C4b-binding protein (C4BP) beta-chain Short Consensus Repeat-2 specifically contributes to the interaction of C4BP with protein S.
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 a-chains and a single 3-chain. Each chain contains Short Consensus Repeats (SCRs). By making chimeras of 13-chain SCRs fused to tissue-type plasminogen activator (tPA chimeras), we found that 13-chain SCR-2 contributed to the interaction of 13-chain SCR-1 with protein S (van de Poel RHL, Meijers JCM, Bouma BN. J Biol Chem 274:15144-15150, 1999). Chimeras containing C4BP a-chains with SCR-1, SCR-l +2 or SCR-l +2+3 replaced by their 13-chain counterpart had affinities for protein S similar to C4BP (Hardig Y, Dahlb¿ck B. J Biol Chem 271:20861-20867, 1996). This was not in agreement with the finding that Beta-chain SCR-2 contributed to the interaction and could be explained by the possibility that alpha-chain SCR-2 in the alpha-chain chimeras contributed comparable with Beta-chain SCR-2 in the tPA chimeras. To investigate this we constructed a tPA chimera containing Beta-chain SCR-1 and alpha-chain SCR-2 (Beta1alpha2). Binding studies showed that Beta1alpha2 had a lower affinity compared with SCR-1 +2, indicating that alpha-chain SCR-2 did not contribute to the interaction. The difference with the alpha-chain chimeras may be explained by the fact that the alpha-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 alpha-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, Beta-chain SCR-2 specifically contributes to the interaction of SCR-1 with protein S.
van de Poel
R H
RH
Thrombosis and Haemostasis Laboratory, Department of Haematology, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands.
Meijers
J C
JC
Dahlbäck
B
B
Bouma
B N
BN
D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology; D001667:Binding, Competitive; D001777:Blood Coagulation / Q000187:drug effects; D018929:Cell Culture Techniques; D003169:Complement Inactivator Proteins; D016384:Consensus Sequence; D006224:Cricetinae; D006023:Glycoproteins; D006801:Humans; D011485:Protein Binding; D017293:Protein S / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011951:Receptors, Complement / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D010959:Tissue Plasminogen Activator / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D000925:Anticoagulants; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D017293:Protein S; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D010959:Tissue Plasminogen Activator