Activation of complement caused by certain pathological conditions can lead to the deposition of the cytolytic membrane attach complex (MAC) on host cells surfaces, but metabolically active nucleated cells are normally resistant to cytolysis by homologous MAC due to various inhibitory and recovery mechanisms. Some disease processes, however (eg. rheumatoid arthritis, several renal diseases, myocardial infarction), results in the deposition of non-lethal amounts of MAC on cell membranes, a condition shown to cause the production may toxic pro-inflammatory mediators which condition shown to cause the production many toxic pro- inflammatory mediators which contribute to disease pathogenesis. CD59 is a widely distributed cell surface protein that inhibits homologous MAC formation by binding to the complement proteins C8 and C9 in the assembling complex. The primary objective of this proposal is to precisely define the interaction between this important anti-inflammatory molecule and its ligands C8 and C9, information which may be useful for the long term objective of designing more potent MAC-inhibitory molecules. Based on preliminary results, the binding site for CD59 on C8 and C9 will be determined by a strategy using peptides which correspond to regions of C8 and C9. Peptides will be synthesized and used to test functional inhibition of CD59 in human erythrocytes, and in direct and competitive binding assays to cell surface and purified CD59. Anti-peptide Abs will be raised to directly assess exposure of putative binding sites during MAC formation. The region(s) of CD59 important for binding and function will be determined by preparing mutant proteins and chimeric CD59-Ly6C.2 proteins; Ly6C.2 is a mouse cell surface protein with a structural, but not functional homology to CD59, and acquisition of MAC-inhibitory activity of Ly6C.2 containing substituted regions of CD59, will indicate regions of CD59 important function. Disulfide bonded cysteine pairs will also be determined. Functional analogues of CD59 from rabbit and horse will be isolated and the genes cloned and sequenced. Since these CD59 analogues inhibit homologous, but not human MAC, sequence comparisons and production of chimeric constructs with CD59 are expected to yield important structure and function information by defining the species- selective site. To further this aim, rat C8a and C9 will also be cloned, as cloning of rat CD59 is currently underway. Animal CD59 analogous could also be very useful for therapeutic evaluation of recombinant CD59 and proteins designed as a result of these proposal, in animal models of disease. Finally, evaluation and development of various expression systems for both soluble and GPI-linked CD59 is proposed which may assist in structure and function studies, but with the eventual goal of economic production for therapeutic evaluation.
