Cell type-specific post-translational modifications of mouse osteopontin are associated with different adhesive properties
Cell type-specific post-translational modifications of mouse osteopontin are associated with different adhesive properties
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DOI：
10.1074/jbc.m703055200
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发表时间：
2007-07-06
影响因子：
4.8
通讯作者：
Sorensen, Esben S.
中科院分区：
文献类型：
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作者：
Christensen, Brian;Kazanecki, Christian C.;Sorensen, Esben S.
骨桥蛋白 (OPN) 是一种高度修饰的整合素结合蛋白，存在于所有体液中。 OPN 的表达与许多不同人类癌症的不良预后密切相关，表明该蛋白在肿瘤发生和转移中具有重要但知之甚少的作用。该蛋白质存在多种不同的亚型，其翻译后修饰程度不同，可能表现出不同的功能特性。这项研究首次研究了转化细胞中 OPN 的翻译后修饰以及这些修饰对细胞生物学的影响。我们通过质谱分析和 Edman 降解相结合，表征了鼠转化成纤维细胞 (FbOPN) 和分化成骨细胞 (ObOPN) 表达的 OPN 的完整磷酸化和糖基化模式。质谱分析显示 FbOPN 和 ObOPN 的质量分别为 34.9 和 35.9 kDa。酶促去磷酸化、序列和质量分析表明，FbOPN 包含大约 4 个磷酸基团，分布在 16 个潜在磷酸化位点上，而 ObOPN 包含类似的 21 个磷酸基团，分布在 27 个位点上。两种异构体中有五个残基被 O-糖基化。这些残基在 FbOPN 中被完全修饰，而在 ObOPN 中单位点被部分糖基化。虽然两种形式的 OPN 都能介导强整合素介导的小鼠 ras 转化成纤维细胞的粘附，但磷酸化程度较低的 FbOPN 介导的 MDA-MD-435 人类肿瘤细胞的结合几乎是重磷酸化 ObOPN 的 6 倍。这些结果有力地支持了这样的假设：不同细胞类型产生的 OPN 磷酸化程度可以调节其功能。
Osteopontin (OPN) is a highly modified integrin-binding protein found in all body fluids. Expression of OPN is strongly correlated with poor prognosis in many different human cancers, suggesting an important but poorly understood role for this protein in tumorigenesis and metastasis. The protein exists in a number of different isoforms differing in the degree of post-translational modifications that are likely to exhibit different functional properties. This study examines for the first time the post-translational modifications of OPN from transformed cells and the effects of these modifications on cell biology. We have characterized the complete phosphorylation and glycosylation patterns of OPN expressed by murineras-transformed fibroblasts(FbOPN) and differentiating osteoblasts (ObOPN) by a combination of mass spectrometric analyses and Edman degradation. Mass spectrometric analysis showed masses of 34.9 and 35.9 kDa for FbOPN and ObOPN, respectively. Enzymatic dephosphorylation, sequence, and mass analyses demonstrated that FbOPN contains approximately four phosphate groups distributed over 16 potential phosphorylation sites, whereas ObOPN contains similar to 21 phosphate groups distributed over 27 sites. Five residues are O-glycosylated in both isoforms. These residues are fully modified in FbOPN, whereas onesite is partially glycosylated in ObOPN. Although both forms of OPN mediated robust integrin-mediated adhesion of mouse ras-transformed fibroblasts, the less phosphorylated FbOPN mediated binding of MDA-MD-435 human tumor cells almost 6-fold more than the heavy phosphorylated ObOPN. These results strongly support the hypothesis that the degree of phosphorylation of OPN produced by different cell types can regulate its function.