Defining sites of interaction in the Ã-chain of C3 for factor H, membrane cofactor protein (MCP), and complement receptor 1 (CR1)

grantor: University of TorontoThe goal of this thesis was to identify sequences in human complement component C3 that contribute to its interaction with several of its regulatory molecules. Several groups have implicated residues within the 727-767 segment at the N-terminus of C3b $\alpha\sp\prime$-chain as being important for its interactions with the regulatory molecules factor H and complement receptor 1 (CR1), as well as the alternative pathway component factor B. This laboratory had previously identified residues 736$\rm\sp{E}$ and 737$\rm\sp{E}$ as being important for both factor B binding and CR1 binding, but mutation of these residues had little or no effect on factor H binding. I conducted a charged residue to alanine mutagenic scan between residues 741 and 767. Recombinant molecules were transiently expressed in COS-1 cells and were examined for their interaction with factor H using a factor I-mediated cleavage assay that was dependent upon prior complex formation between the C3b-equivalent molecule C3(H$\sb2$O) and the I-cofactor, in this case factor H. This approach identified the negatively charged side chains of residues 744$\rm\sp{E}$ and 747$\rm\sp{E}$ as being important for the binding interaction with factor H. The 727-767 series of mutants were further examined for their interactions with soluble forms of CR1 and MCP. In addition to confirming that residues 736,737$\rm\sp{EE}$ play a role in the CR1 interaction, I have further identified residues 747$\rm\sp{E}$ and 754,755$\rm\sp{ED}$ as being similarly important for this interaction. Residues 744$\rm\sp{E}$ and 757,758$\rm\sp{KE}$ also appeared to contribute to this interaction, although to a lesser extent. Thus, although the factor H and CR1 sites of interaction within the 727-767 segment appeared to overlap, the negatively-charged residue contacts required for CR1 binding appeared to be more extensive than those for factor H. None of the mutations had any effect on the binding with MCP, suggesting that the N-terminal $\alpha\sp\prime$-chain segment plays no role in this interaction. (Abstract shortened by UMI.)M.Sc