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Expression of catabolic pathway of asparagine-linked oligosaccharides on plasma membrane and apoptosis

天冬酰胺连接寡糖分解代谢途径在质膜上的表达与细胞凋亡

基本信息

批准号：
14580628
负责人：
ITO Kazuo
金额：
$ 2.24万
依托单位：
Osaka City University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

1.Verification and specification of target glycoproteins deglycosylated by Endo HSTarget glycoprotein(TGP) deglycosylated by Endo HS was detected from attached epithelial cell in the human oral cavity more than detached cell, but not in leukocyte. The protein of molecular weight(MW) of 55K from the attached epithelial cell was specified as TGP55 by in-gel deglycosylation method. The proteins of MW of 20K, 50K and 105〜110K were also specified as TGP20, TGP50 and TGP105〜110.2.Identification of TGP55 and TGP50TGP55 and TGP50 were purified. The N-terminal amino acid was thought to be acetylated. The N-terminal amino acid sequence of deacetylated TGP55 showed high homology with the inner amino acid sequence of human cytokeratin 13, but no proteins having the same N-terminal amino acid sequence was not observed by homology search. The N-terminal amino acid of TGP50 was also blocked. The N-terminal and inner amino acid sequence of TGP50 showed high homology with those of human type I cytokerain 13, indicating that TGP50 is type I cytokeratin 13 or a similar protein to it. Human type I cytokeratin 13 has an asparagine of potential glycosylation site. Endo HS was thought to removing the asparagine-linked oligosaccharide at the site and taking part in the differentiation of the epithelial cell.3.Immunochemical properties and cellular localization of TGP55 analyzed using monoclonal antibodies against TGP55Three monoclonal antibodies against TGP55 were prepared. Each antibody reacted with TGP55 and other proteins of attached and detached epithelial cell, the kinds of proteins were different between attached and detached epithelial cell. The outskirts part of the epithelial cell was stained by the antibodies, indicating that TGP55 localizes around the outskirt part of the epithelial cell and the localization changes after peeling of the epithelial cell.

1.Endo HSTGET糖蛋白(TGP)脱糖作用的验证和鉴定经Endo HS脱糖后的靶糖蛋白(TGP)在口腔附着的上皮细胞中比在分离的细胞中检测到更多，但在白细胞中检测不到。凝胶内脱糖法测得贴壁上皮细胞产生的相对分子质量为55K的蛋白质为TGP55。TGP20、TGP50和TGP105~110.2蛋白的相对分子质量分别为20K、50K和105~110K。对TGP55和TGP50TGP55和TGP50进行了鉴定。N末端的氨基酸被认为是乙酰化的。脱乙酰化的TGP55的N端氨基酸序列与人细胞角蛋白13的内部氨基酸序列具有很高的同源性，但通过同源性搜索没有发现具有相同N端氨基酸序列的蛋白质。TGP50的N端氨基酸也被阻断。TGP50的N末端和内部氨基酸序列与人I型细胞角蛋白13有很高的同源性，表明TGP50是I型细胞角蛋白13或与之类似的蛋白质。人I型细胞角蛋白13含有一个潜在糖基化位点的天冬酰胺。Endo HS被认为能清除天冬酰胺连接的低聚糖，并参与上皮细胞的分化。3.用抗TGP55的单抗分析TGP55的免疫化学性质和细胞定位。每种抗体均可与附着和脱落上皮细胞的TGP55及其他蛋白质发生反应，附着和脱落的上皮细胞表达的蛋白质种类不同。免疫组织化学染色显示，TGP55定位于上皮细胞的外围，剥离后定位发生改变。