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

Stephanie H. Stalnaker, Sana Hashmi, Jae Min Lim, Kazuhiro Aoki, Mindy Porterfield, Gerardo Gutierrez-Sanchez, James Wheeler, James M. Ervasti, Carl Bergmann, Michael Tiemeyer, Lance Wells

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83 Scopus citations


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 work-flows 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.

Original languageEnglish (US)
Pages (from-to)24882-24891
Number of pages10
JournalJournal of Biological Chemistry
Issue number32
StatePublished - Aug 6 2010


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