| dc.identifier.uri | http://hdl.handle.net/1951/56096 | |
| dc.identifier.uri | http://hdl.handle.net/11401/71674 | |
| dc.description.sponsorship | This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. | en_US |
| dc.format | Monograph | |
| dc.format.medium | Electronic Resource | en_US |
| dc.language.iso | en_US | |
| dc.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dc.type | Dissertation | |
| dcterms.abstract | 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-Α1,3-Xyl-Α1,3-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 Β3N-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. | |
| dcterms.available | 2012-05-17T12:21:52Z | |
| dcterms.available | 2015-04-24T14:48:29Z | |
| dcterms.contributor | William J. Lennarz | en_US |
| dcterms.contributor | Robert S. Haltiwanger. Erwin London. | en_US |
| dcterms.contributor | Deborah Brown | en_US |
| dcterms.contributor | Kenneth D. Irvine. | en_US |
| dcterms.creator | Rana, NadiaA. | |
| dcterms.dateAccepted | 2012-05-17T12:21:52Z | |
| dcterms.dateAccepted | 2015-04-24T14:48:29Z | |
| dcterms.dateSubmitted | 2012-05-17T12:21:52Z | |
| dcterms.dateSubmitted | 2015-04-24T14:48:29Z | |
| dcterms.description | Department of Molecular and Cellular Biology | en_US |
| dcterms.format | Monograph | |
| dcterms.format | Application/PDF | en_US |
| dcterms.identifier | http://hdl.handle.net/1951/56096 | |
| dcterms.identifier | Rana_grad.sunysb_0771E_10514.pdf | en_US |
| dcterms.identifier | http://hdl.handle.net/11401/71674 | |
| dcterms.issued | 2011-05-01 | |
| dcterms.language | en_US | |
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Previous issue date: 1 | en |
| dcterms.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dcterms.subject | Biochemistry -- Cellular Biology | |
| dcterms.subject | Fringe, glycans, Mass spectrometry, Notch, O-fucose, O-glucose | |
| dcterms.title | 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 | |
| dcterms.type | Dissertation | |
