MECHANISM OF COMPLEMENT-MEDIATED MEMBRANE DAMAGE

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Complement(C) is a group of serum proteins which constitutes the humoral arm of our immune defense system. Its diverse functions include lysis of certain bacteria, viruses and cells by direct attack on the bacterial, viral or cellular membranes. Membrane attack by C involves the assembly of a multimeric complex of C terminal proteins (C5b-9) on membrane which inserts into the membranes to form channels permeable to ions and small solutes. However, the mechanism of channel formation by these inserted proteins is still unresolved. The long-range goal of the proposed research is to develop a clearer understanding of the mechanism of complement-mediated lysis of cells and of the regulatory processes controlling the efficacy of complement against homologous cells. The specific aims of this project are: 1) to analyze structure/function relationships of the C5b-8 and C5b-9 complexes in terms of depth of penetration into the membrane and cooperativity between individual complexes in forming functional channels, 2) to systematically investigate the effect of temperature and the role of Ca+2 in channel assembly and function, and 3) to investigate the involvement of membrane surface molecules in modulating insertion, and hence lytic efficiency, of membrane-bound terminal complexes on homologous cells. The depth of penetration of the individual proteins in terminal complexes will be determined using membrane-restricted, photo-reactive glycolipid probes anchored at defined positions in either the outer or the inner monolayer of model membranes which will be used as substrates for C attack. The dynamics of channel formation will be studied by comparing the kinetics of insertion with the kinetics of marker release from vesicles as a function of the concentration of inserted complexes. These methods will allow us to evaluate the specific effects of temperature and Ca+2 ions on separate processes (eg. binding, insertion, C9 polymerization) involved in channel assembly. In addition to the physicochemical factors affecting functional assembly of terminal complexes, the influence of membrane factors will also be studied. In particular, the role of terminal protein interaction with membrane surface proteins will be addressed using photosensitive crosslinking reagents to detect associated proteins. The relevance of these associations to insertion of the terminal complex will then be studied.