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Study on structures and functions of CMP-sialic acid modifying enzymes

CMP-唾液酸修饰酶的结构与功能研究

基本信息

批准号：
02680147
负责人：
TAI Tadashi
金额：
$ 1.28万
依托单位：
Tokyo Metropolitan Institute of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02680147/
关键词：
Sialic acid Glycolipid Glycoprotein Transferase Ganglioside ガングリオニド

项目摘要

The aim of this research was to isolate cDNAs encoding mammalian O-Acetyltransferases involved in the synthsis of glycolipids. There are two methods for the isolation of the cDNAs ; (i) an approach that based on sequence information derived from the purified protein or antibodies to the enzymes ; (ii) an approach that does not require sequence of the peptides. In the first year, we checked which procedure is better for the isolation of the cDNAs. We found that (i) the transferase was enriched in the membrane fraction, (ii) several human melanoma cell lines empressed a large amount of O-Ac-GD3 with a specific mouse monoclonal antibody (MAb), which was established by immunizing mice with the purified ganglioside. Based on these results, we have decided to adapt an mammalian cDNA expression cloning scheme. As a next step, we determined the structure of glycolipids and the activities of the glycosyltransferase from a number of host cells. Two cell lines (COS-1 and COP5) were candidates for the host cells. None of these cells expressed O-Ac-GD3. w Moreover, COS-1 cells expressed a number of gangliosides, including GM3 and GD3. Thus, COS-1 cells were selected as a host cells. A CDNA library was prepared from human tumor cells that express a large amount of relevant gangliosides on cell surfaces, using the procedure of B. Seed and the mammalian expression vector pCDM8. Cells were transfered with the plasmid library. The DEAE-dextran method of transfection was used. The transfected cells were added to dishes containing absorbed antibody following the panning technique. We have been trying to isolate positive-cells with the MAb.

本研究的目的是分离编码参与糖脂合成的哺乳动物o -乙酰转移酶的cdna。有两种分离cdna的方法；(i)基于纯化蛋白或酶抗体的序列信息的方法；（ii）不需要肽序列的方法。在第一年，我们检查了哪种方法更适合分离dna。我们发现(i)转移酶在膜部分富集，（ii）几种人类黑色素瘤细胞系用一种特异性的小鼠单克隆抗体（MAb）表达了大量的O-Ac-GD3，该抗体是用纯化的神经节苷脂免疫小鼠建立的。基于这些结果，我们决定采用哺乳动物cDNA表达克隆方案。下一步，我们从许多宿主细胞中测定了糖脂的结构和糖基转移酶的活性。两个细胞系（COS-1和COP5）是宿主细胞的候选细胞系。这些细胞均不表达O-Ac-GD3。w此外，COS-1细胞表达多种神经节苷脂，包括GM3和GD3。因此，选择COS-1细胞作为宿主细胞。利用B. Seed程序和哺乳动物表达载体pCDM8，从细胞表面大量表达相关神经节苷元的人肿瘤细胞中制备CDNA文库。用质粒文库转移细胞。采用deae -葡聚糖法转染。将转染后的细胞加入到含有吸收抗体的培养皿中。我们一直在尝试用单克隆抗体分离阳性细胞。