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Interaction between protein S and complement C4b-binding protein (C4BP). Affinity studies using chimeras containing
R H van de Poel1, J C Meijers, B N Bouma
1Thrombosis and Haemostasis Laboratory, Department of Haematology, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands. r.h.l.vandepoel@med.uu.nl
Insights
Human C4b-binding protein (C4BP) regulates the complement system and protein C pathway. Research shows C4BP beta-chain SCR-2 specifically contributes to binding anticoagulant protein S, impacting its function.
Area of Science:
- Immunology
- Biochemistry
Background:
- Human C4b-binding protein (C4BP) is a key regulator of the complement system.
- C4BP modulates the anticoagulant protein C pathway by binding protein S, reducing its cofactor activity for activated protein C.
- The protein S binding site on C4BP beta-chain was previously localized to SCR-1.
Purpose of the Study:
- To further delineate the specific domains of the C4BP beta-chain involved in protein S binding.
- To elucidate the role of individual short consensus repeats (SCRs) in the interaction between C4BP and protein S.
Main Methods:
- Construction of chimeric proteins fusing different C4BP beta-chain SCRs (SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, SCR-2+3) to tissue-type plasminogen activator.
- Binding assays to quantify the affinity of protein S for the C4BP beta-chain chimeras.
- Functional assays measuring the cofactor activity of protein S in the presence of C4BP constructs.
Main Results:
- Chimeras containing SCR-1 and SCR-2 (SCR-1+2) exhibited a 5-fold higher affinity for protein S compared to SCR-1 alone or SCR-1+3.
- Binding of protein S to constructs containing SCR-1 reduced its cofactor activity, with SCR-1+2 showing more potent inhibition.
- SCR-2 demonstrated a specific contribution to protein S binding, as SCR-3 did not enhance the interaction with SCR-1.
Conclusions:
- The beta-chain SCR-2 of C4BP plays a specific and significant role in the interaction with protein S.
- This finding refines our understanding of C4BP's regulatory mechanism within the complement and protein C pathways.
Abstract:
Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical alpha-chains and a single beta-chain (C4BPbeta), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPbeta. To further localize the protein S binding site, we constructed chimeras containing C4BPbeta SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, beta-chain SCR-2 contributes to the interaction of C4BP with protein S.