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• W130491139 abstract "Asparagine (N)-linked glycosylation is a common post-translational modification that regulates the structure and function of secretory and membrane proteins in eukaryotes. This process is catalysed by the multiprotein complex oligosaccharyltransferase (OTase), which glycosylates selected asparagine residues in nascent polypeptides as they enter theendoplasmic reticulum (ER) through the translocon. N-glycosylation is crucial in protein folding, and the precise glycans present on mature glycoproteins are often vital for their biological functions. Two isoforms of OTase exist in Bakers’ yeast Saccharomyces cerevisiae, determined by the presence of either of the homologous proteins Ost3p or Ost6p. Previous studies have identified that efficient glycosylation of some specific sites requires Ost3p, while others require Ost6p. Ost3p and Ost6p have thioredoxin-like ER soluble domains with grooves adjacent to their redox active sites, and our recent in vitro assays have shown that Ost3p and Ost6p can bind stretches of hydrophobic polypeptide with complementary characteristics to these peptide-binding grooves. A model of Ost3/6p function has been proposed based on this in vivo and structural data in which we hypothesize that Ost3p and Ost6p transiently bind nascent polypeptide to increase the glycosylation efficiency of selected asparagines. In the first part of the thesis, we tested a key prediction of this model using yeast genetics and glycoproteomics and established that this peptide-binding activity of Ost3/6p is physiologically relevant in N-glycosylation in vivo, suggesting that nascent polypeptide binds directly to the grooves in Ost3/6p. Further, we identified the precise sites of interaction between the Gas1p substrate nascent polypeptide and the Ost3/6p peptide binding groove in vivo, using a combination of site-directed mutagenesis and mass spectrometry glycoproteomics. Finally, using our in vitro peptide binding assay, we demonstrated and validated that a stretch of Gas1p we identify as interacting with Ost3/6p in vivo also interacted with Ost6p in vitro. Together, this study advances our understanding of Ost3/6p function in which they transiently bind stretches of nascent polypeptide substrate proximal and N-terminal to specific glycosylation sites, which inhibits local protein folding and thereby increases glycosylation efficiency.In the second part of the thesis, we investigated the in vitro interactions between substrate polypeptide and Ost3/6p by peptide affinity chromatography and Selected Reaction Monitoring (SRM) Mass Spectrometry. We developed SRM-MS for improved relative quantification and sensitivity for detection in conjunction with peptide affinity chromatography. We show that Ost3p and Ost6p bind distinct subsets of peptides from a yeast cell wall glycoprotein substrate, with Ost3p binding peptides rich in aromatic acids and Ost6p binding peptides rich in aliphatic amino acids. Our data supports a model of Ost3/6p function in which they transiently bind complementary hydrophobic stretches of nascent polypeptide substrate to optimally inhibit protein folding, thereby increasing glycosylation efficiency at nearby asparagine residues. In the third part of the thesis, we developed another improved analytical method for sitespecific glycosylation analysis using PNGase F to label glycosylation sites with an asparagines-aspartate conversion that creates a new endoproteinase AspN cleavage site, followed by proteolytic digestion, and detection of peptides and glycopeptides by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). This approach will be useful in site-specific glycosylation analysis in many model systems and clinical application. Overall, these two novel analytical methods introduced in the second and the third part of this thesis will provide advanced knowledge for improved detection and quantification of glycosylation sites in vivo and peptide interactions in vitro.In the fourth part of the thesis, we used a novel strategy for engineering glycoprotein stability to determine the detailed function of Ost3p and Ost6p in vitro. The ER lumenal domain of Ost3p has a single glycosylation sequon, while Ost6p has no glycosylation sequons and contains a cluster of acidic amino acids at the position corresponding to the Ost3p  sequon. We showed that incorporation of targeted like-charged amino acids at N-glycosylation sequons in Ost3/6p increases in vitro stability and activity. Optimal stabilization through minimal number of local point mutations in Ost3p glycosylation sequon showed increased in vitro binding of peptides and these peptides are complementary to the characteristics of the peptide binding groove of Ost3, a key determinant of transient nascent polypeptide tethering by Ost3p, consistent with the function of Ost3p and Ost6p in modulating N-glycosylation substrate selection by OTase activity in vivo.In summary, this thesis has made a significant contribution towards our understanding the molecular mechanisms of glycosylation site selection by OTase activity, the connectivity between glycoprotein folding and N-glycosylation in the endoplasmic reticulum, and opens up potential applications in synthetic glycobiology such as engineering of glycosylation  sites and OTases. Our data provides advance information towards solving the model of the mechanisms by which Ost3/6p increase the efficiency of site-specific N-glycosylation. Here we detailed the mechanism of this regulatory system through mapping the precise sites of interactions between Ost3/6p and Gas1p nascent polypeptide which was not previously identified. This is a novel insight into the mechanisms of co-translocational polypeptide modification in the endoplasmic reticulum." @default.
• W130491139 created "2016-06-24" @default.
• W130491139 creator A5062387146 @default.
• W130491139 date "2015-03-09" @default.
• W130491139 modified "2023-09-27" @default.
• W130491139 title "Molecular mechanisms regulating N-glycosylation site selection by yeast oligosaccharyltransferase: Role of Ost3p and Ost6p" @default.
• W130491139 doi "https://doi.org/10.14264/uql.2015.362" @default.
• W130491139 hasPublicationYear "2015" @default.
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