Christensen Brian, Kazanecki Christian C, Petersen Torben E, Rittling Susan R, Denhardt David T, Sørensen Esben S
Protein Chemistry Laboratory, Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark.
J Biol Chem. 2007 Jul 6;282(27):19463-72. doi: 10.1074/jbc.M703055200. Epub 2007 May 11.
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 murine ras-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 approximately 21 phosphate groups distributed over 27 sites. Five residues are O-glycosylated in both isoforms. These residues are fully modified in FbOPN, whereas one site 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.
骨桥蛋白（OPN）是一种在所有体液中都能找到的高度修饰的整合素结合蛋白。OPN的表达与许多不同人类癌症的不良预后密切相关，这表明该蛋白在肿瘤发生和转移中发挥着重要但尚未完全理解的作用。该蛋白存在多种不同的异构体，其翻译后修饰程度不同，可能表现出不同的功能特性。本研究首次检测了来自转化细胞的OPN的翻译后修饰及其对细胞生物学的影响。我们通过质谱分析和埃德曼降解相结合的方法，对小鼠ras转化的成纤维细胞（FbOPN）和分化中的成骨细胞（ObOPN）表达的OPN的完整磷酸化和糖基化模式进行了表征。质谱分析显示，FbOPN和ObOPN的质量分别为34.9 kDa和35.9 kDa。酶促去磷酸化、序列和质量分析表明，FbOPN含有大约4个磷酸基团，分布在16个潜在的磷酸化位点上，而ObOPN含有大约21个磷酸基团，分布在27个位点上。两种异构体中均有5个残基发生O-糖基化。这些残基在FbOPN中被完全修饰，而在ObOPN中一个位点部分糖基化。尽管两种形式的OPN都介导了小鼠ras转化的成纤维细胞强大的整合素介导的黏附，但磷酸化程度较低的FbOPN介导MDA-MD-435人肿瘤细胞的结合能力几乎是磷酸化程度高的ObOPN的6倍。这些结果有力地支持了以下假设：不同细胞类型产生的OPN的磷酸化程度可以调节其功能。
