Site Mapping and Characterization of O-Glycan Structures on α-Dystroglycan Isolated from Rabbit Skeletal Muscle

• Published in: The Journal of biological chemistry 2010-08, Vol.285 (32), p.24882-24891
• Main Authors: Stalnaker, Stephanie H., Hashmi, Sana, Lim, Jae-Min, Aoki, Kazuhiro, Porterfield, Mindy, Gutierrez-Sanchez, Gerardo, Wheeler, James, Ervasti, James M., Bergmann, Carl, Tiemeyer, Michael, Wells, Lance
• Format: Article
• Language: English
• Subjects: Animals; Carbohydrate Structure; Carbohydrates - chemistry; Dystroglycan; Dystroglycans - chemistry; Genomics and Proteomics; Glycobiology and Extracellular Matrices; Glycoconjugate; Glycoconjugates - chemistry; Glycomics; Glycomics - methods; Glycoprotein; Glycoproteins - metabolism; Glycoproteomics; Glycosylation; Laminin - chemistry; Mannose - chemistry; Mass Spectrometry (MS); Mass Spectrometry - methods; Mice; Muscle, Skeletal - metabolism; Muscular Dystrophy; Polysaccharides - chemistry; Rabbits; Site-mapping; Surface Plasmon Resonance - methods
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.126474

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