C4b-Binding Protein (C4BP) β-Chain Short Consensus Repeat-2 Specifically Contributes to the Interaction of C4BP with Protein S

ABSTRACT: C4b-binding protein (C4BP) regulates the complement system and the anticoagulant activity of protein S. Protein S can bind to C4BP, resulting in a decreased cofactor activity of protein S for anticoagulant activated protein C. C4BP contains several identical α-chains and a single β-chain. Each chain contains Short Consensus Repeats (SCRs). By making chimeras of β-chain SCRs fused to tissue-type plasminogen activator (tPA chimeras), we found that β-chain SCR-2 contributed to the interaction of β-chain SCR-1 with protein S (van de Poel RHL, Meijers JCM, Bouma BN. J Biol Chem 274:15144-15150, 1999). Chimeras containing C4BP α-chains with SCR-1, SCR-1+2 or SCR-1+2+3 replaced by their β-chain counterpart had affinities for protein S similar to C4BP (Härdig Y, Dahlbäck B. J Biol Chem 271:20861-20867, 1996). This was not in agreement with the finding that β-chain SCR-2 contributed to the interaction and could be explained by the possibility that α-chain SCR-2 in the α-chain chimeras contributed comparable with β-chain SCR-2 in the tPA chimeras. To investigate this we constructed a tPA chimera containing β-chain SCR-1 and α-chain SCR-2 (β1α2). Binding studies showed that β1α2 had a lower affinity compared with SCR-1+2, indicating that α-chain SCR-2 did not contribute to the interaction. The difference with the α-chain chimeras may be explained by the fact that the α-chain chimeras were linked by their C-terminal cysteines, resulting in multiple binding sites in a single molecule. Thereby, the effect of a lower affinity of each α-chain chimera may have been masked. The studies performed here help to clarify the apparent inconsistencies in two previous reports about the contribution of the SCR-2 domain in C4BP to protein S binding. In conclusion, β-chain SCR-2 specifically contributes to the interaction of SCR-1 with protein S.