Date of Award

1-1-2011

Document Type

Campus Access Dissertation

Department

Chemistry and Biochemistry

Sub-Department

Chemistry

First Advisor

James M Sodetz

Abstract

The human complement system assists in the elimination of pathogenic organisms. Activation of the system leads to the sequential interaction of complement components C5b, C6, C7, C8 and C9 to form C5b-9n (n = 12-18), a cytolytic, pore-like structure referred to as the membrane attack complex (MAC). C8 contains three genetically distinct proteins (C8α, C8β, C8r); C8α-r is a disulfide linked heterodimer that is non-covalently associated with C8β. Sequence alignment of the MAC components shows homology amongst C6, C7, C8α, C8β and C9. These MAC proteins have homologous N- and C-terminal modules and a central ~40 kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. Recent studies showed the C8α and C8β MACPF domains have a structural fold similar to the bacterial cholesterol-dependent cytolysins (CDCs). CDCs contain a core β-sheet flanked by two α-helical bundles that undergo a major conformational change to form transmembrane β hairpins (TMH1 and TMH2). Alignment of multiple TMHs produces a β-barrel pore that is typical of the CDCs.

Among the MAC components, C9 is unique due to its ability to self-polymerize to form the MAC pore. The first aim of this project is to identify sites of C9-C9 interaction that lead to MAC pore formation. Efforts were made to produce and characterize a soluble form of the C9MACPF domain to determine if, as in C8, the sites of interaction lie within the MACPF region. A customized C9MACPF phage display library was also used to localize binding sites. Unfortunately, C9MACPF could not be produced in soluble form and results from phage display were inconclusive.

The second aim of this project is to use perfringolysin O (PFO), a well-characterized CDC, as a scaffold to create chimeras to that might reveal a role for the TMH regions of C8α and C9 in MAC formation. Chimeras of PFO and C8αTMH2 (PFO/C8αTMH2) or C9TMH2 (PFO/C9TMH2) were produced recombinantly and found to retain several functional characteristics of PFO. These results suggest the TMH2 region of C8α and C9 may function similarly to the membrane inserting TMH2 region of the CDCs.

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