Analysis of Binding Sites in Human C-reactive Protein for Fc RI, Fc RIIA, and C1q by Site-directed Mutagenesis*

Human C-reactive protein (CRP) is a classical, acute phase serum protein synthesized by the liver in response to infection, inflammation, or trauma. CRP binds to microbial antigens and damaged cells, opsonizes particles for phagocytosis and regulates the inflammatory response by the induction of cytokine synthesis. These activities of CRP depend on its ability to activate complement and to bind to Fc receptors (Fc R). The goal of this study was to elucidate amino acid residues important for the interaction of CRP with human Fc RI (CD64) and Fc RIIa (CD32). Several mutations of the CRP structure were studied based on the published crystal structure of CRP. Mutant and wild-type recombinant CRP molecules were expressed in the baculovirus system and their interactions with Fc R and C1q were determined. A previous study by our laboratory identified an amino acid position, Leu, critical for CRP binding to Fc RI and work by others (Agrawal, A., Shrive, A. K., Greenhough, T. J., and Volanakis, J. E. (2001) J. Immunol. 166, 3998–4004) determined several residues important for C1q binding. The amino acid residues important to CRP binding to Fc RIIa were previously unknown. This study newly identifies residues Thr and Asn as important for the binding of CRP to Fc RIIa and Fc RI. Lys, like Leu, was implicated in binding to Fc RI, but not Fc RIIa. Single mutations at amino acid positions Lys, Asp, Thr, Tyr, and Leu affected C1q binding to CRP. These results further identify amino acids involved in the binding sites on CRP for Fc RI, Fc RIIa, and C1q and indicate that these sites are overlapping.