Akiyama S K, Aota S, Yamada K M
Laboratory of Developmental Biology, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892, USA.
Cell Adhes Commun. 1995 Feb;3(1):13-25. doi: 10.3109/15419069509081275.
Previous studies have reached conflicting conclusions about the minimal size and sequences of the fibronectin cell-adhesive domain necessary for retention of high cell adhesive activity. We have expressed a recombinant 20 kDa cell-binding fragment of human fibronectin consisting of the ninth and tenth type III modules, which includes the Arg-Gly-Asp (RGD) cell recognition site and a second cell adhesive domain that acts synergistically with the RGD site. This polypeptide retained a similar activity as a larger 110 kDa fibronectin fragment when used in soluble form in inhibition assays, but it displayed low cell adhesive activity if assayed after direct adsorption to a plastic substrate. However, adhesive function was restored if the fragment was bound to a non-inhibitory anti-fibronectin antibody pre-adsorbed to the plastic substrate. The antibody-bound fragment also promoted cell migration. Both cell spreading and migration were specifically mediated by the alpha 5 beta 1 integrin. Affinity columns containing immobilized 20 kDa cell-binding fragment effectively bound alpha 5-, alpha 3-, and alpha v-containing fibronectin-binding integrins. In contrast, an immobilized 11.5 kDa fragment that contained the RGD sequence but lacked the synergistic sequence was bound only poorly by alpha 5-containing fibronectin receptor integrins, even though the alpha 3- and alpha v-containing integrins bound readily. Our results indicate that the manner in which adhesion proteins are presented to cells is important and that most cell adhesive activity is retained in a minimal 20 kDa segment of fibronectin.
先前的研究对于纤连蛋白细胞黏附结构域保持高细胞黏附活性所需的最小尺寸和序列得出了相互矛盾的结论。我们表达了一种重组的20 kDa人纤连蛋白细胞结合片段，其由第九和第十个III型模块组成，其中包括精氨酸-甘氨酸-天冬氨酸（RGD）细胞识别位点以及一个与RGD位点协同作用的第二细胞黏附结构域。当以可溶性形式用于抑制试验时，该多肽保留了与更大的110 kDa纤连蛋白片段相似的活性，但如果在直接吸附到塑料底物后进行检测，它表现出低细胞黏附活性。然而，如果该片段与预先吸附到塑料底物上的非抑制性抗纤连蛋白抗体结合，则黏附功能得以恢复。抗体结合的片段也促进细胞迁移。细胞铺展和迁移均由α5β1整合素特异性介导。含有固定化20 kDa细胞结合片段的亲和柱有效地结合了含α5、α3和αv的纤连蛋白结合整合素。相比之下，一个固定化的11.5 kDa片段，其包含RGD序列但缺乏协同序列，仅被含α5的纤连蛋白受体整合素微弱结合，尽管含α3和αv的整合素能轻易结合。我们的结果表明，黏附蛋白呈现给细胞的方式很重要，并且大多数细胞黏附活性保留在纤连蛋白最小的20 kDa片段中。
