Incorporation of human complement C8 into the membrane attack complex is mediated by a binding site located within the C8β MACPF domain

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the membrane attack complex (MAC). C8 is an oligomeric protein composed of a disulfide-linked C8 alpha-gamma heterodimer and a noncovalently associated C8 beta chain. C8 alpha and C8 beta are homologous; both contain N- and C-terminal modules and an intervening similar to 40 kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. C8 beta participates in at least two binding interactions. It has a high affinity binding site for C8 alpha, which facilitates its interaction with C8 alpha-gamma. C8 beta also mediates incorporation of C8 into the MAC by binding to C5b-7, an intermediate in the MAC assembly pathway. Little is known about the location or properties of the respective binding sites on C8 beta. In this study, the MACPF domain of C8 beta (beta MACPF) was expressed in Escherichia coli and its role in binding C8 alpha and C5b-7 examined. Recombinant beta MACPF was shown to bind C8 alpha-gamma in solution and form a noncovalent complex (beta MACPF center dot C8 alpha-gamma) that exhibited C8 hemolytic activity. PMACPF was also capable of binding independently to erythrocytes carrying C5b-7. Subsequent addition of C8 alpha-gamma and C9 to these cells produced a hemolytically active MAC. The ability to produce a soluble, recombinant PMACPF that retains the binding functions of UP suggests this segment of C8 beta is an independently folded domain. Furthermore, results indicate the principal binding sites for C8 alpha and C5b-7 are located within this domain, and that C8 beta binding specificity is not determined by the N- and C-terminal modules. (c) 2006 Elsevier Ltd. All rights reserved.