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...

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-β1–]. This modification allowed α-DG to bind laminin-G domain–containing ECM ligands.

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.