Type
Text
Type
Dissertation
Advisor
Haltiwanger, Robert S | Brown, Deborah | Holdener, Bernadette | Majerus, Elaine.
Date
2015-08-01
Keywords
Biochemistry | Chaperone, Folding, O-Fucosylation, Pofut2, TSR
Department
Department of Biochemistry and Cell Biology.
Language
en_US
Source
This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree.
Identifier
http://hdl.handle.net/11401/76932
Publisher
The Graduate School, Stony Brook University: Stony Brook, NY.
Format
application/pdf
Abstract
Protein O-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 O-fucosylation is CXX(S/T)CXXG. Database searches with the consensus sequence predict fifty TSR-containing protein targets for Pofut2. The O-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 O-fucosylation. Pofut2 can distinguish between properly folded and unfolded TSRs in vitro. 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 O-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 O-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. | 143 pages
Recommended Citation
Vasudevan, Deepika, "A Role for O-Fucosylation in the Folding of Thrombospondin Type I Repeats" (2015). Stony Brook Theses and Dissertations Collection, 2006-2020 (closed to submissions). 2805.
https://commons.library.stonybrook.edu/stony-brook-theses-and-dissertations-collection/2805