Glycosaminoglycans: Structure and Biological Functions

James D San Antonio, Thomas Jefferson University, Philadelphia, Pennsylvania, USA Renato V Iozzo, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
Glycosaminoglycans are among the most complex polysaccharide chains that are either covalently linked to protein cores (to form proteoglycans) or free as unsulfated hyaluronan. They exist associated with virtually all cell surfaces and extracellular matrices of higher organisms, where their fine structure facilitates interactions with proteins, which underlie their myriad biological functions.

Secondary article
Article Contents
. Introduction . Fine Structure and Macromolecular Interactions between GAGs and Proteins . Biological Actions . Role in Human Disease . Genetic Defects of Metabolism . Therapeutic Uses

Introduction
Glycosaminoglycans (GAGs) contribute to numerous biological functions such as modulation of enzyme activities, regulation of cell growth, and control of assembly and function of extracellular matrices, largely through noncovalent interactions with proteins. GAGs are classied into six families, each being composed of repeating subunits of uronic acid and hexosamine sugars, and in some cases are highly modied by sulfations, epimerizations and N-acetylations. Details concerning GAG synthesis on proteoglycan (PG) core proteins, and PG structure, function, and genetics are reviewed elsewhere (Iozzo, 1999) and in this Encyclopedia. Although there are probably a hundred or more proteins that exhibit high-anity interactions with heparan sulfate (HS) or heparin (HN) and other GAGs, the specicity of such interactions has been dened for only a small number of them. We will rst review the structural features of GAGs and proteins that mediate their biological interactions, then we will discuss some of the activities arising from such interactions, with special emphasis on the role of GAGs in human health.

Fine Structure and Macromolecular Interactions between GAGs and Proteins

Structural features of protein-binding domains in GAGs
As HS and HN are among the most structurally diverse and biologically active GAGs, their protein-interactive features have been the most thoroughly studied. Fine structural features of HS chains, including dened

sequences, rare modications, domain structures and gross polymer characteristics are each believed to contribute to various classes of interactions with dierent proteins (Lindahl et al., 1998). The rst protein-binding structure in GAGs to be thoroughly characterized was the antithrombin III (ATIII) binding site of HN. In the plasma, the protease inhibitor ATIII neutralizes the serine protease thrombin by forming a 1:1 covalent complex with its active site, thereby inhibiting blood coagulation by preventing thrombin from catalysing the conversion of brinogen to brin, a major component of the blood clot. In the presence of HN, the rate of thrombin inactivation by ATIII is potentiated up to 2000-fold by inducing an allosteric alteration allowing a critical arginine residue of the protease inhibitor to interact more eciently with the active centre of thrombin. The determinants on HN necessary for ATIII binding were located on only about one-third of HN chains, suggesting that a rare domain or sequence might be responsible for the activity. A variety of approaches indicated that the ATIIIbinding site is in fact a pentasaccharide composed of a 6-Osulfated glucosamine in the rst position, a 3-O-sulfated central glucosamine, two N-sulfated glucosamines, and a carboxylated iduronic acid (Figure 1a). Other modications may increase the activity of HN on ATIII, but are not essential for activity. Although HN is among the most widely used of anticoagulants in humans, the in vivo relevance of the ATIII-binding sequence on HN is unknown. This is because HN chains are synthesized as components of serglycin PGs which are made almost exclusively by mast cells. However, it has been shown that the ATIII binding sequence is present on the GAG chains of specic types of HSPGs synthesized by endothelial cells and that, in vivo, anticoagulation-active HSPGs accumulate in the subendothelial extracellular matrix (ECM), suggesting a role for the contributions of native PGs to

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Glycosaminoglycans: Structure and Biological Functions

Figure 1 Structural features underlying GAG protein interactions. (a) ATIII-binding sequence in HN, moieties critical to binding are shown in red. (b) HN-binding sequence in ATIII is in an a-helical region containing a sequence rich in basic amino acids (shown in red and green), asterisk denotes position of residue number 125 (adapted from Jackson et al., 1991). (c) (Upper left panel) Electron micrograph of procollagen molecules (each $ 300 nm) with globular C-termini; (lower left panel) HN-gold binds near procollagen N-terminus, and (right panel) crosslinks two procollagens at binding site. Sequence proposed as the HN-binding domain of type I collagen begins at lysine residues (shown in red) located at position 87 from the N-terminus, is composed of three chains and is rich in basic amino acids (shown in red and green). Artwork by Drew Likens.

antithrombotic properties of blood vessel surfaces and of the ECM. Other molecules including the cytokines interferon-g and interleukin-8 are thought to be retained by cell surface HSPGs for presentation to target cells. These cytokines do not appear to bind a rare sequence in HS, but rather interact with single HS chains by binding regions composed of two domains of consecutive N-sulfated disaccharides that ank one central domain of consecutive N-acetylated disaccharides. However, the binding selectivities of the two cytokines for HS can be distinguished because each preferentially interacts with regions that dier with respect to the size of the central domain of Nacetylated residues. Similarly, most other known proteininteractive sites on HS appear not to rely upon unique or rare sequences, but rather upon domains of about 515 saccharide units containing specic features. For example on HS, a 2-O-sulfated iduronic acid group within an Nsulfated domain is required for binding to broblast growth factor (FGF)-2, whereas a 6-O-sulfated glucosamine is required for binding to platelet-derived growth factor and lipoprotein lipase. Finally, even gross features of GAG structure including domain charge density and exibility may underlie the relatively lower-anity inter2

actions exhibited by HS and other GAGs for some types of matrix molecules including collagens.

Structural features of GAG-binding sequences in proteins

Analyses of the structural features of a large number of HN- and HS-binding proteins have identied motifs responsible for interactions with GAGs (Jackson et al., 1991). Many HN-binding proteins contain one or more clusters of basic amino acids that exactly or closely follow the arrangements X-B-B-X-B-X or X-B-B-B-X-X-B-X, where B represents an amino acid with basic charge, usually arginine or lysine, and X an uncharged or hydrophobic amino acid. Conformational modelling indicates that the candidate HN-binding sequences exist within a-helical domains, with the basic residues clustered in one region on the outside of the helix (Figure 1b). The latter probably facilitates interactions with the anionic sulfate and carboxylate groups of the GAGs. In some cases, but not all, manipulation of the putative HNbinding sequences through site-directed mutagenesis or via chemical modication conrmed the HN-binding function

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Glycosaminoglycans: Structure and Biological Functions

of such consensus sequences. However, mimetic peptides of these consensus sequences often fail to show the highanity HN-binding characteristics of the native proteins, and many proteins that bind to GAGs with high anity do not contain such sequences. To explain these discrepancies, it has been speculated that the three-dimensional arrangement of multiple HN-binding consensus sites within or between HN-binding proteins, and/or the presence of novel HN-binding sites may be responsible for highanity HS or HNprotein interactions. An example of the latter includes those of the chymases, which are enzymes stored in mast cell secretion granules presumably complexed with the PG serglycin. HN-binding function of these enzymes is not carried by consensus-type sequences, but has been ascribed to clusters of basic charge on protein surfaces. Another example includes that of type I collagen, in which the HN-binding site was mapped to a region rich in basic charge, in which none of the three protein chains contain HN-binding consensus sequences (Figure 1c). Typically, then, protein domains responsible for binding HS and HN include cluster(s) of basic amino acids, and may or may not t the proposed consensus sequence model for HN-binding sites.

cell cycle stage and can subsequently block the mitogeninduced S-phase transition. Once in the cytoplasm, HN acts on signal transduction pathways; for example, it strongly inhibits serum, phorbol ester and platelet-derived growth factor-BB induction of MAP (mitogen-activated protein) kinase activity and phosphorylation, as well as suppressing protein kinase C (PKC)-dependent signalling. Nuclear events are also aected by HN, which inhibits the expression of genes essential for cell cycle progression in VSMC, and has been shown to suppress expression of the c-fos, c-myc and c-myb proto-oncogenes, and AP1 transcription factor activation. Inhibition of transcription factor activation and gene expression is thought to be due to a blocking of the transmission of second messenger signals; however, some studies have reported that a specic fraction of HN or HS enters the nucleus and may directly inhibit gene transcription.

Extracellular actions
Extracellularly, HN interacts with matrix molecules known to contain single or multiple HN-binding sites, including bronectin, laminin and various collagens, and in some cases alters the solubility or polymerization state of the matrix ligand. Exogenous GAGs may also inuence the space-lling function thought to be carried by the GAG chains of matrix-associated PGs (Figure 2b). Furthermore, abundant evidence suggests a role for cell surface PGs in cell adhesion, thus, HN and other GAGs often competitively disrupt cell attachment to ECM substrata. In this way, GAGs may also secondarily inuence cell growth through disruption of integrin receptorECM interactions, or by displacement of cell surface HSPGs from their ECM-binding sites, thus interfering with the cellsubstratum attachments required for mitosis (Figure 2c). Finally, GAGs exert signicant but poorly understood eects on PG metabolism. For example, the amount of HS and chondroitin sulfate (CS) synthesized by corneal epithelium is enhanced by the supplementation of these GAGs to the cells in culture. Cartilage matrix synthesis by chondrocytes is stimulated by CS, dermatan sulfate (DS), HN, and HS and inhibited by hyaluronan (HA), and the expression of matrix components during cartilage dierentiation is strongly inuenced by various GAG preparations. GAGs also inuence the expression of other types of matrix components; for example, HN aects the transcription, extracellular deposition and lysyl oxidasemediated crosslinking of type I collagen, and inhibits the expression of proteases important for cell-mediated matrix degradation.

Biological Actions
Intracellular actions
GAGs exhibit a wide variety of potent activities on cell growth, migration, dierentiation, metabolism and adhesion (Jackson et al., 1991). One of the earliest reports of an eect of GAGs on cell growth reported that broblastic mouse L cells in suspension culture exposed to 50 mg mL 2 1 HN were growth inhibited by 73%. Several strong antiproliferative activities of GAGs on a variety of cell types have since been reported. The eects of HN on vascular smooth-muscle cells (VSMC) have been the most extensively studied owing to the relevance of this topic to vascular disease. Although for some cell types HN antiproliferative action may involve displacement of HSor HN-binding growth factors from cell surface receptors, for VSMC HN may also be internalized and act directly in the cytoplasm and nucleus (Figure 2a). HN-binding sites in VSMC are specic, saturable, high-anity, and present at about 100 000 sites/cell. HN is internalized by VSMC by two mechanisms: an initial rapid uptake occurring within 15 min after binding, followed by a slow rate of uptake occurring over several days. Growth-arrested VSMC bind nearly 10 times more HN, and are up to 100 times more sensitive to HN than are their exponentially growing counterparts. These ndings are consistent with the observation that VSMC are more sensitive to growth inhibition by HN if it is added when the cells are released from growth arrest. HN is most eective during the G0/G1

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Glycosaminoglycans: Structure and Biological Functions

Figure 2 Intracellular and extracellular actions of GAGs. (a) Some proposed intracellular activities of GAGs. (b) Electron micrograph of the substantia propria of human cornea showing cross and longitudinal sections of collagen fibrils; GAGs occupy interfibrillar spaces (magnification 90 000). (c) (Top) PG and integrin-mediated cell adhesion to matrix; (bottom) model for disruption of cell-matrix interactions by GAG. (d) Left: carotid artery with restenosis-like response after endothelial denudation; right: carotid artery exposed following injury to HN. Reprinted with permission of the American Heart Association from Guyton et al., (1980). Artwork in (c) by Shawn M. Sweeney.

Role in Human Disease

Vascular disorders
Atherosclerosis is often characterized by the pathological accumulation of lipoproteins at the site of vascular lesions, and PGs are believed to trap or localize low-density lipoproteins (LDLs) to the arterial wall (Iozzo, 1999). For example, complexes of apolipoprotein B-100 (ApoB-100)
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and PGs can be isolated from aortic fatty lesions, and, in vitro, PGs and GAGs from the arterial wall precipitate LDL. The importance of GAG chains to such interactions is demonstrated by the ndings that HN and chondroitin 6sulfate (C-6-S) or C-4-S chains bind directly to LDL. Furthermore, the amount of LDL and C-6-S, but not of HS in the artery correlates positively with the development of fatty lesions. Atherosclerotic lesions contain increased amounts of high-Mr CS molecules, but have CSPGs with

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Glycosaminoglycans: Structure and Biological Functions

fewer GAG chains. The PGs proposed to interact with and trap LDL in the subendothelial basement membrane and in the interstitium of the blood vessel media include perlecan and the large aggregating CSPGs. There is much interest in deciphering how LDL modications that occur in vivo inuence their anities for PGs, and thus their potential for lipid retention by the ECM. For example, circulating LDLs with high anity for CSPG have a greater content of cholesterol, less triglyceride, and a more basic charge density than do LDL with lower avidities for PGs. Although LDL oxidation occurs in atherosclerotic lesions in vivo, this change reduces LDL anity for PGs. On the other hand, proteolysis of the ApoB-100 portion of LDL causes fusion of LDL particles resulting in an enhanced anity for PGs. Finally, lipolysis of LDL can occur in the vascular wall through action of sphingomyelinase and secretory phospholipase A2; consequently LDL has been shown to bind PGs with increased strength (Oorni et al., 1998). Also of interest are mechanisms whereby growth factors and cytokines upregulated during atherogenesis may induce changes in the ne structure of the GAG chains of arterial PGs, potentially increasing their avidities for LDL. Another important component of vascular diseases, including atherosclerosis and restenosis, is the pathological growth of vascular smooth muscle cells (VSMC). GAGs are strong regulators of VSMC growth and therefore are potentially useful in treating these diseases. The eect of HN on VSMC growth in vivo was rst discovered in experiments aimed at determining whether HN may inhibit the response to injury cascade of accelerated atherosclerosis owing to its antithrombotic activity; a dramatic inhibition of VSMC proliferation by HN was observed (Guyton et al., 1980) (Figure 2d). It was next shown that the growth eect of HN on VSMC in vivo is exhibited by either anticoagulant or nonanticoagulant fractions, and that these eects are mimicked by HN or HS on VSMC in vitro. These results implied that HN does not act on VSMC growth via its anticoagulant activity. Further experiments showed the smallest active HN fragment to be a pentasaccharide, and that the 3-O-sulfate on the internal glucosamine residue is sucient but probably not essential for its antiproliferative action. In endothelial HS, domains rich in 2-O-sulfated iduronic acid were shown to mediate their antiproliferative properties. It has been proposed that in the healthy vascular wall, endothelial-derived HS maintains VSMC in a quiescent growth state, but that injuries that result in endothelial denudation remove this paracrine mechanism, resulting in uncontrolled VSMC proliferation and vascular lesion formation. Percutaneous transluminal coronary angioplasty (PTCA) or balloon angioplasty is a widely used surgical technique to treat vascular occlusive lesions in humans. However, a signicant fraction of these procedures fail within one year owing to VSMC proliferative restenosis. Although HN is among the most potent inhibitors of accelerated arterio-

sclerosis in rat models, and is a potent inhibitor of the proliferation of human VSMC in vitro, to date, in human clinical trials HN has been unsuccessful for treatment of intimal hyperplasia following PTCA. Two explanations have been proposed regarding the discrepancies between the data obtained from the testing of HN as an antiproliferative agent in animal models versus the human clinical restenosis trials. Thus, based on results from experiments in animals, it has been speculated that the dosage, route and timing of HN administration have not been optimized in the clinical trials and it may be rapidly metabolized and cleared before reaching its site of action. Another explanation is that, at least in some individuals, HN-resistant VSMC may exist in the blood vessel wall and contribute to vascular restenosis despite HN therapy (San Antonio et al., 1998). Indeed, humans with restenosis contain VSMC that are less sensitive to HN, which led to the speculation that VSMC HN insensitivity might represent a cellular risk factor for restenosis. Ongoing research is attempting to understand the origin and cell biology of HN-resistant VSMC, and to test their potential role in vascular disease.

Cancer
Tumour matrix stromas may play important roles in potentiating tumour growth and metastasis (Iozzo, 1995). For example, the development of the majority of colon carcinomas is accompanied by a marked CS deposition and a change in its ne structure to C-6- and O-sulfation. Increases in perlecan expression are also seen during development of these tumours and of malignant melanomas; its HS chains may potentiate growth factor activity and induce angiogenesis surrounding the tumour, thereby enhancing its growth (Nugent and Iozzo, 1999). Furthermore, the binding selectivity of HS chains for various members of the FGF family can be inuenced by ne structural features such as the patterns of 6-O-sulfation and the abundance of sulfated domains (Lindahl et al., 1998). A pathological role of tumour cell surface PGs has also been suggested. For example, Chinese hamster ovary cells carrying various mutations of PG synthesis were injected into nude mice and tested for their tumorigenic abilities. Mutants that expressed low levels of PGs failed to produce tumours, and of those with normal PG levels but with defects in the synthesis of specic GAG types, the structure of HS, but not of CS, was most important to their tumorigenicity. PGs secreted by normal cells are proposed to play a key barrier function by inhibiting the migration of tumour cells across basement membranes. However, tumour cells have been shown to secrete the enzyme heparatinase, which degrades the HS chains within basement membranes, thereby potentially enabling such malignant cells to breach the basement membrane, enter the circulation, and spread
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Glycosaminoglycans: Structure and Biological Functions

throughout the body. Finally, malignancy is also believed to be promoted by factors secreted by tumour cells that upregulate HA synthesis; the extracellular elaboration of this GAG has been shown to increase the invasiveness of tumour cells into collagenous matrices.

Scrapie and related disorders

Scrapie and other spongiform encephalopathies, as well as Alzheimer disease are characterized by the accumulation of an abnormal isoform of a protease-resistant host protein (PrP) in amyloid plaques. A role for PGs in the pathogenesis of these diseases has been suggested largely because highly sulfated GAGs, including the HS components of perlecan, are found in tissue-derived amyloid deposits. It has been proposed that sulfated GAGs may promote amyloidogenesis by inducing a PrP shift in conformation from the a-helix to the b-sheet structure characteristic of amyloid brils, by inhibiting the proteolysis of PrP, or by targeting PrP to cellular sites that mediate the generation of its protease-resistant form (Caughey, 1994). Finally, it has been observed that HS preparations from healthy organs or those from organs with AA amyloidosis exhibited marked dierences in Osulfation patterns, suggesting that changes in HS ne structure may contribute to amyloidogenesis.

study of the pathogenesis of these disorders and the development of therapies of potential use in humans with MPS. For example, alleviation of the outward signs of MPS I and MPS VII in dogs has been achieved through heterologous bone marrow transplantation, where donor bone marrow cells supply normal enzyme to other tissues (Figure 3c). Another example of a mutation of GAG metabolism in humans is that underlying hereditary multiple exostosis, a disorder characterized by the appearance of multiple cartilage-capped tumours derived from the endochondral growth plate of bone. Two genes associated with this disease encode glycosyltransferases required for HS biosynthesis, although the consequences of these mutations to HS chain polymerization and modication in aected individuals remain unknown.

Therapeutic Uses
HN is among the most commonly prescribed of anticoagulants in medicine, owing to its strong acceleration of ATIII activity. HN is also commonly used in scientic and commercial laboratories in the preparation of anity matrices for the purication of growth factors, transcription factors, and other HN-binding proteins from tissue extracts. Other common uses of GAGs and GAG components are for the treatment of osteoarthritis. Studies suggest some alleviation of arthritic symptoms after oral administration of glucosamine or CS, which are now among the most common over-the-counter nutrient supplements available to the public. Glucosamine is known to be absorbed through the small intestine into the bloodstream, and can be used as a precursor of GAG chains of cartilage; increasing the steady-state levels of glucosamine in arthritic joints may promote GAG biosynthesis by chondrocytes, thereby helping to rebuild joint surfaces. It is believed that CS must rst be hydrolysed by the stomach to its component monosaccharides, which are then absorbed and metabolized by cartilage. Chemically hypersulfated GAGs are also used via direct injection to treat arthritic joints in animals. HA preparations are employed extensively as optically clear, high-viscosity gels for ophthalmic examinations and for surgery such as corneal extractions and transplantations. HA gels are also used to promote the healing of cutaneous wounds such as those of stasis dermatitis, and to help minimize scarring during wound healing. Finally, chemically crosslinked GAGcollagen mixtures are employed as scaolds upon which cells can be seeded to engineer tissue substitutes such as skin for transplantation purposes. In summary, GAGs regulate numerous biological activities including cell growth, adhesion and metabolism, and play roles in human disorders such as atherosclerosis, Alzheimer disease, and cancer. Although the biological

Genetic Defects of Metabolism

As many dierent enzymes contribute to the synthesis, modication and degradation of GAGs, it is no surprise that mutations should be discovered with a basis in defective activities of some of these enzymes. The best known of these are the mucopolysaccharidoses (MPS), which arise from defects in GAG catabolism; individual syndromes may involve defects in any of nine dierent enzymes (Neufeld and Meunzer, 1995). In normal individuals, GAGs are catabolized into their component saccharides by a series of lysosomal exoglycosidases and sulfatases, which act in sequence on the nonreducing termini of the GAG chains. In MPS patients, however, defective GAG catabolism leads to abnormal amounts of GAG accumulated in lysosomes and excreted in the urine. These individuals exhibit other anomalies, with the most signicant being connective-tissue defects (Figure 3a) and in some cases mental retardation. In Hunter syndrome (MPS II), for example, iduronate sulfatase activity is lacking and HS and DS with nonreducing terminal 2-sulfated residues accumulate; whereas in patients with Hurler syndrome (MPS I), defects in a-l -iduronidase activity result in the accumulation of nonsulfated iduronic residues on the nonreducing end of HS or DS chains (Figure 3b). Many types of MPS have also been discovered in dogs, cats, and other animals, which have proved to be valuable for the
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Glycosaminoglycans: Structure and Biological Functions

Figure 3 Defects of GAG catabolism cause the mucopolysaccharidosis (MPS) disorders in humans and animals. (a) Clinical features of a 10-year-old child with MPS I. Note the depressed bridge of the nose, wide-spaced eyes, low-set ears and coarse facial features. She is unable to fully extend her fingers. (b) Two steps in the lysosomal catabolism pathway of HS; further steps exist but are not shown (adapted from Neufeld and Meunzer, 1995). (c) Clinical features of MPS VII in the dog. The dog on the right cannot stand or walk at 6 months of age. He has low-set ears, wide-spaced eyes with corneal clouding and tearing, and malformed ribs. The dog on the left also has MPS VII but received a heterologous bone marrow transplant at 6 weeks of age and continued to stand and walk for 6 years, and had diminished facial dysmorphia, corneal clouding, and rib malformations. The authors gratefully acknowledge Sallie Martinez of Escondido California for the photograph in (a), and Dr Mark Haskins, School of Veterinary Medicine, University of Pennsylvania, for the photograph in (c), which includes unpublished data from his research. Artwork by Drew Likens.

functions of GAGs typically rely upon their interactions with proteins, in general these mechanisms are poorly understood. However, in some cases for the GAGs such interactions have been shown to depend upon specic sequences, ne structural features such as positions of sulfate moieties on their component saccharides, or gross characteristics including polymer negative charge and exibility. For the proteins, domains rich in basic amino acids appear to be necessary to facilitate interactions with

GAGs. Currently, dramatic inroads are being made in dening the biochemistry of protein-interactive domains of GAGs owing to new developments in GAG sequencing technology. Furthermore, eorts are underway to elucidate how GAG chain synthesis and modications are regulated by cells to generate the GAG ne structural variants required to carry out their tissuespecic functions.

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Glycosaminoglycans: Structure and Biological Functions

References
Caughey B (1994) Scrapie-associated PrP accumulation and agent replication: eects of sulphated glycosaminoglycan analogues. Philosophical Transactions of the Royal Society of London B 343: 399404. Guyton JR, Rosenberg RD, Clowes AW and Karnovsky MJ (1980) Inhibition of rat arterial smooth muscle cell proliferation by heparin. I. In vivo studies with anticoagulant and non-anticoagulant heparin. Circulation Research 46: 625634. Iozzo RV (ed.) (1999) Proteoglycans: Structure, Biology, and Molecular Interactions. New York: Marcel Dekker, in press. Iozzo RV (1995) Tumor stroma as a regulator of neoplastic behavior. Laboratory Investigations 73: 157160. Jackson RL, Busch SJ and Cardin AD (1991) Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes. Physiological Reviews 71: 481539. Lindahl U, Kusche-Gullberg M and Kjellen L (1998) Regulated diversity of heparan sulfate. Journal of Biological Chemistry 273: 2497924982. Neufeld EF and Meunzer J (1995) The mucopolysaccharidoses. In: Scriver CR, Beaudet AL, Sly WS and Valle D (eds) The Metabolic and Molecular Bases of Inherited Disease, 7th edn, pp. 24652494. New York: McGraw-Hill. Nugent M and Iozzo RV (1999) Heparan sulfate proteoglycans as modulators of broblast growth factors. International Journal of Biochemistry and Cell Biology, in press. Oorni K, Hakala JK, Annila A, Ala-Korpela M and Kovanen PT (1998) Sphingomyelinase induces aggregation and fusion, but phospholipase A2 only aggregation, of low density lipoprotein (LDL) particles. Journal of Biological Chemistry 273: 2912729134. San Antonio JD, Verrecchio A and Pukac LA (1998) Heparin sensitive and resistant vascular smooth muscle cells: biology and role in restenosis. Connective Tissue Research 37: 87103.

Further Reading
Conrad HE (1998) Heparin-Binding Proteins. San Diego, CA: Academic Press. Esko JD, Rostand KS and Weinke JL (1988) Tumor formation dependent on proteoglycan biosynthesis. Science 241: 10921096. Haskins ME and Giger U (1997) Lysosomal storage diseases. In: Kaneko JJ, Harvey JW and Bruss ML (eds) Clinical Biochemistry of Domestic Animals, 5th edn, pp. 741761. New York: Academic Press. Hay ED (ed.) (1991) Cell Biology of the Extracellular Matrix, 2nd edn. New York: Plenum Press. Iozzo RV (1998) Matrix proteoglycans: from molecular design to cellular function. Annual Reviews of Biochemistry 67: 609652. Kelly GS (1998) What is the role of glucosamine sulfate and chondroitin sulfates in the treatment of degenerative joint disease. Alternative Medicine Reviews 3: 2739. Ottlinger ME, Pukac LP and Karnovsky MJ (1993) Heparin inhibits mitogen-activated protein kinase activation in intact rat vascular smooth muscle cells. Journal of Biological Chemistry 268: 19173 19176. San Antonio JD, Lander AD, Karnovsky MJ and Slayter HS (1994) Mapping the heparin-binding sites on type I collagen monomers and brils. Journal of Cell Biology 125: 11791188. Turnbull JE and Gallagher JT (1991) Distribution of iduronate 2sulphate residues in heparan sulfate. Biochemical Journal 273: 553 559. Watson DJ, Lander AD and Selkoe DJ (1997) Heparin-binding properties of the amyloidogenic peptides Ab and amylin. Dependence on aggregation state and inhibition by Congo red. Journal of Biological Chemistry 272: 3161731624.

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