| dc.identifier.uri | http://hdl.handle.net/11401/76932 | |
| 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 | Protein <italic>O</italic>-fucosyltransferase 2 (Pofut2) is a soluble, ER localized enzyme that adds a fucose residue to specific Serines and Threonines found in Thrombospondin type I repeats (TSRs). TSRs are small, cysteine-rich motifs usually found as tandem repeats. The current consensus sequence for <italic>O</italic>-fucosylation is CXX(S/T)CXXG. Database searches with the consensus sequence predict fifty TSR-containing protein targets for Pofut2. The <italic>O</italic>-fucose on TSRs is extended by the addition of a β 1,3-glucose, catalyzed by β 3-glucosyltransferase (β 3GlcT). Pofut2 knockout mice are early embryonic lethal while β 3GlcT mutations in humans cause a development disorder called Peters plus syndrome. To understand Pofut2 and β 3GlcT phenotypes, it is important to deduce the molecular role of <italic>O</italic>-fucosylation. Pofut2 can distinguish between properly folded and unfolded TSRs <italic>in vitro</italic>. Taken together with its localization to the ER, a protein-folding compartment, we have hypothesized that Pofut2 plays a role in quality control. Eliminating the donor substrate, GDP-fucose, or Pofut2, results in loss of secretion of two targets - ADAMTS13 and ADAMTSL1. In this thesis, I extend these observations to other Pofut2 targets and demonstrate that Pofut2 has a dual role as a chaperone and fucosyltransferase. I show that both the number of tandem TSRs and the primary amino acid sequence influence fucose-dependent secretion. I demonstrate that <italic>O</italic>-fucosylation is both co-translational and post-translational. I show that in the absence of GDP-fucose, Pofut2 binds more tightly to its substrates, providing a potential explanation for why elimination of GDP-fucose results in decreased secretion of target proteins. I also identify several ER-resident proteins that are in complex with Pofut2, potentially assisting in the folding of TSRs and retaining Pofut2 in the ER. Mature TSRs from target proteins show high stoichiometries of O-fucosylation, whereas most cell-associated proteins are aggregated, partially folded and poorly fucosylated. A small portion of cell-associated protein is mostly folded and is nearly fully fucosylated, suggesting that <italic>O</italic>-fucosylation is a marker of properly folded TSRs in the cell. Finally, I establish a direct role for Pofut2 in the folding of TSRs in vitro and determine that both GDP-fucose and enzymatic activity are required for this process. | |
| dcterms.available | 2017-09-20T16:51:28Z | |
| dcterms.contributor | Haltiwanger, Robert S | en_US |
| dcterms.contributor | Brown, Deborah | en_US |
| dcterms.contributor | Holdener, Bernadette | en_US |
| dcterms.contributor | Majerus, Elaine. | en_US |
| dcterms.creator | Vasudevan, Deepika | |
| dcterms.dateAccepted | 2017-09-20T16:51:28Z | |
| dcterms.dateSubmitted | 2017-09-20T16:51:28Z | |
| dcterms.description | Department of Biochemistry and Cell Biology. | en_US |
| dcterms.extent | 143 pg. | en_US |
| dcterms.format | Application/PDF | en_US |
| dcterms.format | Monograph | |
| dcterms.identifier | http://hdl.handle.net/11401/76932 | |
| dcterms.issued | 2015-08-01 | |
| dcterms.language | en_US | |
| dcterms.provenance | Made available in DSpace on 2017-09-20T16:51:28Z (GMT). No. of bitstreams: 1
Vasudevan_grad.sunysb_0771E_11634.pdf: 12079334 bytes, checksum: 301523a3860f08bb04196f1f5598bb70 (MD5)
Previous issue date: 2013 | en |
| dcterms.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dcterms.subject | Chaperone, Folding, O-Fucosylation, Pofut2, TSR | |
| dcterms.subject | Biochemistry | |
| dcterms.title | A Role for O-Fucosylation in the Folding of Thrombospondin Type I Repeats | |
| dcterms.type | Dissertation | |
