The protein S-binding site localized to the central core of C4b-binding protein.
Human C4b-binding protein (C4BP) is a regulator of the classical pathway of the complement system. It appears in two forms in plasma, as free protein and in a noncovalent complex with the vitamin K-dependent coagulation protein, protein S. In the electron microscope C4BP has a spider-like structure with a central core and seven extended tentacles, each of which has a binding site for C4b, although the protein S-binding site has not been unequivocally pinpointed. C4BP was subjected to chymotrypsin digestion which yielded two major fragments, one of 160 kDa representing the central core, and one of 48 kDa representing the cleaved-off tentacles. We have now localized the protein S-binding site to the 160-kDa central core fragment. Using immunoblotting with a panel of polyclonal antisera, the isolated central core was shown to be completely devoid of 48-kDa fragments. The protein S-binding site was susceptible to proteolysis by chymotrypsin, but was protected by a molar excess of protein S included during the proteolysis. The 160-kDa central core fragment consisted of identical, disulfide-linked 25-kDa peptides and a proper disulfide bond arrangement was crucial to protein S binding. Using a direct binding assay it was shown that the isolated central core had the same affinity for protein S as intact C4BP.
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D003169:Complement Inactivator Proteins; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D010446:Peptide Fragments / Q000378:metabolism; D017293:Protein S
D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D004220:Disulfides; D006023:Glycoproteins; D010446:Peptide Fragments; D017293:Protein S; D002918:Chymotrypsin