ANALYSIS OF PROTEIN-S C4B-BINDING PROTEIN INTERACTIONS BY HOMOLOGY MODELING AND INHIBITORY ANTIBODIES

A monoclonal antibody (mAb 6F6) directed against the beta-chain of C4b-binding protein (C4BP) was previously shown to inhibit the binding of protein S to C4BP. To localize the epitope of this antibody, 23 overlapping synthetic peptides (15-mers) covering the entire sequence (1-235) of the beta-chain of C4BP were used. When the immobilized peptides were screened for their ability to bind mAb 6F6, only peptide beta(51-65) showed high-affinity binding. The apparent affinity of mAb 6F6 for immobilized peptide beta-(51-65) was somewhat similar to that for native C4BP with K-d similar to 1 nM for C4BP and similar to 9 nM for peptide beta(51-65). Peptide beta(5 1-65) inhibited the binding of the mAb 6F6 to immobilized C4BP with half-maximal inhibition at 30 mu M peptide. Clotting assays of protein S anticoagulant cofactor activity using a factor Xa-1-stage assay with activated protein C allow measurement of free protein S in solution since only free protein S is active. Studies using such clotting assays showed that preincubation of C4BP with either mAb 6F6 or polyclonal anti-beta(31-45) antibodies inhibited the formation of the complex between C4BP and protein S. Previous studies showed that, although peptide beta(5 1-65) itself does not inhibit complex formation, peptide beta(31-45) does bind directly to protein S and does inhibit protein S binding to C4BP. The three-dimensional structure of the first SCR (residues 2-60) of the C4BP beta-chain was made on the basis of homology modeling. In the computer graphics model, residues 51-60 are spatially proximate to residues 31-45, suggesting that the inhibitory effect of mAb 6F6 on complex formation is most likely due to the antibody's steric hindrance of protein S access to beta-chain residues 31-45. Thus, these anti-beta-chain antibody results are very consistent with the hypothesis that the first SCR of the beta-chain of C4BP provides a binding site for protein S.