权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Molecular biological analysis of the carbohydrate specifically expressed in the nervous system

神经系统中特异性表达的碳水化合物的分子生物学分析

基本信息

批准号：
11680604
负责人：
OKA Shogo
金额：
$ 2.24万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

The HNK-1 carbohydrate epitope, which is recognized by monoclonal antibody HNK-1, is characteristically expressed on a series of cell adhesion molecules and also on some glycolipids in the nervous system over a wide range of species from insect to mammal. The HNK-1 epitope is involved in cell-cell and/or cell-substrate interaction and recognition during the development of the nervous system. The characteristic structural feature of this epitope is the sulfoglucuronyl residue, because the inner structure, Gal β1-4 GlcNAc, is found commonly in various glycoproteins and glycolipids, suggesting that glucuronyltransferase (s) and sulfotransferase (s) are key enzymes in the biosynthesis. We have recently cloned novel glucuronyltransferases (GlcAT-P and GlcAT-S) and a sulfotransferase cDNAs, which are key enzymes of the biosynthesis of the HNK-1 epitope. In the present study, we obtained following results using these cDNAs. 1) In situ hybridization analysis revealed that the different distributions of GlcAT-P and GlcAT-S were observed in each brain region in contrast to the ubiquitous expression of sulfotransferase. 2) Using the stable transformant of C6 glioma cells transfected with GlcAT-P cDNA, we demonstrated not only that the HNK-1 epitope was preferentially expressed on the NCAM and L1 molecules but also that the HNK-1 epitope expressed on L1 was mainly involved in the morphological changes of the cells. 3) To investigate the function of the HNK-1 epitope in vivo, we have generated the mice lacking GlcAT-P.We confirmed homologous recombination in GlcAT-P deficient mice by Southern blot analysis and absence of GlcAT-P expression by Northern blot analysis. Western blot analysis with HNK-1 antibody revealed that almost all of the HNK-1 carobhydrate epitope disappeared in the GlcAT-P deficient mice.

由单克隆抗体HNK-1识别的HNK-1碳水化合物表位在从昆虫到哺乳动物的广泛物种的神经系统中的一系列细胞粘附分子上以及一些糖脂上特征性地表达。HNK-1表位参与神经系统发育过程中的细胞-细胞和/或细胞-底物相互作用和识别。该表位的特征性结构特征是磺基葡萄糖醛酸残基，因为内部结构Gal β1-4 GlcNAc常见于各种糖蛋白和糖脂中，表明葡萄糖醛酸转移酶和磺基转移酶是生物合成中的关键酶。我们最近克隆了新的葡萄糖醛酸转移酶（GlcAT-P和GlcAT-S）和磺基转移酶cDNA，它们是HNK-1表位生物合成的关键酶。在本研究中，我们使用这些cDNA获得了以下结果。1)原位杂交分析表明，不同的分布，GlcAT-P和GlcAT-S观察到在不同的脑区域的磺基转移酶的普遍表达。2)利用稳定转染GlcAT-P cDNA的C6胶质瘤细胞，我们证明HNK-1表位不仅优先表达在NCAM和L1分子上，而且表达在L1上的HNK-1表位主要参与细胞的形态学变化。3)为了研究HNK-1表位在体内的功能，我们已经产生了缺乏GlcAT-P的小鼠。我们通过Southern印迹分析和北方印迹分析证实了GlcAT-P缺陷小鼠中的同源重组和GlcAT-P表达的缺失。用HNK-1抗体进行的Western印迹分析显示，几乎所有的HNK-1碳水化合物表位在GlcAT-P缺陷小鼠中消失。