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

HNK-1碳水化合物表位被单克隆抗体HNK-1识别，在从昆虫到哺乳动物的一系列细胞粘附分子和神经系统的一些糖脂质上具有特征性表达。HNK-1表位在神经系统发育过程中参与细胞-细胞和/或细胞-底物的相互作用和识别。该表位的结构特征为硫代糖醛基残基，其内部结构Gal β1-4 - GlcNAc普遍存在于各种糖蛋白和糖脂中，提示葡萄糖醛基转移酶和硫代糖醛基转移酶是其生物合成的关键酶。我们最近克隆了新的葡萄糖醛基转移酶（GlcAT-P和GlcAT-S）和硫代转移酶cdna，它们是HNK-1表位生物合成的关键酶。在本研究中，我们利用这些cdna获得了以下结果。1)原位杂交分析显示，GlcAT-P和GlcAT-S在各脑区分布不同，而硫转移酶则普遍表达。2)通过转染GlcAT-P cDNA的C6胶质瘤细胞的稳定转化，我们发现HNK-1表位不仅优先表达在NCAM和L1分子上，而且在L1上表达的HNK-1表位主要参与细胞的形态改变。3)为了研究HNK-1表位在体内的功能，我们培育了缺乏GlcAT-P的小鼠。我们用Southern blot分析证实了GlcAT-P缺失小鼠的同源重组，用Northern blot分析证实了GlcAT-P缺失小鼠的同源重组。用HNK-1抗体进行Western blot分析发现，GlcAT-P缺陷小鼠中几乎所有HNK-1碳水化合物表位都消失了。

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.