Structure-function analysis of gC1qR/p33 using deletion mutants show additional binding sites for C1q. (134.69)

Abstract

Abstract Surface expressed or soluble gC1qR has been shown to activate complement and to play a role in various inflammatory processes. By peptide mapping and antibody inhibition studies, we had previously identified a binding site for C1q in a gC1qR domain contained within residues 76-93. However, although mAbs 60.11, which recognizes residues 76-93, can inhibit C1q binding to gC1qR, the inhibition is not complete. Similarly, binding of C1q to a truncated form of gC1qR lacking residues 76-93 is reduced by only 40-50% when compared to the full-length gC1qR. These findings therefore prompted us to look for additional binding sites elsewhere in the molecule. To this end, we generated several recombinant gC1qR proteins by deletion of domains of very high negative charge— and which, based on their location in the crystal structure—were predicted to interact with solution-phase ligands. The capabilities of these mutants to bind to C1q were therefore examined by comparing to the binding of the full-length gC1qR. Analyses of the data reveal the presence of two additional binding sites for C1q—one contained within residues 190-192 and another within residues 144-162. These results not only demonstrate well the value of crystal-based structure-function predictions, but also allow the identification of potential targets for drug design to alleviate complement-mediated inflammation. [Supported by NIH-NIAID Grant R01 AI-060866]