Binding Site for C4b-Binding Protein in Vitamin K-Dependent Protein S Fully Contained in Carboxy-Terminal Laminin-G-type Repeats. A Study Using Recombinant Factor IX-Protein S Chimeras and Surface Pla
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- 作者：Xuhua He ; Lei Shen ; Ann-Christin Malmborg ; Kenneth J. Smith ; Bjö ; rn Dahlbä ; ck ; and Sara Linse
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：March 25, 1997
- 年：1997
- 卷：36
- 期：12
- 页码：3745 - 3754
- 全文大小：213K
- 年卷期：v.36,no.12(March 25, 1997)
- ISSN：1520-4995
文摘
The interaction between vitamin K-dependent protein Sand the C4b-binding protein (C4BP)was studied using surface plasmon resonance and genetic engineering.The affinity, as well as associationand dissociation rates of the complex, was measured for human andbovine protein S at five differentcalcium concentrations. The binding to C4BP of six protein hybridscontaining different parts of coagulationfactor IX and protein S was studied in the absence and presence ofcalcium. The results show thatdissociation of the human protein S-C4BP complex is extremely slow inthe presence of 
10 
M calcium(koff = 7 × 10-6s-1) and the association rate constant iskon = 7 × 4M-1 s-1. Humanand bovine proteinS were found to bind to human C4BP with the same affinity,KD = 0.1 nM, but the rates ofassociationand dissociation were higher for the bovine protein S(kon = 2 × 5M-1 s-1,koff = 2 × 10-5s-1). In theabsence of calcium, the affinity for C4BP was reduced by a factor of 65for human protein S and by afactor of 40 for bovine protein S. The decreased affinity could bemainly attributed to an increasedoff-rate (12-17-fold), while the on-rate decreased 3-4-fold.The studies using chimeric proteins showthat the portion of protein S that is responsible for binding to C4BPis fully contained in the two laminin-G-type repeats, which are homologous to the sex hormone binding globulin(SHBG). All hybrids thatcontain the laminin-G-type repeats bind to C4BP with the same affinityas recombinant protein S, whereashybrids lacking these repeats show no detectable binding to C4BP.The present data also suggest that theeffect of calcium on the C4BP-binding properties is mediated by calciumbinding site(s) in the laminin-G-type repeats.
10 M calcium(koff = 7 × 10-6s-1) and the association rate constant iskon = 7 × 4M-1 s-1. Humanand bovine proteinS were found to bind to human C4BP with the same affinity,KD = 0.1 nM, but the rates ofassociationand dissociation were higher for the bovine protein S(kon = 2 × 5M-1 s-1,koff = 2 × 10-5s-1). In theabsence of calcium, the affinity for C4BP was reduced by a factor of 65for human protein S and by afactor of 40 for bovine protein S. The decreased affinity could bemainly attributed to an increasedoff-rate (12-17-fold), while the on-rate decreased 3-4-fold.The studies using chimeric proteins showthat the portion of protein S that is responsible for binding to C4BPis fully contained in the two laminin-G-type repeats, which are homologous to the sex hormone binding globulin(SHBG). All hybrids thatcontain the laminin-G-type repeats bind to C4BP with the same affinityas recombinant protein S, whereashybrids lacking these repeats show no detectable binding to C4BP.The present data also suggest that theeffect of calcium on the C4BP-binding properties is mediated by calciumbinding site(s) in the laminin-G-type repeats.