The Tyrosine-phosphorylation Mechanism of CagA Protein Secreted from Helicobacter Pylori and The Physiological Significance

幽门螺杆菌分泌的CagA蛋白酪氨酸磷酸化机制及其生理意义

基本信息

批准号：
13670541
负责人：
ASAHI Momoyo
金额：
$ 2.62万
依托单位：
Fukui Prefectural University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Helicobacter pylori CagA is injected into the host cell and tyrosine-phosphorylated. We examined tyrosine-phosphorylation sites of CagA, as well as the function of CagA proteins in vivo and in vitro. After proteolytic digestion of CagA with lysyl endopeptidase, CagA tyrosine-phosphorylation sites were determined using quadropolar time-of flight mass spectrometry analysis. Specific anti-pY CagA polyclonal and anti-CagA monoclonal antibodies were used to examine gastric mucosal biopsy specimens from H. pylori infected patients. Mass spectrometry identified five crucial tyrosine-phosphorylation sites of CagA at Tyr893. Tyr912, Tyr965, Tyr999, and Tyr1033 within the five repeated EPIYA sequences of H.pylori (NCTC11637)-infected AGS cells. CagA protein also had an immunoreceptor tyrosine-based activation motif (ITAM)-like amino acid sequences in the 3' region of the cagA, EPIYATIx_<27>EIYATI, which closely resembled the ITAM. CagA proteins, (i) were localized to the 1% TritonX-100 resistant membrane fraction (lipid rafts), (ii) formed a cluster of phosphorylated CagA protein complexes, (iii) associated with tyrosine-phosphorrylated GIT1/Cat1 (G protein-coupled receptor kinase-interactor 1/ Cool-associated, tyrosine-phosphorylated 1), substrate molecules of receptor type protein-tyrosine phosphatase, (RPTPζ/β), which is the receptor of VacA and (iv) were involved in a delay and negative regulation of VacA-induced signal. Immunohistochemical staining of gastric mucosal biopsy specimens provided strong evidence that tyrosine-phosphorylated CagA is found together with CagA at the luminal surface of gastric foveola in vivo. These findings suggest an important role for CagA containing ITAM-like sequences in the pathogenesis of H.pylori-related disease.

将幽门螺杆菌 CagA 注射到宿主细胞中并进行酪氨酸磷酸化，我们检查了 CagA 的酪氨酸磷酸化位点，以及 CagA 蛋白在体内和体外的功能。使用四极飞行时间质谱分析确定位点。使用 CagA 多克隆和抗 CagA 单克隆抗体检查幽门螺杆菌感染患者的胃粘膜活检标本，并通过质谱法在 5 个重复 EPIYA 中确定了 CagA 的 Tyr893、Tyr965、Tyr999 和 Tyr1033 的五个关键酪氨酸磷酸化位点。幽门螺杆菌 (NCTC11637) 感染的 AGS 序列CagA蛋白在cagA的3'区域也具有基于免疫受体酪氨酸的激活基序(ITAM)样氨基酸序列，EPIYATIx_ 27 EIYATI，其与CagA蛋白非常相似，(i)是定位的。 1% TritonX-100 抗性膜组分（脂筏），(ii) 形成磷酸化 CagA 蛋白复合物簇， (iii) 与酪氨酸磷酸化 GIT1/Cat1（G 蛋白偶联受体激酶相互作用物 1/Cool 相关，酪氨酸磷酸化 1）相关，受体型蛋白酪氨酸磷酸酶的底物分子 (RPTP z/β)，其为VacA 受体和 (iv) 参与 VacA 诱导的胃免疫组织化学染色信号的延迟和负调节。粘膜活检标本提供了强有力的证据，证明酪氨酸磷酸化的 CagA 与体内胃小凹管腔表面的 CagA 一起存在。这些发现表明，含有 ITAM 样序列的 CagA 在幽门螺杆菌相关疾病的发病机制中发挥着重要作用。