Abstract
Vitamin K-dependent protein S, a blood coagulation inhibitor, interacts with the C4b-binding protein (C4BP) in human plasma with high affinity (KD = 0.1 nM). Identification of a portion of protein S that binds to C4BP has been approached using random libraries of 6- and 15-mer peptides displayed on bacteriophage surfaces. Bacteriophage binding to the beta-chain of C4BP were selected in several rounds of affinity purification with intervening amplification in E. coli. Homology searches of the affinity purified peptide sequences against protein S led to the identification of four regions in protein S that were similar to several of the selected peptides. These regions were synthesized as linear peptides and tested in inhibition experiments. Only one distinct peak (around position 450) was observed when the homology scores versus human protein S sequence were averaged over all affinity purified peptides. A synthetic peptide comprising residues 439-460 in human protein S was found to inhibit protein S binding to C4BP. The same result was found with two overlapping peptides (residues 447-468 and 435-468, respectively) in a second set of synthetic peptides. Direct binding of the peptides to C4BP was inferred from titrations monitored by recording the near UV circular dichroism spectra or the polarization of tryptophan fluorescence. The results suggest that residues 447-460 constitute a portion of protein S that is important for the interaction with C4BP. These findings may have implications for patients suffering from thrombosis, due to the lack of free protein S, by directing the design of drugs that disrupt protein S binding to C4BP.
Highlights
Vitamin K-dependent protein S and C4b-binding protein (C4BP)1 form a tight 1:1 molar complex in human plasma [1, 2]
The results suggest that residues 447– 460 constitute a portion of protein S that is important for the interaction with C4BP
Phage-bound peptides were affinity purified against the short consensus repeat (SCR)-1,2-fragment, which contains the protein S binding site of C4BP (Fig. 1)
Summary
Vitamin K-dependent protein S and C4b-binding protein (C4BP) form a tight 1:1 molar complex in human plasma [1, 2]. C4BP is an acute-phase protein, and its concentration can increase as much as 4-fold during the acute-phase inflammatory response It has recently been demonstrated, that it is primarily the concentration of the C4BP isoform lacking the -chain that increases during the acutephase inflammatory response, resulting in a maintained anticoagulation system in a normal individual and indicating a differential regulation of the ␣- and -chains [28]. It is important to identify the domains that are mediating this proteinprotein interaction
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