Date of Award
Campus Access Thesis
Chemistry and Biochemistry
James M Sodetz
The human complement system is part of the innate immune response that acts to
eliminate pathogenic organisms. Complement is activated by three pathways leading to the sequential, non-enzymatic assembly of C5b, C6, C7, C8, and C9 to form the membrane attack complex (MAC). C6, C7, C8α, C8β, and C9 make up the “MAC family” of proteins. MAC family proteins have homologous N and C terminal modules and a central MACPF domain named for sequence similarity between the MAC family of proteins and perforin, a self-polymerizing protein released from cytotoxic T- lymphocytes.
The first aim was to express and characterize MACPF domains of C6 and C7. Based on sequence homology in the MACPF domains of MAC family proteins, it was hypothesized that the MACPF domains of C6 and C7 may hold important sites of interaction with C5b, C7, C8β and C5b-6 or C8β respectively. Despite creation of multiple constructs and various expression conditions, soluble protein with ability to bind to a Ni-NTA column was not able to be produced.
The second aim was to determine if residues in TMH regions of C8αMACPF insert into the membrane when incorporated in the MAC. CDCs are pore forming toxins found in gram-positive bacteria known to insert into target membranes through a well characterized mechanism involving refolding of α-helical bundles into trans-membrane β- hairpins (TMHs) to form a lytic pore.
Structural similarity between CDCs and C8αMACPF suggests that TMH regions of C8αMACPF may insert into the membrane. Fluorescent labeling of residues in C8αMACPF was unable to produce reliable data to indicate membrane insertion.
The final aim used chimeras containing TMH regions of C8β replacing TMH regions of the CDC Perfingolysin O (PFO) to determine if TMH regions from MAC proteins will insert into the membrane as the TMH regions of CDCs are known to. A construct with TMH2 of C8β replacing the TMH2 region of PFO has been expressed as insoluble inclusion bodies. All efforts detailed in this thesis culminate in an effort to gain knowledge of the MACPF domains of MAC family proteins to provide insight into the mechanism through which the MAC forms.
Damour, M. S.(2010). Components of the Membrane Attack Complex (Mac) of Human Complement: Functional Studies of Their Macpf Domains. (Master's thesis). Retrieved from http://scholarcommons.sc.edu/etd/673