In the endoplasmic reticulum (ER), dolichylpyrophosphoryl oligosaccharide (Dol-PP-OS) is the glycan donor substrate for asparagine (N)-linked glycosylation 1). In some eukaryotes, the matured Dol-PP-OS is composed of 14 sugars (three glucose, nine mannose, two N-acetylglucosamine residues) conjugated with the dolichol lipid through a pyrophosphate group.
In the wild type budding yeast (Saccharomyces cerevisiae), the matured Dol-PP-OS is synthesized. The Dol-PP-OS is extracted from the yeast microsomes by differential extractions with organic solvents 2). The glycan moiety of the Dol-PP-OS can be released from dolichol by mild acid hydrolysis for structural analysis by high performance liquid chromatography (HPLC) after fluorescent labeling with 2-aminopyridine 3). |
Category | Biosynthesis & Metabolism |
Protocol Name | Preparation of dolichylpyrophosphoryl oligosaccharides from Saccharomyces cerevisiae |
Authors
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Harada, Yoichiro
Glycometabolome Team, RIKEN Advanced Science Institute
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KeyWords |
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Reagents
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Yeast cells (In this protocol, BY4741 (purchased from Thermo Fisher Scientific Inc., Waltham, MA) haploid cells were used.) |
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YPD medium for yeast culture, autoclaved (YPD: yeast extract, 10 g/L; tryptone, 20 g/L and glucose, 20 g/L) |
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Phosphate-buffered saline (PBS) |
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Lysis buffer (20 mM Hepes-KOH, pH 7.4, 2 mM EDTA, 200 mM sorbitol, 50 mM potassium acetate) |
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Resuspension buffer (20 mM Tris-HCl, pH 7.4 and 250 mM sucrose) |
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Methanol (abbreviated as M) |
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Chloroform (abbreviated as C) |
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Graphitized carbon column (150 mg/3 mL, GL Sciences Inc., Tokyo, Japan) |
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Dowex AG1-X2 (200–400 mesh, acetate form) (Bio-Rad Laboratories, Hercules, CA) |
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Dowex AG50-X8 (200–400 mesh, H+ form) (Bio-Rad Laboratories) |
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Polyprep column (10 mL, Bio-Rad Laboratories) |
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Instruments
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Shaker for yeast culture (Excella E24, New Brunswick Scientific Co., Inc. Edison NJ) |
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Glass beads (0.5 mm, Yasui Kikai Corp., Osaka, Japan) |
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Glass tube with screw cap (PYREX) |
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Bead-beater (Biospec Products Inc., Bartlesville, OK) |
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Centrifuge (Avanti HP-30I, Beckman Coulter, Inc., Brea, CA) and (himac CF8DL, Hitachi, Ltd., Tokyo, Japan) |
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Ultracentrifuge (CP-80WX, Hitachi Ltd.) |
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Probe-type sonicator (or water bath-type sonicator) (VC750, Sonics & Materials, Inc., Newtown, CT) |
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Incubator that can heat up to over 100ºC (NDS-400, EYELA, Tokyo, Japan) |
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Speed Vac (Centrivap 78100, Labconco Corp., Kansas City, MO) |
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Methods |
1. |
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1) |
Pre-culture yeast cells in YPD medium at 30ºC for 18 h. |
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2) |
Prepare six 2-L flasks containing 1 L each of YPD. |
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Add 1 mL of the pre-culture to each flask. |
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Incubate the culture at 200 rpm at 30ºC until an optical density at 600 nm (OD600) reaches 7–10. This incubation step may take 16 h. |
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Preparation of the microsomes from yeast
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Harvest the cells by centrifugation at 3,000 rpm for 5 min at 4ºC and discard the supernatant. |
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2) |
Resuspend the cells with 100 mL of water and centrifuge at 3,000 rpm for 5 min at 4ºC. |
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Resuspend the cells with 100 mL of PBS and centrifuge at 3,000 rpm for 5 min at 4ºC. |
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Resuspend the cell with Lysis buffer and adjust the volume to approximately 150 mL. |
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Transfer the cell suspension to a bead-beater chamber. |
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Add approximately 150 mL of ice-cold glass beads. |
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Lyse the cell by sixteen 10-sec homogenizations separated by 10-sec cooling periods on ice. |
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Filtrate the homogenate into a glass beaker through a funnel fitted with gauzes. |
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Centrifuge the filtrate at 3,000 rpm for 5 min at 4ºC and recover the supernatant. |
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Centrifuge the supernatant at 6,000 × g for 5 min at 4ºC and recover the supernatant. |
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Ultracentrifuge the supernatant at 100,000 × g for 1 h at 4ºC and discard the supernatant. |
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Resuspend the pellet (microsomes) with a small volume of resuspension buffer. |
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The final volume of the microsomes may be 5–10 mL. |
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17) |
If the microsomes become jelly-like substances, pass the microsomes 10–20 times through 18G and 22G needles sequentially. |
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The microsomes may be flash frozen in liquid nitrogen and stored at −80ºC until use (Not recommended though). |
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3. |
Extraction of Dol-PP-OS from the microsomes
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If the microsomes are frozen, thaw on ice slowly. |
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Transfer 2 mL of the microsomes into a glass tube with screw cap. |
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Add 30 mL of ice-cold C/M (2:1, v/v), vortex and sonicate until any aggregate becomes dispersed. |
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Centrifuge at 3,000 rpm for 10 min at 4ºC and discard the supernatant. |
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Dry the pellet under nitrogen stream. |
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Add 30 mL of ice-cold M/W (1:1, v/v) containing 4 mM MgCl2, vortex and sonicate until any aggregate becomes dispersed. |
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Centrifuge at 3,000 rpm for 10 min at 4ºC and discard the supernatant. |
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Dry the pellet under nitrogen stream. |
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Add 4 mL of C/M/W (10:10:3, v/v/v, room temperature; the temperature is IMPORTANT!), vortex, sonicate until any aggregate becomes dispersed and then incubate at 37ºC for 10 min. |
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12) |
Centrifuge at 3,000 rpm for 10 min at room temperature (IMPORTANT!) and save the supernatant. |
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13) |
Repeat 3-11) and 3-12) once and save the supernatant. |
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Combine the first and second supernatants and dry in vacuo. |
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Extract Dol-PP-OS from the dried pellet by addition of 4 mL of C/M/W (10:10:3, v/v/v, room temperature; the temperature is IMPORTANT!) and vortex. |
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Centrifuge at 3,000 rpm for 10 min at room temperature and save the supernatant. |
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Repeat 3-15) and 3-16) once and save the supernatant. |
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Combine the first and second supernatant and dry in vacuo. |
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Dissolve the pellet in a small volume (~ 1 mL) of C/M/W (10:10:3, v/v/v, room temperature). The resultant solution contains the partially purified Dol-PP-OS. |
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Store the partially purified Dol-PP-OS at −80ºC. |
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4. |
Release of glycan from the Dol-PP-OS preparation by mild acid hydrolysis (Optional)
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Transfer various volumes of the Dol-PP-OS preparation to glass tubes with screw cap. |
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Add 500 μL of isopropanol and vortex. |
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Add 500 μL of 40 mM HCl and vortex (proceed the next step even you see some aggregates). |
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Heat the glass tubes at 100ºC for 30 min. |
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Add 1 mL of water, vortex and centrifuge at 3,000 rpm for 10 min at 4ºC. |
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Save the supernatant (it contains the released glycans). |
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Apply the combined supernatant (approximately 3 mL) to a graphitized carbon column. |
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Wash the column with 3 mL of water. |
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Elute the glycan with 2.5 mL of 25% acetonitrile in water. |
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Dissolve the dried samples in water and apply onto a coupled ion exchange chromatography (250 μL each of Dowex AG1-X2 and Dowex AG50-X8, resins should be carefully packed in a Polyprep column). |
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Wash the column with 1.5 mL of water and save the flow-through. |
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Combine all the flow-through fractions and dry in vacuo. |
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Label the glycan with 2-aminopyridine and analyze them by high performance liquid chromatography (HPLC) as described elsewhere. The glycans were quantitated by using PA-isomaltohexaose included in PA-glucose oligomer DP=3-15 (Takara Bio Inc., Otsu, Japan) as a standard. |
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Initial amount | |
Produced amount | Approximately 10 nmol of total DLOs as PA-isomaltohexose. |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2014-08-29 17:18:11 |
- Aebi, M., Bernasconi, R., Clerc, S.. and Molinari, M. (2010) N-glycan structures: recognition and processing in the ER. Trends. Biochem. Sci. 35, 74–82 [PMID : 19853458]
- Gao, N.. and Lehrman, M.A. (2006) Non-radioactive analysis of lipid-linked oligosaccharide compositions by fluorophore-assisted carbohydrate electrophoresis. Methods Enzymol. 415, 3–20 [PMID : 17116464]
- Hase, S., Hara, S., and Matsushima, Y. (1979) Tagging of sugars with a fluorescent compound, 2-aminopyridine. J. Biochem. 85, 217–220 [PMID : 762044]
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