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

Abstract

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.

Fig. 1

Chemical dephosphorylation by HFaq treatment abolishes laminin and virus binding to α-DG in tissues from Wt mice. (A and B) Treated glycoproteins prepared from Wt muscle were subjected to: (A) immunoblotting with antibodies against the α-DG core protein (CORE) or the laminin-binding form α-DG epitope (IIH6) and β-DG (AP83), or to laminin overlay assay; (B) virus overlay assays with γ-inactivated LFV or LCMV cl-13. (C) Wt muscle glycoproteins with and without HFaq-treatment were subjected to a solid-phase laminin-binding assay (n=3). Open circles=treated; closed circles=untreated. Error bars indicate standard deviation. (D) Muscle glycoproteins from Wt and Large^myd (Myd) mice were digested using cocktails of glycosidases that degrade sialylated and/or fucosylated N-glycan, Core1 O-glycan, and O-mannosyl glycan, before (first 4 lanes) and after (last 4 lanes) HFaq treatment. The products were subjected to either immunoblotting with CORE antibody or laminin overlay assay.

Fig. 2

The mucin-like region of α-DG is phosphorylated in an O-mannosylation-dependent manner. (A) Structures of recombinant α-DG constructs used in the study. (B and C) [³²P]-orthophosphate labeling of (B) Fc-Ctrl- or DGFc4-expressing HEK293 cells and (C) DGFc5-expressing cultured cells from CMD patients (WWS, MEB, FCMD) and control humans, and from Wt and Large^myd (Myd) mice. Fc-tagged recombinant α-DG was isolated from the culture medium using protein-A agarose, separated by SDS-PAGE, stained with Coomassie brilliant blue (CBB) and analyzed by phosphor-imaging ([³²P]). Phosphorylation on α-DG required prior O-mannosylation. Asterisks indicate contaminating proteins derived from FBS. (D) IMAC-binding assay testing glycoproteins from Wt and Large^myd mice, and from FCMD, MEB, and control human muscle (SkM).

Fig. 3

NMR analysis of phosphorylated O-glycan on HEK293-produced DGFc4. (A) HMQC spectrum where the assigned cross peaks are labeled with a letter for the subunit designated in (E), and a number for the position on that subunit. The folded cross peaks are indicated in red, and the cross peaks derived from sample impurities are marked by asterisks. (B) TOCSY-HSQC spectrum obtained using a selective excitation pulse at the subunit CH1 proton and a selective TOCSY mixing time of 113 ms. (C) HMBC (green) and HMQC (black) spectra for the assignment of interglycoside linkages. (D) ³¹P/¹H COSY spectrum. (E) Structure of the O-glycan, with the sugar subunits labelled A–C.

Fig. 4

Mapping of phosphorylated trisaccharide on HEK293-produced DGFc4, and characterization of mannosyl phosphorylation. (A) CID-MS/MS spectra from 780-1320 m/z (upper panel) and 375-2000 m/z (lower panel), revealing neutral loss pattern (upper panel) and peptide-derived b and y ions (lower panel) of the selected precursor ions at m/z 1318.63. Full FT mass spectrum is shown in fig. S8. Square: HexNAc; circle: Hexose. (B) Peptide and mononsaccharide unit identification based on fragmentation of the phosphorylated glycopeptide. (C) Glycoproteins prepared from cell lysates of fibroblasts derived from Mucolipidosis II patients and subjected to immunoblotting with CORE antibody, and to laminin overlay assay. (D) Schematic representation of O-mannosyl glycans on α-DG, with proteins involved in its biosynthesis, and diseases in which relevant steps are defective indicated. Shading indicates glycan identified in brain and muscle α-DG.