Voir dire efficacy in highly publicized criminal cases

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 beta1,3-glucose, catalyzed by beta3-glucosyltransferase (beta3GlcT). Pofut2 knockout mice are early embryonic lethal while beta3GlcT mutations in humans cause a development disorder called Peters plus syndrome. To understand Pofut2 and beta3GlcT 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.