Role of CCP2 of the C4b-binding protein β-chain in protein S binding evaluated by mutagenesis and monoclonal antibodies

Abstract

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 α-chains and a unique β-chain, each chain comprising repeating complement control protein (CCP) modules. The binding site for protein S mainly involves the first of the three β-chain CCPs (CCP1). However, recently it has been suggested that CCP2 of the β-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 β-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 β-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 β-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.