Construction of the Man8GlcNAc2 glycosylation Saccharomyces cerevisiae mutant strain

Abstract:

In Saccharomyces cerevisiae，protein glycosylation passed two different N-linked modification pathways after the export of predominantly Man₈GlcNAc₂-containing glycoproteins from ER to the Golgi. The core oligosaccharide undergoes maturation in the Golgi resulting in a Man_8-13GlcNAc₂ structure. Alternatively，core structures may be hypermannosylated with up to 200 mannose residues composing of a backbone of α1，6-mannosyl residues with branched α1，2- and α1，3-mannosyl side chains. Mnn1p and Och1p play an important role in this process. The null disruption of MNN1，OCH1 was replaced by the S. cerevisiae URA3，HIS3，respectively. To characterize the N-glycosylation in the mnn1 och1 mutant，mannoproteins were obtained by hot citrate buffer extraction after the mnn1 och1 cells were crumbled. The extracted mannoprotein was precipitated by ethanol，and further purified by concanavalin A-sepharose 4B. The N-oligomannose saccharides were released from mannoprotein by PNGase F digestion，and then peptides and detergents were removed by passage through ion exchange columns. For desalting，glycans were applied to porous graphitic-carbon cartridge. 2-aminopyridine pyridylaminated sugars were profiled and purified by size fractionation HPLC with Shim-pack clc-NH2 column，and result showed dominantly a single peak. MALDI TOF/MS analysis ofthis peak revealed that its molecular weight was 1796.5Da，which corresponds to the calculated mass of Man₈GlcNAc₂-PA. These results indicated that disruptions of MNN1 and OCH1 eliminated the hypermannosylation of the N-linked glycans，and glycoproteins were glycosylated with a single core type glycan，Man₈GlcNAc₂，in the mnn1 och1 mutant.

Key words:Saccharomyces cerevisiae; Glycosylation; Mannoprotein; Mannosyltransferase