Chimeric and Truncated Forms of Human Complement Protein C8α Reveal Binding Sites for C8β and C8γ within the Membrane Attack Complex/Perforin Region

Abstract

Human C8 is one of five components of the membrane attack complex of complement. It is an oligomeric protein composed of three subunits (C8 alpha, C8 beta, and C8 gamma) that are derived from different genes. C8 alpha and C8 beta are homologous and both contain a pair of tandemly arranged N-terminal modules [thrombospondin type 1 (TSP1) + low-density lipoprotein receptor class A (LDLRA)], an extended middle segment referred to as the membrane attack complex/perforin region (MACPF), and a pair of C-terminal modules [epidermal growth factor (EGF) + TSP1]. During biosynthetic processing, C8 alpha and C8 gamma associate to form a disulfide-linked dimer (C8 alpha-gamma) that binds to C8 beta through a site located on C8 alpha. In this study, the location of binding sites for C8 beta and C8 gamma and the importance of the modules in these interactions were investigated by use of chimeric and truncated forms of C8 alpha in which module pairs were either exchanged for those in C8 beta or deleted. Results show that exchange or deletion of one or both pairs of modules does not abrogate the ability of C8 alpha to form a disulfide-linked dimer when coexpressed with C8 gamma in COS cells. Furthermore, each chimeric and truncated form of C8 alpha-gamma retains the ability to bind C8 beta; however, only those containing the TSP1 + LDLRA modules from C8 alpha are hemolytically active. These results indicate that binding sites for C8 beta and C8 gamma reside within the MACPF region of C8 alpha and that interaction with either subunit is not dependent on the modules. They also suggest that the N-terminal modules in C8 alpha are important for C9 binding and/or expression of C8 activity.

Structure of Complement C6 Suggests a Mechanism for Initiation and Unidirectional, Sequential Assembly of Membrane Attack Complex (MAC)

“…Although the precise functions of the auxiliary domains are unclear, their importance in MAC assembly is supported by several studies. For example, two independent studies (using deletion mutants) demonstrated that the N-terminal modules of C8␣ (TS2 and LR) are strictly required for MACPF formation and hemolytic activity, although deletion of the C-terminal TS3 domain greatly reduced activity (26,27). A study on C9 provided evidence for regulatory roles for the N terminus and TS2 domain; thus, short deletions at the N terminus promoted MACPF formation, and deletions or mutations within the TS2 domain caused nonproductive C9 self-polymerization (28).…”

Loss of NECROTIC SPOTTED LESIONS 1 associates with cell death and defense responses in Arabidopsis thaliana

“…This hypothesis is supported by the biochemical analyses of MACPF domain proteins in mammalian complement components. In mammals, the MACPF domain is required for the intramolecular interaction between homologous complement components C8a and C8b that form part of a trans-membrane pore (Musingarimi et al 2002;Plumb et al 1999). The amphipathic helix-loop-helix (HLH) motif within the MACPF domain, a putative membrane-spanning motif, has been suggested to be important for integration of the final pore structure into the plasma membrane (Peitsch et al 1990).…”

Detection of a polymorphic site of the porcine <i>C8G</i> gene and evaluation of association with haemolytic complement activity

“…C8 is one of five components of the membrane attack complex (MAC) in the terminal pathway of the complement system. It is an oligomeric protein composed of three subunits C8A, C8B, and C8G that are derived from different genes (Plumb et al 1999). The C8A and C8G subunits are bound covalently through a disulphide linkage, whereas the C8B is associated via weaker, non-covalent bonds (Alper et al 1983).…”