Complement inhibitor C4b-binding protein—friend or foe in the innate immune system?

Molecular Immunology 40 (2004) 1333–1346

Complement inhibitor C4b-binding protein—friend or foe in the innate immune system? Anna M. Blom a,∗ , Bruno O. Villoutreix b,1 , Björn Dahlbäck a a

The Wallenberg Laboratory, Department of Clinical Chemistry, University Hospital Malmö, Lund University, S-205 02 Malmö, Sweden b INSERM U428, University of Paris V, 4 Ave. de L’Observatoire, 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; Host–pathogen 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: [email protected] (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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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 structure–function studies as well as the novel functions of C4BP.

2. C4BP—from 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 6–8 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 (Dahlbäck, 1983). C4BP exists in several isoforms having different combinations of ␣- and ␤-chains. The major isoform, which constitutes about 75–80% 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 Dahlbäck, 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.

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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 (Dahlbäck 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 arm–axis 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) (Dahlbäck et al., 1983; Hillarp and Dahlbäck, 1988; Härdig and Dahlbäck, 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.,

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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 Dahlbäck, 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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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 Dahlbäck, 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 Dahlbäck, 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 C4BP—relationships 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 CCP1–3 form a full binding site for C4b (Fig. 2) (Blom et al., 2001a; Fukui et al., 2002). The C4BP–C4b interaction is highly sensitive to ionic strength implying that it is based on ionic–electrostatic 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 CCP1–3 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 CCP1–4 (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 three–four 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

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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 C4BP–C4b 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

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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 CCP1–3 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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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 Dahlbäck, 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) (Härdig and Dahlbäck, 1996; Härdig 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 (Dahlbäck, 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; Zöller et al., 1995).

6. Interaction of the C4BP–PS 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 (Dahlbäck,

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.

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1983; Dahlbäck 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

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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 C4BP–PS 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 (Dahlbäck 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 (Dahlbäck 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 C4BP–PS 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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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 C4BP–CD40 interaction.

7. Activation of B cells by C4BP–CD40 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. CD40–CD40L 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 C4BP–CD40 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 C4BP–CD40 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 host’s complement regulators such as C4BP and FH and subsequently down regulate complement activation. Others produce their own regulators with remarkable similarity to the host’s 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

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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 CCP1–CCP2 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; Åkerström 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 (Berggård 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 CCP1–CCP2 interface of the ␣-chain (Berggård 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). C4BP–Por1B interaction is ionic in nature (inhibited by high salt as well as by heparin), while the C4BP–Por1A 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

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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 CCP1–2 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 CCP1–2 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 (Nordström 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.

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