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OBJECTIVE: This study aimed to elucidate the molecular features of inclusion body myositis (IBM). METHODS: We performed RNA sequencing analysis of muscle biopsy samples from 67 participants, consisting of 58 myositis patients with the pathological finding of CD8-positive T cells invading non-necrotic muscle fibers expressing major histocompatibility complex class I (43 IBM, 6 polymyositis, and 9 unclassifiable myositis), and 9 controls. RESULTS: Cluster analysis, principal component analysis, and pathway analysis showed that differentially expressed genes and pathways identified in IBM and polymyositis were mostly comparable. However, pathways related to cell adhesion molecules were upregulated in IBM as compared with polymyositis and controls (p < 0.01). Notably, CDH1, which encodes the epidermal cell junction protein cadherin 1, was overexpressed in the muscles of IBM, which was validated by another RNA sequencing dataset from previous publications. Western blotting confirmed the presence of mature cadherin 1 protein in the muscles of IBM. Immunohistochemical staining confirmed the positivity for anti-cadherin 1 antibody in the muscles of IBM, whereas there was no muscle fiber positive for anti-cadherin 1 antibody in immune-mediated necrotizing myopathy, antisynthetase syndrome, and controls. The fibers stained with anti-cadherin 1 antibody did not have rimmed vacuoles or abnormal protein accumulation. Experimental skeletal muscle regeneration and differentiation systems showed that CDH1 is expressed during skeletal muscle regeneration and differentiation. INTERPRETATION: CDH1 was detected as a differentially expressed gene, and immunohistochemistry showed that cadherin 1 exists in the muscles of IBM, whereas it was rarely seen in those of other idiopathic inflammatory myopathies. Cadherin 1 upregulation in muscle could provide a valuable clue to the pathological mechanisms of IBM. ANN NEUROL 2022;91:317-328.

DOI： 10.1002/ana.26304

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Dystroglycanopathy is a collective term referring to muscular dystrophies with abnormal glycosylation of dystroglycan. At least 18 causative genes of dystroglycanopathy have been identified, and its clinical symptoms are diverse, ranging from severe congenital to adult-onset limb-girdle types. Moreover, some cases are associated with symptoms involving the central nervous system. In the 2010s, the structure of sugar chains involved in the onset of dystroglycanopathy and the functions of its causative gene products began to be identified as if they were filling the missing pieces of a jigsaw puzzle. In parallel with these discoveries, various dystroglycanopathy model mice had been created, which led to the elucidation of its pathological mechanisms. Then, treatment strategies based on the molecular basis of glycosylation began to be proposed after the latter half of the 2010s. This review briefly explains the sugar chain structure of dystroglycan and the functions of the causative gene products of dystroglycanopathy, followed by introducing the pathological mechanisms involved as revealed from analyses of dystroglycanopathy model mice. Finally, potential therapeutic approaches based on the pathological mechanisms involved are discussed.

DOI： 10.3390/ijms222313162

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OBJECTIVE: Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+ permeable channel and plays a role in mediating intracellular Ca2+ current via mechanical stimuli. This study aimed to examine the expression and role of TRPV2 in adult articular cartilage and development of osteoarthritis (OA). METHODS: We examined TRPV2 expression in mouse and human articular cartilage. We analyzed development of OA in Col2a1-CreERT2 ;Trpv2fl/fl and Trpv2fl/fl littermates in the resection of the medial meniscus and medial collateral ligament model (n = 5 each), the destabilization of the medial meniscus model (n = 5 each), and the aging model (n = 8-9). We examined marker protein expression in these joints, Ca2+ influx by mechanical stimuli, and downstream pathways in vitro. RESULTS: Trpv2 was expressed in mouse and human articular cartilage and ectopic ossification lesions. In all models, Col2a1-CreERT2 ;Trpv2fl/fl mice showed enhanced degradation of articular cartilage accompanied by decreased expression of lubricin/Prg4, and marked formation of periarticular ectopic ossification. Mechanical stress-induced Ca2+ influx was decreased by Trpv2 knockout. Prg4 induction by fluid flow shear stress was diminished in Trpv2 knockout chondrocytes, and this was mediated by the Ca2+ /calmodulin-dependent protein kinase kinase-cyclic adenosine monophosphate response element binding protein axis. Hypertrophic differentiation was enhanced in Trpv2 knockout chondrocytes. Increased activity of calcineurin and nuclear translocation of nuclear factor of activated T cells 1 by fluid flow shear stress or TRP agonist treatment were cancelled by Trpv2 knockout. CONCLUSION: This study shows regulation of articular cartilage by TRPV2 through Prg4 induction and suppression of ectopic ossification.

DOI： 10.1002/art.41684

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BACKGROUND: Amyloid-β (Aβ) induces cerebrovascular damage and is reported to stimulate endothelial cell senescence. We previously demonstrated that angiotensin II (Ang II) promoted vascular senescence. We examined the possible cross-talk between Ang II and Aβ in regulating brain vascular smooth muscle cell (BVSMC) senescence. METHODS: BVSMC were prepared from adult male mice and stimulated with Ang II (0, 0.1, 1, 10, 100 nmol/L) and/or Aβ 1-40 (0, 0.1, 0.3, 0.5, 1, 3, 5 µmol/L) for the indicated times. Cellular senescence was evaluated by senescence-associated β-galactosidase staining. RESULTS: Treatment with Ang II (100 nmol/L) or Aβ (1 µmol/L) at a higher dose increased senescent cells compared with control at 6 days. Treatment with Ang II (10 nmol/L) or Aβ (0.5 µmol/L) at a lower dose had no effect on senescence whereas a combined treatment with lower doses of Ang II and Aβ significantly enhanced senescent cells. This senescence enhanced by lower dose combination was markedly blocked by valsartan (Ang II type 1 receptor inhibitor) or TAK-242 (Aβ receptor TLR4 inhibitor) treatment. Moreover, lower dose combination caused increases in superoxide anion levels and p-ERK expression for 2 days, NF-κB activity, p-IκB, p-IKKα/β, p16 and p53 expression for 4 days, and an obvious decrease in pRb expression. These changes by lower dose combination, except in p-IκB expression and NF-κB activity, were significantly inhibited by pretreatment with U0126 (ERK inhibitor). CONCLUSIONS: Ang II and Aβ synergistically promoted BVSMC senescence at least due to enhancement of the p-ERK-p16-pRb signaling pathway, oxidative stress and NF-κB/IκB activity.

DOI： 10.1093/ajh/hpaa218

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Sialic acid attached to nonreducing ends of glycan chains via different linkages is associated with specific interactions and physiological events. Linkage-specific derivatization of sialic acid is of great interest for distinguishing sialic acids by mass spectrometry, specifically for events governed by sialyl linkage types. In the present study, we demonstrate that α-2,3/8-sialyl linkage-specific amidation of esterified sialyloligosaccharides can be achieved via an intramolecular lactone. The method of lactone-driven ester-to-amide derivatization for sialic acid linkage-specific alkylamidation, termed LEAD-SALSA, employs in-solution ester-to-amide conversion to directly generate stable and sialyl linkage-specific glycan amides from their ester form by mixing with a preferred amine, resulting in the easy assignments of sialyl linkages by comparing the signals of esterified and amidated glycan. Using this approach, we demonstrate the accumulation of altered N-glycans in cardiac muscle tissue during mouse aging. Furthermore, we find that the stability of lactone is important for ester-to-amide conversion based on experiments and density functional theory calculations of reaction energies for lactone formation. By using energy differences of lactone formation, the LEAD-SALSA method can be used not only for the sialyl linkage-specific derivatization but also for distinguishing the branching structure of galactose linked to sialic acid. This simplified and direct sialylglycan discrimination will facilitate important studies on sialylated glycoconjugates.

DOI： 10.1021/acs.analchem.0c02209

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

DOI： 10.1371/journal.pgen.1008826

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Alzheimer's disease (AD) is the most common type of dementia, and its pathogenesis is associated with accumulation of β-amyloid (Aβ) peptides. Aβ is produced from amyloid precursor protein (APP) that is sequentially cleaved by β- and γ-secretases. Therefore, APP processing has been a target in therapeutic strategies for managing AD; however, no effective treatment of AD patients is currently available. Here, to identify endogenous factors that modulate Aβ production, we performed a gene microarray-based transcriptome analysis of neuronal cells derived from human induced pluripotent stem cells, because Aβ production in these cells changes during neuronal differentiation. We found that expression of the glycophosphatidylinositol-specific phospholipase D1 (GPLD1) gene is associated with these changes in Aβ production. GPLD1 overexpression in HEK293 cells increased the secretion of galectin 3-binding protein (GAL3BP), which suppressed Aβ production in an AD model, neuroglioma H4 cells. Mechanistically, GAL3BP suppressed Aβ production by directly interacting with APP and thereby inhibiting APP processing by β-secretase. Furthermore, we show that cells take up extracellularly added GAL3BP via endocytosis and that GAL3BP is localized in close proximity to APP in endosomes where amyloidogenic APP processing takes place. Taken together, our results indicate that GAL3BP may be a suitable target of AD-modifying drugs in future therapeutic strategies for managing AD.

DOI： 10.1074/jbc.RA119.008703

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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.

DOI： 10.1038/s41467-019-14220-z

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Publishing type：Research paper (scientific journal)   Publisher：Cambridge University Press (CUP)  

<title>Abstract</title>
The anabolic effects of androgen on skeletal muscles are thought to be mediated by androgen receptor (AR). Although multiple studies concerning the effects of AR in males have been performed, the molecular mechanisms of AR in skeletal muscles remain unclear. Here we first confirmed that satellite cells from mouse hindlimb muscles express AR. We then generated satellite cell-specific AR knockout mice using <italic>Pax7^CreERT2</italic> and <italic>AR^L2/Y</italic> mice to test whether AR in satellite cells is necessary for muscle regeneration. Surprisingly, we found that muscle regeneration was compromised in both <italic>Pax7^{CreERT2(Fan)/+}</italic> control mice and <italic>Pax7^{CreERT2(Fan)/+};AR^L2/Y</italic> mice compared to <italic>AR^L2/Y</italic> mice. However, <italic>Pax7^{CreERT2(Gaka)/+};AR^L2/Y;R26^tdTomato/+</italic> mice showed no significant differences between control and mutant muscle regeneration. These findings indicate that AR in satellite cells is not essential for muscle regeneration. We propose that <italic>Pax7^{CreERT2(Fan)/+}</italic> control mice should be included in all experiments, because these mice negatively affect the muscle regeneration and show the mild regeneration phenotype.

DOI： 10.1017/exp.2020.14

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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.

DOI： 10.1038/s41467-019-13623-2

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Language：Japanese   Publisher：(一社)日本認知症学会  

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Human induced pluripotent stem cells (hiPSCs) grow indefinitely in culture and have the potential to regenerate various tissues. In the development of cell culture systems, a fragment of laminin-511 (LM511-E8) was found to improve the proliferation of stem cells. The adhesion of undifferentiated cells to LM511-E8 is mainly mediated through integrin α6β1. However, the involvement of non-integrin receptors remains unknown in stem cell culture using LM511-E8. Here, we show that dystroglycan (DG) is strongly expressed in hiPSCs. The fully glycosylated DG is functionally active for laminin binding, and although it has been suggested that LM511-E8 lacks DG binding sites, the fragment does weakly bind to DG. We further identified the DG binding sequence in LM511-E8, using synthetic peptides, of which, hE8A5-20 (human laminin α5 2688-2699: KTLPQLLAKLSI) derived from the laminin coiled-coil domain, exhibited DG binding affinity and cell adhesion activity. Deletion and mutation studies show that LLAKLSI is the active core sequence of hE8A5-20, and that, K2696 is a critical amino acid for DG binding. We further demonstrated that hiPSCs adhere to hE8A5-20-conjugated chitosan matrices. The amino acid sequence of DG binding peptides would be useful to design substrata for culture system of undifferentiated and differentiated stem cells.

DOI： 10.1038/s41598-019-49669-x

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：日本筋学会  

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Multiple system atrophy (MSA) is a fatal adult-onset neurodegenerative disease that is characterized by varying degrees of cerebellar dysfunction and Parkinsonism. The neuropathological hallmark of MSA is alpha-synuclein (AS)-positive glial cytoplasmic inclusions (GCIs). Although severe neuronal loss (NL) is also observed in MSA, neuronal inclusions (NIs) are rare compared to GCIs, such that the pathological mechanism of NL in MSA is unclear. GCIs and NIs are late-stage pathology features relative to AS oligomers and may not represent early pathological changes in MSA. To reveal the early pathology of MSA, it is necessary to examine the early aggregation of AS, i.e., AS oligomers. Here, we adopted a proximity ligation assay (PLA) to examine the distribution of AS oligomers in brain tissue samples from patients with MSA and other diseases. Surprisingly, MSA brains showed a widespread distribution and abundant accumulation of oligomeric AS in neurons as well as oligodendrocytes of the neocortex. In several regions, oligomeric AS signal intensity was higher in cases with MSA than in cases with Parkinson's disease. In contrast to previous studies, AS-PLA revealed abundant AS oligomer accumulation in Purkinje cells in MSA brains, identifying oligomeric AS accumulation as a possible cause of Purkinje cell loss. This wide distribution of AS oligomers in MSA brain neurons has not been described previously and indicates a pathological mechanism of NL in MSA.

DOI： 10.1007/s00401-019-01961-w

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Parkinson's disease (PD) is a neurodegenerative disorder characterized by dopaminergic neuron loss. At present, there are no drugs that stop the progression of PD. As with other multifactorial genetic disorders, genome-wide association studies (GWASs) found multiple risk loci for PD, although their clinical significance remains uncertain. Here, we report the identification of candidate drugs for PD by a method using GWAS data and in silico databases. We identified 57 Food and Drug Administration-approved drug families as candidate neuroprotective drugs for PD. Among them, dabrafenib, which is known as a B-Raf kinase inhibitor and is approved for the treatment of malignant melanoma, showed remarkable cytoprotective effects in neurotoxin-treated SH-SY5Y cells and mice. Dabrafenib was found to inhibit apoptosis, and to enhance the phosphorylation of extracellular signal-regulated kinase (ERK), and inhibit the phosphorylation of c-Jun NH2-terminal kinase. Dabrafenib targets B-Raf, and we confirmed a protein-protein interaction between B-Raf and Rit2, which is coded by RIT2, a PD risk gene in Asians and Caucasians. In RIT2-knockout cells, the phosphorylation of ERK was reduced, and dabrafenib treatment improved the ERK phosphorylation. These data indicated that dabrafenib exerts protective effects against neurotoxicity associated with PD. By using animal model, we confirmed the effectiveness of this in silico screening method. Furthermore, our results suggest that this in silico drug screening system is useful in not only neurodegenerative diseases but also other common diseases such as diabetes mellitus and hypertension.

DOI： 10.1093/hmg/ddy279

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Establishing synaptic contacts between neurons is paramount for nervous system function. This process involves transsynaptic interactions between a host of cell adhesion molecules that act in cooperation with the proteins of the extracellular matrix to specify unique physiological properties of individual synaptic connections. However, understanding of the molecular mechanisms that generate functional diversity in an input-specific fashion is limited. In this study, we identify that major components of the extracellular matrix proteins present in the synaptic cleft-members of the heparan sulfate proteoglycan (HSPG) family-associate with the GPR158/179 group of orphan receptors. Using the mammalian retina as a model system, we demonstrate that the HSPG member Pikachurin, released by photoreceptors, recruits a key post-synaptic signaling complex of downstream ON-bipolar neurons in coordination with the pre-synaptic dystroglycan glycoprotein complex. We further demonstrate that this transsynaptic assembly plays an essential role in synaptic transmission of photoreceptor signals.

DOI： 10.1016/j.celrep.2018.08.068

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α-Dystroglycan (α-DG) is a highly glycosylated cell-surface laminin receptor. Defects in the O-mannosyl glycan of an α-DG with laminin-binding activity can cause α-dystroglycanopathy, a group of congenital muscular dystrophies. In the biosynthetic pathway of functional O-mannosyl glycan, fukutin (FKTN) and fukutin-related protein (FKRP), whose mutated genes underlie α-dystroglycanopathy, sequentially transfer ribitol phosphate (RboP) from CDP-Rbo to form a tandem RboP unit (RboP-RboP) required for the synthesis of the laminin-binding epitope on O-mannosyl glycan. Both RboP- and glycerol phosphate (GroP)-substituted glycoforms have recently been detected in recombinant α-DG. However, it is unclear how GroP is transferred to the O-mannosyl glycan or whether GroP substitution affects the synthesis of the O-mannosyl glycan. Here, we report that, in addition to having RboP transfer activity, FKTN and FKRP can transfer GroP to O-mannosyl glycans by using CDP-glycerol (CDP-Gro) as a donor substrate. Kinetic experiments indicated that CDP-Gro is a less efficient donor substrate for FKTN than is CDP-Rbo. We also show that the GroP-substituted glycoform synthesized by FKTN does not serve as an acceptor substrate for FKRP and that therefore further elongation of the outer glycan chain cannot occur with this glycoform. Finally, CDP-Gro inhibited the RboP transfer activities of both FKTN and FKRP. These results suggest that CDP-Gro inhibits the synthesis of the functional O-mannosyl glycan of α-DG by preventing further elongation of the glycan chain. This is the first report of GroP transferases in mammals.

DOI： 10.1074/jbc.RA118.003197

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Myotube formation by fusion of myoblasts and subsequent elongation of the syncytia is essential for skeletal muscle formation. However, molecules that regulate myotube formation remain elusive. Here we identify PIEZO1, a mechanosensitive Ca2+ channel, as a key regulator of myotube formation. During myotube formation, phosphatidylserine, a phospholipid that resides in the inner leaflet of the plasma membrane, is transiently exposed to cell surface and promotes myoblast fusion. We show that cell surface phosphatidylserine inhibits PIEZO1 and that the inward translocation of phosphatidylserine, which is driven by the phospholipid flippase complex of ATP11A and CDC50A, is required for PIEZO1 activation. PIEZO1-mediated Ca2+ influx promotes RhoA/ROCK-mediated actomyosin assemblies at the lateral cortex of myotubes, thus preventing uncontrolled fusion of myotubes and leading to polarized elongation during myotube formation. These results suggest that cell surface flip-flop of phosphatidylserine acts as a molecular switch for PIEZO1 activation that governs proper morphogenesis during myotube formation.

DOI： 10.1038/s41467-018-04436-w

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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.

DOI： 10.1093/hmg/ddy032

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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.

DOI： 10.1016/j.bbrc.2018.02.162

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The Ror family receptor tyrosine kinases, Ror1 and Ror2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and Ror2 during skeletal muscle regeneration. Using an in vivo skeletal muscle injury model, we show that expression of Ror1 and Ror2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1β, after injury and that inhibition of TNF-α and IL-1β by neutralizing antibodies suppresses expression of Ror1 and Ror2 in injured muscles. Importantly, expression of Ror1, but not Ror2, was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1β induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of Ror1 decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.

DOI： 10.1074/jbc.M117.785709

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A defect in O-mannosyl glycan is the cause of α-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant α-dystroglycan (α-DG) glycosylation. Recently, the entire structure of O-mannosyl glycan, [3GlcAβ1-3Xylα1]n-3GlcAβ1-4Xyl-Rbo5P-1Rbo5P-3GalNAcβ1-3GlcNAcβ1-4 (phospho-6)Manα1-, which is required for the binding of α-DG to extracellular matrix ligands, has been proposed. However, the linkage of the first Xyl residue to ribitol 5-phosphate (Rbo5P) is not clear. TMEM5 is a gene product responsible for α-dystroglycanopathy and was reported as a potential enzyme involved in this linkage formation, although the experimental evidence is still incomplete. Here, we report that TMEM5 is a xylosyltransferase that forms the Xylβ1-4Rbo5P linkage on O-mannosyl glycan. The anomeric configuration and linkage position of the product (β1,4 linkage) was determined by NMR analysis. The introduction of two missense mutations in TMEM5 found in α-dystroglycanopathy patients impaired xylosyltransferase activity. Furthermore, the disruption of the TMEM5 gene by CRISPR/Cas9 abrogated the elongation of the (-3GlcAβ1-3Xylα1-) unit on O-mannosyl glycan. Based on these results, we concluded that TMEM5 acts as a UDP-d-xylose:ribitol-5-phosphate β1,4-xylosyltransferase in the biosynthetic pathway of O-mannosyl glycan.

DOI： 10.1074/jbc.M116.751917

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The dystrophin glycoprotein complex, which connects the cell membrane to the basement membrane, is essential for a variety of biological events, including maintenance of muscle integrity. An O-mannose-type GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man structure of α-dystroglycan (α-DG), a subunit of the complex that is anchored to the cell membrane, interacts directly with laminin in the basement membrane. Reduced glycosylation of α-DG is linked to some types of inherited muscular dystrophy; consistent with this relationship, many disease-related mutations have been detected in genes involved in O-mannosyl glycan synthesis. Defects in protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), a glycosyltransferase that participates in the formation of GlcNAc-β1,2-Man glycan, are causally related to muscle-eye-brain disease (MEB), a congenital muscular dystrophy, although the role of POMGnT1 in postphosphoryl modification of GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man glycan remains elusive. Our crystal structures of POMGnT1 agreed with our previous results showing that the catalytic domain recognizes substrate O-mannosylated proteins via hydrophobic interactions with little sequence specificity. Unexpectedly, we found that the stem domain recognizes the β-linked GlcNAc of O-mannosyl glycan, an enzymatic product of POMGnT1. This interaction may recruit POMGnT1 to a specific site of α-DG to promote GlcNAc-β1,2-Man clustering and also may recruit other enzymes that interact with POMGnT1, e.g., fukutin, which is required for further modification of the GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man glycan. On the basis of our findings, we propose a mechanism for the deficiency in postphosphoryl modification of the glycan observed in POMGnT1-KO mice and MEB patients.

DOI： 10.1073/pnas.1525545113

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Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD) is cytidine diphosphate ribitol (CDP-Rbo) synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases.

DOI： 10.1016/j.celrep.2016.02.017

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

α-Dystroglycanopathy encompasses a group of congenital and limb-girdle-type muscular dystrophies that are caused by abnormal glycosylation of α-dystroglycan. α-Dystroglycanopathy is often associated with brain abnormalities including type II lissencephaly and mental retardation. Currently, around 15 genes have been identified in which mutations cause abnormal glycosylation of α-dystroglycan resulting in disease. Dystroglycan is a highly glycosylated peripheral membrane protein that functions as a cell-surface receptor for proteins in the extracellular matrices and synapses. Unique O-mannosyl glycosylation is necessary for the ligand-binding activities of dystroglycan, and some of α-dystroglycanopathy gene products are involved in the process of α-dystroglycan glycosylation. Studies using animal and cell models for α-dystroglycanopathy have contributed to understanding the pathogenesis of this disease and to establishing therapeutic strategies. In this chapter, we review the structure, modification pathways, and physiological roles of dystroglycan glycosylation, as well as their involvement in human diseases, disease pathogenesis, and therapeutic strategies.

DOI： 10.1007/978-4-431-55678-7_2

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Language：Japanese   Publisher：(公社)日本生化学会  

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α-Dystroglycanopathy (α-DGP) is a group of muscular dystrophy characterized by abnormal glycosylation of α-dystroglycan (α-DG), including Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease, Walker-Warburg syndrome, and congenital muscular dystrophy type 1D (MDC1D), etc. LARGE, the causative gene for MDC1D, encodes a glycosyltransferase to form [-3Xyl-α1,3GlcAβ1-] polymer in the terminal end of the post-phosphoryl moiety, which is essential for α-DG function. It has been proposed that LARGE possesses the great potential to rescue glycosylation defects in α-DGPs regardless of causative genes. However, the in vivo therapeutic benefit of using LARGE activity is controversial. To explore the conditions needed for successful LARGE gene therapy, here we used Large-deficient and fukutin-deficient mouse models for MDC1D and FCMD, respectively. Myofibre-selective LARGE expression via systemic adeno-associated viral gene transfer ameliorated dystrophic pathology of Large-deficient mice even when intervention occurred after disease manifestation. However, the same strategy failed to ameliorate the dystrophic phenotype of fukutin-conditional knockout mice. Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation. Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.

DOI： 10.1038/srep08316

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Language：English   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Defects in dystroglycan glycosylation are associated with a group of muscular dystrophies, termed dystroglycanopathies, that include Fukuyama congenital muscular dystrophy (FCMD). It is widely believed that abnormal glycosylation of dystroglycan leads to disease-causing membrane fragility. We previously generated knock-in mice carrying a founder retrotransposal insertion in fukutin, the gene responsible for FCMD, but these mice did not develop muscular dystrophy, which hindered exploring therapeutic strategies. We hypothesized that dysferlin functions may contribute to muscle cell viability in the knock-in mice; however, pathological interactions between glycosylation abnormalities and dysferlin defects remain unexplored. To investigate contributions of dysferlin deficiency to the pathology of dystroglycanopathy, we have crossed dysferlin-deficient dysferlin(sjl/sjl) mice to the fukutin-knock-in fukutin(Hp/-) and Large-deficient Large(myd/myd) mice, which are phenotypically distinct models of dystroglycanopathy. The fukutin(Hp/-) mice do not show a dystrophic phenotype; however, (dysferlin(sjl/sjl):fukutin(Hp/-)) mice showed a deteriorated phenotype compared with (dysferlin(sjl/sjl):fukutin(Hp/+)) mice. These data indicate that the absence of functional dysferlin in the asymptomatic fukutin(Hp/-) mice triggers disease manifestation and aggravates the dystrophic phenotype. A series of pathological analyses using double mutant mice for Large and dysferlin indicate that the protective effects of dysferlin appear diminished when the dystrophic pathology is severe and also may depend on the amount of dysferlin proteins. Together, our results show that dysferlin exerts protective effects on the fukutin(Hp/-) FCMD mouse model, and the (dysferlin(sjl/sjl):fukutin(Hp/-)) mice will be useful as a novel model for a recently proposed antisense oligonucleotide therapy for FCMD.

DOI： 10.1371/journal.pone.0106721

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Several types of muscular dystrophy are caused by defective linkage between alpha-dystroglycan (alpha-DG) and laminin. Among these, dystroglycanopathy, including Fukuyama-type congenital muscular dystrophy (FCMD), results from abnormal glycosylation of alpha-DG. Recent studies have shown that like-acetylglucosaminyltransferase(LARGE) strongly enhances the laminin-binding activity of alpha-DG. Therefore, restoration of the alpha-DG-laminin linkage by LARGE is considered one of the most promising possible therapies for muscular dystrophy. In this study, we generated transgenic mice that overexpress LARGE (LARGE Tg) and crossed them with dy(2J) mice and fukutin conditional knockout mice, a model for laminin alpha 2-deficient congenital muscular dystrophy (MDC1A) and FCMD, respectively. Remarkably, in both the strains, the transgenic overexpression of LARGE resulted in an aggravation of muscular dystrophy. Using morphometric analyses, we found that the deterioration of muscle pathology was caused by suppression of muscle regeneration. Overexpression of LARGE in C2C12 cells further demonstrated defects in myotube formation. Interestingly, a decreased expression of insulin-like growth factor 1 (IGF-1) was identified in both LARGE Tg mice and LARGE-overexpressing C2C12 myotubes. Supplementing the C2C12 cells with IGF-1 restored the defective myotube formation. Taken together, our findings indicate that the overexpression of LARGE aggravates muscular dystrophy by suppressing the muscle regeneration and this adverse effect is mediated via reduced expression of IGF-1.

DOI： 10.1093/hmg/ddu168

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

ZG16p is a soluble mammalian lectin, the first to be described with a Jacalin-related beta-prism-fold. ZG16p has been reported to bind both to glycosaminoglycans and mannose. To determine the structural basis of the multiple sugar-binding properties, we conducted glycan microarray analyses of human ZG16p. We observed that ZG16p preferentially binds to alpha-mannose-terminating short glycans such as Ser/Thr-linked O-mannose, but not to high mannose-type N-glycans. Among sulfated glycosaminoglycan oligomers examined, chondroitin sulfate B and heparin oligosaccharides showed significant binding. Crystallographic studies of human ZG16p lectin in the presence of selected ligands revealed the mechanism of multiple sugar recognition. Man alpha 1-3Man and Glc beta 1-3Glc bound in different orientations: the nonreducing end of the former and the reducing end of the latter fitted in the canonical shallow mannose binding pocket. Solution NMR analysis using N-15-labeled ZG16p defined the heparin-binding region, which is on an adjacent flat surface of the protein. On-array competitive binding assays suggest that it is possible for ZG16p to bind simultaneously to both types of ligands. Recognition of a broad spectrum of ligands by ZG16p may account for the multiple functions of this lectin in the formation of zymogen granules via glycosaminoglycan binding, and in the recognition of pathogens in the digestive system through alpha-mannose-related recognition.

DOI： 10.1074/jbc.M113.539114

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The heart has a dynamic compensatory mechanism for haemodynamic stress. However, the molecular details of how mechanical forces are transduced in the heart are unclear. Here we show that the transient receptor potential, vanilloid family type 2 (TRPV2) cation channel is critical for the maintenance of cardiac structure and function. Within 4 days of eliminating TRPV2 from hearts of the adult mice, cardiac function declines severely, with disorganization of the intercalated discs that support mechanical coupling with neighbouring myocytes and myocardial conduction defects. After 9 days, cell shortening and Ca2+ handling by single myocytes are impaired in TRPV2-deficient hearts. TRPV2-deficient neonatal cardiomyocytes form no intercalated discs and show no extracellular Ca2+-dependent intracellular Ca2+ increase and insulin-like growth factor (IGF-1) secretion in response to stretch stimulation. We further demonstrate that IGF-1 receptor/PI3K/Akt pathway signalling is significantly downregulated in TRPV2-deficient hearts, and that IGF-1 administration partially prevents chamber dilation and impairment in cardiac pump function in these hearts. Our results improve our understanding of the molecular processes underlying the maintenance of cardiac structure and function.

DOI： 10.1038/ncomms4932

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Language：English   Publishing type：Part of collection (book)   Publisher：Springer Japan  

Abnormal glycosylation of proteins is often associated with human diseases. A group of muscular dystrophy, dystroglycanopathy, is caused by abnormal glycosylation of dystroglycan (DG), a cell-surface laminin receptor. Fukutin (gene symbol, FKTN) and fukutin-related protein (FKRP
gene symbol, FKRP) are putative glycosyltransferases involved in the synthesis of a unique glycan structure of α-DG (Fig. 105.1), which is required for laminin-binding activity. Both genes were originally identified as causative genes of muscular dystrophies: FKTN for Fukuyama congenital muscular dystrophy (FCMD) and FKRP for congenital muscular dystrophy 1C (MDC1C) and limb-girdle type 2I (LGMD2I). Mutations in FKTN or FKRP lead to abnormal glycosylation and subsequent reduction of the ligand-binding activity of α-DG, which is associated with the pathology of the skeletal muscle, cardiac muscle, and the central nervous system. Consistently, studies using genetically modified animals support that fukutin/FKRP-dependent modification plays biologically important roles such as the maintenance of muscle cell membrane integrity, cortical histogenesis, and normal ocular development.

DOI： 10.1007/978-4-431-54240-7_141

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α-synuclein (SNCA) is an established susceptibility gene for Parkinson's disease (PD), one of the most common human neurodegenerative disorders. Increased SNCA is considered to lead to PD and dementia with Lewy bodies. Four single-nucleotide polymorphisms (SNPs) in SNCA 3′ region were prominently associated with PD among different ethnic groups. To examine how these SNPs influence disease susceptibility, we analyzed their potential effects on SNCA gene expression. We found that rs356219 showed allele-specific features. Gel shift assay using nuclear extracts from SH-SY5Y cells showed binding of one or more proteins to the protective allele, rs356219-A. We purified the rs356219-A-protein complex with DNA affinity beads and identified a bound protein using mass spectrometry. This protein, YY1 (Yin Yang 1), is an ubiquitous transcription factor with multiple functions. We next investigated SNCA expression change in SH-SY5Y cells by YY1 transfection. We also analyzed the expression of antisense noncoding RNA (ncRNA) RP11-115D19.1 in SNCA 3′-flanking region, because rs356219 is located in intron of RP11-115D19.1. Little change was observed in SNCA expression levels
however, RP11-115D19.1 expression was prominently stimulated by YY1. In autopsied cortices, positive correlation was observed among RP11-115D19.1, SNCA and YY1 expression levels, suggesting their functional interactions in vivo. Knockdown of RP11-115D19.1 increased SNCA expression significantly in SH-SY5Y cells, suggesting its repressive effect on SNCA expression. Our findings of the protective allele-specific YY1 and antisense ncRNA raised a novel possible mechanism to regulate SNCA expression. © 2013 The Japan Society of Human Genetics.

DOI： 10.1038/jhg.2013.90

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been Implicated in alpha-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 beta-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated alpha-dystroglycan (alpha-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 alpha-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 alpha-dystroglycanopathies. (C) 2013 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2013.09.022

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A group of muscular dystrophies, dystroglycanopathy is caused by abnormalities in post-translational modifications of dystroglycan (DG). To understand better the pathophysiological roles of DG modification and to establish effective clinical treatment for dystroglycanopathy, we here generated two distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identified for Fukuyama congenital muscular dystrophy. The first dystroglycanopathy modelumyofiber-selective fukutin-cKO [muscle creatine kinase (MCK)-fukutin-cKO] miceushowed mild muscular dystrophy. Forced exercise experiments in presymptomatic MCK-fukutin-cKO mice revealed that myofiber membrane fragility triggered disease manifestation. The second dystroglycanopathy modelumuscle precursor cell (MPC)-selective cKO (Myf5-fukutin-cKO) miceuexhibited more severe phenotypes of muscular dystrophy. Using an isolated MPC culture system, we demonstrated, for the first time, that defects in the fukutin-dependent modification of DG lead to impairment of MPC proliferation, differentiation and muscle regeneration. These results suggest that impaired MPC viability contributes to the pathology of dystroglycanopathy. Since our data suggested that frequent cycles of myofiber degeneration/regeneration accelerate substantial and/or functional loss of MPC, we expected that protection from disease-triggering myofiber degeneration provides therapeutic effects even in mouse models with MPC defects; therefore, we restored fukutin expression in myofibers. Adeno-associated virus (AAV)-mediated rescue of fukutin expression that was limited in myofibers successfully ameliorated the severe pathology even after disease progression. In addition, compared with other gene therapy studies, considerably low AAV titers were associated with therapeutic effects. Together, our findings indicated that fukutin-deficient dystroglycanopathy is a regeneration-defective disorder, and gene therapy is a feasible treatment for the wide range of dystroglycanopathy even after disease progression.

DOI： 10.1093/hmg/ddt157

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

alpha-Dystroglycan (alpha-DG) is a membrane-associated glycoprotein that interacts with several extracellular matrix proteins, including laminin and agrin. Aberrant glycosylation of alpha-DG disrupts its interaction with ligands and causes a certain type of muscular dystrophy commonly referred to as dystroglycanopathy. It has been reported that a unique O-mannosyl tetrasaccharide (Neu5Ac-alpha 2,3-Gal-beta 1,4-GlcNAc-beta 1,2-Man) and a phosphodiester-linked modification on O-mannose play important roles in the laminin binding activity of alpha-DG. In this study, we use several dystroglycanopathy mouse models to demonstrate that, in addition to fukutin and LARGE, FKRP (fukutin-related protein) is also involved in the post-phosphoryl modification of O-mannose on alpha-DG. Furthermore, we have found that the glycosylation status of alpha-DG in lung and testis is minimally affected by defects in fukutin, LARGE, or FKRP. alpha-DG prepared from wild-type lung-or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results show that FKRP is involved in post-phosphoryl modification rather than in O-mannosyl tetrasaccharide synthesis. Our data also demonstrate that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of alpha-DG functional expression as a laminin receptor in normal tissues and cells.

DOI： 10.1074/jbc.M111.271767

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common childhood muscular dystrophy in Japan, is caused by alterations in the fukutin gene. Mutations in fukutin cause abnormal glycosylation of alpha-dystroglycan, a cell surface laminin receptor; however, the exact function and pathophysiological role of fukutin are unclear. Although the most prevalent mutation in Japan is a founder retrotransposal insertion, point mutations leading to abnormal glycosylation of alpha-dystroglycan have been reported, both in Japan and elsewhere. To understand better the molecular pathogenesis of fukutin-deficient muscular dystrophies, we constructed 13 disease-causing missense fukutin mutations and examined their pathological impact on cellular localization and alpha-dystroglycan glycosylation. When expressed in C2C12 myoblast cells, wild-type fukutin localizes to the Golgi apparatus, whereas the missense mutants A170E, H172R, H186R, and Y371C instead accumulated in the endoplasmic reticulum. Protein O-mannose beta 1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) also mislocalizes when co-expressed with these missense mutants. The results of nocodazole and brefeldin A experiments suggested that these mutant proteins were not transported to the Golgi via the anterograde pathway. Furthermore, we found that low temperature culture or curcumin treatment corrected the subcellular location of these missense mutants. Expression studies using fukutin-null mouse embryonic stem cells showed that the activity responsible for generating the laminin-binding glycan of alpha-dystroglycan was retained in these mutants. Together, our results suggest that some disease-causing missense mutations cause abnormal folding and localization of fukutin protein, and therefore we propose that folding amelioration directed at correcting the cellular localization may provide a therapeutic benefit to glycosylation-deficient muscular dystrophies.

DOI： 10.1074/jbc.M111.300905

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Language：Japanese   Publisher：11  

Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), and Walker-Warburg syndrome (WWS) are autosomal recessive disorders characterized by congenital muscular dystrophy with structural brain and eye abnormalities. Aberrant glycosylation of α-dystroglycan (α-DG) is a common pathomechanism of these disorders. In addition, genetic and glycobiological evidence has shown that abnormal glycosylation of α-DG is also seen in several forms of congenital and limb-girdle-type muscular dystrophies. These disorders are collectively called "α-dystroglycanopathy" and nowadays, this term is widely accepted because it is useful for illustrating a complicated genotype-phenotype correlation of these disorders. α-DG is a membrane-associated protein that interacts with extracellular matrix proteins such as laminin, and abnormal glycosylation of α-DG results in loss of its laminin-binding activity. A number of serine/threonine residues are present in the mucin-like domain of α-DG and are majorly composed of sugar chains. Among these glycans, an O-mannosyl tetrasaccharide (Neu5Ac-α,2,3-Gal-β1, 4-GlcNAc-β1,2- Man) is important for laminin-binding activity of α-DG. POMT1/2 and POMGnT1, protein products of causative genes of WWS and MEB, respectively, are enzymes that directly catalyze the biosynthesis of this glycan. Recent studies have suggested that a phosphodiester-linked structure on O-mannose is also important for the laminin-binding activity and that mutations in other causative genes of α-dystroglycanopathy, such as fukutin (originally identified as the gene responsible for FCMD) and LARGE, disrupt the post-phosphoryl structure. Here, we review the history of basic and clinical research on α-dystroglycanopathy and refine its clinical spectrum, which should be broadly extended. In addition, we reveal some progress in research on α-dystroglycanopathy including a novel disease mechanism and anti-sense oligonucleotide therapy for FCMD.

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alpha-dystroglycan is a highly O-glycosylated extracellular matrix receptor that is required for anchoring of the basement membrane to the cell surface and for the entry of Old World arenaviruses into cells. Like-acetylglucosaminyltransferase (LARGE) is a key molecule that binds to the N-terminal domain of a-dystroglycan and attaches ligand-binding moieties to phosphorylated O-mannose on a-dystroglycan. Here we show that the LARGE modification required for laminin-and virus-binding occurs on specific Thr residues located at the extreme N terminus of the mucin-like domain of a-dystroglycan. Deletion and mutation analyses demonstrate that the ligand-binding activity of a-dystroglycan is conferred primarily by LARGE modification at Thr-317 and -319, within the highly conserved first 18 amino acids of the mucin-like domain. The importance of these paired residues in laminin-binding and clustering activity on myoblasts and in arenavirus cell entry is confirmed by mutational analysis with full-length dystroglycan. We further demonstrate that a sequence of five amino acids, Thr(317)ProThr(319)ProVal, contains phosphorylated O-glycosylation and, when modified by LARGE is sufficient for laminin-binding. Because the N-terminal region adjacent to the paired Thr residues is removed during posttranslational maturation of dystroglycan, our results demonstrate that the ligand-binding activity resides at the extreme N terminus of mature a-dystroglycan and is crucial for a-dystroglycan to coordinate the assembly of extracellular matrix proteins and to bind arenaviruses on the cell surface.

DOI： 10.1073/pnas.1114836108

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Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene(1-3). In FCMD, the SVA insertion occurs in the 3' untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant messenger RNA (mRNA) splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in patients with FCMD. This region spans part of the 3' end of the fukutin coding region, a proximal part of the 3' UTR and the SVA insertion. Correspondingly, fukutin mRNA transcripts in patients with FCMD and SVA knock-in model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin carboxy (C) terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in cells of patients with FCMD and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of alpha-dystroglycan (alpha-DG) and laminin binding by alpha-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia(4), neutral lipid storage disease(5)) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known(6-8), we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.

DOI： 10.1038/nature10456

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Mutations in the caveolin-3 gene cause autosomal dominant limb-girdle muscular dystrophy 1C (LGMD1C). However, the precise molecular pathogenesis of caveolin-3-related muscular dystrophy remains uncertain. Here, we demonstrate the effect of gene dosage on the severity of the myopathic phenotype in P104L mutant caveolin-3 (mCav3(P104L)) transgenic mice, a model of LGMD1C. We analyzed the endoplasmic reticulum (ER) stress response in the transgenic mice and found upregulated transcription of the molecular chaperone, glucose-regulated protein (GRP78). Moreover, signaling downstream of GRP78 in the myofibers was activated toward apoptosis. However, terminal transferase dUTP nick end labeling assays detected a few apoptotic nuclei in transgenic mouse skeletal muscle, probably due to the transcriptional activation of Dad1, an anti-apoptotic factor in the ER. These findings suggest that the ER stress response caused by mCav3(P104L) plays a role in the pathogenesis of LGMD1C as a toxic gain of function effect.

DOI： 10.1093/hmg/ddr201

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MASSACHUSETTS MEDICAL SOC  

Dystroglycan, which serves as a major extracellular matrix receptor in muscle and the central nervous system, requires extensive O-glycosylation to function. We identified a dystroglycan missense mutation (Thr192. Met) in a woman with limb-girdle muscular dystrophy and cognitive impairment. A mouse model harboring this mutation recapitulates the immunohistochemical and neuromuscular abnormalities observed in the patient. In vitro and in vivo studies showed that the mutation impairs the receptor function of dystroglycan in skeletal muscle and brain by inhibiting the post-translational modification, mediated by the glycosyltransferase LARGE, of the phosphorylated O-mannosyl glycans on a-dystroglycan that is required for high-affinity binding to laminin.

DOI： 10.1056/NEJMoa1006939

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Pikachurin, the most recently identified ligand of dystroglycan, plays a crucial role in the formation of the photoreceptor ribbon synapse. It is known that glycosylation of dystroglycan is necessary for its ligand binding activity, and hypoglycosylation is associated with a group of muscular dystrophies that often involve eye abnormalities. Because little is known about the interaction between pikachurin and dystroglycan and its impact on molecular pathogenesis, here we characterize the interaction using deletion constructs and mouse models of muscular dystrophies with glycosylation defects (Large(myd) and POMGnT1-deficient mice). Pikachurin-dystroglycan binding is calcium-dependent and relatively less sensitive to inhibition by heparin and high NaCl concentration, as compared with other dystroglycan ligand proteins. Using deletion constructs of the laminin globular domains in the pikachurin C terminus, we show that a certain steric structure formed by the second and the third laminin globular domains is necessary for the pikachurin-dystroglycan interaction. Binding assays using dystroglycan deletion constructs and tissue samples from Large-deficient (Large(myd)) mice show that Large-dependent modification of dystroglycan is necessary for pikachurin binding. In addition, the ability of pikachurin to bind to dystroglycan prepared from POMGnT1-deficient mice is severely reduced, suggesting that modification of the GlcNAc-beta 1,2-branch on O-mannose is also necessary for the interaction. Immunofluorescence analysis reveals a disruption of pikachurin localization in the photoreceptor ribbon synapse of these model animals. Together, our data demonstrate that post-translational modification on O-mannose, which is mediated by Large and POMGnT1, is essential for pikachurin binding and proper localization, and suggest that their disruption underlies the molecular pathogenesis of eye abnormalities in a group of muscular dystrophies.

DOI： 10.1074/jbc.M110.116343

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Skeletal muscle basal lamina is linked to the sarcolemma through transmembrane receptors, including integrins and dystroglycan. The function of dystroglycan relies critically on posttranslational glycosylation, a common target shared by a genetically heterogeneous group of muscular dystrophies characterized by alpha-dystroglycan hypoglycosylation. Here we show that both dystroglycan and integrin alpha 7 contribute to force-production of muscles, but that only disruption of dystroglycan causes detachment of the basal lamina from the sarcolemma and renders muscle prone to contraction-induced injury. These phenotypes of dystroglycan-null muscles are recapitulated by Large(myd) muscles, which have an intact dystrophin-glycoprotein complex and lack only the laminin globular domain-binding motif on alpha-dystroglycan. Compromised sarcolemmal integrity is directly shown in Large(myd) muscles and similarly in normal muscles when arenaviruses compete with matrix proteins for binding alpha-dystroglycan. These data provide direct mechanistic insight into how the dystroglycan-linked basal lamina contributes to the maintenance of sarcolemmal integrity and protects muscles from damage.

DOI： 10.1073/pnas.0906545106

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Hypoglycosylation and reduced laminin-binding activity of alpha-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated alpha-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact alpha-dystroglycan, and solid-phase assays determined laminin binding levels to be similar to 50% of normal. In contrast, intact alpha-dystroglycan is undetectable in the dystrophic Large(myd) mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact alpha-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of alpha-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of alpha-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of alpha-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.

DOI： 10.1093/hmg/ddn387

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：F HERNANDEZ  

Expression profiles of sarcospan in muscles with muscular dystrophies are scarcely reported. To examine this, we studied five Fukuyama congenital muscular dystrophy ( FCMD) muscles, five Duchenne muscular dystrophy (DMD) muscles, five disease control and five normal control muscles. Immunoblot showed reactions of sarcospan markedly decreased in FCMD and DMD muscle extracts. Immunohistochemistry of FCMD muscles showed that most large diameter myofibers expressed sarcospan discontinuously at their surface membranes. Immature small diameter FCMD myofibers usually did not express sarcospan. Immunoreactivity of sarcospan in DMD muscles was similarly reduced. With regard to dystroglycans and sarcoglycans, immunohistochemistry of FCMD muscles showed selective deficiency of glycosylated alpha-dystroglycan, together with reduced expression of beta-dystroglycan and alpha-, beta-, gamma-, delta-sarcoglycans. Although the expression of glycosylated alpha-dystroglycan was lost, scattered FCMD myofibers showed positive immunoreaction with an antibody against the core protein of alpha-dystroglycan. The group mean ratios of sarcospan mRNA copy number versus GAPDH mRNA copy number by real-time RT-PCR showed that the ratios between FCMD and normal control groups were not significantly different (P &gt; 0.1 by the two-tailed t test). This study implied either O- linked glycosylation defects of alpha-dystroglycan in the Golgi apparatus of FCMD muscles may lead to decreased expression of sarcoglycan and sarcospan molecules, or selective deficiency of glycosylated alpha-dystroglycan due to impaired glycosylation in FCMD muscles may affect the molecular integrity of the basal lamina of myofibers. This, in turn, leads to decreased expression of sarcoglycans, and finally of sarcospan at the FCMD myofiber surfaces.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix-like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.

DOI： 10.1038/nn.2160

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC beta-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The beta construct beta-AID dominant negative, which disrupts the RIM1-beta association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with b in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.

DOI： 10.1038/nn1904

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The recent identification of mutations in genes encoding demonstrated or putative glycosyltransferases has revealed a novel mechanism for congenital muscular dystrophy. Hypoglycosylated alpha-dystroglyean (alpha-DG) is commonly seen in Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), and Large(myd) mice. POMGnT1 and POMTs, the gene products responsible for MEB and WWS, respectively, synthesize unique O-mannose sugar chains on alpha-DG. The function of fukutin, the gene product responsible for FCMD, remains undetermined. Here we show that flukutin co-localizes with POMGnT1 in the Golgi apparatus. Direct interaction between fukutin and POMGnT1 was confirmed by co-immunoprecipitation and two-hybrid analyses. The transmembrane region of fukutin mediates its localization to the Golgi and participates in the interaction with POMGnT1. Y371C, a missense mutation found in FCMD, retains fukutin in the ER and also redirects POMGnT1 to the ER. Finally, we demonstrate reduced POMGnT1 enzymatic activity in transgenic knock-in mice carrying the retrotransposal insertion in the fukutin gene, the prevalent mutation in FCMD. From these findings, we propose that fukutin forms a complex with POMGnT1 and may modulate its enzymatic activity. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.09.129

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The receptor for lymphocytic choriomeningitis virus (LCMV), the human pathogenic Lassa fever virus (LFV), and clade C New World arenaviruses is et-dystroglycan (alpha-DG), a cell surface receptor for proteins of the extracellular matrix (ECM). Specific posttranslational modification of alpha-DG by the glycosyltransferase LARGE is critical for its function as an ECM receptor. In the present study, we show that LARGE-dependent modification is also crucial for alpha-DG's function as a cellular receptor for arenaviruses. Virus binding involves the mucin-type domain of alpha-DG and depends on modification by LARGE. A crucial role of the LARGE-dependent glycosylation of alpha-DG for virus binding is found for several isolates of LCMV, LFV, and the arenaviruses Mobala and Oliveros. Since the posttranslational modification by LARGE is crucial for alpha-DG recognition by both arenaviruses and the host-derived ligand laminin, it also influences competition between virus and laminin for a-DG. Hence, LARGE-dependent glycosylation of a-DG has important implications for the virus-host cell interaction and the pathogenesis of LFV in humans.

DOI： 10.1128/JVI.79.22.14282-14296.2005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Dystroglycan is a cell-surface matrix receptor that requires LARGE-dependent glycosylation for laminin binding. Although the interaction of dystroglycan with laminin has been well characterized, less is known about the role of dystroglycan glycosylation in the binding and assembly of perlecan. We report reduced perlecan-binding activity and mislocalization of perlecan in the LARGE-deficient Large(myd) mouse. Cell-surface ligand clustering assays show that laminin polymerization promotes perlecan assembly. Solid-phase binding assays provide evidence for the first time of a trimolecular complex formation of dystroglycan, laminin and perlecan. These data suggest functional disruption of the trimolecular complex in glycosylation-deficient muscular dystrophy. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2005.07.059

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CANCER RESEARCH  

Alterations in the basement membrane receptor dystroglycan (DG) are evident in muscular dystrophies and carcinoma cells and characterized by a selective loss or modification of the extracellular alpha-DG subunit. Defects in posttranslational modifications of DG have been identified in some muscular dystrophies, but the underlying modifications in carcinoma cells have not yet been defined. We reveal here multiple posttranslational modifications that modulate the composition and function of DG in normal epithelial cells and carcinoma cells. We show that alpha-DG is shed from the cell surface of normal and tumorigenic epithelial cells through a proteolytic mechanism that does not require direct cleavage of either alpha- or beta-DG. Shedding is dependent on metalloprotease activity and the proprotein convertase furin. Surprisingly, furin is also found to directly process alpha-DG as a proprotein substrate, changing the existing model of DG composition. We also show that the glycosylation of alpha-DG is altered in invasive carcinoma cells, and this modification causes complete loss of laminin binding properties. Together, these data elucidate several novel events regulating the functional composition of DG and reveal defects that arise during cancer progression, providing direction for efforts to restore this link with the basement membrane in carcinoma cells.

DOI： 10.1158/0008-5472.CAN-04-1638

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Several congenital muscular dystrophies caused by defects in known or putative glycosyltransferases are commonly associated with hypoglycosylation of alpha-dystroglycan (alpha-DG) and a marked reduction of its receptor function. We have investigated changes in the processing and function of alpha-DG resulting from genetic manipulation of LARGE, the putative glycosyltransferase mutated both in Large(myd) mice and in humans with congenital muscular dystrophy 1D (MDC1D). Here we show that overexpression of LARGE ameliorates the dystrophic phenotype of Large(myd) mice and induces the synthesis of glycan-enriched alpha-DG with high affinity for extracellular ligands. Notably, LARGE circumvents the alpha-DG glycosylation defect in cells from individuals with genetically distinct types of congenital muscular dystrophy. Gene transfer of LARGE into the cells of individuals with congenital muscular dystrophies restores alpha-DG receptor function, whereby glycan-enriched alpha-DG coordinates the organization of laminin on the cell surface. Our findings indicate that modulation of LARGE expression or activity is a viable therapeutic strategy for glycosyltransferase-deficient congenital muscular dystrophies.

DOI： 10.1038/nm1059

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

Reduced ligand binding activity of alpha-dystroglycan is associated with muscle and central nervous system pathogenesis in a growing number of muscular dystrophies. Posttranslational processing of alpha-dystroglycan is generally accepted to be critical for the expression of functional dystroglycan. Here we show that both the N-terminal domain and a portion of the mucin-like domain of alpha-dystroglycan are essential for high-affinity laminin-receptor function. Posttranslational modification of alpha-dystroglycan by glycosyltransferase, LARGE, occurs within the mucin-like domain, but the N-terminal domain interacts with LARGE, defining an intracellular enzyme-substrate recognition motif necessary to initiate functional glycosylation. Gene replacement in dystroglycan-deficient muscle demonstrates that the dystroglycan C-terminal domain is sufficient only for dystrophin-glycoprotein complex assembly, but to prevent muscle degeneration the expression of a functional dystroglycan through LARGE recognition and glycosylation is required. Therefore, molecular recognition of dystroglycan by LARGE is a key determinant in the biosynthetic pathway to produce mature and functional dystroglycan.

DOI： 10.1016/j.cell.2004.06.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

It has been well established that phosphotylation is an important reaction for the regulation of protein functions. In the N-terminal domain of the alpha-chain of pig gastric H+/K+-ATPase. reversible sequential phosphorylation occurs at Tyr 10 and Tyr 7. In this study, we determined the structure of the peptide involving the residues from Gly 2 to Gly 34 of pig gastric H+/K+-ATPase and investigated the tyrosine phosphorylation-induced conformational change using CD and NMR experiments. The solution structure showed that the N-terminal fragment has a helical conformation. and the peptide adopted two alpha-helices in 50% trifluoroethanol (TFE) solvent, suggesting that the peptide has a high helical propensity under hydrophobic conditions. Furthermore, the CD and NMR data suggested that the structure of the N-terminal fragment becomes more disordered as a result of phosphorylation of Tyr 10. This conformational change induced by the phosphorylation of Tyr 10 might be an advantageous reaction for sequential phosphorylation and may be important for regulating the function of H+/K+-ATPase. (C) 2002 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S0006-291X(02)02794-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Muscle-eye-brain disease (MEB) and Fukuyama congenital muscular dystrophy (FCMD) are congenital muscular dystrophies with associated, similar brain malformations(1,2). The FCMD gene, fukutin, shares some homology with fringe-like glycosyltransferases, and the MEB gene, POMGnT1, seems to be a new glycosyltransferase(3,4). Here we show, in both MEB and FCMD patients, that alpha-dystroglycan is expressed at the muscle membrane, but similar hypoglycosylation in the diseases directly abolishes binding activity of dystroglycan for the ligands laminin, neurexin and agrin. We show that this post-translational biochemical and functional disruption of alpha-dystroglycan is recapitulated in the muscle and central nervous system of mutant myodystrophy (myd) mice. We demonstrate that myd mice have abnormal neuronal migration in cerebral cortex, cerebellum and hippocampus, and show disruption of the basal lamina. In addition, myd mice reveal that dystroglycan targets proteins to functional sites in brain through its interactions with extracellular matrix proteins. These results suggest that at least three distinct mammalian genes function within a convergent post-translational processing pathway during the biosynthesis of dystroglycan, and that abnormal dystroglycan-ligand interactions underlie the pathogenic mechanism of muscular dystrophy with brain abnormalities.

DOI： 10.1038/nature00837

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ELSEVIER SCIENCE BV  

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When pig stomach membrane H,K-ATPase preparations were incubated with 32ATP, Mg2+ and Ca2+, reversible phosphorylation of specific Tyr and Ser residues in the N-terminal a-chain of H,K-ATPase occurred without any detectable phosphorylation in other regions of the chain. Amino acid sequence analysis of purified peptides and others showed a sequential phosphorylation of Tyr-10 and Tyr-7 and sub μM Ca2+ activated phosphorylation of Set-27. The apparent molecular weight of a detergent solubilized Tyr-kinase was estimated to be around 50 kDa by a gel filtration column and the kinase activity detected on a gel band after renaturation. The Ser-kinase present was suggested to be a conventional PKC. H,K-ATPase preparations phosphorylated fusion proteins consisted of maltose binding protein and a peptide portion of the a-chain from Gly-2 to Gln-111. These data and others suggested that N-terminal cytosolic domain is sufficient for phosphorylation by endogenous Tyr-kinase and PKC in the membrane H,K-ATPase preparations.

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When pig stonach membrane H+,K+-ATPase preparations were incubated with [γ-32P]ATP, Mg2+ and Ca2+, reversible phosphorylation of specific Tyr and Ser residues in the N-terminal α-chain of H+, K+-ATPase occurred without any detectable phosphorylation in other regions of the α-chain. Mild tosylphenylalanyl chloromethyl ketone-trypsin treatment followed by reverse-phase column chromatography yielded three radioactive peptide peaks. The first peak contained both Tyr10(32P) and Tyr7(32P) and the second peak contained Tyr10(32P). The third peak contained Ser27(32P). A fusion protein consisted of maltose binding protein and a peptide portion of the α-chain from Gly2 to Gln111 could be phosphorylated by membrane H+, K+-ATPase preparations in the presence of vanadate. Fusion proteins mutated at Tyr10 to Phe or Ser27 to Ala showed little phosphorylation. The data suggested that N-terminal cytosolic domain is sufficient for phosphorylation by intrinsic protein kinases present in the membrane H+,K+-ATPase preparations. The data also suggest that phosphorylation of Tyr10 was prerequisite for the phosphorylation of Tyr7 in the α-chain of H+, K+-ATPase by the tyrosine kinase.

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Language：Japanese   Publisher：(一社)日本神経治療学会  

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Other Link： https://search-tp.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2021&ichushi_jid=J02615&link_issn=&doc_id=20220124260039&doc_link_id=10.15082%2Fjsnt.38.3_274&url=https%3A%2F%2Fdoi.org%2F10.15082%2Fjsnt.38.3_274&type=J-STAGE&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00007_3.gif

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：Japanese   Publisher：(公社)日本薬剤学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publisher：Movement Disorder Society of Japan (MDSJ)  

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Language：Japanese   Publisher：日本結合組織学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

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Language：Japanese   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

<p>Dystroglycanopathy (DGpathy) is a group of muscular dystrophies that is caused by abnormalities in the sugar chains on dystroglycan. We contributed to the understanding of structure, mechanism of modification, and function of sugar chains of dystroglycan. In particular, we found modification with ribitol phosphate, a new type of post-translational modification, and showed that FKTN, FKRP, ISPD, and TMEM5 are involved in ribitol phosphate modification. We also clarified the pathological mechanism using DGpathy model mice and proposed treatment strategies. Here, we discuss the recent advancements related to ribitol phosphate modification and the pathological mechanism and therapeutic strategies for DGpathies.</p>

DOI： 10.4052/tigg.1936.2SJ

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publisher：生命科学系学会合同年次大会運営事務局  

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Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

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Language：Japanese   Publisher：生命科学系学会合同年次大会運営事務局  

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Language：Japanese   Publisher：日本筋学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：FEDERATION AMER SOC EXP BIOL  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：IOS Press  

Muscular dystrophy is a group of genetic disorders characterized by progressive muscle weakness. In the early 2000s, a new classification of muscular dystrophy, dystroglycanopathy, was established. Dystroglycanopathy often associates with abnormalities in the central nervous system. Currently, at least eighteen genes have been identified that are responsible for dystroglycanopathy, and despite its genetic heterogeneity, its common biochemical feature is abnormal glycosylation of alpha-dystroglycan. Abnormal glycosylation of alpha-dystroglycan reduces its binding activities to ligand proteins, including laminins. In just the last few years, remarkable progress has been made in determining the sugar chain structures and gene functions associated with dystroglycanopathy. The normal sugar chain contains tandem structures of ribitol-phosphate, a pentose alcohol that was previously unknown in humans. The dystroglycanopathy genes fukutin, fukutin-related protein (FKRP), and isoprenoid synthase domain-containing protein (ISPD) encode essential enzymes for the synthesis of this structure: fukutin and FKRP transfer ribitol-phosphate onto sugar chains of alpha-dystroglycan, and ISPD synthesizes CDP-ribitol, a donor substrate for fukutin and FKRP. These findings resolved long-standing questions and established a disease subgroup that is ribitol-phosphate deficient, which describes a large population of dystroglycanopathy patients. Here, we review the history of dystroglycanopathy, the properties of the sugar chain structure of alpha-dystroglycan, dystroglycanopathy gene functions, and therapeutic strategies.

DOI： 10.3233/JND-170255

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (trade magazine, newspaper, online media)  

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publisher：(一社)日本神経学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS INC  

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：GAKUSHIN PUBL CO  

Dystroglycan is a highly glycosylated peripheral membrane protein that functions as a cell surface receptor for proteins in the extracellular matrices and synapses. O-Mannosyl glycosylation is necessary for the ligand-binding activities of dystroglycan and a unique "post-phosphoryl moiety" modified via a phosphodiester linkage on the O-mannose likely forms the ligand-binding domain. Several proteins are involved in the process of this modification, the mechanism for which appears highly ordered. In various tissues, dystroglycan plays important physiological roles such as maintenance of muscle cell viability and structural development of the brain. Conversely, abnormal glycosylation causes a group of muscular dystrophy, collectively called "dystroglycanopathy," which is often associated with brain abnormalities including type II lissencephaly and mental retardation. Here, we will be reviewing the structure, modification pathway, and physiological roles of dystroglycan glycosylation as well as their involvement in human diseases.

DOI： 10.4052/tigg.26.41

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Language：Japanese   Publishing type：Book review, literature introduction, etc.   Publisher：Japanese Biochemical Society  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

DOI： 10.1016/j.nmd.2012.06.045

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Language：Japanese   Publisher：(公社)日本小児科学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-BLACKWELL  

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DOI： 10.11477/mf.2425101116

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

DOI： 10.1016/j.nmd.2010.07.019

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

DOI： 10.1016/j.nmd.2010.07.018

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS INC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

DOI： 10.1016/j.nmd.2009.06.176

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Language：Japanese   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：NATURE PUBLISHING GROUP  

Muscular dystrophies are a heterogeneous group of genetic disorders. In addition to genetic information, a combination of various approaches such as the use of genetic animal models, muscle cell biology, and biochemistry has contributed to improving the understanding of the molecular basis of muscular dystrophy&apos;s etiology. Several lines of evidence confirm that the structural linkage between the muscle extracellular matrix and the cytoskeleton is crucial to prevent the progression of muscular dystrophy. The dystrophin-glycoprotein complex links the extracellular matrix to the cytoskeleton, and mutations in the component of this complex cause Duchenne-type or limb-girdle-type muscular dystrophy. Mutations in laminin or collagen VI, muscle matrix proteins, are known to cause a congenital type of muscular dystrophy. Moreover, it is not only the primary genetic defects in the structural or matrix proteins, but also the primary mutations of enzymes involved in the protein glycosylation pathway that are now recognized to disrupt the matrix-cell interaction in a certain group of muscular dystrophies. This group of diseases is caused by the secondary functional defects of dystroglycan, a transmembrane matrix receptor. This review considers recent advances in understanding the molecular pathogenesis of muscular dystrophies that can be caused by the disruption of the cell-matrix linkage.

DOI： 10.1007/s10038-006-0056-7

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS INC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：OXFORD UNIV PRESS INC  

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H/K-ATPase preparations (the G1 membrane) from pig stomach contain both kinases and phosphatases and show reversible phosphorylation of Tyr(7), Tyr(10), anti Ser(27) residues of the alpha-chain of H/K-ATPase. The Tyr-kinase is sensitive to genistein and quercetin and recognized by anti-c-Src antibody. The Ser-kinase is dependent on Ca2+ (K-0.5 = 0.9 mu M), sensitive to a PKC inhibitor, and recognized by antibodies against PBC alpha and PKC beta II. The addition of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS) caused a dramatic increase in the phosphorylation of added synthetic copolymer substrates and permitted the phosphorylation of maltose-binding proteins fused with the N-terminal domain of alpha-chains. The phosphotyrosine phosphatase was inhibited by vanadate. The phosphoserine phosphatase was inhibited by okadaic acid and by inhibitor-2. The presence of protein phosphatase-1 was immunologically detected. Column chromatographic separation of CHAPS-solubilized G1 membrane and others indicate the apparent molecular weight of the Src-kinase to be similar to 60 kDa, the PKC alpha and/or PKC beta II to be similar to 80 kDa, the Tyr-phosphatase to be 200 kDa, and PP-1 to be similar to 35 kDa. These data show that these membrane-bound enzyme systems are in sufficiently close proximity to be responsible for reversible phosphorylation of Tyr(7), Tyr(10) and Ser(27) of the catalytic subunit of membrane H/K-ATPase in parietal cells, the physiological role of which is unknown.

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CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2000024496

Language：English   Presentation type：Poster presentation  

Venue：Paris  

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Language：Japanese   Presentation type：Poster presentation  

Venue：横浜市  

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Language：Japanese   Presentation type：Poster presentation  

Venue：横浜市  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都市  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：京都市  

ジストログリカンはラミニンなどの基底膜分子の細胞膜受容体で、筋組織では機械的な負荷に対して細胞膜の安定性を維持する役割を担っている。また、ジストログリカンの機能不全は筋ジストロフィーなどの疾患につながることも知られている。ジストログリカンがラミニン結合能を発揮するためにはO-マンノース型の糖鎖修飾が必要である。この１０年ほどの間に、ジストログリカンの糖鎖構造、その生合成に関わる糖転移酵素、そして、筋ジストロフィーの原因遺伝子が続々と同定され、ジストログリカンの糖鎖構造の全容が明らかになってきた。ジストログリカン糖鎖の生合成には多くの修飾酵素が関与するが、マンノースキナーゼによるリン酸化、哺乳類では初めて同定されたリビトールリン酸という修飾体、グルクロン酸（GlcA）とキシロース（Xyl）の二糖を単位とするグリコサミノグリカン様の繰り返し構造［（Glc

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Language：Japanese   Presentation type：Poster presentation  

Venue：仙台市  

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Language：Japanese   Presentation type：Poster presentation  

Venue：神戸  

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Language：English   Presentation type：Oral presentation (general)  

Venue：ジュネーブ, スイス  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Symposium, workshop panel (nominated)  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：シャーロット  

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 f

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Presentation type：Symposium, workshop panel (public)  

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Language：English   Presentation type：Poster presentation  

Venue：ナポリ, イタリア  

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Language：English   Presentation type：Poster presentation  

Venue：オタワ, カナダ  

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Language：English   Presentation type：Poster presentation  

Venue：葉山  

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Language：Japanese   Presentation type：Poster presentation  

Venue：横浜  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京都  

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Language：English   Presentation type：Poster presentation  

Venue：ケルン, 独  

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Language：English   Presentation type：Oral presentation (general)  

Venue：ギーセン, 独  

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Language：English   Presentation type：Poster presentation  

Venue：ホノルル, アメリカ  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸  

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Language：Japanese   Presentation type：Poster presentation  

Venue：神戸  

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Language：English   Presentation type：Poster presentation  

Venue：ホノルル, 米国  

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Language：Japanese   Presentation type：Poster presentation  

Venue：長岡  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長岡  

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Language：English   Presentation type：Poster presentation  

Venue：トロント, カナダ  

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：神戸  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：埼玉  

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Language：Japanese   Presentation type：Poster presentation  

Venue：さいたま  

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Language：English   Presentation type：Poster presentation  

Venue：熊本  

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Language：Japanese   Presentation type：Poster presentation  

Venue：さいたま  

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Language：English   Presentation type：Poster presentation  

Venue：千葉  

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Language：English   Presentation type：Poster presentation  

Venue：熊本  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡  

福山型筋ジストロフィ－(FCMD)は、重度の筋ジストロフィ－に脳奇形を伴う常染色体劣性遺伝性神経筋疾患であり、日本に多く、未だ治療法がない。殆どのFCMD患者は、フクチン遺伝子の3&apos;非翻訳領域に約3kbのSVA型レトロトランスポゾンの挿入変異を持つ。この疾患の発症機序は、mRNAの不安定化や転写障害によるものと考えられていたが、詳細は不明であった。今回我々は、FCMDがSVAの挿入により誘導されるスプライシング異常症であることを証明した。この異常スプライシングは、SVA挿入配列内に存在する強力なスプライシング受容部位のexon-trapping機能により、最終エクソンのフクチンをコードする領域内の選択的スプライシング供与部位が活性化されて、引き起こされていた。そこで、異常スプライシングを阻止するアンチセンスオリゴヌクレオチド(AON)を用い

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Language：Japanese   Presentation type：Poster presentation  

Venue：東京  

Aberrant glycosylation of alpha-DG with reduced laminin-binding activity is a biochemical hallmark of a group of muscular dystrophy commonly referred to as dystroglycanopathy. Among causative genes for dystroglycanopathy, it has been reported that fukuti

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡  

福山型筋ジストロフィ－(FCMD)は、重度の筋ジストロフィ－に脳奇形を伴う常染色体劣性遺伝性神経筋疾患であり、日本に多く、未だ治療法がない。殆どのFCMD患者は、フクチン遺伝子の3&apos;非翻訳領域に約3kbのSVA型レトロトランスポゾンの挿入変異を持つ。この疾患の発症機序は、mRNAの不安定化や転写障害によるものと考えられていたが、詳細は不明であった。今回我々は、FCMDがSVAの挿入により誘導されるスプライシング異常症であることを証明した。この異常スプライシングは、SVA挿入配列内に存在する強力なスプライシング受容部位のexon-trapping機能により、最終エクソンのフクチンをコードする領域内の選択的スプライシング供与部位が活性化されて、引き起こされていた。そこで、異常スプライシングを阻止するアンチセンスオリゴヌクレオチド(AON)を用い

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Language：Japanese   Presentation type：Poster presentation  

Venue：千葉  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜  

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Language：English   Presentation type：Oral presentation (general)  

Venue：アルガルベ, ポルトガル  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：千葉  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都  

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Language：English   Presentation type：Poster presentation  

Venue：アルガルベ, ポルトガル  

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Language：English   Presentation type：Poster presentation  

Venue：札幌  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都  

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Language：Japanese  

Venue：大阪  

筋ジストロフィーは、筋萎縮と筋力の低下が進行していく遺伝性疾患で、様々な病型が存在する。我々は、ジストログリカンの糖鎖異常を発症原因とする病型を発見し、糖鎖異常型筋ジストロフィー（ジストログリカノパチー）という、新しい疾患概念を確立させてきた。本症は、重篤な筋病変にくわえ、中枢神経障害や眼病変も伴う、最重症型の筋ジストロフィーで、有効な治療法が存在しない難病である。我々は、種々の組織における糖鎖機能や生合成経路の解明などの研究を通じ、組織形成・機能維持に関するメカニズムと、その破綻がもたらす病態を明らかにし、また、治療法の開発を行ってきた。

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：大阪  

福山型筋ジストロフィーの原因遺伝子フクチンのコンディショナルKOマウスを用いて明らかになった病態機序と遺伝子治療について報告する。

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：福岡  

筋ジストロフィーは、骨格筋の壊死・変性を主病変とし、進行性の筋力低下をみとめる遺伝性疾患の総称で、多くの病型が存在する。近年、基底膜ラミニン受容体であるジストログリカンの糖鎖修飾異常を発症要因とする、一群の筋ジストロフィー（ジストログリカノパチー）が注目されている。現在のところ10種の遺伝子が、ジストログリカノパチーに関与することが知られている。その中のひとつ、福山型筋ジストロフィーは、中枢神経障害を合併する先天性筋ジストロフィーで、本邦の小児期筋ジスの中では、デュシャンヌ型についで多くみられる。我々は、原因遺伝子フクチンを同定し、3&apos;UTRへのレトロトランスポゾン挿入変異が大部分の患者にみられることを報告した。挿入変異によって、スプライシング異常が生じ、フクチン機能が喪失すること、また、フクチンは、ジストログリカンのラミニン結合活性に重要

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡  

福山型筋ジストロフィー(FCMD)は、筋病変に加え、多小脳回を基本とする脳の形成異常(II型滑脳症)や精神発達遅滞といった中枢神経障害を伴う重篤な遺伝性疾患である。疾患原因遺伝子であるfukutinの変異によって、基底膜と細胞骨格を結ぶ膜型の糖タンパク質であるジストログリカンの糖鎖異常が生じることが病因とされるが、その詳細は明らかではない。ジストログリカンの糖鎖についても不明な点が多いが、最近、fukutinが関与するO-マンノシルリン酸化糖鎖という新しい修飾がリガンド結合に関与することが示唆され、注目されている。本研究では、中枢神経におけるfukutin及びジストログリカン糖鎖の生理的役割、さらにFCMD脳病態の解明を目的として、中枢神経選択的にfukutin遺伝子を欠損するconditional knockout(cKO)マウスを作出した。cKO

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡  

ジストログリカンは、ラミニンなどの基底膜分子の受容体として、様々な組織で機能を発揮する糖タンパク質で、その多様な糖鎖構造の中でも、O-マンノース糖鎖上にリン酸ジエステル結合を介して存在する新規の修飾体（ポストリン酸構造）が、リガンド結合に重要であることが明らかになってきた。一方、脳奇形や精神発達遅滞を伴う先天性筋ジストロフィーの中には、ジストログリカンの糖鎖異常を発症要因とする疾患群が存在する。そのひとつである福山型筋ジストロフィーの原因遺伝子として発見されたフクチンは、ポストリン酸構造の修飾に関与していることが示唆されている。本研究では、フクチン依存的な修飾の生理的・病的意義を明らかにするため、フクチンのconditional knock-out (cKO)マウスを作出した。骨格筋でフクチンを欠損するcKOマウスとして、筋線維選択的なMCK-fuk

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Language：English   Presentation type：Oral presentation (general)  

Venue：San Francisco, USA  

Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy.

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鎌倉  

フクチンとFKRPは、ジストログリカンのポストリン酸修飾に関与する

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島  

脳奇形や精神発達遅滞を伴う先天性筋ジストロフィーの中には、ジストログリカンの糖鎖異常を発症要因とする疾患が存在し、それらはジストログリカノパチーと総称されている。ジストログリカンはラミニンやアグリンといった基底膜やシナプス分子の膜受容体であり、そのリガンド結合活性には糖鎖修飾が必須である。リガンド結合や発症に関与する構造として、O-マンノース型糖鎖と、マンノース上にリン酸ジエステル結合を介して存在する新規の修飾体（ポストリン酸構造）が重要であることが明らかになってきた。しかし、ポストリン酸構造の詳細や修飾機序について不明な点は多く残されている。本研究では、複数のジストログリカノパチーのモデルマウスを用いて、ジストログリカノパチー原因遺伝子産物のフクチンとLARGEに加え、FKRPもまたポストリン酸修飾に関与することを新たに明らかにした。また、ジストロ

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Language：English   Presentation type：Poster presentation  

Venue：Perth, Australia  

Aberrant glycosylation of alpha-DG with reduced laminin-binding activity is a biochemical hallmark of a group of muscular dystrophy commonly referred to as dystroglycanopathy. Among causative genes for dystroglycanopathy, it has been reported that fukuti

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Language：English   Presentation type：Poster presentation  

Venue：NewOrleans, USANewOrleans, USA  

Aberrant glycosylation of alpha-DG with reduced laminin-binding activity is a biochemical hallmark of a group of muscular dystrophy commonly referred to as dystroglycanopathy. Among causative genes for dystroglycanopathy, it has been reported that fukuti

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Language：English   Presentation type：Poster presentation  

Venue：New Orleans  

Dystroglycanopathy is a group of congenital and limb-girdle muscular dystrophies. Hypoglycosylation of dystroglycan is a hallmark of these disorders. Eye abnormalities are often associated with severe dystroglycanopathy; however the molecular basis of ophthalmologic phenotype is not completely understood. Pikachurin, the most recently identified ligand of dystroglycan, plays a

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Language：Japanese  

Venue：横浜  

なし

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Language：English   Presentation type：Poster presentation  

Venue：Asilomar, アメリカ  

A group of muscular dystrophies, dystroglycanopathy, is caused by abnormalities in post-translational modifications of dystroglycan (DG). To better understand the pathophysiological roles of DG modification and to establish effective treatment for dystroglycanopathy, we generated 2 distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identifi

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Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：Ascona, スイス  

A group of muscular dystrophies, dystroglycanopathy, is caused by abnormalities in post-translational modifications of dystroglycan (DG). To better understand the pathophysiological roles of DG modification and to establish effective treatment for dystroglycanopathy, we generated 2 distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identifi

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：名古屋  

筋ジストロフィーは、骨格筋の壊死・変性を主病変とする遺伝性疾患の総称で、多くの病型が存在する。中でも、ラミニン受容体であるジストログリカンのO-マンノース型糖鎖異常を発症要因とする疾患群（ジストログリカノパチー）は、筋病変にくわえ、脳奇形（II型滑脳症）や精神発達遅滞などの中枢神経障害と眼症状を伴う独特な特徴をしめす。本邦に多くみられる福山型筋ジストロフィー（原因遺伝子フクチン）は、ジストログリカノパチーとして初めて提唱された疾患である。本疾患群が中枢神経障害を伴う理由として、ジストログリカンに修飾されるO-マンノース型糖鎖とポストリン酸構造と呼ばれる新規の修飾体が、骨格筋のみならず、中枢神経系においても重要な生理的役割を担っているためと考えられる。ポストリン酸構造は、O-マンノース上のリン酸ジエステル結合を介して存在し、リガンド結合に必要な修飾体と

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Language：Japanese   Presentation type：Poster presentation  

Venue：名古屋  

福山型筋ジストロフィー(FCMD)は筋病変のみならず、多小脳回を基本とする脳の形成異常(II型滑脳症)や精神発達遅滞といった中枢神経障害を伴う重篤な遺伝性疾患である。疾患原因遺伝子であるfukutinの変異によって、基底膜と細胞骨格を結ぶ膜型の糖タンパク質であるジストログリカンに糖鎖異常が生じることが病因とされるが、その詳細は明らかではない。ジストログリカンの糖鎖についても不明な点が多いが、最近、fukutinが関与するポストリン酸構造が基底膜や細胞外マトリックスとの結合に関与することが示唆され、注目されている。本研究では、中枢神経におけるfukutin及びジストログリカン糖鎖の生理的役割、さらにFCMD脳病態の解明を目的として、中枢神経選択的にfukutin遺伝子を欠損するconditional knock out(cKO)マウスを作出した。cKO

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜  

ジストログリカノパチーと総称される一群の筋ジストロフィーは、ジストログリカンの糖鎖異常によって発症する。本研究では、ジストログリカンに修飾される糖鎖の病態生理的意義の解明と、治療法の開発を目的に、ジストログリカノパチーとして最初に見出された福山型筋ジストロフィーの原因遺伝子フクチンのconditional knock-out （cKO）マウスを2種類作出した。筋線維選択的なMCK-fukutin-cKOマウスは、非常に軽症の筋ジストロフィー病変を示した。未発症のMCK-fukutin-cKOマウスを用いた強制運動負荷実験の結果から、細胞膜の脆弱化が発症の引き金になることが示された。一方、筋前駆細胞選択的なMyf5-fukutin-cKOマウスは、より重篤な病態を示した。単離した筋前駆細胞を用いた増殖・分化実験の結果から、フクチン依存のジストログリカン

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：京都  

筋ジストロフィーは、筋力が進行的に低下する遺伝性疾患の総称で、多くの病型が存在する。中でも、福山型筋ジストロフィーをはじめとする、ジストログリカンの糖鎖異常を発症要因とする疾患群は、ジストログリカノパチーと総称され近年注目されている。我々は、ジストログリカノパチーの病態機序を明らかにし、治療法を開発するため、福山型筋ジストロフィーの原因遺伝子フクチンの変異マウスを作出してきた。筋管選択的、あるいは筋前駆細胞選択的な２種の異なるフクチンコンディショナルノックアウト（cKO）マウスの比較から、細胞膜の脆弱化が発症の引き金になること、そして、筋前駆細胞と筋再生の異常が病態の重篤度に関与することが明らかになった。一方、福山型患者で多くみられるレトロトランスポゾン挿入変異をノックインしたマウスでは、ジストログリカンの糖鎖異常が認められたものの、正常型糖鎖もわず

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京  

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Language：English   Presentation type：Poster presentation  

Venue：二－ス, フランス  

Dystroglycanopathy (DGpathy) is caused by abnormal glycosylation of dystroglycan (DG). To establish effective treatment, we generated 2 distinct conditional knock-out (cKO) mice for fukutin, one of the causative genes for DGpathy. Myofiber-selective fukutin-cKO mice showed mild muscular dystrophy whereas muscle precursor cell (MPC)-selective cKO mice exhibited severe phenotypes

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名古屋  

筋ジストロフィーは、骨格筋の壊死・変性を主病変とし、進行性の筋力低下をともなう遺伝性疾患の総称で、多くの病型が存在する。中でも、基底膜ラミニン受容体のジストログリカンの糖鎖異常を発症要因とする疾患群は、ジストログリカノパチーと総称される。福山型筋ジストロフィーは、最初に同定されたジストログリカノパチーで、原因遺伝子フクチンの変異によって発症する。我々は、フクチンのコンディショナルノックアウト (cKO)マウスを作出し、筋細胞膜の脆弱化が発症の引き金になること、筋前駆細胞活性の低下が病態重篤度に関与することを明らかにしてきた。しかし、ジストログリカノパチーに有効な治療法は未だに確立されていない。本研究では、重篤な筋ジストロフィー病態を示す筋前駆細胞選択的フクチンcKOマウス（Myf5-fukutin-cKO）を疾患モデルとし、筋線維限定的なフクチン遺伝

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Language：Japanese   Presentation type：Poster presentation  

Venue：福岡  

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Language：English   Presentation type：Poster presentation  

Venue：シカゴ, 米国  

Dystroglycanopathy (DGpathy) is caused by abnormal glycosylation of dystroglycan (DG). To establish effective treatment, we generated 2 distinct conditional knock-out (cKO) mice for fukutin, one of the causative genes for DGpathy. Myofiber-selective fukutin-cKO mice showed mild muscular dystrophy whereas muscle precursor cell (MPC)-selective cKO mice exhibited severe phenotypes

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Language：Japanese   Presentation type：Poster presentation  

Venue：神戸  

筋ジストロフィーは筋の壊死・変性を主病変とし、進行性の筋力低下を伴う遺伝性疾患で、原因遺伝子や遺伝形式等によって多くの病型に分けられる。福山型筋ジストロフィー(FCMD)は、精神遅滞・脳形成不全を伴う重篤な筋ジストロフィーで、本邦における小児期筋ジストロフィーの中では、デュシャンヌ型筋ジストロフィー(DMD)に次いで頻度が高い。我々は以前FCMDの原因遺伝子fukutinを同定した。fukutin変異によって、細胞外マトリックスと細胞骨格を結ぶ膜タンパク質であるジストログリカンの糖鎖に異常が生じ、ラミニンなどの細胞外基質との結合能が低下する。FCMD以外にも糖鎖異常を病因とする筋ジストロフィーが知られており、ジストログリカノパチーと総称されるが、発症機序について不明な点は多い。本研究ではfukutinの生理的役割とジストログリカノパチー病態の解明を目

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：淡路  

Alpha-dystroglycan (α-DG) is a cell surface receptor for extracellular matrix proteins such as laminins. A novel post-translational modification on O-mannose termed post-phosphoryl modification is essential for the ligand-binding activity of α-DG. Abnormal glycosylation of α-DG is associated with a group of muscular dystrophy that is often accompanied by abnormalities in brain

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Language：English   Presentation type：Poster presentation  

Venue：Boston, USA  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：神戸  

ジストログリカンは細胞外マトリックスと細胞骨格を結ぶ膜タンパク質で、基底膜や細胞膜の物理的安定性の維持など、様々な組織で重要な役割を担っている。ジストログリカンは、多段階におよぶ糖鎖修飾やプロセシングなどの翻訳後修飾を経て成熟し、ラミニンなどのマトリックス分子結合能を獲得する。一方、糖鎖関連遺伝子の変異に起因するジストログリカンの糖鎖異常は、ジストログリカノパチーと総称される、中枢神経障害をともなう筋ジストロフィーの発症に関わることも知られている。最近、ジストログリカノパチー原因遺伝子が続々と同定され、また、原因遺伝子産物の機能解析から、ジストログリカンのユニークな翻訳後修飾の機序が徐々に明らかになってきた。本講演では、まず、ERやゴルジ体といった各々の修飾オルガネラにおける、ジストログリカン翻訳後修飾の機序を概説する。次に、ジストログリカノパチー遺

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Language：Japanese   Presentation type：Poster presentation  

Venue：神戸  

骨格筋は運動において必要不可欠な組織であり、加齢に伴う筋力低下は筋組織に存在する筋組織特異的幹細胞（衛星細胞）の増殖能の低下に起因すると考えられる。しかし、衛星細胞の増殖を制御する分子機構は未だ不明な点が多い。本研究では衛星細胞の増殖機構を解明するため、カルディオトキシンによる筋損傷修復モデルマウスおよびマウス筋芽細胞株C2C12細胞を用いて解析を行った。筋損傷修復モデルマウスの解析から、修復早期の筋組織では受容体型チロシンキナーゼRor1の発現が亢進し、また、それに先行して炎症性サイトカインTNFαの発現が亢進することが確認され、TNFαによるRor1の発現誘導の可能性が示唆された。実際、低濃度血清培地（分化培地）で培養したC2C12細胞をTNFαにより刺激した際にRor1の発現誘導が観察された。TNFαによるRor1の発現誘導は、NF-κB経路の

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Language：Japanese   Presentation type：Poster presentation  

Venue：東京  

福山型筋ジストロフィーをはじめとする、ジストログリカンの糖鎖異常を発症要因とする疾患群は、ジストログリカノパチーと総称される。我々は、ジストログリカノパチーの病態機序の解明と治療法の開発を目的に、福山型筋ジストロフィーの原因遺伝子フクチンの変異マウスを作出してきた。筋管選択的、筋前駆細胞選択的な２種の異なるコンディショナルノックアウト（cKO）マウスの比較から、細胞膜の脆弱化が発症の引き金になること、筋前駆細胞と筋再生の異常が病態の重篤度に関与することが明らかになった。一方、福山型患者で多くみられるレトロトランスポゾン挿入変異をノックインしたマウスでは、ジストログリカンの糖鎖異常が認められたものの、正常型糖鎖もわずかながら残存しており、発症に至らなかった。これらの疾患モデル研究より明らかになった病態機序を考慮すると、部分的な糖鎖回復でも治療効果が得ら

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Language：Japanese   Presentation type：Poster presentation  

Venue：東京  

Dystroglycanopathy is a group of muscular dystrophy caused by abnormal glycosylation of dystroglycan. Here, we explored the conditions needed for successful LARGE gene therapy for dystroglycanopathy. Our data show that myofiber-selective LARGE expression

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：東京  

筋ジストロフィーは、骨格筋の壊死・変性を主病変とする遺伝性疾患の総称で、多くの病型が存在する。中でも、ラミニン受容体であるジストログリカンのO-マンノース型糖鎖異常を発症要因とする疾患群（ジストログリカノパチー）は、筋病変にくわえ、脳奇形（II型滑脳症）や精神発達遅滞などの中枢神経障害と眼症状を伴う独特な特徴をしめす。本邦に多くみられる福山型筋ジストロフィー（原因遺伝子フクチン）は、ジストログリカノパチーとして初めて提唱された疾患である。本疾患群が中枢神経障害を伴う理由として、ジストログリカンに修飾されるO

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：新潟  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：神戸  

Muscular dystrophy is a heterogeneous group of genetic disorders that is characterized by the progressive loss of muscle strength and integrity. Dystroglycanopathy is a group of muscular dystrophy, which is commonly characterized by abnormal glycosylation of dystroglycan. Dystroglycan is a cell surface receptor for extracellular matrix proteins such as laminins, and a novel pos

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Language：English   Presentation type：Poster presentation  

Venue：Kobe  

The Skeletal muscle has a remarkable ability to regenerate following injury. In adult muscle, satellite cells play a central role in mediating regeneration of damaged muscle by acting as myogenic stem cells. Following injury, quiescent satellite cells are activated, and proliferate and differentiate to supply myoblasts. However, it remains elusive about the mechanisms regulatin

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Language：English   Presentation type：Poster presentation  

Venue：ホノルル, 米国  

Dystroglycan is a highly glycosylated peripheral membrane protein that functions as a cell surface receptor for proteins in the extracellular matrices and synapses. Abnormal glycosylation of dystroglycan causes several forms of muscular dystrophy, collectively called dystroglycanopathy. To understand the pathophysiological roles of dystroglycan glycosylation and to establish ef

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Language：Japanese   Presentation type：Poster presentation  

Venue：横浜  

骨格筋は高い再生能を有し、損傷が生じても速やかに再生し、その機能を回復する。成体骨格筋には、筋幹細胞として機能する筋衛星細胞と呼ばれる単核の細胞が存在する。筋組織に損傷が生じると静止期にある筋衛星細胞は活性化され、増殖を開始し、筋芽細胞へと分化することで筋再生に寄与する。また、損傷がなくとも、筋衛星細胞は必要に応じて活性化され増殖を再開し、筋組織の恒常性維持に働くことが知られている。損傷修復や恒常性維持の過程で、活性化された筋衛星細胞の一部は、未分化状態を維持し静止期に至ることにより、筋衛星細胞の枯渇が防がれている。しかし、骨格筋における筋衛星細胞の活性化及び維持の分子機構は未だ不明な点が多い。Rorファミリー受容体型チロシンキナーゼ（Ror1、Ror2）は、 発生過程の様々な組織で発現し、組織形成において重要な役割を担っている。我々は、成体マウス骨

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Language：English   Presentation type：Poster presentation  

Venue：ホノルル, 米国  

Fukuyama congenital muscular dystrophy (FCMD) is characterized by congenital muscular dystrophy in combination with abnormalities of the central nervous system (brain malformation characterized by micropolygyria of the cerebrum and cerebellum, type II lissencephaly) and severe mental retardation. Mutations in the fukutin gene, the FCMD responsible gene, cause abnormal glycosyla

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Language：Japanese   Presentation type：Poster presentation  

Venue：横浜  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：横浜  

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Language：English   Presentation type：Poster presentation  

Venue：ボルチモア, アメリカ合衆国  

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Language：English   Presentation type：Poster presentation  

Venue：バンクーバー, カナダ  

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Language：Japanese   Presentation type：Poster presentation  

Venue：東京  

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Language：English   Presentation type：Oral presentation (general)  

Venue：グラナダ, スペイン  

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Language：English   Presentation type：Poster presentation  

Venue：神戸  

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Language：Japanese   Presentation type：Poster presentation  

Venue：東京  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：神戸  

筋ジストロフィーは、筋力が進行的に低下する遺伝性疾患で、様々な病型が存在する。中でも、福山型筋ジストロフィーをはじめとする、ジストログリカンの糖鎖異常を発症要因とする疾患群は、ジストログリカノパチーと総称されている。ジストログリカンは細胞外で基底膜成分のラミニンと、細胞内でジストロフィンと結合しており、基底膜－細胞膜の連携を担う糖タンパク質である。ジストログリカンがラミニンと結合するためには、ポストリン酸糖鎖とよばれるユニークな修飾体が必要であるが、ジストログリカノパチーでは、この修飾異常に伴うラミニン結合能の低下によって基底膜－細胞膜の連携が破綻し、発症すると考えられている。ここ数年、新たなジストログリカノパチー遺伝子や糖鎖構造が徐々に明らかになり、ポストリン酸糖鎖が骨格筋の恒常性維持に関わる翻訳後修飾体として着目されている。我々は、福山型筋ジスト

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：淡路  

Fukuyama congenital muscular dystrophy (FCMD) is characterized by congenital muscular dystrophy in combination with abnormalities of the central nervous system (brain malformation characterized by micropolygyria of the cerebrum and cerebellum, type II lissencephaly) and severe mental retardation. Mutations in the fukutin gene, the FCMD responsible gene, cause abnormal glycosyla

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸  

骨格筋は再生能の高い組織であり、再生過程では骨格筋特異的な組織幹細胞である衛星細胞が必須の役割を担っている。通常、衛星細胞は静止期にあるが、骨格筋損傷後には活性化し細胞増殖を開始する。増殖した衛星細胞は筋芽細胞に分化し、最終的に新たな筋線維形成に寄与することで骨格筋の損傷部位の修復に貢献する。これまでに衛星細胞は骨格筋再生に必須であることが報告されてきているが、その制御機構については不明な点も多い。また、骨格筋常在性の間葉系幹細胞であるFibro/adipogenic progenitor cells(FAPs)が骨格筋再生時の衛星細胞の機能を制御することが近年報告されている。FAPsは筋疾患で観察される線維化や脂肪化の原因の一つとして考えられており、その機能制御機構は重要と考えられているが、未解明な点が多い。これらのことから、骨格筋再生メカニズムを

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Language：English   Presentation type：Poster presentation  

Venue：京都  

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Language：English   Presentation type：Oral presentation (general)  

Venue：神戸  

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Language：English   Presentation type：Poster presentation  

Venue：オーランド, アメリカ  

Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. -dystroglycan (-DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pen

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Language：Japanese   Presentation type：Poster presentation  

Venue：東京  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸  

【目的】ジストログリカン（DG）は、基底膜成分のラミニンやシナプス分子の膜受容体で、リガンドとの結合にはO-マンノース(O-Man)型の糖鎖修飾が必要である。O-Man型糖鎖の末端には、キシロース（Xyl）とグルクロン酸（GlcA）からなるリピート構造があり、直接のリガンド結合部位として機能している。また、O-Man型糖鎖の異常は、ジストログリカン異常症（DG異常症）と総称される筋ジストロフィーの原因になることが知られている。しかし、ジストログリカンの糖鎖構造の全容や、DG異常症原因遺伝子fukutin、fukutin-related protein (FKRP), isoprenoid synthase domain containing (ISPD)などの機能は不明だった。本研究では、DG異常症の発症機序の理解と治療法への道筋を拓くことを目的に、

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：大阪  

筋ジストロフィーは徐々に筋力が失われていく遺伝性疾患の総称で、様々な病型が存在するものの、いずれも有効な治療法は存在しない難病である。ジストログリカン異常症（DG異常症）は、中枢神経障害も伴う最重篤型の筋ジストロフィーのひとつで、ジストログリカンに修飾される糖鎖の異常が発症要因とされる(1)。現在18種類のDG異常症にかかわる遺伝子が知られている。日本で最も多くみられるDG異常症は福山型筋ジストロフィーで、原因遺伝子はフクチン（fukutin）である(2)。欧米では肢帯型筋ジストロフィー2Iが多くみられ、フクチンと相同性のあるfukutin-related protein (FKRP)が原因遺伝子として知られている(3)。ジストログリカン（DG）は、基底膜成分のラミニンやシナプス分子の膜受容体で、リガンドとの結合にはO-マンノース型の糖鎖修飾が必要で

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Language：English   Presentation type：Poster presentation  

Venue：Chicago, USA  

Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pen

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Language：English   Presentation type：Poster presentation  

Venue：東京  

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Language：Japanese   Presentation type：Poster presentation  

Venue：神戸市  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：西宮市  

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Language：English   Presentation type：Symposium, workshop panel (nominated)  

Venue：神戸  

筋ジストロフィーは進行性の筋力の低下をみとめる遺伝性疾患の総称で様々な病型が存在する。1990年代に筋ジストロフィー原因遺伝子の機能が明らかになりはじめ、基底膜と細胞膜の連携が筋の恒常性維持に重要であることが確立されてきた。2000年代にはいると基底膜受容体であるジストログリカンの糖鎖異常を認める疾患が続々と同定され、ジストログリカン異常症という新たな疾患概念が樹立された。糖鎖に異常が生じると基底膜との結合活性が消失する。しかし、発症に直結する糖鎖構造やその修飾に関わる酵素は長年の間不明であった。我々は、ジストログリカン糖鎖の中に、キシリトール系糖アルコールのリビトールが存在し、リビトールリン酸タンデム構造を形成していることを発見した。更に、筋ジストロフィー原因遺伝子で機能未知であったIsoprenoid synthase domain-contai

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：横浜  

糖鎖修飾は様々な生命機能に重要な翻訳後修飾で、その異常は時に重篤な疾患を導く。ジストログリカンは基底膜やシナプス分子の受容体で、糖鎖修飾はリガンド結合活性に不可欠であり、その異常は筋ジストロフィーや滑脳症の原因になる。しかしながら、ジストログリカン糖鎖の構造や修飾酵素の全貌は明らかにされていなかった。今回、我々は、ジストログリカン糖鎖の中に、キシリトール系糖アルコールのリビトールが存在し、リビトールリン酸タンデム構造を形成していることを発見した。更に、筋ジストロフィー原因遺伝子で機能未知であったIsoprenoid synthase domain-containing (ISPD)、フクチン、Fukutin-related protein (FKRP)が、リビトールリン酸修飾に関わる酵素であることを同定した。ISPDはリビトールリン酸の供与体となるC

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：シンガポール, シンガポール  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸  

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Language：Japanese   Presentation type：Oral presentation (general)  

Venue：熱海  

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Language：English   Presentation type：Poster presentation  

Venue：神戸  

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