often the first indication of the presence of glycans. Some glycoconjugates of
very high molecular weight, such as mucins and proteoglycans, do not enter ordinary gels or
migrate as smears. Agarose gels or combination polyacrylamide-agarose gels may be of some
help in this situation. Many analytical options to verify a positive carbohydrate staining (e.g.,
PAS) reaction are available depending on the type and quantity of sample (Figure 38.1).
Treatment of the proteins with endoglycosidases (e.g., peptide-N-glycosidase F, endoglycosidase
F2, endoglycosidase H) may result in a mobility change of one or more of the bands on the gel,
indicating the presence of N-glycans. O-sialoglycoprotease can be used for the identification of
glycoproteins containing clustered sialylated O-glycans (mucins), which are specifically
degraded by this protease. Treatment with sialidases or glycosaminoglycan lyases may also
produce a shift in mobility. Various eliminases and hydrolases are available to further define the
class and/or structures of GAG chains (see Chapter 11). However, detection of all glycans by
these treatments cannot be assured due to the possibility of structures being resistant to the
enzymes used. Complete removal of N- and O-glycans can be achieved by chemical treatments
(e.g., hydrazinolysis, β-elimination, or hydrogen fluoride treatment); usually, peptide damage
precludes further analysis using gel electrophoresis.
Figure 38.1. Basic strategies for the detection of carbohydrates in glycoconjugates.
Glycolipids
Mixtures of glycolipids can be fractionated by TLC. Staining of TLC plates with carbohydrate-
reactive reagents allows detection of individual glycolipids as bands. Using different reagents, it
is possible to recognize gangliosides (resorcinol-HCl detects sialic acids) from those TLC bands
that only contain neutral monosaccharides (orcinol-sulfuric acid detects all monosaccharides).
Reagents are available for detection of sulfate and phosphate groups as well. However, a
particular glycolipid present in small quantity may be masked by other lipids that are typically
present in larger quantities in tissue extracts (e.g., neutral lipids, cholesterol, phospholipids,
sulfatides, and glycerolipids). For this reason, some prepurification of the crude extract is usually
preferred (e.g., Folch partitioning and diethylaminoethyl [DEAE] column chromatography).
These procedures separate nonpolar or nonionic lipids from polar lipids (e.g.,
glycosphingolipids) and those that contain charged groups (i.e., gangliosides, phospholipids, and
sulfatides). In the resulting enriched mixture, glycolipids can now be detected more easily by
carbohydrate-reactive reagents. It is also common practice to evaluate the shifts produced in the
migration of a band following a chemical or enzymatic treatment. Depending on the presence of
a particular structural feature, TLC plates can also be overlaid with monoclonal antibodies,
lectins, or even intact microorganisms (see Chapter 28). Detailed features can be recognized by
running the TLC in a second dimension following a specific treatment (e.g., exposure of a plate
to ammonia vapors after the first run, followed by evaporation of excess ammonia, and a second
run in a second dimension can indicate which bands contain base-labile O-acyl esters).
Glycolipids can be further separated according to the number of negative charges (e.g., by
column chromatography on DEAE eluting with a gradient of salts in chloroform/methanol/water
mixtures) or according to their polarity (e.g., by column chromatography on silica gel eluting
with a solvent mixture of increasing polarity). Each group can be further fractionated with more
specific chromatographic approaches, including highresolution HPLC.
GPI Anchors
GPI-anchored proteins, with their lipid, protein, and carbohydrate moieties, have unique
physicochemical properties that can be exploited for detection purposes. Triton X-114 at low
temperature extracts soluble and integral proteins as well as GPI-anchored proteins. When the
solution is warmed, two phases separate, and GPI anchors and other amphiphylic proteins remain
associated with the detergent-enriched phase. GPI-specific phospholipases alter the partitioning
between the phases. Cleavage also can be assessed by analyzing samples by SDS-PAGE, since
removal of the GPI anchor causes a shift in molecular mass.
Plant and Bacterial Polysaccharides
This family of carbohydrates comprises a large variety of structures, including homo- and
heteropolysaccharides, neutral and ionic polysaccharides, linear and branched structures, and
widespread molecular sizes ranging from a few monosaccharide units to thousands (see Chapters
20 and 21). These polysaccharides are typically extracted with water, salts, chaotropic agents, or
detergents and isolated by precipitation with alcohols. Detection is based on refractive index or
colorimetric reactions, since sample quantity is not usually a limitation.
Monosaccharide Composition Analysis

Once it has been determined that one or more of the components in a given sample are
glycosylated, the next step is to ascertain the total carbohydrate content of the glycoconjugate
and elucidate what type of monosaccharide residues are present. Some qualitative information
may already be available based on the results of enzymatic treatments, lectin blotting, or specific
chemical reactions. Simple colorimetric reactions can be used to determine the total amount of
hexose, hexuronic acid, or hexosamine in the sample. While these approaches only require
common reagents and a spectrophotometer, the total carbohydrate content may not be accurately
obtained due to errors arising from the lack of specificity and/or sensitivity of the assays. Some
colorimetric reactions have been adapted to suit HPLC monitoring of the product, increasing the
sensitivity of the assay considerably.
Quantitative monosaccharide analysis provides precise molar ratios of individual sugars, and
may suggest the presence of specific oligosaccharide classes (e.g., N- vs. O-glycans). It can also
be used as a measure of production consistency (quality control) for recombinant glycoprotein
therapeutics. Monosaccharide composition analysis involves the following steps: cleavage of all
glycosidic linkages, fractionation of the resulting monosaccharides, detection of each
monosaccharide, and quantification of the signal obtained. Several methods exist for this purpose
(Figure 38.2). Since the early 1960s, a variety of gas-liquid chromatography (GLC) methods
have been developed to quantify monosaccharides. Some use flame-ionization detection (FID),
but the most useful ones involve coupling GLC and mass spectrometry (MS) for positive peak
identification. By the 1970s, GLC-MS was used as a major tool for determining carbohydrate
compositions. Peracetylated alditol acetates and per-O-trimethylsilyl ethers, obtained after
cleaving the glycosidic linkages and subsequent derivatization, were the most widely used
derivatives in this type of analysis. Incorporation of an optically pure chiral aglycone (e.g., a [
]-2-butyl group), in combination with trimethylsilylation, allows the GLC separation of the d-
and l-pair of isomers, and thus determination of absolute configuration, of each monosaccharide.
In the 1980s, with the advent of HPLC, pre- and postcolumn derivatization methods were
developed to quantify monosaccharides. In the early 1990s, these methods were supplanted by
high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD),
since this method does not require monosaccharide derivatization. The result of a
monosaccharide composition analysis of bovine fetuin by HPAEC-PAD is shown in Figure 38.3.
In the mid 1990s, fluorescent derivatives of monosaccharides produced by reductive amination
(e.g., 2-amino-benzamide [2-AB], 2-amino-pyridine [2-AP], and 8-amino-1,3,6-naphthalene
trisulfonic acid [ANTS]) became popular in conjunction with reverse-phase HPLC with on-line
fluorescence detection, gel electrophoresis (fluorophore-assisted carbohydrate electrophoresis
[FACE]), or high-performance capillary electrophoresis (HPCE). For example, tagging sialic
acids with a fluorescent compound (1,2-diamino-4,5-methylene-dioxy-benzene [DMB]) has
allowed an increase in detection sensitivity to the femtomole range.
Figure 38.2. Approaches for monosaccharide composition analysis of glycoconjugates: A

historic perspective. (For abbreviations, see Table 38.3.)
Figure 38.3. Monosaccharide composition analysis of bovine fetuin by HPAEC-PAD: (A)
neutral and amino sugars; (B) sialic acids. Two 50-µg samples of fetuin were submitted to strong
(A) and mild (B) acid hydrolysis, respectively. After evaporation of excess acid, the hydrolyzates
were redissolved in water and injected onto a CarboPac PA-1 anion-exchange HPLC column.
Elution was by sodium hydroxide and detection by pulsed amperometry. Qualitative and
quantitative evaluation of the chromatograms was based on analysis of elution times and PAD
responses of monosaccharide standards in separate experiments. Chromatogram A indicates that
bovine fetuin contains GalN(Ac), GlcN(Ac), Gal, and Man; i.e., sugars that are compatible with
the presence of both N- and O-glycans in the glycoprotein. (The acetyl groups of GlcNAc and
GalNAc are cleaved off during strong acid hydrolysis; thus, these monosaccharides are retrieved
as free amino sugars, GlcNH2 and GalNH2.) As for the sialic acids (chromatogram B), bovine
fetuin contains predominantly Neu5Ac, along with a few percent of Neu5Gc. The total
carbohydrate content of the investigated batch of fetuin was determined to be 15% (by weight).
The high sensitivity of current instrumentation also makes possible the composition analysis of
glycoproteins blotted onto polyvinylidene difluoride (PVDF) (not nitrocellulose) membranes and
cut out as specific bands. The methods employed are basically the same as those described
above, with the advantage that the hydrolyzate is easily recovered, leaving any peptide or protein
bound on the membrane. Sequential analyses are also possible using the same piece of PVDF
(e.g., sialic acids are first released with mild acid, and the resulting first supernatant is analyzed;
strong acid is added to release the rest of the monosaccharides, which are recovered in the
second supernatant). A summary of the most common approaches for monosaccharide
composition analysis is presented in Table 38.3.
Table 38.3. Commonly used methods for monosaccharide composition

analysis
Method Derivatization Instrumentation Limit of Expertise Information obtained

and materials detection needed
required
GLC- complete GLC instrument nmole range medium type and quantity of
FID derivatization to with flame monosaccharides in
produce volatile ionization detector comparison with
compounds known standards
derivatization capillary column nmole range absolute
with chiral configuration(d/l) of
aglycone monosaccharides
GLC- complete GLC instrument 10 pmoles high type and quantity of
MS derivatization to coupled to a mass monosaccharides in
produce volatile spectrometer with comparison with
compounds EI source known standards
capillary column sensitivity can unknowns can be
be increased to identified from their
100 fmoles MS fragmentation
with selective patterns
ion monitoring
HPLC fluorescent HPLC instrument fmole-pmole medium type and quantity of
tagging of the with on-line range monosaccharides in
reducing end fluorescence comparison with
detector known standards
HPLC column
HPAEC- not needed HPLC instrument 5 50 pmoles medium type and quantity of
PAD coupled to pulsed (depending on monosaccharides in
amperometric response comparison with
detector factor) known standards
CarboPac PA-1 or
PA-10 column
(EI) Electron impact; (FID) flame ionization detection; (GLC) gas-liquid chromatography;
(HPAEC) high pH anion-exchange chromatography; (MS) mass spectrometry; (PAD) pulsed
amperometric detection.
Release, Profiling, and Fractionation of Glycans
Once the presence, quantity, and composition of carbohydrate have been unambiguously
established, the next challenge is to determine how many structurally different glycans are
present in the glycoconjugate. The approach varies depending on the glycoconjugate in question,
but the answer is generally attained by some type of chromatographic profiling.
Glycosphingolipids are readily profiled by HPTLC on silica plates or HPLC on Iatrobeads
support. Since these profiling strategies have been extensively used, comparison with literature
data and known standards may reveal a considerable amount of information about the molecules
under investigation. Selective digests of glycoproteins (e.g., with trypsin) are profiled by reverse-
phase HPLC to separate glycopeptides according to the peptide structure. Adequate monitoring
(e.g., fraction collection, followed by carbohydrate detection and MS; or LC-MS on-line) allows
identification of the peak(s) that corresponds (correspond) to glycopeptides. Polysaccharides of
different sorts, intact proteoglycans, and glycoproteins have been profiled successfully with
different size-exclusion chromatography (SEC) strategies. The heterogeneity of the glycans
themselves can be explored by direct MS analysis (see below) of the intact glycoconjugate or
may require the release of the glycans from the aglycone.
The next step in the analysis is to release the glycan from the glycoconjugate. When
carbohydrates are released prior to chromatographic profiling, it is necessary to consider (1) the
need for a quantitative release procedure that neither destroys nor structurally alters the
carbohydrate and (2) the loss of information about the way the carbohydrate is attached to the
aglycone.
Release of Oligosaccharides from Glycoconjugates
The glycan moiety of glycosphingolipids can be cleaved enzymatically (by endoglyco-

ceramidase) or chemically (by ozonolysis). The free oligosaccharides are then profiled by HPLC,
HPAEC, HPCE, or FACE. Chemical approaches suitable for release of glycans from a protein
include hydrazinolysis to release both N-glycans and O-glycans or, under controlled conditions,
to cleave selectively only the O-glycans, and β-elimination (alkaline borohydride treatment), a
procedure that under carefully controlled conditions releases only O-glycans. N-linked glycans
can also be released with PNGase F or PNGase A (see Detection of Carbohydrates above). Endo
H may be used for the selective release of high-mannose and hybrid type structures. GPI anchors
are usually cleaved from the protein by proteolysis, deacylated, and analyzed with or without
dephosphorylation and nitrous acid treatment. The oligosaccharides obtained in this manner are
profiled by HPLC. GAGs, because of their large size, are not analyzed intact but rather cleaved
into small fragments by chemical (nitrous acid deamination) or enzymatic (lyase) methods.
Those fragments are identified and quantified by HPLC.
Profiling of Oligosaccharides
Glycans released from a glycoconjugate usually constitute a complex mixture. Even when only
one glycosylation site in the protein is occupied, it can bear different glycan species in individual
molecules. This microheterogeneity accounts for the large number of species obtained.
Chromatographic profiles can be used for comparative studies and to obtain a preliminary
indication of the number, relative quantities, and types of oligosaccharide structures present in a
glycoprotein.
Profiling strategies are chosen based on the quantity of sample available. For large amounts (>50
µg), HPAEC-PAD or other HPLC-based profiling is feasible if the mixture contains about 20 50
glycan species. The HPAEC-PAD profile of the oligosaccharides released from bovine fetuin is
shown in Figure 38.4 as an example. Individual fractions can be recovered for further structural
analysis by MS or NMR. Alternatively, one may wish to increase the sensitivity of the detection,
allowing use of only a small fraction of the glycan mixture for analytical profiling purposes.
Figure 38.4. HPAEC-PAD profile of the N- and O-glycans obtained from bovine fetuin (250 µg)
by automated hydrazinolysis. The glycan mixture was dissolved in water, injected onto a
CarboPac PA-1 anion-exchange HPLC column, and eluted with a linear gradient (20 250 mm)
of sodium acetate in 100 mm sodium hydroxide. The elution positions of the various glycans
correspond to mono- and disialyl O-glycans and a range of mono-, di-, tri- and tetrasialyl N-
glycans. Within a group of glycans with the same charge, differences in elution times reflect
different degrees of branching, and/or a different linkage position and/or branch location of a
sialic acid and/or galactose residue. (The glycan fraction marked by an asterisk was reduced with
borohydride and analyzed by 1H NMR spectroscopy; see Figure 38.5.)
Figure 38.5. 1H NMR spectrum of a mixture of two trisialyl triantennary N-type
oligosaccharide-alditols obtained from bovine fetuin by hydrazinolysis, followed by purification
on HPAEC (see Figure 38.4) and subsequent reduction with sodium borohydride. The spectrum
was recorded at 500 MHz, using a solution of 100 µg of the glycan mixture in 0.7 ml of D2O in a
5-mm NMR probe at pH 6.5 and 23°C. The structures of the two glycans are given in the inset.
Please note that they differ only in the linkage position of sialic acid to galactose in the Manα1
6 branch. The numbers in the spectrum refer to the corresponding residues in the structures.
Assignments are given for the structural-reporter group signals, including those of the anomeric
protons (H-1 signals), the Man H-2, Gal H-3, Neu5Ac H-3eq and H-3ax, and N-acetyl amino
sugar methyl signals; signals marked by an asterisk (for residues 4 and 6 ) refer to the
component with Neu5Ac in α2 3 linkage to Gal-6 . The H-1 signals to the left of the residual
water (HDO) peak are indicative of the presence of α-linked monosaccharides in the glycan(s),
whereas those to the right of HDO originate from β-linked monosaccharide residues in the
structure(s).
Chemical radioactive labeling (e.g., reduction of the reducing end with sodium borotritide or
mild periodate oxidation of the sialic acid glycerol side chain followed by reduction with sodium
borotritide) can be used for enhancement of detection sensitivity. Fluorescent tagging (with 2-
AB or 2-amino-antranillic acid [2-AA]) achieves similar sensitivity, with the advantage that the
cleanup is easier and samples are amenable to a variety of chromatographic separations and
structural analysis techniques. If a label is introduced at the reducing end, it is possible to use
sequential exoglycosidase treatments and look for shifts of the oligosaccharide in a
chromatographic system (e.g., paper chromatography, HPLC, TLC) as an indication of
susceptibility to the enzyme. Comparison with known standards treated in the same manner
allows tentative glycan identification. However, well-characterized standards of known structure
are difficult to obtain in a pure form and there are always "peaks" in a chromatogram that appear
at unusual elution times. It is very important to note that separation technology should not be
confused with actual structural analysis.
Once the number and relative ratios of glycan forms present in a glycoprotein are known, it is
possible to calculate from the analytical chromatography result whether a sufficient quantity of
individual glycans can be prepared to completely identify their structures by physicochemical
approaches (i.e., NMR and MS). Quantity of purified material permitting, the best choice is to
perform 1H NMR analysis first. Because this method is nondestructive, the same sample can
later be used for other, destructive approaches (e.g., MS and methylation analysis). The
limitations on the use of NMR spectroscopy are the cost of equipment and the level of expertise
required for interpreting NMR spectra. However, access to high-field (i.e., 500 MHz and above)
NMR spectrometers fitted with very sensitive probes (e.g., Nano-NMR probes) allows one to
obtain 1H NMR profiles of individual HPLC fractions using minute quantities of sample (2 5
nmoles per oligosaccharide). As an example, the 1H NMR spectrum of a mixture of two trisialyl
triantennary oligosaccharides obtained from bovine fetuin is shown in Figure 38.5.
Linkage Analysis
The structural analysis of a complex carbohydrate is a labor-intensive and time-consuming
endeavor, and multiple iterative methods are often needed to obtain a complete picture of the
structure. A listing of the most common approaches for carbohydrate linkage analysis is
presented in Table 38.4.
Table 38.4. Methods for carbohydrate linkage analysis
Method Derivatization Instrumentation Limit of Expertise Information

and materials detection needed obtained
required
Exoglycosidase depending on specific enzymes fmole-pmole low presence of

digestions quantity range, residues in α or β
available, a depending on linkages to specific
fluroescent tag analytical positions of the
may need to be approach (i.e., underlying
introduced at tagged vs. saccharide: limited
the reducing nontagged to a few enzymes
end glycans) of high specificity
HPLC, HPLTC,
HPCE
instruments
appropriate
detectors
Methylation complete gas-liquid 10 pmoles high type and quantity of
analysis derivatization to chromatograph residues with
produce volatile coupled to a specific positions
compounds mass substituted:
spectrometer identification
with requires correlation
of retention times
and fragmentation
patterns; standards
are required
capillary column sensitivity can ring size (p or f) for
be increased each
to 100 fmoles monosaccharide
with selective
ion
monitoring
MS complete mass fmole-pmole very high linkage type and
derivatization spectrometer range position might be
or tagging of with FAB, inferred from
the reducing LSIMS, or ESI specific
end source and fragmentations
MS/MS across sugar rings
capabilities
NMR not needed NMR anomericity: very high anomericity of each
spectrometer 10 100 pmole monosaccharide
operating at 500 quantities are residue obtained
MHz or above sufficient with from the chemical
microscale shift and coupling
probes (i.e., constant of H-1
Nano-NMR
probe)
linkage linkage positions
position: deduced from two-
nmoles are dimensional
required even HMBC experiment
in microscale
probes
(CI) Chemical ionization; (EI) electron impact; (ESI) electrospray ionization; (FAB) fast-atom
bombardment; (HMBC) heteronuclear multiple-bond correlation; (HPCE) high-per-formance
capillary electrophoresis; (HPTLC) high-performance thin-layer chromatography; (LSIMS)
liquid secondary ion mass spectrometry; (MS) mass spectrometry; (MS/MS) tandem mass
spectrometry.
Determination of Linkage Positions
The next question en route to the complete structural characterization of an isolated

oligosaccharide concerns the linkages of the individual units to one another. Methylation
analysis, an ingenious approach for determining linkage positions, was developed in the late
1960s and early 1970s (Figure 38.6). The principle of this method is to introduce on each free
hydroxyl group of the native oligosaccharide a stable substituent (methyl group). The glycosidic
linkages are then cleaved, producing individual monosaccharide residues with new free hydroxyl
groups that appear at the positions that were previously involved in a linkage. The partially
methylated monosaccharides were originally analyzed by cellulose column chromatography and
identified by a laborious process. Today, the monosaccharides are derivatized to produce volatile
molecules amenable to GLC-MS analysis. The most common derivatization strategy involves
reduction of the monosaccharides to produce alcohols at C-1 (eliminating the formation of ring
structures) followed by derivatization (e.g., acetylation) of free hydroxyl groups to produce
volatile molecules. Individual components of the mixture of partially methylated (methyl groups
mark the hydroxyl groups that were originally free), partially acetylated (acetyl groups mark
hydroxyl groups originally involved in glycosidic linkages and ring structures) monosaccharide
alditols can be identified by GLC-MS.
Figure 38.6. Methylation analysis: A chemical approach to elucidating the glycosidic linkage
positions in glycans.
Traditional MS approaches are based on the fragmentation of organic molecules under some
kind of impact, followed by the differentiation of the resulting particles according to their mass-
to-charge ratio. In methylation analysis, peaks are identified by a combination of their retention
time (relative time of elution from the GLC column) and their electron impact (EI)-MS
fragmentation pattern. The EI fragmentation patterns of similarly substituted monosaccharides of
the same class (e.g., aldohexoses) are the same. Thus, definitive identification requires, in
addition to the analysis of the MS pattern, the comparison of retention times with those of known
standards (i.e., all 2,3,4-tri-O-methyl-hexoses produce the same EI-MS spectrum, but
peracetylated 2,3,4-tri-O-methylgalactitol elutes later than peracetylated 2,3,4-tri-O-
methylglucitol). This type of analysis indicates which residues are terminal (i.e., they are
methylated at every position except OH-1 and OH-5) and how each monosaccharide is
substituted; the latter reveals, inter alia, the occurrence of branching points. Methylation analysis
also allows the determination of the ring size (p or f) for each monosaccharide. However,
methylation analysis neither indicates which residues are attached to each other (i.e., does not
provide sequence information) nor if a particular linkage is of the α or β configuration.
Determination of Anomericity
The next step is to determine the linkage configuration of each residue (anomericity, α or β).
Some information in this regard may already be available from NMR profiling (see above), and
more can be obtained from sequential exoglycosidase digestions. Cleavage by α or β
exoglycosidases indicates the anomericity of specific terminal sugar residues. Cleavage by
specific endoglycosidases can give added information regarding internal regions of the chain.
Some glycosidases will cleave linkages of a given residue only when they are present at a
specific position of the underlying monosaccharide, allowing detailed structural conclusions. At
the present time, the number of such enzymes available is limited.
If sufficient quantities of carbohydrate are available, the anomericity of a particular

monosaccharide residue in a glycan is more reliably determined by 1H NMR spectroscopy. The
anomeric resonances (H-1 signals) appear in a clear region of the spectrum and show
characteristic doublets with a splitting (coupling constant) that is significantly larger for β
anomers than for α anomers (see Figure 38.5). Thus, a first glance of the NMR spectrum
typically indicates how many residues there are (counting the anomeric signals) and how many
of them belong to each anomer type. Since the exact positions (chemical shifts) at which those
H-1 signals appear in the NMR spectrum are more or less characteristic of the type of
monosaccharide and its substitution pattern, more careful study of the spectrum may define
which monosaccharide residues are present and in favorable cases provide the entire primary
structure of the glycan through application of the structural-reporter group concept, especially if
the 1H NMR spectra of well-characterized glycans of related structure are available for
comparison.
Sequence Analysis
A summary of the most common approaches for carbohydrate sequence analysis is presented in
Table 38.5.
Table 38.5. Methods for carbohydrate sequence analysis
Method Derivatization Instrumentation and Limit of Expertise Information obtained

materials required detection needed
MS may or may not mass spectrometer pmole range high sequence deduced
be needed with MALDI source from the mass
depending on and TOF analyzer decrements produced
quantity available by exoglycosidases
complete mass spectrometer pmoles for very high sequence deduced
derivatization with FAB, LSIMS, ESI from the fragmentation
and/or tagging of or ESI source and patterns of daughter
the reducing end MS/MS capabilities ions through several
generations
nmoles for
FAB and
LSIMS
NMR not necessary NMR spectrometer 1 10 nmoles very high sequence may be
(in inferred by comparison
microscale with standards
probes) (structural-reporter
group concept)
10 100 sequence
nmoles (in unambiguously
5-mm deduced from two-
probes) dimensional HMBC
experiment
(MALDI) Matrix-assisted laser desorption ionization; (TOF) time-of-flight. For other

abbreviations, see Table 38.4.
NMR Spectroscopy
NMR spectroscopy is the only technique that has the potential for de novo full structural
characterization of a glycan, with little or no assistance from other methods, provided a sufficient
amount of relatively pure material is available. Complete structural elucidation requires the full
assignment of both the 1H and 13C NMR spectra of the oligosaccharide. This task typically is
accomplished by a combination of two-dimensional NMR techniques such as correlated
spectroscopy (COSY) and total correlation spectroscopy (TOCSY) for 1H, then heteronuclear
single-quantum coherence (HSQC) for 13C. The result of a TOCSY experiment allows
assignment of the 1H signals of individual monosaccharide residues. The key experiment for
sequencing is the two-dimensional heteronuclear multiple-bond correlation (HMBC) experiment,
which detects a coupling between the anomeric proton and the carbon atom on the opposite side
of the glycosidic linkage. The HMBC spectrum allows monosaccharide fragments to be pieced
together by linkages of fully defined position and anomericity. However, in instances where
there is not enough sample for these two-dimensional 1H13C NMR experiments (HMBC is not a
very sensitive technique), data provided by other techniques are required to complete the
structural picture. A less rigorous NMR approach for glycan sequencing relies exclusively on
two-dimensional 1H NMR spectroscopy, using through-space effects (nuclear Overhauser effects
[NOEs]) as sole source of evidence for linking position and sequence.
Mass Spectrometry
The analysis of intact derivatized oligosaccharides using various MS approaches also provides
sequence information (Figure 38.7). Initially, trimethylsilyl and acetyl derivatives of
oligosaccharides were analyzed by GLC-MS (using short columns) and direct-inlet EI-MS.
However, this approach was limited to relatively small glycans; it was replaced by soft ionization
techniques, i.e., fast atom bombardment (FAB)-MS and liquid secondary ion mass spectrometry
(LSIMS), in the late 1970s to early 1980s. These techniques yield molecular ions and only some
fragmentation (at "labile points" such as the glycosidic linkages of hexosamines). The spectra
can be interpreted to obtain some sequence information of carbohydrates much larger than those
amenable to EI- and chemical ionization (CI)-MS. In some instances, sequence information of
native glycoconjugate molecules (e.g., glycosphingolipids, GPIs) can be obtained because their
physical properties make them good producers of ions on the surface of the matrix used for the
analysis. The combined strategy, for linkage and sequence analysis, requires the permethylation
of 1 20 nmoles of starting material, with 1 2 nmoles used for the FAB or LSIMS analysis and
the remainder for GLC-MS. LSIMS or FAB ionization can be combined with two mass
analyzers in tandem (MS/MS). The ions selected with the first mass analyzer are then collided
with a neutral gas in a cell. The collision produces fragmentation (CID, collision-induced
dissociation), and those fragments are analyzed by the second mass analyzer, providing more
detailed structural information. These MS techniques are capable of analyzing mixtures of
oligosaccharides and provide (1) the molecular weight of the oligosaccharides, (2) the
heterogeneity of the sample, (3) structural information, for example, on branching, and (4)
structural information on individual components (using MS/MS).
Figure 38.7. Approaches to analyzing intact, underivatized or derivatized, oligosaccharades
using mass spectrometric methods: A historic perspective. (For abbreviations, see Tables 38.4
and 38.5.)
In laboratories specializing in this technology, MS and MS/MS analyses are becoming the
methods of choice for glycan profiling as well. Molecules are separated according to their mass-
to-charge ratio (m/z), allowing differentiation between oligosaccharides with very similar
properties that are difficult to separate chromatographically. Other derivatization strategies can
be used for CID analysis, such as coupling of the reducing end with nonpolar tags (e.g., the p-
aminobenzoyl ethyl ester [ABEE] and p-aminobenzoyl octyl ester [ABOE] groups).
Newer ionization approaches introduced in the 1990s include electrospray ionization (ESI) and
matrix-assisted laser-desorption ionization (MALDI). Their advantages are higher sensitivity,
high mass measurement capabilities, and the ability in some cases to analyze underivatized
oligosaccharides. Today, ESI-MS has mostly replaced FAB-MS for peptide and glycopeptide
mapping purposes. Although MALDI-time of flight [TOF]-MS still lags behind other MS
approaches in terms of resolution and mass accuracy, its results are far better than those obtained
by SDS-PAGE or SEC. A few femtomoles of a glycoprotein are enough to obtain a global
molecular weight. Furthermore, the MALDI-TOF technique is much less sensitive to the
presence of salts than FAB-MS or LSIMS. Enzymatic incubations can be carried out directly on
the MALDI target, allowing detection of shifts in their mass-to-charge ratio, which indicate how
many glycans are present in a protein. In addition, instrumentation has become more affordable
with the advancement of technology (today, benchtop instruments perform tasks that were
limited to large and expensive magnetic sector mass spectrometers only a few years ago). The
combined use of MALDI mass spectrometry and enzymatic treatments for the sequence analysis
of the N-glycans of bovine fetuin is illustrated in Figure 38.8.
Figure 38.8. MALDI-TOF mass spectra of N-glycan mixtures obtained from 5 µg of bovine
fetuin after treatment at pH 7.5 with (A) PNGase F and neuraminidase; (B) PNGase F,
neuraminidase, and β1 4 galactosidase; (C) PNGase F, neuraminidase, β1 4 galactosidase, and
β-N-acetylglucosaminidase. The m/z values measured for the various peaks are compatible with
the monosodium adducts of oligosaccharides with the given schematic structures (for
explanation of symbolic notation, see Chapter 1).
Mapping of Glycosylation Sites
If each of the glycans isolated from a glycoconjugate is analyzed using the appropriate
combination of approaches, a complete picture of the structures present can be put together. The
ultimate level of glycoprotein primary structure analysis involves the detailed determination of
the particular glycans present at each glycosylation site. This could be accomplished, for
instance, by production and mapping of peptides, identification and preparative isolation of those
peptides containing sugars, and the study of each peak using the methods described above.
Alternatively, current state-of-the-art direct MS analysis makes it possible to accomplish this
task at the level of the intact glycoconjugate.
Future Directions
Historically, the level of detail to which glycan primary structure could be assessed was
determined by the available technology. This trend will likely continue, and eventually new
techniques may emerge that allow total glycan analysis to be performed on intact
glycoconjugates. Eliminating the need for prior isolation of individual glycans will eliminate
selective and nonselective losses and the production of artifacts inherent to degradative
procedures. The contribution from a variety of disciplines will be required for such technologies
to materialize and come to fruition. To put together the pieces of the puzzle provided by primary
structural analysis promises to be as interesting a challenge for the generations to come as it is
for today's carbohydrate chemists.
References
1. Townsend R.R. and Hotchkiss A.T. Jr., eds. 1997. Techniques in glycobiology. Marcel
Dekker, New York.
2. Jackson P. and Gallagher J.T., eds. 1997. A laboratory guide to glycoconjugate analysis.
BioMethods , vol. 9. Birkhäuser, Basel, Switzerland.
3. Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A., Struhl K.,
Albright L.M., Coen D.M., and Varki A., eds. 1996. Preparation and analysis of
glycoconjugates. In Current protocols in molecular biology , unit 17. Wiley, New York.
4. Verbert A., ed. 1995. Methods on glycoconjugates: A laboratory manual. Harwood,

Singapore.
5. El Rassi Z., ed. 1995. Carbohydrate analysis: High performance liquid chromatography and
capillary electrophoresis. J. Chromatogr. Libr. , vol. 58. Elsevier, Amsterdam.
6. Lennarz W.J. and Hart G., eds. 1994. Guide to techniques in glycobiology . Methods Enzymol
., vol. 230. Academic Press, San Diego.
7. Fukuda M., ed. 1994. Glycobiology: A practical approach. IRL Press, Oxford, United
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8. Hounsell E.F., ed. 1993. Glycoprotein analysis in biomedicine. Methods Mol. Biol. , vol. 14.
Humana Press, Totowa, New Jersey.
9. McCloskey J.A., ed. 1990. Mass spectrometry. Methods Enzymol. , vol. 193. Academic Press,
San Diego.
10. Bierman C.J. and McGinnis G.D. 1989. Analysis of carbohydrates by GLC and MS . CRC
Press, Boca Raton, Florida.
11. Chaplin M.F. and Kennedy J.F. 1987. Carbohydrate analysis: A practical approach. IRL
Press, Oxford, United Kingdom.
12. Ginsburg V., ed. 1989. Complex carbohydrates, Part F . Methods Enzymol., vol. 179.
13. Ginsburg V., ed. 1987. Complex carbohydrates, Part E. Methods Enzymol., vol. 138.
14. Ginsburg V., ed. 1982. Complex carbohydrates, Part D. Methods Enzymol., vol. 83.
15. Ginsburg V., ed. 1978. Complex carbohydrates, Part C. Methods Enzymol., vol. 50.
16. Ginsburg V., ed. 1972. Complex carbohydrates, Part B . Methods Enzymol., vol. 28.
17. Rao V.S.R., Qasba P.K., Balaji P.V., and Chandrasekaran R. 1998. Conformation of
carbohydrates. Harwood, Singapore.
39. Chemical and Enzymatic Synthesis of Glycans
Primary contributions to this chapter were made by O. Hindsgaul (University of Alberta,
Canada, and The Burnham Institute, La Jolla, California).
THIS CHAPTER ADDRESSES THE SYNTHESIS of oligosaccharides on a preparative scale

(greater than milligram quantities) in the laboratory. Both organic chemical and enzymatic
approaches are presented. Emphasis is placed on the scope and limitations of the existing
methods, and an effort is made to provide an understanding of the amount of experimental work
required to produce well-defined glycan samples for biological studies.
Background (1 3)
The interest of organic chemists in the synthesis of oligosaccharides arises from the challenge of
stereospecifically assembling large highly functionalized complex molecules in an efficient
manner. The interest of glycobiologists, on the other hand, is in obtaining samples of pure well-
characterized oligosaccharides for use in biological studies. Such studies almost always probe
the role of a glycan in some biological recognition process such as the binding of bacteria,
toxins, or viruses to mammalian cell surface glycans or the specific recognition of a glycoprotein
or glycolipid by a cell surface receptor. The glycans in question are often very large, but the
protein-combining sites tend to be more limited in size and usually recognize epitopes presented
by as few as 1 4 sugar units. For this reason, this chapter focuses on the synthesis of small
oligosaccharides.
Mammalian glycans are formed from only ten monosaccharides, which are considered the
"building blocks" (see Chapter 2). These are d-Glc, d-Gal, d-Man, d-Xyl, l-Fuc, d-GlcNAc, d-
GalNAc, d-GlcA, l-IdoA, and Neu5Ac. Additional structural diversity can be present in glycans
through acylation, sulfation, and phosphorylation. Each of these monosaccharides can in
principle form glycosidic linkages with either the α or β configuration, although not all of the
possible anomers have been discovered. In bacteria, a seemingly unending list of
monosaccharides that includes deoxy and amino sugars is combined to produce highly complex
and synthetically challenging molecules (see Chapter 21).
Oligosaccharide Synthesis Is More Difficult Than DNA or

Peptide Synthesis (4 5)
The challenge of oligosaccharide synthesis is easily appreciated by comparing it with the much
more developed areas of DNA and peptide synthesis, which are commonly performed on the
solid phase using commercial instruments.
The general scheme used in the solid-phase synthesis of DNA oligomers is shown in Figure 39.1.
A partially protected first nucleoside is immobilized by attachment to a solid support through its
3 -OH group. Its 5 -dimethoxytrityl group is then cleaved under mildly acidic conditions. An
activated form of the next nucleoside, usually a 3 -phosphite derivative, is then added to make
the 3 5 phosphite. The phosphite is then oxidized to the phosphate, usually with iodine in
pyridine/water, and the phosphodiester linkage is completed. Removal of the dimethoxytrityl
group then allows the cycle to be repeated with the next nucleoside derivative. The key features
of the chemistry are that all of the required derivatized nucleosides are commercially available or
are prepared in two to three steps. The time for cleavage of the dimethoxytrityl group is 3
minutes, that for phosphite coupling is 5 minutes, and that for oxidation is 2 minutes. Because all
of the nucleosides couple at the same rate, there is no need to monitor each reaction. The total
cycle time is under 20 minutes and the yield is near 99%, and thus many cycles can be
performed. Clearly, the chemistry has been extensively developed and is extremely efficient,
simplified by the fact that there are only two OH groups involved a nonhindered primary 5 -
OH group and a secondary 3 -OH group and the linkage is always between the 3 -OH on one
residue and the 5 -OH on the next residue. The final oligonucleotide is obtained after cleavage
from the solid support (usually glass) by base treatment, which also removes the protecting
groups on the nucleoside base and on the phosphodiester.
Figure 39.1. Synthetic protocol for the solid-phase synthesis of DNA oligomers (A) and peptide
oligomers (B). (DMT) Dimethoxytrityl; (B) base (e.g., uracil); (Fmoc)
fluorenemethyloxycarbonyl.
A general scheme for the solid-phase synthesis of peptides is shown in Figure 39.1. The most
commonly used protocol involves amino acids that have their side-chain functional groups
(amines, carboxylates, etc.) protected with acid-labile protecting groups (such as Boc groups)
and the amino acid nitrogen protected with a base-labile Fmoc group. In the "normal direction,"
the Fmoc-protected carboxy-terminal amino acid is coupled to a solid support through its
carboxyl group using an acid-labile linker. The Fmoc-protecting group is cleaved with a base,
e.g., morpholine, to expose a free reactive amine. The next amino acid is then added, either as a
preactivated ester (such as a pentafluorophenyl ester) or as the free acid in the presence of a
condensing agent such as a carbodiimide. Catalysts are often added, and the completion of the
reaction can be monitored colorimetrically for the disappearance of the basicity of unreacted
amino groups. When the reaction is complete, the Fmoc group of the newly added amino acid is
cleaved and the next cycle is initiated. Unlike the case of DNA synthesis, different amino acids
can react at different rates, and some sequences are particularly problematic, which is the reason
colorimetric monitoring is desirable. With modern techniques, racemization is virtually
eliminated, but reaction yields for the formation of the amide bonds are more commonly under
97%, and side reactions occur at the level of a few percent. In practice, a cycle can take from one
to several hours, and after cleavage from the resin by acid treatment, which also cleaves the side-
chain-protecting groups, the purity of the product peptide must be evaluated by analytical
chromatography. Preparative HPLC is generally performed as a final purification step. Synthetic
peptide synthesis is generally considered efficient for up to about 30 cycles.
All of the required natural protected nucleosides and Fmoc-protected amino acids for DNA and
peptide synthesis are commercially available, as are the prepackaged reagents, solvents, and
instruments that add reagents using pumps, which perform the washing and cleavage steps. The
synthesis of DNA and peptides is therefore common in nonchemical laboratories.
Complexities and Challenges in the Chemical Synthesis of

Oligosaccharides (1 3)
The above two cases contrast sharply with that of the chemical synthesis of oligosaccharides,
which is far more complex. The differences are outlined in Figure 39.2, which shows the
synthesis of a simple disaccharide made up of two glucose units. The mammalian
monosaccharide building blocks all have either three or four OH groups. If a specific
disaccharide sequence, such as a 1 4-linkage, is required, the remaining OH groups must be
protected. Once this has been accomplished, the second sugar (the glycosyl donor) is added in an
activated form, with a leaving group "L" at the anomeric center. The glycosyl donor then couples
with the glycosyl acceptor having a single free-OH group. Formation of the glycosidic linkage
creates a new asymmetric center which can have either the α or β configuration. Clearly,
mixtures are not wanted; therefore, stereospecific methods are required to produce the correct
anomer of the target disaccharide.
In normal cases, a glycosylation reaction takes 2 48 hours. Individual sugars can react at very
different rates and give a very different ratio depending on the free-OH group that is being
glycosylated. Yields are generally in the 60 80% range, with anomeric ratios rarely better than
20:1 and often 6:1 or worse. With such yields, chromatographic purification is essential at each
step. Even more severe limitations exist if longer oligosaccharides are required. As seen in
Figure 39.2, after the second sugar has been added to the first, seven potential sites are available
for the addition of the next sugar to form a trisaccharide. In the case shown, a single generic
protecting group "P" was used (for simplicity of presentation) such that no single OH group in
the product disaccharide can be selectively removed for chain extension. If the next sugar must
be added to the 2 -OH group, for example, then a glycosyl donor with its 2-OH group
differentially protected must be used as the glycosyl donor. In other words, four differentially
protected acceptors and four differentially protected donors must be prepared to be able to make
each of the possible triglucosides. The situation clearly gets much more complex if a
tetrasaccharide is envisioned and other monosaccharides are also involved. Very few of the
required building blocks are commercially available.
Figure 39.2. Synthetic protocol for a hypothetical Glc1-4Glc disaccharide. P is a generic
hydroxyl-protecting group.
The challenge of oligosaccharide synthesis then reduces to the requirement for elaborate OH
group protection strategies and the need for stereospecific methods for glycosylation. As noted
above, even in well-precedented systems, the yields and α/β ratios are poor by comparison with
that of DNA and peptide synthesis, and they are also difficult to predict in new sequences. It is
for these reasons that only 18 papers have appeared to date reporting the solid-phase synthesis of
oligosaccharides (for review, see Reference 3).
A Representative Trisaccharide Synthesis (6)

Figure 39.3 shows the synthetic scheme followed for the preparation of a glycosyltransferase
acceptor that is used to measure the activity of the metastasis-associated enzyme N-
acetylglucosaminyltransferase-V (see Chapters 17 and 35) in crude tissue extracts. This
represents a highly efficient solution synthesis of a trisaccharide in a very well precedented
system and is presented so that the reader can get a feel for an actual oligosaccharide synthesis
procedure. The scheme requires 17 steps from commercially available starting materials; 8
intermediate compounds require purification by chromatography and typically 100 mg of the
final compound is obtained. Initially, the development of the synthetic scheme required about 3
months of work for an experienced postdoctoral fellow. To repeat the sequence now takes about
1 month of full-time work.
Figure 39.3. Synthetic scheme used for the preparation of trisaccharide that is used in a
glycosyltransferase assay.
Enzymatic Synthesis of Oligosaccharides (7 9)
The very high current level of interest in using enzymes to synthesize oligosaccharides on a
preparative scale is in direct response to the severe limitations still present in the chemical
approaches. Both glycosyltransferases (the enzymes that biosynthesize the oligosaccharides) and
glycosidases (the enzymes that hydrolyze them) have been used. The attractiveness of enzymatic
synthesis is that protecting groups are not required and that stereochemically defined glycosidic
linkages, not α/β mixtures, are always formed.
Synthesis of Oligosaccharides Catalyzed by Glycosidases (10 12)
Glycosidases (see Chapter 18) were used well ahead of glycosyltransferases (see Chapter 17) for
the preparative synthesis of oligosaccharides. This is because these enzymes are stable and easy
to isolate, and they were therefore more generally available. Glycosidases normally catalyze the
hydrolysis of glycosidic linkages and must therefore be "coaxed" into providing useful yields of
oligosaccharides.
Most frequently, glycosidases are used in "transglycosylation" reactions where they transfer a
monosaccharide from another readily available and inexpensive glycoside. For some
glycosidases, the reactions are very regiospecific, but for others, mixtures are obtained. Figure
39.4 illustrates the use of lactose as the "glycosyl donor" for three different β-galactosidases that
have different specificities. The enzymes cleave the terminal β-galactosidase residues from
lactose, but, under appropriate experimental conditions involving high concentrations of added
acceptors, yields of more than 20% of new Gal GlcNAc disaccharides can be obtained.
Hydrolysis (transfer to water) always competes and thus limits the yields, but all of the new
disaccharides have exclusively the β configuration.
Figure 39.4. Examples of the use of β-galactosidases in transglycosylation reactions to produce

disaccharides on a preparative scale. (a) Enzyme from bovine testes; (b) enzyme from
Lactobacillus bifidus; (c) enzyme from Escherichia coli.
Another strategy used to increase yields in glycosidase-mediated oligosaccharide synthesis is to

use p-nitrophenyl glycosides as thermodynamically high-energy donors. This is illustrated in
Figure 39.5 where a β-hexosaminidase was used to synthesize the very linkage it normally
hydrolyzes. Transfer of GlcNAc, from its p-nitrophenyl glycoside to free GlcNAc, resulted in a
55% yield of the 1-4-linked chitobiose derivative and a 22% yield of the 1-6-linked disaccharide.
This reaction also illustrates the main limitation of glycosidase-catalyzed oligosaccharide
synthesis: Mixtures of positional isomers are frequently, and unpredictably, obtained. In fact, this
lack of regioselectivity has been used in the generation of oligosaccharide libraries using
glycosidases.
Figure 39.5. Example of a single glycosidase producing two different disaccharides on
transglycosylation using a p-nitrophenyl glycoside as the donor.
Synthesis of Oligosaccharides Using Glycosyltransferases (7 9,13 14)
Glycosyltransferases transfer monosaccharide residues from their activated forms, the sugar
nucleotides, to growing oligosaccharide chains (Figure 39.6). Since oligosaccharides are
biosynthesized by glycosyltransferases in vivo, it stands to reason that this class of enzymes can
be used also for their in vitro preparative synthesis. This requires access to both the sugar
nucleotides and the enzymes themselves, and this has until recently been a major impediment.
The mammalian sugar nucleotides are UDP-Glc, UDP-Gal, UDP-Xyl, UDP-GlcNAc, UDP-
GalNAc, UDP-GlcA, GDP-Man, GDP-Fuc, and CMP-Neu5Ac, and each exists in only one
anomeric form. The sugars can be transferred with either retention or inversion of the anomeric
configuration, inversion being the more common.
Figure 39.6. Use of two sequential glycosyltransferase reactions in the synthesis of the Lewis X
(LeX) trisaccharide.
All of the mammalian sugar nucleotides are now commercially available, although they are
usually expensive. For very large scales where economy is of concern, the sugar nucleotides can
be produced in situ using very elegant enzyme recycling systems beginning with the free sugars
themselves and high-energy drivers such as phosphoenol pyruvate (see Chapter 41). Until
recently, only a bovine β1-4 GalT was commercially available in amounts sufficient to prepare
milligram quantities of oligosaccharide (Figure 39.6). The tremendous progress made in the
cloning of glycosyltransferases, however, has now yielded the sequences of scores of enzymes,
and several recombinant galactosyltransferases, fucosyltransferases, and sialyltransferases have
appeared on the market.
For glycosyltransferase-assisted oligosaccharide synthesis, the acceptor, donor, and enzyme are
simply mixed in a buffer where the enzyme activity is at its optimum. Manganese ions are
frequently required. The product oligosaccharide that is formed is controlled by the specificity of
the enzyme so that protection is not required. The reaction also produces a nucleotide by-
product, e.g., UDP in the reaction of a β1-4 GalT (Figure 39.6). The nucleotide by-product
usually inhibits the enzyme, and alkaline phosphatase is sometimes added to destroy it.
Most glycosyltransferases do not act on simple monosaccharides but, as in vivo, require more
elaborate structures for recognition and transfer, and the enzymes have a specified order of
addition. For example, the Lewis X trisaccharide (Figure 39.6) must be prepared first by the
addition of galactose to GlcNAc. Only then can the fucose residue be added to the GlcNAc
residue.
Future Prospects (14 21)
The move toward solid-phase synthesis is long overdue. Despite the tremendous problems ahead,
new methods for solid-phase synthesis are slowly appearing that are truly different and hold
promise for simplifying oligosaccharide assembly. These methods should become sufficiently
robust to permit the assembly of oligosaccharide and other carbohydrate libraries, an area of
research that is in its infancy.
Numerous examples have appeared describing the use of glycosyltransferases in the synthesis of
natural oligosaccharide sequences. The enzyme specificities are sometimes relaxed, and with
sufficient enzyme available, analogs of the natural oligosaccharides can also be produced,
particularly those containing deoxy sugars. The limitation of glycosyltransferases in synthesis
will be their inability to prepare sequences not found in nature. The use of glycosyltransferases
to "cap-off" oligosaccharides immobilized on the solid phase promises to further speed up the
preparation of biologically active glycans.
References
1. H. Paulsen. 1982. Advances in selective chemical synthesis of oligosaccharides Angew. Chem.
Int. Ed. Engl. 21: 155-173.
2. Khan S.H. and Hindsgaul O. 1994. Chemical synthesis of oligosaccharides. In Molecular

glycobiology: Frontiers in molecular biology (ed. Fukuda M. and Hindsgaul O.), pp. 206 223.
Oxford University Press, Oxford, United Kingdom.
3. Y. Ito. 1998. Solid phase oligosaccharide synthesis and related technologies Curr. Opin.
Chem. Biol. 2: 701-708. (PubMed)
4. M.H. Caruthers. 1991. Chemical synthesis of DNA and DNA analogues Accts. Chem. Res. 24:
278-284.
5. D.A. Wellings and E. Atheton. 1997. Standard Fmoc protocols Methods Enzymol. 289: 44-67.
(PubMed)
6. O. Kanie, S.C. Crawley, M.M. Palcic, and O. Hindsgaul. 1994. Key involvement of all three
GlcNAc hydroxyl groups in the recognition of β-D-GlcpNAc-1-2-α-D-Manp-1-6-β-D-Glcp-OR
by N-acetylglucosaminyl-transferase-V Bioorg. Med. Chem. Lett. 2: 1231-1241. (PubMed)
7. W. Klaffke and O. van Noortlaan. 1994. Application of enzymes in the synthesis of

saccharides and activated sugars Carbohydr. Europe 10: 9-17.
8. C.H. Wong, R.L. Halcomb, Y. Ichikawa, and T. Kajimoto. 1995. Enzymes in organic
synthesis: Application to the problems of carbohydrate recognition (part 1) Angew. Chem. Int.
Ed. Engl. 34: 412-432.
9. C.H. Wong, R.L. Halcomb, Y. Ichikawa, and T. Kajimoto. 1995. Enzymes in organic
synthesis: Application to the problems of carbohydrate recognition (part 2) Angew. Chem. Int.
Ed. Engl. 34: 521-546.
10. Nilsson K.G.I. 1996. Synthesis with glycosidases. In Modern methods in carbohydrate
synthesis (ed. Khan S.H. and O Neill R.A.), pp. 518 547. Harwood Academic Press.
11. S. Singh, J. Packwood, C.J. Samuel, P. Critchley, and D.H.G. Crout. 1995. Glycosidase-
catalysed oligosaccharide synthesis: Preparation of N-acetylchitooliogsaccharides using the β-N-
acetylhexosaminidase of Aspergillus oryzae Carbohydr. Res. 279: 293-305. (PubMed)
12. T. Murata and T. Usui. 1997. Preparation of oligosaccharide units library and its utilization
Biosci. Biotechnol. Biochem. 61: 1059-1066. (PubMed)
13. P. Sears and C.H. Wong. 1998. Enzyme action in glycoprotein synthesis Cell. Mol. Life Sci.
54: 223-252. (PubMed)
14. M.M. Palcic and O. Hindsgaul. 1996. Glycosyltransferases in the synthesis of

oligosaccharide Analogs. 1996 Trends Glycosci. Glycotechnol. 8: 37-49.
15. S.J. Danishefsky, K.F. McClure, J.T. Randolph, and R.B. Ruggeri. 1993. A strategy for the
solid phase synthesis of oligosaccharides Science 260: 1307-1309. (PubMed)
16. Y. Ito, O. Kanie, and T. Ogawa. 1996. Orthogonal glycosylation strategy for rapid assembly
of oligosaccharides on a polymer support Angew. Chem. Int. Ed. Engl. 35: 2510-2512.
17. Y. Ito and T. Ogawa. 1997. Intramolecular aglycon delivery on polymer support: Gatekeeper
monitored glycosylation J. Am. Chem. Soc. 119: 5562-5566.
18. O. Kanie, F. Barresi, Y. Ding, J. Labbe, A. Otter, L.S. Forsberg, B. Ernst, and O. Hindsgaul.
1995. A strategy of "random-glycosylation" for the production of oligosaccharide libraries
Angew. Chemie 34: 2720-2722.
19. Y. Ding, O. Kanie, J. Labbe, M.M. Palcic, B. Ernst, and O. Hindsgaul. 1995. Synthesis of
biological activity of oligosaccharide libraries Adv. Exp. Med. Biol. 376: 261-269. (PubMed)
20. R. Liang, L. Yan, J. Loebach, M. Ge, Y. Uozumi, K. Sekanina, N. Horan, J. Gildersleeve, C.

Thompson, A. Smith, K. Biswis, W.C. Still, and D. Kahne. 1996. Parallel synthesis and
screening of a solid phase carbohydrate library Science 274: 1520-1522. (PubMed)
21. O. Blixt and T. Norberg. 1998. Solid-phase synthesis of a sialyl LeX tetrasaccharide on a
Sepharose matrix J. Org. Chem. 63: 2705-2710. (PubMed)
40. Natural and Synthetic Inhibitors of Glycosylation
Diego).
INHIBITORS OF GLYCOSYLTRANSFERASES and glycosidases provide important tools for

studying glycosylation in cells, tissues, and whole organisms. This chapter discusses various
types of inhibitors, including natural products, substrate-based tight-binding inhibitors, glycoside
primers, and one example of a rationally designed inhibitor. The use of these compounds for
studying reaction chemistry and glycoconjugate biology and their use as potential therapeutics
are also covered.
Introduction
Chapters 31 33 described various natural and induced mutants with defects in glycosylation.
These genetic experiments have helped define genes encoding various transferases and
glycosidases, and in some cases, alternate biosynthetic pathways have been uncovered. Mutants
also provide insights into the function of glycosylation in cells and tissues as well as models for
human in-born errors in metabolism and disease. However, one limitation of studying mutants is
that the analyses are usually restricted to the cell or organism from which the mutant strain was
isolated. In addition, many mutations are lethal in animals, which makes the study of the gene in
adult animals more difficult.
Inhibitors provide another approach for studying glycosylation that avoids some of the problems
associated with studying mutants. Many of these compounds are small molecules that are taken
up readily by a variety of cell types, thus circumventing the cumbersome task of isolating similar
mutants from different organisms or cells. Some inhibitors block glycosyltransferases (e.g.,
substrate analogs), whereas others act on processing enzymes (e.g., the alkaloids). Some act on
central steps in metabolism (e.g., chlorate blocks the formation of PAPS) and therefore have
pleiotropic effects on different pathways of glycan synthesis. Others are highly specific and
affect only a single enzyme reaction (e.g., neuraminidase inhibitors). Some of the compounds
can be absorbed through the gut, which provides an opportunity for designing drugs to treat
human diseases and disorders correlated with altered glycosylation.
Glycosylation inhibitors arise naturally in plants, fungi, and bacteria, probably as part of a
chemical defense strategy against other competing organisms in the same econiche. Other
compounds have been synthesized based on the known substrate preferences for a particular
enzyme or its X-ray crystal structure. In either case, inhibitors act as leads for producing
additional analogs, often with increased activity or altered specificity.
This chapter describes several classes of inhibitors that act on glycosyltransferases and
glycosidases in vertebrate cells (Table 40.1). Because the field is quite large, it is impossible to
review all of the literature in the area. Therefore, only those inhibitors that act on specific
enzymes or metabolic pathways and that illustrate certain basic concepts are discussed. Agents
that block protein/carbohydrate interactions are discussed in Chapter 41 and elsewhere. Chapter
21 briefly discusses the aminoglycoside antibiotics.
Table 40.1. Classes of inhibitors
Class of Target
inhibitor
Metabolic steps involved in formation of common intermediates such as PAPS or

inhibitors nucleotide sugars
Tunicamycin N-linked glycosylation through inhibition of Dol-PP-GlcNAc formation;
peptidoglycan biosynthesis through inhibition of undecaprenyl-PP-GlcNAc
assembly
Plant alkaloids N-linked glycosylation through inhibition of processing glycosidases
Substrate specific glycosyltransferases or glycosidases
analogs
Glycoside glycosylation pathways by diverting the assembly of glycans from endogenous
primers acceptors to exogenous primers
Indirect Inhibitors and Metabolic Poisons (1 15)
A number of inhibitors have been described that block glycosylation by interfering with
metabolism of common precursors or by interfering with intracellular transport activities. Some
of these compounds act indirectly by interfering with the transit of proteins between the ER,
Golgi, and trans-Golgi network (e.g., the fungal metabolite, Brefeldin A). Brefeldin A causes
retrograde transport of Golgi components located proximal to the trans-Golgi network. Thus,
treating cells with Brefeldin A separates enzymes located in the trans-Golgi network from those
found in the ER and Golgi and uncouples the assembly of the core structures of some glycans
from later reactions, such as sialylation or sulfation. In proteoglycan biosynthesis, Brefeldin A
blocks the formation of chondroitin sulfate entirely but allows some residual heparan sulfate
synthesis to continue, suggesting that the enzymes involved in heparan sulfate formation spread
throughout the Golgi and the trans-Golgi network, whereas those involved in chondroitin
synthesis are almost entirely located in the trans-Golgi network. This approach provides a
snapshot of the particular system under study. Care should be taken in extrapolating the effects
of Brefeldin A in one system to another, because the localization of the enzymes varies
considerably in different cell types.
Some inhibitors act at key steps in intermediary metabolism where precursors involved in
glycosylation are formed. For example, a glutamine analog, 6-diazo-5-oxo-l-norleucine, blocks
glutamine:fructose-6-phosphate amidotransferase, the enzyme that forms glucosamine from
fructose and glutamine (see Chapter 6). Depressing glucosamine production in this way has a
pleiotropic effect on the assembly of glycans, since all of the major families of glycans contain
GlcNAc or GalNAc. The effect of 6-diazo-5-oxo-l-norleucine on glycosylation can be partially
reversed by feeding cells glucosamine or by the salvage of glucosamine from the turnover of
glycoprotein and proteoglycans.
Chlorate is another type of general inhibitor that blocks sulfation. The chlorate anion (ClO42-) is
an analog of sulfate (SO42-) and forms an abortive complex with the sulfurylase involved in the
formation of phosphoadenosine-5 -phosphosulfate, the active sulfate donor for all known
sulfation reactions. Thus, treating cells with chlorate (usually 10 30 mm) inhibits sulfation by
more than 90%, but the effect is not specific for any particular class of glycan or sulfation
reaction (e.g., tyrosine sulfation is also affected). The effect of chlorate can be overcome with
inorganic sulfate, consistent with its being a competitive inhibitor.
A number of sugar analogs have been made with the hope that they might exhibit selective
inhibition of glycosylation. 2-deoxyglucose and fluorinated analogs of sugars (3-deoxy-3-fluoro-
and 4-deoxy-4-fluoroglucosamine, 2-deoxy-2-fluoro-glucose, and 2-deoxy-2-fluoro-mannose)
inhibit glycoprotein biosynthesis as measured by incorporation of labeled [3H]glucosamine or
[3H]fucose, but the mechanism underlying the inhibitory effect is unclear in many cases. Early
studies of 2-deoxyglucose showed that the analog was converted to UDP-2-deoxyglucose as well
as to GDP-2-deoxyglucose and dolichyl-P-2-deoxyglucose. Inhibition of glycoprotein formation
occurs apparently due to accumulation of various dolichol oligosaccharides containing 2-
deoxyglucose, which cannot be elongated or transferred to glycoproteins normally.
Unfortunately, many of these compounds may have other effects as well due to their conversion
into other reactants through intermediary metabolism.
Several of the glycosyltransferases will tolerate adducts appended to the donor nucleotide sugar.
For example, one of the α3-fucosyltransferases will transfer a fucose analog on GDP containing
bulky substituents (even another sugar!) at the C6 position. This observation makes it possible to
modify the structure of oligosaccharides on the surface of a cell with purified enzymes and
synthetic donors. Another approach consists of using mannosamine analogs in which an acyl,
alkyl, or ketone group is covalently linked to the amino group in place of acetate (Figure 40.1).
H H
The mannosamine analogs are incorporated into the nucleotide precursor pools and end up in
novel sialic acid analogs on cell surface glycoproteins and glycolipids. When ketone-containing
derivatives are incorporated, the cell surface glycoconjugates become sensitive to reagents that
react with carbonyl groups.
Figure 40.1. Mannosamine analogs. Several derivatives of mannosamine containing different

functionalities linked to the amino group are taken up by cells, converted to the corresponding
derivative of CMP-sialic acid, and transferred to glycoproteins and glycolipids.
Tunicamycin: Inhibition of DOL-PP-GlcNAc Assembly (4, 6,16 H H H H

22)
H
Tunicamycin was first identified in Streptomyces lysosuperificus, and related compounds were
found later in other microorganisms. Tunicamycin derives its name from its antiviral activity,
which was due to inhibition of viral coat (or "tunica") formation. Tunicamycin belongs to a class
of nucleoside antibiotics composed of uridine, an 11-carbon disaccharide called
aminodeoxydialdose (tunicamine; Figure 40.2), and a fatty acid of variable length (13 17
H H
carbons), branching, and unsaturation.
Figure 40.2. Structure of tunicamycin. Tunicamycin consists of uridine conjugated to the

dialdose, tunicamine. The compound is part of a family of related agents that vary in the length,
branching, and degree of unsaturation of the fatty acid amide (R) linked to tunicamine.
Tunicamycin inhibits N-glycosylation in eukaryotes by blocking the transfer of GlcNAc-1-P

from UDP-GlcNAc to dolichyl-P (catalyzed by GlcNAc phosphototransferase, GPT), thereby
decreasing dolichyl-PP-GlcNAc. Other GlcNAc transferase reactions are not inhibited (e.g.,
GlcNAcTI-V; see Chapters 7 and 16), but the transfer of GlcNAc-1-P to undecaprenyl-P and the
H H H H
formation of undecaprenyl-PP-MurNAc pentapeptide involved in bacterial peptidoglycan

biosynthesis are sensitive to tunicamycin (see Chapter 21). Tunicamycin acts as a tight binding
H H
competitive inhibitor presumably because it resembles the donor nucleotide sugar. Indeed, the Ki
value for tunicamycin is about 5 × 10-8m, whereas the Km value for UDP-GlcNAc is
approximately 3 × 10-6m. Another compound called amphomycin inhibits
mannosylphosphoryldolichol synthesis by forming a complex with dolichylmonophosphate.
However, this compound is a lipopeptide and apparently forms complexes with the glycosyl-
carrier lipid, preventing its participation in the enzymatic reaction.
The actual amount of tunicamycin needed to inhibit glycosylation varies in different cells (0.1
10 µg/ml), possibly due to variable uptake and culture conditions or differences in the level of
expression of the phosphotransferase. Tunicamycin is cytotoxic to cells, and resistant mutants
overproduce GPT. Similarly, transfection of cells with the cloned GPT confers resistance,
suggesting that the variable dose of inhibitor required in different cells may reflect variation in
enzyme levels.
Tunicamycin has been used extensively for studying the role of N-glycans in glycoprotein
maturation, secretion, and function, as documented by more than 2500 papers citing its use since
its first discovery in 1973. Recently, the drug was shown to induce apoptosis preferentially in
cancer cells, presumably due to alterations in glycosylation of various cell surface receptors and
signaling molecules. Although the mechanism underlying its apoptosis-inducing activity may be
complex, the observation suggests the possibility of a chemotherapeutic treatment based on
inhibiting glycosylation (see Chapter 35).
H H
Plant Alkaloids: Natural Inhibitors of Glycosidases (4, 6,23 H H H H
32)
H
Plant alkaloids block glycosylation by inhibiting the processing glycosidases involved in N-

glycan formation (Table 40.2). Unlike tunicamycin, which blocks glycosylation of glycoproteins
H H
entirely, the alkaloids inhibit the trimming reactions that occur after the Glc3Man9GlcNAc2
oligosaccharide is attached to a glycoprotein (see Chapter 7). Thus, the inhibitory alkaloids
H H
generally do not interfere with protein folding, but the glycoproteins that appear on the cell
surface lack the characteristic termini found on mature oligosaccharides.
Table 40.2. Examples of alkaloids that inhibit glycosidases involved in N-
linked glycan biosynthesis
One class of alkaloids inhibits the α-glucosidases involved in the initial processing of the N-
glycans and in quality control of protein folding (see Chapter 22). This class includes
H H
castanospermine (from the seed of the Australian chestnut tree, Castanosperum australe), which
inhibits both α-glucosidases I and II; australine (also from C. australe), which preferentially
inhibits α-glucosidase I; and deoxynojirimycin (from Streptomyces species), which preferentially
inhibits α-glucosidase II. As expected, castanospermine and australine cause accumulation of
fully glucosylated chains, whereas deoxynojirimycin results in chains containing one to two
glucose residues. Treating cells with these inhibitors revealed that some trimming of the
mannose residues can occur independently of removal of the glucose residues (see Chapter 7).
H H
Swainsonine was first discovered in plants from the western United States (Astragalus species,
aka locoweed) and Australia (Swainsona canescens), and it was later found in the fungus
Rhizoctonia leguminocola that infects red clover. Consumption of these plants causes a severe
abnormality called locoism, which includes accumulation of glycoproteins in the lymph nodes.
Thus, swainsonine is part of a chemical defense strategy used by plants against grazing animals
(and probably insects). Swainsonine inhibits α-mannosidase II, causing the accumulation of
high-mannose oligosaccharides (Man4GlcNAc2 and Man5GlcNAc2) and hybrid-type chains at the
expense of complex oligosaccharides. It also inhibits the lysosomal α-mannosidase. Mannostatin
A works in a similar way, but differs significantly in structure from swainsonine (Table 40.2).
H H
Other mannosidase inhibitors include deoxymannojirimycin and kifunensin, which selectively

inhibit α-mannosidase I. As expected, these agents cause the accumulation of Man7 9GlcNAc2
oligosaccharides on glycoproteins.
All of these inhibitors have in common polyhydroxylated ring systems that mimic the orientation
of hydroxyl groups in the natural substrates, but a strict correlation between stereochemistry and
enzyme target (α-glucosidase vs. α-mannosidase) does not exist. The compounds contain
nitrogen, usually in place of the ring oxygen. One idea is that the nitrogen in the protonated state
may mimic the positive charge on the ring oxygen that arises from delocalization of charge from
the tentative carbocation at C1 generated during the hydrolysis reaction. Although no crystal
structures for the processing glycosidases are available, the structure of the glucoamylase that
removes glucose residues from starch has been solved with and without deoxynojirimycin in the
active site. The crystal structure shows a glutamate residue that forms a salt bridge with the
protonated form of the ring nitrogen. Thus, the relatively low pK of the amino group makes the
substrate look like the transition state of the hydrolytic reaction.
Alkylated and acylated analogs of the alkaloids have been made and shown to have interesting
and useful properties. N-butylation of deoxynojirimycin actually converts the glucosidase
inhibitor into an inhibitor of glycolipid biosynthesis, but the mechanism of inhibition is
unknown. Alkylation of the amino group or acylation of the hydroxyl groups can raise the
potency of the compound, presumably by facilitating uptake across the plasma and Golgi
membranes. Some of these compounds have shown positive effects as drugs for treating
diabetes, cancer, and HIV infection. N-hydroxyethyl-deoxynojirimycin (Miglitol ) inhibits the
disaccharidases in the intestine involved in digestion of sucrose and other dietary sugars, thus
lowering the amount that is available for absorption and consequently decreasing blood sugar
levels. Swainsonine and its carbonoyloxy analogs have potent antitumor activity in mice and
humans, suggesting that these inhibitors may provide novel chemotherapeutic approaches for
treating cancer.
Substrate Analogs: Directed Synthesis of Inhibitors (33 H
44)
H
A number of inhibitors have been developed based on the concept that substrate analogs might
act as tight-binding inhibitors of specific transferases. Analogs of acceptors have the advantage
that they should be more selective for specific enzymes than nucleotide sugar donors, which are
used by multiple transferases. The general strategy is to modify the acceptor hydroxyl group or
hydroxyls in the immediate vicinity (Table 40.3). About half of the compounds that have been
H H
made lack inhibitory activity, presumably because removal or alkylation of the targeted hydroxyl
group prevents binding of the analog to the enzyme. In other cases, the analogs exhibit Ki values
in the approximate range of the Km values for the parent substrate. As one might expect, the
analogs usually act competitively with respect to the unmodified substrate, but in a few cases, the
inhibition pattern was more complex, suggesting possible binding outside the active site.
Table 40.3. Synthetic substrate-based inhibitors of glycosyltransferases
In addition to acceptor analogs, a number of nucleotide sugar derivatives have been made.
Several of these exhibit strong inhibitory activity, but because they contain the negatively
charged pyrophosphate linkage between the nucleoside and the sugar, they cannot cross the
plasma membrane and therefore lack activity in vivo. "Bisubstrate" analogs consist of the
nucleoside sugar donor covalently linked to the acceptor substrate by way of a neutral bridging
group. This arrangement potentially generates inhibitors whose binding characteristics should
reflect the product of the affinity constants for each substrate (approximated by the product of
the individual Km values). The two bisubstrates that have been made have low Ki values (2 16
µm) in the range of the Km values for the nucleotide sugar donor, suggesting that the correct
geometry for the bridging group may have not been attained. More analogs need to be made to
determine if these compounds will have useful properties in vivo.
A certain amount of serendipity is needed to obtain active compounds using these synthetic
approaches, and the synthesis of di-, tri-, and tetrasaccharides with the desired modifications is
rather labor intensive. Nevertheless, the approach has yielded insights into the binding and
reactivity of the enzymes, and substrate analogs with selectivity for particular enzymes have
been developed in this way. Because many of the transferases have now been purified and
cloned, we can look forward to more detailed kinetic and crystallographic studies, which will
provide clues for deriving mechanism-based inhibitors in the future.
Glycoside Primers: Mimicking What Already Works (45 H
56)
H
The utility of any glycosyltransferase inhibitor ultimately depends on its ability to cross the
plasma membrane and enter the Golgi where the glycosyltransferases reside. Unfortunately,
many of the compounds described above lack activity in live cells, presumably because their
polarity prevents their uptake. Okayama et al. 25 years ago found that d-xylose in β-linkage to a
hydrophobic aglycone was taken up rather efficiently and inhibited the assembly of
glycosaminoglycans on proteoglycans. Inhibition was caused by "priming" of chains on the
added xyloside, which diverted the assembly process from the endogenous core proteins. In
general, cells incubated with xylosides secrete large amounts of individual glycosaminoglycan
chains and accumulate proteoglycans containing truncated chains. The enormous success of β-d
xylosides in altering proteoglycan biosynthesis suggested that other glycosides might act as
"primers" as well (Table 40.4). Subsequent studies have shown that α-N-acetylgalactosaminides
H H
prime oligosaccharides found on mucins and inhibit O-glycosylation of glycoproteins. Other

active glycosides include β-glucosides, β-galactosides, β-N-acetylglucosaminides, and even
disaccharides and trisaccharides. These compounds require conjugation to appropriate aglycones
and acetylation to neutralize the polar hydroxyl groups on the sugars. Cells contain several
carboxylesterases that remove the acetyl groups. Apparently this can occur in a way that makes
the compounds available to the transferases in the Golgi.
Table 40.4. Examples of glycoside primers
Glycoside Pathway affected
Xylβ-O-R glycosaminoglycans
glycolipids
Galβ-O-R glycosaminoglycans
GalNAcα-O-R O-glycans found on glycoproteins and mucins
GlcNAcβ-O-R polylactosamines
Peracetylated Galβ4GlcNAcβ-O-R Lewis X
Peracetylated GlcNAcβ3Galβ-O-R Lewis X
Priming by glycosides occurs in a concentration-dependent manner, but the efficiency varies

widely among different compounds and cell types. These variations may relate to the relative
abundance of endogenous substrates, enzyme concentration and composition, the solubility of
different glycosides, their susceptibility to hydrolysis, their uptake across the plasma membrane
and into the Golgi, and their relative affinity for the glycosyltransferases. The type of chain made
on a given primer also depends on concentration and aglycone structure, which may reflect
selective partitioning of primers into different intracellular compartments or into different
branches of biosynthetic pathways.
Glycoside primers act as inhibitors of glycoprotein and proteoglycan assembly by subverting the
cellular machinery for making oligosaccharides on endogenous proteins. Like priming, inhibition
occurs in a dose-dependent fashion, but the blockade is rarely complete, probably due to the
inability of glycosides to completely mimic the endogenous core glycoprotein substrates. It is
important to remember that (1) glycosides inhibit the formation of entire classes of
glycoconjugates, but the effects on individual proteins may vary, and (2) inhibition occurs in the
midst of priming. The primed material may also have a composition different from that of the
oligosaccharides produced on core proteins. Thus, any effect on a biological phenomenon may
be due to the decline in glycoprotein or proteoglycan assembly or the increase in free
oligosaccharide chains. (3) The possibility should always be considered that the enhanced rate of
synthesis of oligosaccharides on primers may deplete pools of nucleotide sugars and thus alter
the assembly of other classes of glycoconjugates besides those that are generated on the primer.
For example, xylosides will prime compounds resembling intermediates in glycosaminoglycan
biosynthesis, an unusual compound containing GalNAc in α-linkage to the tetrasaccharide core
found in chondroitin sulfate and heparan sulfate, a compound related to ganglioside GM3,
Xylβ4Xyl-R, GlcA(3S)Xyl-R, in addition to free glycosaminoglycan chains. Although this lack
of specificity may seem to be a disadvantage, the products that have been found have revealed
previously undescribed pathways in cells.
Primers represent starting points for making analogs that might have the properties of tight-
binding inhibitors described in the previous section. Many of the compounds described in Table
H
40.3 could be converted to permeable acylated glycosides and tested in live cells for inhibitory
H
activity. Active compounds might have the potential of becoming carbohydrate-based drugs for
treating diseases and disorders dependent on glycosylation.
Priming of oligosaccharides may have beneficial effects as well. Xylosides, for example, can be
absorbed through the gut, and when consumed at sufficient concentration, they exhibit
antithrombotic activity. Many glycosides occur naturally, since various organisms (especially
plants) produce hydrophobic compounds as part of chemical defense and conjugate them to
sugars in order to render them soluble. Thus, the human diet may contain various types of
glycosides with interesting (and unknown) biological activities.
Inhibitors of Glycolipids and GPI Anchors (57 H

62)
H
Reagents have been described that can alter the assembly of glycolipids in cells. As mentioned
above, xylosides have a mild effect on glycolipid formation, possibly due to the assembly of a
GM3-like compound (Neu5Acα3Galβ4Xylβ-O-R) on the primer. In glycolipid biosynthesis,
many of the intermediates behave like the synthetic glycosides described above in the sense that
cells will take them up and use them as primers. Glucosylceramide is a naturally occurring
intermediate, and it will give rise to more complex glycolipids when fed to cells. On the basis of
this observation, an analog containing a reactive exocyclic epoxide group was prepared. The
compound inhibits glycolipid formation (IC50 ~8 µm), presumably by reaction of the epoxide
with a nucleophile in the active site of lactosylceramide synthase.
As mentioned above, the α-glucosidase inhibitor, N-butyldeoxynojirimycin, has been used to

inhibit glycosphingolipid formation because it inhibits glucosylceramide synthesis. However, the
sphingolipid analogs shown in Figure 40.3 are more potent inhibitors. Lengthening the
H H
hydrocarbon chain from 10 to 16 carbons further enhances efficacy. These compounds can cause
almost complete depletion of glycolipids in cells, and they require only micromolar amounts for
activity.
Figure 40.3. Inhibitors of glycosphingolipid formation. These commercially available analogs
are potent inhibitors of the glucosyltransferase that initiates glycosphingolipid formation.
Inhibitors of GPI anchor formation have also been described. Mannosamine inhibits GPI anchor
formation both in Trypanosoma brucei and in mammalian cells by formation of ManNH2-Man-
GlcN-PI. Apparently, mannosamine in its activated form (GDP-ManNH2) is used as an analog of
mannose in the second mannosyltransferase reaction, but the ManNH2-Man-GlcN-PI
intermediate will not act as a substrate for the next α2 mannosyltransferase (see Chapter 10).
H H
Another class of inhibitors is based on fatty acid analogs that only trypanosomes incorporate into
GPI anchors. Trypanosomes, unlike their mammalian hosts, incorporate myristic acid into GPI
anchors by exchanging myristic acid for other fatty acids in the phosphatidylinositol moiety. By
making a series of heteroatom-containing analogs, one was found that was highly toxic to
trypanosomes in culture and nontoxic to mammalian cells (10-[propoxy]decanoic acid). A
reagent such as this has the potential of becoming a drug for treating trypanosomiasis, which is
endemic in sub-Saharan regions of Africa.
Neuraminidase Inhibitors: Rational Design from X-ray Crystal

Structures (63 66)
H H
Studies of the influenza neuraminidase exemplify the power of rationally designed drugs. The
crystal structure for influenza neuraminidase was obtained in 1983, and since then many other
sialidases have been characterized from other sources. Even before the crystal structure had been
obtained, an inhibitor for neuraminidase was deduced by assuming that the hydrolysis reaction
probably involved a transition state with a carbocation intermediate at C2. This would result in
C2 and C3 adopting a trigonal planar (sp2) configuration, and therefore compounds that
mimicked this geometry might have inhibitory activity. Indeed, Neu5Ac-2-ene (Figure 40.4) has
H H
a micromolar Ki value. Interestingly, this compound works on most sialidases, but not on the
trypanosome trans-sialidase, and only weakly on bacterial sialidases.
Figure 40.4. Structure of influenza neuraminidase inhibitors. The analog Neu5Ac-2-ene is
thought to resemble the transition state in hydrolysis, and the addition of the guanidinium group
permits higher-affinity binding to the active site.
Visual inspection of the influenza enzyme with the inhibitor bound showed that two glutamate
residues lined a pocket near O4 of the sialic acid residue and formed hydrogen bonds with O4
(Figure 40.5). The pocket was fairly open, suggesting that a bulkier substituent at this position
H H
might be tolerated, at least sterically. A substrate analog was produced containing a positively
charged guanidinium group instead of O4, and binding studies showed that the analog had a Ki
value of 10-11m (Figure 40.4). The higher affinity was presumably due to an additional salt
H H
bridge formed between the charged guanidinium group and the carboxylates lining the pocket.
Interestingly, the analog does not work on the bacterial enzyme because the pocket is filled with
an arginine group and only one aspartate residue lines the pocket.
Figure 40.5. Binding site for sialic acid in influenza neuraminidase. The crystal structure
showed the orientation of active-site residues of the enzyme that interact with the substrate. The
O4 pocket is at the top of the figure. The two conserved glutamate residues that line the pocket
are shown in white. (Reprinted, with permission, from [64] Taylor 1996 [© Current Biology
Ltd.].)
Subsequent studies have attempted to use noncarbohydrate analogs based on the similarity of
benzoic acid to the partially planar ring of the proposed intermediate in hydrolysis. Reasonably
good inhibitors were found in this way, as long as hydrogen bond donors and acceptors were
added to the ring. The best inhibitor contained a guanidinium group in the same position relative
to the carboxylate anion, but the crystal data showed that the analog actually bound to the
enzyme in an orientation very different from that of sialic acid. As the crystal structures for other
sialidases are solved, the design of species-specific analogs may be possible. This rational
approach for making inhibitors has great potential not only for neuraminidase inhibitors, but also
for the design of compounds that might block the activity of various glycosyltransferases.
Future Directions
The various types of inhibitors described in this chapter were generated with the hope of
studying the mechanism of action of individual enzymes and as probes to alter glycosylation in
cells and tissues. Progress in this area has been modest in comparison with the development of
inhibitors for other metabolic pathways. In part, this reflects the relatively recent cloning of
many of the transferases, the sparse information on reaction mechanisms, and inherent
difficulties in preparing carbohydrate-based analogs. Nevertheless, the available agents have
been useful tools, and second-generation derivatives have had unusual properties that may prove
of therapeutic value. As more crystal structures for glycosyltransferases emerge, improvements
in inhibitor design and action should be forthcoming, which in turn should lead to compounds
with greater specificity. Armed with enzyme-specific inhibitors, glycobiologists will be able to
explore the biological function of the individual enzymes in normal and pathophysiology.
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amino analogues of the acceptor α-l-Fuc p-(1 2)-β-d-Gal p-OR by the blood-group A and B
gene-specified glycosyltransferases Carbohydr. Res. 251: 33-67. (PubMed) H H
40. A.C. Helland, O. Hindsgaul, M.M. Palcic, C.L. Stults, and B.A. Macher. 1995. Methyl 3-
amino-3-deoxy-β-d-galactopyranosyl-(1 4)-2-acetamido-2-deoxy-β-d-glucopyranoside: An
inhibitor of UDP-D-galactose: β-d-galactopyranosyl-(1 4)-2-acetamido-2-deoxy-d-glucose
(1 3)-α-d-galactopyranosyltransferase Carbohydr. Res. 276: 91-98. (PubMed) H H
41. F. Reck, M. Springer, E. Meinjohanns, H. Paulsen, I. Brockhausen, and H. Schachter. 1995.

Synthetic substrate analogues for UDP-GlcNAc: Manα1-3R β1-2-N-
acetylglucosaminyltransferase. 1. Substrate specificity and inhibitors for the enzyme Glycoconj.
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42. H. Hashimoto, T. Endo, and Y. Kajihara. 1997. Synthesis of the first tricomponent
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43. P.P. Lu, O. Hindsgaul, H. Li, and M.M. Palcic. 1997. Synthesis and evaluation of eight
aminodeoxy trisaccharide inhibitors for N-acetylglucosaminyltransferase-V Carbohydr Res. 303:
283-291. (PubMed)
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44. B.W. Murray, V. Wittmann, M.D. Burkart, S.C. Hung, and C.H. Wong. 1997. Mechanism of
human α-1,3-fucosyltransferase V: Glycosidic cleavage occurs prior to nucleophilic attack
Biochemistry 36: 823-831. (PubMed) H H
45. M. Okayama, K. Kimata, and S. Suzuki. 1973. The influence of p-nitrophenyl β-d-xyloside
on the synthesis of proteochondroitin sulfate by slices of embryonic chick cartilage J. Biochem.
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46. H.C. Robinson, M.J. Brett, P.J. Tralaggan, D.A. Lowther, and M. Okayama. 1975. The effect
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embryonic chicken cartilage Biochem. J. 148: 25-34. (PubMed) (Full Text in PMC)
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47. S.F. Kuan, J.C. Byrd, C. Basbaum, and Y.S. Kim. 1989. Inhibition of mucin glycosylation by
aryl-N-acetyl-α-galactosaminides in human colon cancer cells J. Biol. Chem. 264: 19271-19277.
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48. D. Zhuang, A. Grey, M. Harris-Brandts, E. Higgins, M.A. Kashem, and J.W. Dennis. 1991.
Characterization of O-linked oligosaccharide biosynthesis in cultured cells using paranitrophenyl
α-d-GalNAc as an acceptor Glycobiology 1: 425-433. (PubMed) H H
49. T.A. Fritz, F.N. Lugemwa, A.K. Sarkar, and J.D. Esko. 1994. Biosynthesis of heparan sulfate
on β-d-xylosides depends on aglycone structure J. Biol. Chem. 269: 300-307. (PubMed) H H
50. D.C.A. Neville, R.A. Field, and M.A.J. Ferguson. 1995. Hydrophobic glycosides of N-
acetylglucosamine can act as primers for polylactosamine synthesis and can affect glycolipid
synthesis in vivo Biochem. J. 307: 791-797. (PubMed) (Full Text in PMC)
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51. H.H. Freeze and J.R. Etchison. 1996. A new side of xylosides and their close relatives: Co-
localization mapping glycosyltransferases in the functional Golgi Trends Glycosci. Glycotechnol.
8: 65-77.
52. N.B. Martin, P. Masson, C. Sepulchre, J. Theveniaux, J. Millet, and F. Bellamy. 1996.
Pharmacologic and biochemical profiles of new venous antithrombotic β-d-xyloside derivatives:
Potential antiathero/thrombotic drugs Semin. Thromb. Hemost. 22: 247-254. (PubMed) H H
53. A. Pörtner, J.R. Etchison, D. Sampath, and H.H. Freeze. 1996. Human melanoma and
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galactosyltransferase Glycobiology 6: 7-13. (PubMed) H H
54. Y. Miura and T. Yamagata. 1997. Glycosylation of lactosylceramide analogs in animal cells:
Amphipathic disaccharide primers for glycosphingolipid synthesis Biochem. Biophys. Res.
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55. A.K. Sarkar, K.S. Rostand, R.K. Jain, K.L. Matta, and J.D. Esko. 1997. Fucosylation of
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56. W.H. Taylor, A. Sinha, I. Khan, S.T. McDaniel, and J.D. Esko. 1998. Primers of
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57. R.R. Vunnam and N.S. Radin. 1980. Analogs of ceramide that inhibit glucocerebroside
synthetase in mouse brain Chem. Phys. Lipids 26: 265-278. (PubMed)
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58. T.L. Doering, J. Raper, L.U. Buxbaum, S.P. Adams, J.I. Gordon, G.W. Hart, and P.T.
Englund. 1991. An analog of myristic acid with selective toxicity for African trypanosomes
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59. Y.T. Pan, T. Kamitani, C. Bhuvaneswaran, Y. Hallaq, C.D. Warren, E.T. Yeh, and A.D.
Elbein. 1992. Inhibition of glycosylphosphatidylinositol anchor formation by mannosamine J.
Biol. Chem. 267: 21250-21255. (PubMed)
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60. H.H. Freeze, D. Sampath, and A. Varki. 1993. α-and β-xylosides alter glycolipid synthesis in
human melanoma and Chinese hamster ovary cells J. Biol. Chem. 268: 1618-1627. (PubMed) H H
61. C. Zacharias, G. van Echten-Deckert, M. Plewe, R.R. Schmidt, and K. Sandhoff. 1994. A
truncated epoxy-glucosylceramide uncouples glycosphingolipid biosynthesis by decreasing
lactosylceramide synthase activity J. Biol. Chem. 269: 13313-13317. (PubMed)
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62. A. Abe, N.S. Radin, J.A. Shayman, L.L. Wotring, R.E. Zipkin, R. Sivakumar, J.M. Ruggieri,
K.G. Carson, and B. Ganem. 1995. Structural and stereochemical studies of potent inhibitors of
glucosylceramide synthase and tumor cell growth J. Lipid. Res. 36: 611-621. (PubMed)
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63. M. von Itzstein, W.Y. Wu, G.B. Kok, M.S. Pegg, J.C. Dyason, B. Jin, T. Van Phan, M.L.
Smythe, H.F. White, S.W. Oliver, P.M. Colman, J.N. Varghese, D.M. Ryan, J.M. Woods, R.C.
Bethell, V.J. Hotham, J.M. Cameron, and C.R. Penn. 1993. Rational design of potent sialidase-
based inhibitors of influenza virus replication Nature 363: 418-423. (PubMed)
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64. G. Taylor. 1996. Sialidases: Structures, biological significance and therapeutic potential
Curr. Opin. Struct. Biol. 6: 830-837. (PubMed)
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of protein-carbohydrate interactions Curr. Opin. Struct. Biol. 6: 703-709. (PubMed)
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66. P. Chand, Y.S. Babu, S. Bantia, N. Chu, L.B. Cole, P.L. Kotian, W.G. Laver, J.A.
Montgomery, V.P. Pathak, S.L. Petty, D.P. Shrout, D.A. Walsh, and G.M. Walsh. 1997. Design
and synthesis of benzoic acid derivatives as influenza neuraminidase inhibitors using structure-
based drug design J. Med. Chem.. 40: 4030-4052. (PubMed)
H H
41. Glycobiology in Biotechnology and Medicine
Primary contributions to this chapter were made by J.C. Paulson (Ths Scripps Research
Institute, La Jolla, California), A. Varki (University of California at San Diego), and J.D. Esko
(University of California at San Diego).
THIS CHAPTER PROVIDES AN OVERVIEW of the increasing importance of glycobiology

and carbohydrate chemistry in modern biotechnology and the pharmaceutical industry. The
carbohydrates of therapeutic recombinant glycoproteins have important roles in determining
their pharmacokinetic properties. Important biological interactions and biological functions
mediated by glycans are also being targeted for therapeutic manipulation in vivo. Examples of
carbohydrate-based therapeutics in development include inhibitors of microbial pathogens and
their toxins, cancer vaccines, and drugs designed to suppress the immune system for treatment of
inflammation and transplant rejection.
Therapeutic Glycoproteins (1 H
9)
H
The most important class of biotechnology products to date are therapeutic glycoproteins. These
include erythropoietin, granulocyte macrophage-colony-stimulating factor, and tissue
plasminogen activator, which together generate sales of 3 5 billion dollars worldwide. In
addition, approximately 60 recombinant glycoproteins are currently in development as
therapeutic agents. Therapeutic glycoproteins are typically produced as recombinant products in
cell culture systems or in transgenic animals. Control of glycosylation takes on major importance
during the development of these drugs, since the glycan chains have dramatic effects on stability,
action, and pharmacodynamics in intact organisms. In most cases, glycosylation must be
optimized to ensure prolonged circulatory half-life in the blood. Manipulation of glycans to
promote targeting to specific tissues and cell types has also been an essential element of drug
design for several successful therapeutic glycoproteins.
Optimizing Glycans of Therapeutic Glycoproteins for Prolonged Serum Half-

life (1 7)
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A well-studied example is that of erythropoietin, perhaps the single most successful

biotechnology product to date. Erythropoietin interacts with a membrane-signal-transducing
receptor, inducing proliferation and differentiation of erythroid progenitors, and thus, it has great
value in treating anemias caused by bone marrow suppression, e.g., after chemotherapy. Natural
and recombinant forms of erythropoietin carry four sialylated complex-type N-glycans. Although
the in vitro activity of deglycosylated erythropoietin is equal to fully glycosylated erythropoietin,
the in vivo activity of the deglycosylated form is less than 10% of the glycosylated
erythropoietin, because poorly glycosylated forms of erythropoietin are rapidly cleared by
filtration in the kidney. Furthermore, undersialylated erythropoietin is rapidly cleared by
Gal/GlcNAc/Man receptors in hepatocytes and macrophages. Both processes can be reduced by
having fully sialylated chains and by increasing the number of tetra-antennary chains.
Decreasing the rate of clearance of erythropoietin, in turn, increases its in vivo activity nearly
tenfold. Erythropoietin is perhaps unusual, since it is small enough to be cleared by the kidney if
underglycosylated. For most glycoprotein therapeutics, minimizing clearance by the
Gal/GlcNAc/Man receptors by having fully sialylated chains is the most important consideration
(see Chapter 25).
H H
Because the glycans can have dramatic effects on the properties of these drugs, it is important to
ensure that glycosylation is controlled during their production to satisfy regulatory requirements
for batch-to-batch product consistency. Changes in culture pH, the availability of precursors and
nutrients, and the presence or absence of various cytokines and hormones can each affect the
extent of glycosylation, the degree of branching, and the completeness of sialylation and
sulfation. The presence of sialidases and other glycosidases either secreted or released by dead
cells can cause degradation of the previously intact product. Thus, during the development of
each glycoprotein drug, considerable effort is devoted to defining appropriate conditions for
reproducibly producing homogeneous preparations of recombinant glycoproteins.
Targeting of Lysosomal Enzymes in Enzyme Replacement Therapy (10 13) H H
Unlike most therapeutic glycoproteins that interact with target receptors on the surface of cells,
lysosomal enzymes developed for enzyme replacement therapy must be delivered intracellularly
to lysosomes, their site of action. These drugs are intended to treat genetic defects for
deficiencies in individual lysosomal enzymes leading to pathological accumulation of their
substrates in inclusion bodies inside the cells (see lysosomal storage disorders, Chapter 18). H H
During the normal biosynthesis of lysosomal enzymes, the N-linked glycans become modified
with Man-6-P residues that target them to lysosomes through the Man-6-P receptor (see Chapter H
23). The challenge for enzyme replacement therapy is to get the enzymes targeted properly to
H
lysosomes where they can degrade accumulated substrate.
A special case is enzyme replacement therapy for Gaucher's disease, a glucocerebrosidase

deficiency. Exogenous enzyme has been targeted to lysosomes of macrophages through the cell
surface mannose receptor (see Chapter 25). This approach required that the recombinant enzyme
H H
be produced with N-glycans containing terminal mannose residues. This was accomplished by
cleaving the N-glycans of the enzymes produced in mammalian cells with glycosidases
(sialidase, galactosidase, and hexosaminidase). Alternatively, the enzyme can be produced in
baculovirus-infected insect cells, which elaborate N-glycans with terminal mannose residues.
The commercial success of glucocerebrosidase has stimulated the development of lysosomal
enzymes for treatment of other lysosomal storage diseases.
Complex Carbohydrate-based Therapeutics (14 H

20)
H
An increasing number of therapeutics aimed at modulating processes mediated by carbohydrate

groups of glycoproteins and glycolipids are currently in development. Advances in the enzymatic
production of carbohydrates are available to facilitate the production and commercialization of
such compounds.
Inhibitors of Pathogenic Microbes and Toxins
As discussed in Chapter 28, many microbes and toxins bind to mammalian tissues by
H H
recognizing specific carbohydrate ligands. Thus, small soluble glycans or glycan mimics can be
used to block the initial attachment of microbes and toxins to cell surfaces (or block their
release), and thus prevent or suppress infection. Because many of these organisms naturally gain
access through the airways or gut, the carbohydrate-based drugs can be delivered orally or
bronchially without the requirement of being distributed systemically. Examples of such
applications currently under study are listed in Table 41.1 and Table 41.2. Notable for their
H H H H
advanced stage of development are inhibitors of influenza virus sialidase (Table 41.3). The
H H
sialidase is required for the release of newly produced virus from sialic acid receptors on the cell
surface. Therefore, inhibition of the sialidase stops further replication of the virus and appears to
reduce the severity and duration of the disease.
Human breast milk oligosaccharides are believed to be natural antagonists of intestinal infection
in infants and to promote the growth of beneficial gut flora. Some companies have begun to
market nontherapeutic nutritional products that are "fortified" with oligosaccharides to impart the
putative beneficial properties provided by these natural oligosaccharides. More defined
therapeutic applications are also being evaluated (Table 41.1).
H H
Table 41.1. Possible carbohydrate antagonists of microbial adhesion
Proposed indication (microbe) Carbohydrate Company
Ulcers (H. pylori) sialyllactose Neose

Otitis media (Haemophilus influenzae) lacto-N-sialyltetrose a Neose
Kidney failure (E. coli toxin) Galα1 4Gal Synsorb Biotech
Diarrhea (C. difficile toxin) Galα1 3Gal Synsorb Biotech
Diarrhea (V. cholerae toxin) GM1 oligosaccharide Synsorb Biotech
Table 41.2. Examples of glycan therapeutics being developed to enhance the

immune system
Indication Carbohydrate Company
Immune stimulation: prevent Betafectin PGG- glucan Betafectin Alpha Beta

infection after upper GI
surgery by stimulating
macrophage scavengers
Anticancer vaccine: melanoma, GM2/GD2 ganglioside- conjugated to Bristol Meyers
breast cancer keyhole limpet hemocyanin Squibb/Progenics
Anticancer vaccine: breast sialyl-Tn conjugated to keyhole limpet Chiron/BioMira

cancer hemocyanin
Targeted immunotoxic conjugates of α-Gal carbohydrates with Sangstaat Medical
reactions: cancer, prostectomy, tissue-specific ligands to induce Corporation
ovarectomy immunotoxic reactions with endogenous
anti-α-Gal antibodies in recipient's blood.
Table 41.3. Examples of enzyme-based inhibitors as potential therapeutics
Clinical situation Drug/Mechanism Company
Influenza A infection GS4104 (neuraminidase inhibitor) Glaxo

Wellcome/Biota
Influenza A infection Zanamivir (Relenza) (neuraminidase Gilead/Hoffman
inhibitor) LaRoche
Tumors and hepatitis swainsonine (α-mannosidase II GlycoDesign
inhibitor)
Glycolipid accumulation in glycosyltransferase inhibitors Oxford
lysosomal storage diseases GlycoSciences
Diabetes Acarbose (intestinal α-glucosidase Bayer
inhibitor)
Flea infestation in pets Lufenuron (Program ) (chitin Ciba-Geigy
synthesis inhibitor)
Bacterial Vaccines
Conjugate vaccines with oligosaccharides coupled to carrier proteins are proving to be highly
effective. For example, Haemophilus influenzae type b causes an acute lower respiratory
infection among young children. As recently as a decade ago, approximately 20,000 children in
the United States suffered infections. In the early 1990s, a conjugated form of a Hib-derived
oligosaccharide coupled to a protein carrier was shown to provide an effective vaccine.
The Hib vaccine is now routinely given to infants in developed countries and has resulted in a
more than 95% decrease in incidence of Hib infections in vaccinated populations. In addition,
new vaccines are being developed against conjugated components of the capsular
polysaccharides of Neisseria meningitidis and Streptococcus pneumoniae and the preliminary
studies look promising.
Cancer Vaccines
Several carbohydrate-based vaccines are under development to treat cancer. Some of these
vaccines are based on ganglioside immunogens present on certain types of cancer cells (e.g.,
gangliosides GM2 and GD2 on melanomas and breast cancer). These vaccines are composed of
carbohydrate haptens conjugated to a protein carrier (Table 41.2). In one case, a breast cancer
H H
antigen, sialylTn (Sialylα2 6GalNAcα-) is synthesized chemically and conjugated to a protein

carrier (keyhole limpet hemocyanin). SialylTn is found on cancer mucins, and its expression
correlates with progression to metastatic breast cancer (see Chapter 35).
H H
Xenotransplantation: Acute Transplant Rejection Mediated by
Carbohydrates (21 23)H H
As discussed in Chapter 16, a variety of glycans, including the classical A and B blood group
H H
determinants, can act as barriers to blood transfusion and transplantation of organs. Rejection of
mismatched blood or organs occurs because hosts have a high titer of preexisting antibodies
against the carbohydrate epitopes, presumably as a prior reaction to related structures found on
bacteria or other microbes. In the case of the ABO blood groups, incompatibility is routinely
managed by blood and tissue typing and finding an appropriate donor for the recipient. However,
one approach to circumvent blood group incompatibility is to use immobilized A and B blood
groups, which under some circumstances could allow mismatched blood to be used for
transfusions. In practice, such circumstances are rare.
A related problem is found in xenotransplantation, transplantation of organs between species,

which is actively being pursued as a solution for the shortage of organs for human recipients.
The animal donors of preference are pigs, since many porcine organs resemble those of humans
in size, physiology, and structure. Unlike humans and certain other primates, pigs and most other
mammals produce a terminal Galα1,3Gal linear epitope on glycoproteins and glycolipids.
Humans have naturally occurring high-titer antibodies in their blood directed toward the Galα1
3Gal epitope, resulting in hyperacute rejection of porcine organ transplants, which is due to
reaction of the antibodies with this antigen on enodothelial cells of blood vessels. Attempts to
prevent this reaction include blood filtration over glycan affinity columns to remove
xenoreactive antibodies and blockade of the interaction by infusing soluble competing αGal
oligosaccharides. Ultimately, producing transgenic pigs lacking the reactive Gal α1 3
transferase may solve this problem. Of course, there are other carbohydrate differences between
humans and pigs (e.g., absence of Neu5Gc in humans, see Chapter 15) that could also be of
H H
concern.
Inhibition of Selectin-mediated Leukocyte Trafficking (24 26) H H
If specific glycan-protein interactions in vivo are responsible for selective cell-cell interactions
and a resulting pathology, then infusion of small-molecule analogs or mimics of the natural
ligand could be theoretically useful as a therapeutic. The best studied example is the selectin-
mediated recruitment of neutrophils (and other leukocytes) into sites of inflammation or
ischemia/reperfusion injury, which involves specific selectin-glycan interactions occurring in the
vascular system (see Chapter 26). Approaches taken include the use of sialyl Lewis X derivatives
H H
or small-molecule selectin antagonists. Simple monovalent compounds have already proven

effective in various animal inflammatory models. This finding is somewhat surprising since the
binding constant for the monovalent ligands is much poorer than that for the physiological
ligand. Nevertheless, preliminary studies in animals indicate that sialyl Lewis X derivatives are
probably effective at submillimolar concentrations, suggesting that other low-affinity ligands for
cellular lectins might prove to be effective antagonists as well.
An alternate approach for interfering with selectin-carbohydrate interactions consists of finding

specific inhibitors of the enzymes involved in forming endogenous sialyl Lewis X determinants
in vivo. Thus, considerable effort is under way to design inhibitors of the FucT VII enzyme
responsible for generating most natural selectin ligands (also see Chapter 40). Other therapeutics
H H
under development based on modulating protein-carbohydrate interactions include small-

molecule inhibitors of heparin-growth factor interactions in cancer, inflammation, and
angiogenesis. A particularly appealing aspect of this work is that antagonists discovered in this
way can be used to treat both acute and chronic disorders.
Large-scale Chemoenzymatic Synthesis of Complex Glycans (27) H H
A limiting factor in developing carbohydrate-based compounds for clinical application is the

high cost and complexity of producing glycans in adequate quantities. Advances in cost-effective
technology for enzymatic synthesis appear to be promising solutions to this problem (for the
generic principles involved, see Figure 41.1). This approach uses recombinant
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glycosyltransferases, relatively inexpensive precursors, and recycling reactions to regenerate the

most costly reagents (nucleotide sugars). The reactions tend to be efficient and specific, giving a
single carbohydrate product with very high yields, thus allowing efficient recovery of product
from reaction mixtures. By using selected glycosyltransferases in sequence, the approach is
suitable for the synthesis of most complex carbohydrates (e.g., see Figure 41.2). At least four
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compounds have been produced at the 1 10-kg scale using this approach.
Another application of the enzymatic synthesis technology is to produce analogs of lead

compounds. Some glycosyltransferases, kinases, and phosphorylases will utilize alternate
substrates with low efficiency. However, the reactions can be carried out for long periods of
time, resulting in the generation of specific oligosaccharides containing one or more functional
group modifications. For example, the β1 4 galactosyltransferases will use a number of UDP-
Gal analogs lacking hydroxyl groups or containing amino groups. Although the overall yields are
low, the generation of analogs in sufficient quantity for testing purposes is feasible and much
less laborious than standard orthogonal blocking strategies used in conventional carbohydrate
synthesis (see Chapter 39).
H H
Figure 41.1. Generic principle for sugar nucleotide cycles (SNC).

Figure 41.2. Example of a synthesis of a simple sialyloligosaccharide using in situ regeneration
of CMP-NeuAc.
Future Directions (20, 35 H H

39)
H
Medical applications of glycobiology have to date focused largely on roles of carbohydrates

known for several decades, including mediating adhesion of pathogens and toxins to host cells,
the appearance of tumor-associated antigens, the regulation of blood coagulation, and the
circulatory half-life of plasma glycoproteins. More recently, novel families of carbohydrate-
specific cell adhesion proteins have been identified that are being recognized to mediate
important functions in the immune system. Although the carbohydrate ligands of the selectin
family of leukocyte adhesion proteins were only identified in this decade, several companies are
developing compounds to block selectin-mediated functions as novel agents to treat acute and
chronic inflammation. It is likely that additional therapeutic opportunities will emerge as the
roles of newly described and yet to be discovered carbohydrate-binding proteins are elucidated.
Although the approaches to modulating biological systems involving carbohydrates are varied,
most involve the use of glycans or glycomimetics to modulate the activity of a carbohydrate-
binding protein. A promising alternative is to use inhibitors of the enzymes that degrade or
synthesize carbohydrates as a way of modulating the functions or properties they impart (see
Table 41.3). Sialic acid analogs being developed as potent inhibitors of influenza virus sialidase
H H
are showing promising results in clinical trials and may be available within a few years (see
Chapter 40). Other glycosidase inhibitors being evaluated target endogenous glycosidases. For
H H
exmple, Acarbose, an intestinal α-glucosidase inhibitor, is being used to treat diabetes mellitus.
Inhibition of α-glucosidase in the gut blocks the uptake of glucose from glucose-containing
polysaccharides in the diet (e.g., dietary glycogen) and thereby reduces the load of exogenous
glucose derived from a meal. Glucosylceramide synthetase is an example of a
glycosyltransferase that is a target for inhibitor development since inhibition of this enzyme
might decrease the accumulation of glycosphingolipids in lysosomal storage disorders. Another
example already on the consumer product market is Lufenuron, a benzoylphenyl urea chitin
synthesis inhibitor that is effective against certain insects such as fleas. When adult female fleas
feed on an animal that has been treated with Lufenuron, egg development does not occur
normally, since chitin is not deposited correctly. Thus, the life cycle of the flea is interrupted.
Such creative approaches to modulating the many structural and biological functions of
carbohydrates set the stage for creating products of commercial importance even as the myriad
of functions of this class of compounds are still being discovered.
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Role of antennary structure of N-linked sugar chains in renal handling of recombinant human
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Inc., New York.
Glossary: Commonly Used Terms
Acetal An organic compound derived from a hemiacetal by reaction with an alcohol. If the
hemiacetal is a sugar, the acetal is a glycoside.
Adhesin A protein on the surface of bacteria, viruses, or parasites that binds to a ligand present
on the surface of a higher eukaryotic cell.
Aglycone Noncarbohydrate portion of a glycoconjugate or glycoside that is glycosidally linked

to the glycan through the reducing terminal sugar.
Aldose A monosaccharide with an aldehyde group or potential aldehydic carbonyl group (by
definition, this is the C-1 position).
Amino Sugar A monosaccharide in which an alcoholic hydroxyl group is replaced by an amino

group.
Anomericity The α or β configuration of the glycosidic bond of a sugar to another sugar or an

aglycone.
Anomers Stereoisomers of a monosaccharide that differ only in configuration at the anomeric

carbon of the ring structure (i.e., the C-1 position in a cyclic hemiacetal).
Antenna A branch of an oligosaccharide emanating from a "core" structure.
Asn-linked Oligosaccharide See N-glycan.
C-type Lectins A class of Ca++-dependent lectins recognizable by a characteristic sequence

comprising their carbohydrate recognition domain.
Capsule A protective extracellular polysaccharide coat surrounding certain bacteria. Presence of

a capsular polysaccharide is often associated with virulence.
Carbohydrate A generic term used interchangeably in this book with sugar, saccharide, or
glycan. Includes monosaccharides, oligosaccharides, and polysaccharides as well as derivatives
of these compounds.
Carbohydrate Recognition Domain The domain of a polypeptide that is specifically involved

in binding to carbohydrate; in lectins, often a highly evolutionarily conserved region of the
polypeptide.
Ceramide The common lipid component of glycosphingolipids, composed of a long-chain basic

alcohol (sphingosine) and an amide-linked fatty acid.
Chondroitin Sulfate A type of glycosaminoglycan defined by the disaccharide unit (GalNAcβ1-

4GlcAβ1-3)n, modified with ester-linked sulfate at certain positions and typically found
covalently linked to a proteoglycan core protein.
C-type Lectins . A class of Ca++-dependent lectins distinguishable by the amino acid sequence
of their carbohydrate recognition domains.
Cerebroside A glycolipid composed of ceramide with an attached galactose
(galactosylceramide) or glucose (glucosylceramide).
Deoxy Sugar A monosaccharide in which an alcoholic hydroxyl group is replaced by a

hydrogen atom.
Dermatan Sulfate A modified form of chondroitin sulfate in which a portion of the β-

glucuronate residues are epimerized to α-iduronates.
Dolichol A polyisoprenoid lipid carrier utilized during the assembly of N-glycans and GPI
anchors.
β-Elimination Base-catalyzed, nonhydrolytic cleavage of an O-linked glycan attached to the

hydroxyl moiety of a serine or threonine residue within a protein or peptide.
Endoglycosidase An enzyme that catalyzes the cleavage of an internal glycosidic linkage in an

oligosaccharide or polysaccharide.
Endotoxin See Lipopolysaccharide.
Epimerase An enzyme that catalyzes racemization of a chiral center in a sugar.
Epimers Two isomeric monosaccharides differing only in the configuration of a single chiral
carbon. For example, mannose is the C-2 epimer of glucose.
Exoglycosidase An enzyme that cleaves a monosaccharide from the outer (nonreducing) end of
an oligosaccharide, polysaccharide, or glycoconjugate.
Exotoxins Heat-labile, proteinaceous toxins secreted by bacteria that cause illness.
Extracellular Matrix A complex array of secreted molecules including glycoproteins,

proteoglycans, and/or polysaccharides and structural proteins. In plants, the extracellular matrix
is also referred to as the cell wall.
Furanose Five-membered (four carbons and one oxygen, i.e., an oxygen heterocycle) ring form
of a monosaccharide named after the structural similarity to the compound furan.
Galectins S-type (sulfhydryl-dependent) β-galactoside-binding lectins, usually occurring in a

soluble form, expressed by a wide variety of animal cell types and distinguishable by
distinguishable by the amino acid sequence of their carbohydrate recognition domains.
Ganglioside Anionic glycosphingolipid containing one or more residues of sialic acid.
Glycan A generic term for any sugar or assembly of sugars, in free form or attached to another
molecule, used interchangeably in this book with saccharide or carbohydrate.
Glycation The nonenzymatic, chemical modification of proteins by addition of carbohydrate,

usually through a Schiff-base reaction with the amino group of the side chain of lysine and
subsequent Amadori rearrangement to give a stable conjugate. Not to be confused with
(enzymatic) glycosylation.
Glycobiology Study of the structure, chemistry, biosynthesis, and biological functions of glycans
and their derivatives.
Glycocalyx The cell coat structure consisting of glycans and glycoconjugates surrounding
animal cells that is seen as an electron-dense layer by electron microscopy.
Glycoconjugate A molecule in which one or more glycan units are covalently linked to a
noncarbohydrate entity.
Glycoforms Different molecular forms of a glycoprotein, resulting from variable glycan

structure and/or glycan attachment site occupancy.
Glycolipid General term denoting a molecule containing a saccharide linked to a lipid aglycone.
In higher organisms, most glycolipids are glycosphingolipids, but glyceroglycolipids and other
types exist.
Glycomimetics Noncarbohydrate compounds that mimic the properties of saccharides.
Glycopeptide A peptide having one or more covalently attached glycan units.
Glycophospholipid Anchor (Glycosylphosphatidylinositol, GPI Anchor) A membrane anchor

consisting of a glycan bridge between phosphatidylinositol and a phosphoethanolamine in amide
linkage to the carboxyl terminus of a protein.
Glycoprotein A protein with one or more covalently bound glycans.
Glycosaminoglycans Polysaccharide side-chains of proteoglycans or free complex

polysaccharides composed of linear disaccharide repeating units, each composed of a
hexosamine and a hexose or a hexuronic acid (see heparin, heparan sulfate, chondroitin sulfate,
dermatan sulfate, and hyaluronan).
Glycosidase An enzyme that catalyzes the hydrolysis of glycosidic bonds in a glycan. See
exoglycosidase and endoglycosidase.
Glycoside A glycan containing at least one glycosidic linkage to another glycan or an aglycone.
Glycosidic Linkage Linkage of a monosaccharide to another residue via the anomeric hydroxyl
group. The linkage generally results from the reaction of a hemiacetal with an alcohol (e.g., a
hydroxyl group on another monosaccharide or amino acid) to form an acetal.
Glycosphingolipid Glycolipid containing a glycan glycosidically attached to the primary

hydroxyl group of ceramide.
Glycosylation The enzyme-catalyzed covalent attachment of a carbohydrate to a polypeptide,

lipid, polynucleotide, carbohydrate, or other organic compound, generally catalyzed by
glycosyltransferases, utilizing specific sugar nucleotide donor substrates.
Glycosyltransferase Enzyme that catalyzes transfer of a sugar from a sugar nucleotide donor to
a substrate.
Glycotypes Cell-type-specific glycoforms of a polypeptide.

Hemagglutinin A lectin that recognizes carbohydrates on the surface of red blood cells and
causes agglutination (aggregation).
Hemiacetal A compound formed by reaction of an aldehyde with an alcohol group, as in ring

closure of an aldose.
Hemiketal A compound formed by reaction of a ketone with an alcohol group, as in ring closure
of a ketose.
Heparan sulfates Glycosaminoglycans defined by the disaccharide unit (GlcNAcα1-4GlcAβ1-

4/IdoAα1-4)n, containing N- and O-sulfate esters at various positions, and typically found
covalently linked to a proteoglycan core protein.
Heparin A type of heparan sulfate made by mast cells that has the highest amount of iduronic
acid and of N- and O-sulfate residues.
Heteropolysaccharide A polysaccharide containing more than one type of monosaccharide.
Hexose A 6-carbon monosaccharide typically with an aldehyde (or potential aldehyde) at the C-1
position (aldohexose) and hydroxyl groups at all other positions. Common examples in
vertebrate glycans are mannose, glucose, and galactose.
Hexosamine Hexose with an amino group in place of the hydroxyl group at the C-2 position.
Common examples found in vertebrate glycans are the N-acetylated forms, N-acetylglucosamine
and N-acetylgalactosamine.
Homopolysaccharide A polysaccharide composed of only one type of monosaccharide.
Hyaluronan A glycosaminoglycan defined by the disaccharide unit (GlcNAcβ1-4GlcAβ1-3)n

that is neither sulfated nor covalently linked to protein, referred to in older literature as
hyaluronic acid.
Hydrazinolysis A chemical method that uses hydrazine to cleave amide bonds, e.g., the
glycosylamine linkage between a sugar residue and asparagine or the acetamide bond in N-
acetylhexosamines.
I-type Lectins A class of lectins belonging to the immunoglobulin superfamily.
Keratan Sulfate A polylactosamine [Galβ1-4GlcNAcβ1-3]n with sulfate esters at C-6 of

GlcNAc and galactose residues, found as a side chain of a keratan sulfate proteoglycan.
Ketose A monosaccharide with a ketone group or a potential ketonic carbonyl group (typically at
the C-2 position in natural compounds).
Ketal An organic compound derived from a hemiketal by reaction with an alcohol. If the
hemiketal is a sugar, the ketal is a glycoside.
Lectin A protein (other than an anticarbohydrate antibody) that specifically recognizes and binds
to glycans without catalyzing a modification of the glycan.
Ligand A molecule that is recognized by a specific receptor. In the case of lectins, the ligands
are partly or completely glycan-based and are sometimes called counterreceptors.
Lipopolysaccharide A bacterial polysaccharide linked to a lipid moiety containing glucosamine
rather than glycerol (lipid A) that makes up the major portion of the outer leaflet of the outer
membrane of gram-negative bacteria. A major determinant of antigenic specificity, also known
as heat-stable toxin or endotoxin.
Lysozyme An endo-β-N-acetylhexosaminidase that cleaves the polysaccharide backbone of

bacterial peptidoglycan.
Methylation Analysis A method for carbohydrate structure analysis based on the acid stability
of methyl ethers and the acid lability of glycosidic linkages; used to determine the linkage
positions of monosaccharide residues in an oligosaccharide chain.
Microheterogeneity Structural variations in the glycan at any given glycosylation site on a

protein (one source of glycoforms).
Monosaccharide Carbohydrate that cannot be hydrolyzed into a simpler carbohydrate. The

building block of oligosaccharides and polysaccharides. Simple monosaccharides are
polyhydroxyaldehydes or polyhydroxyketones with three or more carbon atoms.
Mucin Large glycoprotein with a high content of serine, threonine, and proline residues and
numerous O-linked saccharides, often occurring in clusters on the polypeptide.
Mucopolysaccharide An out-of-date term replaced by the term, glycosaminoglycan. Still used

as a group name for human disorders ("mucopolysaccharidoses") involving glycosaminoglycan
accumulation due to genetic deficiency in certain lysosomal enzymes.
N-acetyllactosamine A disaccharide with the sequence Galβ1-4GlcNAc.
N-glycan (N-linked Oligosaccharide, N-linked Glycan) Glycan covalently linked to an

asparagine residue of a polypeptide chain in the consensus sequence: -Asn-X-Ser/Thr. Unless
otherwise stated, the term N-glycan is used generically in this book to denote the most common
linkage region, Manβ1-4GlcNAcβ1-4GlcNAcβ1-N-Asn.
Nonreducing Terminus (Nonreducing End) Outermost end of an oligosaccharide or

polysaccharide chain, opposite to that of the reducing end.
Oligosaccharide Linear or branched chain of monosaccharides attached to one another via

glycosidic linkages. The number of monosaccharide units can vary; the term polysaccharide is
usually reserved for large glycans with repeating units.
O-glycan (O-linked Oligosaccharide, O-linked Glycan) A glycan glycosidically linked to the

hydroxyl group of the amino acids serine, threonine, tyrosine, or hydroxylysine. Unless
otherwise stated, the term O-glycan is used in this book to denote the common linkage
GalNAcα1-O-Ser/Thr.
Peptidoglycan A bacterial polysaccharide consisting of MurNAcβ1-4GlcNAcβ1-4 repeat units,

covalently cross-linked to short peptides. Also known as murein, peptidoglycan represents the
major structural component of the periplasm.
Periodate Oxidation A selective chemical reaction for carbohydrates resulting in cleavage of
C C bonds with vicinal hydroxyl groups. A technique useful in determining the structure of
glycans.
Pili (Fimbriae) Hair-like appendages on the surface of some bacteria that often contain
adhesins.
Polylactosamine (Poly-N-Acetyllactosamine) . Repeating units of N-acetyllactosamines

[Galβ1-4GlcNAcβ1-3] n , of variable length (sometimes called PolyLacNAc).
Polyisoprenoid A lipid polymer composed of repeating units of the unsaturated 5-carbon

isoprene unit. See Dolichol.
Polysaccharide . Glycan composed of repeating monosaccharides, generally greater than ten

monosaccharide units in length.
Polysialic Acid A homopolymer of sialic acids abundant in the brain and fish eggs and found on
certain pathogenic bacteria.
Proteoglycan Any protein with one or more covalently attached glycosaminoglycan chains.
P-type Lectins Class of lectins that recognize mannose-6-phosphate (also called M6P receptors).
Pyranose Six-membered (five carbons and one oxygen, i.e, an oxygen heterocycle) ring form of
a monosaccharide; the most common form found for hexoses and pentoses. The name is based
on the structural similarity to the compound "pyran."
Receptor A protein that binds to a ligand and initiates signal transmission or other cellular
activity. In this book, most receptors are lectins (i.e., they recognize glycans). In microbiology,
the terminology is reversed: Adhesins or agglutinins on the microbes bind to receptors which are
glycans on the host cell.
Reducing Terminus (Reducing End) End of a glycan that has reducing power because it is
unattached to an aglycone and is thus a hemiacetal. In a glycoconjugate, reducing terminus is
also used as a synonym for a potential reducing terminus, referring to the end of a glycan
covalently attached to the aglycone by a glycosidic bond (i.e., it would have reducing power if it
were released).
Saccharide A generic term for any carbohydrate or assembly of carbohydrates, in free form or
attached to another molecule, used interchangeably in this book with carbohydrate and glycan.
Selectin A C-type (Ca++-dependent) lectin expressed by cells in the vasculature and bloodstream.
The three known selectins are L-selectin/CD62L (expressed by most leukocytes), E-
selectin/CD62E (expressed by cytokine-activated endothelial cells), and P-selectin/CD62P
(expressed by activated endothelial cells and platelets).
Ser/Thr-linked Oligosaccharide See O-glycan.
Sugar A generic term often used to refer to any carbohydrate, but most frequently to low-
molecular-weight carbohydrates that are sweet in taste. Table sugar, sucrose, is a non-reducing
disaccharide (Fruβ2-1αGlc). Oligosaccharides are sometimes called "sugar chains" and
individual monosaccharides in a sugar chain are sometimes referred to as "sugar residues."
Sugar Nucleotide Activated forms of monosaccharides, such as UDP-Gal, GDP-Fuc, and CMP-
Sia, typically used as donor substrates by glycosyltransferases.
Sugar Nucleotide Transporter Membrane-bound proteins that specifically transport sugar

nucleotides from the cytosol into the lumen of intracellular organelles (e.g., the Golgi).
Teichoic Acid A complex polymer consisting of either phosphoglycerol- or phosphoribitol-

carrying carbohydrates or amino acids, found on the surface of gram-positive bacteria.
Undecaprenol (Bactoprenol, C55 Isoprenoid) A polyisoprenoid lipid carrier for glycan

synthesis in bacteria.
Essentials of Glycobiology
Ajit Varki, Editor

Richard Cummings, Editor
Jeffrey Esko, Editor
Hudson Freeze, Editor
Gerald Hart, Editor
Jamey Marth, Editor
Contributions from: Maarten Chrispeels
Ole Hindsgaul
James C. Paulson
Adriana Manzi
Leland Powell
Herman van Halbeek
Developmental Editor: Kaaren Janssen
Project Coordinators: MaryInez CozzaSialiano
Production Editor: Dorothy Brown
Desktop Editor: Danny deBruin
Book Design: Denise Weiss
Cover Design: Tony Urgo
Cold Spring Harbor Laboratory Press Cold Spring Harbor, New York
ISBN 0-87969-559-5 (cloth). -- ISBN 0-87969-560-9 (printed hardcover)
1999 by The Consortium of Glycobiology Editors, La Jolla, California
Library of Congress Cataloging-in-Publication Data
The essentials of glycobiology / edited by Ajit Varki ... et al.].
p. cm.
Includes bibliographical references and index.
ISBN 0-87969-559-5 (cloth). -- ISBN 0-87969-560-9 (printed hardcover)
1. Glycoproteins. 2. Glycosylation. I. Varki, Ajit.
QP552.G59E87 1999
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Richard Cummings, Professor of Biochemistry Ed Miller Endowed Professor of Biochemistry

Jeffrey Esko, Professor of Cellular Molecular Medicine Associate Director, Glycobiology

Hudson Freeze, Professor of Glycobiology The Burnham Institute, La Jolla, California

Ole Hindsgaul, Professor of Chemistry University of Alberta, Edmonton, Canada

Adriana Manzi, Director, Analytical Research Development/Quality Control Nextran Inc., an

Jamey Marth, Professor of Medicine HHMI Associate Investigator Member, Glycobiology

Herman van Halbeek, Director, Glycotechnology Core Resource Member, Glycobiology

Washington University School of Medicine

THIS CHAPTER PROVIDES HISTORICAL BACKGROUND to the emergence of the field of

Monosaccharides Are the Basic Structural Units of Glycans (5)

Glycans Can Constitute a Major Portion of a Glycoconjugate (2)

Monosaccharides Generate More Linkage Variation Than

Common Monosaccharide Units of Animal Glycoconjugates (5, 7)

This limited set of monosaccharides seems to dominate the glycobiology of higher

A variety of modifications of oligosaccharides enhance the diversity of oligosaccharides

Details regarding the structural depiction of monosaccharides, linkages, and

Major Classes of Glycoconjugates and Oligosaccharides (8 12)

Topological Issues Relevant to the Biosynthesis of Glycans (10,12 22)

Nuclear and Cytoplasmic Glycosylation Is Common (10)

Outer Structures Are Often Shared among Classes of Glycans

Microheterogeneity: A Common Feature of Protein

Turnover and Degradation of Glycans (24, 25)

Tools Used to Study Glycosylation (26 36)

Genetic Glycosylation Defects in Cultured Cells and Intact

Biological Roles of Glycans Appear to Be Diverse (4, 7,41 50)

Glycosylation Changes in Development, Differentiation,

Glycobiology in Biotechnology and Medicine (23,58 61)

4. G. Hart. 1992. Glycosylation Curr. Opin. Cell Biol. 4: 1017-1023. (PubMed)

35. A.L. Burlingame. 1996. Characterization of protein glycosylation by mass spectrometry

38. J. Jaeken, H. Carchon, and H. Stibler. 1993. The carbohydrate-deficient glycoprotein

47. J. Dénarié, F. Debellé, and J.C. Promé. 1996. Rhizobium lipo-chitooligosaccharide

57. M. Costache, P.A. Apoil, A. Cailleau, A. Elmgren, G. Larson, S. Henry, A. Blancher, D.

FOLLOWING AN INTRODUCTION ON GENERAL CARBOHYDRATE

In general, carbohydrates contain a number of monosaccharides linked together as oligomers or

Ring Conformation of Sugars

Chemical Reactions of Monosaccharides

The interconversion of the α and β anomeric forms of a monosaccharide (mentioned above)

Figure 2.9. Oxidized forms of d-glucose.

Figure 2.12. Glycoside formation: conversion of hemiacetal into acetal.

Oligosaccharides, Polysaccharides, and Glycoconjugates

Figure 2.13. Structures of disaccharides.

The term homopolysaccharide is used to indicate a carbohydrate polymer that is composed of

3. El Khadem H.S. 1988. Carbohydrate chemistry Monosaccharides and their oligomers.

THIS CHAPTER PROVIDES A COMPARATIVE OVERVIEW of the patterns of glycosylation

Relatively Little Is Known about Glycan Diversity in Nature (1 8)

Evolution of the N-Glycan Processing Pathway (9 18)

Figure 3.1. Dominant pathways of N-glycan processing among different taxa.

Evolutionary Distribution of O-Glycans (19 21)

Homologs of the O-GalNAc:Ser/Thr transferases that initiate O-glycans in vertebrates (see

Diversity of Sialic Acids in Evolution (25 27)

Evolutionary Distribution of Glycosaminoglycan Chains (1, 2)

Evolutionary Distribution of Glycophospholipid Anchors (6, 7, 28, 29)

Evolutionary Distribution of O-linked N-Acetylglucosamine (30, 31)

Molecular Mimicry of Animal Glycans by Pathogens (32 40)

Interspecies and Intraspecies Differences in Glycosylation (41 52)

As discussed in Chapter 5, two classes of biological roles involve recognition of specific

Why Do Widely Expressed Glycosyltransferases Have Limited

1. C. Cassaro and C. Dietrich. 1977. Distribution of sulfated mucopolysaccharides in

3. R. Kornfeld and S. Kornfeld. 1985. Assembly of asparagine-linked oligosaccharides Annu.

4. Y. Kishimoto. 1986. Phylogenetic development of myelin glycosphingolipids Chem. Phys.

5. H. Rahmann, R. Hilbig, and F. Geiser. 1986. Brain gangliosides in monotremes, marsupials