Mutational analysis of the active site and antibody epitopes of the complement-inhibitory glycoprotein, CD59
Bodian DL, Davis SJ, Morgan BP, Rushmere NK. (1997), J Exp Med. 185, 507-16
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 mechanism of action is not well understood, CD59 is thought to prevent assembly of the MAC by binding to the C8 and/or C9 proteins of the nascent complex. Here a systematic, structure-based mutational approach has been used to determine the region(s) of CD59 required for its protective activity. Analysis of 16 CD59 mutants with single, highly nonconservative substitutions suggests that CD59 has a single active site that includes Trp-40, Arg-53, and Glu-56 of the glycosylated, membrane-distal face of the disk-like extra-cellular domain and, possibly, Asp-24 positioned at the edge of the domain. The putative active site includes residues conserved across species, consistent with the lack of strict homologous restriction previously observed in studies of CD59 function. 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.
The epitopes of anti-CD59 function-blocking mAb cluster in the region of the proposed active site of CD59. Residues whose mutation disrupted the binding of each antibody (inverse shaded black in Table 1) are shaded black and those whose mutation led to reduced levels of binding (shaded gray in Table 1) are shaded gray. Only antibodies HC1 and MEM43/5 have no CD59-blocking ability. In the last panel, all of the residues that were mutated are shaded gray except for the visible residues whose mutation disrupted the function of CD59 which are shaded black and labeled. In each panel the protein face shown is the one containing the proposed active site shown in the same view as in Fig. 3 A. The experimental data are superimposed on the lowest energy NMR structure (19) drawn using Rasmol (46).