Metabolic Labeling of Glycoproteins with Radioactive Sugars

Martin D. Snider1

1 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 7.8
DOI:  10.1002/0471143030.cb0708s13
Online Posting Date:  February, 2002
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Abstract

This unit describes methods for preparation of glycoproteins metabolically labeled with radioactive sugars, sulfate, and phosphate. Methods for liberation of both N‐ and O‐linked glycans are also described. These protocols can be used to generate materials for characterization of glycoprotein glycans from cultured cells.

     
 
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Table of Contents

  • Basic Protocol 1: Pulse‐Chase Labeling with Radioactive Precursors
  • Alternate Protocol 1: Long‐Term Labeling with Radioactive Precursor
  • Support Protocol 1: Enzymatic Release of N‐Linked Glycans from Glycoproteins
  • Support Protocol 2: Release of O‐Linked Glycans from Glycoproteins
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Pulse‐Chase Labeling with Radioactive Precursors

  Materials
  • Growth medium: tissue culture medium supplemented with dialyzed serum (see recipe; use the serum concentration required for growth of the cultured cells being studied)
  • Tissue culture cells
  • recipeLabeling medium (see recipe)
  • Radioactive precursor: [35S]sulfate, [32P]orthophosphate, or sugar labeled with either 3H or 14C
  • Phosphate‐buffered saline (PBS; appendix 2A), ice cold
  • Additional reagents and equipment for experiments optimizing label incorporation (see )

Alternate Protocol 1: Long‐Term Labeling with Radioactive Precursor

  • Disposable plastic syringe and 0.2‐µm syringe filter

Support Protocol 1: Enzymatic Release of N‐Linked Glycans from Glycoproteins

  Materials
  • Sample (e.g., see protocol 1 or protocol 2)
  • 1% (w/v) SDS/0.1 M EDTA/0.5 M 2‐mercaptoethanol
  • 200 mM sodium phosphate, pH 8.6 ( appendix 2A)
  • 10% (w/v) NP‐40
  • Peptide N‐glycosidase F (PNGase F, N‐glycanase) from F. meningosepticumi or recombinant enzyme expressed in E. coli (Glyko or Roche Diagnostics)
  • Additional reagents and equipment for immunoprecipitation (unit 7.2)

Support Protocol 2: Release of O‐Linked Glycans from Glycoproteins

  Materials
  • Sample (e.g., see protocol 1 or protocol 2)
  • 20% (w/v) trichloroacetic acid (i.e., 20 g TCA in 89.4 ml H 2O), ice cold
  • 80% (v/v) aqueous acetone, ice cold
  • 1 M NaBH 4/50 mM NaOH
  • 4 M acetic acid
  • Cation exchange resin: AG 50‐X8 (Bio‐Rad), H+ form
  • Methanol
  • Nitrogen gas
  • Disposable chromatography column (e.g., Bio‐Rad Poly‐Prep columns)
  • Glass test tubes
  • Lyophilizer or SpeedVac evaporator
NOTE: Steps to apply only for whole cells or cell extracts. For immunoprecipitates on a solid support, begin at step .
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Figures

Videos

Literature Cited

Literature Cited
   Carlson, D.M. 1968. Structures and immunochemical properties of oligosaccharides isolated from pig submaxillary mucins. J. Biol. Chem. 243:616‐626.
   Chu, F.K. 1986. Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N‐glycosidase F. J. Biol. Chem. 261:172‐177.
   Diaz, S. and Varki, A. 1995. Metabolic radiolabeling of animal cell glycoconjugates. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, H.L. Ploegh, D.W. Speicher, and P. Wingfield, eds.) pp. 12.2.1‐12.2.15. John Wiley and Sons, New York.
   Elbein, A.D. 1987. Glycosylation inhibitors for N‐linked glycoproteins. Methods Enzymol 138:661‐709.
   Kingsley, D.M., Kozarsky, K.F., Hobbie, L., and Krieger, M. 1986. Reversible defects in O‐linked glycosylation and LDL receptor expression in a UPD‐Gal/UDP‐GalNAc 4‐epimerase deficient mutant. Cell 44:749‐759.
   Kornfeld, R. and Kornfeld, S. 1985. Assembly of asparagine‐linked oligosaccharides. Annu. Rev. Biochem. 54:631‐664.
   Lidholt, K. 1997. Biosynthesis of glycosaminoglycans in mammalian cells and in bacteria. Biochem. Soc. Trans. 25:866‐870.
   Perez‐Vilar, J. and Hill, R.L. 1999. The structure and assembly of secreted mucins. J. Biol. Chem. 274:31751‐31754.
   Rearick, J.I., Chapman, A., and Kornfeld, S. 1981. Glucose starvation alters lipid‐linked oligosaccharide biosynthesis in Chinese hamster ovary cells. J. Biol. Chem. 256:6255‐6261.
   Snider, M. D. and Rogers, O. C. 1986. Membrane traffic in animal cells: Cellular glycoproteins return to the site of Golgi mannosidase I. J. Cell Biol. 103:265‐275.
   Tarentino, A.L., Gomez, C.M., and Plummer, T.H. Jr. 1985. Deglycosylation of asparagine‐linked glycans by peptide:N‐glycosidase F. Biochemistry 24:4665‐4671.
Key References
   Diaz and Varki, 1995. See above.
  Methods for glycoprotein labeling in cultured cells.
   Yurchenco, P.D., Ceccarini, C., and Atkinson, P.H. 1978. Labeling complex carbohydrates of animal cells with monosaccharides. Methods Enzymol. 50:175‐204.
  An excellent discussion of monosaccharide incorporation, with methods for determining the specific activity of incorporated label.
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