Inhibition of N‐Linked Glycosylation

Leland D. Powell1

1 University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.10A
DOI:  10.1002/0471142727.mb1710as32
Online Posting Date:  May, 2001
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Abstract

Treatment of cells with inhibitors of the enzymes that synthesize N‐linked oligosaccharide chains results in production of glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of this class of oligosaccharides on specific proteins or intact cells. This unit describes the use of inhibitors to prevent N‐linked glycosylation of proteins in cultured cells. First, the optimal concentration of inhibitor for the experiment (i.e., highest nontoxic concentration) is determined by monitoring [35S]methionine incorporation as a measure of protein biosynthesis. The inhibitor's ability to inhibit oligosaccharide processing is then determined by analyzing cells labeled with [HH]mannose using TCA precipitation or endo H digestion (UNIT 13). Further suggestions are given on how to use methods for identifying a specific glycoprotein (if available) to measure the effect of the inhibitor on its N‐linked oligosaccharide chains. A support protocol details a method for concentrating proteins by acetone precipitation.

     
 
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Table of Contents

  • Support Protocol 1: Acetone Precipitation
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cultured cell line, either adherent or suspension
  • Complete culture medium
  • recipeInhibitor of N‐linked glycosylation (one or more of the following: tunicamycin, deoxynojirimycin, castanospermine, deoxymannojirimycin, or swainsonine; see reagents and solutions and Table 17.10.2).
  • Solvent used for making inhibitor solution (see reagents and solutions)
  • Multiply deficient medium (MDM; unit 17.4) without glucose
  • [3H]mannose (5 to 20 Ci/mmol)
  • Phosphate‐buffered saline (PBS; appendix 22), ice‐cold
  • Lysis buffer (unit 10.16) without 1% bovine hemoglobin
  • 0.5 U/ml endoglycosidase H (endo H) and recipeendo H digestion buffer ( reagents and solutions)
  • 20% (w/v) SDS
  • Sephacryl S‐200 column ( 97.80.4711Table 10.9.2)
  • 25 mM ammonium formate/0.1% (w/v) SDS
  • 24‐well tissue culture plate
  • 100‐mm tissue culture plates
  • Disposable plastic scraper or rubber policeman
  • 1.5‐ml, 15‐ml, or 50‐ml conical polypropylene centrifuge tube
  • Additional reagents and equipment for tissue culture (Chapter 9 introduction), counting viable cells (unit 11.5), short‐term labeling of suspension or adherent cells with [35S]methionine (unit 10.18), metabolic radiolabeling of glycoconjugates (unit 17.4), lysis of cells (as for immunoprecipitation; unit 10.16), TCA precipitation (unit 10.18), endo H digestion (unit 17.13), gel‐filtration chromatography (unit 10.9), and one‐dimensional gel electrophoresis of proteins (unit 10.2).
    Table 7.0.2   MaterialsRange of Inhibitor Concentrations for Dilution Series

    Inhibitor Stock solution Stock added to first dilution (µl) Concentration range
    Tunicamycin 1 mg/ml 80 0.15‐10 µg/ml
    Swainsonine 1 mg/ml 80 0.15‐10 µg/ml
    Deoxynojirimycin 400 mM 200 0.15‐10 mM
    Deoxymannojirimycin 400 mM 200 0.15‐10 mM
    Castanospermine 400 mM 200 0.15‐10 mM

Support Protocol 1: Acetone Precipitation

  Additional Materials
  • 100% acetone (HPLC or ACS grade), −20°C
  • 1.5‐ml, 15‐ml, or 50‐ml conical polypropylene or other centrifuge tubes
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Figures

Videos

Literature Cited

Literature Cited
   Elbein, A.D. 1987. Inhibitors of the biosynthesis and processing of N‐linked oligosaccharide chains. Annu. Rev. Biochem. 56:497‐534.
   Fuhrmann, U., Bause, E., Legler, G., and Ploegh, H. 1984. Novel mannosidase inhibitor blocking conversion of high mannose to complex oligosaccharides. Nature (Lond.) 307:755‐758.
   Guarnaccia, S.P., Shaper, J.H., and Schnaar, R.L. 1987. Tunicamycin inhibits ganglioside biosynthesis in neuronal cells. Proc. Natl. Acad. Sci. U.S.A. 80:1551‐1555.
   Hart, G.W. and Lennarz, W.J. 1978. Effects of tunicamycin on the biosynthesis of glycosaminoglycans by embryonic chick cornea. J. Biol. Chem. 253:5795‐5801.
   Hubbard, S.C. and Ivatt, R.J. 1981. Synthesis and processing of asparagine‐linked oligosaccharides. Annu. Rev. Biochem. 50:555‐583.
   Kobata, A. and Takasaki, S. 1992. Structure and biosynthesis of cell surface carbohydrates. In Cell Surface Carbohydrates and Cell Development (M. Fukuda, ed.) pp.1‐24. CRC Press, Boca Raton, Fla.
   Lubas, W.A. and Spiro, R.G. 1987. Golgi endo‐α‐D‐mannosidase from rat liver, a novel N‐linked carbohydrate unit processing enzyme. J. Biol. Chem. 262:3775‐3781.
   McDowell, W. and Schwarz, R.T. 1988. Dissecting glycoprotein biosynthesis by the use of specific inhibitors. Biochimie (Paris) 70:1535‐1549.
   Pan, Y.T., Hori, H., Saul, R., Sanford, B.A., Molyneux, R.J., and Elbein, A.D., 1983. Castanospermine inhibits the processing of the oligosaccharide protion of the influenza viral hemagglutinin. Biochemistry 22:3975‐3984.
   Romero, P., Friedlander, P., and Herscovics, A. 1985. Deoxynojirimycin inhibits the formation of Glc3Man9GlcNAc2‐PP‐dolichol in intestinal epithelial cells in cultures. FEBS Lett. 183:29‐32.
   Saunier, B., Kilker, R.D., Tkacz, J.S., Quaroni, A., and Herscovics, A. 1982. Inhibition of N‐linked complex oligosaccharide formation by 1‐deoxynojirimycin, an inhibitor of processing glucosidases. J. Biol. Chem. 257:14155‐14161.
   Tkacz, J.S. and Lampen, J.O. 1975. Tunicamycin inhibition of polyisoprenyl N‐acetylglucosaminyl pyrophosphate formation in calf liver microsomes. Biochem. Biophys. Res. Commun. 65:248‐55.
   Tulsiani, D.R.P., Harris, T.M., and Touster, O. 1982. Swainsonine inhibits the biosynthesis of complex glycoproteins by inhibition of Golgi mannosidase II. J. Biol. Chem. 257:7936‐7939.
   Tulsiani, D.R.P. and Touster, O. 1983. Swainsonine causes the production of hybrid glycoproteins by human skin fibroblasts and rat liver Golgi preparations. J. Biol. Chem. 258:7578‐7585.
   Yusuf, H.K.M., Pohlentz, G., Schwarzmann, G., and Sandhoff, K. 1983. Ganglioside biosynthesis in Golgi apparatus of rat liver. Eur. J. Biochem. 134:47‐54.
Key References
   Elbein, A.D. 1987. See above.
  Useful general reviews.
   McDowell, W. and Schwarz, R.T. 1988. See above.
  Useful general reviews.
   Hubbard, S.C. and Ivatt, R.J. 1981. See above.
  Good review on early work on the processing pathway.
   Kobata, A. and Takasaki, S. 1992. See above.
  Good review of recent work on some of the complexities in oligosaccharide processing.
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