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1.
Elife ; 32014 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-25279699

RESUMO

Dystroglycan is a cell membrane receptor that organizes the basement membrane by binding ligands in the extracellular matrix. Proper glycosylation of the α-dystroglycan (α-DG) subunit is essential for these activities, and lack thereof results in neuromuscular disease. Currently, neither the glycan synthesis pathway nor the roles of many known or putative glycosyltransferases that are essential for this process are well understood. Here we show that FKRP, FKTN, TMEM5 and B4GAT1 (formerly known as B3GNT1) localize to the Golgi and contribute to the O-mannosyl post-phosphorylation modification of α-DG. Moreover, we assigned B4GAT1 a function as a xylose ß1,4-glucuronyltransferase. Nuclear magnetic resonance studies confirmed that a glucuronic acid ß1,4-xylose disaccharide synthesized by B4GAT1 acts as an acceptor primer that can be elongated by LARGE with the ligand-binding heteropolysaccharide. Our findings greatly broaden the understanding of α-DG glycosylation and provide mechanistic insight into why mutations in B4GAT1 disrupt dystroglycan function and cause disease.


Assuntos
Distroglicanas/metabolismo , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/metabolismo , Animais , Linhagem Celular , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Ácido Glucurônico/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutação/genética , Fosforilação , Transporte Proteico , Frações Subcelulares/enzimologia , Especificidade por Substrato , Xilose/metabolismo
2.
J Biol Chem ; 288(4): 2132-42, 2013 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-23223448

RESUMO

Dystroglycan (DG) is a cell surface receptor for extracellular matrix proteins and is involved in cell polarity, matrix organization, and mechanical stability of tissues. Previous studies documented loss of DG protein expression and glycosylation in a variety of cancer types, but the underlying mechanisms and the functional consequences with respect to cancer progression remain unclear. Here, we show that the level of expression of the ßDG subunit as well as the glycosylation status of the αDG subunit inversely correlate with the Gleason scores of prostate cancers; furthermore, we show that the functional glycosylation of αDG is substantially reduced in prostate cancer metastases. Additionally, we demonstrate that LARGE2 (GYLTL1B), a gene not previously implicated in cancer, regulates functional αDG glycosylation in prostate cancer cell lines; knockdown of LARGE2 resulted in hypoglycosylation of αDG and loss of its ability to bind laminin-111 while overexpression restored ligand binding and diminished growth and migration of an aggressive prostate cancer cell line. Finally, our analysis of LARGE2 expression in human cancer specimens reveals that LARGE2 is significantly down-regulated in the context of prostate cancer, and that its reduction correlates with disease progression. Our results describe a novel molecular mechanism to account for the commonly observed hypoglycosylation of αDG in prostate cancer.


Assuntos
Distroglicanas/genética , Distroglicanas/fisiologia , Regulação Neoplásica da Expressão Gênica , Glicosiltransferases/genética , Glicosiltransferases/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Neoplasias da Próstata/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Separação Celular , Progressão da Doença , Epitélio/metabolismo , Matriz Extracelular/metabolismo , Citometria de Fluxo , Glicosilação , Humanos , Imuno-Histoquímica/métodos , Laminina/metabolismo , Masculino , Microscopia de Fluorescência/métodos , Invasividade Neoplásica , RNA Interferente Pequeno/metabolismo
3.
N Engl J Med ; 364(10): 939-46, 2011 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-21388311

RESUMO

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 α-dystroglycan that is required for high-affinity binding to laminin.


Assuntos
Distroglicanas/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Mutação de Sentido Incorreto , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Linhagem , Fenótipo , Análise de Sequência de DNA
4.
J Clin Invest ; 120(12): 4366-74, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21060153

RESUMO

Mutations in the dysferlin gene underlie a group of autosomal recessive muscle-wasting disorders denoted as dysferlinopathies. Dysferlin has been shown to play roles in muscle membrane repair and muscle regeneration, both of which require vesicle-membrane fusion. However, the mechanism by which muscle becomes dystrophic in these disorders remains poorly understood. Although muscle inflammation is widely recognized in dysferlinopathy and dysferlin is expressed in immune cells, the contribution of the immune system to the pathology of dysferlinopathy remains to be fully explored. Here, we show that the complement system plays an important role in muscle pathology in dysferlinopathy. Dysferlin deficiency led to increased expression of complement factors in muscle, while muscle-specific transgenic expression of dysferlin normalized the expression of complement factors and eliminated the dystrophic phenotype present in dysferlin-null mice. Furthermore, genetic disruption of the central component (C3) of the complement system ameliorated muscle pathology in dysferlin-deficient mice but had no significant beneficial effect in a genetically distinct model of muscular dystrophy, mdx mice. These results demonstrate that complement-mediated muscle injury is central to the pathogenesis of dysferlinopathy and suggest that targeting the complement system might serve as a therapeutic approach for this disease.


Assuntos
Complemento C3/deficiência , Complemento C3/genética , Proteínas de Membrana/deficiência , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Animais , Disferlina , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Camundongos Transgênicos , Contração Muscular , Músculo Esquelético/imunologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/imunologia , Distrofia Muscular Animal/fisiopatologia
5.
PLoS One ; 5(3): e9915, 2010 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-20369005

RESUMO

BACKGROUND: Alpha-dystroglycan (alpha-DG) is a cell surface receptor providing a molecular link between the extracellular matrix (ECM) and the actin-based cytoskeleton. During its biosynthesis, alpha-DG undergoes specific and unusual O-glycosylation crucial for its function as a high-affinity cellular receptor for ECM proteins. METHODOLOGY/PRINCIPAL FINDINGS: We report that expression of functionally glycosylated alpha-DG during thymic development is tightly regulated in developing T cells and largely confined to CD4(-)CD8(-) double negative (DN) thymocytes. Ablation of DG in T cells had no effect on proliferation, migration or effector function but did reduce the size of the thymus due to a significant loss in absolute numbers of thymocytes. While numbers of DN thymocytes appeared normal, a marked reduction in CD4(+)CD8(+) double positive (DP) thymocytes occurred. In the periphery mature naïve T cells deficient in DG showed both normal proliferation in response to allogeneic cells and normal migration, effector and memory T cell function when tested in acute infection of mice with either lymphocytic choriomeningitis virus (LCMV) or influenza virus. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that DG function is modulated by glycosylation during T cell development in vivo and that DG is essential for normal development and differentiation of T cells.


Assuntos
Distroglicanas/química , Distroglicanas/metabolismo , Glicosilação , Timo/citologia , Actinas/química , Animais , Ciclo Celular , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Citometria de Fluxo/métodos , Humanos , Vírus da Coriomeningite Linfocítica/metabolismo , Camundongos , Orthomyxoviridae/metabolismo , Linfócitos T/citologia
6.
Hum Genet ; 121(6): 685-90, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17436019

RESUMO

Intragenic homozygous deletions in the Large gene are associated with a severe neuromuscular phenotype in the myodystrophy (myd) mouse. These mutations result in a virtual lack of glycosylation of alpha-dystroglycan. Compound heterozygous LARGE mutations have been reported in a single human patient, manifesting with mild congenital muscular dystrophy (CMD) and severe mental retardation. These mutations are likely to retain some residual LARGE glycosyltransferase activity as indicated by residual alpha-dystroglycan glycosylation in patient cells. We hypothesized that more severe LARGE mutations are associated with a more severe CMD phenotype in humans. Here we report a 63-kb intragenic LARGE deletion in a family with Walker-Warburg syndrome (WWS), which is characterized by CMD, and severe structural brain and eye malformations. This finding demonstrates that LARGE gene mutations can give rise to a wide clinical spectrum, similar as for other genes that have a role in the post-translational modification of the alpha-dystroglycan protein.


Assuntos
Distrofias Musculares/congênito , Distrofias Musculares/genética , N-Acetilglucosaminiltransferases/genética , Sequência de Bases , Encéfalo/anormalidades , Consanguinidade , Análise Mutacional de DNA , Distroglicanas/química , Distroglicanas/metabolismo , Éxons , Anormalidades do Olho/genética , Feminino , Dosagem de Genes , Ligação Genética , Glicosilação , Humanos , Lactente , Recém-Nascido , Masculino , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Linhagem , Fenótipo , Processamento de Proteína Pós-Traducional , Deleção de Sequência , Síndrome
7.
Pediatr Neurol ; 31(2): 114-8, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15301830

RESUMO

Möbius syndrome is a rare congenital disease characterized by the paralysis of the facial nerve, accompanied by impaired ocular abduction. We have performed an extensive mutation analysis on a recently identified positional candidate gene, PLEXIN-D1, for Möbius syndrome 2 mapping to chromosome 3q21-q22. Southern analysis of patients from the Möbius syndrome 2 family and 41 isolated Möbius syndrome patients did not reveal chromosomal abnormalities in the PLEXIN-D1 gene. Direct sequencing of deoxyribonucleic acid from familial patients, and single-strand conformational polymorphism analysis of PLEXIN-D1 in 41 isolated patients identified 18 nucleotide changes. Seventeen of these 18 changes could be dismissed as polymorphisms, as they did not co-segregate with the disease, or were present in a control group. A single nucleotide change identified in intron 29 of an isolated Möbius syndrome patient could not be identified in a control group. However, the position of this nucleotide change makes it highly unlikely that it could be causative for Möbius syndrome in this patient because it does not affect known splicing sequences. Likewise, reverse transcriptase polymerase chain reaction analysis in patients from the Möbius syndrome 2 family did not reveal splicing aberrations, and revealed bi-allelic expression, ruling out the possibility of promoter disrupting mutations. Taken together, these results lead to the exclusion of the PLEXIN-D1 gene as the causative gene in Möbius syndrome 2, and in isolated Möbius syndrome.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Análise Mutacional de DNA/métodos , Glicoproteínas de Membrana/genética , Síndrome de Möbius/genética , Proteínas do Tecido Nervoso/genética , Southern Blotting , Moléculas de Adesão Celular Neuronais/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/química , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
Eur J Paediatr Neurol ; 7(3): 129-37, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12788039

RESUMO

Walker-Warburg syndrome (WWS) is an autosomal recessive disorder characterized by the combined involvement of the central nervous and skeletal muscle systems. Although the molecular basis of WWS remains unknown, defects in the muscle fibre basal lamina are characteristic of other forms of congenital muscular dystrophy (CMD). In agreement with this, some forms of CMD, due to glycosyltransferase defects, display a reduction in the immunolabelling of alpha-dystroglycan, whilst beta-dystroglycan labelling appears normal. Here we describe an almost complete absence of alpha-dystroglycan using both immunohistochemistry and immunoblotting in two patients with WWS. In addition, there was a mild reduction of laminin-alpha 2. In contrast, immunohistochemical labelling of perlecan and collagen VI was normal. Linkage analysis excluded the recently identified POMT1 locus, responsible for a proportion of WWS cases. These results confirm that WWS is a genetically heterogeneous condition and suggest that disruption of the alpha-dystroglycan/laminin-alpha 2 axis in the basal lamina may play a role in the degeneration of muscle fibres in WWS-also in cases not due to POMT1 defects.


Assuntos
Doenças do Sistema Nervoso Central/complicações , Doenças do Sistema Nervoso Central/metabolismo , Proteínas do Citoesqueleto/metabolismo , Manosiltransferases/metabolismo , Glicoproteínas de Membrana/metabolismo , Distrofias Musculares/complicações , Distrofias Musculares/metabolismo , Membrana Basal/metabolismo , Membrana Basal/patologia , Western Blotting , Doenças do Sistema Nervoso Central/genética , Pré-Escolar , Colágeno Tipo VI/metabolismo , Distroglicanas , Feminino , Ligação Genética/genética , Glicosilação , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Laminina/deficiência , Masculino , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Distrofias Musculares/genética , Proteínas Quinases/genética , Proteínas de Schizosaccharomyces pombe , Síndrome
9.
Am J Hum Genet ; 71(5): 1033-43, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12369018

RESUMO

Walker-Warburg syndrome (WWS) is an autosomal recessive developmental disorder characterized by congenital muscular dystrophy and complex brain and eye abnormalities. A similar combination of symptoms is presented by two other human diseases, muscle-eye-brain disease (MEB) and Fukuyama congenital muscular dystrophy (FCMD). Although the genes underlying FCMD (Fukutin) and MEB (POMGnT1) have been cloned, loci for WWS have remained elusive. The protein products of POMGnT1 and Fukutin have both been implicated in protein glycosylation. To unravel the genetic basis of WWS, we first performed a genomewide linkage analysis in 10 consanguineous families with WWS. The results indicated the existence of at least three WWS loci. Subsequently, we adopted a candidate-gene approach in combination with homozygosity mapping in 15 consanguineous families with WWS. Candidate genes were selected on the basis of the role of the FCMD and MEB genes. Since POMGnT1 encodes an O-mannoside N-acetylglucosaminyltransferase, we analyzed the possible implication of O-mannosyl glycan synthesis in WWS. Analysis of the locus for O-mannosyltransferase 1 (POMT1) revealed homozygosity in 5 of 15 families. Sequencing of the POMT1 gene revealed mutations in 6 of the 30 unrelated patients with WWS. Of the five mutations identified, two are nonsense mutations, two are frameshift mutations, and one is a missense mutation. Immunohistochemical analysis of muscle from patients with POMT1 mutations corroborated the O-mannosylation defect, as judged by the absence of glycosylation of alpha-dystroglycan. The implication of O-mannosylation in MEB and WWS suggests new lines of study in understanding the molecular basis of neuronal migration.


Assuntos
Anormalidades Múltiplas/genética , Manosiltransferases/genética , Anormalidades Múltiplas/embriologia , Anormalidades Múltiplas/enzimologia , Encéfalo/anormalidades , Encéfalo/embriologia , Pré-Escolar , Mapeamento Cromossômico , Proteínas do Citoesqueleto/metabolismo , Análise Mutacional de DNA , Distroglicanas , Anormalidades do Olho/genética , Feminino , Morte Fetal , Glicosilação , Humanos , Imuno-Histoquímica , Lactente , Masculino , Glicoproteínas de Membrana/metabolismo , Dados de Sequência Molecular , Linhagem , Análise de Sequência de DNA
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