The amino-terminal module of the C4b-binding protein beta-chain contains the protein S-binding site.
Human C4b-binding protein (C4BP) is composed of multiple alpha-chains associated with a single beta-chain. Each chain is composed of homologous, tandemly arranged repeats of so-called short consensus repeats (SCRs). We have previously shown that the three SCR modules of the beta-chain contain a high affinity binding site for anticoagulant vitamin K-dependent protein S. On the basis of experiments using synthetic peptides, residues 31-45 of the amino-terminal SCR (SCR-1) in the beta-chain were suggested to be involved in protein S binding, but it is not known whether SCR-1 contains the entire protein S-binding site. To address this question, two different truncated forms of the beta-chain (beta1,2 and beta2, 3) were expressed in a prokaryotic expression system. The beta1,2 construct (SCR-1 + SCR-2) contained the high affinity binding site for protein S in contrast to beta2,3 (SCR-2 + SCR-3), which did not bind protein S. Unfortunately, it was not possible to express SCR-1 alone in this system. To further elucidate whether the protein S-binding site is fully contained in SCR-1 or whether SCR-2 is also required, recombinant alpha/beta-chain chimeras were constructed. These chimeras were composed of alpha-chains with one, two, or three of the amino-terminal SCR modules replaced by the beta-chain counterpart and were expressed in a eukaryotic expression system. All recombinant variants were retained within the cells and could be extracted in biologically active forms. The three alpha/beta-chain chimeras bound protein S equally well, with a Ka of approximately 2.3 x 10(8) +/- 0.2 M-1 as compared with 2.1 x 10(8) +/- 0.3 M-1 for plasma-purified C4BP. These results show that the entire protein S-binding site on C4BP is contained within beta-chain SCR-1.
Härdig
Y
Y
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, S-205 02 Malmö, Sweden.
Dahlbäck
B
B
D001483:Base Sequence; D001665:Binding Sites; D015935:Complement C4b / Q000378:metabolism; D003169:Complement Inactivator Proteins; D017931:DNA Primers / Q000737:chemistry; D006023:Glycoproteins; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D011485:Protein Binding; D017293:Protein S / Q000378:metabolism*; D011951:Receptors, Complement / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship
D003169:Complement Inactivator Proteins; D017931:DNA Primers; D006023:Glycoproteins; D008024:Ligands; D017293:Protein S; D011951:Receptors, Complement; D011993:Recombinant Fusion Proteins; D015935:Complement C4b