The short amino acid sequence Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhesive function.
The short amino acid sequence Pro-His-Ser-Arg-Asn in human fibronectin enhances cell-adhesive function.
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发表时间：
1994-10
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通讯作者：
S. Aota;M. Nomizu;K. Yamada
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作者：
S. Aota;M. Nomizu;K. Yamada
纤连蛋白（FN）的中央细胞粘附结构域中的协同位点显著增强由纤连蛋白的α 5 β 1整联蛋白受体介导的细胞粘附。我们的特点是一个关键的最小序列需要使用定点诱变和同源性扫描使用分子内嵌合体的协同活性。在大肠杆菌表达系统中表达由第9和第10个III型FN重复序列组成的FN的最小细胞结合结构域。当使用FN介导的粘附幼仓鼠肾或HT-1080细胞的竞争性抑制测定法测定时，该蛋白质保留高生物活性。相反，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重复片段替换第8重复的各个部分，我们将协同区域定位到第9重复的中心。当第9个重复序列中的一个很短的肽序列Pro-His-Ser-Arg-Asn（PHSRN）取代第8个重复序列中的同源五肽位点时，重组蛋白的活性显著增强。进一步的诱变分析表明，该五肽序列的精氨酸残基对于功能是重要的。我们还确定了一个较弱的相邻协同区域以外的PHSRN区域。抑制FN介导的粘附的抗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不表现出竞争性抑制活性，也不与含有可溶性RGD的肽协同作用。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重复的相应位点时，它介导了粘附活性的增强。这些结果鉴定了协同增强FN RGD序列的细胞粘附活性的五肽位点。
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.