Topology and structure of the C1q-binding site on C-reactive protein

The host defense functions of human C-reactive protein (CRP) depend to a great extent on its ability to activate the classical complement pathway. The aim of this study was to define the topology and structure of the CRP site that binds C1q, the recognition protein of the classical pathway. We have previously reported that residue Asp(112) of CRP plays a major role in the formation of the C1q-binding site, while the neighboring Lys(114) hinders C1q binding. The three-dimensional structure of CRP shows the presence of a deep, extended cleft in each protomer on the face of the pentamer opposite that containing the phosphocholine-binding sites. Asp(112) is part of this marked cleft that is deep at its origin but becomes wider and shallower close to the inner edge of the protomer and the central pore of the pentamer. The shallow end of the pocket is bounded by the 112-114 loop, residues 86-92 (the inner loop), the C terminus of the protomer, and the C terminus of the pentraxin alpha-helix 169-176, particularly Tyr(175). Mutational analysis of residues participating in the formation of this pocket demonstrates that Asp(112) and Tyr(175) are important contact residues for C1q binding, that Glu(88) influences the conformational change in C1q necessary for complement activation, and that Asn(158) and His(38) probably contribute to the correct geometry of the binding site. Thus, it appears that the pocket at the open end of the cleft is the C1q-binding site of CRP.     

Regulation of complement activation by C-reactive protein: targeting of the inhibitory activity of C4b-binding protein

C-reactive protein (CRP) is the major acute phase protein in humans. It has been shown that CRP interacts with factor H, an inhibitor of the alternative pathway of complement, and now we demonstrate binding of CRP to the fluid-phase inhibitor of the classical pathway, C4b-binding protein (C4BP). C4BP bound to directly immobilized recombinant CRP as well as CRP attached to phosphorylcholine. The binding was sensitive to ionic strength and was enhanced in the presence of calcium. C4BP lacking beta-chain and protein S, which is a form of C4BP increasing upon inflammation, bound CRP with higher affinity than the C4BP-protein S complex. The binding could not be blocked with mAbs directed against peripheral parts of the alpha-chains of C4BP while the isolated central core of C4BP obtained by partial proteolytic digestion bound CRP, indicating that the binding site for CRP is localized in the central core of the C4BP molecule. Furthermore, we found complexes in serum from a patient with an elevated CRP level and trace amounts of CRP were also identified in a plasma-derived C4BP preparation. We were also able to detect C4BP-CRP complexes in solution and established that C4BP retains full complement regulatory activity in the presence of CRP. In addition, we found that C4BP can compete with C1q for binding to immobilized CRP and that it inhibits complement activation locally. We hypothesize that CRP limits excessive complement activation on targets via its interactions with both factor H and C4BP.     

Localization of the phosphocholine-binding sites on C-reactive protein by immunoelectron microscopy

C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed.     

Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fcgamma receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficiently down-regulated at CRP levels > 150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.     

Protein A--a new ligand for human C-reactive protein

Phosphorylcholine (PC) is a classical ligand of C-reactive protein (CRP), a clinically important acute phase protein. In search of new ligands, CRPs were affinity-purified from several pathological samples, which exhibited distinct molecular variants induced in different diseases. Both glycosylated and non-glycosylated CRPs showed calcium-independent differential-binding to Staphylococcus aureus cell-surface Protein A. CRP possesses separate binding sites for Protein A and PC with different binding constants. We have demonstrated that Protein A is another ligand in addition to PC establishing an extended definition of CRP. Protein A binding may impart immunomodulatory roles of CRP in combating microorganisms or other foreign materials.     

Heme interacts with c1q and inhibits the classical complement pathway

C1q is the recognition subunit of the first component of the classical complement pathway. It participates in clearance of immune complexes and apoptotic cells as well as in defense against pathogens. Inappropriate activation of the complement contributes to cellular and tissue damage in different pathologies, urging the need for the development of therapeutic agents that are able to inhibit the complement system. In this study, we report heme as an inhibitor of C1q. Exposure of C1q to heme significantly reduced the activation of the classical complement pathway, mediated by C-reactive protein (CRP) and IgG. Interaction analyses revealed that heme reduces the binding of C1q to CRP and IgG. Furthermore, we demonstrated that the inhibition of C1q interactions results from a direct binding of heme to C1q. Formation of complex of heme with C1q caused changes in the mechanism of recognition of IgG and CRP. Taken together, our data suggest that heme is a natural negative regulator of the classical complement pathway at the level of C1q. Heme may play a role at sites of excessive tissue damage and hemolysis where large amounts of free heme are released.     

Mapping the binding areas of human C-reactive protein for phosphorylcholine and polycationic compounds. Relationship between the two types of binding sites.

We developed a fluorescence-based assay method for determining ligand binding activities of C-reactive protein (CRP) in solution. Using this method, we compared the phosphorylcholine (PC)- and polycation-based binding activities of human CRP. The PC-based binding required calcium, whereas a polycation (e.g. poly-l-lysine) was bound in the presence of either calcium or EDTA, the binding being stronger in the presence of EDTA. The published crystallographic structures of CRP and the CRP.PC complex show it to be a ring-shaped pentamer with a single PC-binding site per subunit facing the same direction. As expected from such a structure, binding affinity of a ligand increased tremendously when multiple PC residues were present on a macromolecular structure. In addition to PC-related structures, certain sugar phosphates (e.g. galactose 6-phosphate) are bound near the PC-binding site, and one of the sugar hydroxyl groups appears to interact with CRP. The best small ligands for the polycationic binding site were Lys-Lys and Lys4. Because of the presence of multiple Lys-Lys sequences, polylysines have tremendously enhanced affinity. Although PC inhibits both PC- and polycation-based binding, none of the amines that inhibit polylysine binding inhibits PC binding, suggesting that the PC and polycationic binding sites do not overlap.     

Characteristics of the binding of human C-reactive protein (CRP) to laminin

Human CRP binds to the basement membrane protein laminin in vitro in a Ca2+-dependent manner via the phosphorylcholine (PC) binding site of C-reactive protein (CRP). The binding was saturable at a molar ratio of 4 (CRP/laminin). The specificity of the binding was shown by inhibition of binding of labeled CRP to laminin by unlabeled CRP, but not by human IgG. Specific binding was optimal in the presence of 5 mM Ca2+, but did not occur in the absence of Ca2+ or in the presence of EDTA. The binding of Ca2+ to CRP causes a conformational change in the molecule, which is required for binding to PC and to laminin. The PC binding site of CRP was implicated in the binding to laminin on the basis of inhibition by both soluble PC and anti-idiotypic mAbs directed to the TEPC-15 PC-binding idiotype found on mouse antibodies to PC. In addition, mouse mAbs specific for the CRP PC binding site displayed decreased reactivity with CRP already bound to laminin. The binding of CRP to laminin provides a possible explanation for selective deposition of CRP at inflamed sites. The CRP-laminin interaction may serve as a means of concentrating CRP at sites of tissue damage so that the CRP might function as a ligand for leukocytes, an event that will result in removal of necrotic tissue and cell debris.     

Heterocomplexes of mannose-binding lectin and the pentraxins PTX3 or serum amyloid P component trigger cross-activation of the complement system

The long pentraxin 3 (PTX3), serum amyloid P component (SAP), and C-reactive protein belong to the pentraxin family of pattern recognition molecules involved in tissue homeostasis and innate immunity. They interact with C1q from the classical complement pathway. Whether this also occurs via the analogous mannose-binding lectin (MBL) from the lectin complement pathway is unknown. Thus, we investigated the possible interaction between MBL and the pentraxins. We report that MBL bound PTX3 and SAP partly via its collagen-like domain but not C-reactive protein. MBL-PTX3 complex formation resulted in recruitment of C1q, but this was not seen for the MBL-SAP complex. However, both MBL-PTX3 and MBL-SAP complexes enhanced C4 and C3 deposition and opsonophagocytosis of Candida albicans by polymorphonuclear leukocytes. Interaction between MBL and PTX3 led to communication between the lectin and classical complement pathways via recruitment of C1q, whereas SAP-enhanced complement activation occurs via a hitherto unknown mechanism. Taken together, MBL-pentraxin heterocomplexes trigger cross-activation of the complement system.     

Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway

Human fibrinogen (Fg) binds to surface proteins expressed by many pathogenic bacteria and has been implicated in different host-pathogen interactions, but the role of bound Fg remains unclear. Here, we analyse the role of Fg bound to Streptococcus pyogenes M protein, a major virulence factor that confers resistance to phagocytosis. Studies of the M5 system showed that a chromosomal mutant lacking the Fg-binding region was completely unable to resist phagocytosis, indicating that bound Fg plays a key role in virulence. Deposition of complement on S. pyogenes occurred via the classical pathway even under non-immune conditions, but was blocked by M5-bound Fg, which reduced the amount of classical pathway C3 convertase on the bacterial surface. This property of M protein-bound Fg may explain its role in phagocytosis resistance. Previous studies have shown that many M proteins do not bind Fg, but interfere with complement deposition and phagocytosis by recruiting human C4b-binding protein (C4BP), an inhibitor of the classical pathway. Thus, all M proteins may share ability to recruit a human plasma protein, Fg or C4BP, which inhibits complement deposition via the classical pathway. Our data identify a novel function for surface-bound Fg and allow us to propose a unifying mechanism by which M proteins interfere with innate immunity.     

C-reactive protein binds to phosphorylated carbohydrates

C-reactive protein (CRP) is a major acute phase protein in man. In order to more fully understand the physiological role of this serum protein, we have demonstrated high avidity binding for a defined chemically synthesized carbo-hydrate ligand which represents the repeating disaccharide of lipophosphoglycan, the major surface glycoconjugate of the unicellular parasite Leishmania donovani. Increasing the number of phosphorylated disaccharides in a molecule from one up to seven did not increase the avidity for CRP, however increasing this to 10 potential CRP binding sites did. In order to define the important features of this complex and variable structure for CRP binding we competed CRP binding to whole Leishmania parasites with amino, sulfated, phosphorylated, and unsubstituted monosaccharides, of which only phosphorylated monosaccharides were able to inhibit. Both the carbohydrate and the position of phosphorylation influenced the avidity for CRP. Synthetic oligosaccharides and phospho-oligosaccharides of various lengths and conformations were used to define the structural requirements for CRP recognition. The optimum structure for recognition of a single phosphate group was between two monosaccharide pyranose rings, and within a linear rather than a cyclic molecule. This stresses the importance of the interaction of the CRP binding site with both the carbohydrate and the phosphate group. CRP function may be mediated via the recognition of large arrays of phosphorylated carbohydrates as are characteristic of the surface of microorganisms.     

Role of the property of C-reactive protein to activate the classical pathway of complement in protecting mice from pneumococcal infection

C-reactive protein (CRP) is not an acute-phase protein in mice, and therefore, mice are widely used to investigate the functions of human CRP. It has been shown that CRP protects mice from pneumococcal infection, and an active complement system is required for full protection. In this study, we assessed the contribution of CRP's ability of activating the classical pathway of complement in the protection of mice from lethal infection with virulent Streptococcus pneumoniae type 3. We used two CRP mutants, Y175A and K114A. The Y175A CRP does not bind C1q and does not activate complement in human serum. The K114A CRP binds C1q and activates complement more efficiently than wild-type CRP. Passively administered, both CRP mutants and the wild-type CRP protected mice from infection equally. Infected mice injected with wild-type or mutant CRP had reduced bacteremia, resulting in lower mortality and increased longevity compared with mice that did not receive CRP. Thus, the protection of mice was independent of CRP-mediated activation of the classical pathway of complement. To confirm that human CRP does not differentiate between human and mouse complement, we analyzed the binding of human CRP to mouse C1q. Surprisingly, CRP did not react with mouse C1q, although both mutant and wild-type CRP activated mouse C3, indicating species specificity of CRP-C1q interaction. We conclude that the mouse is an unfit animal for exploring CRP-mediated activation of the classical complement pathway, and that the characteristic of CRP to activate the classical complement pathway has no role in protecting mice from infection.     

Complement activation and antibody binding by pneumolysin via a region of the toxin homologous to a human acute-phase protein

Pneumolysin, a membrane-damaging toxin, is known to activate the classical complement pathway. We have shown that 1 microgram ml-1 of pneumolysin can activate complement, which is a much lower level than observed previously. We have identified two distinct regions of pneumolysin which show homology with a contiguous sequence within acute-phase proteins, including human C-reactive protein (CRP). Site-directed mutagenesis of the pneumolysin gene was used to change residues common to pneumolysin and CRP. Some of the modified toxins had a reduced ability both to activate complement and bind antibody. We suggest that the ability of pneumolysin to activate complement is related to its ability to bind the Fc portion of immunoglobulin G.     

Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis

C-reactive protein (CRP) is an acute-phase reactant that is found bound to cells at sites of inflammation. We have passively sensitized HEp-2 cells for CRP binding and examined the effect of this treatment on complement activation and cell lysis. When cells were treated with protamine sulfate and CRP and were incubated with normal human serum in a 4-hr 51Cr-release assay, no significant lysis was noted. In contrast, HEp-2 cells treated with antibody and normal human serum were lysed. The consumption of complement components in normal human serum after incubation with cells treated with protamine and CRP was measured by hemolytic assays. CRP-treated cells consumed over 80% of C1, C4, and C2 and about 40% of C3 present. No significant consumption of C5 through C9 components was observed. Cells treated with antibody and complement showed consumption of C1 through C9. Cells were also sensitized for CRP binding by using diazophenylphosphocholine. This treatment also led to CRP binding and activation of the early classical pathway (C1, C4, C2, and to a lesser extent C3). The components of the membrane attack complex (C5 through C9) were not activated. Both a mouse monoclonal IgM and a human IgG antibody to phosphocholine activated the entire classical pathway. These results indicate that CRP activation of the classical complement pathway is restricted to the early part of the pathway. In the absence of activation of the membrane attack complex, complement-mediated cell lysis cannot occur.     

The interaction of C-reactive protein and serum amyloid P component with nuclear antigens

The pentraxins are a family of proteins characterized by cyclic pentameric structure, calcium-dependent ligand binding and sequence homology. The two main representatives of this family are the serum proteins, C-reactive protein (CRP) and serum amyloid P component (SAP). In man CRP is an acute phase reactant which increases up to 1000 fold during the acute phase response whereas SAP is a constitutive protein expressed at about 30 g/ml. These proteins activate complement through the classical pathway and participate in opsonization of particulate antigens and bacteria. In the past several years it has been determined that both of these pentraxins interact with nuclear antigens including chromatin and small nuclear ribonucleoproteins (snRNPs). Both CRP and SAP have nuclear transport signals which facilitate their entry into the nuclei of intact cells. Furthermore, these pentraxins have been shown to affect the clearance of nuclear antigens in vivo. It is now believed that one of the major functions of the pentraxins could be to interact with the nuclear antigens released from apoptotic or necrotic cells. This interaction could mitigate against deposition of these antigens in tissue and autoimmune reactivity.Abbreviations CRP C-reactive protein - HSA human serum albumin - PC phosphocholine - SAP serum amyloid P component - snRNP small nuclear ribonucleoprotein - SLE systemic lupus erythematosus     

Regulation of complement activation by C-reactive protein: targeting the complement inhibitory activity of factor H by an interaction with short consensus repeat domains 7 and 8-11.

C-reactive protein (CRP) is a major acute phase protein whose functions are not totally clear. In this study, we examined the interaction of CRP with factor H (FH), a key regulator of the alternative pathway (AP) of complement. Using the surface plasmon resonance technique and a panel of recombinantly expressed FH constructs, we observed that CRP binds to two closely located regions on short consensus repeat (SCR) domains 7 and 8-11 of FH. Also FH-like protein 1 (FHL-1), an alternatively spliced product of the FH gene, bound to CRP with its most C-terminal domain (SCR 7). The binding reactions were calcium-dependent and partially inhibited by heparin. In accordance with the finding that CRP binding sites on FH were distinct from the C3b binding sites, CRP preserved the ability of FH to promote factor I-mediated cleavage of C3b. We propose that the function of CRP is to target functionally active FH and FHL-1 to injured self tissues. Thereby, CRP could restrict excessive complement attack in tissues while allowing a temporarily enhanced AP activity against invading microbes in blood.     

Collectin CL-P1 utilizes C-reactive protein for complement activation

BackgroundC-reactive protein (CRP) is a plasma pentraxin family protein that is massively induced as part of the innate immune response to infection and tissue injury. CRP and other pentraxin proteins can activate a complement pathway through C1q, collectins, or on microbe surfaces. It has been found that a lectin-like oxidized LDL receptor 1 (LOX-1), which is an endothelial scavenger receptor (SR) having a C-type lectin-like domain, interacts with CRP to activate the complement pathway using C1q. However it remains elusive whether other lectins or SRs are involved in CRP-mediated complement activation and the downstream effect of the complement activation is also unknown.MethodsWe prepared CHO/ldlA7 cells expressing collectin placenta-1 (CL-P1) and studied the interaction of CRP with cells. We further used ELISA for testing binding between proteins. We tested for C3 fragment deposition and terminal complement complex (TCC) formation on HEK293 cells expressing CL-P1.ResultsHere, we demonstrated that CL-P1 bound CRP in a charge dependent manner and the interaction of CRP with CL-P1 mediated a classical complement activation pathway through C1q and additionally drove an amplification pathway using properdin. However, CRP also recruits complement factor H (CFH) on CL-P1 expressing cell surfaces, to inhibit the formation of a terminal complement complex in normal complement serum conditions.General SignificanceThe interaction of collectin CL-P1 with CFH might be key for preventing attack on “self” as a result of complement activation induced by the CL-P1 and CRP interaction.     

Inhibition of antibody responses to phosphocholine by C-reactive protein

C-reactive protein (CRP) is an acute phase serum protein in man that binds to the cell wall C-polysaccharide (PnC) of Streptococcus pneumoniae via phosphocholine (PC) determinants. We have previously shown that in mice CRP increases splenic clearance of PnC-coated autologous erythrocytes and S. pneumoniae, and increases survival after pneumococcal infection. Because CRP alters clearance of particulate PnC antigens, we tested its effect on immunization with pneumococci. Pretreatment of mice with 50 to 200 micrograms CRP 30 min before immunization with serotype 3 S. pneumoniae resulted in dose-dependent inhibition of the antibody response to PC. Both serum hemagglutinin and splenic PFC against PC were decreased in CRP-treated mice tested from 1 to 10 days after injection of antigen. CRP treatment had no effect on the antibody response to the serotype 3 capsular polysaccharide, another T-independent antigen. To determine whether CRP inhibition was related to altered processing of particulate antigen, mice were immunized with horse red blood cells (HRBC) conjugated with PC or PnC and the PFC responses to PC and HRBC were determined. CRP treatment resulted in specific inhibition of the PFC response to PC in both cases without affecting the response to HRBC. These results indicate that inhibition of the antibody response by CRP is not the result of altered antigen localization and processing, and that CRP may prevent immunization by masking determinants on bacterial or other surfaces.     

Binding of C-reactive protein to the pneumococcal capsule or cell wall results in differential localization of C3 and stimulation of phagocytosis

C-reactive protein (CRP) is a serum protein that shows rapid increases of as much as 1000-fold in concentration in response to infection, traumatic injury, or inflammation. CRP reacts with the phosphocholine moiety of pneumococcal cell wall C-polysaccharide, and this reaction can lead to complement activation in vitro and protection against pneumococcal infection in vivo. We have previously studied the chemiluminescence response of human neutrophils to Streptococcus pneumoniae as a measure of in vitro opsonophagocytosis by CRP and complement. CRP in the presence of complement was an effective opsonin for S. pneumoniae serotype 27 (Pn27), but not for serotypes 3 or 6. Because Pn27 differs from most serotypes of S. pneumoniae in containing phosphocholine in its capsular polysaccharide, we have determined the sites of CRP and C3 fixation to Pn27 and S. pneumoniae serotype 4 (Pn4), and related these to the ability of CRP and complement to opsonize these serotypes in vitro. By using a chemiluminescence (CL) assay to measure opsonophagocytosis, CRP was shown to enhance the response of human neutrophils and monocytes to Pn27 in the presence of normal human serum. The CL response of neutrophils and monocytes to Pn4 was not affected by the addition of CRP to serum. The addition of anti-capsular antibody to Pn4 and Pn27 enhanced the CL responses of both neutrophils and monocytes to both bacteria. The localization of bound CRP and C3 on Pn4 and Pn27 was determined by immunoelectron microscopy. CRP bound to Pn4 only in the cell wall region and C3 was located in this area whether or not CRP was present. Anti-capsular antibody deposited C3 in the capsule of Pn4. In contrast, Pn27 bound CRP throughout the capsule and cell wall areas. C3 was deposited in the cell wall region of Pn27 by serum alone and in the cell wall region and capsule when CRP or anti-capsular antibody was present. Because C3 fixation to the capsule was consistently associated with enhanced responses by phagocytic cells, it appears that the site of CRP binding and subsequent complement activation may be critical in the opsonophagocytosis of S. pneumoniae. These findings extend the correlation between capsular C3 and opsonization to a nonimmune system. By using CRP and different pneumococcal serotypes we have shown that the same molecules that are effective in the stimulation of phagocytic cells when bound to the capsule are not effective when bound to the cell wall.     

Interaction of rabbit C-reactive protein with phospholipid monolayers studied by microfluorescence film balance with an externally applied electric field.

C-reactive protein (CRP) is one of the most characteristic acute-phase proteins in humans and many other animals. It binds to phosphorylcholine in a calcium-dependent manner. In addition, CRP activates the complement systems via the classical pathway. The interaction between rabbit CRP (rCRP) and model biological membrane is studied using dimyristoylphosphatidylethanolamine and dipalmitoylphosphatidylcholine monolayers. Observations with fluorescence microscopy indicate that rCRP is more likely to be incorporated in the liquid phase of monolayers. Such incorporation does not depend on the presence of calcium and is not inhibited by phosphocholine. The area occupied by the protein when incorporated into the monolayer was estimated. The dipole moment density of the protein crossing the air/water interface was measured by applying an external electric field. Our results indicate that calcium binding leads to a conformational change in CPR, which might modify the orientation of CRP in the monolayer. In addition, a negative charge or negative difference in dipole moment density facilitates the incorporation of CPR into the monolayer.