Akira Togayachi

National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan

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Publications (66)263.93 Total impact

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    ABSTRACT: AimsWisteria floribunda agglutinin (WFA)-positive human Mac-2-binding protein (WFA+-M2BP) is a new glycol marker related to liver fibrosis. The aim of the present study was to evaluate WFA+-M2BP as a predictor of hepatocellular carcinoma (HCC) development in patients with chronic hepatitis C.Methods This case–control study included 14 patients with chronic hepatitis C who developed HCC and 52controls, matched for age, gender, and fibrosis stage. WFA+-M2BP was measured at biopsy and follow-up. Time zero was set at the date of liver biopsy.ResultsWFA+-M2BP increased stepwise with progression of liver fibrosis (p < 0.001). Cumulative incidence of HCC development was significantly higher in patients with WFA+-M2BP ≥4.2 (p < 0.001) or in those with time-course changes in WFA+-M2BP (ΔWFA+-M2BP/year) ≥0.3 (p = 0.03). Multivariate analyses demonstrated that WFA+-M2BP ≥4.2 [hazard ratio (HR): 4.1, 95% confidence interval (CI): 1.1–15, p = 0.04], ΔWFA+-M2BP/year ≥0.3 (HR: 5.5, 95% CI: 1.5–19, p = 0.008), and AFP ≥10 ng/ml (HR: 4.7, 95% CI: 1.1–19, p = 0.03) were independent predictive factors of HCC development. Based on these data, we developed a simple scoring system to predict HCC development using these three factors. Using these scores, patients were classified into four groups; cumulative incidence of HCC development significantly increased with increasing scores (p < 0.001).ConclusionsWFA+-M2BP measurements and time-course changes in WFA+-M2BP can be used to identify patients at high risk of HCC development. Real-time monitoring of WFA+-M2BP can be a novel predictor of HCC development. This article is protected by copyright. All rights reserved.
    Hepatology Research 01/2015; · 2.07 Impact Factor
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    ABSTRACT: Histopathological classification of lung cancer has important implications in the application of clinical practice guidelines and the prediction of patient prognosis. Thus, we focused on discovering glycobiomarker candidates to classify the types of lung cancer tissue. First, we performed lectin microarray analysis of lung cancer tissue specimens and cell lines and identified Aleuria aurantia lectin (AAL), Hippeastrum hybrid lectin (HHL), and Concanavalia ensiformis agglutinin (ConA) as lectin probes specific to non-small cell lung carcinoma (NSCLC). LC-MS-based analysis was performed for the comprehensive identification of glycoproteins and N-linked glycosylation sites using lectin affinity capture of NSCLC-specific glycoforms of glycoproteins. This analysis identified 1092 AAL-bound glycoproteins (316 gene symbols) and 948 HHL/ConA-bound glycoproteins (279 gene symbols). The lectin microarray-assisted verification using 15 lung cancer cell lines revealed the NSCLC-specific expression of fibronectin. The glycosylation profiling of fibronectin indicated that the peanut agglutinin (PNA) signal appeared to differentiate two NSCLC types, adenocarcinoma and large cell carcinoma, whereas the protein expression level was similar between these types. Our glycoproteomics approach together with the concurrent use of an antibody and lectin is applicable to the quantitative and qualitative monitoring of variations in glycosylation of fibronectin specific to certain types of lung cancer tissue.
    Journal of Proteome Research 09/2014; · 5.06 Impact Factor
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    ABSTRACT: In this study, we selected 181 nematode glycogenes that are orthologous to human glycogenes and examined their RNAi phenotypes. The results are deposited in the C. elegans Glycogene Database (CGGDB) at AIST, Tsukuba, Japan. The most prominent RNAi phenotypes observed are disruptions of cell cycle progression in germline mitosis/meiosis and in early embryonic cell mitosis. Along with the previously reported roles of chondroitin proteoglycans, glycosphingolipids and GPI-anchored proteins in cell cycle progression, we show for the first time that the inhibition of the functions of N-glycan synthesis genes (cytoplasmic alg genes) resulted in abnormal germline formation, ER stress, and small body size (Sma) phenotypes. The results provide additional information on the roles of glycoconjugates in the cell cycle progression mechanisms of germline and embryonic cells.
    Glycobiology 08/2014; · 3.75 Impact Factor
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    ABSTRACT: The Wisteria floribunda agglutinin-positive human Mac-2-binding protein (WFA+-M2BP) was recently shown to be a liver fibrosis glycobiomarker with a unique fibrosis-related glyco-alteration. We evaluated the ability of WFA+-M2BP to predict the development of hepatocellular carcinoma (HCC) in patients who were infected with the hepatitis C virus (HCV). A total of 707 patients who had been admitted to our hospital with chronic HCV infection without other potential risk factors were evaluated to determine the ability of WFA+-M2BP to predict the development of HCC; factors evaluated included age, sex, viral load, genotypes, fibrosis stage, aspartate and alanine aminotransferase levels, bilirubin, albumin, platelet count, alpha-fetoprotein (AFP), WFA+-M2BP and the response to interferon therapy. Serum WFA+-M2BP levels were significantly increased according to the progression of liver fibrosis stage (p < 0.001). In each distinctive stage of fibrosis (F0/1, F2, F3, and F4), the risk of development of HCC was increased according to the elevation of WFA+-M2BP. Multivariate analysis identified age ≥ 57 years, F4, AFP ≥ 20 ng/mL, WFA+-M2BP ≥ 4, and WFA+-M2BP 1 - 4 as well as the response to interferon (no therapy vs. SVR) as independent risk factors for the development of HCC. The time-dependent areas under the receiver operating characteristic curve demonstrated that the WFA+-M2BP assay predicted the development of HCC with higher diagnostic accuracy than AFP. Conclusion: WFA+-M2BP can be applied as a useful surrogate marker for the risk of HCC development, in addition to liver biopsy. (Hepatology 2014;)
    Hepatology 07/2014; · 11.19 Impact Factor
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    ABSTRACT: Carbohydrate structures, including Lewis X (Lex), which is not synthesized in mutant mice that lack α1,3-fucosyltransferase 9 (Fut9−/−), are involved in cell–cell recognition and inflammation. However, immunological alteration in Fut9−/− mice has not been studied. Thus, the inflammatory response of Fut9−/− mice was examined using the highly neurovirulent mouse hepatitis virus (MHV) JHMV srr7 strain. Pathological study revealed that inflammation induced in the brains of Fut9−/− mice after infection was more extensive compared with that of wild-type mice, although viral titers obtained from the brains of mutant mice were lower than those of wild-type mice. Furthermore, the reduction in cell numbers in the spleens of wild-type mice after infection was not observed in the infected Fut9−/− mice. Although there were no clear differences in the levels of cytokines examined in the brains between Fut9−/− and wild-type mice except for interferon-β (IFN-β) expression, some of those in the spleens, including interferon-γ (IFN-γ), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1), showed higher levels in Fut9−/− than in wild-type mice. Furthermore, Fut9−/− mice were refractory to the in vivo inoculation of endotoxin (LPS) compared with wild-type mice. These results indicate that Lex structures are involved in host responses against viral or bacterial challenges.
    Pathology International 05/2014; 64(5). · 1.59 Impact Factor
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    ABSTRACT: Recently, a novel marker, hyperglycosylated Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA(+)-M2BP), was developed for liver fibrosis using the glycan "sugar chain"-based immunoassay; however, the feasibility of WFA(+)-M2BP for assessing liver fibrosis has not been proven with clinical samples of hepatitis. Serum WFA(+)-M2BP values were evaluated in 200 patients with chronic liver disease who underwent histological examination of liver fibrosis. The diagnostic accuracy of WFA(+)-M2BP values was compared with various fibrosis markers, such as ultrasound based-virtual touch tissue quantification (VTTQ), magnetic resonance imaging based-liver-to-major psoas muscle intensity ratio (LMR), and serum markers, including hyaluronic acid, type 4 collagen, and aspartate transaminase to platelet ratio index (APRI). Serum WFA(+)-M2BP levels in patients with fibrosis grades F0, F1, F2, F3, and F4 had cutoff indices 1.62, 1.82, 3.02, 3.32, and 3.67, respectively, and there were significant differences between fibrosis stages F1 and F2, and between F2 and F3 (P < 0.01). The area under the receiver operating characteristic curves for the diagnosis of fibrosis (F ≥ 3) using serum WFA(+)-M2BP values (0.812) was almost comparable to that using VTTQ examination (0.814), but was superior to the other surrogate markers, including LMR index (0.766), APRI (0.694), hyaluronic acid (0.683), and type 4 collagen (0.625) (P < 0.01 each). Serum WFA(+)-M2BP values based on a glycan-based immunoassay is an accurate, reliable, and reproducible method for the assessment of liver fibrosis. This approach could be clinically feasible for evaluation of beneficial therapy through the quantification of liver fibrosis in hepatitis patients if this measurement application is commercially realized.
    Journal of Gastroenterology 03/2014; · 4.02 Impact Factor
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    ABSTRACT: Epithelial ovarian cancer (EOC) is often asymptomatic and thus diagnosed at advanced stages with a poor prognosis. False-negative results for the conventional marker CA125 frequently occur in cases of clear cell carcinoma (CCC), a type of EOC; therefore, it is necessary to develop biomarkers with greater sensitivity. We previously reported a strategy to discover glyco-biomarker candidates by combined lectin microarray and IGOT-LC/MS analysis. We have now optimized this strategy for discovering EOC biomarkers. Glycopeptides possessing cancerous glycans were enriched from the ascites fluids and culture supernatants of cancer cell lines with a fucose-binding lectin, AAL. IGOT-LC/MS analysis of CCC samples yielded 144 candidate glycoproteins. We selected WFA by lectin microarray as the optimal lectin to distinguish EOC from gastric and colon cancer. The candidates were narrowed by western analysis of the WFA-bound fraction of ascites fluids. One of the final candidates, WFA-reactive ceruloplasmin, produced higher signals in the ascites fluids of EOC patients, including CCC, in comparison to the benign samples, while CA125 levels were comparable in the sandwich ELISA. Thus, our glycoproteomic strategy featuring efficient enrichment of glycans with disease-related alterations is applicable to various diseases.
    Journal of Proteome Research 02/2014; · 5.06 Impact Factor
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    ABSTRACT: The importance of diagnosis and therapies for liver cirrhosis (LC) is indisputable. Thus, a reliable method for monitoring the progression of liver fibrosis and resultant LC is urgently needed. Previously, using a lectin-assisted glycoproteomic method, we identified 26 serum glycoproteins as promising glycobiomarker candidates for monitoring the progression of liver diseases [Kaji et al. J. Proteome Res. (2013)]. In this study, we identified colony stimulating factor 1 receptor (CSF1R) as a promising LC marker candidate, and then established Wisteria floribunda agglutinin (WFA)-reactive CSF1R (WFA(+)-CSF1R) as a novel possible glycobiomarker candidate by utilizing a glycoproteomics-based strategy [Narimatsu et al. FEBS J (2010)]. The serum level of WFA(+)-CSF1R in patients with hepatitis C virus (HCV)-infected liver disease was measured by an antibody-lectin sandwich ELISA. In a proof-of-concept experiment of the strategy preceding to future clinical studies, LC patients showed a high serum WFA(+)-CSF1R level in selected samples (P = 1.3E-17). This result suggests WFA(+)-CSF1R is a possible biomarker candidate for evaluation of LC. Our results verified feasibility of this strategy for glycobiomarker development.
    Journal of Proteome Research 01/2014; · 5.06 Impact Factor
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    ABSTRACT: Lewis X [Le(X), Galβ1-4(Fucα1-3)GlcNAc] is a carbohydrate epitope that is present at the nonreducing terminus of sugar chains of glycoproteins and glycolipids, and is abundantly expressed in several stem cell populations. Le(X) antigen can be used in conjunction with fluorescence-activated cell sorting to isolate neurosphere-forming neural stem cells (NSCs) from embryonic mouse brains. However, its function in the maintenance and differentiation of stem cells remains largely unknown. In this study, we examined mice deficient for fucosyltransferase 9 (Fut9), which is thought to synthesize most, if not all, of the Le(X) moieties in the brain. We found that the number of NSCs was increased in the brain of Fut9(-/-) embryos, suggesting that Fut9-synthesized Le(X) is dispensable for the maintenance of NSCs. Another α1,3-fucosyltransferase gene, fucosyltransferase 10 (Fut10), is expressed in the ventricular zone (VZ) of the embryonic brain. Overexpression of Fut10 enhanced the self-renewal of NSCs. Conversely, suppression of Fut10 expression induced the differentiation of NSCs and embryonic stem cells. In addition, knockdown of Fut10 expression in the cortical VZ of the embryonic brain by in utero electroporation of Fut10-miRNAs impaired the radial migration of neural precursor cells. Our data suggest that Fut10 is a unique α1,3-fucosyltransferase with stringent substrate specificity, and that its activity is required to maintain stem cells in an undifferentiated state.
    Journal of Biological Chemistry 08/2013; · 4.60 Impact Factor
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    ABSTRACT: We previously proposed a high-throughput strategy to discover serological biomarker candidates of cancer. This strategy focuses on a series of candidate glycoproteins that are specifically expressed in the original tissues (cells) of the target cancer and that carry glycan structures associated with carcinogenesis [Narimatsu H. et al. FEBS J. (2010)]. Here, we examined the effectiveness of our strategy in identifying biomarkers to assess progression of liver fibrosis and for the early-detection of hepatocellular carcinoma (HCC). Based on the results of lectin array analyses in culture media of hepatoma cell lines, we captured glycopeptides carrying AAL-ligands (fucosylated glycans) or DSA-ligands (branched glycans) from a digest of culture media proteins and sera from HCC patients with a background of liver cirrhosis (LC). Glycoproteins were identified by the IGOT-LC-MS method. In all, 21 candidates were selected from 744 AAL-bound glycoproteins for further verification according to (i) their abundance in serum, (ii) specific expression in liver, and (iii) the availability of antibodies to the glycoproteins. All selected candidates showed enhancement of AAL-reactivity in sera of HCC patients compared with that of healthy volunteers (HV). These results indicate that our glycoproteomic strategy is effective for identifying multiple glyco-biomarker candidates in a high-throughput manner.
    Journal of Proteome Research 04/2013; · 5.06 Impact Factor
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    ABSTRACT: Fucose (Fuc)-containing glycoconjugates play important roles in numerous physiological and pathological processes. Given the biological importance of post-translational glycosylation, a specific and robust strategy for the identification of fucosylated glycoproteins is highly desirable. In this study, we demonstrate an alternative way of labeling of fucosylated structures by metabolic engineering, using a chemoenzymatic approach. In this approach, the activities of Bacteroides fragilis 9343 L-fucokinase/guanosine-5'-diphosphate-Fuc pyrophosphorylase and human α1,3-fucosyltransferase 9 are combined in a Namalwa cellular model. Interestingly, this system could be applied to labeling of alkyne-modified fucosylated glycoproteins. N-Glycan site mapping and identification were done using an in vitro selective chemical ligation reaction and isotope-coded glycosylation site-specific tagging, subsequent to liquid chromatography-tandem mass spectrometry analysis. This work illustrates the use of a click chemistry-based strategy combined with a glycoproteomic technique to get further insight into the pattern of Fuc-mediated biological processes and functions.
    Glycobiology 12/2011; 22(5):630-7. · 3.75 Impact Factor
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    ABSTRACT: It is known that mutant mice of the beta-1,3-N-acetylglucosaminyltransferase gene (beta3Gn-T5) respond well to T-cell dependent and independent antigens. Here, we examined the effectiveness of anti-ganglioside antibody generation by immunization of beta3Gn-T5 mutant mice with liposome-embedded glycosphingolipids such as GD1a and GT1b. Consequently, the mutant mice showed a more efficient generation of anti-GD1a or anti-GT1b antibodies than wild-type mice in an enzyme-linked immunosorbent assay using sera during immunization. Thus, the beta3Gn-T5 deficient mutant mice proved more responsive than wild-type mice to not only protein antigens, but also to carbohydrates in glycolipids. Furthermore, about 50% of monoclonal antibodies generated using splenocytes of the immunized mutant mice were of the IgG class. Besides general high responsiveness to proteins and glycolipids, it could be expected that the mutant mice of beta3Gn-T5 would be useful in the generation of monoclonal antibodies towards lacto-/neolacto-series glycolipids, since these mutants lack lacto-/neolacto-series glycolipids. In fact, they showed a good serum response in immuno-fluorescence assay with cultured living cells when immunized by glycolipids extracted from ovarian cancer cell lines. These results suggested that beta3Gn-T5 mutant mice are useful for the generation of anti-glycolipid antigens with lacto-/neolacto-core structures expressed in cancer cells.
    Nagoya journal of medical science 08/2011; 73(3-4):137-46. · 0.80 Impact Factor
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    ABSTRACT: Chondroitin sulfate (CS) is a glycosaminoglycan, consisting of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues, and plays important roles in development and homeostasis of organs and tissues. Here, we generated and analyzed mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). Csgalnact1(-/-) mice were viable and fertile but exhibited slight dwarfism. Biochemically, the level of CS in Csgalnact1(-/-) cartilage was reduced to ∼50% that of wild-type cartilage, whereas its chain length was similar to wild-type mice, indicating that CSGalNAcT-1 participates in the CS chain initiation as suggested in the previous study (Sakai, K., Kimata, K., Sato, T., Gotoh, M., Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) J. Biol. Chem. 282, 4152-4161). Histologically, the growth plate of Csgalnact1(-/-) mice contained shorter and slightly disorganized chondrocyte columns with a reduced volume of the extracellular matrix principally in the proliferative layer. Immunohistochemical analysis revealed that the level of both aggrecan and link protein 1 were decreased in Csgalnact1(-/-) cartilage. Western blot analysis demonstrated an increase in processed forms of aggrecan core protein. These results suggest that CSGalNAcT-1 is required for normal levels of CS biosynthesis in cartilage. Our observations suggest that CSGalNAcT-1 is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence causes a rapid catabolism of aggrecan.
    Journal of Biological Chemistry 02/2011; 286(7):5803-12. · 4.60 Impact Factor
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    ABSTRACT: Chondroitin sulfate (CS) is a glycosaminoglycan, consisting of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues, and plays important roles in development and homeostasis of organs and tissues. Here, we generated and analyzed mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). Csgalnact1−/− mice were viable and fertile but exhibited slight dwarfism. Biochemically, the level of CS in Csgalnact1−/− cartilage was reduced to ∼50% that of wild-type cartilage, whereas its chain length was similar to wild-type mice, indicating that CSGalNAcT-1 participates in the CS chain initiation as suggested in the previous study (Sakai, K., Kimata, K., Sato, T., Gotoh, M., Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) J. Biol. Chem. 282, 4152–4161). Histologically, the growth plate of Csgalnact1−/− mice contained shorter and slightly disorganized chondrocyte columns with a reduced volume of the extracellular matrix principally in the proliferative layer. Immunohistochemical analysis revealed that the level of both aggrecan and link protein 1 were decreased in Csgalnact1−/− cartilage. Western blot analysis demonstrated an increase in processed forms of aggrecan core protein. These results suggest that CSGalNAcT-1 is required for normal levels of CS biosynthesis in cartilage. Our observations suggest that CSGalNAcT-1 is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence causes a rapid catabolism of aggrecan.
    Journal of Biological Chemistry 02/2011; 286(7):5803-5812. · 4.60 Impact Factor
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    ABSTRACT: Chondroitin sulfate (CS) is a glycosaminoglycan, consisting of repeating disaccharide units of N-acetylgalactosamine and glucuronic acid residues, and plays important roles in development and homeostasis of organs and tissues. Here, we generated and analyzed mice lacking chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGalNAcT-1). Csgalnact1(-/-) mice were viable and fertile but exhibited slight dwarfism. Biochemically, the level of CS in Csgalnact1(-/-) cartilage was reduced to ∼50% that of wild-type cartilage, whereas its chain length was similar to wild-type mice, indicating that CSGalNAcT-1 participates in the CS chain initiation as suggested in the previous study (Sakai, K., Kimata, K., Sato, T., Gotoh, M., Narimatsu, H., Shinomiya, K., and Watanabe, H. (2007) J. Biol. Chem. 282, 4152-4161). Histologically, the growth plate of Csgalnact1(-/-) mice contained shorter and slightly disorganized chondrocyte columns with a reduced volume of the extracellular matrix principally in the proliferative layer. Immunohistochemical analysis revealed that the level of both aggrecan and link protein 1 were decreased in Csgalnact1(-/-) cartilage. Western blot analysis demonstrated an increase in processed forms of aggrecan core protein. These results suggest that CSGalNAcT-1 is required for normal levels of CS biosynthesis in cartilage. Our observations suggest that CSGalNAcT-1 is necessary for normal levels of endochondral ossification, and the decrease in CS amount in the growth plate by its absence causes a rapid catabolism of aggrecan.
    Journal of Biological Chemistry 02/2011; 286(7):5803-12. · 4.60 Impact Factor
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    ABSTRACT: A novel member of the human ppGalNAc-T family, ppGalNAc-T20, was identified and characterized. Amino acid alignment revealed a high sequence identity between ppGalNAc-T20 and -T10. In the GalNAc transfer assay towards mucin-derived peptide substrates, the recombinant ppGalNAc-T20 demonstrated to be a typical glycopeptide GalNAc-transferase that exhibits activity towards mono-GalNAc-glycosylated peptide EA2 derived from rat submandibular gland mucin but no activity towards non-modified EA2. The in vitro catalytic property of ppGalNAc-T20 was compared with that of ppGalNAc-T10 to show different acceptor substrate specificities and kinetic constants. The ppGalNAc-T20 transcript was found exclusively in testis and brain. In situ hybridization further reveals that ppGalNAc-T20 was specifically localized in primary and secondary spermatocytes of the two meiotic periods, suggesting that it may involve in O-glycosylation during mouse spermatogenesis.
    Biochemical and Biophysical Research Communications 10/2010; 402(4):680-6. · 2.28 Impact Factor
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    ABSTRACT: In a previous study, we demonstrated that beta1,3-N-acetylglucosaminyltransferase 5 (B3gnt5) is a lactotriaosylceramide (Lc(3)Cer) synthase that synthesizes a precursor structure for lacto/neolacto-series glycosphingolipids (GSLs) in in vitro experiments. Here, we generated B3gnt5-deficient (B3gnt5(-/-)) mice to investigate the in vivo biological functions of lacto/neolacto-series GSLs. In biochemical analyses, lacto/neolacto-series GSLs were confirmed to be absent and no Lc(3)Cer synthase activity was detected in the tissues of these mice. These results demonstrate that beta3GnT5 is the sole enzyme synthesizing Lc(3)Cer in vivo. Ganglioside GM1, known as a glycosphingolipid-enriched microdomain (GEM) marker, was found to be up-regulated in B3gnt5(-/-) B cells by flow cytometry and fluorescence microscopy. However, no difference in the amount of GM1 was observed by TLC-immunoblotting analysis. The GEM-stained puncta on the surface of B3gnt5(-/-) resting B cells were brighter and larger than those of WT cells. These results suggest that structural alteration of GEM occurs in B3gnt5(-/-) B cells. We next examined whether BCR signaling-related proteins, such as BCR, CD19, and the signaling molecule Lyn, had moved into or out of the GEM fraction. In B3gnt5(-/-) B cells, these molecules were enriched in the GEM fraction or adjacent fraction. Moreover, B3gnt5(-/-) B cells were more sensitive to the induction of intracellular phosphorylation signals on BCR stimulation and proliferated more vigorously than WT B cells. Together, these results suggest that lacto/neolacto-series GSLs play an important role in clustering of GEMs and tether-specific proteins, such as BCR, CD19, and related signaling molecules to the GEMs.
    Proceedings of the National Academy of Sciences 06/2010; 107(26):11900-5. · 9.81 Impact Factor
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    ABSTRACT: The polylactosamine structure is a fundamental structure of carbohydrate chains and carries a lot of biofunctional carbohydrate epitopes. To investigate the biological function of polylactosamine chains, here we generated and analyzed knockout mice lacking the gene B3gnt2, which encodes a major polylactosamine synthase. In beta1,3-N-acetylglucosaminyltransferase (B3gnt2) B3gnt2-deficient (B3gnt2-/-) mice, the number of polylactosamine structures was markedly lower than in wild-type mice. Flow cytometry, LEL lectin-blotting, and glycan analysis by metabolic labeling demonstrated that the amount of polylactosamine chains on N-glycans was greatly reduced in the tissues of B3gnt2-/- mice. We examined whether immunological abnormalities were present in B3gnt2-/- mice. We screened polylactosamine-carrying molecules of wild-type mice by lectin microarray analysis and found that polylactosamine was present on CD28 and CD19, two established immune co-stimulatory molecules. Polylactosamine levels on these molecules were lower in B3gnt2-/- mice than in wild-type mice. B3gnt2-/- T cells were more sensitive to the induction of intracellular Ca2+ flux on stimulation with anti-CD3epsilon/CD28 antibodies and proliferated more strongly than wild-type T cells. B3gnt2-/- B cells also showed hyperproliferation on BCR stimulation. These results showed that hyperactivation of lymphocytes occurred due to a lack of polylactosamine on receptor molecules in B3gnt2-/- mice. This finding indicates that polylactosamine has an important role in immunological biofunctions. We can therefore attempt to identify the in vivo biological function of glycans using glycogene-deficient mice.
    Methods in enzymology 01/2010; 479:185-204. · 1.90 Impact Factor
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    ABSTRACT: The polylactosamine structure is a fundamental structure of carbohydrate chains and carries a lot of biofunctional carbohydrate epitopes. To investigate the biological function of polylactosamine chains, here we generated and analyzed knockout mice lacking the gene B3gnt2, which encodes a major polylactosamine synthase. In β1,3-N-acetylglucosaminyltransferase (B3gnt2) B3gnt2-deficient (B3gnt2−/−) mice, the number of polylactosamine structures was markedly lower than in wild-type mice. Flow cytometry, LEL lectin-blotting, and glycan analysis by metabolic labeling demonstrated that the amount of polylactosamine chains on N-glycans was greatly reduced in the tissues of B3gnt2−/− mice. We examined whether immunological abnormalities were present in B3gnt2−/− mice. We screened polylactosamine-carrying molecules of wild-type mice by lectin microarray analysis and found that polylactosamine was present on CD28 and CD19, two established immune co-stimulatory molecules. Polylactosamine levels on these molecules were lower in B3gnt2−/− mice than in wild-type mice. B3gnt2−/− T cells were more sensitive to the induction of intracellular Ca2+ flux on stimulation with anti-CD3ε/CD28 antibodies and proliferated more strongly than wild-type T cells. B3gnt2−/− B cells also showed hyperproliferation on BCR stimulation. These results showed that hyperactivation of lymphocytes occurred due to a lack of polylactosamine on receptor molecules in B3gnt2−/− mice. This finding indicates that polylactosamine has an important role in immunological biofunctions. We can therefore attempt to identify the in vivo biological function of glycans using glycogene-deficient mice.
    Methods in Enzymology - METH ENZYMOLOGY. 01/2010; 479:185-204.
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    Biochemical and Biophysical Research Communications 06/2009; 384(2):275. · 2.28 Impact Factor

Publication Stats

2k Citations
263.93 Total Impact Points

Institutions

  • 2001–2014
    • National Institute of Advanced Industrial Science and Technology
      • Research Center for Medical Glycoscience
      Tsukuba, Ibaraki, Japan
  • 2003
    • Aichi Medical University
      • Institute for Molecular Science of Medicine
      Japan
  • 1998–2003
    • Soka University
      • Institute of Life Science
      Edo, Tōkyō, Japan