This project focuses on molecular details of the assembly and function of human C5b-9, the cytolytic complex of terminal complement proteins C5b, C6, C7, C8 and C9. Emphasis will be on characterizing the structure, function and molecular genetics of human C8 so as to understand its role in C5b-9 formation and function, the significance of its highly peculiar structure, its evolutionary relationship to other terminal components and the nature of the molecular defect in C8 deficiencies at both the protein and gene levels.
Specific aims to be pursued include characterizing the genes encoding the alpha, beta, and gamma subunits of human C8. Emphasis will be on chromosomal assignments, determining gene size, identifying physical linkages, mapping of genomic clones, determining exonintron organization, characterizing possible regulatory regions and identifying evolutionary relationships. Results will provide the foundation for analysis of gene defects in C8 deficiencies and for future studies of C8 regulation. Structure-function studies will identify segments in alpha, beta and C9 that are involved in beta-alpha and alpha-C9 interactions. Experiments will explore the possibility that conserved structural domains in these homologous proteins mediate beta-alpha-C9 co- polymerization in solution and that such interactions may constitute the underlying mechanism of C5b-9 assembly. Related experiments will focus on C5-beta and alpha-gamma in order to further understand the basis for recognition amongst terminal components. Approaches to be used include protein fragmentation, isolation of functional domains and chemical modifications. In other experiments, segments of alpha and beta that interact directly with target membranes will be radiolabeled by photosensitive, membrane-restricted probes, isolated, characterized and compared to those segments predicted to be membrane-interacting based on amino acid sequence. Accessibility of these segments in tact C8 will be evaluated to test the hypothesis that such segments are inaccessible prior to association with C5b-7. Experiments will also explore the utility of structural analogues of C5b-9 as cytolytic agents. Conjugates of Fab-C8 that are directed against cell-surface antigens will be tested for their ability to mediate C9 binding and produce membrane damage. Such analogues could be useful as therapeutic agents. Understanding protein- protein and protein-membrane interactions during C5b-9 assembly is of general biological importance because it is a unique system that functions as an endogenous mediator of cell killing.
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