Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly.
Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly.
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DOI：
10.1083/jcb.114.6.1295
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发表时间：
1991-09
影响因子：
7.8
通讯作者：
Yamada, K M
中科院分区：
文献类型：
--
作者：
Nagai, T;Yamakawa, N;Aota, S;Yamada, S S;Akiyama, S K;Olden, K;Yamada, K M
定点诱变研究表明，除了最小的Arg-Gly-Asp（RGD）序列外，纤连蛋白中央细胞结合结构域中的其他肽信息是其完全粘附活性所必需的。通过蛋白质化学和免疫学方法，使用粘附、迁移和基质组装的生物测定，进一步分析了这第二个协同位点的性质。将来自细胞结合结构域的片段共价偶联到平板上，并将其在介导BHK细胞铺展中的比摩尔活性与完整的纤连蛋白进行比较。从人血浆纤连蛋白的胰凝乳蛋白酶消化物中纯化的37-kD片段与完整的纤连蛋白具有基本相同的比摩尔活性。相反，其他片段如缺少37-kD片段的NH 2-末端序列的11.5-kD片段仅具有基于摩尔的差的铺展活性。此外，在纤连蛋白介导的细胞扩散的竞争性抑制试验中，37 kD片段的活性比GRGDS合成肽高约325倍（摩尔）。使用37-kD蛋白作为免疫原产生mAb，并表征其表位。两个单独的单克隆抗体，一个结合接近的RGD位点和其他网站约15 kD远离RGD位点，单独抑制BHK细胞的纤连蛋白上的扩展超过90%。相反，在这两个位点之间结合的抗体具有最小的抑制活性。在BHK细胞的细胞扩散试验中发现具有抑制作用的抗体还抑制纤连蛋白介导的细胞扩散和人HT-1080细胞的迁移，这些功能也依赖于α 5 β 1整联蛋白（纤连蛋白受体）的功能。组装成细胞外基质的内源性合成的纤连蛋白并没有显着抑制大多数的抗37-kD单克隆抗体，但强烈抑制抗体结合接近的RGD位点或推定的协同作用位点。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依赖于α 5 β 1纤连蛋白受体的多种功能的完全生物活性是至关重要的。该位点由比先前预期更靠近RGD位点的mAb定位。
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.