Binding site for vitamin K-dependent protein S on complement C4b-binding protein

Protein S and C4b-binding protein (C4bp) form a plasma complex, but the binding mechanism remains unclear. Prior research has shown that protein S associates with C4bp, a complement regulatory protein. However, no prior work had resolved the exact location of the protein S-binding site on C4bp. This gap motivated the current study to identify the specific region on C4bp responsible for protein S binding. Understanding this interaction is important for comprehending complement regulation. The complement system plays a central role in innate immunity. C4bp exists in multiple isoforms, including C4bp-high and C4bp-low, which differ in molecular weight and protein S binding. No prior work had resolved whether these isoforms differ in structure or binding capacity. This uncertainty drove the investigation into structural differences and functional implications.

The aim of this study was to identify the region on C4b-binding protein (C4bp) that binds to protein S. Researchers focused on structural and functional differences between C4bp isoforms. They hypothesized that the protein S-binding site resides in a specific region of C4bp. The specific problem addressed was the lack of clarity on how protein S interacts with C4bp. The motivation stemmed from the need to understand complement regulation and plasma protein interactions. The study sought to determine whether C4bp-high and C4bp-low differ in structure or binding capacity. Researchers also aimed to identify the exact amino acid sequence involved in protein S binding. This could clarify the functional differences between C4bp isoforms.

The study used monoclonal anti-C4bp-IgGs to identify the protein S-binding site. C4bp was digested with chymotrypsin to produce NH2-terminal arm fragments and a COOH-terminal core fragment. The COOH-terminal fragment was tested for its ability to inhibit protein S binding. A monoclonal antibody (MFbp16) was used to isolate a peptide from the COOH-terminal fragment. Affinity chromatography with MFbp16-Sepharose was used to purify a peptide of Mr 2,500. The sequence of this peptide was determined using amino acid analysis. The binding of protein S to the peptide was tested in the presence of C4bp. Structural differences between C4bp-high and C4bp-low were also compared using binding assays.

The COOH-terminal fragment of C4bp inhibited protein S binding in a dose-dependent manner. A monoclonal antibody (MFbp16) bound to the COOH-terminal fragment and blocked protein S binding. Affinity chromatography isolated a peptide of Mr 2,500 from the COOH-terminal fragment. Protein S bound to this peptide, and this binding was inhibited by C4bp. The peptide sequence was identified as Ser447-Tyr467 near the COOH terminus of C4bp. This region appears to be the protein S-binding site on C4bp. C4bp-low did not bind to MFbp16, suggesting it lacks the protein S-binding site. C4bp-low dissociates into identical subunits when reduced, indicating structural differences.

The protein S-binding site on C4b-binding protein (C4bp) resides in the COOH-terminal region. The sequence Ser447-Tyr467 was identified as the binding site based on peptide binding assays. C4bp-high contains this binding site, while C4bp-low does not. This suggests structural differences between C4bp isoforms. The interchain disulfide bond region appears closer to the NH2-terminal end than the binding site. These findings clarify the functional differences between C4bp-high and C4bp-low. The study supports the hypothesis that the COOH-terminal region is critical for protein S binding. The results provide insight into the structural basis of protein S-C4bp interactions.