Identification of mutations in rat CD59 that increase the complement regulatory activity

Formation of the membrane attack complex (MAC) of complement on host cells is inhibited by the glycosylphosphatidylinositol- (GPI-) anchored glycoprot ein CD59. Published data on the active site of human CD59 are confusing. To clarify these data, we set out to elucidate the active site of a nonprimat e CD59 molecule by site-directed mutagenesis. We also undertook to investig ate a region of potential species selectivity, and to this end rat CD59 was chosen for all mutations. Our investigations confirmed the proposal that t he active site of CD59 is the major hydrophobic groove, with mutations Y36A , W40A, and L54A ablating complement inhibitory function of CD59. Other mut ations reducing the function of rat CD59 were I56E, D24A, and D24R. Importa ntly, mutations at one residue increased the function of rat CD59. The K48E mutation significantly increased function against human rat or rabbit seru m, whereas the K48A mutation increased function against human serum alone. A similar mutation in human CD59 (N48E) had no effect on activity against h uman or rat serum but completely abolished all activity against rabbit seru m. These findings suggest that the ex-helix of human CD59, adjacent to the hydrophobic groove, influences the interaction between human CD59 and rabbi t C8, C9, or both.