[Construction of the Man8 GlcNAc2 glycosylation Saccharomyces cerevisiae mutant strain].
In Saccharomyces cerevisiae, protein glycosylation passed two different N-linked modification pathways after the export of predominantly Man8 GlcNAc2-containing glycoproteins from ER to the Golgi. The core oligosaccharide undergoes maturation in the Golgi resulting in a Man8-13 GlcNAc2 structure. Alternatively, core structures may be hypermannosylated with up to 200 mannose residues composing of a backbone of alpha1,6-mannosyl residues with branched alpha1, 2- and alpha1,3-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 cle-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 Man8 GlcNAc2-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, Man8 GlcNAc2, in the mnn1 och1 mutant.
Zhou
Jun-Gang
JG
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. zhoujungang@tom.com
Zhang
Hou-Cheng
HC
Wang
Peng
P
Qi
Qing-Sheng
QS
D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D006031:Glycosylation; D008364:Mannosyltransferases / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008562:Membrane Glycoproteins; C075636:OCH1 protein, S cerevisiae; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; C025363:mannoproteins; C086604:MNN1 protein, S cerevisiae; D008364:Mannosyltransferases