O -Mannosyl Phosphorylation of Alpha-Dystroglycan Is Required for Laminin Binding

Abstract

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...

B4GALNT2 (GALGT2) Gene Therapy Reduces Skeletal Muscle Pathology in the FKRP P448L Mouse Model of Limb Girdle Muscular Dystrophy 2I

“…present in its mucin-like domain, 2 and mutations in well more than a dozen genes that control a dystroglycan glycosylation give rise to congenital or limb girdle forms of MD. 3,4 In their most severe forms, including WWS, Fukuyama congenital MD, and muscle eye brain disease, dystroglycanopathies not only affect skeletal muscle but also eye and brain development, leading to lissencephalic changes in the cerebral cortex due to defects in the glial limitans-pial basement membrane and to ocular malformations that can include retinal detachment.…”

Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins

“…1A). So far this pathway has only been associated with a few O-Man glycosites (Thr317, Thr319, and Thr379) in α-DG (20,26), and the POMGnT2 enzyme has only been tested with a peptide derived from the site at Thr317. Moreover, we did not identify glycosites in these regions in the present study despite finding many of the expected O-Man glycosites in the N-terminal region of the mucin domain of α-DG (Fig.…”

“…An alternative elongation pathway, which has so far only been identified at one specific O-Man site in α-DG, involves elongation of O-Man by the UDP-GlcNAc: Manα1-O-Ser/Thr β1,4GlcNAc-transferase (POMGnT2/GTDC2) (19). This pathway involves further elongation by β3GalNAc-T2 with subsequent phosphorylation of the mannose residue by POMK (SGK196) which is extended with GlcA and xylose by like-acetylglucosaminyltransferase to form the structure required for DG function (19,20).…”

Congenital muscular dystrophy type 1D (MDC1D) due to a large intragenic insertion/deletion, involving intron 10 of the LARGE gene

“…A recent study suggests that LARGE participates in postphosphoryl glycosylation of a novel phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant aDG, which is required for laminin binding. 10 Evidence from cell culture and a fukutin transgenic mouse suggests that fukutin physically interacts with POMGnT1 and can cause reduced POMGnT1 activity when abnormal. 11 To investigate whether LARGE may also function by influencing the first steps of the O-mannosyl pathway, we assessed the activity levels of POMT and POMGnT1 in cultured lymphoblasts from one of our patients.…”