Department of Biochemistry, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima City, Fukushima 960-1295, Japan.
Department of Biochemistry, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima City, Fukushima 960-1295, Japan
Biochem J. 2018 May 9;475(9):1583-1595. doi: 10.1042/BCJ20170205.
Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr/Thr/Thr/Thr/Thr) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr/Thr or Thr/Thr/Thr had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation.
骨桥蛋白 (OPN) 是一种细胞外糖基化磷蛋白，通过与几种整合素受体相互作用促进细胞黏附。我们之前报道过，在富含苏氨酸/脯氨酸区域中缺乏五个 O-糖基化位点（Thr/Thr/Thr/Thr/Thr）的 OPN 突变体与野生型相比，细胞黏附活性和磷酸化增加。然而，O-糖基化在 OPN 细胞黏附活性和磷酸化中的作用仍需阐明。在这里，我们表明 OPN 富含苏氨酸/脯氨酸区域中的特异性 O-糖基化独立地和/或协同地影响其细胞黏附活性和磷酸化。使用定点突变，我们发现 OPN 突变体 Thr/Thr 或 Thr/Thr/Thr 的替换集分别降低和增加细胞黏附活性。相比之下，单个突变引入 O-糖基化位点对 OPN 细胞黏附活性没有影响。使用针对 αvβ3 和 β1 整合素的功能阻断抗体以及过表达 αvβ3 整合素的 A549 细胞进行的黏附测定表明，特异性 O-糖基化影响 OPN 与两种整合素的结合。使用 phos-tag 和 LC-MS/MS 进行的磷酸化分析表明，磷酸化水平和位点受 O-糖基化状态的影响，尽管 OPN 中的 O-糖基化位点数量与磷酸化水平无关。此外，对具有特异性 O-糖基化的 OPN 突变体中磷酸化水平与细胞黏附活性之间的相关性分析表明，它们并不总是相关的。这些结果提供了确凿的证据，证明了特异性 O-糖基化对 OPN 细胞黏附活性和磷酸化的新型调节机制。
