Alpha-dystroglycan 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 post-translational modification, and defective binding with laminin underlies a subclass of congenital muscular dystrophy. Here, using mass spectrometry-and NMR-based structural analyses, we identified a phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant alpha-dystroglycan, 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 post-phosphoryl modification of this phosphorylated O-linked mannose, and that this modification is mediated by the likeacetylglucosaminyltransferase (LARGE) protein. Our findings expand our understanding of the mechanisms that underlie congenital muscular dystrophy.Diverse post-translational modifications influence the structure and function of many proteins. Dystroglycan (DG) is a membrane protein that requires extensive post-translational processing in order to function as an extracellular matrix receptor. It is comprised of an extracellular α-* To whom correspondence should be addressed. kevin-campbell@uiowa.edu.Supporting Online Material www.sciencemag.org Materials and Methods Figs. S1 to S12 Table S1 NIH Public Access DG subunit and a transmembrane β-DG subunit (1). α-DG serves as a receptor for extracellular matrix laminin G domain-containing ligands such as laminin (1) and agrin (2) in both muscle and brain, and these interactions depend on an unidentified post-translational α-DG modification. α-DG is also the cellular receptor for lymphocytic choriomeningitis virus (LCMV), Lassa fever virus (LFV), and clade C New World arenaviruses (3,4). Although the binding sites for LCMV and LFV on α-DG have not yet been identified, they are thought to overlap with the modification recognized by laminin (5,6).Glycosyltransferase-mediated glycosylation is one form of post-translational modification that can modulate protein structure and function. The main forms in mammals are N-and Oglycosylation, and these are distinguished by how the oligosaccharide moiety links to the amino acid. Mutations in six known or putative glycosyltransferase genes-POMT1 (7), POMT2 (8), POMGnT1 (9), fukutin (10), FKRP (11), and LARGE (12)-have been identified in patients with congenital muscular dystrophy (CMD). These disorders cover a spectrum of abnormalities affecting the brain, eye, and skeletal muscle, and show a dramatic gradient of phenotypic severity ranging from the most devastating in Walker-Warburg syndrome (WWS; OMIM# 236670), to less severe in muscle-eye-brain disease (MEB; OMIM# 253280) and Fukuyama CMD (FCMD; OMIM# 253800), and to mild limb-girdle muscular dystrophies. In these diseases, the ability of α-DG to bind laminin is markedly reduced (13), suggesting that these (putative) glycosyltransferases participa...

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, ␣-dystroglycan (␣-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown ␣-DG to be modified by both O-GalNAc-and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle ␣-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on ␣-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from ␣-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.Defects in protein glycosylation related to human disease were first reported in the 1980s, and since then, about 40 various types of congenital disorders of glycosylation have been reported (1). The term congenital disorders of glycosylation was first used to describe alterations of the N-glycosylation pathway and was later expanded to include the O-glycosylation pathways (1-3). The importance and complexity of O-linked glycosylation have only recently begun to be appreciated (1, 3, 4). In particular, mutations in genes encoding (putative) glycosyltransferases, which catalyze the addition and extension of O-linked mannose-initiated glycans, have garnered increased attention in the last decade given that they are causative for several forms of congenital muscular dystrophy (5, 6).The most common forms of O-glycosylation on secretory proteins are the mucin-like O-GalNAc structures that are initiated by polypeptide N-␣-acetylgalactosaminyltransferases in the endoplasmic reticulum-Golgi intermediate compartment and/or early cis-Golgi (7). Additionally, other O-linked structures are initiated with alternative monosaccharides, such as O-mannose, O-glucose, O-fucose, O-xylose, and O-GlcNAc onSer/Thr residues and the O-galactose modification of hydroxylysine residues in collagen domains (4). The diversity of O-mannosylated proteins in mammals, although quite abundant in some tissues (ϳ30% of O-glycans released from mouse brains (8)), has not been well characterized. The only clearly identified mamma...

Congenital muscular dystrophy (CMD) 3 is a heterogeneous group of inherited neuromuscular disorders characterized by severe muscle weakness, ocular and neuronal migration abnormalities, and variable mental retardation (1). Within recent years, it has become increasing clear through genetic studies that hypoglycosylation of the protein dystroglycan (DG) is a commonality in many forms of CMD (the so-called dystroglycanopathies). DG is post-translationally cleaved into an extracellular ␣-DG subunit and a transmembrane ␤-DG subunit (2). ␣-DG is a key component of the dystrophin-glycoprotein complex that serves as a link between the cytoskeleton of cells and the extracellular matrix by binding to proteins such as laminin (3). Interaction between ␣-DG and its extracellular ligands requires ␣-DG to be properly post-translationally modified through the addition of O-linked oligosaccharides, specifically O-mannose (4, 5). To date, mutations in six genes that encode determined or predicted glycosyltransferases have been shown to result in varying forms of CMD in which the post-translational processing of ␣-DG is affected (4 -6). The six mutated genes and their original resulting form of CMD are as follows: POMT1 (protein O-mannosyltransferase 1) and POMT2, Walker-Warburg syndrome (7,8); POMGnT1 (protein Olinked mannose ␤1,2-N-acetylglucosaminyltransferase 1), muscle-eye-brain disease (9); fukutin, Fukuyama congenital muscular dystrophy (10); FKRP (fukutin-related protein), congenital muscular dystrophy 1C (11); and LARGE, congenital muscular dystrophy 1D (12). Recent work has demonstrated that selected mutations in some of these genes can cause various forms of CMD that are likely dependent on the severity of the mutation on enzymatic activity and stability (13). Abnormal glycosylation of ␣-DG appears to be a commonality among all of the aforementioned forms of CMD. Although expression of ␣-DG appears not to be grossly affected, the ability of ␣-DG to be recognized by monoclonal antibodies IIH6 and VIA4 1 is eliminated, as is the ability of ␣-DG to properly bind its ligands (14).␣-DG is composed of a central mucin-like region that is extensively heterogeneously glycosylated with glycan chains that are initiated by both O-

Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O-mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure—a ribitol in a phosphodiester linkage—for the moiety on which TMEM5, B4GAT1, and LARGE act to generate the functional receptor for ECM proteins having LG domains.DOI: http://dx.doi.org/10.7554/eLife.14473.001

Recent studies highlighted an emerging possibility of using Drosophila as a model system for investigating the mechanisms of human congenital muscular dystrophies, called dystroglycanopathies, resulting from the abnormal glycosylation of alpha-dystroglycan. Several of these diseases are associated with defects in O-mannosylation, one of the most prominent types of alpha-dystroglycan glycosylation mediated by two protein O-mannosyltransferases. Drosophila appears to possess homologs of all essential components of the mammalian dystroglycan-mediated pathway; however, the glycosylation of Drosophila Dystroglycan (DG) has not yet been explored. In this study, we characterized the glycosylation of Drosophila DG using a combination of glycosidase treatments, lectin blots, trypsin digestion, and mass spectrometry analyses. Our results demonstrated that DG extracellular domain is O-mannosylated in vivo. We found that the concurrent in vivo activity of the two Drosophila protein O-mannosyltransferases, Rotated Abdomen and Twisted, is required for O-mannosylation of DG. While our experiments unambiguously determined some O-mannose sites far outside of the mucin-type domain of DG, they also provided evidence that DG bears a significant amount of O-mannosylation within its central region including the mucin-type domain, and that O-mannose can compete with O-GalNAc glycosylation of DG. We found that Rotated Abdomen and Twisted could potentiate in vivo the dominant-negative effect of DG extracellular domain expression on crossvein development, which suggests that O-mannosylation can modulate the ligand-binding activity of DG. Taken together these results demonstrated that O-mannosylation of Dystroglycan is an evolutionarily ancient mechanism conserved between Drosophila and humans, suggesting that Drosophila can be a suitable model system for studying molecular and genetic mechanisms underlying human dystroglycanopathies.