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1.
Glycobiology ; 30(10): 817-829, 2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32149355

RESUMO

Mutations in multiple genes required for proper O-mannosylation of α-dystroglycan are causal for congenital/limb-girdle muscular dystrophies and abnormal brain development in mammals. Previously, we and others further elucidated the functional O-mannose glycan structure that is terminated by matriglycan, [(-GlcA-ß3-Xyl-α3-)n]. This repeating disaccharide serves as a receptor for proteins in the extracellular matrix. Here, we demonstrate in vitro that HNK-1 sulfotransferase (HNK-1ST/carbohydrate sulfotransferase) sulfates terminal glucuronyl residues of matriglycan at the 3-hydroxyl and prevents further matriglycan polymerization by the LARGE1 glycosyltransferase. While α-dystroglycan isolated from mouse heart and kidney is susceptible to exoglycosidase digestion of matriglycan, the functional, lower molecular weight α-dystroglycan detected in brain, where HNK-1ST expression is elevated, is resistant. Removal of the sulfate cap by a sulfatase facilitated dual-glycosidase digestion. Our data strongly support a tissue specific mechanism in which HNK-1ST regulates polymer length by competing with LARGE for the 3-position on the nonreducing GlcA of matriglycan.


Assuntos
Distroglicanas/metabolismo , Ácido Glucurônico/metabolismo , Sulfotransferases/metabolismo , Animais , Distroglicanas/química , Ácido Glucurônico/química , Glicosilação , Camundongos , Sulfotransferases/química , Sulfotransferases/isolamento & purificação
2.
Mol Ther ; 28(2): 664-676, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31843448

RESUMO

Patients with α-dystroglycanopathies, a subgroup of rare congenital muscular dystrophies, present with a spectrum of clinical manifestations that includes muscular dystrophy as well as CNS and ocular abnormalities. Although patients with α-dystroglycanopathies are genetically heterogeneous, they share a common defect of aberrant post-translational glycosylation modification of the dystroglycan alpha-subunit, which renders it defective in binding to several extracellular ligands such as laminin-211 in skeletal muscles, agrin in neuromuscular junctions, neurexin in the CNS, and pikachurin in the eye, leading to various symptoms. The genetic heterogeneity associated with the development of α-dystroglycanopathies poses significant challenges to developing a generalized treatment to address the spectrum of genetic defects. Here, we propose the development of a bispecific antibody (biAb) that functions as a surrogate molecular linker to reconnect laminin-211 and the dystroglycan beta-subunit to ameliorate sarcolemmal fragility, a primary pathology in patients with α-dystroglycan-related muscular dystrophies. We show that the treatment of LARGEmyd-3J mice, an α-dystroglycanopathy model, with the biAb improved muscle function and protected muscles from exercise-induced damage. These results demonstrate the viability of a biAb that binds to laminin-211 and dystroglycan simultaneously as a potential treatment for patients with α-dystroglycanopathy.


Assuntos
Anticorpos Biespecíficos/farmacologia , Distroglicanas/metabolismo , Laminina/metabolismo , Síndrome de Walker-Warburg/metabolismo , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/metabolismo , Modelos Animais de Doenças , Distroglicanas/imunologia , Expressão Gênica , Humanos , Imuno-Histoquímica , Injeções Intramusculares , Laminina/genética , Laminina/imunologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas/genética , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Síndrome de Walker-Warburg/tratamento farmacológico , Síndrome de Walker-Warburg/etiologia
3.
ACS Biomater Sci Eng ; 4(2): 558-565, 2018 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33418745

RESUMO

Hyaluronic acid (HA) microgels were investigated as biocompatible and biodegradable reagents for facilitating endosomolysis in human cells. Employing inverse emulsion templates, HA microgels were prepared by cross-linking aqueous sodium hyaluronate droplets with divinyl sulfone (DVS). Introduction of ether sulfone cross-links was confirmed by infrared (IR) spectroscopy and elemental analysis. The degree of cross-linking of the microgels was estimated using high performance liquid chromatography (HPLC). The size distribution of the water-dispersible HA microgels was studied by laser diffraction analysis, and the gel morphology was investigated using scanning electron microscopy (SEM). Aqueous microgel suspensions were found to be well-tolerated in human cells at concentrations of up to 100 µg/mL. Endosome-rupturing properties of the HA microgels were evaluated in vitro using calcein internalization and Cre protein delivery assays. The results of this study serve as a proof-of-principle for the utility of cross-linked HA microgels as a new class of biocompatible and biodegradable endosomolytic reagents.

4.
Nat Chem Biol ; 12(10): 810-4, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27526028

RESUMO

Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by α-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-ß1,3-xylose-α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native α-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin-G-like (LG) domains 4 and 5 (LG4 and LG5) of laminin-α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-ß1,3-xylose disaccharide repeat straddles a Ca(2+) ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca(2+)-bound water molecules. The chelating binding mode accounts for the high affinity of this protein-carbohydrate interaction. These results reveal a previously uncharacterized mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy.


Assuntos
Distroglicanas/química , Laminina/química , Sítios de Ligação , Modelos Moleculares , Estrutura Molecular
5.
Glycobiology ; 25(7): 702-13, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25882296

RESUMO

Associations between cells and the basement membrane are critical for a variety of biological events including cell proliferation, cell migration, cell differentiation and the maintenance of tissue integrity. Dystroglycan is a highly glycosylated basement membrane receptor, and is involved in physiological processes that maintain integrity of the skeletal muscle, as well as development and function of the central nervous system. Aberrant O-glycosylation of the α subunit of this protein, and a concomitant loss of dystroglycan's ability to function as a receptor for extracellular matrix (ECM) ligands that bear laminin globular (LG) domains, occurs in several congenital/limb-girdle muscular dystrophies (also referred to as dystroglycanopathies). Recent genetic studies revealed that mutations in DAG1 (which encodes dystroglycan) and at least 17 other genes disrupt the ECM receptor function of dystroglycan and cause disease. Here, we summarize recent advances in our understanding of the enzymatic functions of two of these disease genes: the like-glycosyltransferase (LARGE) and protein O-mannose kinase (POMK, previously referred to as SGK196). In addition, we discuss the structure of the glycan that directly binds the ECM ligands and the mechanisms by which this functional motif is linked to dystroglycan. In light of the fact that dystroglycan functions as a matrix receptor and the polysaccharide synthesized by LARGE is the binding motif for matrix proteins, we propose to name this novel polysaccharide structure matriglycan.


Assuntos
Membrana Basal/química , Distroglicanas/química , Matriz Extracelular/química , Polissacarídeos/química , Mutação
6.
Nature ; 503(7474): 136-40, 2013 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-24132234

RESUMO

The dense glycan coat that surrounds every cell is essential for cellular development and physiological function, and it is becoming appreciated that its composition is highly dynamic. Post-translational addition of the polysaccharide repeating unit [-3-xylose-α1,3-glucuronic acid-ß1-]n by like-acetylglucosaminyltransferase (LARGE) is required for the glycoprotein dystroglycan to function as a receptor for proteins in the extracellular matrix. Reductions in the amount of [-3-xylose-α1,3-glucuronic acid-ß1-]n (hereafter referred to as LARGE-glycan) on dystroglycan result in heterogeneous forms of muscular dystrophy. However, neither patient nor mouse studies has revealed a clear correlation between glycosylation status and phenotype. This disparity can be attributed to our lack of knowledge of the cellular function of the LARGE-glycan repeat. Here we show that coordinated upregulation of Large and dystroglycan in differentiating mouse muscle facilitates rapid extension of LARGE-glycan repeat chains. Using synthesized LARGE-glycan repeats we show a direct correlation between LARGE-glycan extension and its binding capacity for extracellular matrix ligands. Blocking Large upregulation during muscle regeneration results in the synthesis of dystroglycan with minimal LARGE-glycan repeats in association with a less compact basement membrane, immature neuromuscular junctions and dysfunctional muscle predisposed to dystrophy. This was consistent with the finding that patients with increased clinical severity of disease have fewer LARGE-glycan repeats. Our results reveal that the LARGE-glycan of dystroglycan serves as a tunable extracellular matrix protein scaffold, the extension of which is required for normal skeletal muscle function.


Assuntos
Distroglicanas/química , Distroglicanas/metabolismo , Matriz Extracelular/metabolismo , Distrofias Musculares/prevenção & controle , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Animais , Membrana Basal/metabolismo , Membrana Basal/patologia , Diferenciação Celular , Linhagem Celular , Matriz Extracelular/química , Feminino , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Desenvolvimento Muscular , Músculos/metabolismo , Músculos/patologia , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Mioblastos , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/genética , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Fenótipo , Polissacarídeos/química
7.
Science ; 341(6148): 896-9, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23929950

RESUMO

Phosphorylated O-mannosyl trisaccharide [N-acetylgalactosamine-ß3-N-acetylglucosamine-ß4-(phosphate-6-)mannose] is required for dystroglycan to bind laminin-G domain-containing extracellular proteins with high affinity in muscle and brain. However, the enzymes that produce this structure have not been fully elucidated. We found that glycosyltransferase-like domain-containing 2 (GTDC2) is a protein O-linked mannose ß 1,4-N-acetylglucosaminyltransferase whose product could be extended by ß 1,3-N-acetylgalactosaminyltransferase2 (B3GALNT2) to form the O-mannosyl trisaccharide. Furthermore, we identified SGK196 as an atypical kinase that phosphorylated the 6-position of O-mannose, specifically after the mannose had been modified by both GTDC2 and B3GALNT2. These findings suggest how mutations in GTDC2, B3GALNT2, and SGK196 disrupt dystroglycan receptor function and lead to congenital muscular dystrophy.


Assuntos
Distroglicanas/metabolismo , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Células HEK293 , Humanos , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Proteínas Quinases/genética , Trissacarídeos/metabolismo
8.
Glycobiology ; 23(3): 295-302, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23125099

RESUMO

LARGE-dependent modification enables α-dystroglycan (α-DG) to bind to its extracellular matrix ligands. Mutations in the LARGE gene and several others involved in O-mannosyl glycan synthesis have been identified in congenital and limb-girdle muscular dystrophies that are characterized by perturbed glycosylation and reduced ligand-binding affinity of α-DG. LARGE is a bifunctional glycosyltransferase that alternately transfers xylose and glucuronic acid, thereby generating the heteropolysaccharides on α-DG that confer its ligand binding. Although the LARGE paralog LARGE2 (also referred to as GYLTL1B) has likewise been shown to enhance the functional modification of α-DG in cultured cells, its enzymatic activities have not been identified. Here, we report that LARGE2 is also a bifunctional glycosyltransferase and compare its properties with those of LARGE. By means of a high-performance liquid chromatography-based enzymatic assay, we demonstrate that like LARGE, LARGE2 has xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, as well as polymerizing activity. Notably, however, the pH optima of the Xyl-T and GlcA-T of LARGE2 are distinct from one another and also from those of LARGE. Our results suggest that LARGE and LARGE2 catalyze the same glycosylation reactions for the functional modification of α-DG, but that they have different biochemical properties.


Assuntos
Distroglicanas/metabolismo , Glicosiltransferases/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Animais , Células CHO , Domínio Catalítico , Cricetinae , Cricetulus , Ácido Glucurônico/metabolismo , Glicosiltransferases/química , Concentração de Íons de Hidrogênio , Cinética , Camundongos , N-Acetilglucosaminiltransferases/química , Multimerização Proteica , Xilose/metabolismo
9.
Nat Genet ; 44(5): 575-80, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22522420

RESUMO

Walker-Warburg syndrome (WWS) is clinically defined as congenital muscular dystrophy that is accompanied by a variety of brain and eye malformations. It represents the most severe clinical phenotype in a spectrum of diseases associated with abnormal post-translational processing of a-dystroglycan that share a defect in laminin-binding glycan synthesis1. Although mutations in six genes have been identified as causes of WWS, only half of all individuals with the disease can currently be diagnosed on this basis2. A cell fusion complementation assay in fibroblasts from undiagnosed individuals with WWS was used to identify five new complementation groups. Further evaluation of one group by linkage analysis and targeted sequencing identified recessive mutations in the ISPD gene (encoding isoprenoid synthase domain containing). The pathogenicity of the identified ISPD mutations was shown by complementation of fibroblasts with wild-type ISPD. Finally, we show that recessive mutations in ISPD abolish the initial step in laminin-binding glycan synthesis by disrupting dystroglycan O-mannosylation. This establishes a new mechanism for WWS pathophysiology.


Assuntos
Distroglicanas/metabolismo , Manose/metabolismo , Manosiltransferases/metabolismo , Mutação/genética , Nucleotidiltransferases/genética , Síndrome de Walker-Warburg/genética , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Teste de Complementação Genética , Glicosilação , Humanos , Lactente , Laminina/metabolismo , Manosiltransferases/genética , Polissacarídeos/metabolismo , Pele/citologia , Pele/metabolismo
10.
Science ; 335(6064): 93-6, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22223806

RESUMO

Posttranslational modification of alpha-dystroglycan (α-DG) by the like-acetylglucosaminyltransferase (LARGE) is required for it to function as an extracellular matrix (ECM) receptor. Mutations in the LARGE gene have been identified in congenital muscular dystrophy patients with brain abnormalities. However, the precise function of LARGE remains unclear. Here we found that LARGE could act as a bifunctional glycosyltransferase, with both xylosyltransferase and glucuronyltransferase activities, which produced repeating units of [-3-xylose-α1,3-glucuronic acid-ß1-]. This modification allowed α-DG to bind laminin-G domain-containing ECM ligands.


Assuntos
Distroglicanas/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Polissacarídeos/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Configuração de Carboidratos , Domínio Catalítico , Cricetinae , Distroglicanas/química , Ácido Glucurônico/metabolismo , Glucuronosiltransferase/metabolismo , Glicosaminoglicanos/metabolismo , Glicosilação , Células HEK293 , Humanos , Laminina/metabolismo , Ligantes , Camundongos , Mutação , N-Acetilglucosaminiltransferases/química , N-Acetilglucosaminiltransferases/genética , Pentosiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Xilose/metabolismo , UDP Xilose-Proteína Xilosiltransferase
11.
Proc Natl Acad Sci U S A ; 108(42): 17426-31, 2011 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-21987822

RESUMO

α-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 α-dystroglycan and attaches ligand-binding moieties to phosphorylated O-mannose on α-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 α-dystroglycan. Deletion and mutation analyses demonstrate that the ligand-binding activity of α-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 α-dystroglycan and is crucial for α-dystroglycan to coordinate the assembly of extracellular matrix proteins and to bind arenaviruses on the cell surface.


Assuntos
Infecções por Arenaviridae/etiologia , Infecções por Arenaviridae/metabolismo , Distroglicanas/metabolismo , Laminina/metabolismo , Vírus da Coriomeningite Linfocítica , N-Acetilglucosaminiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Distroglicanas/química , Distroglicanas/genética , Glicosilação , Células HEK293 , Humanos , Vírus da Coriomeningite Linfocítica/patogenicidade , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese , Mioblastos/metabolismo , Fosforilação , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina/química , Internalização do Vírus
12.
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
13.
Science ; 327(5961): 88-92, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20044576

RESUMO

Alpha-dystroglycan (alpha-DG) is a cell-surface glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin-G domains and certain arenaviruses. Receptor binding is thought to be mediated by a posttranslational modification, and defective binding with laminin underlies a subclass of congenital muscular dystrophy. Using mass spectrometry- and nuclear magnetic resonance (NMR)-based structural analyses, we identified a phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant alpha-DG, which was required for laminin binding. We demonstrated that patients with muscle-eye-brain disease and Fukuyama congenital muscular dystrophy, as well as mice with myodystrophy, commonly have defects in a postphosphoryl modification of this phosphorylated O-linked mannose, and that this modification is mediated by the like-acetylglucosaminyltransferase (LARGE) protein. These findings expand our understanding of the mechanisms that underlie congenital muscular dystrophy.


Assuntos
Distroglicanas/metabolismo , Laminina/metabolismo , Manose/metabolismo , Animais , Configuração de Carboidratos , Linhagem Celular , Distroglicanas/química , Glicosilação , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Distrofia Muscular Animal/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
14.
Proc Natl Acad Sci U S A ; 106(31): 12573-9, 2009 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-19633189

RESUMO

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 alpha7 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.


Assuntos
Membrana Basal/fisiologia , Distroglicanas/fisiologia , Laminina/fisiologia , Sarcolema/fisiologia , Animais , Sítios de Ligação , Distroglicanas/química , Glicosilação , Integrinas/fisiologia , Laminina/química , Vírus da Coriomeningite Linfocítica , Camundongos , Distrofia Muscular Animal/etiologia
15.
J Biol Chem ; 284(17): 11279-84, 2009 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-19244252

RESUMO

The interaction between epithelial cells and the extracellular matrix is crucial for tissue architecture and function and is compromised during cancer progression. Dystroglycan is a membrane receptor that mediates interactions between cells and basement membranes in various epithelia. In many epithelium-derived cancers, beta-dystroglycan is expressed, but alpha-dystroglycan is not detected. Here we report that alpha-dystroglycan is correctly expressed and trafficked to the cell membrane but lacks laminin binding as a result of the silencing of the like-acetylglucosaminyltransferase (LARGE) gene in a cohort of highly metastatic epithelial cell lines derived from breast, cervical, and lung cancers. Exogenous expression of LARGE in these cancer cells restores the normal glycosylation and laminin binding of alpha-dystroglycan, leading to enhanced cell adhesion and reduced cell migration in vitro. Our findings demonstrate that LARGE repression is responsible for the defects in dystroglycan-mediated cell adhesion that are observed in epithelium-derived cancer cells and point to a defect of dystroglycan glycosylation as a factor in cancer progression.


Assuntos
Distroglicanas/metabolismo , Epitélio/patologia , Regulação Neoplásica da Expressão Gênica , Laminina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Neoplasias Cutâneas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Inativação Gênica , Glicosilação , Células HeLa , Humanos , Modelos Biológicos , Metástase Neoplásica , Ligação Proteica
16.
Cell ; 117(7): 953-64, 2004 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-15210115

RESUMO

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.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Glicosiltransferases/metabolismo , Glicoproteínas de Membrana/metabolismo , Adenoviridae/genética , Animais , Western Blotting , Células Cultivadas , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Distroglicanas , Glicosilação , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Coelhos , Receptores de Laminina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Células-Tronco/citologia
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