N-Linked Protein Glycosylation. Functional characterization of the eukaryotic oligosaccharyltransferases

N-linked protein glycosylation is a highly conserved and an essential protein modification found throughout the three domains of life. In animal, plants and fungi the preassembled oligosaccharide consisting of two N-acetylglucosamines, nine mannoses and three glucoses is transferred en bloc from the lipid carrier to the asparagine side chains within N-X-S/T consensus sequences on nascent polypeptides in the lumen of the endoplasmic reticulum (ER). This transfer is catalysed by the central enzyme of the pathway, oligosaccharyltransferase (OST). After the transfer, the oligosaccharide is modified in a species, tissue and cell-specific manner, resulting in a vast array of different glycan structures. The first chapter of this thesis offers an overview of the N-linked protein glycosylation pathway. The biosynthesis of the lipid-linked oligosaccharide (LLO) is described first followed by the description of the OST and the biological relevance of this modification. Furthermore, diverse analytical tools and methods used to help explore and characterize the pathway, with the emphasis on the quantitative mass spectrometric approaches, are discussed. In the second chapter, we coupled parallel reaction monitoring (PRM) mass spectrometry method to stable isotope labelling (SILAC) to measure N-linked glycosylation occupancy of yeast glycoproteins. The OST from yeast Saccharomyces cerevisiae is a multi-subunit protein complex. Using our SILAC-PRM method, we further explored the roles of the two non-essential OST subunits, Ost3p and Ost6p, and provided insights into the mechanisms that regulate site-specific N-glycosylation by the OST. The investigation of the substrate specificity of the single subunit OSTs from Trypanosoma brucei is reported in chapter three. The effect of different oligosaccharide substrate structures on the function of TbOSTs was studied in yeast using genetic manipulations of the LLO biosynthesis. Additionally, structure to function coupled to SILAC-PRM analyses were performed to identify regions in the OST proteins that influence the specificity of different paralogues for the LLO as well as for the polypeptide substrate. In order to functionally characterize WBP1, an essential subunit of yeast OST, we performed a mutagenesis study reported in the fourth chapter. We have used reverse genetics approach for generating point mutations in the conserved residues of Wbp1p and identified novel mutants that alter glycosylation by influencing complex assembly. Concluding remarks and future perspectives complete the thesis in the fifth chapter