Tunicamycin

Authors：

Category

Biosynthesis & Metabolism

Protocol Name

Tunicamycin

Authors

Yong Ma, Bruce
Research Center for Glycobiotechnology, Ritsumeikan University

KeyWords

oligosaccharides tunicamycin N-linked glycosylation

Reagents

●	Cultured cell line, either adherent or suspension
●	Complete culture medium appropriate for cell line
●	Tunicamycin (stock solution 1mg/mL), an inhibitor of N-linked glycosylation: Dissolve tunicamycin (mw 840) in dimethylsulfoxide (DMSO), dimethylformamide (DMF), 95% ethanol, or 25 mM NaOH to 1 mg/mL. Stable ~1 year at −20°C.
●	Solvent used for making inhibitor solution
●	Multiply deficient medium without glucose
●	[³H]mannose (5 to 20 Ci/mmol)
●	[³⁵S]methionine (> 800 Ci/mmol)
●	Metabolic radiolabeling of glycoconjugates
●	Phosphate-buffered saline (PBS)
●	Lysis buffer
●	0.5 U/mL endoglycosidase H (endo H) and endo H digestion buffer [Note] All chemicals for TCA precipitation, gel electrophoresis, immunoprecipitation and immunoblotting.

Instruments

●	37°C, 5% CO₂ incubator for cell culture
●	Laser confocal microscopy (e.g. FV1000, Olympus, Tokyo, Japan)
●	Image analyzer for chemiluminescence (e.g. LAS-4000, Fujifilm, Tokyo, Japan)
●	Centrifuges
●	24-well and 100-mm tissue culture plates
●	1.5-mL, 15-mL, or 50-mL conical polypropylene centrifuge tube
●	Additional regents and equipment for counting viable cells
●	Autoradiography

Methods

Determine optimal tunicamycin concentration

1)	For tunicamycin tested, set up 15 wells of a 24-well tissue culture plate with 1.8 mL cells/well (1–5 × 10⁴ cells/well). Incubate 24 h.	Comment 1

2)	While cells are incubating, make a series of 1:1 (v:v) dilutions of tunicamycin as follows: Place 80 μL of tunicamycin stock solution (1 mg/mL) in a sterile microcentrifuge tube and dilute to 800 μL with complete culture medium. Transfer 400 μL to a second tube and dilute with 400 μL complete culture medium. Repeat for a total of seven tubes (tunicamycin concentration range 0.15–10 μg/mL). As a control, prepare an identical series of dilutions of the solvent used for making the tunicamycin stock solution.	Comment 0

3)	Add 200 μL tunicamycin-containing medium from each dilution tube to a well of the tissue culture plate (from step 2) containing cells (2 mL final). Add 200 μL complete culture medium alone to the remaining well (as a zero-inhibitor control point).	Comment 1

4)	Incubate plate 24 h. Store the unused tunicamycin-containing medium (200 μL/tube) at 4°C until used in step 6.	Comment 0

5)	Perform short-term labeling of cells with [³⁵S]methionine using 0.2 mCi/mL label and 2 mL final volume per well.	Comment 0

6)	Add the remaining 200 μL of each dilution of tunicamycin (from step 4) to the wells and incubate an additional 4 h.	Comment 0

7)	Harvest cells and determine the incorporation of [³⁵S]methionine into macromolecules by TCA precipitation.	Comment 0

Plot label incorporation versus tunicamycin concentration.

Comment 1

Label cells in the presence of tunicamycin for inhibiting N-linked glycosylation

1)	Split a new cell sample into complete medium in a 100-mm tissue culture plate and incubate 24 h. The cell density should be 5 × 10⁵ to 2.5 × 10⁶ cells/plate.	Comment 0

2)	Add tunicamycin to the optimal concentration determined above. Set up a control plate containing same quantity of stock solution solvent but no tunicamycin. Incubate 24 h.	Comment 0

3)	Wash cells (using the same procedure as employed during short-term labeling, step 1–5) and, to each plate, add sufficient glucose-free MDM to cover the cells (~5 mL for a 100-mm plate).	Comment 0

4)	Add tunicamycin or solvent as in step 2-2. Add [³H]mannose to 0.02 to 0.1 mCi/mL. Incubate 4 to 12 h.	Comment 1

Harvest adherent cells by scraping with a disposable scraper into ice-cold PBS. Harvest suspension cells by centrifuging several minutes at 300 × g, 4°C, and resuspend in ice-cold PBS.

Comment 0

Measure incorporated macromolecular radioactivity

1)	Determine the amount of incorporated macromolecular radioactivity by TCA precipitation.	Comment 0

2)	If desired, and if methods are available for purifying and identifying a specific glycoprotein from cells rediolabeled with [³⁵S]methionine, examine the effect of a given tunicamycin by SDS-PAGE and autoradiography.	Comment 0

3)	Instead of autoradiography, immunoprecipitation together with immunoblotting can be used for analyzing the inhibition of N-linked glycosylation by tunicamycin.	Comment 0

Instead of autoradiography, confocal microscopy can be used for studying the role of tunicamycin on the subcellular localization of cargo transport lectins in glycoprotein quality control.

Comment 0

Discussion

The optimal concentration of tunicamycin for the experiment (i.e., highest nontoxic concentration) is determined by monitoring [³⁵S]methionine incorporation as a measure of protein biosynthesis.

The tunicamycin’s ability to inhibit oligosaccharide processing is then determined by analyzing cells labeled with [³H]mannose using TCA precipitation or endoglycosidase H digestion.

Tunicamycin also blocks the assembly of type II keratan sulfate chains, because these glycans also utilize GlcNAc-PP-dolichol. Glycolipid biosynthesis may also be inhibited, although the mechanism underlying this has not been established.

Each N-linked carbohydrate chain contributes ~2000 to 4000 Da to a protein’s mass, so synthesis of a protein in the presence of tunicamycin will result in faster mobility on SDS-PAGE and generally show less size heterogeneity, and proteins will not shift to a faster mobility on SDS-PAGE after PNGase F treatment.

Figure & Legends

Fig. 1. Effects of N-glycosylation inhibitor treatment on the I-MBP/LAMP-1 interaction

HEK293 cells were treated with the inhibitors for 24 h at 37°C prior to transfection. After transfection, the cells were cultured in the presence of inhibitors again for 24 h under the same conditions prior to harvesting for immunoprecipitation and immunoblotting.

This figure was originally published in J Biol Chem. Nonaka M, Ma BY et al. "Subcellular localization and physiological significance of intracellular mannan-binding protein" 2007 282(24):17908–20. © the American Society for Biochemistry and Molecular Biology.

Fig. 2. Carbohydrate-dependent subcellular localization of I-MBP

MBP-GFP-transfected human hepatoma HLF cells were treated for 24 h at 37°C with 1 μg/mL tunicamycin, which prevents N-linked glycosylation. Confocal microscopy showed that the subcellular localization of I-MBP exhibited an equally diffuse cytoplasmic distribution (Fig. 2B), in contrast to the punctate structures observed in the control cells (Fig. 2A).

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Date of registration：2015-02-03 14:33:49