Molecular Immunology 40 (2004) 13331346

Complement inhibitor C4b-binding proteinfriend or

foe in the innate immune system?
Anna M. Blom a, , Bruno O. Villoutreix b,1 , Bjrn Dahlbck a
a

The Wallenberg Laboratory, Department of Clinical Chemistry, University Hospital Malm, Lund University, S-205 02 Malm, Sweden
b INSERM U428, University of Paris V, 4 Ave. de LObservatoire, 75006 Paris, France
Received 9 December 2003; received in revised form 9 December 2003; accepted 11 December 2003

Abstract
The complement system constitutes an important component of the defence against foreign organisms, functioning both in innate
and adaptive immune systems. It is potentially harmful also to the own organism and is therefore tightly regulated by a number of
membrane-bound and soluble factors. C4b-binding protein (C4BP) is a potent circulating soluble inhibitor of the classical and lectin
pathways of complement. In recent years, the relationships between the structure of C4BP and its functions have been elucidated using a
combination of computer-based molecular analysis and recombinant DNA technologies. Moreover, two novel functions have recently been
ascribed to C4BP. One is the ability of C4BP to localize complement regulatory activity to the surface of apoptotic cells via its interaction
with the membrane-binding vitamin K-dependent protein S. The other is the ability of C4BP to act as a survival factor for B cells due to an
interaction with CD40. The complement regulatory activity of C4BP is not only beneficial because it is also explored by pathogens such
as Neisseria gonorrhoeae, Bordetella pertussis, Streptococcus pyogenes, Escherichia coli K1, and Candida albicans, that bind C4BP to
their surfaces. This contributes to the serum resistance and the pathogenicity of these bacteria. In this review, the structural requirements
and functional importance of the interactions between C4BP and its various ligands are discussed.
2004 Elsevier Ltd. All rights reserved.
Keywords: Complement; C4b-binding protein; Protein S; Coagulation; B cells; CD40; Hostpathogen interactions

1. Regulators of the complement system

The innate immune system is able to provide protection
against pathogens without previous exposure and immunization. The complement system is a key component of the innate immune system. It consists of more than 30 proteins and
not only guards against invading microorganisms with the
help of its opsonic, inflammatory and lytic activities, but it
also enhances the adaptive immunity (Fearon, 1998; Fearon
and Locksley, 1996; Hoffmann et al., 1999) and participates
in the process of clearance of apoptotic cells (Fishelson
Abbreviations: C3b, activated complement factor 3; C4b, activated
complement factor 4; C4BP, C4b-binding protein; CCP, complement control protein domain; CR1, complement receptor 1; DAF, decay accelerating factor; EGF, epidermal growth factor; Gla, -carboxyglutamic
acid; FH, factor H; FI, factor I; LG, laminin G-type domain; LOS,
lipooligosacharide; LRP, low density lipoprotein receptor-related protein;
OmpA, outer membrane protein A; PS, protein S; por1A/1B, porin 1A/1B;
SAP, serum amyloid P component
Corresponding author. Tel.: +46-40-33-72-28; fax: +46-40-33-70-44.
E-mail address: anna.blom@klkemi.mas.lu.se (A.M. Blom).
1 Tel.: +33-6-11-72-93-74; fax: +33-1-44-07-17-72.
0161-5890/$ see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.molimm.2003.12.002

et al., 2001). The complement system can be activated via

three major routes: the classical, the alternative and the
lectin pathway (Walport, 2001) (Fig. 1). Independent of the
way the complement is initiated it proceeds to proteolytic
activation of the major complement protein C3 and the subsequent assembly of the membrane attack complex (MAC).
The complement cascade is potentially destructive to
the organism and is therefore tightly regulated by several
inhibitors, which protect the own tissues of the host. The
majority of the complement inhibitors are regulating the
activity of the C3-convertases, which are crucial enzymatic
complexes of all three pathways of complement whose
functions are to activate C3. The C4bC2a complex is the
C3-convertase of the classical and lectin pathways, whereas
C3bBb is the corresponding convertase of the alternative
pathway. The complement inhibitors are either located on
cell membranes or present in soluble form in different body
fluids. Many of the complement inhibitors are composed
almost exclusively of complement control protein (CCP)
domains (between 4 and 35), also known as short consensus
repeats (SCRs) or sushi domains (Barlow et al., 1993). The
CCP-containing proteins inhibit the C3-convertases either

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A.M. Blom et al. / Molecular Immunology 40 (2004) 13331346

ical importance in vivo of these functions has not been elucidated. To date, no cases of C4BP deficiency have been
described, which could otherwise have shed more light on
the physiological function of C4BP. Knock-out mice lacking C4BP are not yet described but when produced they will
hopefully provide insights into functional importance of this
unique protein. In this review, we will discuss recent results
of structurefunction studies as well as the novel functions
of C4BP.

2. C4BPfrom gene to protein

Fig. 1. Three pathways by which human complement system can be activated. There are several physiological effects accompanying complement
activation: removal of apoptotic cells, opsonisation of pathogens and immune complexes for phagocytosis, release of anaphylatoxins and lysis.

by increasing the dissociation of the enzyme complexes (acceleration of decay) or by being cofactors in the proteolytic
degradation of C3b or C4b by the serine proteinase factor
I (FI).
There are two major soluble CCP-containing complement inhibitors in plasma, C4b-binding protein (C4BP)
that inhibits the classical and lectin pathways, and factor
H (FH) that controls the alternative route. In the recent
years, FH-like molecules were identified but their roles in
the inhibition of complement are unknown (Zipfel et al.,
2002). Membrane-associated CCP-containing complement
inhibitors include the transmembrane complement receptors
1 (CR1) and 2 (CR2) and membrane cofactor protein (MCP)
as well as GPI-anchored decay accelerating factor (DAF).
All these inhibitors are encoded by genes localized on chromosome 1, and are referred to as family of Regulators of
Complement Activation (RCA) (Hourcade et al., 1992).
C4BP is the only circulating complement inhibitor with
a polymeric structure, the molecule being composed of 68
identical -chains and a single unique -chain. It is unique
in that it circulates in complex with the vitamin K-dependent
protein S, which provides the C4BP with the ability to
interact with negatively charged phospholipid membranes.
Apart from having a protective role for the host organism
as inhibitor of complement, C4BP can also be captured on
the surface of a number of human pathogens, which renders them resistant to complement and contributes to their
pathogenic potential. Thus, depending on the circumstances
where C4BP is located the molecule can be considered as
a friend or foe. In the recent years, several novel functions
have been ascribed to C4BP, such as the regulation of complement on apoptotic cells and binding to and stimulation
of CD40 on certain immune cells. However, the physiolog-

C4BP is a large glycoprotein (570 kDa) with an estimated

plasma concentration of 200 mg/l (Dahlbck, 1983). C4BP
exists in several isoforms having different combinations of
- and -chains. The major isoform, which constitutes about
7580% of C4BP in plasma, is composed of seven identical
-chains and one -chain, the chains being linked together
in their C-terminal parts (Fig. 2) (Hillarp and Dahlbck,
1990; Scharfstein et al., 1978). Other less abundant forms
are composed of six -chains and one -chain or exclusively
of seven -chains (Hillarp et al., 1989; Sanchez Corral et al.,
1995). The isoform pattern can be modified by factors with
differential effects on the expression of the two genes coding
for - and -chains (Sanchez Corral et al., 1995).
The - and -chains contain eight and three CCP domains, respectively. The C-terminal extensions (60 amino
acid residues long) of both - and -chains contain two
cysteine residues each and an amphiphatic -helix region,
which is required for intracellular polymerisation of the
molecule (Kask et al., 2002). The cysteins link the different
chains by disulphide bridges. The C4BP molecule is assembled in endoplasmatic reticulum. The -chain subunit is not
required for the polymerisation of the -chains, which in

Fig. 2. Schematic representation of C4BP with indicated binding sites

for various ligands. C4BP is a polymer of seven identical -chains that
harbour in their N-terminal parts binding sites for several ligands. The
unique -chain present in 70% of molecules forms high affinity complex
with anticoagulant PS.

A.M. Blom et al. / Molecular Immunology 40 (2004) 13331346

principle is similar to what has been shown for the J-subunit

of hexameric IgM (Niles et al., 1995). However, the -chain
when expressed alone (i.e. without the -chain) is retained
in the cells and degraded (unpublished observation). When
analysed by electron microscopy, C4BP displays a spider or
octopus-like shape with each of the extended tentacles being
formed by an -chain (Dahlbck et al., 1983). Synchrotron
X-ray scattering and hydrodynamic analysis suggested
C4BP in solution to behave as a bundle of seven extended
arms being held together at their C-termini and having
an average armaxis angle of 10 (Perkins et al., 1986).
The -chain contains a high affinity binding site for the
vitamin K-dependent protein S and all -chain-containing
C4BP molecules in plasma circulate in complex with protein S (PS) (Dahlbck et al., 1983; Hillarp and Dahlbck,
1988; Hrdig and Dahlbck, 1996). Protein S contains a
Gla-domain, which binds negatively charged phospholipids
that help localize the C4BP-protein S complex to certain cell
membranes.
The genes encoding the C4BP -chain (C4BPA) and
-chain (C4BPB) are located in the RCA gene cluster on
the long arm of chromosome 1 in the vicinity of other
CCP-containing complement inhibitors (Andersson et al.,
1990). The two genes are only 4 kb apart and arranged
head to tail, which supports the hypothesis that the two
genes are the result of a gene duplication event. C4BP is
mainly synthesized in the liver, but there are reports of
secondary sites of synthesis such as monocytes, in which
C4BP mRNA was detected and shown to increase upon
stimulation with interferon and (Lappin and Whaley,
1990). In addition, mRNA for the -chain, but not for the
-chain, has been detected in human ovary, the functional
significance of which is unknown (Criado Garcia et al.,

1335

1999). The plasma level of C4BP appear to be hormonally

regulated as it increases during pregnancy and in women
using oral contraceptives (Malm et al., 1988b). The C4BP
plasma concentrations in premature and term newborns
are 5 and 20% of adult levels, respectively (Malm et al.,
1988a; Melissari et al., 1988; Moalic et al., 1988). C4BP is
an acute phase reactant, its plasma level being elevated up
to four-fold during inflammation (Barnum and Dahlbck,
1990; Boerger et al., 1987; Saeki et al., 1989). During the
acute phase, the synthesis of the -chains, but not of the
-chain, increases and as a consequence, the plasma levels
of the isoforms of C4BP lacking -chain increase whereas
the level of -chain-containing C4BP remains unaffected
(Garcia de Frutos et al., 1994). This is consistent with results
from in vitro experiments demonstrating the expression of
- and -chains to be regulated in different ways by cytokines in the liver-derived cell line Hep3B (Criado Garcia
et al., 1995). A number of questions concerning regulation
of C4BP biosynthesis both in health and disease remain
unanswered.

3. C4BP in other species

C4BP was analyzed at the cDNA level in several species.
Perhaps the most relevant, due to existence of knock-out
technology, was analysis of mouse C4BP. Mouse C4BP
-chain is relatively similar to its human counterpart but
lacks two CCP domains (five and six) and the two cysteine
residues in the C-terminal region (Fig. 3) (Kristensen et al.,
1987). However, the protein is able to form non-covalent
polymers (Kaidoh et al., 1981), similar to what we have observed for human -chains in which the two cysteines were

Fig. 3. C4BP in various species. cDNA coding for - and -chain of C4BP were cloned from several species. Positions of potential N-linked carbohydrates
are denoted with a vertical line and a filled circle. The cysteine residues that are present in the C-terminal extensions are indicated by vertical lines.

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A.M. Blom et al. / Molecular Immunology 40 (2004) 13331346

removed by mutagenesis (Kask et al., 2002). Mouse C4BP

contains no -chain because the mouse -chain gene has
evolved into a pseudogene (Rodriguez de Cordoba et al.,
1994). Bovine, rat and rabbit -chains are similar to the
human -chain in being composed of eight CCPs (Garcia
de Frutos and Dahlbck, 1995; Hillarp et al., 1994; Hillarp
et al., 1997). However, the bovine -chain has three cysteines in C-terminal extension as opposed to two in human
protein. The consequence of this is unknown as the bovine
C4BP has never been purified and characterized. Interestingly, in contrast to mouse, rat -chain is similar to its human counterpart and rat C4BP is able to form a complex
with PS (Hillarp et al., 1997). Bovine -chain has during the
evolution lost its most N-terminal CCP domain and thereby
the ability to interact with PS (Hillarp et al., 1994). Cloning
of rabbit -chain was unsuccessful, implying that the rabbit gene also has evolved into pseudogene (Garcia de Frutos
and Dahlbck, 1995). Comparison of structural elements in
-chains of all the species in which it was cloned revealed
that CCP2, three and eight are the most conserved ones.
Guinea-pig serum contains C4BP that is able to inhibit complement but cDNA from this species has not been cloned
(Burge et al., 1981).

4. Inhibition of complement by C4BPrelationships

between structure and function
C4BP is best known as inhibitor of the classical,
antibody-dependent complement pathway where it controls
C4b-mediated reactions in at least three ways. First, C4BP
acts as a cofactor to the serine proteinase factor I, in the proteolytic inactivation of C4b, which prevents the formation
and reconstitution of the classical C3-convertase (C4bC2a)
(Scharfstein et al., 1978). The mechanism by which C4BP
operates as a cofactor to FI is not fully understood but it
appears that C4b changes its conformation upon binding to
C4BP and becomes susceptible to proteolytic cleavage by
FI. Second, C4BP prevents the assembly of the classical
C3-convertase by binding nascent C4b and finally it accelerates the natural decay of the complex (Gigli et al., 1979).
C4BP also acts as FI cofactor in the cleavage of C3b in the
fluid phase thereby inhibiting to some extent the alternative
pathway of complement as well (Blom et al., 2003a; Seya
et al., 1985, 1995). However, C4BP does not seem to be an
inhibitor of the assembled alternative C3-convertase since it
can not inhibit the fluid phase alternative pathway C3 convertase (Seya et al., 1985) and it does not reduce the hemolytic
activity of cell-bound C3b unless present at very high concentration (Blom et al., 2003a; Fujita and Nussenzweig,
1979). Therefore C4BP is not able to fully replace FH, which
is the major fluid phase inhibitor of the alternative pathway.
Most of the effects that C4BP has on the complement
system are mediated by interaction with C4b. At physiological ionic strength, one C4BP molecule is able to interact with four C4b molecules each having KD in micromolar

range (Blom et al., 2003b; Ziccardi et al., 1984). C4BP binds

weakly to C4c but has no affinity for C4d and C4 (Ziccardi
et al., 1984). Recent studies using a panel of C4BP mutants with systematically removed individual CCP domains
as well as monomeric -chains of various lengths showed
that CCP13 form a full binding site for C4b (Fig. 2) (Blom
et al., 2001a; Fukui et al., 2002). The C4BPC4b interaction
is highly sensitive to ionic strength implying that it is based
on ionicelectrostatic interactions between amino acids from
both molecules (Blom et al., 2000a). A cluster of positively
charged amino acids on interface between CCP1 and CCP2
was identified as part of binding site for C4b (Blom et al.,
1999).
Binding of C4b is a prerequisite for the cofactor activity of C4BP and it was demonstrated that the CCP13
region of the -chains contains features required for the
cofactor activity in the cleavage of C4b molecules (Blom
et al., 2001a) and for prevention of assembly and decay of
the classical pathway C3-convertase (Blom et al., 2000b,
2001a). A recent study described two mutants of C4BP,
K126Q/K128Q and F144S/F149S, clustered on -chain
CCP3, which selectively lost their ability to act as cofactors
in the cleavage of both C4b and C3b. Both mutants showed
the same binding affinity for C4b/C3b and had the same
inhibitory effect on formation and decay of the classical
pathway C3-convertase as the wild type C4BP. It appears
that C4b and C3b do not undergo the same conformational
changes upon binding to these C4BP mutants as during the
interaction with the wild type C4BP, which then results in
the observed loss of the cofactor activity.
For the other complement regulators, CR1, MCP, DAF
and FH, the involvement of individual CCP domains in
complement regulatory function and binding of C4b/C3b
have also been investigated. In the case of DAF (four CCPs),
it was shown that the classical pathway C3 convertase
regulatory function resides within CCP2 and CCP3, while
regulation of the alternative pathway requires CCP1, CCP2
and CCP3 (Brodbeck et al., 1996). In MCP (four CCPs),
sites for C4b/C3b interaction have been mapped primarily
to CCP2, CCP3 and CCP4 (Adams et al., 1991; Iwata et al.,
1995). In FH (20 CCPs), there are three C3b-binding sites
localized to CCP14 (Alsenz et al., 1984; Gordon et al.,
1995; Kuhn et al., 1995; Kuhn and Zipfel, 1996), CCP12-14
and CCP19-20 (Jokiranta et al., 2000). CR1 (28 CCPs) is
organized into four repeats each consisting of seven CCP
units. Full ligand binding (C4b, C3b) and functional activity
requires the first four CCPs in each repeat (Klickstein et al.,
1988; Krych et al., 1991, 1994, 1998). Taken together, the
analysis of C4BP and reports on other complement regulators suggest that a basic C3b-/C4b-binding unit consists of
threefour CCP domains.
C4BP binds heparin with a binding site being located
on each of the -chains (Hessing et al., 1990; Sahu and
Pangburn, 1993). Due to the polymeric structure of C4BP
with multiple binding sites the overall avidity of the binding is high. The physiological relevance of the interaction

A.M. Blom et al. / Molecular Immunology 40 (2004) 13331346

between C4BP and heparin and related molecules of heparan sulfate present on cell surfaces is unclear at present.
Screening of a number of malignant cell lines resulted in
the observation that the ovarian adenocarcinoma cell lines
SK-OV-3, Caov-3 and SW626 were capable of binding
C4BP (Holmberg et al., 2001). Functional tests showed that
tumour cell-bound C4BP retained its cofactor activity for
FI-mediated inactivation of C4b thus increasing the control
of classical pathway activation on the surfaces of these cells.
C4BP binding moiety was not identified and it is possible
that binding was mediated by heparan sulphate present on
the cell surface. The C4BPC4b interaction can be inhibited
by heparin, suggesting that the C4b and heparin-binding
sites overlap (Villoutreix et al., 1999). It was shown that
heparin binding ability of C4BP is abolished by the removal
of CCP2 and by insertion of two alanines between CCP1 and
CCP2. In contrast to the dramatic effects on C4b binding,
deletion of CCP3 and CCP1 had only minor effects on heparin binding, suggesting CCP2 to be the most important for

1337

the interaction (Fig. 2) (Blom et al., 2001a). Furthermore,

the binding was diminished in the C4BP mutants lacking
positively charged amino acids at the interface of CCP1 and
CCP2, which also form a C4b-binding site (Blom et al.,
1999).
C4BP was demonstrated to interact with low-density
lipoprotein receptor-related protein, which is endocytic receptor involved in catabolism of several plasma proteins
with heparin-binding properties (Westein et al., 2002). The
binding appears to be mediated by heparin binding site on
-chain of C4BP (Fig. 4) since the recombinant molecule
composed exclusively of -chains binds LRP and the monoclonal antibody directed against the heparin binding region
blocks the interaction. In cellular degradation experiments,
LRP expressing cells bound and degraded C4BP and the
initial clearance of C4BP in mice was delayed upon injection of receptor-associated protein (Westein et al., 2002).
Therefore, LRP may at least in part mediate catabolism of
C4BP.

Fig. 4. Binding sites on -chain of C4BP. To the left is shown 3D homology-based model of the human -chain with indicated potential glycosylation sites
and highlighted residues (in red) corresponding to peptides shown to block the interaction with OmpA of E. coli (Prasadarao et al., 2002). Other peptides
that were tested but did not have an effect are denoted in several colours. On the right: enlarged view on CCP13 with indicated amino acids (blue)
involved in binding of C4b, C3b, heparin and M proteins of S. pyogenes. Also, amino acids specifically required for FI cofactor activity are marked in red.

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A.M. Blom et al. / Molecular Immunology 40 (2004) 13331346

5. Localization of the protein S-binding site

on the -chain of C4BP
The binding site for PS on C4BP is fully contained in
the -chain (Hillarp and Dahlbck, 1988). Using chimeric
molecules composed of a variable number of -chain CCP
domains expressed together with CCP domains from the
-chains it was shown that the binding site for PS is located on the -chain CCP1 (Fig. 5) (Hrdig and Dahlbck,
1996; Hrdig et al., 1993). Later on, some contribution from
CCP2 was also demonstrated (van de Poel et al., 1999). To
elucidate the structural background for the involvement of
CCP2 in the PS binding, a number of additional recombinant
-chain variants were tested. The mutations covered most
of the possible binding surfaces of CCP2 and they all bound
equally well as recombinant wild type to PS (Webb et al.,
2003b). Taken together, the results suggest that the role of
CCP2 in protein S binding is to direct and stabilise CCP1
rather than to directly be part of the binding site. Based on
structural prediction of the -chain followed by the site directed mutagenesis, residues Ile16 , Val18 , Val31 and Ile33 re-

siding at the CCP1 surface were defined as crucial for PS

binding with secondary contributions from Leu38 and Val39
(Webb et al., 2001). In addition, positively charged Lys41
and Lys42 contributed slightly to the interaction. The fact
that mostly hydrophobic amino acids are involved in this interaction is in full agreement with the observation that the
binding is not sensitive to ionic strength (Blom et al., 1998)
and has a very high affinity (KD = 0.2 nM). Interestingly,
PS bound to C4BP is unable to participate in the anticoagulant protein C system (Dahlbck, 1986). It is imperative that
the balance between free and bound PS is maintained at stable levels as lack of free PS leads to thrombosis (Simmonds
et al., 1997; Zller et al., 1995).

6. Interaction of the C4BPPS complex with

apoptotic cells
In plasma, all -chain containing C4BP circulates in a
high affinity complex with PS and the molar excess of PS
(about 30% of total PS) constitutes the free form (Dahlbck,

Fig. 5. Interaction between C4BP -chain and PS. Top (a): PS is composed of several domains and theoretical model structures for several regions
have been reported. The region comprising the Gla (membrane binding domain), thrombin sensitive region (TSR) and EGF1 has been predicted. The
conformation of the TSR is still not clear and this segment could adopt several conformation but does not seem directly involved in membrane binding.
The three other EGF domains can be predicted by homology modelling but the exact organisation of this segment is not known. The SHBG-like region of
PS is formed by two laminin-like domains (LG domain in green and white), and both domains are important for binding of C4BP. The SHBG-like region
was originally predicted using the laminin X-ray structure but was recently refined using the X-ray structure of Gas6 (unpublished data). Segments of the
SHBG-like region of PS proposed as important for the interaction with C4BP from peptide studies, site directed mutagenesis or targeted N-glycosylation
are shown in red. Three Asn residues in the SHBG-like region are glycosylated and it seems that these sugars are not important for C4BP binding. Asp
292 is likely to be involved in calcium binding and it is known that this ion is important for interaction with C4BP. Top (b): model for the first CCP
module (magenta) of the C4BP -chain with a region rich in solvent exposed hydrophobic residues (in yellow) that has been proposed to play a crucial
role in the interaction with PS. C4BP N47 and N54 are glycosylated but it seems that sugar molecules do not play any role in the binding process.
Bottom (c): several docking algorithms have been used to dock C4BP on the surface of PS (unpublished data). In this figure, the orientation of PS is
similar to the one presented above and the colour coding remains (LG2 in white, LG1 in green and regions potentially involved in C4BP binding are in
red). Several models for the complex are presented and are presently under investigation.

A.M. Blom et al. / Molecular Immunology 40 (2004) 13331346

1983; Dahlbck and Stenflo, 1981). PS is a 75 kDa glycoprotein synthesized mainly by hepatocytes, but also endothelial cells and testicular Leydig cells (Fair and Marlar,
1986; Fair et al., 1986; Malm et al., 1994). PS is a multidomain protein beginning at the N-terminus with a Gla domain
(contains -carboxyglutamic acid), which is followed by
the thrombin sensitive region, four epidermal growth factor
(EGF) domains and the C-terminal sex hormone binding
globule (SHBG)-like region. This latter part, which does
not bind steroid hormones, comprises two laminin G-type
domains (LG domains). The Gla domain binds calcium ions
and interacts with negatively charged phospholipids such
as phosphatidylserine (Nelsestuen et al., 1978; Schwalbe
et al., 1989) while the SHBG-region interacts with C4BP
(He et al., 1997; Van Wijnen et al., 1998). The Gla domain
of PS provides the complex with the ability to interact with
negatively charged phospholipids (Schwalbe et al., 1990),
a property, which recently was demonstrated to be involved
in the localization of C4BP to this type of phospholipid on
the surface of apoptotic cells.
Apoptosis is under physiological conditions a wellregulated process, characterized by a lack of induction of
inflammatory responses. The process of removal of apoptotic cells is remarkably complex including a number of
receptors on the phagocytes, various bridging molecules
and several eat me markers on dying cells (Savill et al.,
2002). Among the proteins that bind to apoptotic cells there
are several complement components, most importantly C1q
(Nauta et al., 2002). Binding of the early components of
the classical pathway is thought to be very important in the
clearance since deficiencies of these components are strong
risk factors for the development of systemic lupus erythematosus (SLE) (Walport, 2002). SLE is characterized by the
presence of autoantibodies against cell components associated with apoptotic cells, which are normally not exposed
for prolonged time to the immune system (Casciola-Rosen
et al., 1994). The hypothesis of a potential role of C1q in
clearance of apoptotic cells was further strengthened when
C1q and mannan-binding lectin (MBL) were shown to
stimulate uptake of apoptotic cells by macrophages (Ogden
et al., 2001). In addition, the collagenous tails of C1q and
MBL bind to calreticulin on the macrophage resulting in a
signal via the surface molecule CD91 for ingestion of the
apoptotic cell by macropinocytosis (Ogden et al., 2001).
Once these early components have bound they may have the
potential to activate the complement system which could
further lead to the generation of pro-inflammatory mediators such as C5a, the deposition of C3 and formation of
the membrane attack complex. However, apoptosis is under
physiological conditions characterized by a lack of induction of inflammatory responses in the surrounding tissues
suggesting that the cell are protected from assembly of later
complement components, and that anaphylatoxin release is
prohibited. This suggests the presence of a strong complement inhibitor on the surface of apoptotic cells. It has been
shown that C-reactive protein (CRP) binds apoptotic cells

1339

Fig. 6. Proposed role of C4BP in removal of apoptotic cells. In early

apoptosis, negatively charged phospholipids are exposed and C4BP localized to cell surface via PS. C4BP inhibits the classical pathway that would
otherwise be initiated after binding of C1. C1 interacts with yet unknown
receptors on macrophages and facilitates phagocytosis of the cell.

and augments deposition of initial complement components

of the classical pathway but prevents assembly of terminal
complement components (Gershov et al., 2000).
The C4BPPS complex provides another mechanism
by which apoptotic cells are protected from complement
activation and MAC assembly. In the membrane of viable
cells there is an asymmetric distribution of phospholipids
between the outer and inner leaflet, the negatively charged
phosphatidylserine being predominantly localized in the
inner leaflet (Fig. 6). Early in the process of apoptosis,
phosphatidylserine is transferred to the outer leaflet. Such
surfaces bind vitamin K-dependent coagulation factors such
as PS. The physiological role of the complex between C4BP
and PS has remained an enigma but it has been suggested that
PS, due to its high affinity for negatively charged phospholipids, localizes C4BP to areas where such phospholipids are
exposed (Dahlbck and Stenflo, 1981). When this hypothesis was tested it was demonstrated that the complex binds to
synthetic phospholipid vesicles (Schwalbe et al., 1990), but
not to platelet-derived microparticles even though free PS
efficiently binds to these particles (Dahlbck et al., 1992).
Using Jurkat T-cells and neutrophiles, apoptosis-dependent
binding of C4BP to the cells in the presence, but not absence
of PS was demonstrated (Webb et al., 2002). The binding
was dependent on calcium and was blocked by monoclonal
antibodies directed against the Gla-domain of PS. The C4BP
that was bound via PS to the apoptotic cells was able to interact with the complement protein C4b, suggesting that it
was able to inhibit complement. The PS-mediated binding of
C4BP to apoptotic cells was not cell type-specific, supporting a physiological role of the C4BPPS complex in regulation of complement on the surface of apoptotic cells (Webb
et al., 2003a). Recently, free PS was suggested to work as
one of the factors that stimulates phagocytosis (Anderson
et al., 2003). The possible mechanism of this activity would
be that PS interacts simultaneously with a Gla-domain with
negatively charged phospholipids on surface of apoptotic
cells and as of yet unknown receptors on macrophages. The

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A.M. Blom et al. / Molecular Immunology 40 (2004) 13331346

authors did not address the question whether PS in complex

with C4BP could mediate phagocytosis.

in progress. Also, studies using C4BP knock-out mice will

be of great value to determine physiological importance of
C4BPCD40 interaction.

7. Activation of B cells by C4BPCD40 interaction

CD40 is a cell surface receptor that belongs to the tumor necrosis factor (TNF) receptor family and was first
identified and functionally characterized on B cells but
it is also present on monocytes, dendritic cells, endothelial and epithelial cells. The classical activator of CD40
is the CD40 ligand (CD40L), which is present on activated CD4+ T-cells, mast cells, basophils, eosinophils
and in some conditions of B cells, NK cells, monocytes
and dendritic cells. CD40CD40L has a pivotal role in
T-cell-dependent B cell responses but may also play a more
general role in immune regulation (Banchereau et al., 1994).
Engagement of B cell CD40 with CD40 ligand results in
the up-regulation of the surface expression of a number of
markers, proliferation and rescue of germinal center B cells
from apoptosis (Calderhead et al., 2000). In addition, CD40
ligation in the presence of interleukin 4 and 13 induces
immunoglobulin isotype switching to IgE (Oettgen, 2000).
The interaction is crucial for isotype switching as shown by
the fact that patients with hyper-IgM syndrome often carry
mutations in CD40 or CD40L (Schneider, 2000).
It has been recently demonstrated that C4BP binds directly
to CD40 on human peripheral blood B cells and several B
cell lines (Brodeur et al., 2003). Binding of C4BP to CD40,
although at a site that does not overlap to a large extent with
that used by CD40L, induces proliferation, up-regulation of
CD54 (ICAM-1) and CD86 as well as IL4-dependent IgE
isotype switching in normal B cells (Brodeur et al., 2003).
These effects were not observed for B cells from patients
with CD40 or IKK/NEMO (relevant signalling pathway)
deficiencies confirming that the effects are mediated by a direct C4BPCD40 interaction. The concentrations of C4BP
used in the study were five to eight-fold lower than physiological levels in the blood. However, the circulating B cells
are most probably not important target for C4BP partly because only a very small fraction of B cells is present in
circulation and the high C4BP concentration would lead to
constant stimulation. Also, other proteins present in plasma
may compete with binding of C4BP such as SAP. SAP is
known to bind C-terminal region of C4BP -chains, a region
that is homologous to p23, a bovine serum protein first identified as CD40 ligand (Morio et al., 1995). However, there
are other locations where C4BPCD40 interaction could be
of importance. For instance, it was demonstrated that C4BP
co-localizes with B cells but not T-cells in the secondary
lymphoid follicles in human tonsils (Brodeur et al., 2003).
The selective presence of C4BP in B cell areas suggests that
C4BP is either carried by B cells transmigrating from circulation or produced locally. The interaction is mediated by
the -chain of C4BP and detailed studies of structural requirements for the interaction for both C4BP and CD40 are

8. Binding of C4BP to pathogenic bacteria renders

them resistant to complement attack
Infectious agents such as viruses, bacteria and parasites
are constantly developing strategies to avoid clearance and
destruction by the complement system. When the strategy
to avoid recognition by the complement fails, a number
of pathogens employ complement inhibitors for protection.
Some pathogens are able to hijack hosts complement regulators such as C4BP and FH and subsequently down regulate
complement activation. Others produce their own regulators
with remarkable similarity to the hosts own proteins (for review see Lindahl et al., 2000). C4BP has been demonstrated
to bind to a number of microorganisms and their number
is constantly increasing. In some cases it was possible to
directly correlate the binding with resistance of bacteria to
complement-mediated killing. Inhibition of complement by
C4BP leads to decreased opsonisation of the bacteria with
C3b, which in turn results in decrease in phagocytosis that is
the major weapon against the pathogens (Fig. 7). The number of pathogens (bacteria, yeast, parasites) that are able to
bind C4BP and/or FH and similar molecules is increasing
and it can be speculated that all pathogens that must at some
stage survive contact with blood are able to protect themselves by this mechanism. Another aspect of the observation
that most of complement inhibitors bind to pathogens is that
one has to consider it while planning to use complement
inhibitors as protection during xenotransplantation.
8.1. Streptococcus pyogenes
Streptococcus pyogenes (group A Streptococcus) is one of
the most common causes of bacterial infections in humans
and can bring about a wide array of illnesses such as pharyngitis (strep throat), skin infection impetigo, necrotising
fasciitis, septicemia and toxic shock syndrome sometimes
followed by serious sequelae such as rheumatic fever or
glomerulonephritis. Important virulence factors, M proteins
form fibrillar coiled-coil dimers on the streptococcal surface
and have been studied extensively due to their important
ability to inhibit phagocytosis allowing bacteria to multiply
in blood (Fischetti, 1989; Lancefield, 1962). A remarkable
property of M proteins is their ability to bind a number of human plasma proteins such as immunoglobulins (Frithz et al.,
1989; Heath and Cleary, 1989; Johnsson et al., 1994;
Stenberg et al., 1994), complement inhibitors (Kotarsky
et al., 1998), fibrinogen, fibronectin (Katerov et al., 1998)
and albumin (Frick et al., 1994). C4BP was also shown
to bind with high affinity to streptococcal M proteins
(Thern et al., 1995). Studies of several different M proteins
showed that the binding site for C4BP is localized to the

A.M. Blom et al. / Molecular Immunology 40 (2004) 13331346

1341

Fig. 7. Pathogens capturing C4BP are protected from complement mediated lysis and phagocytosis. C4BP bound to the surface of a pathogen inhibits
classical C3-convertase and serves as FI cofactor in cleavage of C4b both in solution and surface-bound, which leads to decrease in opsonisation and
less efficient phagocytosis.

hypervariable N-terminal region of the M proteins (Johnsson

et al., 1996). This finding implies that the interaction with
C4BP is of physiological importance, since the ability to
bind C4BP has been retained in spite of extensive sequence
variation in the hypervariable region. M proteins interact
with CCP1CCP2 of -chain and their binding site overlaps to some extent with the binding site for C4b (Fig. 2)
(Accardo et al., 1996; Blom et al., 2000a). The interaction
appears to be based essentially on non-ionic/hydrophobic
contacts, which yields binding of high affinity. The interaction with C4BP is restricted to primates (Accardo et al.,
1996; kerstrm et al., 1991), a finding that may be related to the fact that S. pyogenes normally causes disease
only in humans. Most importantly, the ability to bind C4BP
was recently correlated with phagocytosis resistance of
these bacteria (Carlsson et al., 2003; Morfeldt et al., 2001).
It appears that deposition of complement on S. pyogenes
occurs almost exclusively via the classical pathway, even
under non-immune conditions, but is down regulated by
bacteria-bound C4BP, providing an explanation for the ability of bound C4BP to inhibit phagocytosis (Carlsson et al.,
2003).
8.2. Bordetella pertussis
Filamentous hemagglutinin from Bordetella pertussis, an
etiologic factor of a whooping cough, is another surface protein known to interact with C4BP (Berggrd et al., 1997).

There is however, at least one more as yet elusive component

on the bacterial surface that contributes to this interaction.
The binding is very similar to that between C4BP and C4b
and may be an example of a molecular mimicry. The interaction is based on ionic interactions and requires a cluster
of charged amino acids at the CCP1CCP2 interface of the
-chain (Berggrd et al., 2001).
8.3. Neisseria gonorrhoeae
Neisseria gonorrhoeae is a human-specific pathogen that
causes the sexually transmitted disease gonorrhea. The bacteria colonize mucosal surfaces of the urethra, endocervix,
conjunctiva, fallopian tube, rectum and pharynx. Occasionally, gonococci disseminate systematically to cause severe
diseases including bacteremia, which may lead to purulent
arthritis, pelvic inflammatory disease and endocarditis. N.
gonorrhoeae have devised several ways of protection against
complement attack. Apart from binding FH to sialylated
lipooligosaccharide (LOS) and porin Por1A (Ram et al.,
1998a, b) they also employ porin molecules (Por1A and
Por1B) to bind C4BP (Ram et al., 2001). C4BPPor1B interaction is ionic in nature (inhibited by high salt as well as by
heparin), while the C4BPPor1A bond is hydrophobic. Only
recombinant C4BP mutant molecules that contain -chain
CCP1 bind both Por1A and Por1B gonococci, implying that
CCP1 contains porin-binding sites (Fig. 2). Using allelic
exchange to construct strains with hybrid porin molecules it

1342

A.M. Blom et al. / Molecular Immunology 40 (2004) 13331346

was demonstrated that the N-terminal loop (loop 1) of Por1A

and Por1B loops 5 and 7 together are required for C4BP
binding. Furthermore, pilC subunit of type IV pili from N.
gonorrhoeae bind CCP12 of human C4BP (Blom et al.,
2001b). Inhibition of C4BP binding to serum resistant Por1A
and Por1B strains in a serum bactericidal assay using Fab
fragments against C4BP CCP1 results in complete killing of
otherwise fully serum resistant strains, underscoring the role
of C4BP in mediating gonococcal serum resistance (Ram
et al., 2001). Interestingly, it appears that LOS structure
can impact binding of C4BP to gonococcal porins. Strains
with no hexose substitutions on the HepI chain do not bind
C4BP as well as isogenic mutants with one to four hexoses
branching from HepI. Consistent with decreased C4BP binding, decreased resistance to complement-mediated killing
was observed in serum bactericidal assays (unpublished
data).

albicans can also cause life threatening systemic infections

especially in immunocompromised and granulocytopenic
patients (Fisher-Hoch and Hutwagner, 1995). Invasive infections with C. albicans are difficult to diagnose and to
treat and with mortality of a hematogeneously disseminated
infection up to 40%. C. albicans activates all three pathways of the complement, but it is unclear how the yeast
evades toxic effects of the activated system. Both yeast and
hyphal forms of C. albicans capture complement alternative
pathway regulators factor H and FHL-1 (Meri et al., 2002)
and now the same was demonstrated for C4BP (Meri et al.,
2004). In hyphae, a prominent binding site was identified at
the tip, which has for a long time been considered important
for tissue penetration and pathogenesis. The binding is mediated by CCP12 of C4BP -chain and as FHL-1 competes
with the binding of C4BP it appears that these two related
complement regulators share the same ligand/receptor on
the surface of Candida (Meri et al., 2004).

8.4. Escherichia coli

8.6. Moraxella catarrhalis
Furthermore, Escherichia coli K1 responsible for meningitis in neonates binds C4BP (Prasadarao et al., 2002). Due
to the need of certain threshold levels of bacteremia for the
development of meningitis, the bacteria must have a capacity to resist serum bactericidal activity in order to multiply.
At first it was suggested that the K1 capsular polysaccharide,
which is a polymer of sialic acid, is necessary for survival
of E. coli in the blood (Kim et al., 1992). It was subsequently shown using outer membrane protein OmpA+ and
OmpA E. coli strains that OmpA confers serum resistance
both in vivo and in vitro (Weiser and Gotschlich, 1991).
The mechanism by which the resistance was conferred
was unclear until it was demonstrated that CCP3 of C4BP
-chain interacts hydrophobically with the N-terminal part
of outer membrane protein A molecule (Figs. 2 and 3)
(Prasadarao et al., 2002). The binding of C4BP to OmpA
was not significantly inhibited in the presence of either C4b
or heparin and was not salt sensitive, implying that it is hydrophobic in nature. A compelling observation in this study
was that the synthetic peptides corresponding to CCP3
sequences block the binding of C4BP to OmpA and also
significantly enhance the serum bactericidal activity. Furthermore, an antibody directed to N-terminal part of OmpA
increased bactericidal activity of human serum. Therefore,
the N-terminus of OmpA could be a suitable target for the
construction of an effective vaccine that would nullify the
binding of C4BP in order to permit complement attack.
8.5. Candida albicans
Candida albicans is the most common human pathogenic
yeast causing cutaneous and mucocutaneous candidiasis
(Pfaller and Wenzel, 1992). In healthy individuals the cellular form of the yeast is often present as a commensal
and may reside harmlessly on the skin, in oral cavity and
urogenital as well as gastrointestinal tracts. However, C.

Moraxella catarrhalis was considered to be a harmless

commensal in the respiratory tract, but is now acknowledged
as an important mucosal pathogen. It is the third leading
bacterial cause of acute otitis media in children after Streptococcus pneumoniae and Haemophilus influenzae and is
also a common cause of sinusitis and lower respiratory tract
infections in adults with chronic obstructive pulmonary disease (Murphy, 1996).It has been recently shown that C4BP
binds to ubiquitous surface proteins 1 and 2 (Usp1, Usp2)
of Moraxella with Usp2 being the major binder (Nordstrm
et al., 2004). It has been previously demonstrated that Usp2
mediates serum resistance of the bacteria, which could be
due to the binding of C4BP. It has been suggested previously
that Moraxella binds vitronectin, which could also contribute
to resistance against serum (McMichael et al., 1998). The
binding sites for Usp2 on C4BP includes CCP2 and CCP7
and is based on ionic interactions. The KD of interaction between single arm of C4BP and recombinant, purified Usp2
was determined by Biacore to be 1.1 M but the affinity observed in physiological conditions should be much higher
due to polymeric nature of C4BP yielding multiple binding
sites.

9. Concluding remarks
C4BP is a fascinating protein with a number of important
functions some of which are probably still waiting to be
discovered. C4BP is important for inhibition of unwanted
or excessive complement activation, controlled removal of
apoptotic cells without evoking inflammatory reactions as
well as survival of B cells. However, a number of human
pathogens have developed the ability to capture C4BP to
their surface and protect themselves from adverse effects of
complement system.

A.M. Blom et al. / Molecular Immunology 40 (2004) 13331346

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