C3 plays a critical role in both pathways of complement activation due to its ability to bind to numerous other complement proteins. In addition, its interactions with several cell surface receptors make it a key participant in phagocytic and immunoregulatory processes, while its interactions with proteins from foreign pathogens may provide a mechanism by which these microorganisms evade complement neutralization. The elucidation of the molecular features related to these C3 associated functions requires further analysis of its structure. It is the purpose of this project to study the interactions of human C3 with CR1, CR2, CR3, H, B, I and P by engineering chimeric and mutated C3 molecules based on the structure and function of C3 from other species. Studies from our laboratory suggest that it is possible to identify the structural features of C3 that are important for its functions by comparing the C3 amino acid sequences between species, """"""""natural analogs"""""""", and correlating this information with the ability of these C3s to bind the different C3- binding proteins. First, we will purify C3 from Human (Hu), Xenopus (Xe), and Trout (Tr) plasma and test their ability to react with human and autologous complement proteins CR1, CR2, CR3, H, B, I, and P. The binding of human and autologous C3-binding proteins to different C3s will be analyzed by direct binding ELISA, by binding to C3 fragment-coated erythrocytes, and by inhibition assays. To correlate the functional conservation of the C3-ligand interactions with the C3 structure we will obtain the primary structure of Tr and Xe C3 by cloning and sequencing their cDNA. To evaluate the substrate specificity of factor I and its dependence on the various cofactors in mediating the different C3b cleavages we will mutate the factor I cleavage sites in human C3 and analyze the cleavage of the mutated C3b fragment by I in the presence of H, CR1 or CR2. To characterize the CR1, CR2, H and B binding domain(s) in C3b and the functions associated with each domain we will engineer chimeric molecules by exchanging the ligand binding domains between Hu, Xe, and Tr C3 and CVF. To localize the CR3 binding site(s) in C3 we will generate C3 fragments, by enzymatic/chemical degradation and cDNA expression, and synthetic peptides and testing their ability to bind to CR3 and/or to inhibit the binding of iC3b to CR3. Our preliminary experiments have suggested that the residues His1431 and Ser1432 are involved in the binding of C3 peptides to properdin. We will assess the role of these residues in C3b-P interaction by mutating His1431 and Ser1432 of human C3 to residues N and Q found in a2-macroglobulin; a2- macroglobulin does not bind P and differs from Hu C3, within amino acids 1424-1432, only in these two residues. The biological role of C3-CR1/CR2 and C3-B/P interactions will be analyzed in vitro and in vivo by evaluating the ability of synthetic and/or expressed C3 fragments to interfere with the antibody responses and to inhibit the alternative pathway mediated injury of skeletal muscle using an ischemia/reperfusion mouse model, respectively. The proposed studies, in addition to the phylogenetic data on C3, will provide basic information on the structural features of C3 as they relate to its multiple functions and assist in the design of specific complement inhibitors which may become medically important.
| Mastellos, Dimitrios C; Reis, Edimara S; Yancopoulou, Despina et al. (2018) Expanding Complement Therapeutics for the Treatment of Paroxysmal Nocturnal Hemoglobinuria. Semin Hematol 55:167-175 |
| Reis, Edimara S; Mastellos, Dimitrios C; Ricklin, Daniel et al. (2018) Complement in cancer: untangling an intricate relationship. Nat Rev Immunol 18:5-18 |
| Huber-Lang, Markus; Lambris, John D; Ward, Peter A (2018) Innate immune responses to trauma. Nat Immunol 19:327-341 |
| Ricklin, Daniel; Mastellos, Dimitrios C; Reis, Edimara S et al. (2018) The renaissance of complement therapeutics. Nat Rev Nephrol 14:26-47 |
| Kajikawa, Tetsuhiro; Briones, Ruel A; Resuello, Ranillo R G et al. (2017) Safety and Efficacy of the Complement Inhibitor AMY-101 in a Natural Model of Periodontitis in Non-human Primates. Mol Ther Methods Clin Dev 6:207-215 |
| Harder, Markus J; Kuhn, Nadine; Schrezenmeier, Hubert et al. (2017) Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation. Blood 129:970-980 |
| Primikyri, Alexandra; Papanastasiou, Malvina; Sarigiannis, Yiannis et al. (2017) Method development and validation for the quantitation of the complement inhibitor Cp40 in human and cynomolgus monkey plasma by UPLC-ESI-MS. J Chromatogr B Analyt Technol Biomed Life Sci 1041-1042:19-26 |
| Papanastasiou, Malvina; Koutsogiannaki, Sophia; Sarigiannis, Yiannis et al. (2017) Structural Implications for the Formation and Function of the Complement Effector Protein iC3b. J Immunol 198:3326-3335 |
| Xue, Xiaoguang; Wu, Jin; Ricklin, Daniel et al. (2017) Regulator-dependent mechanisms of C3b processing by factor I allow differentiation of immune responses. Nat Struct Mol Biol 24:643-651 |
| Hajishengallis, George; Reis, Edimara S; Mastellos, Dimitrios C et al. (2017) Novel mechanisms and functions of complement. Nat Immunol 18:1288-1298 |
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