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The acute-phase protein C-reactive protein (CRP) recruits C1q to the surface of damaged cells and thereby initiates complement activation. However, CRP also recruits complement inhibitors, such as C4b-binding protein (C4bp) and factor H, which both block complement progression at the level of C3 and inhibits inflammation. To define how CRP modulates the classic complement pathway, we studied the interaction of CRP with the classic pathway inhibitor C4bp. Monomeric CRP (mCRP), but not pentameric CRP (pCRP), binds C4bp and enhances degradation of C4b and C3b. Both C1q, the initiator, and C4bp, the inhibitor of the classic pathway, compete for mCRP binding, and this competition adjusts the local balance of activation and inhibition. After attachment of pCRP to the surface of necrotic rat myocytes, generation of mCRP was demonstrated over a period of 18 h. Similarly, a biological role for mCRP, C1q, and C4bp in the disease setting of acute myocardial infarction was revealed. In this inflamed tissue, mCRP, pCRP, C4bp, C1q, and C4d were detected in acetone-fixed and in unfixed tissue. Protein levels were enhanced 6 h to 5 d after infarction. Thus, mCRP bound to damaged cardiomyocytes recruits C1q to activate and also C4bp to control the classic complement pathway.

C-reactive protein (CRP) is a pattern recognition molecule that binds several microbial and host ligands. Ligand-bound CRP activates the complement system via the classical pathway. Previously, we identified human complement factor H-related protein 4 (CFHR4), a member of the factor H protein family, as a CRP binding protein. Here, we investigated the molecular basis and the functional relevance of the interaction of CFHR4 with native CRP. Using recombinantly expressed CFHR4 fragments, the CRP binding site was localized to the first short consensus repeat (SCR) domain of CFHR4. Peptide arrays identified residues 35-41 of CFHR4 to be involved in CRP binding. Substitutions of the positively charged amino acids of this motif resulted in strongly reduced CRP binding. Sequence comparisons revealed that such a motif is not present in the related SCR6 domain of factor H, or in the homologous domains of the four other CFHR proteins. Homology modelling based on SCR6 of factor H showed that the CRP binding site is surface exposed on SCR1 of CFHR4. CFHR4-bound CRP was able to activate complement, determined by C3 fragment deposition. Recombinant CFHR4 proteins with mutations in the identified binding site showed reduced CRP binding, which in turn resulted in reduced complement activation. In summary, these data reveal the molecular basis of the specific interaction of CFHR4 with native CRP and suggest a role for CFHR4 in enhancing opsonization via CRP binding., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Complement forms the first defense line of innate immunity and has an important role in the non-inflammatory clearance of apoptotic and necrotic cells. Factor H is one essential complement inhibitor that binds to the acute phase reactant C-reactive protein (CRP). By using recombinant proteins, calcium-independent binding of Factor H to monomeric CRP (mCRP), but not to pentameric CRP (pCRP), was shown. In addition to the two known CRP-binding sites, a novel third site was localized within the C-terminus. This region is frequently mutated in the hemolytic uremic syndrome and the mutant proteins show reduced mCRP binding. In this study, we show that mCRP directs Factor H to the surface of apoptotic and necrotic endothelial cells and identify phosphocholine as one binding moiety for this complex. Factor H-mCRP complexes enhance C3b inactivation both in the fluid phase and on the surface of damaged cells and inhibit the production of pro-inflammatory cytokines. By recruiting the soluble complement inhibitor Factor H to the surface of damaged cells, mCRP blocks the progression of the complement cascade beyond the step of the C3 convertase, prevents the formation of inflammatory activation products, and thus contributes to the safe removal of opsonized damaged cells and particles.

Human complement factor H-related protein 4 (CFHR4) is a plasma glycoprotein which appears in two isoforms. CFHR4 is a member of the factor H protein family, and shares structural similarity and sequence homology with the other CFHR proteins and with the complement regulator factor H. Given the structural and sequence similarity, we hypothesized that similar to factor H, CFHR4 binds to C-reactive protein (CRP). We have recombinantly expressed the two CFHR4 isoforms and analyzed their binding to both native and denatured, monomeric CRP. Here, we show that both CFHR4 isoforms bind in the presence of calcium to native pentameric CRP, but not to modified CRP. This is in contrast to factor H, which binds to modified CRP independent of calcium. Comparison of the two CFHR4 isoforms and a recombinant CFHR4 fragment for CRP binding indicates that the first domain of CFHR4 is relevant for this interaction. Interaction of the native proteins was demonstrated by co-precipitation of CFHR4 and CRP from serum of sepsis patients with elevated CRP levels. CFHR4 bound to necrotic cells and was localized in necrotic tumor tissue as demonstrated by immunohistological analyses. In addition, CFHR4 facilitated binding of native CRP to the surface of necrotic cells. Altogether these data identify CFHR4 as a novel ligand for native CRP, and suggest a role for CFHR4 in opsonization of necrotic cells.