Inhibition of N‐Linked Glycosylation

Leland D. Powell1

1 University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.14
DOI:  10.1002/0471142735.im0814s09
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 ability of the inhibitor to hinder oligosaccharide processing is then determined by analyzing cells labeled with [3H]mannose using TCA precipitation or endo H digestion. 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
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 5A.
  • Cultured cell line, either adherent or suspension
  • Complete culture medium appropriate for cell line
  • recipeInhibitor of N‐linked glycosylation (one or more of the following: tunicamycin, deoxynojirimycin, castanospermine, deoxymannojirimycin, or swainsonine; see recipe and Table 8.14.2)
  • recipeSolvent used for making inhibitor solution (see Inhibitor stock solutions recipe)
  • Multiply deficient medium (MDM; unit 8.13) without glucose
  • [3H]mannose (5 to 20 Ci/mmol)
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • Lysis buffer (unit 8.3) without 1% bovine hemoglobin
  • 0.5 U/ml endoglycosidase H (endo H) and recipeendo H digestion buffer (see recipe)
  • 20% (w/v) SDS
  • Sephacryl S‐200 (Pharmacia Biotech) column, calibrated and equilibrated
  • 25 mM ammonium formate/0.1% (w/v) SDS
  • 24‐well tissue culture plate
  • 100‐mm tissue culture plates
  • Sorvall H‐1000B rotor, or equivalent
  • 1.5‐ml, 15‐ml, or 50‐ml conical polypropylene centrifuge tube
  • Additional reagents and equipment for counting viable cells ( appendix 2A), short‐term labeling of suspension or adherent cells with [35S]methionine (unit 8.12), metabolic radiolabeling of glycoconjugates (unit 8.13), lysis of cells (for immunoprecipitation; unit 8.3), TCA precipitation (unit 8.12), endo H digestion (unit 8.15), gel‐filtration chromatography (unit 9.2), one‐dimensional gel electrophoresis of proteins (unit 8.4), and autoradiography ( appendix 3A)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Acetone Precipitation

  Additional Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • 100% acetone (HPLC or ACS grade), −20°C
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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 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. 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. 65:248‐255.
   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 review.
   McDowell, W. and Schwarz, R.T. 1988. See above.
  Useful general review.
   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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