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碳水化合物表位在一系列细胞粘附分子上，以及在神经系统中的某些糖脂上的特征表达，从昆虫到哺乳动物的广泛物种。 HNK-1表位参与神经系统发展过程中的细胞细胞和/或细胞 - 基底相互作用和识别。该表位的特征结构特征是磺核居住地，因为内部结构GALβ1-4GlcNAC通常在各种糖蛋白和糖醇中发现，这表明糖苷隆转移酶（S）和磺胺转移酶（S）和磺胺转移酶（S）是键合酶（S）。我们最近克隆了新颖的糖苷隆转移酶（GLCAT-P和GLCAT-S）和硫代转移酶CDNA，它们是HNK-1发作的生物合成的关键酶。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，与硫代转移酶无处不在的表达相反，在每个大脑区域中观察到Glcat-P和Glcat-S的不同分布。 2）使用用Glcat-P cDNA翻译的C6神经胶质瘤细胞的稳定转化剂，我们不仅证明了HNK-1表位优选在NCAM和L1分子上表达，而且还表明在L1上表达的HNK-1表位主要与细胞的形态变化有关。 3）为了研究体内HNK-1表位的功能，我们已经产生了缺乏Glcat-P的小鼠。我们通过Southern blot分析和缺少GLCAT-P表达通过Northern Blot分析证实了Glcat-P缺乏小鼠的同源重组。 HNK-1抗体的蛋白质印迹分析表明，GLCAT-P缺乏小鼠中几乎所有HNK-1 Carobhydrate表位消失了。