{"@context":"http://iiif.io/api/presentation/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/manifest.json","@type":"sc:Manifest","label":"On the Fringe of Notch: Mapping O-fucose and O-glucose glycans at specific sites on the extracellular domains of Notch receptors using nano-LC-ESI-MS/MS","metadata":[{"label":"dc.description.sponsorship","value":"This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree."},{"label":"dc.format","value":"Monograph"},{"label":"dc.format.medium","value":"Electronic Resource"},{"label":"dc.identifier.uri","value":"http://hdl.handle.net/11401/71674"},{"label":"dc.language.iso","value":"en_US"},{"label":"dc.publisher","value":"The Graduate School, Stony Brook University: Stony Brook, NY."},{"label":"dcterms.abstract","value":"Notch is an essential cell surface receptor whose proper functioning is critical during development in metazoans. Notch can be modified with two unique forms of O-linked glycosylation: O-fucose and O-glucose.  Protein O-fucosyltransferase1 (Pofut1, OFUT1 in flies) and Protein O-glucosyltransferase (Poglut, Rumi in flies) are the glycosyltransferases that add O-fucose and O-glucose monosaccharides to Notch, respectively.  Loss of Pofut1/Ofut1 or Poglut/rumi in mice or flies phenocopies Notch-null phenotypes, suggesting both forms of O-glycosylation are essential for Notch function.  To gain insights into how these essential modifications impart functionality to the receptor, it is vital to identify sites of O-glycosylation, and structures of glycans at each of these sites.        The extracellular domain (ECD) of mouse Notch1 contains 36-tandem epidermal growth factor-like (EGF) repeats, 16 of which contain the consensus sequence for O-glucosylation (C<super>1</super>XSXPC<super>2</super>).  Using nanoLC-ESI-MS/MS to determine occupancy at these 16 sites, I found most predicted sites were efficiently modified, though efficiency of modifying O-glucose sites was cell type-dependent, and some sites were under-glucosylated.  O-Glucose was elongated to Xyl-&#913;1,3-Xyl-&#913;1,3-Glc at all sites. In addition, a novel non-traditional consensus site was found to be efficiently O-glucosylated at EGF 9, leading to a revision of the consensus sequence for O-glucosylation to allow Alanine N-terminal to Cysteine 2: C<super>1</super>X<underline>S</underline>X(P/A)C<super>2</super>.      The ECD of Drosophila Notch (dN) also contains 36 EGF repeats, the majority of which contain consensus sequences for O-fucosylation (C<super>2</super>XXXX(<underline>S/T</underline>)C<super>3</super>) and/or O-glucosylation (C1X<underline>S</underline>X(P/A)C2).  O-Fucose moieties can be further extended to a disaccharide by Fringe, a &#914;3N-acetylglucosaminyltransferase and known modulator of Notch.  O-Glucose moieties can be elongated by a xylosyltransferase to a disaccharide. I have mapped both O-fucose and O-glucose glycans on the ECD of dN over-expressed in S2 cells co-transfected with or without Fringe using nanoLC-ESI-MS/MS. I have also compared relative levels of O-fucosylation and Fringe elongation, as well as O-glucosylation and subsequent xylosylation, at specific sites using Multiple Reaction Monitoring (MRM), a semi-quantitative method that allows relative quantitation of glycoforms at a specific site.  My results suggest the predicted glycosylation sites are very efficiently modified with the O-glycan monosaccharides and that extent of elongation of either O-linked modification is substoichiometric and site-specific."},{"label":"dcterms.available","value":"2015-04-24T14:48:29Z"},{"label":"dcterms.contributor","value":"Kenneth D. Irvine."},{"label":"dcterms.creator","value":"Rana, NadiaA."},{"label":"dcterms.dateAccepted","value":"2012-05-17T12:21:52Z"},{"label":"dcterms.dateSubmitted","value":"2012-05-17T12:21:52Z"},{"label":"dcterms.description","value":"Department of Molecular and Cellular Biology"},{"label":"dcterms.format","value":"Application/PDF"},{"label":"dcterms.identifier","value":"http://hdl.handle.net/1951/56096"},{"label":"dcterms.issued","value":"2011-05-01"},{"label":"dcterms.language","value":"en_US"},{"label":"dcterms.provenance","value":"Made available in DSpace on 2012-05-17T12:21:52Z (GMT). No. of bitstreams: 1\nRana_grad.sunysb_0771E_10514.pdf: 8710658 bytes, checksum: 8cabf69c3e6252e69fbe1f7bdacee4af (MD5)\n  Previous issue date:    1"},{"label":"dcterms.publisher","value":"The Graduate School, Stony Brook University: Stony Brook, NY."},{"label":"dcterms.subject","value":"Fringe, glycans, Mass spectrometry, Notch, O-fucose, O-glucose"},{"label":"dcterms.title","value":"On the Fringe of Notch: Mapping O-fucose and O-glucose glycans at specific sites on the extracellular domains of Notch receptors using nano-LC-ESI-MS/MS"},{"label":"dcterms.type","value":"Dissertation"},{"label":"dc.type","value":"Dissertation"}],"description":"This manifest was generated dynamically","viewingDirection":"left-to-right","sequences":[{"@type":"sc:Sequence","canvases":[{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json","@type":"sc:Canvas","label":"Page 1","height":1650,"width":1275,"images":[{"@type":"oa:Annotation","motivation":"sc:painting","resource":{"@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/56%2F20%2F27%2F56202710785804340734338630322315235763/full/full/0/default.jpg","@type":"dctypes:Image","format":"image/jpeg","height":1650,"width":1275,"service":{"@context":"http://iiif.io/api/image/2/context.json","@id":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/56%2F20%2F27%2F56202710785804340734338630322315235763","profile":"http://iiif.io/api/image/2/level2.json"}},"on":"https://repo.library.stonybrook.edu/cantaloupe/iiif/2/canvas/page-1.json"}]}]}]}