Role of CCP2 of the C4b-binding protein beta-chain in protein S binding evaluated by mutagenesis and monoclonal antibodies.
Complement regulator C4b-binding protein (C4BP) and the anticoagulant vitamin K-dependent protein S form a high affinity complex in human plasma. C4BP is composed of seven alpha-chains and a unique beta-chain, each chain comprising repeating complement control protein (CCP) modules. The binding site for protein S mainly involves the first of the three beta-chain CCPs (CCP1). However, recently it has been suggested that CCP2 of the beta-chain also contributes to the binding of protein S. To elucidate the structural background for the involvement of CCP2 in the protein S binding, several recombinant beta-chain CCP1-2 variants having mutations in CCP2 were expressed and tested for protein S binding. Mutations were chosen based on analysis of a homology model of the beta-chain and included R60A/R101A, D66A, L105A, F114A/I116A and H108A. All mutant proteins bound equally well as recombinant wild type to protein S. Several monoclonal antibodies against the beta-chain CCP2 were raised and their influence on protein S binding characterized. Taken together, the results suggest that the role of CCP2 in protein S binding is to orient and stabilize CCP1 rather than to be directly part of the binding site.
Webb
Joanna H
JH
Division of Clinical Chemistry, Department of Laboratory Medicine, Lund University, Sweden.
Villoutreix
Bruno O
BO
Dahlbäck
Björn
B
Blom
Anna M
AM
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001665:Binding Sites; D003169:Complement Inactivator Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006023:Glycoproteins; D006801:Humans; D016296:Mutagenesis; D010446:Peptide Fragments / Q000378:metabolism; D017354:Point Mutation; D017293:Protein S / Q000378:metabolism*; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D010446:Peptide Fragments; D017293:Protein S