Molecular biological studies of HNK-1 carbohydrate epitope

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。HNK-1抗原表位由硫酸化的三糖基组成，硫酸化的三糖基是糖脂和糖蛋白共有的，硫酸化的三糖基是3GlcAβ1-3Galβ1-4GlcNAc。我们从大鼠脑中分离到一种新的葡萄糖醛酸转移酶（GlcAT-P），它是HNK-1抗原表位合成的关键酶。从cDNA序列推断的一级结构预测的II型跨膜蛋白与347个氨基酸，并没有检测到的相似性与任何其他蛋白质的已知功能，包括葡萄糖醛酸转移酶的肝脏和嗅觉上皮。在COS-1细胞中表达该酶的可溶性重组形式产生活性葡糖醛酸基转移酶。将GlcAT-PcDNA转染COS-1细胞，不仅诱导细胞表面HNK-1表位的表达，而且细胞形态发生明显变化，提示HNK-1表位与细胞-基质相互作用有关。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细胞表现出明显的细胞聚集率降低。我们还提供了NCAM-NCAM嗜同性结合受HNK-1碳水化合物表位的存在负调控的证据。这些证据表明，HNK-1表位表达的细胞粘附分子，如NCAM和L1负调控他们的嗜同性相互作用，并导致减少细胞-细胞粘附。

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.