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Molecular mechanism of fibronectin-induced chemotactic migration

纤连蛋白诱导趋化迁移的分子机制

基本信息

批准号：
04680173
负责人：
FUKAI Fumio
金额：
$ 1.22万
依托单位：
Faculty of Pharmaceutical Sciences, Science University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

We have shown that proteolytic degradation of FN yields biological activities not present in intact FN.We found that 21K Fib 2 fragment has a chemotactic activity at extremely low concentrations that intact FN hardly show the activity. Adipocyte differentiation of ST-13 preadipocytes is inhibited by intact FN, but the 24K fragment originated fromtheamino-terminal fibrin-binding domain of FN molecule dramatically stimulated the differentiation. The mode of proteolytic degradation of intact FN may be an important determinant for activation of FN function. First, we examined effects of inflammatory proteases (plasmin, neutrophil cathepsin G and elastase) and matrix metalloproteinases (stromelysin, matrilysin, gelatinase A, and interstitial collagenase) on the release of active FN fragments. Gelatinase A was found to be the most active in generating chemotactic Fib 2 fragments. Differentiation stimuratory Fib 1 fragments were effectively released by neutrophil elastase. In addition to such … More indirect action of proteases on the chemotactic migration, some intracellular Ca^<2+> dependentcysteine proteases were shown to involve directly in the chemotactic migration in response to FN.Second, a cell surface receptor protein for the 21K Fib 2 fragment was tried to isolate by an affinity column using 21K Fib 2 fragment-fixed gel. Washing the column with GRGDSP peptide, BSA solution, and then the Fib 2 fragment-depleted FN fragments mixture, after applying the ^<125>I-labed NIH-L13 cell extract, enabled to remove exclusively the proteins binding nonspecifically to the affinity gel. As a result, one sharp radioactive peak could be eluted with the 21K Fib 2 fragment, in which three protein bands with Mr.of 30kDa, 60kDa, and >200kDa were detected by aotoradiography. Cross-linking of the 21K Fib 2 fragment and cell surface receptor with bireactive agent DSS also showed that the Fib 2 fragment binds to proteins with the same Mrs. These proteins would be expected to be receptors mediating the Fib 2 fragment effects. Less

我们已经证明FN的蛋白水解降解产生了完整FN中不存在的生物活性。我们发现21 K Fib 2片段在极低浓度下具有趋化活性，而完整FN几乎不显示该活性。完整的FN可抑制ST-13前脂肪细胞向脂肪细胞的分化，而FN氨基端纤维蛋白结合区的24 K片段可显著促进ST-13前脂肪细胞向脂肪细胞的分化。完整FN的蛋白水解降解模式可能是FN功能激活的重要决定因素。首先，我们研究了炎性蛋白酶（纤溶酶，中性粒细胞组织蛋白酶G和弹性蛋白酶）和基质金属蛋白酶（基质溶解素，基质溶解素，明胶酶A，和间质胶原酶）的活性FN片段的释放的影响。明胶酶A被发现在产生趋化性Fib 2片段中是最活跃的。中性粒细胞弹性蛋白酶可有效地释放分化刺激性Fib 1片段。除了这种 ...更多信息蛋白酶对趋化性迁移的间接作用，一些细胞内Ca^<2+>依赖的半胱氨酸蛋白酶被证明直接参与了FN对趋化性迁移的反应。第二，尝试用21 K Fib 2片段固定的凝胶通过亲和柱分离21 K Fib 2片段的细胞表面受体蛋白。用GRGDSP肽、BSA溶液洗涤柱，然后用Fib 2片段去除的FN片段混合物洗涤柱，在应用13 <125>I标记的NIH-L13细胞提取物后，能够专门去除非特异性结合亲和凝胶的蛋白质。结果表明，21 KFib-2片段可洗脱出一个放射性峰，放射自显影显示出Mr为30 kDa、60 kDa和> 200 kDa的三条蛋白带。用双反应剂DSS交联21 K Fib 2片段和细胞表面受体也表明Fib 2片段与具有相同Mrs的蛋白质结合。这些蛋白质预期是介导Fib 2片段效应的受体。少