Isolation and characterization of the kininogen-binding protein p33 from endothelial cells - Identity with the gC1q receptor
Isolation and characterization of the kininogen-binding protein p33 from endothelial cells - Identity with the gC1q receptor
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DOI：
10.1074/jbc.271.22.13040
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发表时间：
1996-05-31
影响因子：
4.8
通讯作者：
MullerEsterl, W
中科院分区：
文献类型：
--
作者：
Herwald, H;Dedio, J;MullerEsterl, W
激肽原是血管活性激肽的前体蛋白，与血小板、中性粒细胞和内皮细胞特异性、可逆性和饱和性结合。激肽原的两个结构域暴露主要的细胞结合位点：H-和L-激肽原共享的结构域D3和仅存在于H-激肽原中的结构域D5（H）。先前我们已经将激肽原细胞结合位点分别定位于D3的27个残基（“LDC 27”）和D5（H）的20个残基（“HKH 20”）（Herwald，H.，哈桑A. A. K.，Godovae-Zimmermann，J.，Schmaier，A. H、和Muller-Esterl，W.（1995）J.Biol.Chem.270，14634-14642; Hasan，A. A. K.，Cines，D. B.，Herwald，H.，Schmaier，A. H、和Muller-Esterl，W.等人（1995）J. Biol. Chen. 270，19256-19261）。细胞表面暴露的相应激肽原受体位点仍不清楚。使用非离子去污剂，Nonidet P-40，我们已经能够溶解激肽原结合位点从内皮细胞系，EA.hy926，在其功能活性形式。对HKH 20（一种代表D5（H）细胞结合位点的合成肽）上溶解的激肽原结合位点进行亲和层析，使我们能够分离出一种33-kDa蛋白（“p33”），该蛋白特异性且可逆地结合H-激肽原，K-D（表观解离常数）为9 +/-2 nM。随后通过NH 2-末端氨基酸序列分析的SDS电泳鉴定了激肽原结合蛋白p33作为gC 1 q受体（“gC 1 qR”），一种与补体成分C1 q的球状结构域相互作用的外源性膜蛋白。纯化的p33以中等亲和力结合C1 q，KD = 240 +/- 10 nM。相应的cDNA在大肠杆菌中的重组表达表明，p33结合H-激肽原，而不是L-激肽原。肽HKH 20而不是肽LDC 27以浓度依赖性方式抑制H-激肽原与重组p33的结合，表明H-激肽原通过结构域D5（H）与p33结合。重组p33能有效抑制H-激肽原与EA.hy926细胞的结合。因子XII，而不是前激肽释放酶，与H-激肽原竞争结合p33。这些研究结果表明，内皮结合蛋白介导的激肽产生途径的内皮细胞表面上的关键组件的组装，从而连接的早期事件的激肽形成和补体激活。
Kininogens, the precursor proteins of the vasoactive kinins, bind specifically, reversibly, and saturably to platelets, neutrophils, and endothelial cells. Two domains of the kininogens expose major cell binding sites: domain D3 that is shared by H- and L-kininogen and domain D5(H) that is exclusively present in H-kininogen. Previously we have mapped the kininogen cell binding sites to 27 residues of D3 (''LDC27'') and 20 residues of D5(H) (''HKH20''), respectively (Herwald, H., Hasan, A. A. K., Godovae-Zimmermann, J., Schmaier, A. H., and Muller-Esterl, W. (1995) J. Biol. Chem. 270, 14634-14642; Hasan, A. A. K., Cines, D. B., Herwald, H., Schmaier, A. H., and Muller-Esterl, W. (1995) J. Biol. Chen. 270, 19256-19261). The corresponding kininogen acceptor site(s) exposed by the cell surfaces are still poorly defined. Using a non-ionic detergent, Nonidet P-40, we have been able to solubilize kininogen binding sites from an endothelial cell line, EA.hy926, in their functionally active form. Affinity chromatography of the solubilized kininogen binding sites on HKH20, a synthetic peptide representing the D5(H) cell binding site, allowed us to isolate a 33-kDa protein (''p33'') that binds specifically and reversibly to H-kininogen with a K-D (apparent dissociation constant) of 9 +/- 2 nM. Preparative SDS electrophoresis followed by NH2-terminal amino acid sequence analysis identified the kininogen-binding protein p33 as the gC1q receptor (''gC1qR''), an extrinsic membrane protein that interacts with the globular domains of the complement component C1q. The purified p33 binds C1q with moderate affinity, K-D = 240 +/- 10 nM. Recombinant expression of the corresponding cDNA in Escherichia coli demonstrated that p33 binds H-kininogen, but not L-kininogen. Peptide HKH20 but not peptide LDC27 inhibited binding of H-kininogen to the recombinant p33 in a concentration-dependent manner, indicating that H-kininogen binds to p33 via domain D5(H). Recombinant p33 efficiently inhibited the binding of H-kininogen to EA.hy926 cells. Factor XII, but not prekallikrein, competed with H-kininogen binding to p33. These findings suggest that an endothelial binding protein mediates the assembly of critical components of the kinin-generating pathway on the surface of endothelial cells, thereby linking the early events of kinin formation and complement activation.