Murphy-Ullrich J E, Lightner V A, Aukhil I, Yan Y Z, Erickson H P, Höök M
Department of Biochemistry, University of Alabama, Birmingham 35294.
J Cell Biol. 1991 Nov;115(4):1127-36. doi: 10.1083/jcb.115.4.1127.
Tenascin, together with thrombospondin and SPARC, form a family of matrix proteins that, when added to bovine aortic endothelial cells, caused a dose-dependent reduction in the number of focal adhesion-positive cells to approximately 50% of albumin-treated controls. For tenascin, a maximum response was obtained with 20-60 micrograms/ml of protein. The reduction in focal adhesions in tenascin-treated spread cells was observed 10 min after addition of the adhesion modulator, reached the maximum by 45 min, and persisted for at least 4 h in the continued presence of tenascin. This effect was fully reversible, was independent of de novo protein synthesis, and was neutralized by a polyclonal antibody to tenascin. Monoclonal antibodies to specific domains of tenascin (mAbs 81C6 and 127) were used to localize the active site to the alternatively spliced segment of tenascin. Furthermore, a recombinant protein corresponding to the alternatively spliced segment (fibronectin type III domains 6-12) was expressed in Escherichia coli and was active in causing loss of focal adhesions, whereas a recombinant form of a domain (domain 3) containing the RGD sequence had no activity. Chondroitin-6-sulfate effectively neutralized tenascin activity, whereas dermatan sulfate and chondroitin-4-sulfate were less active and heparan sulfate and heparin were essentially inactive. Studies suggest that galactosaminoglycans neutralize tenascin activity through interactions with cell surface molecules. Overall, our results demonstrate that tenascin, thrombospondin, and SPARC, acting as soluble ligands, are able to provoke the loss of focal adhesions in well-spread endothelial cells.
腱生蛋白与血小板反应蛋白和富含半胱氨酸的酸性分泌蛋白形成了一个基质蛋白家族。当将它们添加到牛主动脉内皮细胞中时，会导致粘着斑阳性细胞数量呈剂量依赖性减少，降至白蛋白处理对照组的约50%。对于腱生蛋白，20 - 60微克/毫升的蛋白可获得最大反应。在添加粘着调节剂10分钟后，观察到腱生蛋白处理的铺展细胞中粘着斑减少，45分钟时达到最大值，并在腱生蛋白持续存在的情况下至少持续4小时。这种效应是完全可逆的，与从头合成蛋白无关，并且被腱生蛋白的多克隆抗体中和。针对腱生蛋白特定结构域的单克隆抗体（单克隆抗体81C6和127）用于将活性位点定位到腱生蛋白的可变剪接片段。此外，与可变剪接片段相对应的重组蛋白（纤连蛋白III型结构域6 - 12）在大肠杆菌中表达，并在导致粘着斑丧失方面具有活性，而含有RGD序列的结构域（结构域3）的重组形式则无活性。硫酸软骨素-6有效地中和了腱生蛋白的活性，而硫酸皮肤素和硫酸软骨素-4的活性较低，硫酸乙酰肝素和肝素基本无活性。研究表明，氨基聚糖通过与细胞表面分子相互作用来中和腱生蛋白的活性。总体而言，我们的结果表明，腱生蛋白、血小板反应蛋白和富含半胱氨酸的酸性分泌蛋白作为可溶性配体，能够引发铺展良好的内皮细胞中粘着斑的丧失。
