Molecular biological studies of HNK-1 carbohydrate epitope

HNK-1碳水化合物表位的分子生物学研究

基本信息

批准号：
09307053
负责人：
KAWASAKI Toshisuke
金额：
$ 25.47万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09307053/
关键词：
HNK-1 epitope 3-sulfoglucuronic acid glucuronyltransferase cell adhesion molecule COS-1 cells sulfotransferase Extra Cellular Matrix C6 glioma 基質特異性スフィンゴミエリンホスファチジルイノシトール N-グリコシド型糖鎖 cDNAクローニング II型膜貫通タンパク質ラット脳 NCAM 細胞間相互作

项目摘要

The HNK-1 carbohydrate epitope 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 HNK-1 epitope has been demonstrated to be composed of the sulfated trisaccharides, sulfate-3GlcAβ1-3Galβ1-4GlcNAc, which is shared with glycolipids and glycoproteins We isolated a novel glucuronyltransferase (GlcAT-P) from rat brain which is a key enzyme of the biosynthesis of the HNK-1 epitope on glycoproteins. The primary structure deduced from the cDNA sequence predicted a type II transmembrane protein with 347 amino acids and had no detectable similarity with any other proteins of known functions, including glucuronyltransferases of the liver and olfactory epithelium. Expression of a soluble recombinant form of the enzyme in COS-1 cells produced an active glucuronyltransferase. Transfection of GlcAT-P cDNA into COS-1 cells induced not only expression of the HNK-1 epitope on the cell surface but also marked morphological changes of the cells, suggesting that the HNK-1 epitope associates with the cell-substratum interaction. In addition, cell aggregation analysis revealed that the parental C6 cells showed a strong tendenecy to aggregation in time-dependent manner,whereas C6/GlcAT-P cells showed a markedly reduced rate of cell aggregation. We also provided the evidence that NCAM-NCAM homophilic binding is negatively regulated by the presence of HNK-1 carbohydrate epitope. These lines of evidence suggested that the HNK-1 epitope expressed on cell adhesion molecules such as NCAM and L1 negatively regulated their homophilic interactions and resulted in reducing the cell-cell adhesion.

HNK-1表位的特征是在一系列细胞粘附分子以及神经系统中的某些糖脂上表达，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。 HNK-1表位已证明是由硫酸盐-3GLCAβ1-3GALβ1-4GLCNAC组成的，它与糖脂蛋白和糖蛋白共享，我们分离了一种新型的糖cocoyltrantransferase（Glcat-P），这是一个eye Zyme，这是一个新型的glcococonlantransferlase（Glcat-P）在糖蛋白上。从cDNA序列推导的主要结构预测了具有347氨基酸的II型跨膜蛋白，并且与已知功能的任何其他蛋白质（包括肝脏和嗅觉上皮的糖coconlanslansferase）没有可检测到的相似性。 COS-1细胞中酶的固体重组形式的表达产生了活性糖隆转移酶。将Glcat-P cDNA转染到CoS-1细胞中不仅诱导了HNK-1表位在细胞表面上的表达，而且还标志着细胞的形态变化，这表明HNK-1表位与细胞 - 肌间相互作用相关。此外，细胞聚集分析表明，亲本的C6细胞以时间依赖性方式表现出强烈的聚集倾向，而C6/Glcat-P细胞显示出细胞聚集的速率明显降低。我们还提供了一个证据，表明NCAM-NCAM均电结合受HNK-1碳水化合物表位的负面调节。这些证据线表明，在NCAM和L1等细胞粘附分子上表达的HNK-1表位对其均电相互作用负调节，并导致细胞细胞粘附。