A Region of Vitamin K-dependent Protein S That Binds to C4b Binding Protein (C4BP) Identified Using Bacteriophage Peptide Display Libraries

Abstract

Vitamin K-dependent protein S, a blood coagulation inhibitor, interacts with the C4b-binding protein (C4BP) in human plasma with high affinity (K D ‫؍‬ 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 ␤-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.

Structural Requirements of Anticoagulant Protein S for Its Binding to the Complement Regulator C4b-binding Protein

“…Synthetic Peptides-Five peptides (Table I) with acetylated NH 2 termini and amidated COOH termini were synthesized on a MilliGen 9050 Plus synthesizer and purified by high pressure liquid chromatography, as described previously (39).…”

“…In the present study, we have continued characterizing the binding site in protein S for C4BP, focusing on the region encompassing residues 447-460, which, based on phage display experimentation, was suggested to be involved in C4BP binding (39). Using Ala scanning mutagenesis, peptide inhibition assays, surface plasmon resonance, and monoclonal epitope mapping, the involvement of this region in C4BP binding was elucidated.…”

Dependence on Vitamin K-dependent Protein S for Eukaryotic Cell Secretion of the β-Chain of C4b-binding Protein

“…The binding site in PS for C4BP is located in the SHBG region of PS, and both laminin G domains are required (6). The binding site in PS has not been narrowed down further, even though there are several studies suggesting different regions of the SHBG domain to be of importance (7,8).…”

“…The binding site in PS for C4BP is located in the SHBG region of PS, and both laminin G domains are required (6). The binding site in PS has not been narrowed down further, even though there are several studies suggesting different regions of the SHBG domain to be of importance (7,8).C4BP acts as a cofactor for the down-regulation of the complement cascade and shares structural features with several other proteins participating in this system. It is composed of multiple disulfide-linked subunits (␣-and ␤-chains), each containing repeats of complement control protein (CCP) domains (9).…”

Vitamin K-Dependent Protein S Localizing Complement Regulator C4b-Binding Protein to the Surface of Apoptotic Cells

“…Protein S contains multiple domains, including an N-terminal ␥-carboxy glutamic acid (Gla)-rich domain, which binds Ca 2ϩ and is responsible for the interaction with negatively charged phospholipids, a thrombin sensitive region, four epidermal growth factor (EGF)-like domains, and two laminin G-type domains. The G domains are involved in the interaction with C4BP, especially residues 605-614 (28,29), 420 -434 (30,31), and 447-460 (32). C4BP is an important regulator of the classical pathway of complement.…”