MUTATIONAL ANALYSIS OF THE ACTIVE-SITE AND ANTIBODY EPITOPES OF THE COMPLEMENT-INHIBITORY GLYCOPROTEIN, CD59

The Ly-6 superfamily of cell surface molecules includes CD59, a potent regulator of the complement system that protects host cells from the cytolytic action of the membrane attack complex (MAC). Although its me chanism of action is not well understood, CD59 is thought to prevent a ssembly of the MAC by binding to the C8 and/or C9 proteins of the nasc ent complex. Here a systematic, structure-based mutational approach ha s been used to determine the region(s) of CD59 required for its protec tive activity. Analysis of 16 CD59 mutants with single, highly noncons ervative substitutions suggests that CD59 has a single active site tha t includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-di stal face of the disk-like extracellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site include s residues conserved across species, consistent with the lack of stric t homologous restriction previously observed in studies of CD59 functi on. Competition and mutational analyses of the epitopes of eight CD59- blocking and non-blocking monoclonal antibodies confirmed the location of the active site. Additional experiments showed that the expression and function of CD59 are both glycosylation independent.