Aota S, Nomizu M, Yamada K M
Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1994 Oct 7;269(40):24756-61.
Synergistic sites in the central cell-adhesive domain of fibronectin (FN) substantially enhance cell adhesion mediated by the alpha 5 beta 1 integrin receptor for fibronectin. We characterized a critical minimal sequence needed for synergistic activity using site-directed mutagenesis and homology scanning using intramolecular chimeras. The minimal cell-binding domain of FN consisting of the 9th and 10th type III FN repeat was expressed in an Escherichia coli expression system. This protein retained high biological activity when assayed using a competitive inhibition assay for FN-mediated adhesion of baby hamster kidney or HT-1080 cells. In contrast, a construct consisting of the 8th and 10th repeat displayed very low biological activity. By replacing various portions of the 8th repeat with homologous 9th repeat segments, we mapped the synergistic region to the center of the 9th repeat. When a very short peptide sequence, Pro-His-Ser-Arg-Asn (PHSRN), from the 9th repeat was substituted for the homologous pentapeptide site in the 8th repeat sequence, the recombinant protein showed markedly enhanced activity. Further mutagenesis analysis suggested that the arginine residue of this pentapeptide sequence is important for function. We also identified a weaker adjacent synergy region other than the PHSRN region. Epitope mapping of an anti-FN monoclonal antibody that inhibits FN-mediated adhesion identified the same critical regions. A synthetic peptide containing the PHSRN sequence showed neither competitive inhibitory activity in solution nor synergy with a soluble RGD-containing peptide. However, when the same synthetic peptide was positioned via a covalent bond at the corresponding site of the normally inactive 8th repeat, it mediated an enhancement of adhesive activity. These results identify a pentapeptide site that synergistically enhances the cell-adhesive activity of the FN RGD sequence.
纤连蛋白（FN）中央细胞黏附结构域中的协同位点可显著增强由纤连蛋白的α5β1整合素受体介导的细胞黏附。我们使用定点诱变和分子内嵌合体的同源性扫描来表征协同活性所需的关键最小序列。由第9和第10个III型纤连蛋白重复序列组成的FN最小细胞结合结构域在大肠杆菌表达系统中表达。当使用竞争性抑制试验检测FN介导的幼仓鼠肾细胞或HT - 1080细胞黏附时，该蛋白保留了高生物活性。相比之下，由第8和第10个重复序列组成的构建体显示出非常低的生物活性。通过用同源的第9个重复序列片段替换第8个重复序列的各个部分，我们将协同区域定位到第9个重复序列的中心。当来自第9个重复序列的非常短的肽序列Pro - His - Ser - Arg - Asn（PHSRN）替代第8个重复序列中的同源五肽位点时，重组蛋白显示出明显增强的活性。进一步的诱变分析表明，该五肽序列的精氨酸残基对功能很重要。我们还确定了除PHSRN区域之外的一个较弱的相邻协同区域。抑制FN介导黏附的抗FN单克隆抗体的表位作图确定了相同的关键区域。含有PHSRN序列的合成肽在溶液中既没有竞争性抑制活性，也不与含可溶性RGD的肽协同。然而，当相同的合成肽通过共价键定位在通常无活性的第8个重复序列的相应位点时，它介导了黏附活性的增强。这些结果确定了一个五肽位点，该位点协同增强了FN RGD序列的细胞黏附活性。
