The objective of this project is to identify and define structural features of terminal complement proteins which mediate their interactions with each other and with target cell membranes during the cytolytic process. This will be accomplished through a detailed analysis of structure-function relationships in the eighth component of human complement (C8). Studies of C8 interactions will focus on the final two steps leading to formation of the cytolytic C5b-9 complex, namely C5b-7-C8-C5b-8-C9-C5b-9. Evidence suggests the alpha-gamma and beta subunits of C8 may be products of different genes; therefore, initial experiments will examine the chemical basis for the high affinity between these two proteins. Subsequent experiments will use chemical modifications to characterize structural domains on beta which mediate interaction of C8 with C5b-7 while parallel studies using chemical crosslinkers will attempt to define the C8 binding site itself. Other experiments will examine the C9 binding site on C5b-8 and by using chemical modifications, crosslinkers and synthetic C8-C9 dimers, attempt to define the role of C8 in mediating C9 incorporation into this complex. Regarding C8 interaction with target membranes, those structural domains inserted in the lipid bilayer during lysis will be identified using a photosensitive, membrane-restricted probe. Intra-membrane peptides will be isolated and characterized to establish if they contain extended sequences of hydrophobic residues analogous to integral membrane proteins. Occurrence of such sequences could explain the mechanism by which these proteins function. Concurrent with these studies, amino acid sequencing of alpha, beta and gamma will be performed using conventional methods and a parallel approach of oligonucleotide sequencing of cloned cDNA inserts isolated from an existing human liver cDNA library. With sequence information, highly specific oligonucleotide probes can be prepared and used to begin long-term studies of alpha-gamma and beta gene structure. Availability of complete sequences will also contribute substantially to defining structure-function correlates in C8. Using hepatocyte cell-cultures and cell-free systems, other experiments will investigate the biosynthesis of C8. These will focus on the existence of a single-chain alpha-gamma precursor, the occurrence of pre- and pro-peptides in alpha-gamma and beta, glycosylation and secretion of each and whether association of subunits to form C8 is a pre- or post-secretory event. Because this project is concerned with understanding the mechanism and regulation of an intrinsic, physiological process for destroying cells, its biological significance relates to potential improvements in the treatment of infectious diseases and cancer.
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