Investigating the pathway of asparagine-linked glycoprotein biosynthesis

Abstract

The biosynthesis of asparagine-linked glycoproteins, highly conserved throughout all eukaryotes, requires a dolichylpyrophosphate-linked tetradecasaccharide precursor (Dol-PP-GlcNAc2Man9Glc3), from which the tetradecasaccharide is transferred co-translationally to nascent polypeptides in the lumen of the ER by the multimeric membrane-bound enzyme, oligosaccharyl transferase (OT). The saccharide donor is assembled by a series of membrane-bound enzymes, which together comprise the dolichol pathway. Despite over two decades of genetic and bioinformatics approaches that have identified the vast majority of dolichol pathway genes in yeast, the roles of two mannosyltransferases in the pathway, Alg2 and Algl 1, remained ambiguous. This thesis describes the biochemical studies that were carried out to clarify these roles. The substrate specificity of Algl, the first mannosyltransferase in the pathway, was studied, and this enzyme was also used as a tool to prepare Man1 ,4-GlcNAc2-PP-Dol from synthetic GlcNAc2-PP-Dol. Access to this trisaccharide intermediate facilitated the characterization of Alg2 function, proposed to be involved in addition of the second and/or third mannose.

(cont.) A cell membrane fraction isolated from E. coli overexpressing thioredoxin-tagged Alg2 was used to demonstrate that this enzyme carries out an al 1,3-mannosylation, followed by an al,6-mannosylation, to form the branched pentasaccharide intermediate of the dolichol pathway. Having the means to access this intermediate chemoenzymatically, it was thus possible to define the function of Algl 1, which had similarly been proposed to catalyze addition of the fourth and/or fifth mannose. Using the same procedure, TRX-Algll 1 was shown to catalyze two sequential al 1,2-mannosylations onto the a 1,3-branch of the pentasaccharide substrate to afford the heptasaccharide intermediate. The elucidation of the dual function of each of these enzymes thus completes the identification of the entire ensemble of glycosyltransferases that comprise the dolichol pathway. Finally, peptidyl mimics based on the consensus site for glycosylation by OT on nascent polypeptides, Asn-Xaa-Thr/Ser, were designed and evaluated.

(cont.) Both substrate-based peptide isosteres and product-based neoglycoconjugates were used to investigate the conformational and stereoelectronic preferences of OT binding. Neoglycoconjugates also showed promise as inhibitors of the deglycosylating enzyme Peptide: N-glycanase (PNGase), which aids in the degradation of misfolded proteins in the secretory pathway.