人胃上皮细胞中幽门螺杆菌CagA相关未知磷酸化蛋白和磷酸化位点分析

Helicobacter pylori CagA-related Unknown Phosphorylated Proteins and Phosphorylation Sites in Human Gastric Epithelial Cells

背景：细胞毒素相关基因A蛋白（CagA）是Ⅰ型幽门螺杆菌（H.pylori）的主要毒力因子。H.pylori胃癌、胃炎相关株和CagA缺失（△CagA）株的蛋白质组学研究尚未发现CagA相关生物标记蛋白。目的：分析H.pyloriCagA阳性株和△CagA株作用后人胃上皮细胞中差异表达的未知磷酸化蛋白和磷酸化位点,为研究CagA的致病机制提供线索。方法：以金属离子亲和吸附富集技术富集经H.pylori标准株和△CagA株作用4h的人胃腺癌细胞株AGS的磷酸化蛋白,双向电泳（2-DE）分离蛋白,飞行时间质谱（TOF-MS）技术鉴定差异蛋白点。结果：H.pylori标准株作用后,AGS细胞FAM50A蛋白276位丝氨酸发生磷酸化,PQBP1蛋白227～251序列中有磷酸化位点。与H.pylori标准株相比,△CagA株作用于AGS细胞可引起至少13种未知磷酸化蛋白的变化,其质谱图中均出现中性丢失峰,其中2种表达量增加,4种表达量降低,6种消失,1种新发生磷酸化。结论：CagA阳性H.pylori感染可致AGS细胞FAM50A和PQBP1蛋白发生磷酸化。所发现的13种未知磷酸化蛋白为揭示CagA的致病机制提供了线索。

Background： Cytotoxin-associated gene A protein （CagA） is the main virulence factor of type Ⅰ Helicobacter pylori （H. pylori）. Proteomics studies of gastric cancer- and gastritis-related H. pylori strains and CagA deleted （△CagA） strains have not yet found any biomarker protein of these CagA-related diseases. Aims： To investigate the differentially expressed unknown phosphorylated proteins and phosphorylation sites in human gastric epithelial cell infected by CagA-positive or △CagA H. priori strains, so as to explore the pathogenic mechanism of CagA. Methods： The phosphorylated proteins of human gastric adenocarcinoma cell line AGS infected with H. pylori standard strain and △CagA strain for 4 hours were extracted and enriched by metal ion affinity adsorption enrichment technique. The proteins in sample were separated by 2- dimensional electrophoresis （2-DE）, and the significantly differentially expressed protein spots were identified by time-offlight mass spectrometry （TOF-MS）. Results： When infected with H. pylori standard strain, two phosphorylated proteins were detected in AGS cells, the 276 site serine of FAMSOA was phosphorylated, and the 227-251 sequence of PQBP1 had phosphorylation site. Compared with H. pylori standard strain, at least 13 unknown differentially expressed phosphorylated proteins with neutral-loss peptides were found in △CagA strain infected AGS cells, among them, 2 were up-regulated, 4 down-regulated, 6 absent and 1 newly phosphorylated. Conclusions： FAM50A and PQBP1 proteins are phosphorylated in AGS cells infected with CagA-positive H. pylori strain. The thirteen unknown phosphorylated proteins might provide clues for clarifying the pathogenic mechanism of CagA.