Interaction of fibrin(ogen) with the endothelial cell receptor VE-Cadherin:: Mapping of the receptor-binding site in the NH2-terminal portions of the fibrin β chains
Interaction of fibrin(ogen) with the endothelial cell receptor VE-Cadherin:: Mapping of the receptor-binding site in the NH2-terminal portions of the fibrin β chains
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DOI：
10.1021/bi0160314
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发表时间：
2002-03-26
期刊：
影响因子：
2.9
通讯作者：
Medved, L
中科院分区：
文献类型：
--
作者：
Gorlatov, S;Medved, L
纤维蛋白与内皮细胞的相互作用刺激毛细管形成，从而促进血管生成。这种相互作用通过内皮细胞受体VE-钙粘蛋白和纤维蛋白β链15-42区发生[Bach，T.L.，et al.(1998)J.Biol.化学。272,30719-30728]。为了阐明这种相互作用的机制，我们在大肠杆菌中表达了含有β15-42区或VE-cad(1-2)和VE-cad(1-4)片段的重组纤维蛋白原片段，这些片段分别包含VE-钙粘蛋白的两个和四个胞外NH2末端结构域，并通过表面等离子共振和ELISA法检测了它们之间的相互作用。重组的Bbeta1-57或Bbeta1-片段以及从后者片段通过凝血酶处理去除纤维蛋白肽B而制备的Beta15-57或Beta15-都不能与重组VE-钙粘蛋白片段结合。同时，通过Cys65二硫键连接以模拟纤维蛋白中P链二聚体排列的二聚体重组凝血酶处理的(Beta15-66)(2)片段与VE-cad(1-4)结合良好，但与VE-cad(I-2)结合不好；未处理的(Bbeta1-66)(2)二聚体未观察到结合。接下来，我们突变了二聚体His16、Arg17、Pro18和Asp20中的几个残基，并通过配基印迹和表面等离子共振测试了凝血酶处理的突变体与VE-cad(1-4)的相互作用。与H16A和R17Q单突变体以及H16P、P18V双突变体没有结合，而P18A和D20N单突变体与VE-cad(1-4)结合的亲和力与凝血酶处理的野生型二聚体相同。这些结果表明，纤维蛋白中的VE-钙粘蛋白结合部位包括两条纤维蛋白β链的NH2末端区域，His 16和Arg17对结合是关键的，VE-钙粘蛋白的第三和/或第四个胞外结构域是发生结合所必需的。
Interaction of fibrin with endothelial cells stimulates capillary tube fori-nation thus promoting angiogenesis. This interaction occurs via endothelial cell receptor VE-cadherin and fibrin beta chain 15-42 regions [Bach, T. L., et al. (1998) J. Biol. Chem. 272, 30719-30728]. To clarify the mechanism of this interaction, we expressed in Escherichia coli a number of recombinant fibrin(ogen) fragments containing the beta15-42 region or the VE-cad(1-2) and VE-cad(1-4) fragments encompassing two and four extracellular NH2-terminal domains of VE-cadherin, respectively, and tested interaction between them by surface plasmon resonance and ELISA. Neither the recombinant Bbeta1-57 or Bbeta1-64 fragments, nor beta15-57 or beta15-64 prepared from the latter fragments by thrombin treatment to remove fibrinopeptides B, bound the recombinant VE-cadherin fragments. At the same time, a dimeric recombinant thrombintreated (beta15-66)(2) fragment, which had been disulfide-linked via Cys65 to mimic the dimeric arrangement of the P chains in fibrin, bound VE-cad(1-4) well, but not VE-cad(I-2); no binding was observed with the untreated (Bbeta1-66)(2) dimer. We next mutated several residues in the dimer, His16, Arg17, Pro18, and Asp20, and tested the interaction of the thrombin-treated mutants with VE-cad(1-4) by ligand blotting and surface plasmon resonance. No binding was observed with the H16A and R17Q single mutants and the H16P, P18V double mutant while the P18A and D20N single mutants bound VE-cad(1-4) with the same affinity as the thrombin-treated wild-type dimer. These results indicate that the VE-cadherin binding site in fibrin includes NH2-terminal regions of both fibrin beta-chains, that His 16 and Arg17 are critical for the binding, and that the third and/or fourth extracellular domains of VE-cadherin are required for the binding to occur.