Christensen Brian, Nielsen Mette S, Haselmann Kim F, Petersen Torben E, Sørensen Esben S
Protein Chemistry Laboratory, Department of Molecular Biology, Science Park, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark.
Biochem J. 2005 Aug 15;390(Pt 1):285-92. doi: 10.1042/BJ20050341.
OPN (osteopontin) is an integrin-binding highly phosphorylated glycoprotein, recognized as a key molecule in a multitude of biological processes such as bone mineralization, cancer metastasis, cell-mediated immune response, inflammation and cell survival. A significant regulation of OPN function is mediated through PTM (post-translational modification). Using a combination of Edman degradation and MS analyses, we have characterized the complete phosphorylation and glycosylation pattern of native human OPN. A total of 36 phosphoresidues have been localized in the sequence of OPN. There are 29 phosphorylations (Ser8, Ser10, Ser11, Ser46, Ser47, Thr50, Ser60, Ser62, Ser65, Ser83, Ser86, Ser89, Ser92, Ser104, Ser110, Ser113, Thr169, Ser179, Ser208, Ser218, Ser238, Ser247, Ser254, Ser259, Ser264, Ser275, Ser287, Ser292 and Ser294) located in the target sequence of MGCK (mammary gland casein kinase) also known as the Golgi kinase (S/T-X-E/S(P)/D). Six phosphorylations (Ser101, Ser107, Ser175, Ser199, Ser212 and Ser251) are located in the target sequence of CKII (casein kinase II) [S-X-X-E/S(P)/D] and a single phosphorylation, Ser203, is not positioned in the motif of either MGCK or CKII. The 36 phosphoresidues represent the maximal degree of modification since variability at many sites was seen. Five threonine residues are O-glycosylated (Thr118, Thr122, Thr127, Thr131 and Thr136) and two potential sites for N-glycosylation (Asn63 and Asn90) are not occupied in human milk OPN. The phosphorylations are arranged in clusters of three to five phosphoresidues and the regions containing the glycosylations and the RGD (Arg-Gly-Asp) integrin-binding sequence are devoid of phosphorylations. Knowledge about the positions and nature of PTMs in OPN will allow a rational experimental design of functional studies aimed at understanding the structural and functional interdependences in diverse biological processes in which OPN is a key molecule.
骨桥蛋白（OPN）是一种整合素结合的高度磷酸化糖蛋白，被认为是多种生物过程中的关键分子，如骨矿化、癌症转移、细胞介导的免疫反应、炎症和细胞存活。OPN功能的显著调节是通过翻译后修饰（PTM）介导的。我们结合使用埃德曼降解法和质谱分析，对天然人OPN的完整磷酸化和糖基化模式进行了表征。共有36个磷酸化残基定位在OPN序列中。有29个磷酸化位点（Ser8、Ser10、Ser11、Ser46、Ser47、Thr50、Ser60、Ser62、Ser65、Ser83、Ser86、Ser89、Ser92、Ser104、Ser110、Ser113、Thr169、Ser179、Ser208、Ser218、Ser238、Ser247、Ser254、Ser259、Ser264、Ser275、Ser287、Ser292和Ser294）位于乳腺酪蛋白激酶（MGCK）（也称为高尔基体激酶）（S/T-X-E/S(P)/D）的靶序列中。六个磷酸化位点（Ser101、Ser107、Ser175、Ser199、Ser212和Ser251）位于酪蛋白激酶II（CKII）（S-X-X-E/S(P)/D）的靶序列中，单个磷酸化位点Ser203不在MGCK或CKII的基序中。由于在许多位点观察到变异性，这36个磷酸化残基代表了最大修饰程度。五个苏氨酸残基发生O-糖基化（Thr118、Thr122、Thr127、Thr131和Thr136），两个人乳OPN中的N-糖基化潜在位点（Asn63和Asn90）未被占据。磷酸化以三到五个磷酸化残基的簇形式排列，包含糖基化和RGD（精氨酸-甘氨酸-天冬氨酸）整合素结合序列的区域没有磷酸化。了解OPN中PTM的位置和性质将有助于合理设计功能研究的实验，旨在理解OPN作为关键分子参与的各种生物过程中的结构和功能相互依赖性。
