Nagai T, Yamakawa N, Aota S, Yamada S S, Akiyama S K, Olden K, Yamada K M
Howard University Cancer Center, Washington, DC 20060.
J Cell Biol. 1991 Sep;114(6):1295-305. doi: 10.1083/jcb.114.6.1295.
Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.
定点诱变研究表明，纤连蛋白中央细胞结合结构域中除了最小的精氨酸 - 甘氨酸 - 天冬氨酸（RGD）序列外，还需要其他肽段信息才能实现其完全的黏附活性。利用黏附、迁移和基质组装的生物学检测方法，通过蛋白质化学和免疫学方法进一步分析了这个第二个协同位点的性质。将源自细胞结合结构域的片段共价偶联到平板上，并将它们介导BHK细胞铺展的比活性与完整纤连蛋白的比活性进行比较。从人血浆纤连蛋白的胰凝乳蛋白酶消化物中纯化得到的一个37-kD片段，其比活性与完整纤连蛋白基本相同。相比之下，其他片段，如一个缺少37-kD片段NH2末端序列的11.5-kD片段，在摩尔基础上的铺展活性很差。此外，在纤连蛋白介导的细胞铺展的竞争性抑制试验中，37-kD片段在摩尔基础上比GRGDS合成肽的活性高约325倍。以37-kD蛋白作为免疫原制备单克隆抗体（mAb）并对其表位进行了鉴定。两种不同的单克隆抗体，一种结合在靠近RGD位点处，另一种结合在距RGD位点约15 kD的位点处，它们分别抑制BHK细胞在纤连蛋白上的铺展超过90%。相比之下，结合在这两个位点之间的抗体具有最小的抑制活性。在BHK细胞的细胞铺展试验中发现具有抑制作用的抗体，也抑制了纤连蛋白介导的人HT-1080细胞的铺展和迁移，这些功能也依赖于α5β1整合素（纤连蛋白受体）的功能。内源性合成的纤连蛋白组装成细胞外基质，大多数抗37-kD单克隆抗体对此没有明显抑制作用，但只有结合在靠近RGD位点或假定协同位点的抗体才会强烈抑制。这些结果表明，纤连蛋白上远离RGD位点的第二个位点对于其在依赖α5β1纤连蛋白受体的多种功能中的完全生物学活性至关重要。该位点通过单克隆抗体定位在比先前预期更靠近RGD位点的位置。
