POSTTRANSLATIONAL MODIFICATIONS OF BOVINE OSTEOPONTIN - IDENTIFICATION OF 28 PHOSPHORYLATION AND 3 O-GLYCOSYLATION SITES
POSTTRANSLATIONAL MODIFICATIONS OF BOVINE OSTEOPONTIN - IDENTIFICATION OF 28 PHOSPHORYLATION AND 3 O-GLYCOSYLATION SITES
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DOI：
10.1002/pro.5560041009
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发表时间：
1995-10-01
期刊：
影响因子：
8
通讯作者：
PETERSEN, TE
中科院分区：
文献类型：
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作者：
SORENSEN, ES;HOJRUP, P;PETERSEN, TE
骨桥蛋白 (OPN) 是一种多磷酸化糖蛋白，存在于骨骼和其他正常和恶性组织以及生理液尿液和牛奶中。本研究表明，牛乳骨桥蛋白的 27 个丝氨酸残基和 1 个苏氨酸残基被磷酸化。通过结合氨基酸分析、S-乙基半胱氨酸衍生的磷酸肽的序列分析和质谱分析来鉴定磷酸氨基酸。 25 个磷酸丝氨酸和 1 个磷酸苏氨酸位于 Ser/Thr-X-Glu/Ser(P)/Asp 基序中，2 个磷酸丝氨酸位于 Ser-X-X-Glu/Ser(P) 序列中。这些序列基序分别与乳腺酪蛋白激酶和酪蛋白激酶II的识别序列相同。对磷酸化模式的检查表明，磷酸化聚集成大约三个一组，跨越 11-32 个氨基酸的非磷酸化区域。这种模式可能在 OPN 涉及与 Ca2+ 和无机钙盐相互作用的多种功能中很重要。此外，在蛋白质富含苏氨酸和脯氨酸的区域中还鉴定出了三个 O-糖基化苏氨酸（Thr 115、Thr 124 和 Thr 129）。牛骨桥蛋白中存在三个假定的 N-糖基化位点（Asn 63、Asn 85 和 Asn 193），但序列和质谱分析表明，这些天冬酰胺在牛乳腺骨桥蛋白中均未糖基化。比对分析表明，牛骨桥蛋白中的大部分磷酸化位点以及所有三个 O-糖基化位点在其他哺乳动物序列中都是保守的。这种丝氨酸的保守性，即使是在蛋白质的保守性较差的区域中，也表明骨桥蛋白在特定位点的磷酸化对于蛋白质的功能至关重要。
Osteopontin (OPN) is a multiphosphorylated glycoprotein found in bone and other normal and malignant tissues, as well as in the physiological fluids urine and milk. The present study demonstrates that bovine milk osteopontin is phosphorylated at 27 serine residues and 1 threonine residue. Phosphoamino acids were identified by a combination of amino acid analysis, sequence analysis of S-ethylcysteine-derivatized phosphopeptides, and mass spectrometric analysis. Twenty-five phosphoserines and one phosphothreonine were located in Ser/Thr-X-Glu/Ser(P)/Asp motifs, and two phosphoserines were found in the sequence Ser-X-X-Glu/Ser(P). These sequence motifs are identical with the recognition sequences of mammary gland casein kinase and casein kinase II, respectively. Examination of the phosphorylation pattern revealed that the phosphorylations were clustered in groups of approximately three spanned by unphosphorylated regions of 11-32 amino acids. This pattern is probably of importance in the multiple functions of OPN involving interaction with Ca2+ and inorganic calcium salts. Furthermore, three O-glycosylated threonines (Thr 115, Thr 124, and Thr 129) have been identified in a threonine- and proline-rich region of the protein. Three putative N-glycosylation sites (Asn 63, Asn 85, and Asn 193) are present in bovine osteopontin, but sequence and mass spectrometric analysis showed that none of these asparagines were glycosylated in bovine mammary gland osteopontin. Alignment analysis showed that the majority of the phosphorylation sites in bovine osteopontin as well as all three O-glycosylation sites were conserved in other mammalian sequences. This conservation of serines, even in otherwise less well-conserved regions of the protein, indicates that the phosphorylation of osteopontin at specific sites is essential for the function of the protein.