Mitsutaka Ogawa
Nagoya University
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Featured researches published by Mitsutaka Ogawa.
Archives of Biochemistry and Biophysics | 1968
Tatsuo Sugiyama; N. Nakayama; Mitsutaka Ogawa; Takashi Akazawa
Abstract Fraction I protein was purified from spinach leaves by Sephadex gel filtration and DEAE-cellulose column chromatography. To study the role of SH-groups in the enzyme molecule, p -chloromercuribenzoate (PCMB) titration of SH-groups was carried out with a parallel determination of enzyme activity. It was found that the rate and extent of reaction with PCMB was identical in the presence or absence of urea (4.5 m ), sodium dodecyl sulfate (2 × 10 −2 m ), and ribulose 1,5-diphosphate (RuDP) (1.1 × 10 −4 m ). The total number of SH-groups per mole protein was 96, in agreement with chemical data. Approximately 10 SH-groups were blocked before an appreciable loss of the RuDP-carboxylase activity occurred, and complete inhibition of enzyme activity was associated with the blocking of about 30 SH-groups. The possible role of SH-groups in the structural rigidity of the protein molecule was suggested by the finding that proteolytic digestibility (chymotrypsin and Nagarse) of the protein was greatly enhanced by PCMB pretreatment as measured by decrements of RuDP-carboxylase activity. Full restoration of the enzyme activity by the addition of cysteine to the PCMB-inactivated enzyme protein was accompanied by restoration of resistance to proteolytic attack. That the molecule is restored to a conformation possibly identical with that of the native protein in tertiary structure was supported by electron microscopic observations of the reconstituted protein.
Biochimica et Biophysica Acta | 2012
Mitsutaka Ogawa; Hidenori Mizofuchi; Yuki Kobayashi; Genta Tsuzuki; Mayumi Yamamoto; Shuichi Wada; Kazuo Kamemura
BACKGROUND O-Linked β-N-acetylglucosaminylation (O-GlcNAcylation) on the Ser/Thr residue of nucleocytoplasmic proteins is a dynamic post-translational modification found in multicellular organisms. More than 500 proteins involved in a wide range of cellular functions, including cell cycle, transcription, epigenesis, and glucose sensing, are modified with O-GlcNAc. Although it has been suggested that O-GlcNAcylation is involved in the differentiation of cells in a lineage-specific manner, its role in skeletal myogenesis is unknown. METHODS AND RESULTS A myogenesis-dependent drastic decrease in the levels of O-GlcNAcylation was found in mouse C2C12 myoblasts. The global decrease in O-GlcNAcylation was observed at the earlier stage of myogenesis, prior to myoblast fusion. Genetic or pharmacological inactivation of O-GlcNAcase blocked both the myogenesis-dependent global decrease in O-GlcNAcylation and myoblast fusion. Although inactivation of O-GlcNAcase affected neither cell-cycle exit nor cell survival in response to myogenic stimulus, it perturbed the expression of myogenic regulatory factors. While the expression of myod and myf5 in response to myogenic induction was not affected, that of myogenin and mrf4 was severely inhibited by the inactivation of O-GlcNAcase. CONCLUSION These results indicate that the terminal differentiation program of skeletal myogenesis is negatively regulated by O-GlcNAcylation. GENERAL SIGNIFICANCE O-GlcNAcylation is involved in differentiation in a cell lineage-dependent manner, and a decrease in O-GlcNAcylation may have a common role in the differentiation of cells of muscle lineage.
Biochemical and Biophysical Research Communications | 2013
Mitsutaka Ogawa; Naosuke Nakamura; Yoshiaki Nakayama; Akira Kurosaka; Hiroshi Manya; Motoi Kanagawa; Tamao Endo; Koichi Furukawa; Tetsuya Okajima
Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker-Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.
Biochemical and Biophysical Research Communications | 2013
Mitsutaka Ogawa; Yuki Sakakibara; Kazuo Kamemura
Previously, we demonstrated that the expression of myogenin, a critical transcription factor for myogenesis, is negatively regulated by O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation in mouse C2C12 cells. In this study, we found that Mef2 family proteins, especially Mef2D which is a crucial transcriptional activator of myogenin, are O-GlcNAc glycosylated. Between the two splice variants of Mef2D, Mef2D1a rather than Mef2D1b appears to drive the initiation of myogenin expression in the early stage of myogenesis. A deletion mutant analysis showed that Mef2D1a is glycosylated both in its DNA-binding and transactivation domains. A significant decrease in the glycosylation of Mef2D was observed in response to myogenic stimulus in C2C12 cells. Inhibition of the myogenesis-dependent decrease in the glycosylation of Mef2D suppressed its recruitment to the myogenin promoter. These results indicate that the expression of myogenin is regulated, at least in part, by the decreased glycosylation-dependent recruitment of Mef2D to the promoter region, and this is one of the negative regulatory mechanisms of skeletal myogenesis by O-GlcNAc glycosylation.
eLife | 2017
Shogo Sawaguchi; Shweta Varshney; Mitsutaka Ogawa; Yuta Sakaidani; Hirokazu Yagi; Kyosuke Takeshita; Toyoaki Murohara; Koichi Kato; Subha Sundaram; Pamela Stanley; Tetsuya Okajima
The glycosyltransferase EOGT transfers O-GlcNAc to a consensus site in epidermal growth factor-like (EGF) repeats of a limited number of secreted and membrane proteins, including Notch receptors. In EOGT-deficient cells, the binding of DLL1 and DLL4, but not JAG1, canonical Notch ligands was reduced, and ligand-induced Notch signaling was impaired. Mutagenesis of O-GlcNAc sites on NOTCH1 also resulted in decreased binding of DLL4. EOGT functions were investigated in retinal angiogenesis that depends on Notch signaling. Global or endothelial cell-specific deletion of Eogt resulted in defective retinal angiogenesis, with a mild phenotype similar to that caused by reduced Notch signaling in retina. Combined deficiency of different Notch1 mutant alleles exacerbated the abnormalities in Eogt−/− retina, and Notch target gene expression was decreased in Eogt−/−endothelial cells. Thus, O-GlcNAc on EGF repeats of Notch receptors mediates ligand-induced Notch signaling required in endothelial cells for optimal vascular development. DOI: http://dx.doi.org/10.7554/eLife.24419.001
Journal of Biological Chemistry | 2015
Mitsutaka Ogawa; Shogo Sawaguchi; Takami Kawai; Daita Nadano; Tsukasa Matsuda; Hirokazu Yagi; Koichi Kato; Koichi Furukawa; Tetsuya Okajima
Background: EOGT (epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine) mutations have been identified in patients with Adams-Oliver syndrome (AOS). Results: O-Linked N-acetylglucosaminylation (O-GlcNAcylation) of EGF domains in the endoplasmic reticulum (ER) is impaired in all EOGT variants associated with AOS. Conclusion: AOS-causative EOGT mutations affect ER O-GlcNAcylation. Significance: Impaired EOGT glycosyltransferase activity and a consequent reduction in O-GlcNAcylation underlie the etiology of EOGT-related AOS. Epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) is an endoplasmic reticulum (ER)-resident O-linked N-acetylglucosamine (O-GlcNAc) transferase that acts on EGF domain-containing proteins such as Notch receptors. Recently, mutations in EOGT have been reported in patients with Adams-Oliver syndrome (AOS). Here, we have characterized enzymatic properties of mouse EOGT and EOGT mutants associated with AOS. Simultaneous expression of EOGT with Notch1 EGF repeats in human embryonic kidney 293T (HEK293T) cells led to immunoreactivity with the CTD110.6 antibody in the ER. Consistent with the GlcNAc modification in the ER, the enzymatic properties of EOGT are distinct from those of Golgi-resident GlcNAc transferases; the pH optimum of EOGT ranges from 7.0 to 7.5, and the Km value for UDP N-acetylglucosamine (UDP-GlcNAc) is 25 μm. Despite the relatively low Km value for UDP-GlcNAc, EOGT-catalyzed GlcNAcylation depends on the hexosamine pathway, as revealed by the increased O-GlcNAcylation of Notch1 EGF repeats upon supplementation with hexosamine, suggesting differential regulation of the luminal UDP-GlcNAc concentration in the ER and Golgi. As compared with wild-type EOGT, O-GlcNAcylation in the ER is nearly abolished in HEK293T cells exogenously expressing EOGT variants associated with AOS. Introduction of the W207S mutation resulted in degradation of the protein via the ubiquitin-proteasome pathway, although the stability and ER localization of EOGTR377Q were not affected. Importantly, the interaction between UDP-GlcNAc and EOGTR377Q was impaired without adversely affecting the acceptor substrate interaction. These results suggest that impaired glycosyltransferase activity in mutant EOGT proteins and the consequent defective O-GlcNAcylation in the ER constitute the molecular basis for AOS.
FEBS Letters | 2012
Kazuo Kamemura; Mitsutaka Ogawa; Saki Ohkubo; Yasuhiro Ohtsuka; Yu Shitara; Tohru Komiya; Satoko Maeda; Akihiro Ito; Minoru Yoshida
Mitochondria perform multiple functions critical to the maintenance of cellular homeostasis. Here we report that the downregulation of histone deacetylase 6 (HDAC6) causes a reduction in the net activity of mitochondrial enzymes, including respiratory complex II and citrate synthase. HDAC6 deacetylase and ubiquitin‐binding activities were both required for recovery of reduced mitochondrial metabolic activity due to the loss of HDAC6. Hsp90, a substrate of HDAC6, localizes to mitochondria and partly mediates the regulation of mitochondrial metabolic activity by HDAC6. Our finding suggests that HDAC6 regulates mitochondrial metabolism and might serve as a cellular homeostasis surveillance factor.
World Journal of Biological Chemistry | 2014
Mitsutaka Ogawa; Koichi Furukawa; Tetsuya Okajima
The O-linked β-N-acetylglucosamine (O-GlcNAc)ylation of cytoplasmic and nuclear proteins regulates basic cellular functions and is involved in the etiology of neurodegeneration and diabetes. Intracellular O-GlcNAcylation is catalyzed by a single O-GlcNAc transferase, O-GlcNAc transferase (OGT). Recently, an atypical O-GlcNAc transferase, extracellular O-linked β-N-acetylglucosamine (EOGT), which is responsible for the modification of extracellular O-GlcNAc, was identified. Although both OGT and EOGT are regulated through the common hexosamine biosynthesis pathway, EOGT localizes to the lumen of the endoplasmic reticulum and transfers GlcNAc to epidermal growth factor-like domains in an OGT-independent manner. In Drosophila, loss of Eogt gives phenotypes similar to those caused by defects in the apical extracellular matrix. Dumpy, a membrane-anchored apical extracellular matrix protein, was identified as a major O-GlcNAcylated protein, and EOGT mediates Dumpy-dependent cell adhesion. In mammals, extracellular O-GlcNAc was detected on extracellular proteins including heparan sulfate proteoglycan 2, Nell1, laminin subunit alpha-5, Pamr1, and transmembrane proteins, including Notch receptors. Although the physiological function of O-GlcNAc in mammals has not yet been elucidated, exome sequencing identified homozygous EOGT mutations in patients with Adams-Oliver syndrome, a rare congenital disorder characterized by aplasia cutis congenita and terminal transverse limb defects. This review summarizes the current knowledge of extracellular O-GlcNAc and its implications in the pathological processes in Adams-Oliver syndrome.
Journal of Biological Chemistry | 2016
Kei Kaneko; Yuki Ohkawa; Noboru Hashimoto; Yuhsuke Ohmi; Norihiro Kotani; Koichi Honke; Mitsutaka Ogawa; Tetsuya Okajima; Keiko Furukawa; Koichi Furukawa
To investigate mechanisms for increased malignant properties in malignant melanomas by ganglioside GD3, enzyme-mediated activation of radical sources and subsequent mass spectrometry were performed using an anti-GD3 antibody and GD3-positive (GD3+) and GD3-negative (GD3-) melanoma cell lines. Neogenin, defined as a GD3-neighbored molecule, was largely localized in lipid/rafts in GD3+ cells. Silencing of neogenin resulted in the reduction of cell growth and invasion activity. Physical association between GD3 and neogenin was demonstrated by immunoblotting of the immunoprecipitates with anti-neogenin antibody from GD3+ cell lysates. The intracytoplasmic domain of neogenin (Ne-ICD) was detected in GD3+ cells at higher levels than in GD3− cells when cells were treated by a proteasome inhibitor but not when simultaneously treated with a γ-secretase inhibitor. Exogenous GD3 also induced increased Ne-ICD in GD3− cells. Overexpression of Ne-ICD in GD3− cells resulted in the increased cell growth and invasion activity, suggesting that Ne-ICD plays a role as a transcriptional factor to drive malignant properties of melanomas after cleavage with γ-secretase. γ-Secretase was found in lipid/rafts in GD3+ cells. Accordingly, immunocyto-staining revealed that GD3, neogenin, and γ-secretase were co-localized at the leading edge of GD3+ cells. All these results suggested that GD3 recruits γ-secretase to lipid/rafts, allowing efficient cleavage of neogenin. ChIP-sequencing was performed to identify candidates of target genes of Ne-ICD. Some of them actually showed increased expression after expression of Ne-ICD, probably exerting malignant phenotypes of melanomas under GD3 expression.
Biochimica et Biophysica Acta | 2015
Mitsutaka Ogawa; Shogo Sawaguchi; Koichi Furukawa; Tetsuya Okajima
BACKGROUND O-linked β-N-acetylglucosamine (O-GlcNAc) modification of epidermal growth factor (EGF) domains catalyzed by EGF domain O-GlcNAc transferase (EOGT) is the first example of GlcNAc modification in the lumen of the endoplasmic reticulum (ER). SCOPE OF REVIEW This review summarizes current knowledge on the EOGT-catalyzed O-GlcNAc modification of EGF domains obtained through biochemical characterization, genetic analysis in Drosophila, and identification of human EOGT mutation. Additionally, this review discusses GTDC2-another ER protein homologous to EOGT that catalyzes the GlcNAc modification of O-mannosylated α-dystroglycan-and other components of the biosynthetic pathway involved in GlcNAc modification in the ER lumen. MAJOR CONCLUSIONS GlcNAc modification in the ER lumen has been identified as a novel type of protein modification that regulates specific protein function. Moreover, abnormal GlcNAc modification in the ER lumen is responsible for Adams-Oliver syndrome and Walker-Warburg syndrome. GENERAL SIGNIFICANCE Elucidation of the biological function of GlcNAc modification in the ER lumen will provide new insights into the unique roles of O-glycans, whose importance has been demonstrated in multifunctional glycoproteins such as Notch receptors and α-dystroglyan.