title: Analysis of binding sites in human C-reactive protein for Fc{gamma}RI, Fc{gamma}RIIA, and C1q by site-directed mutagenesis.
abstract: 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 Fcgamma receptors (FcgammaR). The goal of this study was to elucidate amino acid residues important for the interaction of CRP with human FcgammaRI (CD64) and FcgammaRIIa (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 FcgammaR and C1q were determined. A previous study by our laboratory identified an amino acid position, Leu(176), critical for CRP binding to FcgammaRI 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 FcgammaRIIa were previously unknown. This study newly identifies residues Thr(173) and Asn(186) as important for the binding of CRP to FcgammaRIIa and FcgammaRI. Lys(114), like Leu(176), was implicated in binding to FcgammaRI, but not FcgammaRIIa. Single mutations at amino acid positions Lys(114), Asp(169), Thr(173), Tyr(175), and Leu(176) affected C1q binding to CRP. These results further identify amino acids involved in the binding sites on CRP for FcgammaRI, FcgammaRIIa, and C1q and indicate that these sites are overlapping.
authors.affiliation: Department of Veterans Affairs Medical Center and the University of New Mexico, School of Medicine, Albuquerque, New Mexico 87108, USA.
authors.forename: Ranhy
authors.initials: R
authors.lastname: Bang
authors.forename: Lorraine
authors.initials: L
authors.lastname: Marnell
authors.forename: Carolyn
authors.initials: C
authors.lastname: Mold
authors.forename: Mary-Pat
authors.initials: MP
authors.lastname: Stein
authors.forename: Kevin T Du
authors.initials: KT
authors.lastname: Clos
authors.forename: Corinn
authors.initials: C
authors.lastname: Chivington-Buck
authors.forename: Terry W Du
authors.initials: TW
authors.lastname: Clos
mesh_terms: D000483:Alleles; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D015922:Complement C1q / Q000737:chemistry* / Q000235:genetics*; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007313:Insecta; D020014:K562 Cells; D007930:Leucine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017452:Receptors, IgG / Q000737:chemistry* / Q000235:genetics*; D011971:Receptors, Immunologic / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
chemical_list: D015703:Antigens, CD; C071581:CRP protein, human; C080568:Fc gamma receptor IIA; D007074:Immunoglobulin G; D017452:Receptors, IgG; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D015922:Complement C1q; D007930:Leucine; D008239:Lysine