Free oligosaccharides (fOSs) generated from high mannose type glycans are known to be accumulated in the cytosol of eukaryote. The fOSs have shown to be generated by two distinct pathways in mammalian cells. One pathway generates fOSs from lipid-linked oligosaccharides in the ER lumen. Alternatively, fOSs can be released from misfolded glycoproteins by the action of the cytosolic peptide:N-glycanase (PNGase) during endoplasmic reticulum associated degradation (ERAD) [Suzuki and Funakoshi. (2006) Glycoconj J]. Meanwhile, in budding yeast, all of the cytosolic fOSs are generated from misfolded glycoproteins in PNGase dependent manner (Fig. 1). Therefore, the structural and quantitative analysis of yeast fOSs are powerful method for monitoring behavior of misfolded glycoproteins that undergo endoplasmic reticulum (ER)-associated degradation (ERAD) in the cells [Hirayama et al. (2010) JBC]. Here, detail protocols for isolation and structural analysis in the cytosol of yeast cells are described. |
Category | Biosynthesis & Metabolism |
Protocol Name | Purification and Analysis of Free Oligosaccharides in Saccharomyces cerevisiae |
Authors
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Hirayama, Hiroto
Glycometabolome Team, Systems Glycobiology Research Group, Chemical Biology Department, RIKEN
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KeyWords |
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Reagents
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YPD (2 % peptone, 1 % yeast extract, and 2 % glucose) |
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100 % ethanol (Wako Pure Chemical Industries, Ltd., Osaka, Japan) |
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100 % acetonitrile (HPLC grade, Wako Pure Chemical Industries, Ltd.) |
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AG1-X2 (200 – 400 mesh; acetate form; Bio-Rad Laboratories, Hercules, CA) |
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AG50-X8 (200 – 400 mesh; H+ form; Bio-Rad Laboratories) |
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2 - aminopyridine (special grade for fluorescence labeling; Wako Pure Chemical Industries, Ltd.) |
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Dimethylamine-Borane (Wako Pure Chemical Industries, Ltd.) |
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Phenol (Wako Pure Chemical Industries, Ltd) |
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Chloroform (Wako Pure Chemical Industries, Ltd.) |
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Instruments
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InertSep GC column (150 mg/3 mL; GL Sciences Inc., Tokyo, Japan) |
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NH2P-50 4E column (4.6 × 250 mm; Showa Denko K. K., Tokyo, Japan) |
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Speed Vac concentrator (EYELA, Tokyo, Japan) |
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HPLC system (LaChrom Elite; Hitachi High-Tech Science Corporation, Tokyo, Japan) |
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Methods |
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Extraction of free oligosaccharides from yeast cells
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Grow over night culture (5 mL scale). |
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Dilute saturated cells to OD600 = 0.5 with YPD, and grow to OD600 = 2.0 (3–4 h) at 30˚C (50 mL scale). |
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Harvest the cells and resuspend 100 OD600 unit cells in 500 μL of 20 mM Tris-HCl (pH 8.0). |
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Further add 500 μL of 100% EtOH. |
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Mix and centrifuge at top speed for 5 min at 4˚C. |
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Transfer the supernatant to new tube, then lyophilize by Speed Vac or equivalent. |
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Desalting and purification of free oligosaccharides
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Resuspend lyophilized sample in 500 μL of DW. |
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Apply samples onto AG1-X2 and AG50-X8 (resin volume, 500 μL each) and wash the column with 3 mL water, then collect flow-through. |
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Apply the flow-through onto an equilibrated InertSep GC column. |
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Wash the column with 3 mL water (×2 times). |
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Elute purified free oligosaccharides with 3 mL of 25 % acetonitrile. |
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Evaporate samples to dryness by Speed Vac or equivalent. |
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Fluorescent labeling and HPLC analysis of free oligosaccharides
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Pyridylamino labeling and size-fractionation HPLC analysis of free oligosaccharides are carried out as described in “Fluorescent labeling of glycans and HPLC analysis” written by Shin-ichi Nakakita and Shunji Natsuka in glycoPOD. |
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Notes | If significant amount of glucose oligomers (derived from β-1,6-glucan in the cellwall) are contaminated in samples, the oligomers can be removed by treatment with an endo-1,6-β-glucanase as described previously [Hirayama et al., (2010) JBC] . |
Initial amount | |
Produced amount | Approximately 500 pmol of fOSs (Hex5-12HexNac2 forms glycans) |
Figure & Legends |
Figure & Legends
Fig. 1. Formation/catabolic pathway of fOSs in yeast.
On the luminal side of the ER, misfolded glycoproteins are recognized by the ERAD and retrotranslocated from the ER to the cytosol
(1). In the cytosol, Peptide:N-glycanase (Png1) cleaves N-glycans from misfolded glycoproteins and form fOSs. Several misfolded glycoproteins traffic between ER and Golgi and further mannosylated by a Golgi-localized mannosyltransferase, Och1, before their degradation (2). fOSs are trimmed a sole cytosolic α-mannosidase, Ams1, giving rise to M5 form fOSs.
This figure was originally published by Glycobiology. 21 (10): 1341–1348. 2011 “Metabolism of fOSs is facilitated in the och1Δ mutant of Saccharomyces cerevisiae” Hirayama H. and Suzuki T. Oxford University press
Fig. 2. The elution profile of yeast fOSs by size-fractionation HPLC (Shodex Asahipak NH2P-50).
Hex5–Hex12 represents Hex5HexNAc2–Hex12HexNAc2, respectively. The arrowheads indicate the elution position of PA-isomaltooligosaccharides (PA-glucose oligomer) for elution standard. *, non-specific peak. |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2014-12-02 17:22:52 |
- Suzuki T., Funakoshi Y., (2006) Free N-linked oligosaccharide chains: formation and degradation. Glycoconj J. 23,291–302 [PMID : 16897173]
- Hirayama H., Seino J., Kitajima T., Jigami Y., Suzuki T., (2010) Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae. J. Biol. Chem. 285, 12390–404 [PMID : 20150426]
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