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

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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.