Abstract
The glycosylation of glycoproteins and glycolipids is important for central nervous system development and function. Although the roles of several carbohydrate epitopes in the central nervous system, including polysialic acid, the human natural killer-1 (HNK-1) carbohydrate, alpha2,3-sialic acid, and oligomannosides, have been investigated, those of the glycan backbone structures, such as Galbeta1-4GlcNAc and Galbeta1-3GlcNAc, are not fully examined. Here we report the generation of mice deficient in beta4-galactosyltransferase-II (beta4GalT-II). This galactosyltransferase transfers Gal from UDP-Gal to a nonreducing terminal GlcNAc to synthesize the Gal beta1-4GlcNAc structure, and it is strongly expressed in the central nervous system. In behavioral tests, the beta4GalT-II(-/-) mice showed normal spontaneous activity in a novel environment, but impaired spatial learning/memory and motor coordination/learning. Immunohistochemistry showed that the amount of HNK-1 carbohydrate was markedly decreased in the brain of beta4GalT-II(-/-) mice, whereas the expression of polysialic acid was not affected. Furthermore, mice deficient in glucuronyltransferase (GlcAT-P), which is responsible for the biosynthesis of the HNK-1 carbohydrate, also showed impaired spatial learning/memory as described in our previous report, although their motor coordination/learning was normal as shown in this study. Histological examination showed abnormal alignment and reduced number of Purkinje cells in the cerebellum of beta4GalT-II(-/-) mice. These results suggest that the Galbeta1-4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by beta4GalT-II and that the glycans synthesized by beta4GalT-II have essential roles in higher brain functions, including some that are HNK-1-dependent and some that are not.
Highlights
Port, and clearance from circulation, and cell-surface glycans participate in cell-cell and cell-extracellular matrix interactions
The amount of human natural killer-1 (HNK-1) carbohydrate was markedly decreased in the 4GalT-IIϪ/Ϫ brain, but polysialic acid (PSA) expression was not affected. These results suggest that the Gal1– 4GlcNAc structure in the HNK-1 carbohydrate is mainly synthesized by 4GalT-II and that glycans synthesized by 4GalT-II have essential roles in higher brain functions, including ones that are HNK-1 carbohydrate-dependent and ones that are independent of HNK-1
The amount of HNK-1 carbohydrate was markedly decreased in the cerebral cortex and hippocampus, moderately decreased in the cerebellar lobules, and unchanged in the cerebellar nuclei of the 4GalT-IIϪ/Ϫ mice compared with their 4GalT-IIϩ/ϩ littermates
Summary
Generation of 4GalT-IIϪ/Ϫ Mice—The strategy for the 4GalT-II gene targeting was to replace exons 1 and 2 and part of exon 3 with the neomycin resistance gene [20]. The number of Purkinje cells was significantly reduced in the 4th and 5th cerebellar lobules of the 4GalT-IIϪ/Ϫ mice compared with 4GalT-IIϩ/Ϫ mice (Fig. 3D, lower). In the 11-week-old 4GalT-IIϪ/Ϫ mouse brain (Fig. 4B), the HNK-1 reactivity was barely detectable only in the DG of the hippocampus, layers III–IV of the cerebral cortex, and the cerebellar lobule. In the cerebellar nuclei of the 4GalT-IIϪ/Ϫ mice, no reduction of the HNK-1 carbohydrate was apparent, in contrast to the other brain regions To assess their spontaneous activity in a novel environment, the mice were subjected to an open field test for 3 days. The greatest distance moved was recorded during the first phase of the trial (except for day 1), and this activity gradually decreased This behavioral pattern did not significantly differ between the 4GalTIIϩ/Ϫ and 4GalT-IIϪ/Ϫ mice.
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