In the cytosol of the eukaryotic cells, peptide:N-glycanase (PNGase) releases high mannose type N-glycans from misfolded glycoproteins [ref 1]. The released glycans are designated as free oligosaccharides (fOSs). After generation of the fOSs, they are subsequently processed by endo-β-N-acetylglucosaminidase (ENGase) and cytosolic mannosidase Man2C1 [ref 1]. After the processing, the fOSs are transported into the lysosomes for further degradation [ref 1, 2].
In this protocol, neutral fOSs are extracted from mammalian cultured cells with 75% ethanol and purified with a coupled ion exchange chromatography, followed by graphitized carbon column. |
| Category | Biosynthesis & Metabolism |
| Protocol Name | Preparation of neutral free oligosaccharides from mammalian cultured cells |
Authors
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Yoichiro Harada
Glycometabolome Team, RIKEN Advanced Science Institute
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Reagents
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Phosphate-buffered saline (PBS) |
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Lysis buffer (20 mM Tris-HCl, pH7.4 and 10 mM EDTA) |
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Graphitized carbon column (150 mg/3 ml, GL-Science, Tokyo, Japan) |
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Dowex AG1-X2 (200–400 mesh, acetate form) (Bio-Rad) |
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Dowex AG50-X8 (200–400 mesh, H+ form) (Bio-Rad) |
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Polyprep column (Bio-rad) |
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Instruments
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Centrifuge (Avanti HP-30I, Beckman Coulter) and (himac CF8DL, Hitachi) |
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Speed Vac (Centrivap 78100, Labconco) |
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| Methods |
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1. |
Extraction of total fOSs from mammalian cultured cells |
| 1) |
Grow cells of your interest to 0.5 – 1 x 107 cells on a 10-cm dish. |
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Wash the cells twice with 5 ml of PBS. |
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Scrape the cells in 2 ml of PBS. |
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Centrifuge at 1,000 rpm for 5 min at 4ºC. |
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| 6) |
Add 300 μl of lysis buffer and resuspend. |
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Add 900 μl of ice-cold ethanol and vortex. |
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Centrifuge at 15,000 rpm for 15 min at 4ºC. |
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| 10) |
Dry the supernatant with Speed Vac. |
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2. |
Purification of the neutral fOSs |
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Resuspend the dried pellet with 1 ml of water. The solution becomes milky. If aggregates are formed, sonicate until dispersed completely. |
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Centrifuge at 15,000 rpm for 15 min at 4ºC and load the supernatant onto a coupled ion exchange chromatography (250 μl each of Dowex AG1-X2 and Dowex AG50-X8). |
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| 4) |
Repeat 2-(1), 2-(2) and 2-(3) twice. |
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Combine all the flow-through fractions. |
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Load the combined flow-through fractions onto a graphitized carbon column. |
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Wash the column with 3 ml of water. |
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| 8) |
Elute the bound fOSs with 2.5 ml of 25% acetonitrile. |
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| Notes | Complete extraction of total fOSs from mammalian cells can be achieved by a single extraction of the cells with 75% ethanol. Procedures for purification of the fOSs are based on the previous report [ref. 3]. |
| Initial amount | |
| Produced amount | Approximately 1-3 nmol in total. |
| Copyrights |
 Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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| Date of registration：2012-03-14 09:52:43 |
- Suzuki T. (2007) Cytoplasmic peptide:N-glycanase and catabolic pathway for free N-glycans in the cytosol. Semin. Cell. Dev. Biol. 17: 762-769 [PMID : 17950635]
- Chantret I. and Moore SE. (2008) Free oligosaccharide regulation during mammalian protein N-glycosylation. Glycobiology 18: 210-224 [PMID : 18218706]
- Hirayama H., et al. (2010) Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae. J. Biol. Chem. 285:12390-12404. [PMID : 20150426]
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