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The molecular system of Golgi retentior of glycosyltransferases

糖基转移酶高尔基体保留分子系统

基本信息

批准号：
12680620
负责人：
NISHIHARA Shoko
金额：
$ 2.56万
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：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680620/
关键词：
glycosyltransferase focosyltransferase Golgi apparatus H enzyme FUT1 Se enzyme FUT2

项目摘要

Glycosylation of proteins and lipids is performed in the Golgi apparatus by glycosyl-transferases. They 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. The population of resident glycosyltransferases and sorting components must be retained in the Golgi apparatus in spite of the large flow of proteins and lipids through to other organelles in the cell. The aim of this project is to elucidate the molecular mechanisms underlying Golgi retention of 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 were 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 H enzyme (FUT1), Se enzyme (FUT2), Lewis type α1,3/1,4 fucosyltransferase (FUT3), α2,3 sialyl-transferases (ST3GaII and ST3GaIIV) and β1,4gaqlactosyltransferase 1 (β1,4GaIT1) express in the right hemicolon. Each gene was cloned in-frame with the GAL4 sequence encoding the DNA binding domain into pDBLeu as the bait. We carried out screening and obtained ε-COP protein, gp25L2 protein and β-tubulin as the candidate molecules interacting with FUT1 and FUT2.

蛋白质和脂类的糖基化是在高尔基体中通过糖基转移酶完成的。它们都是II型膜蛋白，由N-末端细胞质区域、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。尽管蛋白质和脂类大量流向细胞中的其他细胞器，但驻留糖基转移酶和分选组分的群体必须保留在高尔基体中。本项目的目的是阐明高尔基体保留糖基转移的分子机制。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识别两种蛋白质之间的相互作用。从手术标本和构建在GAL4激活结构域载体pPC86中的cDNA文库中提取人非癌右半结肠组织的cDNAs。我们已报道H酶(FUT1)、Se酶(FUT2)、刘易斯α1，3/1，4岩藻糖基转移酶(FUT3)、α2，3唾液酸基转移酶(ST3GaII和ST3GaIIV)和β1，4半乳糖基转移酶1(β1，4GaIT1)在右半结肠表达。以编码DNA结合区的GAL4序列为诱饵，将每个基因克隆到pDBLeu中。我们进行了筛选，获得了ε-COP蛋白、gp25L2蛋白和β-微管蛋白作为与FUT1和FUT2相互作用的候选分子。