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Kazuhiro Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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Crystal structures of Fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy.
Nature Communications, 2020Co-Authors: Naoyuki Kuwabara, Kazuhiro Kobayashi, Tatsushi Toda, Rieko Imae, Hiroki Tsumoto, Hiroshi Manya, Mamoru Mizuno, Motoi Kanagawa, Tomohiro Tanaka, Toshiya SendaAbstract:α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and Fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity. Fukutin-related protein (FKRP) catalyses the addition of ribitol-phosphate (RboP) to the O-mannosyl glycan of α-dystroglycan and mutations in FKRP cause dystroglycanopathy. Here the authors provide insights into its oligomerization and recognition of the substrates, CDP-Rbo and the RboP-(phospho-)core M3 glycan, by determining the crystal structures of human FKRP.
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Crystal structures of Fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy.
Nature communications, 2020Co-Authors: Naoyuki Kuwabara, Kazuhiro Kobayashi, Tatsushi Toda, Rieko Imae, Hiroki Tsumoto, Hiroshi Manya, Mamoru Mizuno, Motoi Kanagawa, Tomohiro Tanaka, Toshiya SendaAbstract:α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and Fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.
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Elimination of Fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure.
Nature Communications, 2019Co-Authors: Yoshihiro Ujihara, Kazuhiro Kobayashi, Tatsushi Toda, Motoi Kanagawa, Satoshi Mohri, Satomi Takatsu, Keiji Naruse, Yuki KatanosakaAbstract:Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the Fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal Fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.
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Temporal requirement of dystroglycan glycosylation during brain development and rescue of severe cortical dysplasia via gene delivery in the fetal stage.
Human Molecular Genetics, 2018Co-Authors: Atsushi Sudo, Kazuhiro Kobayashi, Motoi Kanagawa, Chiyomi Ito, Mai Kondo, Mitsuharu Endo, Yasuhiro Minami, Atsu Aiba, Tatsushi TodaAbstract:Congenital muscular dystrophies (CMDs) are characterized by progressive weakness and degeneration of skeletal muscle. In several forms of CMD, abnormal glycosylation of α-dystroglycan (α-DG) results in conditions collectively known as dystroglycanopathies, which are associated with central nervous system involvement. We recently demonstrated that Fukutin, the gene responsible for Fukuyama congenital muscular dystrophy, encodes the ribitol-phosphate transferase essential for dystroglycan function. Brain pathology in patients with dystroglycanopathy typically includes cobblestone lissencephaly, mental retardation, and refractory epilepsy; however, some patients exhibit average intelligence, with few or almost no structural defects. Currently, there is no effective treatment for dystroglycanopathy, and the mechanisms underlying the generation of this broad clinical spectrum remain unknown. Here, we analysed four distinct mouse models of dystroglycanopathy: two brain-selective Fukutin conditional knockout strains (neuronal stem cell-selective Nestin-Fukutin-cKO and forebrain-selective Emx1-Fukutin-cKO), a FukutinHp strain with the founder retrotransposal insertion in the Fukutin gene, and a spontaneous Large-mutant Largemyd strain. These models exhibit variations in the severity of brain pathology, replicating the clinical heterogeneity of dystroglycanopathy. Immunofluorescence analysis of the developing cortex suggested that residual glycosylation of α-DG at embryonic day 13.5 (E13.5), when cortical dysplasia is not yet apparent, may contribute to subsequent phenotypic heterogeneity. Surprisingly, delivery of Fukutin or Large into the brains of mice at E12.5 prevented severe brain malformation in Emx1-Fukutin-cKO and Largemyd/myd mice, respectively. These findings indicate that spatiotemporal persistence of functionally glycosylated α-DG may be crucial for brain development and modulation of glycosylation during the fetal stage could be a potential therapeutic strategy for dystroglycanopathy.
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Cell endogenous activities of Fukutin and FKRP coexist with the ribitol xylosyltransferase, TMEM5
Biochemical and Biophysical Research Communications, 2018Co-Authors: Ryuta Nishihara, Kazuhiro Kobayashi, Rieko Imae, Hiroki Tsumoto, Hiroshi Manya, Mamoru Mizuno, Motoi Kanagawa, Tamao Endo, Tatsushi TodaAbstract:Dystroglycanopathies are a group of muscular dystrophies that are caused by abnormal glycosylation of dystroglycan; currently 18 causative genes are known. Functions of the dystroglycanopathy genes Fukutin, Fukutin-related protein (FKRP), and transmembrane protein 5 (TMEM5) were most recently identified; Fukutin and FKRP are ribitol-phosphate transferases and TMEM5 is a ribitol xylosyltransferase. In this study, we show that Fukutin, FKRP, and TMEM5 form a complex while maintaining each of their enzyme activities. Immunoprecipitation and immunofluorescence experiments demonstrated protein interactions between these 3 proteins. A protein complex consisting of endogenous Fukutin and FKRP, and exogenously expressed TMEM5 exerts activities of each enzyme. Our data showed for the first time that endogenous Fukutin and FKRP enzyme activities coexist with TMEM5 enzyme activity, and suggest the possibility that formation of this enzyme complex may contribute to specific and prompt biosynthesis of glycans that are required for dystroglycan function.
Tatsushi Toda - One of the best experts on this subject based on the ideXlab platform.
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Crystal structures of Fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy.
Nature communications, 2020Co-Authors: Naoyuki Kuwabara, Kazuhiro Kobayashi, Tatsushi Toda, Rieko Imae, Hiroki Tsumoto, Hiroshi Manya, Mamoru Mizuno, Motoi Kanagawa, Tomohiro Tanaka, Toshiya SendaAbstract:α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and Fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.
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Crystal structures of Fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy.
Nature Communications, 2020Co-Authors: Naoyuki Kuwabara, Kazuhiro Kobayashi, Tatsushi Toda, Rieko Imae, Hiroki Tsumoto, Hiroshi Manya, Mamoru Mizuno, Motoi Kanagawa, Tomohiro Tanaka, Toshiya SendaAbstract:α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and Fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity. Fukutin-related protein (FKRP) catalyses the addition of ribitol-phosphate (RboP) to the O-mannosyl glycan of α-dystroglycan and mutations in FKRP cause dystroglycanopathy. Here the authors provide insights into its oligomerization and recognition of the substrates, CDP-Rbo and the RboP-(phospho-)core M3 glycan, by determining the crystal structures of human FKRP.
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Elimination of Fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure.
Nature Communications, 2019Co-Authors: Yoshihiro Ujihara, Kazuhiro Kobayashi, Tatsushi Toda, Motoi Kanagawa, Satoshi Mohri, Satomi Takatsu, Keiji Naruse, Yuki KatanosakaAbstract:Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the Fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal Fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.
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Temporal requirement of dystroglycan glycosylation during brain development and rescue of severe cortical dysplasia via gene delivery in the fetal stage.
Human Molecular Genetics, 2018Co-Authors: Atsushi Sudo, Kazuhiro Kobayashi, Motoi Kanagawa, Chiyomi Ito, Mai Kondo, Mitsuharu Endo, Yasuhiro Minami, Atsu Aiba, Tatsushi TodaAbstract:Congenital muscular dystrophies (CMDs) are characterized by progressive weakness and degeneration of skeletal muscle. In several forms of CMD, abnormal glycosylation of α-dystroglycan (α-DG) results in conditions collectively known as dystroglycanopathies, which are associated with central nervous system involvement. We recently demonstrated that Fukutin, the gene responsible for Fukuyama congenital muscular dystrophy, encodes the ribitol-phosphate transferase essential for dystroglycan function. Brain pathology in patients with dystroglycanopathy typically includes cobblestone lissencephaly, mental retardation, and refractory epilepsy; however, some patients exhibit average intelligence, with few or almost no structural defects. Currently, there is no effective treatment for dystroglycanopathy, and the mechanisms underlying the generation of this broad clinical spectrum remain unknown. Here, we analysed four distinct mouse models of dystroglycanopathy: two brain-selective Fukutin conditional knockout strains (neuronal stem cell-selective Nestin-Fukutin-cKO and forebrain-selective Emx1-Fukutin-cKO), a FukutinHp strain with the founder retrotransposal insertion in the Fukutin gene, and a spontaneous Large-mutant Largemyd strain. These models exhibit variations in the severity of brain pathology, replicating the clinical heterogeneity of dystroglycanopathy. Immunofluorescence analysis of the developing cortex suggested that residual glycosylation of α-DG at embryonic day 13.5 (E13.5), when cortical dysplasia is not yet apparent, may contribute to subsequent phenotypic heterogeneity. Surprisingly, delivery of Fukutin or Large into the brains of mice at E12.5 prevented severe brain malformation in Emx1-Fukutin-cKO and Largemyd/myd mice, respectively. These findings indicate that spatiotemporal persistence of functionally glycosylated α-DG may be crucial for brain development and modulation of glycosylation during the fetal stage could be a potential therapeutic strategy for dystroglycanopathy.
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Cell endogenous activities of Fukutin and FKRP coexist with the ribitol xylosyltransferase, TMEM5
Biochemical and Biophysical Research Communications, 2018Co-Authors: Ryuta Nishihara, Kazuhiro Kobayashi, Rieko Imae, Hiroki Tsumoto, Hiroshi Manya, Mamoru Mizuno, Motoi Kanagawa, Tamao Endo, Tatsushi TodaAbstract:Dystroglycanopathies are a group of muscular dystrophies that are caused by abnormal glycosylation of dystroglycan; currently 18 causative genes are known. Functions of the dystroglycanopathy genes Fukutin, Fukutin-related protein (FKRP), and transmembrane protein 5 (TMEM5) were most recently identified; Fukutin and FKRP are ribitol-phosphate transferases and TMEM5 is a ribitol xylosyltransferase. In this study, we show that Fukutin, FKRP, and TMEM5 form a complex while maintaining each of their enzyme activities. Immunoprecipitation and immunofluorescence experiments demonstrated protein interactions between these 3 proteins. A protein complex consisting of endogenous Fukutin and FKRP, and exogenously expressed TMEM5 exerts activities of each enzyme. Our data showed for the first time that endogenous Fukutin and FKRP enzyme activities coexist with TMEM5 enzyme activity, and suggest the possibility that formation of this enzyme complex may contribute to specific and prompt biosynthesis of glycans that are required for dystroglycan function.
Motoi Kanagawa - One of the best experts on this subject based on the ideXlab platform.
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Crystal structures of Fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy.
Nature communications, 2020Co-Authors: Naoyuki Kuwabara, Kazuhiro Kobayashi, Tatsushi Toda, Rieko Imae, Hiroki Tsumoto, Hiroshi Manya, Mamoru Mizuno, Motoi Kanagawa, Tomohiro Tanaka, Toshiya SendaAbstract:α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and Fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.
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Crystal structures of Fukutin-related protein (FKRP), a ribitol-phosphate transferase related to muscular dystrophy.
Nature Communications, 2020Co-Authors: Naoyuki Kuwabara, Kazuhiro Kobayashi, Tatsushi Toda, Rieko Imae, Hiroki Tsumoto, Hiroshi Manya, Mamoru Mizuno, Motoi Kanagawa, Tomohiro Tanaka, Toshiya SendaAbstract:α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and Fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity. Fukutin-related protein (FKRP) catalyses the addition of ribitol-phosphate (RboP) to the O-mannosyl glycan of α-dystroglycan and mutations in FKRP cause dystroglycanopathy. Here the authors provide insights into its oligomerization and recognition of the substrates, CDP-Rbo and the RboP-(phospho-)core M3 glycan, by determining the crystal structures of human FKRP.
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Elimination of Fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure.
Nature Communications, 2019Co-Authors: Yoshihiro Ujihara, Kazuhiro Kobayashi, Tatsushi Toda, Motoi Kanagawa, Satoshi Mohri, Satomi Takatsu, Keiji Naruse, Yuki KatanosakaAbstract:Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the Fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal Fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.
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Temporal requirement of dystroglycan glycosylation during brain development and rescue of severe cortical dysplasia via gene delivery in the fetal stage.
Human Molecular Genetics, 2018Co-Authors: Atsushi Sudo, Kazuhiro Kobayashi, Motoi Kanagawa, Chiyomi Ito, Mai Kondo, Mitsuharu Endo, Yasuhiro Minami, Atsu Aiba, Tatsushi TodaAbstract:Congenital muscular dystrophies (CMDs) are characterized by progressive weakness and degeneration of skeletal muscle. In several forms of CMD, abnormal glycosylation of α-dystroglycan (α-DG) results in conditions collectively known as dystroglycanopathies, which are associated with central nervous system involvement. We recently demonstrated that Fukutin, the gene responsible for Fukuyama congenital muscular dystrophy, encodes the ribitol-phosphate transferase essential for dystroglycan function. Brain pathology in patients with dystroglycanopathy typically includes cobblestone lissencephaly, mental retardation, and refractory epilepsy; however, some patients exhibit average intelligence, with few or almost no structural defects. Currently, there is no effective treatment for dystroglycanopathy, and the mechanisms underlying the generation of this broad clinical spectrum remain unknown. Here, we analysed four distinct mouse models of dystroglycanopathy: two brain-selective Fukutin conditional knockout strains (neuronal stem cell-selective Nestin-Fukutin-cKO and forebrain-selective Emx1-Fukutin-cKO), a FukutinHp strain with the founder retrotransposal insertion in the Fukutin gene, and a spontaneous Large-mutant Largemyd strain. These models exhibit variations in the severity of brain pathology, replicating the clinical heterogeneity of dystroglycanopathy. Immunofluorescence analysis of the developing cortex suggested that residual glycosylation of α-DG at embryonic day 13.5 (E13.5), when cortical dysplasia is not yet apparent, may contribute to subsequent phenotypic heterogeneity. Surprisingly, delivery of Fukutin or Large into the brains of mice at E12.5 prevented severe brain malformation in Emx1-Fukutin-cKO and Largemyd/myd mice, respectively. These findings indicate that spatiotemporal persistence of functionally glycosylated α-DG may be crucial for brain development and modulation of glycosylation during the fetal stage could be a potential therapeutic strategy for dystroglycanopathy.
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Cell endogenous activities of Fukutin and FKRP coexist with the ribitol xylosyltransferase, TMEM5
Biochemical and Biophysical Research Communications, 2018Co-Authors: Ryuta Nishihara, Kazuhiro Kobayashi, Rieko Imae, Hiroki Tsumoto, Hiroshi Manya, Mamoru Mizuno, Motoi Kanagawa, Tamao Endo, Tatsushi TodaAbstract:Dystroglycanopathies are a group of muscular dystrophies that are caused by abnormal glycosylation of dystroglycan; currently 18 causative genes are known. Functions of the dystroglycanopathy genes Fukutin, Fukutin-related protein (FKRP), and transmembrane protein 5 (TMEM5) were most recently identified; Fukutin and FKRP are ribitol-phosphate transferases and TMEM5 is a ribitol xylosyltransferase. In this study, we show that Fukutin, FKRP, and TMEM5 form a complex while maintaining each of their enzyme activities. Immunoprecipitation and immunofluorescence experiments demonstrated protein interactions between these 3 proteins. A protein complex consisting of endogenous Fukutin and FKRP, and exogenously expressed TMEM5 exerts activities of each enzyme. Our data showed for the first time that endogenous Fukutin and FKRP enzyme activities coexist with TMEM5 enzyme activity, and suggest the possibility that formation of this enzyme complex may contribute to specific and prompt biosynthesis of glycans that are required for dystroglycan function.
Makio Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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Roles of Fukutin, the Gene Responsible for Fukuyama-Type Congenital Muscular Dystrophy, in Neurons: Possible Involvement in Synaptic Function and Neuronal Migration
2013Co-Authors: Atsuko Hiroi, Noriyuki Shibata, Tomoko Yamamoto, Makiko Osawa, Makio KobayashiAbstract:© 2011 Fukutin The Japan is a Society gene responsible of Histochemistry for Fukuyama-type and Cy- congenital muscular dystrophy (FCMD), accompanying ocular and brain malformations represented by cobblestone lissencephaly. Fukutin is related to basement membrane formation via the glycosylation of α-dystoglycan (α-DG), and astrocytes play a crucial role in the pathogenesis of the brain lesion. On the other hand, its precise function in neurons is unknown. In this experiment, the roles of Fukutin in mature and immature neurons were examined using brains from control subjects and FCMD patients and cultured neuronal cell lines. In quantitative PCR, the expression level of Fukutin looked different depending on the region of the brain examined. A similar tendency in DG expression appears to indicate a relation between Fukutin and α-DG in mature neurons. An increase of DG mRNA and core α-DG in the FCMD cerebrum also supports the relation. In immunohistochemistry, dot-like positive reactions for VIA4-1, one of the antibodies detecting the glycosylated α-DG, in Purkinje cells suggest that Fukutin is related to at least a post-synaptic function via the glycosylation of α-DG. As for immature neurons, VIA4-1 was predominantly positive in cells before and during migration with expression of Fukutin, which suggest a participation of Fukutin in neuronal migration via the glycosylatio
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post transcriptional regulation of Fukutin in an astrocytoma cell line
International Journal of Experimental Pathology, 2012Co-Authors: Tomoko Yamamoto, Atsuko Hiroi, Noriyuki Shibata, Yoichiro Kato, Makiko Osawa, Makio KobayashiAbstract:Fukutin is the gene responsible for Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive disease associated with central nervous system (CNS) and eye anomalies. Fukutin is involved in basement membrane formation via the glycosylation of α-dystroglycan (α-DG), and hypoglycosylation of α-DG provokes the muscular, CNS and eye lesions of FCMD. Astrocytes play an important role in the pathogenesis of the CNS lesions, but the post-transcriptional regulation of Fukutin mRNA has not been elucidated. In this study, we investigated the characteristics of Fukutin mRNA using an astrocytoma cell line that expresses Fukutin and glycosylated α-DG. The glycosylation of α-DG was considered to be increased by over-expression of Fukutin and decreased by knockdown of Fukutin. Knockdown of Musashi-1, one of the RNA-binding proteins involved in the regulation of neuronal differentiation, induced a decrease in Fukutin mRNA. Immunoprecipitation and ELISA-based RNA-binding assay demonstrated possible binding between Fukutin mRNA and Musashi-1 protein. A relationship between Fukutin mRNA and vimentin protein was also proposed. In situ hybridization for Fukutin mRNA showed a positive cytoplasmic reaction including cytoplasmic processes. From these results, Fukutin mRNA is suggested to be a localized mRNA up-regulated by Musashi-1 and to be a component of a mRNA-protein complex which includes Musashi-1 and (presumably) vimentin proteins.
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Roles of Fukutin, the gene responsible for fukuyama-type congenital muscular dystrophy, in neurons: possible involvement in synaptic function and neuronal migration.
ACTA HISTOCHEMICA ET CYTOCHEMICA, 2011Co-Authors: Atsuko Hiroi, Noriyuki Shibata, Tomoko Yamamoto, Makiko Osawa, Makio KobayashiAbstract:Fukutin is a gene responsible for Fukuyama-type congenital muscular dystrophy (FCMD), accompanying ocular and brain malformations represented by cobblestone lissencephaly. Fukutin is related to basement membrane formation via the glycosylation of α-dystoglycan (α-DG), and astrocytes play a crucial role in the pathogenesis of the brain lesion. On the other hand, its precise function in neurons is unknown. In this experiment, the roles of Fukutin in mature and immature neurons were examined using brains from control subjects and FCMD patients and cultured neuronal cell lines. In quantitative PCR, the expression level of Fukutin looked different depending on the region of the brain examined. A similar tendency in DG expression appears to indicate a relation between Fukutin and α-DG in mature neurons. An increase of DG mRNA and core α-DG in the FCMD cerebrum also supports the relation. In immunohistochemistry, dot-like positive reactions for VIA4-1, one of the antibodies detecting the glycosylated α-DG, in Purkinje cells suggest that Fukutin is related to at least a post-synaptic function via the glycosylation of α-DG. As for immature neurons, VIA4-1 was predominantly positive in cells before and during migration with expression of Fukutin, which suggest a participation of Fukutin in neuronal migration via the glycosylation of α-DG. Moreover, Fukutin may prevent neuronal differentiation, because its expression was significantly lower in the adult cerebrum and in differentiated cultured cells. A knockdown of Fukutin was considered to induce differentiation in cultured cells. Fukutin seems to be necessary to keep migrating neurons immature during migration, and also to support migration via α-DG.
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Functions of Fukutin, a Gene Responsible for Fukuyama Type Congenital Muscular Dystrophy, in Neuromuscular System and Other Somatic Organs
Central Nervous System Agents in Medicinal Chemistry, 2010Co-Authors: Tomoko Yamamoto, Noriyuki Shibata, Makiko Osawa, Yoshiaki Saito, Makio KobayashiAbstract:Fukuyama type congenital muscular dystrophy (FCMD) is an autosomal recessive disease, exhibiting muscular dystrophy, and central nervous system (CNS) and ocular malformations. It is included in alpha-dystroglycanopathy, a group of muscular dystrophy showing reduced glycosylation of alpha-dystroglycan. alpha-Dystroglycan is one of the components of dystrophin-glycoprotein complex linking extracellular and intracellular proteins. The sugar chains of alpha-dystroglycan are receptors for extracellular matrix proteins such as laminin. Fukutin, a gene responsible for FCMD, is presumably related to the glycosylation of alpha-dystroglycan like other causative genes of alpha-dystroglycanopathy. The CNS lesion of FCMD is characterized by cobblestone lissencephaly, associated with decreased glycosylation of alpha-dystroglycan in the glia limitans where the basement membrane is formed. Astrocytes whose endfeet form the glia limitans seem to be greatly involved in the genesis of the CNS lesion. Fukutin is probably necessary for astrocytic function. Other components of the CNS may also need Fukutin, such as migration and synaptic function in neurons. However, roles of Fukutin in oligodendroglia, microglia, leptomeninges and capillaries are unknown at present. Fukutin is expressed in various somatic organs as well, and appears to work differently between epithelial cells and astrocytes. In the molecular level, since the dystrophin-glycoprotein complex is linked to cell signaling pathways involving c-src and c-jun, Fukutin may be able to affect cell proliferation/survival. Fukutin was localized in the nucleus on cancer cell lines. With the consideration that mutations of Fukutin give rise to wide spectrum of the clinical phenotype, more unknown functions of Fukutin besides the glycosylation of alpha-dystroglycan can be suggested. Trials for novel treatments including gene therapy are in progress in muscular dystrophies. Toward effective therapies with minimal side effects, precise evaluation of the pathomechanism of FCMD and the function of Fukutin would be required.
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A role of Fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy, in cancer cells: a possible role to suppress cell proliferation.
International Journal of Experimental Pathology, 2008Co-Authors: Tomoko Yamamoto, Noriyuki Shibata, Yoichiro Kato, Makiko Osawa, Tatsuo Sawada, Makio KobayashiAbstract:Fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy (FCMD), is presumably related to the glycosylation of α-dystroglycan (α-DG), involved in basement membrane formation. Hypoglycosylation of α-DG plays a key role for the pathogenesis of FCMD. On the other hand, Fukutin and α-DG are also expressed in various non-neuromuscular tissues. Recently, a role of α-DG as a cancer suppressor has been proposed, because of a decrease of glycosylated α-DG in cancers. In this study, function of Fukutin was investigated in two cancer cell lines, focusing on whether Fukutin is involved in the glycosylation of α-DG in cancer cells and has any possible roles related to a cancer suppressor. Localization of Fukutin and a result of laminin-binding assay after RNA interference suggest that Fukutin may be involved in the glycosylation of α-DG in a small portion in these cancer cell lines. In Western blotting and immuno-electron microscopy, localization of Fukutin in the nucleus was suggested in addition to the Golgi apparatus and/or endoplasmic reticulum. Immunohistochemically, there were more Ki-67-positive cells and more nuclear staining of phosphorylated c-jun after knockdown of Fukutin in two cell lines. Fukutin appears to suppress cell proliferation through a system involving c-jun, although it is unclear this process is related to α-DG or not at present. The result may propose a possibility of another function of Fukutin in addition to the glycosylation of α-DG in cancer cells.
Tomoko Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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Roles of Fukutin, the Gene Responsible for Fukuyama-Type Congenital Muscular Dystrophy, in Neurons: Possible Involvement in Synaptic Function and Neuronal Migration
2013Co-Authors: Atsuko Hiroi, Noriyuki Shibata, Tomoko Yamamoto, Makiko Osawa, Makio KobayashiAbstract:© 2011 Fukutin The Japan is a Society gene responsible of Histochemistry for Fukuyama-type and Cy- congenital muscular dystrophy (FCMD), accompanying ocular and brain malformations represented by cobblestone lissencephaly. Fukutin is related to basement membrane formation via the glycosylation of α-dystoglycan (α-DG), and astrocytes play a crucial role in the pathogenesis of the brain lesion. On the other hand, its precise function in neurons is unknown. In this experiment, the roles of Fukutin in mature and immature neurons were examined using brains from control subjects and FCMD patients and cultured neuronal cell lines. In quantitative PCR, the expression level of Fukutin looked different depending on the region of the brain examined. A similar tendency in DG expression appears to indicate a relation between Fukutin and α-DG in mature neurons. An increase of DG mRNA and core α-DG in the FCMD cerebrum also supports the relation. In immunohistochemistry, dot-like positive reactions for VIA4-1, one of the antibodies detecting the glycosylated α-DG, in Purkinje cells suggest that Fukutin is related to at least a post-synaptic function via the glycosylation of α-DG. As for immature neurons, VIA4-1 was predominantly positive in cells before and during migration with expression of Fukutin, which suggest a participation of Fukutin in neuronal migration via the glycosylatio
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post transcriptional regulation of Fukutin in an astrocytoma cell line
International Journal of Experimental Pathology, 2012Co-Authors: Tomoko Yamamoto, Atsuko Hiroi, Noriyuki Shibata, Yoichiro Kato, Makiko Osawa, Makio KobayashiAbstract:Fukutin is the gene responsible for Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive disease associated with central nervous system (CNS) and eye anomalies. Fukutin is involved in basement membrane formation via the glycosylation of α-dystroglycan (α-DG), and hypoglycosylation of α-DG provokes the muscular, CNS and eye lesions of FCMD. Astrocytes play an important role in the pathogenesis of the CNS lesions, but the post-transcriptional regulation of Fukutin mRNA has not been elucidated. In this study, we investigated the characteristics of Fukutin mRNA using an astrocytoma cell line that expresses Fukutin and glycosylated α-DG. The glycosylation of α-DG was considered to be increased by over-expression of Fukutin and decreased by knockdown of Fukutin. Knockdown of Musashi-1, one of the RNA-binding proteins involved in the regulation of neuronal differentiation, induced a decrease in Fukutin mRNA. Immunoprecipitation and ELISA-based RNA-binding assay demonstrated possible binding between Fukutin mRNA and Musashi-1 protein. A relationship between Fukutin mRNA and vimentin protein was also proposed. In situ hybridization for Fukutin mRNA showed a positive cytoplasmic reaction including cytoplasmic processes. From these results, Fukutin mRNA is suggested to be a localized mRNA up-regulated by Musashi-1 and to be a component of a mRNA-protein complex which includes Musashi-1 and (presumably) vimentin proteins.
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Roles of Fukutin, the gene responsible for fukuyama-type congenital muscular dystrophy, in neurons: possible involvement in synaptic function and neuronal migration.
ACTA HISTOCHEMICA ET CYTOCHEMICA, 2011Co-Authors: Atsuko Hiroi, Noriyuki Shibata, Tomoko Yamamoto, Makiko Osawa, Makio KobayashiAbstract:Fukutin is a gene responsible for Fukuyama-type congenital muscular dystrophy (FCMD), accompanying ocular and brain malformations represented by cobblestone lissencephaly. Fukutin is related to basement membrane formation via the glycosylation of α-dystoglycan (α-DG), and astrocytes play a crucial role in the pathogenesis of the brain lesion. On the other hand, its precise function in neurons is unknown. In this experiment, the roles of Fukutin in mature and immature neurons were examined using brains from control subjects and FCMD patients and cultured neuronal cell lines. In quantitative PCR, the expression level of Fukutin looked different depending on the region of the brain examined. A similar tendency in DG expression appears to indicate a relation between Fukutin and α-DG in mature neurons. An increase of DG mRNA and core α-DG in the FCMD cerebrum also supports the relation. In immunohistochemistry, dot-like positive reactions for VIA4-1, one of the antibodies detecting the glycosylated α-DG, in Purkinje cells suggest that Fukutin is related to at least a post-synaptic function via the glycosylation of α-DG. As for immature neurons, VIA4-1 was predominantly positive in cells before and during migration with expression of Fukutin, which suggest a participation of Fukutin in neuronal migration via the glycosylation of α-DG. Moreover, Fukutin may prevent neuronal differentiation, because its expression was significantly lower in the adult cerebrum and in differentiated cultured cells. A knockdown of Fukutin was considered to induce differentiation in cultured cells. Fukutin seems to be necessary to keep migrating neurons immature during migration, and also to support migration via α-DG.
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Functions of Fukutin, a Gene Responsible for Fukuyama Type Congenital Muscular Dystrophy, in Neuromuscular System and Other Somatic Organs
Central Nervous System Agents in Medicinal Chemistry, 2010Co-Authors: Tomoko Yamamoto, Noriyuki Shibata, Makiko Osawa, Yoshiaki Saito, Makio KobayashiAbstract:Fukuyama type congenital muscular dystrophy (FCMD) is an autosomal recessive disease, exhibiting muscular dystrophy, and central nervous system (CNS) and ocular malformations. It is included in alpha-dystroglycanopathy, a group of muscular dystrophy showing reduced glycosylation of alpha-dystroglycan. alpha-Dystroglycan is one of the components of dystrophin-glycoprotein complex linking extracellular and intracellular proteins. The sugar chains of alpha-dystroglycan are receptors for extracellular matrix proteins such as laminin. Fukutin, a gene responsible for FCMD, is presumably related to the glycosylation of alpha-dystroglycan like other causative genes of alpha-dystroglycanopathy. The CNS lesion of FCMD is characterized by cobblestone lissencephaly, associated with decreased glycosylation of alpha-dystroglycan in the glia limitans where the basement membrane is formed. Astrocytes whose endfeet form the glia limitans seem to be greatly involved in the genesis of the CNS lesion. Fukutin is probably necessary for astrocytic function. Other components of the CNS may also need Fukutin, such as migration and synaptic function in neurons. However, roles of Fukutin in oligodendroglia, microglia, leptomeninges and capillaries are unknown at present. Fukutin is expressed in various somatic organs as well, and appears to work differently between epithelial cells and astrocytes. In the molecular level, since the dystrophin-glycoprotein complex is linked to cell signaling pathways involving c-src and c-jun, Fukutin may be able to affect cell proliferation/survival. Fukutin was localized in the nucleus on cancer cell lines. With the consideration that mutations of Fukutin give rise to wide spectrum of the clinical phenotype, more unknown functions of Fukutin besides the glycosylation of alpha-dystroglycan can be suggested. Trials for novel treatments including gene therapy are in progress in muscular dystrophies. Toward effective therapies with minimal side effects, precise evaluation of the pathomechanism of FCMD and the function of Fukutin would be required.
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A role of Fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy, in cancer cells: a possible role to suppress cell proliferation.
International Journal of Experimental Pathology, 2008Co-Authors: Tomoko Yamamoto, Noriyuki Shibata, Yoichiro Kato, Makiko Osawa, Tatsuo Sawada, Makio KobayashiAbstract:Fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy (FCMD), is presumably related to the glycosylation of α-dystroglycan (α-DG), involved in basement membrane formation. Hypoglycosylation of α-DG plays a key role for the pathogenesis of FCMD. On the other hand, Fukutin and α-DG are also expressed in various non-neuromuscular tissues. Recently, a role of α-DG as a cancer suppressor has been proposed, because of a decrease of glycosylated α-DG in cancers. In this study, function of Fukutin was investigated in two cancer cell lines, focusing on whether Fukutin is involved in the glycosylation of α-DG in cancer cells and has any possible roles related to a cancer suppressor. Localization of Fukutin and a result of laminin-binding assay after RNA interference suggest that Fukutin may be involved in the glycosylation of α-DG in a small portion in these cancer cell lines. In Western blotting and immuno-electron microscopy, localization of Fukutin in the nucleus was suggested in addition to the Golgi apparatus and/or endoplasmic reticulum. Immunohistochemically, there were more Ki-67-positive cells and more nuclear staining of phosphorylated c-jun after knockdown of Fukutin in two cell lines. Fukutin appears to suppress cell proliferation through a system involving c-jun, although it is unclear this process is related to α-DG or not at present. The result may propose a possibility of another function of Fukutin in addition to the glycosylation of α-DG in cancer cells.
