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The molecular mechanisms underlying Golgi retention of glycosyltransferases

高尔基体保留糖基转移酶的分子机制

基本信息

批准号：
10680590
负责人：
NISHIHARA Shoko
金额：
$ 2.5万
依托单位：
Soka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680590/
关键词：
glycosyltransferase Golgi membrane fucosyltransferase ガラクトース転移酵素

项目摘要

Glycosyltransferases in the Golgi apparatus are type-II membrane proteins which consist of an N-terminal cytoplasmic region, a trans membrane region, a stem region and a c-terminal catalytic region facing to lumen of Golgi. In order to elucidate the molecular mechanisms underlying Golgi retention of glycosyl-transferases, we carried out the following two experiments.(1) Molecular behavior of mutant Lewis enzymes in vivo : The expression of type-1 Lewis antigens on erythrocytes and in digestive organs is determined by a Lewis type α(1, 3/1, 4)fucosyltransferase (Lewis enzyme) encoded by the FUT3 gene (Lewis gene) . We have classified the Lewis alleles in the Japanese population into four types, the wild-type allele (Le) and three mutated alleles, le1, le2, and le3. We carried out an extensive study on the biological properties of the three mutant Lewis enzymes using native tissues. The missense mutation, Leu20 to Arg, in the transmembrane domain reduces retention of the le3 enzyme in th … More e Golgi membrane resulting in an apparent reduction of enzyme activity as revealed by the lack of Lewis antigen synthesis. So the le3 enzyme can not synthesize Lewis-active glycolipids, which result in the Lewis antigen-negative phenotype of erythrocytes.(2) Cloning and identification of genes encoding proteins interacting with glycosyltransferases : The two-hybrid system is an in vivo yeast-based system that identifies interaction between two proteins by reconstituting active transcription factor dimmers. cDNAs have been prepared from human non-cancerous right hemi-colon tissues obtained as a surgical sample and a cDNA library constructed in the GAL4 activation domain vector pPC86. We have reported that Lewis type α1, 3/1, 4 fucosyltransferase (FUT3) , H enzyme, Se enzyme, α2,3sialyltransferases (ST3Gal I and ST3Gal IV) and β1,4galactosyl-transferase I(β1,4Galt I) are all expressed in the normal right hemi-colon. Their genes have been cloned in frame with the GAL4 sequence encoding the DNA-binding domain into pDBLeu as the "bait". We carried out screening and obtained clones as the candidate molecules interacting with glycosyltransferases. Less

高尔基体中的糖基转移酶是由N端细胞质区域、跨膜区域、茎区域和面向高尔基体腔的c末端催化区域组成的II型膜蛋白。为了阐明高尔基体保留糖基转移酶的分子机制，我们进行了以下两个实验：(1)突变的刘易斯酶在体内的分子行为：1型刘易斯抗原在红细胞和消化器官中的表达是由FUT3基因(刘易斯基因)编码的刘易斯类型α(1，3/1，4)岩藻糖基转移酶(刘易斯酶)决定的。我们将日本人群中的Lewis等位基因分为四种类型，野生型等位基因(Le)和三种突变等位基因LE1、Le2和LE3。我们利用天然组织对三种突变的Lewis酶的生物学特性进行了广泛的研究。跨膜区的错义突变，Leu20到Arg，减少了LE3酶在TH…中的保留较多的E高尔基膜导致酶活性明显降低，这表现为缺乏Lewis抗原的合成。因此，LE3酶不能合成Lewis活性糖脂，从而导致红细胞Lewis抗原阴性表型。(2)糖基转移酶相互作用蛋白编码基因的克隆和鉴定：双杂交系统是一个基于酵母的体内系统，通过重组活性转录因子二聚体来鉴定两种蛋白之间的相互作用。从手术标本和构建在GAL4激活域载体pPC86中的人非癌右半结肠组织中提取了cDNA。我们已经报道，在正常右半结肠中，刘易斯α1，3/1，4岩藻糖基转移酶、H酶、Se酶、α2，3唾液酸基转移酶(ST3GalI和ST3GalIV)和β1，4半乳糖基转移酶I(β1，4GalI)均有表达。它们的基因与编码DNA结合区的GAL4序列一起被克隆到pDBLeu中作为诱饵。我们进行了筛选，获得了与糖基转移酶相互作用的候选分子克隆。较少