Detection of autoantibodies against the globular domain of human C1q in the sera of systemic lupus erythematosus patients

The anti-C1q antibodies present in systemic lupus erythematosus (SLE) patients' sera are associated with renal involvement and the titer of these autoantibodies correlates with the clinical activity of the disease. It has previously been shown that anti-C1q antibodies bind neo-epitopes within the collagen region of human C1q. Evidence that these polyclonal autoantibodies recognize epitopes within the globular domain (gC1q) of the molecule has not been documented. In this study, we screened, using ELISA, a number of sera from SLE patients for the presence of anti-gC1q autoantibodies using recombinant globular head regions of individual A (ghA), B (ghB) and C (ghC) chains of human C1q. The recombinant proteins were used as test antigens to determine the levels of autoantibodies directed against ghA, ghB and ghC. SLE sera, containing high levels of anti-C1q antibodies, showed differentially increased binding towards ghA and ghB, which suggested that the gC1q domain can also be target of anti-C1q antibodies generated in SLE patients. Such antibodies can have severe pathophysiological consequences since these are likely to further impair the ability of C1q to clear immune complexes.     

New insight into the autoimmunogenicity of the complement protein C1q

C1q along with its physiological role in maintenance of homeostasis and normal function of the immune system is involved in pathological conditions associated with repetitive generation of anti-C1q autoantibodies. The time and events that cause their first appearance are still unknown. We addressed this issue by analyzing the immunogenicity of C1q in two target groups—one of non-diseased humans and the other of lupus nephritis (LN) patients whose autoimmune disorder is associated with high titers of anti-C1q autoantibodies. The non-diseased humans were represented by pregnant women because the sex hormones are thought to be involved in triggering autoimmune pathologies by their ability to tip the balance of female adaptive immune response to production of antibodies.We screened, using ELISA, 31 sera from healthy pregnant women for the presence of IgM and IgG classes of autoantibodies, recognizing epitopes within the native C1q molecule, its collagen-like region (CLR) and globular head fragment (gC1q). The latter was represented by recombinant analogs of the three globular fragments of A, B and C chains, comprising C1q-ghA, ghB and ghC. We did not find IgM antibodies for all test-antigens which suggest that the natural IgM antibodies are not involved in triggering autoimmunity to C1q. Still more, we did not detect anti-CLR antibodies which have been proved pathogenic in already manifested LN. We completed the analysis with comparative epitope mapping of gC1q and we found similar immunogenic behavior in both target groups—ghA and ghC contained the immunodominant epitopes. This implies that the initial immune response to C1q might occur when the molecule has interacted with its ligands via ghB as part of gC1q. The presence of anti-gC1q in both healthy and diseased humans also implies that these antibodies, unlike anti-CLR, may have a contribution to an onset of autoimmunity.     

Anti-C1q autoantibodies specific against the globular domain of the C1qB-chain from patient with lupus nephritis inhibit C1q binding to IgG and CRP

Lupus nephritis is one of the most severe manifestations of systemic lupus erythematosus. Higher titers of serum anti-C1q autoantibodies correlate with disease activity in patients with lupus nephritis. Anti-C1q autoantibodies have been shown to bind neo-epitopes within the collagen region of human C1q. In a preliminary study, we recently reported that the anti-C1q autoantibodies could also recognize epitopes within the globular domain (gC1q) of the C1q molecule. Here, 38 sera from patients with renal biopsy-proven lupus nephritis were screened for the presence of anti-gC1q autoantibodies, using recombinant globular head regions of individual A (ghA), B (ghB) and C (ghC) chains of human C1q. We isolated anti-gC1q autoantibodies from three selected patients. Human C1q was pre-incubated with increasing concentrations of the isolated anti-ghA, anti-ghB or anti-ghC autoantibodies and its binding to different C1q target molecules such as IgG and CRP was then evaluated. Anti-ghB, but not anti-ghA and anti-ghC autoantibodies, markedly inhibited C1q interaction with IgG as well as CRP. These results appear to suggest that the anti-ghB autoantibodies may partially induce acquired functional C1q deficiency and thus may interfere with the biological function of C1q.     

C1q and its growing family

C1q is the target recognition protein of the classical complement pathway and a major connecting link between innate and acquired immunity. As a charge pattern recognition molecule of innate immunity, C1q can engage a broad range of self and non-self ligands via its heterotrimeric globular (gC1q) domain and thus trigger the classical pathway. The trimeric gC1q signature domain has been identified in a variety of non-complement proteins that can be grouped together as a C1q family. The X-ray crystal structures of the gC1q domain of a few members of the C1q family reveal a compact jelly-roll beta-sandwich fold similar to that of the multifunctional tumor necrosis factor (TNF) ligand family, hence the C1q and TNF superfamily. This review is an update on the structural and functional aspects of the gC1q domain of human C1q. We also mention the diverse range of proteins that utilize a gC1q domain in order to reflect on its importance as a versatile scaffold to support a variety of functions.     

C1q and tumor necrosis factor superfamily: modularity and versatility

C1q is the target recognition protein of the classical complement pathway and a major connecting link between innate and acquired immunity. As a charge pattern recognition molecule of innate immunity, C1q can engage a broad range of ligands via its globular (gC1q) domain and modulate immune cells, probably via its collagen region. The gC1q signature domain, also found in many non-complement proteins, has a compact jelly-roll beta-sandwich fold similar to that of the multifunctional tumor necrosis factor (TNF) ligand family. The members of this newly designated 'C1q and TNF superfamily' are involved in processes as diverse as host defense, inflammation, apoptosis, autoimmunity, cell differentiation, organogenesis, hibernation and insulin-resistant obesity. This review is an attempt to draw structural and functional parallels between the members of the C1q and TNF superfamily.     

gC1q-R/p33, a member of a new class of multifunctional and multicompartmental cellular proteins, is involved in inflammation and infection

Human gC1q‐R (p33, p32, C1qBP, TAP) is a ubiquitously expressed, multiligand‐binding, multicompartmental cellular protein involved in various ligand‐mediated cellular responses. Although expressed on the surface of cells, an intriguing feature of the membrane‐associated form of gC1q‐R is that its translated amino acid sequence does not predict the presence of either a sequence motif compatible with a transmembrane segment or a consensus site for a glycosylphosphatidylinositol anchor. Moreover, the N‐terminal sequence of the pre‐pro‐protein of gC1q‐R contains a motif that targets the molecule to the mitochondria and as such was deemed unlikely to be expressed on the surface. However, several lines of experimental evidence clearly show that gC1q‐R is present in all compartments of the cell, including the extracellular cell surface. First, surface labeling of B lymphocytes with the membrane‐impermeable reagent sulfosuccinimidyl 6‐(biotinamido)hexanoate shows specific biotin incorporation into the surface‐expressed but not the intracellular form of gC1q‐R. Second, FACS and confocal laser scanning microscopic analyses using anti‐gC1q‐R IgG mAb 60.11 or 74.5.2, and the fluorophore Alexa 488‐conjugated F(ab′)² goat anti‐mouse IgG as a probe, demonstrated specific staining of Raji cells (>95% viable). Three‐dimensional analyses of the same cells by confocal microscopy showed staining distribution that was consistent with surface expression. Third, endothelial gC1q‐R, which is associated with the urokinase plasminogen activator receptor, and cytokeratin 1 bind ¹²⁵I‐high molecular weight kininogen in a specific manner, and the binding is inhibited dose‐dependently by mAb 74.5.2 recognizing gC1q‐R residues 204–218. Fourth, native gC1q‐R purified from Raji cell membranes but not intracellular gC1q‐R is glycosylated, as evidenced by a positive periodic acid Schiff stain as well as sensitivity to digestion with endoglycosidase H and F. Finally, cross‐linking experiments using C1q as a ligand indicate that both cC1q‐R and gC1q‐R are co‐immunoprecipitated with anti‐C1q. Taken together, the evidence accumulated to date supports the concept that in addition to its intracellular localization, gC1q‐R is expressed on the cell surface and can serve as a binding site for plasma and microbial proteins, but also challenges the existing paradigm that mitochondrial proteins never leave their designated compartment. It is therefore proposed that gC1q‐R belongs to a growing list of a class of proteins initially targeted to the mitochondria but then exported to different compartments of the cell through specific mechanisms which have yet to be identified. The designation ‘multifunctional and multicompartmental cellular proteins’ is proposed for this class of proteins. This work was supported in part by grant RPG‐95068‐06‐CIM from the American Cancer Society. We thank Dr Jolyon Jesty for providing thrombin and prothrombin, and Weibing Zhang for expert technical assistance.     

Staphylococcus aureus protein A recognizes platelet gC1qR/p33: a novel mechanism for staphylococcal interactions with platelets

The adhesion of Staphylococcus aureus to platelets is a major determinant of virulence in the pathogenesis of endocarditis. Molecular mechanisms mediating S. aureus interactions with platelets, however, are incompletely understood. The present study describes the interaction between S. aureus protein A and gC1qR/p33, a multifunctional, ubiquitously distributed cellular protein, initially described as a binding site for the globular heads of C1q. Suspensions of fixed S. aureus or purified protein A, chemically cross-linked to agarose support beads, were found to capture native gC1qR from whole platelets. Moreover, biotinylated protein A bound specifically to fixed, adherent, human platelets. This interaction was inhibited by unlabeled protein A, soluble recombinant gC1qR (rgC1qR), or anti-gC1qR antibody F(ab')(2) fragments. The interaction between protein A and platelet gC1qR was underscored by studies illustrating preferential recognition of the protein A-bearing S. aureus Cowan I strain by gC1qR compared to recognition of the protein A-deficient Wood 46 strain, as well as inhibition of S. aureus Cowan I strain adhesion to immobilized platelets by soluble protein A. Further characterization of the protein A-gC1qR interaction by solid-phase enzyme-linked immunosorbent assay techniques measuring biotinylated gC1qR binding to immobilized protein A revealed specific binding that was inhibited by soluble protein A with a 50% inhibitory concentration of (3.3 +/- 0.7) x 10(-7) M (mean +/- standard deviation; n = 3). Rabbit immunoglobulin G (IgG) also prevented gC1qR-protein A interactions, and inactivation of protein A tyrosil residues by hyperiodination, previously reported to prevent the binding of IgG Fc, but not Fab, domains to protein A, abrogated gC1qR binding. These results suggest similar protein A structural requirements for gC1qR and IgG Fc binding. Further studies of structure and function using a truncated gC1qR mutant lacking amino acids 74 to 95 demonstrated that the protein A binding domain lies outside of the gC1qR amino-terminal alpha helix, which contains binding sites for the globular heads of C1q. In conclusion, the data implicate the platelet gC1qR as a novel cellular binding site for staphylococcal protein A and suggest an additional mechanism for bacterial cell adhesion to sites of vascular injury and thrombosis.     

Structural biology of the C1 complex of complement unveils the mechanisms of its activation and proteolytic activity

C1 is the multimolecular protease that triggers activation of the classical pathway of complement, a major element of antimicrobial host defense also involved in immune tolerance and various pathologies. This 790,000 Da complex is formed from the association of a recognition protein, C1q, and a catalytic subunit, the Ca2+-dependent tetramer C1s-C1r-C1r-C1s comprising two copies of each of the modular proteases C1r and C1s. Early studies mainly based on biochemical analysis and electron microscopy of C1 and its isolated components have allowed for characterization of their domain structure and led to a low-resolution model of the C1 complex in which the elongated C1s-C1r-C1r-C1s tetramer folds into a more compact, "8-shaped" conformation upon interaction with C1q. A major strategy used over the past years has been to dissect the C1 proteins into modular segments to characterize their function and solve their structure by either X-ray crystallography or nuclear magnetic resonance spectroscopy (NMR). The purpose of this review is to focus on this information, with particular emphasis on the architecture of the C1 complex and the mechanisms underlying its activation and proteolytic activity.     

gC1q-R/p33: structure-function predictions from the crystal structure

Human gC1q-R (p33) is a multicompartmental cellular protein expressed on various types of cells and tissues. Although originally isolated as a receptor for C1q by virtue of its specificity for the globular heads of that molecule, a large body of evidence has now been accumulated which shows that in addition to C1q, gC1q-R can serve as a receptor for diverse ligands including proteins of the intrinsic coagulation/bradykinin forming cascade, as well as antigens of cellular, bacterial, and viral origin. Furthermore, since gC1q-R has been shown to regulate the functions of protein kinase C (PKC), it is postulated that gC1q-R-induced signaling cascade may involve activation of PKC. These data collectively therefore suggest that gC1q-R plays an important role in blood coagulation, inflammation, and infection. However, although significant progress has been made in unraveling the molecular, biochemical, and structural features of this molecule, and data in support of its biological relevance is accumulating, it is still unclear as to how the molecule is anchored on the membrane since its sequence is devoid of a classical transmembrane domain or a glycosylphosphatidylinositol (GPI) anchor. Furthermore, while recombinant gC1q-R can bind to cell surfaces suggesting that it may bind directly to the phospholipid bilayer, our recent experiments show that, at least in vitro, gC1q-R does not bind to unilamellar vesicle preparations of either phosphatidylcholine (PC) or phosphatidylserine: phosphatidylcholine. This work was therefore undertaken to analyze the three-dimensional structure of gC1q-R in order to identify unique structural features that may serve not only to anchor the protein but also to explain its affinity for such a diversity of plasma as well as microbial and viral ligands.     

Identification of the gC1qR sites for the HIV-1 viral envelope protein gp41 and the HCV core protein: Implications in viral-specific pathogenesis and therapy

A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogen-associated pattern recognition receptor (PRR). This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host’s immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4⁺ T cells, thereby rendering them susceptible to autologous destruction by NKp44 receptor expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 11 gC1qR deletion mutants. The results obtained from these studies have identified two major HCV core protein sites on a domain of gC1qR comprising of residues 144–148 and 196–202. Domain 196–202 in turn, is located in the last half of the larger gC1qR segment encoded by exons IV–VI (residues 159–282), which was proposed previously to contain the site for HCV core protein. The major gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174–180. Interestingly, gC1qR residues 174–180 also constitute the cell surface-binding site for soluble gC1qR (sgC1qR), which can bind to the cell surface in an autocrine/paracrine manner via surface expressed fibrinogen or other membrane molecules. The identification of the sites for these viral ligands should therefore provide additional targets for the design of peptide-based or antigen-based therapeutic strategies.     

Zinc induces exposure of hydrophobic sites in the C-terminal domain of gC1q-R/p33

Endothelial cells and platelets are known to express gC1q-R on their surface. In addition to C1q, endothelial cell gC1q-R has been shown to bind high molecular weight kininogen (HK) and factor XII (FXII). However, unlike C1q, whose interaction with gC1q-R does not require divalent ions, the binding of HK to gC1q-R is absolutely dependent on the presence of zinc. However, the mechanism by which zinc modulates this interaction is not fully understood. To investigate the role of zinc, binding studies were done using the hydrophobic dye, bis-ANS. The fluorescence intensity of bis-ANS, greatly increases and the emission maximum is blue-shifted from 525 to 485nm upon binding to hydrophobic sites on proteins. In this report, we show that a blue-shift in emission maximum is also observed when bis-ANS binds to gC1q-R in the presence but not in the absence of zinc suggesting that zinc induces exposure of hydrophobic sites in the molecule. The binding of bis-ANS to gC1q-R is specific, dose-dependent, and reversible. In the presence of zinc, this binding is abrogated by monoclonal antibody 74.5.2 directed against gC1q-R residues 204-218. This segment of gC1q-R, which corresponds to the beta6 strand in the crystal structure, has been shown previously to be the binding site for HK. A similar trend in zinc-induced gC1q-R binding was also observed using the hydrophobic matrix octyl-Sepharose. Taken together, our data suggest that zinc can induce the exposure of hydrophobic sites in the C-terminal domain of gC1q-R involved in binding to HK/FXII.     

Recent progress in the understanding of the structure-function relationships of the globular head regions of C1q

The first step in the activation of the classical pathway of complement cascade by immune complexes involves the binding of the C-terminal globular head regions of C1q to the Fc regions of IgG or IgM, each globular head being composed of the C-terminal halves of one A-, one B- and one C-chain. Recent studies using recombinant forms of globular region appear to suggest that each globular head of C1q may be composed of three, structurally and functionally, independent domains/modules. The heterotrimeric organisation thus could offer functional flexibility and versatility to the whole C1q molecule. The crystal structure of an adipocyte-specific serum protein, Acrp-30, has revealed the existence of a structural fold shared by members of a new C1q/tumor necrosis factor (TNF) superfamily, characterized by a distinctive globular domain. The protein members seem to be active as self-assembling noncovalent trimers, whose individual chains fold as compact ‘jellyroll' b sandwiches. The recognition of a C1q/TNF superfamily, which has wide-ranging functions, highlights the possibility that the globular regions of C1q may fulfill more binding functions than previously envisaged.     

Expression of gC1q-R/p33 and its major ligands in human atherosclerotic lesions

A growing body of evidence supports the hypothesis that atherosclerosis has an inflammatory component, and that immune mechanisms, including complement activation, are likely to be involved. gC1q-R/p33 (gC1q-R) is a multifunctional and multicompartmental cellular protein, which is postulated to play a role in inflammation and thrombosis by interacting with C1q and high molecular weight kininogen (HK). To examine the expression of gC1q-R and its major ligands, C1q and HK, in human atherosclerotic lesions, sections of carotid arteries removed during endarterectomy and coronary arteries obtained at autopsy were stained with specific polyclonal or monoclonal antibodies. Control sections were stained with irrelevant rabbit IgG or isotype matched murine monoclonal antibody (MOPC), respectively. Tissue sections were counterstained with hematoxylin and examined by light microscopy. Specific staining for gC1q-R, C1q, and HK was observed in and around atherosclerotic lesions. In contrast to control antibodies, antibodies directed against gC1q-R reacted with endothelial cells, foam cells, smooth muscle cells, and inflammatory cells present in the intima and media of atherosclerotic lesions. In addition, the necrotic central core of advanced lesions with calcifications, fibrin, and lipids, stained intensely for gC1q-R, and negligibly with control antibodies. HK demonstrated a similar staining pattern, whereas C1q was most heavily expressed in the fibrous cap and necrotic core of atherosclerotic lesions. The localization of gC1q-R and its ligands C1q and HK in atherosclerotic lesions, and the previously described ability of gC1q-R to modulate complement, kinin, and coagulation cascades, suggest that gC1q-R may play an important role in promoting inflammation and thrombosis in atherosclerotic lesions.     

Soluble gC1q-R/p33, a cell protein that binds to the globular "heads" of C1q, effectively inhibits the growth of HIV-1 strains in cell cultures

C1q and the outer envelope protein of HIV, gp120, have several structural and functional similarities. Therefore, it is plausible to assume that proteins that are able to interact with C1q may also interact with isolated gp120 as well as the whole HIV-1 virus. Based on this hypothesis, we studied the potential ability of the recombinant form of the 33-kDa protein, which binds to the globular "heads" of C1q (gC1q-R/p33), to inhibit the growth of different HIV-1 strains in cell cultures. gC1q-R/p33 was found to effectively and dose-dependently inhibit the production of one T-lymphotropic (X4) and one macrophage-tropic (R5) strain in human T cell lines (MT-4 and H9) and human monocyte-derived macrophage cultures, respectively. At a concentration range of 5-25 microg/ml, gC1q-R caused a marked and prolonged suppression of virus production. The extent of inhibition was enhanced when gC1q-R was first incubated with and then removed from the target cell cultures before virus infection, compared to that when the cells were infected with gC1q-R-HIV mixtures. The extent of inhibition was comparable to that of the Leu3a anti-CD4 antibody. Addition of gC1q-R to the cell cultures on day 1 or 2 after infection induced markedly less inhibition of HIV-1 growth than pretreatment of the cells just before or together with the infective HIV strains. In ELISA experiments, gC1q-R did not bind to a solid-phase recombinant gp120 while strong and dose-dependent binding of gC1q-R to solid-phase CD4 was observed. Our present findings indicate that gC1q-R is an effective inhibitor of HIV-1 infection, which prevents viral entry by blocking the interaction between CD4 and gp120. Since gC1q-R is a human protein, it is most probably not antigenic in humans. It would seem logical, therefore, to consider gC1q-R or its fragments involved in the CD4 binding as potential therapeutic agents.     

Structural biology of C1: dissection of a complex molecular machinery

The classical pathway of complement is initiated by the C1 complex, a multimolecular protease comprising a recognition subunit (C1q) and two modular serine proteases (C1r and C1s) associated as a Ca²⁺‐dependent tetramer (C1s‐C1r‐C1r‐C1s). Early studies have allowed identification of specialized functional domains in these proteins and have led to low‐resolution models of the C1 complex. The objective of current studies is to gain deeper insights into the structure of C1, and the strategy used for this purpose mainly consists of dissecting the C1 components into modular fragments, in order to solve their three‐dimensional structure and establish the structural correlates of their function. The aim of this article is to provide an overview of the structural and functional information generated by this approach, with particular emphasis on the domains involved in the assembly, the recognition function, and the highly specific proteolytic properties of C1. The original work cited in this review was supported in part by the Commissariat à l’Energie Atomique (CEA), the Centre National de la Recherche Scientifique, and the European Union Biotechnology programme.     

The receptor for the globular "heads" of C1q, gC1q-R, binds to fibrinogen/fibrin and impairs its polymerization

The 33-kDa cellular C1q binding protein, designated gC1q-R was previously shown to bind a number of plasma proteins involved in the coagulation and kinin systems. This study demonstrates the interaction between recombinant gC1q-R and fibrinogen. Using enzyme-linked immunosorbent assays, biotinylated gC1q-R was found to bind to microplate-immobilized fibrinogen in a manner which was specific and inhibited by excess soluble fibrinogen or polyclonal antibodies directed against either gC1q-R or fibrinogen. Moreover, gC1q-R inhibited fibrin polymerization in a dose-dependent manner. Reptilase induced fibrin clot formation was completely inhibited by gC1q-R at a 2:1 molar ratio (gC1q-R:fibrinogen), and repolymerization of thrombin induced fibrin monomers was similarly abrogated. At equivalent molar concentrations, gC1q-R appeared to be a more potent inhibitor of fibrin polymerization than fibrinogen, a well-known inhibitor. Moreover, in the presence of both gC1q-R and soluble fibrinogen, the effect of each inhibitor on fibrin polymerization was additive. When plasmin derived fibrinogen degradation products, including the C-terminal D domain (D-100) or the N-terminal E domain, were immobilized on microtiter plates, gC1q-R bound to fibrinogen fragment D-100, but not to fragment E. Further digestion of fibrinogen fragment D-100 by plasmin to fragment D-60 resulted in loss of gC1q-R binding. Thus, gC1q-R binds to the D domain of fibrinogen/fibrin, and the carboxyterminal segment of at least the fibrinogen/fibrin gamma chain appears important for this interaction. These observations may suggest a potential role for gC1q-R in modulating fibrin formation particularly at local sites of immune injury or inflammation.     

The Receptor for the Globular “Heads” of C1q, gC1q-R, Binds to Fibrinogen/Fibrin and Impairs Its Polymerization

The 33-kDa cellular C1q binding protein, designated gC1q-R was previously shown to bind a number of plasma proteins involved in the coagulation and kinin systems. This study demonstrates the interaction between recombinant gC1q-R and fibrinogen. Using enzyme-linked immunosorbent assays, biotinylated gC1q-R was found to bind to microplate-immobilized fibrinogen in a manner which was specific and inhibited by excess soluble fibrinogen or polyclonal antibodies directed against either gC1q-R or fibrinogen. Moreover, gC1q-R inhibited fibrin polymerization in a dose-dependent manner. Reptilase induced fibrin clot formation was completely inhibited by gC1q-R at a 2:1 molar ratio (gC1q-R:fibrinogen), and repolymerization of thrombin induced fibrin monomers was similarly abrogated. At equivalent molar concentrations, gC1q-R appeared to be a more potent inhibitor of fibrin polymerization than fibrinogen, a well-known inhibitor. Moreover, in the presence of both gC1q-R and soluble fibrinogen, the effect of each inhibitor on fibrin polymerization was additive. When plasmin derived fibrinogen degradation products, including the C-terminal D domain (D-100) or the N-terminal E domain, were immobilized on microtiter plates, gC1q-R bound to fibrinogen fragment D-100, but not to fragment E. Further digestion of fibrinogen fragment D-100 by plasmin to fragment D-60 resulted in loss of gC1q-R binding. Thus, gC1q-R binds to the D domain of fibrinogen/fibrin, and the carboxyterminal segment of at least the fibrinogen/fibrin γ chain appears important for this interaction. These observations may suggest a potential role for gC1q-R in modulating fibrin formation particularly at local sites of immune injury or inflammation.     

A hantavirus causing hemorrhagic fever with renal syndrome requires gC1qR/p32 for efficient cell binding and infection

Hantaan virus (HTNV) is a pathogenic hantavirus that causes hemorrhagic fever with renal syndrome (HFRS). HTNV infection is mediated by αvβ3 integrin. We used protein blots of Vero E6 cell homogenates to demonstrate that radiolabeled HTNV virions bind to gC1qR/p32, the acidic 32-kDa protein known as the receptor for the globular head domain of complement C1q. RNAi-mediated suppression of gC1qR/p32 markedly reduced HTNV binding and infection in human lung epithelial A549 cells. Conversely, transient expression of either simian or human gC1qR/p32 rendered non-permissive CHO cells susceptible to HTNV infection. These results suggest an important role for gC1qR/p32 in HTNV infection and pathogenesis.     

The contribution of gC1qR/p33 in infection and inflammation

Human gC1qR/p33 is a multi-compartmental and multi-functional cellular protein expressed on a wide range of tissues and cell types including lymphocytes, endothelial cells, dendritic cells, and platelets. Although originally isolated as a receptor for C1q by virtue of its affinity (K(d)=15-50 nM), and specificity for the globular heads of this molecule, a large body of evidence has now been accumulated which shows that in addition to C1q, gC1qR can serve as a receptor for diverse proinflammatory ligands including proteins of the plasma kinin-forming system, most notably high molecular weight kininogen (HK; K(d)=9 nM). In addition, gC1qR has been reported to recognize and bind a number of functional antigens of viral and bacterial origin. It is its ability to interact with microbial antigens and its potential to serve as a cellular protein for bacterial attachment and/or entry that has been the focus of our laboratory in the past few years. On the surface of activated platelets, gC1qR has been shown to serve as a binding site for Staphylococcus aureus and this binding is mediated by protein A. Since the binding of S. aureus to platelets is postulated to play a major role in the pathogenesis of endocarditis, gC1qR may provide a suitable surface for the initial adhesion of the bacterium. Recent data also demonstrate that the exosporium of Bacillus cereus, a member of a genus of aerobic, Gram-positive, spore-forming rod-like bacilli, which includes the deadly Bacillus anthracis, contains a binding site for gC1qR. Therefore, by virtue of its ability to recognize plasma proteins such as C1q and HK, as well as bacterial and viral antigens, cell-surface gC1qR not only is able to generate proinflammatory byproducts from the complement and kinin/kallikrein systems, but also can be an efficient vehicle and platform for a plethora of pathogenic microorganisms.