title: The binding site of human C4b-binding protein on complement C4 is localized in the alpha'-chain.
abstract: C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. We now demonstrate that C4b contains a binding site for C4BP, which is localized on the alpha'-chain of C4b. SDS-PAGE of C C4 and C4b both under reducing and nonreducing conditions was followed by a radiolabeled C4BP ligand blotting procedure. It was demonstrated that the C4BP binding site on C4b is localized on the alpha'-chain. In addition, we found C4BP binding to the alpha-chain of C4, which suggests that the binding site for C4BP becomes available after reduction of the C4 molecule. Direct binding of C4BP to the alpha- and alpha'-chains of C4 and C4b was demonstrated in a radio-labeled C4BP binding assay with the reduced and alkylated isolated chains. mAb against the alpha'-chain of C4b were prepared, characterized, and evaluated for their ability to block the binding of 125I-C4BP to C4b. Two mAb specific for the alpha'-chain of C4b were found that completely abolished C4BP binding to intact C4b. Other mAb recognizing both the alpha- and alpha'-chain of C4 and C4b demonstrated only minor inhibitory effect on the binding of C4BP to C4b. In conclusion, we have localized the C4BP binding site on the alpha'-chain of C4b and have demonstrated that this binding can be inhibited by mAb specific for the alpha'-chain.
authors.affiliation: Department of Haematology, University Hospital Utrecht, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Hessing
authors.forename: C
authors.initials: C
authors.lastname: van 'T Veer
authors.forename: B N
authors.initials: BN
authors.lastname: Bouma
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D003181:Complement C4 / Q000378:metabolism* / Q000648:ultrastructure; D015935:Complement C4b / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight
chemical_list: D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003181:Complement C4; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D046911:Macromolecular Substances; D015935:Complement C4b