van Dijk S, D'Errico J A, Somerman M J, Farach-Carson M C, Butler W T
Department of Basic Sciences, University of Texas Houston Health Science Center.
J Bone Miner Res. 1993 Dec;8(12):1499-506. doi: 10.1002/jbmr.5650081213.
The bone sialoprotein osteopontin (OPN) promotes cell attachment and spreading through its RGD (Arg-Gly-Asp) sequence. To study additional regions of OPN involved in cell attachment, peptides of rat OPN were evaluated for their capacity to mediate cell binding to wells in vitro. Human gingival fibroblasts were incubated on microtiter plates coated with either OPN or OPN peptides. A peptide of M(r) 28 kD, obtained after digestion with endoproteinase Arg-C and isolated by reversed-phase HPLC, enhanced cell attachment to a similar degree as OPN. Sequence analysis showed that the amino terminus of the 28 kD peptide starts at Ser142 and therefore does not contain the RGD cell attachment sequence (residues 128-130). Cell attachment mediated through both OPN and the 28 kD peptide was blocked by the addition of GRGDSPA peptides or LM-609, a monoclonal antibody to the integrin alpha V beta 3, a receptor for vitronectin. A variant peptide, GRG-ESPA, did not alter cell attachment. Based on these observations, we conclude that (1) binding of OPN and the 28 kD peptide to fibroblasts involves binding to alpha V beta 3, (2) a site other than the RGD sequence on OPN is also involved in binding to integrins, and (3) the binding of this second site to alpha V beta 3 is inhibited by RGD-containing peptides.
骨唾液蛋白骨桥蛋白（OPN）通过其RGD（精氨酸-甘氨酸-天冬氨酸）序列促进细胞黏附和铺展。为了研究OPN中参与细胞黏附的其他区域，对大鼠OPN的肽段进行了体外介导细胞与孔结合能力的评估。将人牙龈成纤维细胞接种在包被有OPN或OPN肽段的微量滴定板上。用内肽酶Arg-C消化后经反相高效液相色谱分离得到的一种分子量为28 kD的肽段，其增强细胞黏附的程度与OPN相似。序列分析表明，28 kD肽段的氨基末端始于Ser142，因此不包含RGD细胞黏附序列（第128 - 130位氨基酸残基）。通过添加GRGDSPA肽段或LM - 609（一种针对玻连蛋白受体整合素αVβ3的单克隆抗体）可阻断由OPN和28 kD肽段介导的细胞黏附。变异肽段GRG - ESPA不会改变细胞黏附。基于这些观察结果，我们得出以下结论：（1）OPN和28 kD肽段与成纤维细胞的结合涉及与αVβ3的结合；（2）OPN上除RGD序列之外的一个位点也参与与整合素的结合；（3）这个第二位点与αVβ3的结合受到含RGD肽段的抑制。
