Inhibition of N‐Linked Glycosylation

Leland D. Powell1

1 University of California, San Diego, La Jolla
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 12.3
DOI:  10.1002/0471140864.ps1203s00
Online Posting Date:  May, 2001
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Abstract

This unit describes the use of inhibitors in cultured cells to prevent N‐linked glycosylation of proteins to yield glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of oligosaccharides on specific proteins or intact 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 [3H]mannose using TCA precipitation or endo H digestion. 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
  • Inhibitor of N‐linked glycosylation (one or more of the following: tunicamycin, deoxynojirimycin, castanospermine, deoxymannojirimycin, or swainsonine; see recipe for recipeinhibitor stock solutions and Table 12.3.2)
    Table 2.3.2   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

  • recipeSolvent used for making inhibitor solution (see recipe)
  • Multiply deficient medium (MDM; unit 12.2) supplemented with 200 µM glucose
  • [3H]mannose (5 to 20 Ci/mmol)
  • Phosphate‐buffered saline (PBS; appendix 2E), ice cold
  • recipeLysis buffer (see recipe)
  • Sepharose, activated
  • 0.5 U/ml endoglycosidase H (endo H) and recipeendo H digestion buffer (see recipe)
  • 20% (w/v) SDS ( appendix 2E)
  • Sephacryl S‐200 column (unit 8.3), calibrated and equilibrated in 25 mM ammonium formate/0.1% SDS
  • 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
  • Centrifuges and appropriate rotors: low‐speed and ultracentrifuge
  • 1.5‐ml, 15‐ml, or 50‐ml conical polypropylene centrifuge tube
  • Additional reagents and equipment for metabolic radiolabeling of glycoconjugates (unit 12.2), gel‐filtration chromatography (unit 8.3), and one‐dimensional SDS‐PAGE (unit 10.1)

Support Protocol 1: Acetone Precipitation

  • 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 portion 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 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 of early work on 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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