Maeda T, Hashino K, Oyama R, Titani K, Sekiguchi K
Institute for Comprehensive Medical Science, Fujita Health University School of Medicine, Aichi.
J Biochem. 1991 Sep;110(3):381-7. doi: 10.1093/oxfordjournals.jbchem.a123590.
An artificial cell adhesive protein could be engineered by grafting the RGDS tetrapeptide, the core sequence of the major cell adhesive site of fibronectin, to a truncated form of Staphylococcal protein A (tSPA) via cassette mutagenesis of the tSPA expression vector pRIT2T [T. Maeda et al. (1989) J. Biol. Chem. 264, 15165-15168]. We synthesized a panel of tSPA derivatives grafted with various RGDS-containing oligopeptides to address the problem of how the cell adhesive activity of the resulting tSPA derivatives was affected by the length and amino acid sequence of the grafted oligopeptides and by the sites on tSPA where the extra oligopeptides were inserted. The results showed that (i) the amino acid residues flanking the RGDS core sequence played a key role in modulating the cell adhesive activity of the grafted RGDS signal; (ii) at least two sites on tSPA, each corresponding to on e of the two HindIII sites of pRIT2T, were competent in sustaining the cell adhesive activity of the grafted signal; and (iii) the divalent tSPA containing the RGDS signal at both sites was more active than monovalent derivatives containing only one signal at either site. These results provide a strategic basis for engineering of artificial cell adhesive proteins by grafting the RGDS signal.
通过对葡萄球菌蛋白A（tSPA）表达载体pRIT2T进行盒式诱变[前田哲等（1989年）《生物化学杂志》264卷，15165 - 15168页]，将纤连蛋白主要细胞粘附位点的核心序列RGDS四肽嫁接到截短形式的葡萄球菌蛋白A上，可构建一种人工细胞粘附蛋白。我们合成了一组嫁接着各种含RGDS寡肽的tSPA衍生物，以解决所产生的tSPA衍生物的细胞粘附活性如何受嫁接寡肽的长度和氨基酸序列以及tSPA上插入额外寡肽的位点影响的问题。结果表明：（i）RGDS核心序列两侧的氨基酸残基在调节嫁接的RGDS信号的细胞粘附活性中起关键作用；（ii）tSPA上至少有两个位点，每个位点对应于pRIT2T的两个HindIII位点之一，能够维持嫁接信号的细胞粘附活性；（iii）在两个位点都含有RGDS信号的二价tSPA比在任何一个位点仅含有一个信号的单价衍生物更具活性。这些结果为通过嫁接RGDS信号构建人工细胞粘附蛋白提供了策略依据。
