Tags

Type your tag names separated by a space and hit enter

[Construction of the Man8 GlcNAc2 glycosylation Saccharomyces cerevisiae mutant strain].
Wei Sheng Wu Xue Bao. 2007 Oct; 47(5):785-9.WS

Abstract

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.

Authors+Show Affiliations

Zhou JG
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China. zhoujungang@tom.com
Zhang HC
No affiliation info available
Wang P
No affiliation info available
Qi QS
No affiliation info available

MeSH

Base SequenceChromatography, High Pressure LiquidGlycosylationMannosyltransferasesMembrane GlycoproteinsMolecular Sequence DataOligosaccharidesSaccharomyces cerevisiaeSaccharomyces cerevisiae ProteinsSpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

chi

PubMed ID

18062249

Citation

Zhou, Jun-Gang, et al. "[Construction of the Man8 GlcNAc2 Glycosylation Saccharomyces Cerevisiae Mutant Strain]." Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica, vol. 47, no. 5, 2007, pp. 785-9.
Zhou JG, Zhang HC, Wang P, et al. [Construction of the Man8 GlcNAc2 glycosylation Saccharomyces cerevisiae mutant strain]. Wei Sheng Wu Xue Bao. 2007;47(5):785-9.
Zhou, J. G., Zhang, H. C., Wang, P., & Qi, Q. S. (2007). [Construction of the Man8 GlcNAc2 glycosylation Saccharomyces cerevisiae mutant strain]. Wei Sheng Wu Xue Bao = Acta Microbiologica Sinica, 47(5), 785-9.
Zhou JG, et al. [Construction of the Man8 GlcNAc2 Glycosylation Saccharomyces Cerevisiae Mutant Strain]. Wei Sheng Wu Xue Bao. 2007;47(5):785-9. PubMed PMID: 18062249.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - [Construction of the Man8 GlcNAc2 glycosylation Saccharomyces cerevisiae mutant strain]. AU - Zhou,Jun-Gang, AU - Zhang,Hou-Cheng, AU - Wang,Peng, AU - Qi,Qing-Sheng, PY - 2007/12/8/pubmed PY - 2009/5/13/medline PY - 2007/12/8/entrez SP - 785 EP - 9 JF - Wei sheng wu xue bao = Acta microbiologica Sinica JO - Wei Sheng Wu Xue Bao VL - 47 IS - 5 N2 - 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. SN - 0001-6209 UR - https://www.unboundmedicine.com/medline/citation/18062249/[Construction_of_the_Man8_GlcNAc2_glycosylation_Saccharomyces_cerevisiae_mutant_strain]_ DB - PRIME DP - Unbound Medicine ER -