The Experts below are selected from a list of 339 Experts worldwide ranked by ideXlab platform
Motoi Kanagawa - One of the best experts on this subject based on the ideXlab platform.
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Crystal structures of Fukutin-Related Protein (FKRP), a ribitol-phosphate transferase Related to muscular dystrophy.
Nature communications, 2020Co-Authors: Naoyuki Kuwabara, Kazuhiro Kobayashi, Tatsushi Toda, Rieko Imae, Hiroki Tsumoto, Mamoru Mizuno, Motoi Kanagawa, Tomohiro Tanaka, Toshiya SendaAbstract:α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane Protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and Fukutin-Related Protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.
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cdp glycerol inhibits the synthesis of the functional o mannosyl glycan of α dystroglycan
Journal of Biological Chemistry, 2018Co-Authors: Rieko Imae, Kazuhiro Kobayashi, Tatsushi Toda, Hiroki Tsumoto, Mamoru Mizuno, Motoi Kanagawa, Tomohiro Tanaka, Hiroshi Manya, Kenji OsumiAbstract: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.
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Cell endogenous activities of Fukutin and FKRP coexist with the ribitol xylosyltransferase, TMEM5
Biochemical and Biophysical Research Communications, 2018Co-Authors: Ryuta Nishihara, Kazuhiro Kobayashi, Rieko Imae, Hiroki Tsumoto, Mamoru Mizuno, Motoi Kanagawa, Tamao Endo, Tatsushi TodaAbstract:Dystroglycanopathies are a group of muscular dystrophies that are caused by abnormal glycosylation of dystroglycan; currently 18 causative genes are known. Functions of the dystroglycanopathy genes Fukutin, Fukutin-Related Protein (FKRP), and transmembrane Protein 5 (TMEM5) were most recently identified; Fukutin and FKRP are ribitol-phosphate transferases and TMEM5 is a ribitol xylosyltransferase. In this study, we show that Fukutin, FKRP, and TMEM5 form a complex while maintaining each of their enzyme activities. Immunoprecipitation and immunofluorescence experiments demonstrated Protein interactions between these 3 Proteins. A Protein complex consisting of endogenous Fukutin and FKRP, and exogenously expressed TMEM5 exerts activities of each enzyme. Our data showed for the first time that endogenous Fukutin and FKRP enzyme activities coexist with TMEM5 enzyme activity, and suggest the possibility that formation of this enzyme complex may contribute to specific and prompt biosynthesis of glycans that are required for dystroglycan function.
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identification of a post translational modification with ribitol phosphate and its defect in muscular dystrophy
Cell Reports, 2016Co-Authors: Motoi Kanagawa, Kazuhiro Kobayashi, Mamoru Mizuno, Atsushi Kuga, Michiko Tajiri, Yoshiki Yamaguchi, Keiko Akasakamanya, Junichi Furukawa, Hiroko KawakamiAbstract:Summary 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.
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G.P.11 Fukutin-Related Protein (FKRP) is involved in the post-phosphoryl modification of α-dystroglycan
Neuromuscular Disorders, 2012Co-Authors: Atsushi Kuga, Kazuhiro Kobayashi, Motoi Kanagawa, Tamao Endo, Atsushi Sudo, Y.m. Chan, Michiko Tajiri, Y. WadaAbstract:Abstract Aberrant glycosylation of α -dystroglycan ( α -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 Fukutin and LARGE are involved in phosphodiester-linked modification of O-mannose on α -DG. Fukutin-Related Protein (FKRP) is a responsible gene of dystroglycanopathy, however its precise function is still unknown. In this study, we use several dystroglycanopathy mouse models to demonstrate that FKRP is also involved in the phosphodiester-linked modification. Furthermore, we have found that the glycosylation status of α -DG in lung and testis is minimally affected by defects in Fukutin, LARGE or FKRP. α -DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results suggest that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of α -DG functional expression as a laminin receptor in normal tissues and cells.
Kazuhiro Kobayashi - One of the best experts on this subject based on the ideXlab platform.
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Crystal structures of Fukutin-Related Protein (FKRP), a ribitol-phosphate transferase Related to muscular dystrophy.
Nature communications, 2020Co-Authors: Naoyuki Kuwabara, Kazuhiro Kobayashi, Tatsushi Toda, Rieko Imae, Hiroki Tsumoto, Mamoru Mizuno, Motoi Kanagawa, Tomohiro Tanaka, Toshiya SendaAbstract:α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane Protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and Fukutin-Related Protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.
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cdp glycerol inhibits the synthesis of the functional o mannosyl glycan of α dystroglycan
Journal of Biological Chemistry, 2018Co-Authors: Rieko Imae, Kazuhiro Kobayashi, Tatsushi Toda, Hiroki Tsumoto, Mamoru Mizuno, Motoi Kanagawa, Tomohiro Tanaka, Hiroshi Manya, Kenji OsumiAbstract: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.
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Cell endogenous activities of Fukutin and FKRP coexist with the ribitol xylosyltransferase, TMEM5
Biochemical and Biophysical Research Communications, 2018Co-Authors: Ryuta Nishihara, Kazuhiro Kobayashi, Rieko Imae, Hiroki Tsumoto, Mamoru Mizuno, Motoi Kanagawa, Tamao Endo, Tatsushi TodaAbstract:Dystroglycanopathies are a group of muscular dystrophies that are caused by abnormal glycosylation of dystroglycan; currently 18 causative genes are known. Functions of the dystroglycanopathy genes Fukutin, Fukutin-Related Protein (FKRP), and transmembrane Protein 5 (TMEM5) were most recently identified; Fukutin and FKRP are ribitol-phosphate transferases and TMEM5 is a ribitol xylosyltransferase. In this study, we show that Fukutin, FKRP, and TMEM5 form a complex while maintaining each of their enzyme activities. Immunoprecipitation and immunofluorescence experiments demonstrated Protein interactions between these 3 Proteins. A Protein complex consisting of endogenous Fukutin and FKRP, and exogenously expressed TMEM5 exerts activities of each enzyme. Our data showed for the first time that endogenous Fukutin and FKRP enzyme activities coexist with TMEM5 enzyme activity, and suggest the possibility that formation of this enzyme complex may contribute to specific and prompt biosynthesis of glycans that are required for dystroglycan function.
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identification of a post translational modification with ribitol phosphate and its defect in muscular dystrophy
Cell Reports, 2016Co-Authors: Motoi Kanagawa, Kazuhiro Kobayashi, Mamoru Mizuno, Atsushi Kuga, Michiko Tajiri, Yoshiki Yamaguchi, Keiko Akasakamanya, Junichi Furukawa, Hiroko KawakamiAbstract:Summary 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.
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G.P.11 Fukutin-Related Protein (FKRP) is involved in the post-phosphoryl modification of α-dystroglycan
Neuromuscular Disorders, 2012Co-Authors: Atsushi Kuga, Kazuhiro Kobayashi, Motoi Kanagawa, Tamao Endo, Atsushi Sudo, Y.m. Chan, Michiko Tajiri, Y. WadaAbstract:Abstract Aberrant glycosylation of α -dystroglycan ( α -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 Fukutin and LARGE are involved in phosphodiester-linked modification of O-mannose on α -DG. Fukutin-Related Protein (FKRP) is a responsible gene of dystroglycanopathy, however its precise function is still unknown. In this study, we use several dystroglycanopathy mouse models to demonstrate that FKRP is also involved in the phosphodiester-linked modification. Furthermore, we have found that the glycosylation status of α -DG in lung and testis is minimally affected by defects in Fukutin, LARGE or FKRP. α -DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results suggest that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of α -DG functional expression as a laminin receptor in normal tissues and cells.
Derek J Blake - One of the best experts on this subject based on the ideXlab platform.
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Fukutin Related Protein is essential for mouse muscle brain and eye development and mutation recapitulates the wide clinical spectrums of dystroglycanopathies
Human Molecular Genetics, 2010Co-Authors: Yiumo Michael Chan, Derek J Blake, Jignya Ashar, Elizabeth Keramarisvrantsis, Hart G W Lidov, James Norton, Natalia Zinchenko, Helen E Gruber, Randy J Thresher, Jeffrey RosenfeldAbstract:Mutations in Fukutin-Related Protein (FKRP) cause a common subset of muscular dystrophies characterized by aberrant glycosylation of alpha-dystroglycan (α-DG), collectively known as dystroglycanopathies. The clinical variations associated with FKRP mutations range from mild limb-girdle muscular dystrophy type 2I with predominantly muscle phenotypes to severe Walker–Warburg syndrome and muscle–eye–brain disease with striking structural brain and eye defects. In the present study, we have generated animal models and demonstrated that ablation of FKRP functions is embryonic lethal and that the homozygous-null embryos die before reaching E12.5. The homozygous knock-in mouse carrying the missense P448L mutation almost completely lacks functional glycosylation of α-DG in muscles and brain, validating the essential role of FKRP in the functional glycosylation of α-DG. However, the knock-in mouse survives and develops a wide range of structural abnormalities in the central nervous system, characteristics of neuronal migration defects. The brain and eye defects are highly reminiscent of the phenotypes seen in severe dystroglycanopathy patients. In addition, skeletal muscles develop progressive muscular dystrophy. Our results confirm that post-translational modifications of α-DG are essential for normal development of the brain and eyes. In addition, both the mutation itself and the levels of FKRP expression are equally critical for the survival of the animals. The exceptionally wide clinical spectrums recapitulated in the P448L mice also suggest the involvement of other factors in the disease progression. The mutant mouse represents a valuable model to further elucidate the functions of FKRP and develop therapies for FKRP-Related muscular dystrophies.
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Mutations alter secretion of Fukutin-Related Protein.
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2010Co-Authors: Allen Zillmer, Derek J Blake, Hanns Lochmüller, Judy C. Vachris, Yiumo Michael ChanAbstract:Mutations in the Fukutin-Related Protein (FKRP) gene cause limb-girdle muscular dystrophy type 2I (LGMD2I) as well as other severe muscle disorders, including Walker-Warburg syndrome, muscle-eye-brain disease, and congenital muscular dystrophy type 1C. The FKRP gene encodes a putative glycosyltransferase, but its precise localization and functions have yet to be determined. In the present study, we demonstrated that normal FKRP is secreted into culture medium and mutations alter the pattern of secretion in CHO cells. L276I mutation associated with mild disease phenotype was shown to reduce the level of secretion whereas P448L and C318Y mutations associated with severe disease phenotype almost abolished the secretion. However, a truncated FKRP mutant Protein lacking the entire C-terminal 185 amino acids due to the E310X nonsense mutation was able to secrete as efficiently as the normal FKRP. The N-terminal signal peptide sequence is apparently cleaved from the secreted FKRP Proteins. Alteration of the secretion pathway by different mutations and spontaneous read-through of nonsense mutation may contribute to wide variations in phenotypes associated with FKRP-Related diseases.
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Fukutin-Related Protein localizes to the Golgi apparatus and mutations lead to mislocalization in muscle in vivo.
Muscle & Nerve, 2007Co-Authors: Elizabeth Keramaris-vrantsis, Christopher T Esapa, Derek J Blake, Matthew A Benson, Allen Zillmer, Timothy Doran, Jignya Ashar, Jeffrey RosenfeldAbstract:Mutations in the Fukutin-Related Protein gene (FKRP) are associated with a spectrum of diseases from mild limb-girdle muscular dystrophy type 2I to severe congenital muscular dystrophy type 1C, muscle–eye–brain disease (MEB), and Walker–Warburg syndrome (WWS). The effect of mutations on the transportation of the mutant Proteins may constitute the underlying mechanisms for the pathogenesis of these diseases. Here we examined the subcellular localization of mouse and human normal and mutant FKRP Proteins in cells and in muscle in vivo. Both normal human and mouse FKRPs localize in part of the Golgi apparatus in muscle fibers. Mutations in the FKRP gene invariably altered the localization of the Protein, leading to endoplasmic reticulum retention within cells and diminished Golgi localization in muscle fibers. Our results therefore suggest that an individual missense point mutation can confer at least two independent effects on the Protein, causing (1) reduction or loss of the presumed glycosyltransferase activity directly and (2) mislocalization that could further alter the function of the Protein. The complexity of the effect of individual missense point mutations may partly explain the wide variation of the FKRP-Related myopathies.
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Fukutin Related Protein mutations that cause congenital muscular dystrophy result in er retention of the mutant Protein in cultured cells
Human Molecular Genetics, 2005Co-Authors: Christopher T Esapa, R Jeffrey A Mcilhinney, Derek J BlakeAbstract:: Mutations in the gene encoding Fukutin-Related Protein (FKRP) cause a spectrum of diseases including congenital muscular dystrophy type 1C (MDC1C), limb girdle muscular dystrophy 2I (LGMD2I) and congenital muscular dystrophies (CMDs) with brain malformations and mental retardation. Although these diseases are associated with abnormal dystroglycan processing, the cellular consequences of the idiosyncratic FKRP mutations have not been determined. Here we show, in cultured cells, that FKRP mutants associated with the more severe disease phenotypes (S221R, A455D, P448L) are retained in the endoplasmic reticulum (ER), whereas the wild-type Protein and the mutant L276I that causes LGMD2I are found predominantly in the Golgi apparatus. The ER-retained Proteins have a shorter half-life than the wild-type FKRP and are preferentially degraded by the proteasome. Furthermore, calnexin binds preferentially to the ER-retained mutants suggesting that it may participate in the quality control pathway for FKRP. These data provide the first evidence that the ER-retention of mutant FKRP may play a role in the pathogenesis of CMD and potentially explain why the allelic disorder LGMD2I is milder, because the mutated Protein is able to reach the Golgi apparatus.
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fkrp gene mutations cause congenital muscular dystrophy mental retardation and cerebellar cysts
Neurology, 2003Co-Authors: Haluk Topaloglu, Derek J Blake, Silvia Torelli, S. C. Brown, Y Yuva, M Brockington, Beril Talim, Goknur Haliloglu, F. MuntoniAbstract:Background: Congenital muscular dystrophies (CMD) are autosomal recessive disorders that present within the first 6 months of life with hypotonia and a dystrophic muscle biopsy. CNS involvement is present in some forms. The Fukutin-Related Protein gene ( FKRP ) is mutated in a severe form of CMD (MDC1C) and a milder limb girdle dystrophy (LGMD2I). Both forms have secondary deficiencies of laminin α2 and α-dystroglycan immunostaining. Structural brain involvement has not been observed in patients with FKRP gene mutations. Methods: The authors studied two unRelated patients who had a pattern of muscle involvement identical to MDC1C, mental retardation, and cerebellar cysts on cranial MRI. The FKRP gene was analyzed along with the skeletal muscle expression of laminin α2 and α-dystroglycan. Results: The muscle biopsy of both patients showed severe dystrophic findings, a reduction in laminin α2, and profound depletion of α-dystroglycan. Both patients had homozygous FKRP gene mutations not previously reported (C663A [Ser221Arg] and C981A [Pro315Thr]). Conclusions: Mutations within the FKRP gene can result in CMD associated with mental retardation and cerebellar cysts. This adds structural brain defects to the already wide spectrum of abnormalities caused by FKRP mutations. The severe depletion of α-dystroglycan expression suggests that FKRP is involved in the processing of α-dystroglycan.
Susan C. Brown - One of the best experts on this subject based on the ideXlab platform.
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Fukutin-Related Protein Alters the Deposition of Laminin in the Eye and Brain
Journal of Neuroscience, 2011Co-Authors: M.r. Ackroyd, Francesco Muntoni, M. Kaluarachchi, C. Whitmore, Sarah Prior, Margareta Nikolic, Ulrike Mayer, Susan C. BrownAbstract:Mutations in Fukutin-Related Protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement. The defining feature of this group of disorders is the hypoglycosylation of α-dystroglycan and its inability to effectively bind extracellular matrix ligands such as laminin α2. However, α-dystroglycan has the potential to interact with a number of laminin isoforms many of which are basement membrane/tissue specific and developmentally regulated. To further investigate this we evaluated laminin α-chain expression in the cerebral cortex and eye of our FKRP knock-down mouse (FKRPKD). These mice showed a marked disturbance in the deposition of laminin α-chains including α1, α2, α4, and α5, although only laminin α1- and γ1-chain mRNA expression was significantly upregulated relative to controls. Moreover, there was a diffuse pattern of laminin deposition below the pial surface which corRelated with an abrupt termination of many of the radial glial cells. This along with the pial basement membrane defects, contributed to the abnormal positioning of both early- and late-born neurons. Defects in the inner limiting membrane of the eye were associated with a reduction of laminin α1 demonstrating the involvement of the α-dystroglycan:laminin α1 axis in the disease process. These observations demonstrate for the first time that a reduction in Fkrp influences the ability of tissue-specific forms of α-dystroglycan to direct the deposition of several laminin isoforms in the formation of different basement membranes.
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Reduced expression of Fukutin Related Protein in mice results in a model for Fukutin Related Protein associated muscular dystrophies.
Brain, 2008Co-Authors: M.r. Ackroyd, L Skordis, M. Kaluarachchi, J. Godwin, S. Prior, M. Fidanboylu, Richard J. Piercy, Susan C. BrownAbstract:Mutations in Fukutin Related Protein (FKRP) are responsible for a common group of muscular dystrophies ranging from adult onset limb girdle muscular dystrophies to severe congenital forms with associated structural brain involvement, including Muscle Eye Brain disease. A common feature of these disorders is the variable reduction in the glycosylation of skeletal muscle -dystroglycan. In order to gain insight into the pathogenesis and clinical variability, we have generated two lines of mice, the first containing a missense mutation and a neomycin cassette, FKRP-Neo(Tyr307Asn) and the second containing the FKRPTyr307Asn mutation alone. We have previously associated this missense mutation with a severe muscleeyebrain phenotype in several families. Homozygote Fkrp-Neo(Tyr307Asn) mice die soon after birth and show a reduction in the laminin-binding epitope of -dystroglycan in muscle, eye and brain, and have reduced levels of FKRP transcript. Homozygous Fkrp(Tyr307Asn) mice showed no discernible phenotype up to 6 months of age, contrary to the severe clinical course observed in patients with the same mutation. These results suggest the generation of a mouse model for FKRP Related muscular dystrophy requires a knock-down rather than a knock-in strategy in order to give rise to a disease phenotype.
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Mutated Fukutin-Related Protein (FKRP) localises as wild type in differentiated muscle cells.
Experimental Cell Research, 2005Co-Authors: N Dolatshad, Francesco Muntoni, Silvia Torelli, Martin Brockington, L Skordis, U Wever, Dominic J. Wells, Susan C. BrownAbstract:The mechanism of disease in forms of congenital and limb girdle muscular dystrophy linked to mutations in the gene encoding for Fukutin-Related Protein (FKRP) has previously been associated with the mis-localisation of FKRP from the Golgi apparatus. In the present report, we have transfected V5-tagged Fukutin-Related Protein expression constructs into differentiated C2C12 myotubes and the tibialis anterior of normal mice. The transfection of either wild type (WT) or several mutant constructs (P448L, C318Y, L276I) into myotubes consistently showed clear co-localisation with GM130, a Golgi marker. In contrast, whilst WT and the L276I localised to the Golgi of Cos-7 cells, the P448L and C318Y was mis-localised in the majority of these undifferentiated cells. The injection of the same constructs into the tibialis anterior of mice resulted in similar localisation of both the WT and all the mutants. Immunolabelling of FKRP in the muscle of MDC1C and LGMD2I patients was found to be indistinguishable from normal controls. Overall, these data suggest that retention in the endoplasmic reticulum of FKRP is not the main mechanism of disease but that this may instead relate to a disruption of the functional activity of this putative enzyme with its substrate(s) in the Golgi.
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Abnormalities in α-Dystroglycan Expression in MDC1C and LGMD2I Muscular Dystrophies
The American journal of pathology, 2004Co-Authors: Susan C. Brown, Silvia Torelli, Martin Brockington, Stephan Kroger, L Feng, Y Yuva, Louise V B Anderson, Cecilia Jimenez, Isabella Ugo, Kate BushbyAbstract:We recently identified mutations in the Fukutin Related Protein (FKRP) gene in patients with congenital muscular dystrophy type 1C (MDC1C) and limb girdle muscular dystrophy type 2I (LGMD2I). The sarcolemma of these patients typically displays an immunocytochemical reduction of α-dystroglycan. In this report we extend these observations and report a clear correlation between the residual expression of α-dystroglycan and the phenotype. Three broad categories were identified. Patients at the severe end of the clinical spectrum (MDC1C) were compound heterozygote between a null allele and a missense mutation or carried two missense mutations and displayed a profound depletion of α-dystroglycan. Patients with LGMD with a Duchenne-like severity typically had a moderate reduction in α-dystroglycan and were compound heterozygotes between a common C826A (Leu276Ileu) FKRP mutation and either a missense or a nonsense mutation. Individuals with the milder form of LGMD2I were almost invariably homozygous for the Leu276Ile FKRP mutation and showed a variable but subtle alteration in α-dystroglycan immunolabeling. Our data therefore suggest a correlation between a reduction in α-dystroglycan, the mutation and the clinical phenotype in MDC1C and LGMD2I which supports the hypothesis that dystroglycan plays a central role in the pathogenesis of these disorders.
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functional requirements for Fukutin Related Protein in the golgi apparatus
Human Molecular Genetics, 2002Co-Authors: Christopher T Esapa, Francesco Muntoni, Martin Brockington, Susan C. Brown, Matthew A Benson, Jorn E Schroder, Enca Martinrendon, Stephan Kroger, Derek J BlakeAbstract:Two forms of congenital muscular dystrophy (CMD), Fukuyama CMD and CMD type 1C (MDC1C) are caused by mutations in the genes encoding two putative glycosyltransferases, Fukutin and Fukutin-Related Protein (FKRP). Additionally, mutations in the FKRP gene also cause limb-girdle muscular dystrophy type 2I (LGMD2I), a considerably milder allelic variant than MDC1C. All of these diseases are associated with secondary changes in muscle alpha-dystroglycan expression. To elucidate the function of FKRP and Fukutin and examine the effects of MDC1C patient mutations, we have determined the mechanism for the subcellular location of each Protein. FKRP and Fukutin are targeted to the medial-Golgi apparatus through their N-termini and transmembrane domains. Overexpression of FKRP in CHO cells alters the post-translational processing of alpha- and beta-dystroglycan inhibiting maturation of the two isoforms. Mutations in the DxD motif in the putative active site of the Protein or in the Golgi-targeting sequence, which cause FKRP to be inefficiently trafficked to the Golgi apparatus, did not alter dystroglycan processing in vitro. The P448L mutation in FKRP that causes congenital muscular dystrophy changes a conserved amino acid resulting in the mislocalization of the mutant Protein in the cell that is unable to alter dystroglycan processing. Our data show that FKRP and Fukutin are Golgi-resident Proteins and that FKRP is required for the post-translational modi cation of dystroglycan. Aberrant processing of dystroglycan caused by a mislocalized FKRP mutant could be a novel mechanism that causes congenital muscular dystrophy.
Atsushi Kuga - One of the best experts on this subject based on the ideXlab platform.
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identification of a post translational modification with ribitol phosphate and its defect in muscular dystrophy
Cell Reports, 2016Co-Authors: Motoi Kanagawa, Kazuhiro Kobayashi, Mamoru Mizuno, Atsushi Kuga, Michiko Tajiri, Yoshiki Yamaguchi, Keiko Akasakamanya, Junichi Furukawa, Hiroko KawakamiAbstract:Summary 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.
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G.P.11 Fukutin-Related Protein (FKRP) is involved in the post-phosphoryl modification of α-dystroglycan
Neuromuscular Disorders, 2012Co-Authors: Atsushi Kuga, Kazuhiro Kobayashi, Motoi Kanagawa, Tamao Endo, Atsushi Sudo, Y.m. Chan, Michiko Tajiri, Y. WadaAbstract:Abstract Aberrant glycosylation of α -dystroglycan ( α -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 Fukutin and LARGE are involved in phosphodiester-linked modification of O-mannose on α -DG. Fukutin-Related Protein (FKRP) is a responsible gene of dystroglycanopathy, however its precise function is still unknown. In this study, we use several dystroglycanopathy mouse models to demonstrate that FKRP is also involved in the phosphodiester-linked modification. Furthermore, we have found that the glycosylation status of α -DG in lung and testis is minimally affected by defects in Fukutin, LARGE or FKRP. α -DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results suggest that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of α -DG functional expression as a laminin receptor in normal tissues and cells.
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absence of post phosphoryl modification in dystroglycanopathy mouse models and wild type tissues expressing non laminin binding form of α dystroglycan
Journal of Biological Chemistry, 2012Co-Authors: Atsushi Kuga, Kazuhiro Kobayashi, Motoi Kanagawa, Atsushi Sudo, Michiko Tajiri, Yiumo Michael Chan, Yamato Kikkawa, Motoyoshi Nomizu, Tamao EndoAbstract:Abstract α-Dystroglycan (α-DG) is a membrane-associated glycoProtein that interacts with several extracellular matrix Proteins, including laminin and agrin. Aberrant glycosylation of α-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-α2,3-Gal-β1,4-GlcNAc-β1,2-Man) and a phosphodiester-linked modification on O-mannose play important roles in the laminin binding activity of α-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 α-DG. Furthermore, we have found that the glycosylation status of α-DG in lung and testis is minimally affected by defects in Fukutin, LARGE, or FKRP. α-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 α-DG functional expression as a laminin receptor in normal tissues and cells.
