Hashino K, Uemori Y, Kimizuka F, Kato I, Titani K
Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Shiga.
J Biochem. 1996 Apr;119(4):604-9. doi: 10.1093/oxfordjournals.jbchem.a021285.
The binding of fibronectin to fibronectin receptor was studied using a recombinant 31-kDa cell-binding domain fragment of fibronectin (C279), which consisted of three type III repeats (III8-III9-III10). Fibronectin receptor in several cell lysates was bound to a column of C279-immobilized Sepharose HP and obtained in a highly purified form by elution with a synthetic peptide, GRGDSP. alpha 5 beta 1-Integrin was detected in the GRGDSP-eluted fraction by immunoblotting. The cell-adhesive activity of C279 was inhibited by GRGDSP peptide, an anti-integrin a5 subunit antibody, and an anti-integrin beta 1 subunit antibody. The cell adhesion of fusion proteins of the 31-kDa fragment with biologically interesting polypeptides (heparin-binding domain of fibronectin, and basic fibroblast growth factor) was also studied. In the presence of an anti-integrin a5 subunit antibody, human fibrosarcoma HT-1080 cells attached to the fusion protein containing fibroblast growth factor, giving rise to changes the morphology of the attached cells. The cell adhesion of C279 was inhibited by GRGDSP peptide but that of the fusion protein with the heparin-binding domain of fibronectin was not completely inhibited by the peptide. These results suggest that these biologically interesting polypeptides contribute to the cell adhesion of the fusion proteins.
使用纤连蛋白的重组31 kDa细胞结合域片段（C279）研究了纤连蛋白与纤连蛋白受体的结合，该片段由三个III型重复序列（III8 - III9 - III10）组成。几种细胞裂解物中的纤连蛋白受体与固定有C279的琼脂糖凝胶HP柱结合，并通过用合成肽GRGDSP洗脱以高度纯化的形式获得。通过免疫印迹在GRGDSP洗脱的级分中检测到α5β1整合素。C279的细胞粘附活性受到GRGDSP肽、抗整合素α5亚基抗体和抗整合素β1亚基抗体的抑制。还研究了31 kDa片段与具有生物学意义的多肽（纤连蛋白的肝素结合域和碱性成纤维细胞生长因子）的融合蛋白的细胞粘附。在存在抗整合素α5亚基抗体的情况下，人纤维肉瘤HT - 1080细胞附着于含有成纤维细胞生长因子的融合蛋白，导致附着细胞的形态发生变化。C279的细胞粘附受到GRGDSP肽的抑制，但含有纤连蛋白肝素结合域的融合蛋白的细胞粘附并未被该肽完全抑制。这些结果表明，这些具有生物学意义的多肽有助于融合蛋白的细胞粘附。
