Binding studies of human complement protein C8gamma

C8gamma is a subunit of complement protein C8, a component of the membrane attack complex (MAC). C8 is a heterotrimer, with subunits arranged as a disulfide-linked C8alpha-gamma dimer that is noncovalently associated with C8beta. C8gamma, a 22 kDa protein, has the distinction of being the only lipocalin among the complement proteins. Lipocalins are a family of extracellular beta-barrel proteins that typically bind small, hydrophobic ligands at the open end of the barrel. The structure of C8gamma bound to the MACPF domain of C8alpha (alphaMACPF) has been solved. The core MACPF domain shares structural similarities with bacterial pore-forming toxins (PFT). Domains within PFTs bind specific ligands on cell membranes including lipid, cholesterol, carbohydrate and GPI anchored receptors. Structural similarities between alphaMACPF-gamma and PFTs suggest that C8gamma may also have a membrane binding function.;The objective of this project was to determine if C8gamma interacts with natural or model membranes. First, it was determined if C8gamma could bind to sheep red blood cells (RBC). Using Western blot analyses, C8gamma, C8alpha-gamma, and C8 were observed to bind to sheep RBCs. Results from competition assays suggest the binding sites on C8gamma and C8alpha-gamma for RBCs are similar. Second, the ability of C8gamma to bind to lipid vesicles (liposomes) was investigated. C8gamma was observed to bind to phosphatidylcholine (PC) liposomes using gel filtration chromatography. Surface plasmon resonance (SPR) studies showed that C8gamma has a much greater affinity for liposomes than do C8alpha-gamma or C8. Competition assays using C8gamma and the C8alpha indel peptide suggest that C8gamma interaction with the liposome surface occurs through the top loops of the C8gamma beta-barrel. C8gamma also bound to PC liposomes containing lipopolysaccharides (LPS) and with greater affinity than to PC liposomes alone. Third, the ability of C8gamma to interact with sheep RBCs and E. coli cells was examined using confocal fluorescence microscopy. Fluorescently labeled C8gamma could not be seen to interact with either cell type, however aggregation of RBCs and E. coli cells was observed in the presence of either unlabeled C8gamma or fluorophore-conjugated C8gamma. The final aim was to determine if C8gamma could bind an array of different lipids immobilized on a commercial lipid strip. Ligand blotting did not detect C8gamma binding to any of the lipids. Together, these findings show that C8gamma can bind lipid and that binding likely involves the loops at the barrel opening.