Inisghts into O-mannosylation revealed by glycoproteomics
Hammond, Stephanie Lauren
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O-mannosylation accounts for approximately 30% of the reported O-linked glycan structures detected from mouse brain. However, O-mannosylation has rarely been observed on other mammalian proteins beside alpha-dystroglycan (α-DG). Previous studies have shown α-DG to be modified by both O-GalNAc and rare O-mannose initiated glycan structures but no sites of attachment have been mapped. Mutations in genes encoding known and putative glycosyltransferases involved in the O-mannosylation of α-DG have been implicated in several forms of Congenital Muscular Dystrophy (CMD). Thus this project sought to provide insights into global O-mannosylation and α-DG modification. Using tandem mass spectrometry approaches we were able to identify 21 sites of modification on 91 glycopeptides derived from α-DG. Additionally, through our glycomic analysis we were able to characterize 34 O-glycan structures, 9 O-mannose and 25 O-GalNAc initiated structures, from mouse models of CMD. This analysis of mouse brains allowed us to determine that POMGnT1 is essential for the extension of classical and branched O-mannose initiated glycan structures, that loss of LARGE has no impact on these observed structures or on O-GalNAc structures, and that additional O-mannose modified proteins besides α-DG exist in mouse brains. All the work outlined in this dissertation is based on the development and application of glycomic and glycoproteomic workflows.