Phosphorylation site analysis of Semliki forest virus nonstructural protein 3.
Phosphorylation site analysis of Semliki forest virus nonstructural protein 3.
复制标题
Semliki森林病毒非结构蛋白3的磷酸化位点分析。
DOI：
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发表时间：
2000
影响因子：
4.8
通讯作者：
J. Saarinen
中科院分区：
文献类型：
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作者：
H. Vihinen;J. Saarinen
非结构蛋白3(NSP3)是甲型病毒核糖核酸复制复合体的一个重要亚基，但其特定功能(S)尚未明确。以前，已经证明Semliki森林病毒NSP3(482个氨基酸)是一种磷酸蛋白，在本研究中，我们已经定位了它的主要磷酸化位点。使用的质谱学方法包括前驱体离子扫描、基质辅助激光解吸/电离质谱法与靶上碱性磷酸酶消化结合使用，以及串联质谱法。应用二维肽图谱分离NSP3的胰酶(32)-P标记的磷酸肽。然后对放射性标记的多肽进行Edman测序和磷酸氨基酸分析。此外，放射性标记的NSP3先后被溴化氰和胰酶切割，并用微型铁络合亲和层析富集磷酸多肽。通过结合这些方法，我们发现NSP3在丝氨酸残基320、327、332、335、356、359、362和367上被磷酸化，而在多肽Gly(338)-Lys(415)上被高度磷酸化，后者携带7-12个磷酸盐分布在其13个潜在的磷酸化位点上。通过构建无磷酸化的NSP3衍生物证实了这些分析结果。这是首次确定甲型病毒非结构蛋白的磷酸化位点，但该方法可用于一般的磷蛋白分析。
Nonstructural protein 3 (Nsp3) is an essential subunit of the alphavirus RNA replication complex, although its specific function(s) has yet to be well defined. Previously, it has been shown that Semliki Forest virus Nsp3 (482 amino acids) is a phosphoprotein, and, in the present study, we have mapped its major phosphorylation sites. Mass spectrometric methods utilized included precursor ion scanning, matrix-assisted laser desorption/ionization mass spectrometry used in conjunction with on-target alkaline phosphatase digestions, and tandem mass spectrometry. Two-dimensional peptide mapping was applied to separate tryptic (32)P-labeled phosphopeptides of Nsp3. Radiolabeled peptides were then subjected to Edman sequencing, and phosphoamino acid analysis. In addition, radiolabeled Nsp3 was cleaved successively with cyanogen bromide and trypsin, and microscale iron-chelate affinity chromatography was used to enrich phosphopeptides. By combining these methods, we showed that Nsp3 is phosphorylated on serine residues 320, 327, 332, 335, 356, 359, 362, and 367, and is heavily phosphorylated on peptide Gly(338)-Lys(415), which carries 7-12 phosphates distributed over its 13 potential phosphorylation sites. These analytical findings were corroborated by constructing a Nsp3 derivative devoid of phosphorylation. The results represent the first determination of phosphorylation sites of an alphavirus nonstructural protein, but the approach can be utilized in phosphoprotein analysis in general.