Molecular biological studies of HNK-1 carbohydrate epitope

HNK-1碳水化合物表位具有特征性地表达在一系列细胞黏附分子上，也表达在从昆虫到哺乳动物的多种物种的神经系统中的一些糖脂上。HNK-1表位参与神经系统发育过程中细胞-细胞和/或细胞-底物的相互作用和识别。HNK1表位由硫酸盐三糖、硫酸盐-3GlcAβ1-3Galβ1-4GlcNAc组成，与糖脂和糖蛋白共有。根据该序列推导出的一级结构预测了一个由347个氨基酸组成的II型跨膜蛋白，与任何其他已知功能的蛋白没有发现相似之处，包括肝脏和嗅觉上皮的葡萄糖醛酸基转移酶。该酶的可溶性重组形式在COS-1细胞中的表达产生了活性的葡萄糖醛酸基转移酶。将GlcAT-P基因导入COS-1细胞，不仅诱导了细胞表面HNK-1表位的表达，而且细胞形态发生了明显的变化，提示HNK-1表位与细胞-底物相互作用有关。此外，细胞聚集分析显示，亲代C6细胞表现出强烈的时间依赖性聚集倾向，而C6/GlcAT-P细胞的细胞聚集率明显降低。我们还提供了NCAM-NCAM亲和性结合受HNK-1碳水化合物表位存在的负调控的证据。这些证据表明，表达在NCAM和L1等细胞黏附分子上的HNK-1表位负性调节它们的亲和性相互作用，导致细胞与细胞之间的黏附减少。

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.