Nagata T, Todescan R, Goldberg H A, Zhang Q, Sodek J
Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Ontario, Canada.
Biochem Biophys Res Commun. 1989 Nov 30;165(1):234-40. doi: 10.1016/0006-291x(89)91059-0.
Secreted phosphoprotein I (SPPI) is a prominent structural protein in mineralized connective tissues. Rat bone cells in culture produce several forms of SPPI that differ in post-translational modifications such as phosphorylation and sulphation. To determine the significance of protein sulphation in bone formation, the synthesis of SPPI was studied in vitro using rat bone marrow cells (RBMC) which form bone-like tissue when grown in the presence of dexamethasone (Dex) and beta-glycerophosphate (beta-GP). In the presence of 10(-7) M Dex SPPI expression was stimulated 4-5-fold. Radiolabelling multilayered RBMCs for 48 h with [35S]-methionine, Na2[35SO4], or Na3[32PO4] revealed that two major phosphorylated forms of SPPI were secreted into the culture medium: a highly phosphorylated form migrating at 44 kDa on 15% SDS-PAGE and a less phosphorylated 55 kDa form. In the mineralized tissue formed in the presence of Dex and beta-GP, both forms of SPPI, in addition to proteoglycans and a 67 kDa protein, incorporated significant amounts of [35SO4]. Sulphation of SPPI was not observed in the absence of mineral formation, indicating that the sulphation of SPPI is closely associated with mineralization and that it can be used as a sensitive and specific marker for the osteoblastic phenotype.
分泌型磷蛋白I（SPPI）是矿化结缔组织中的一种重要结构蛋白。培养的大鼠骨细胞产生几种不同形式的SPPI，它们在翻译后修饰如磷酸化和硫酸化方面存在差异。为了确定蛋白质硫酸化在骨形成中的意义，利用大鼠骨髓细胞（RBMC）在体外研究了SPPI的合成，当RBMC在地塞米松（Dex）和β-甘油磷酸酯（β-GP）存在下生长时会形成类骨组织。在10^(-7) M Dex存在的情况下，SPPI表达被刺激了4 - 5倍。用[35S]-甲硫氨酸、Na2[35SO4]或Na3[32PO4]对多层RBMC进行48小时的放射性标记显示，两种主要的磷酸化形式的SPPI被分泌到培养基中：一种在15% SDS-PAGE上迁移率为44 kDa的高度磷酸化形式和一种磷酸化程度较低的55 kDa形式。在Dex和β-GP存在下形成的矿化组织中，除了蛋白聚糖和一种67 kDa的蛋白质外，两种形式的SPPI都掺入了大量的[35SO4]。在没有矿化形成的情况下未观察到SPPI的硫酸化，这表明SPPI的硫酸化与矿化密切相关，并且它可以用作成骨细胞表型的敏感和特异性标志物。
