The main goal of this project is to elucidate structural and mechanistic details about the assembly and regulation of the membrane attack complex of complement (MAC). The mechanism of killing by complement involves the formation of a cytolytic pore-forming complex on the membrane of target cells. The insertion of a complex containing components C5b, C6, C7 and C8 is followed by the attachment of C9 which polymerizes to form a pore in the membrane. The process is species specific. Human blood and vascular cells contain a lipid-anchored protein, CD59, that protects them from attack by homologous complement. The previous grant period was devoted largely to characterizing the physical and functional properties of this protein. It was shown that CD59 binds to neo-epitopes on C8-alpha and C9, thereby restricting the assembly of the C9 polymer. The present proposal aims to identify specific residues of C8-alpha and C9 that are recognized by CD59 and vice versa (aims 1 and 2) and to examine the kinetic mechanism of these interactions (aim 4). The sites of interaction of C9 with itself and with C8-alpha will also be localized in an effort to better understand the assembly of the MAC (aim 3).
These aims will be approached through a combination of physical and molecular biological approaches. A fifth aim of lower priority involves a collaborative effort to clarify the role of CD59 as an accessory ligand to CD2 in augmenting T-cell response to antigen presenting cells. These studies are designed to provide new insights into the associative interactions and conformational changes that underlie the expression of the pore-forming function of the C5b-9 complex and the regulation of this process by cell surface CD59.
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