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A Role for O-Fucosylation in the Folding of Thrombospondin Type I Repeats

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dc.contributor.advisor Haltiwanger, Robert S en_US
dc.contributor.author Vasudevan, Deepika en_US
dc.contributor.other Department of Biochemistry and Cell Biology. en_US
dc.date.accessioned 2017-09-20T16:51:28Z
dc.date.available 2017-09-20T16:51:28Z
dc.date.issued 2015-08-01 en_US
dc.identifier.uri http://hdl.handle.net/11401/76932 en_US
dc.description 143 pg. en_US
dc.description.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 &beta; 1,3-glucose, catalyzed by &beta; 3-glucosyltransferase (&beta; 3GlcT). Pofut2 knockout mice are early embryonic lethal while &beta; 3GlcT mutations in humans cause a development disorder called Peters plus syndrome. To understand Pofut2 and &beta; 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. en_US
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 en_US
dc.format.medium Electronic Resource en_US
dc.language.iso en_US en_US
dc.publisher The Graduate School, Stony Brook University: Stony Brook, NY. en_US
dc.subject.lcsh Biochemistry en_US
dc.subject.other Chaperone, Folding, O-Fucosylation, Pofut2, TSR en_US
dc.title A Role for O-Fucosylation in the Folding of Thrombospondin Type I Repeats en_US
dc.type Dissertation en_US
dc.mimetype Application/PDF en_US
dc.contributor.committeemember Brown, Deborah en_US
dc.contributor.committeemember Holdener, Bernadette en_US
dc.contributor.committeemember Majerus, Elaine. en_US


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