title: A protein S binding site on C4b-binding protein involves beta chain residues 31-45.
abstract: C4b-binding protein (C4BP) down-regulates the anticoagulant cofactor activity of protein S in the protein C pathway since free protein S but not the protein S:C4BP complex is anticoagulantly active. To identify beta chain residues responsible for binding protein S, synthetic overlapping pentadecapeptides covering the entire 1-235 sequence were tested as inhibitors of complex formation. The peptide comprising residues 31-45 (VCIKGYHLVGKKTLF) from the first short consensus repeat domain inhibited the binding of C4BP to protein S with half-maximal inhibition at 20-45 microM, and studies suggested the sequence of YxLVG was crucial. Peptide beta(31-45) specifically inhibited the APC cofactor activity of purified protein S in Xa-1-stage coagulation assays with 50% inhibition at 15 microM peptide. Peptide beta(31-45) and related peptides such as beta(34-42) inhibited the binding of protein S to an antipeptide monoclonal antibody made against residues 420-434 of protein S (monoclonal antibody LJ-56). Polyclonal anti-beta(31-45) peptide antibodies inhibited complex formation. Dose-dependent binding studies showed that protein S bound directly to immobilized peptide beta(31-45). These results show that residues 31-45 of the C4BP beta chain provide a binding site for protein S, and they suggest that the C4BP beta chain residues 34-42 are located near residues 420-434 of protein S in the protein S:C4BP complex.
authors.affiliation: Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
authors.forename: J A
authors.initials: JA
authors.lastname: Fernández
authors.forename: J H
authors.initials: JH
authors.lastname: Griffin
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015935:Complement C4b / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D016384:Consensus Sequence; D006023:Glycoproteins; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000494:pharmacology*; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D017293:Protein S / Q000302:isolation & purification / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010455:Peptides; D017293:Protein S; D015935:Complement C4b