Liao Y F, Wieder K G, Classen J M, Van De Water L
Center for Engineering in Medicine and Surgical Services, Massachusetts General Hospital and Harvard Medical School, the Shriners Burns Hospital, Boston, Massachusetts 02114, USA.
J Biol Chem. 1999 Jun 18;274(25):17876-84. doi: 10.1074/jbc.274.25.17876.
Alternative splicing of the fibronectin gene transcript gives rise to a group of adhesive glycoproteins showing restricted spatial and temporal expression during embryonic development, tumor growth, and tissue repair. Alternative splicing occurs in three segments termed EIIIB, EIIIA, and V. The EIIIA (or ED-A) segment of fibronectin is expressed prominently but transiently in healing wounds coincident with fibroblast expression of an activation marker, smooth muscle cell alpha-actin. A monoclonal antibody (IST-9) to the EIIIA segment blocks transforming growth factor-beta-mediated smooth muscle cell alpha-actin expression by fibroblasts in culture. A second monoclonal antibody (DH1) blocks chondrocyte condensation in chicken embryos. We find that IST-9 and DH1 react with human, rat, and chicken but not with mouse or frog EIIIA, suggesting that His44 may be important for antibody binding. A series of deletion mutants of rat EIIIA, constructed as glutathione S-transferase fusion proteins, do not react with either IST-9, DH1, or a third monoclonal antibody (3E2). Mutations of pairs of amino acids to alanine have little effect, except for either (Val34Thr35) or (Tyr36Ser37), which are located in a beta strand upstream from His44. For these double mutants, the binding to all three monoclonal antibodies is markedly reduced. By contrast, single mutants at Thr35, Tyr36, or Ser37 retain full activity, suggesting that the epitope for these antibodies is determined in part by conformation. Alanine-scanning mutagenesis of rat EIIIA demonstrates the importance of Ile43 and His44 for binding. Mutation of frog EIIIA (normally Val43Lys44) to rat (Ile43His44) is sufficient to restore fully IST-9 binding and much of the activity of DH1 and 3E2. Our findings demonstrate that the function-blocking antibodies, IST-9 and DH1, bind to the Ile43 and His44 residues in a conformationally dependent fashion, implicating the loop region encompassing both residues as critical for mediating EIIIA function.
纤连蛋白基因转录本的可变剪接产生了一组粘附糖蛋白，它们在胚胎发育、肿瘤生长和组织修复过程中表现出有限的时空表达。可变剪接发生在三个片段中，称为EIIIB、EIIIA和V。纤连蛋白的EIIIA（或ED-A）片段在愈合伤口中显著但短暂地表达，这与成纤维细胞表达激活标志物平滑肌细胞α-肌动蛋白同时发生。一种针对EIIIA片段的单克隆抗体（IST-9）可阻断培养的成纤维细胞中转化生长因子-β介导的平滑肌细胞α-肌动蛋白表达。第二种单克隆抗体（DH1）可阻断鸡胚中的软骨细胞凝聚。我们发现IST-9和DH1与人、大鼠和鸡的EIIIA反应，但不与小鼠或青蛙的EIIIA反应，这表明His44可能对抗体结合很重要。一系列构建为谷胱甘肽S-转移酶融合蛋白的大鼠EIIIA缺失突变体，不与IST-9、DH1或第三种单克隆抗体（3E2）反应。将氨基酸对突变为丙氨酸影响不大，除了位于His44上游β链中的（Val34Thr35）或（Tyr36Ser37）。对于这些双突变体，与所有三种单克隆抗体的结合明显减少。相比之下，Thr35、Tyr36或Ser37处的单突变体保留了全部活性，这表明这些抗体的表位部分由构象决定。大鼠EIIIA的丙氨酸扫描诱变证明了Ile43和His44对结合的重要性。将青蛙EIIIA（通常为Val43Lys44）突变为大鼠（Ile43His44）足以完全恢复IST-9结合以及DH1和3E2的大部分活性。我们的研究结果表明，功能阻断抗体IST-9和DH1以构象依赖性方式结合到Ile43和His44残基上，这意味着包含这两个残基的环区域对于介导EIIIA功能至关重要。
