RESUMO
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.
Assuntos
Antígenos CD/química , Antígenos CD/genética , Complemento C1q/química , Complemento C1q/genética , Mutagênese Sítio-Dirigida , Receptores de IgG/química , Receptores de IgG/genética , Alelos , Animais , Baculoviridae/genética , Sítios de Ligação , Células COS , Linhagem Celular , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunoglobulina G/química , Insetos , Células K562 , Leucina/química , Lisina/química , Modelos Moleculares , Mutagênese , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Imunológicos/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , TransfecçãoRESUMO
The basic issue of recognizing and delimiting characters derived from different levels of biological organization, including molecular, cellular, morphological and behavioral levels, has been addressed previously in the cladistic literature. But when considering new sources of information (such as the proliferating Evo-Devo data), it is critical to review traditional theoretical and methodological approaches to their interpretation. This is especially important because the conclusions of a phylogenetic analysis are dependent upon the initial recognition and definition of the characters, the basic units of comparison in phylogeny reconstruction. This chapter explores the role of recent Evo-Devo studies in systematics and attempts to place the Evo-Devo literature into a systematics context.
