Site mapping and characterization of O-glycan structures on alpha-dystroglycan isolated from rabbit skeletal muscle

Abstract

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, alpha-dystroglycan (alpha-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown alpha-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 alpha-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on alpha-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 alpha-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.

FIGURE 1.

Purified, functionally glycosylated α-DG from rabbit skeletal muscle. a, silver staining following SDS-PAGE of purified α-DG (lane 1). Lanes 2 and 3 represent mock or glycoside (N-glycosidase F, sialidase, endo-O-glycosidase, β(1–4)-galactosidase, and β-N-acetylglucosaminidase)-treated α-DG. b, Western blot analysis following SDS-PAGE of purified α-DG with the glycan-dependent anti-α-DG monoclonal VIA4₁ and IIH6, which specifically recognizes fully glycosylated, functionally active α-DG. c, protein sequence derived from the dystroglycan gene with the capitalized boldface sequence representing the predicted mature α-DG protein. Peptides assigned by tandem mass spectrometry are underlined. Sites detected to be modified by GalNAc are highlighted in blue; sites of O-mannosylation are highlighted with red; residues observed to be modified by both GalNAc and mannose are green. Sites of potential modification are highlighted similarly and distinguished by striking through the modified residue. d, untreated α-DG (a), α-DG treated with β-galactosidase and sialidase (a + b), or glycosidases alone (b, enzymes without α-DG present) binding to immobilized laminin-1 as measured by surface plasmon resonance.

FIGURE 2.

O-Man- and O-GalNAc-initiated glycans of α-DG. a, full MS scan of the released and permethylated O-linked glycan structures on α-DG purified from rabbit skeletal muscle. b, total ion mapping (TIM) profile of the released O-linked glycans allowing for quantifying prevalence of each structure as well as confirmation of structure by fragmentation. c, MS/MS fragmentation at m/z 1256 demonstrates the presence of an O-GalNAc initiated structure SA-Gal-(SA)-GalNAc. d, fragmentation of m/z 1100 demonstrates the presence of an O-Man-initiated structure SA-Gal-GlcNAc-Man.

FIGURE 3.

Assignment of an O-GalNAc α-DG glycopeptide. From the full scan acquired at 49.33 min (a), a peak at 1018.3 m/z was selected for fragmentation (b). The resulting MS/MS of 1018.3 m/z yielded the neutral loss of two terminal SA residues, which was then followed by MS³ fragmentation indicating the loss of a Gal residue followed by a reducing end GalNAc. The combined glycan structure was determined to belong to a peptide with 1087.6 m/z. From examining the MS/MS spectra (not shown) and the neutral loss-triggered MS³ spectra (c), the site of post-translational modification to the serine within the peptide IRTTTSVGPR is assigned.

FIGURE 4.

Assignment of an O-Man α-DG glycopeptide. From the full scan acquired at 52.38 min (a), a peak at 895.8 m/z was selected for fragmentation. b, resulting MS/MS of 895.8 m/z yielded the neutral loss of terminal SA residue, which was then followed by MS³ fragmentation indicating the neutral loss of a Gal-GalNAc residue followed by a reducing end Man. The combined glycan structure was determined to belong to a peptide with 971.5 m/z. From examining the MS/MS spectra (data not shown) and the neutral loss-triggered MS³ spectra (c), the site of post-translational modification to the serine within the peptide LETASPPTR is assigned.