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Articles by R Ito
Total Records ( 4 ) for R Ito
  H Ihara , S Hanashima , T Okada , R Ito , Y Yamaguchi , N Taniguchi and Y. Ikeda

FUT8, a eukaryotic 1,6-fucosyltransferase, catalyzes the transfer of a fucosyl residue from guanine nucleotide diphosphate-β-l-fucose to the innermost GlcNAc of an asparagine-linked oligosaccharide (N-glycan). The catalytic domain of FUT8 is structurally similar to that of NodZ, a bacterial 1,6-fucosyltransferase, which acts on a chitooligosaccharide in the synthesis of Nod factor. While the substrate specificities for the nucleotide sugar and the N-glycan have been determined, it is not known whether FUT8 is able to fucosylate other sugar chains such as chitooligosaccharides. The present study was conducted to investigate the action of FUT8 on chitooligosaccharides that are not generally thought to be a substrate in mammals, and the results indicate that FUT8 is able to fucosylate such structures in a manner comparable to NodZ. Surprisingly, structural analyses of the fucosylated products by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance indicated that FUT8 does not utilize the reducing terminal GlcNAc for fucose transfer but shows a preference for the third GlcNAc residue from the nonreducing terminus of the acceptor. These findings suggest that FUT8 catalyzes the fucosylation of chitooligosaccharide analogous to NodZ, but that a nonreducing terminal chitotriose structure is required for the reaction. The substrate recognition by which FUT8 selects the position to fucosylate might be distinct from that for NodZ and could be due to structural factor requirements which are inherent in FUT8.

  T Okada , H Ihara , R Ito , M Nakano , K Matsumoto , Y Yamaguchi , N Taniguchi and Y. Ikeda

The baculovirus–insect cell expression system is in widespread use for expressing post-translationally modified proteins. As a result, it is potentially applicable for the production of glycoproteins for therapeutic and diagnostic purposes. For practical use, however, remodeling of the biosynthetic pathway of host-cell N-glycosylation is required because insect cells produce paucimannosidic glycoforms, which are different from the typical mammalian glycoform, due to trimming of the non-reducing terminal β1,2-GlcNAc residue of the core structure by a specific β-N-acetylglucosaminidase. In order to establish a cell line which could be used as a host for the baculovirus-based production of glycoproteins with mammalian-type N-glycosylation, we prepared and characterized Spodoptera frugiperda Sf21 cells that had been transfected with the rat cDNA for β1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of a bisecting GlcNAc. As evidenced by structural analyses of N-glycans prepared from whole cells and the expressed recombinant glycoproteins, the introduction of GnT-III led to the production of bisected hybrid-type N-glycans in which the β1,2-GlcNAc residue at the 1,3-mannosyl branch is completely retained and which has the potential to be present in mammalian cells. These results and other related findings suggest that bisected oligosaccharides are highly resistant to β-N-acetylglucosaminidase activity of the S. frugiperda fused lobes gene product, or other related enzymes, which was confirmed in Sf21 cells. Our present study demonstrates that GnT-III transfection has the potential to be an effective approach in humanizing the N-glycosylation of lepidopteran insect cells, thereby providing a possible preliminary step for the generation of complex-type glycoforms if the presence of a bisecting GlcNAc can be tolerated.

  Y Watanabe , T Takahashi , A Okajima , M Shiokawa , N Ishii , I Katano , R Ito , M Ito , M Minegishi , N Minegishi , S Tsuchiya and K. Sugamura

‘Humanized mice’ are anticipated to be a valuable tool for studying the human immune system, but the reconstituted human immune cells have not yet been well characterized. Here, we extensively investigated the differentiation and functions of human B and T cells in a supra-immunodeficient mouse strain, NOD/shi-scid/cnull (NOG) reconstituted with CD34+ hematopoietic stem cells obtained from umbilical cord blood. In these hu-HSC NOG mice, the development of human B cells was partially blocked, and a significant number of B-cell progenitors accumulated in the spleen. The mature CD19+IgM+IgD+ human B cells of the hu-HSC NOG mice could produce IgG in vivo and in vitro by antigenic stimulation. In contrast, although human T cells with an apparently normal phenotype developed, most of them could neither proliferate nor produce IL-2 in response to antigenic stimulation by anti-CD3 and anti-CD28 antibodies in vitro. The positive selection of human T cells in the thymus was sufficiently functional, if not complete, and mainly mediated by mouse class II, suggesting that the human T cells lost their function in the periphery. We found that multiple mechanisms were involved in the T-cell abnormalities. Collectively, our results demonstrate that further improvements are necessary before humanized mice with a functional human immune system are achieved.

  Y Hayashi , M Yamamoto , H Mizoguchi , C Watanabe , R Ito , S Yamamoto , X. y Sun and Y. Murata

Multiple bioactive peptides, including glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2, are derived from the glucagon gene (Gcg). In the present study, we disrupted Gcg by introduction of GFP cDNA and established a knock-in mouse line. Gcggfp/gfp mice that lack most, if not all, of Gcg-derived peptides were born in an expected Mendelian ratio without gross abnormalities. Gcggfp/gfp mice showed lower blood glucose levels at 2 wk of age, but those in adult Gcggfp/gfp mice were not significantly different from those in Gcg+/+ and Gcggfp/+ mice, even after starvation for 16 h. Serum insulin levels in Gcggfp/gfp mice were lower than in Gcg+/+ and Gcggfp/+ on ad libitum feeding, but no significant differences were observed on starvation. Islet -cells and intestinal L-cells were readily visualized in Gcggfp/gfp and Gcggfp/+ mice under fluorescence. The Gcggfp/gfp postnatally developed hyperplasia of islet -cells, whereas the population of intestinal L-cells was not increased. In the Gcggfp/gfp, expression of Aristaless-related homeobox (Arx) was markedly increased in pancreas but not in intestine and suggested involvement of Arx in differential regulation of proliferation of Gcg-expressing cells. These results illustrated that Gcg-derived peptides are dispensable for survival and maintaining normoglycemia in adult mice and that Gcg-derived peptides differentially regulate proliferation/differentiation of -cells and L-cells. The present model is useful for analyzing glucose/energy metabolism in the absence of Gcg-derived peptides. It is useful also for analysis of the development, differentiation, and function of Gcg-expressing cells, because such cells are readily visualized by fluorescence in this model.

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