...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >MOCARHAGIN, A NOVEL COBRA VENOM METALLOPROTEINASE, CLEAVES THE PLATELET VON WILLEBRAND FACTOR RECEPTOR GLYCOPROTEIN IB-ALPHA - IDENTIFICATION OF THE SULFATED TYROSINE/ANIONIC SEQUENCE TYR-276-GLU-282 OF GLYCOPROTEIN IB-ALPHA AS A BINDING SITE FOR VON
【24h】

MOCARHAGIN, A NOVEL COBRA VENOM METALLOPROTEINASE, CLEAVES THE PLATELET VON WILLEBRAND FACTOR RECEPTOR GLYCOPROTEIN IB-ALPHA - IDENTIFICATION OF THE SULFATED TYROSINE/ANIONIC SEQUENCE TYR-276-GLU-282 OF GLYCOPROTEIN IB-ALPHA AS A BINDING SITE FOR VON

机译：MOCARHAGIN是一种新型眼镜蛇毒金属蛋白酶，可清除血小板冯·威勒伯兰因子受体糖蛋白IB-Alpha-硫酸酪氨酸/阴离子序列的鉴定TYR-276-GLU-282糖蛋白聚糖的抗原肽

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Platelet adhesion to the subendothelium is the initiating event in hemostasis and thrombosis and involves the binding of von Willebrand factor (vWF) by the platelet membrane glycoprotein (GP) Ib-IX complex. The alpha-chain of GP Ib contains binding sites for both vWF and alpha-thrombin within a 45-kDa N-terminal tryptic fragment. In the present study, we have further delineated these sites using smaller proteolytic fragments and functional antibodies. Mocarhagin, a cobra venom metalloproteinase, generates the fragment His-1-Glu-282, while cathepsin G, a neutrophil granule serine protease, generates a slightly smaller fragment, His-1-Leu-275, His-1-Glu-282 was as effective as intact soluble GP Ib alpha (glycocalicin) in inhibiting botrocetin-dependent binding of VWF to washed platelets (IC50 similar to 0.3 mu M), whereas His-1-Leu-275 was an order of magnitude less effective (IC50 similar to 3 mu M). Residues Tyr-276-Glu-282 (YDYYPEE) are part of an anionic region homologous to thrombin-binding molecules such as hirudin, In ligand blot analysis, thrombin blotted the His-1-Glu-282 fragment, but not His-1-Leu-275. The three tyrosine residues within Tyr-276-Gln-282 meet the consensus criteria for O-sulfation. A method was developed to distinguish O-sulfated from nonsulfated tyrosine residues based on differences in the UV absorbance spectra. Residues Tyr-276-Glu-282 were isolated from glycocalicin by proteolysis with mocarhagin and cathepsin G. Ion spray mass spectrometry confirmed that Tyr-278 and Tyr-279 were O-sulfated to at least 90%, whereas Tyr-276 was only similar to 50% O-sulfated. Four anti-GP Ib alpha monoclonal antibodies (SZ2, ES85, C34, and VM16d) were found to be modulator-specific, strongly inhibiting botrocetin-dependent binding of VWF, but having less or no effect on ristocetin-dependent VWF binding. These antibodies also inhibited the binding of thrombin to fixed platelets. Immunoprecipitation with GP Ib alpha fragments defined the epitopes for these antibodies as SZ2 (Tyr-276-Glu-282), ES85 (Asp-283-Arg-293), C34 (His-1-Glu-282), and VM16d (His-1-Leu-275). An antibody which inhibited ristocetin-dependent, as well as botrocetin-dependent, VWF binding but had no effect on thrombin binding (AK2) had an epitope within His-1-Leu-275. These findings indicate that the sulfated tyrosine/anionic GP Ib alpha residues Tyr-276-Glu-282 are important for the binding of thrombin and the botrocetin-dependent binding of VWF, but that VWF also interacts with residues within His-1-Leu-275.
机译：血小板粘附于内皮下膜是止血和血栓形成的起始事件，并涉及血小板膜糖蛋白（GP）Ib-IX复合物与von Willebrand因子（vWF）的结合。 GP Ib的α链在45 kDa N末端胰蛋白酶片段内包含vWF和α凝血酶的结合位点。在本研究中，我们使用较小的蛋白水解片段和功能性抗体进一步描述了这些位点。眼镜蛇毒金属蛋白酶Mocarhagin生成片段His-1-Glu-282，而中性粒细胞丝氨酸蛋白酶组织蛋白酶G生成的片段稍小，His-1-Leu-275，His-1-Glu-282与完整的可溶性GP Ibα（糖精）一样有效，可抑制Botrocetin依赖性的VWF与洗涤后的血小板结合（IC50类似于0.3μM），而His-1-Leu-275的效力要低一个数量级（IC50类似于3微米）。残基Tyr-276-Glu-282（YDYYPEE）是与凝血酶结合分子（例如水rud素）同源的阴离子区域的一部分。在配体印迹分析中，凝血酶印迹了His-1-Glu-282片段，但没有印迹His-1 Leu-275。 Tyr-276-Gln-282中的三个酪氨酸残基符合O-硫酸化的共识标准。根据紫外线吸收光谱的差异，开发了一种区分O-硫酸化和非硫酸化酪氨酸残基的方法。残留的Tyr-276-Glu-282是通过用Mocarhagin和组织蛋白酶G的蛋白水解作用从糖胺素中分离得到的。离子喷雾质谱法证实Tyr-278和Tyr-279被O-硫酸化至至少90％，而Tyr-276仅相似到50％的O被硫酸化。发现四种抗GP Ibα单克隆抗体（SZ2，ES85，C34和VM16d）是调节剂特异性的，强烈抑制VWF的Botrocetin依赖性结合，但对ristocetin依赖性VWF结合的影响很小或没有。这些抗体也抑制凝血酶与固定血小板的结合。 GP Ibα片段的免疫沉淀将这些抗体的表位定义为SZ2（Tyr-276-Glu-282），ES85（Asp-283-Arg-293），C34（His-1-Glu-282）和VM16d（His -1-Leu-275）。抑制猪降血脂素依赖性以及与肠胃菌素依赖性的VWF结合但对凝血酶结合（AK2）无影响的抗体在His-1-Leu-275中具有一个表位。这些发现表明，硫酸化的酪氨酸/阴离子GP Ibα残基Tyr-276-Glu-282对凝血酶的结合和VWF的依赖于trotrocetin的结合很重要，但是VWF也与His-1-Leu-中的残基相互作用275。

著录项

相似文献

  • 外文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号