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Tumor suppressor function of laminin-binding alpha-dystroglycan requires a distinct beta3-N-acetylglucosaminyltransferase.

ABSTRACT: Alpha-dystroglycan (alpha-DG) represents a highly glycosylated cell surface molecule that is expressed in the epithelial cell-basement membrane (BM) interface and plays an essential role in epithelium development and tissue organization. The alpha-DG-mediated epithelial cell-BM interaction is often impaired in invasive carcinomas, yet roles and underlying mechanisms of such an impaired interaction in tumor progression remain unclear. We report here a suppressor function of laminin-binding glycans on alpha-DG in tumor progression. In aggressive prostate and breast carcinoma cell lines, laminin-binding glycans are dramatically decreased, although the amount of alpha-DG and beta-dystroglycan is maintained. The decrease of laminin-binding glycans and consequent increased cell migration were associated with the decreased expression of beta3-N-acetylglucosaminyltransferase-1 (beta3GnT1). Forced expression of beta3GnT1 in aggressive cancer cells restored the laminin-binding glycans and decreased tumor formation. beta3GnT1 was found to be required for laminin-binding glycan synthesis through formation of a complex with LARGE, thus regulating the function of LARGE. Interaction of the laminin-binding glycans with laminin and other adhesive molecules in BM attenuates tumor cell migratory potential by antagonizing ERK/AKT phosphorylation induced by the components in the ECM. These results identify a previously undescribed role of carbohydrate-dependent cell-BM interaction in tumor suppression and its control by beta3GnT1 and LARGE.

SUBMITTER: Bao X

PROVIDER: S-EPMC2707272 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Enhanced laminin binding by alpha-dystroglycan after enzymatic deglycosylation.

Project description:Carbohydrate modifications are clearly important to the function of alpha-dystroglycan but their composition and structure remain poorly understood. In the present study, we describe experiments aimed at identifying the alpha-dystroglycan oligosaccharides important for its binding to laminin-1 and carbohydrate-dependent mAbs (monoclonal antibodies) IIH6 and VIA4(1). We digested highly purified skeletal muscle alpha-dystroglycan with an array of linkage-specific endo- and exoglycosidases, which were verified for action on alpha-dystroglycan by loss/gain of reactivity for lectins with defined glyco-epitopes. Notably, digestion with a combination of Arthrobacter ureafaciens sialidase, beta(1-4)galactosidase and beta-N-acetylglucosaminidase substantially degraded SiaAalpha2-3Galbeta1-4GlcNAcbeta1-2Man glycans on highly purified alpha-dystroglycan that nonetheless exhibited enhanced IIH6, VIA4(1) and laminin-1 binding activity. Additional results indicate that alpha-dystroglycan is probably modified with other anionic sugars besides sialic acid and suggest that rare alpha-linked GlcNAc moieties may block its complete deglycosylation with currently available enzymes.

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O-mannosyl phosphorylation of alpha-dystroglycan is required for laminin binding.

Project description: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.

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High degree of conservation of the enzymes synthesizing the laminin-binding glycoepitope of α-dystroglycan.

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Project description: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.

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Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of alpha-dystroglycan.

Project description: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.

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Human natural killer-1 sulfotransferase (HNK-1ST)-induced sulfate transfer regulates laminin-binding glycans on α-dystroglycan.

Project description:Retinoic acid (RA) is a well established anti-tumor agent inducing differentiation in various cancer cells. Recently, a robust up-regulation of human natural killer-1 sulfotransferase (HNK-1ST) was found in several subsets of melanoma cells during RA-mediated differentiation. However, the molecular mechanism underlying the tumor suppression mediated by HNK-1ST remains unclear. Here, we show that HNK-1ST changed the glycosylation state and reduced the ligand binding activity of α-dystroglycan (α-DG) in RA-treated S91 melanoma cells, which contributed to an attenuation of cell migration. Knockdown of HNK-1ST restored the glycosylation of α-DG and the migration of RA-treated S91 cells, indicating that HNK-1ST functions through glycans on α-DG. Using CHO-K1 cells, we provide direct evidence that HNK-1ST but not other homologous sulfotransferases (C4ST1 and GalNAc4ST1) suppresses the glycosylation of α-DG. The activity-abolished mutant of HNK-1ST did not show the α-DG-modulating function, indicating that the sulfotransferase activity of HNK-1ST is essential. Finally, the HNK-1ST-dependent incorporation of [(35)S]sulfate groups was detected on α-DG. These findings suggest a novel role for HNK-1ST as a tumor suppressor controlling the functional glycans on α-DG and the importance of sulfate transfer in the glycosylation of α-DG.

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Structure of the C-terminal laminin G-like domain pair of the laminin alpha2 chain harbouring binding sites for alpha-dystroglycan and heparin.

Project description:The laminins are large heterotrimeric glycoproteins with fundamental roles in basement membrane architecture and function. The C-terminus of the laminin alpha chain contains a tandem of five laminin G-like (LG) domains. We report the 2.0 A crystal structure of the laminin alpha2 LG4-LG5 domain pair, which harbours binding sites for heparin and the cell surface receptor alpha-dystroglycan, and is 41% identical to the laminin alpha1 E3 fragment. LG4 and LG5 are arranged in a V-shaped fashion related by a 110 degrees rotation about an axis passing near the domain termini. An extended N-terminal segment is disulfide bonded to LG5 and stabilizes the domain pair. Two calcium ions, one each in LG4 and LG5, are located 65 A apart at the tips of the domains opposite the polypeptide termini. An extensive basic surface region between the calcium sites is proposed to bind alpha-dystroglycan and heparin. The LG4-LG5 structure was used to construct a model of the laminin LG1-LG5 tandem and interpret missense mutations underlying protein S deficiency.

| S-EPMC310212 | biostudies-literature

Structural basis of laminin binding to the LARGE glycans on dystroglycan.

Project description: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-β1,3-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-β1,3-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.

| S-EPMC5030134 | biostudies-literature

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