CD59, Disulphide-Locked Human C9 and Horse C9 Inhibit Human Membrane Attack Complex Assembly by Similar Mechanisms

Five plasma proteins, C5b, C6, C7, C8 and C9, assemble in a step-wise manner to form the membrane attack complex (MAC) which inserts into target cell membranes to cause lysis. The membrane regulator CD59 binds nascent C5b-8, preventing C9 recruitment and polymerisation into the lytic pore. A disulphide-locked C9 ('C9lock'; C9_F262C/V405C) lacked haemolytic activity in standard assays because the unfolding required for pore formation was prevented, while horse C9 (HoC9) lacked haemolytic activity suggested to be a consequence of species incompatibility in MAC assembly. In this study, we compared the impact of soluble CD59 (sCD59), C9lock and HoC9 on MAC assembly. C9lock and sCD59 were generated recombinantly, while HoC9 and human C9 (HuC9) were affinity-purified from serum. Binding and haemolytic assays were used to identify and compare the modes of action of MAC binding and inhibition by sCD59, C9lock and HoC9. We show that sCD59, C9lock and HoC9 all inhibited human serum mediated haemolysis in both classical and alternative pathways. In reactive lysis assays, all three inhibitors bound immobilised C5b-8 but not C5b-7 intermediates on ELISA wells and gpE, and competitively blocked C9-mediated lysis of gpE. Each of the inhibitors also bound mouse and rat C5b-8 sites on gpE and blocked human C9-mediated lysis. This work clarifies the functional differences between HoC9 and human C9 and highlights the mechanistic similarities of the diverse MAC inhibitors (C9lock, sCD59 and HoC9). These agents not only provide useful tools for analysis of MAC assembly but also signpost novel strategies for specific MAC inhibition in conditions where MAC formation contributes to pathology.