Metabolic Radiolabeling of Animal Cell Glycoconjugates

Sandra Diaz1, Ajit Varki1

1 University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.13
DOI:  10.1002/0471142735.im0813s09
Online Posting Date:  May, 2001
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Abstract

This unit describes metabolic labeling techniques that provide specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. In the Basic Protocol, cells in culture are grown through several population doublings in complete medium supplemented with radiolabeled glycoconjugate precursors to reach a steady‐state level of incorporation. In the alternate protocols, cells are cultured for a short period of time in a deficient medium that contains a high concentration of radiolabeled precursor. A pulse or pulse‐chase labeling procedure is provided to analyze precursor‐product relationships. With the sequential pulse‐labeling method described here, it is possible to obtain quantities of labeled glycoconjugates with the use of a minimum amount of labeled precursor by using the same batch of medium to pulse‐label a series of cultures. A support protocol describes the preparation of multiply deficient medium (MDM) for use in making appropriate deficient media.

     
 
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Table of Contents

  • Basic Protocol 1: Steady‐State Labeling with Radioactive Precursors
  • Alternate Protocol 1: Pulse or Pulse‐Chase Labeling with Radioactive Precursors
  • Alternate Protocol 2: Sequential Pulse or Pulse‐Chase Labeling with Reuse of Radioactively Labeled Medium
  • Support Protocol 1: Preparation and Supplementation of Multiply Deficient Medium (MDM)
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Steady‐State Labeling with Radioactive Precursors

  Materials
  • Radioactive precursor: 3H‐ or 14C‐labeled monosaccharide, [35S]sulfate, [3 H]acetate, or [32P]orthophosphate; at highest available specific activity
  • Complete tissue culture medium appropriate for long‐term growth of tissue culture cell line, supplemented as necessary
  • Established tissue culture cell line, either suspension or monolayer
  • Phosphate‐buffered saline (PBS), pH 7.2, ice cold
  • Disposable 50‐ml vacuum‐suction filter device: filter flask fitted with 0.22‐µm filter
  • Disposable 0.22‐µm filter attached to sterile plastic syringe, both with Luer‐Lok fittings
  • Tissue culture plates or flasks
  • Screw‐cap centrifuge tubes
  • Tabletop centrifuge, 4°C
  • Additional reagents and equipment for trypsinization (unit 10.2)

Alternate Protocol 1: Pulse or Pulse‐Chase Labeling with Radioactive Precursors

  Additional Materials
  • protocol 4Multiply deficient medium (MDM; Table 8.13.1 and protocol 4support protocol), supplemented as appropriate
  • Fetal calf serum GIBCO/BRL), dialyzed ( appendix 3A) against sterile 0.15 M NaCl (glucose concentration ∼250 µM)

Alternate Protocol 2: Sequential Pulse or Pulse‐Chase Labeling with Reuse of Radioactively Labeled Medium

  Additional Materials
  • protocol 4Multiply deficient medium (MDM; Table 8.13.1 and protocol 4support protocol), supplemented as appropriate
  • Fetal calf serum GIBCO/BRL), dialyzed ( appendix 3A) against sterile 0.15 M NaCl (glucose concentration ∼250 µM)

Support Protocol 1: Preparation and Supplementation of Multiply Deficient Medium (MDM)

  Additional Materials
  • Stock solutions for multiply deficient medium (MDM; Table 8.13.1)
  • 100× stock solutions for reconstituting MDM (Table 8.13.2)
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Figures

Videos

Literature Cited

Literature Cited
   Cummings, R.D., Merkle, R.K., and Stults, N.L. 1989. Separation and analysis of glycoprotein oligosaccharides. Methods Cell Biol. 32:141‐183.
   Diaz, S. and Varki, A. 1985. Metabolic labeling of sialic acids in tissue culture cell lines: Methods to identify substituted and modified radioactive neuraminic acids. Anal. Biochem. 150:32‐46.
   Esko, J.D., Elgavish, A., Prasthofer, T., Taylor, W.H., and Weinke, J.L. 1986. Sulfate transport‐deficient mutants of Chinese hamster ovary cells. Sulfation of glycosaminoglycans dependent on cysteine. J. Biol. Chem. 261:15725‐15733.
   Goldberg, D. and Kornfeld, S. 1981. The phosphorylation of beta‐glucuronidase oligosaccharides in mouse P388D1 cells. J. Biol. Chem. 256:13060‐13067.
   Gould, G.W. and Bell, G.I. 1990. Facilitative glucose transporters: An expanding family. Trends Biochem. Sci. 15:18‐23.
   Hardy, M.R., Townsend, R.R., and Lee, Y.C. 1988. Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection. Anal. Biochem. 170:54‐62.
   Hart, G.W., Haltiwanger, R.S., Holt, G.D., and Kelly, W.G. 1989. Glycosylation in the nucleus and cytoplasm. Annu. Rev. Biochem. 58:841‐874.
   Hirschberg, C.B. and Snider, M.D. 1987. Topography of glycosylation in the rough endoplasmic reticulum and Golgi apparatus. Annu. Rev. Biochem. 56:63‐87.
   Kim, J.J. and Conrad, E.H. 1976. Kinetics of mucopolysaccharide and glycoprotein biosynthesis by chick embryo chondrocytes. Effect of D‐glucose concentration in the culture medium. J. Biol. Chem. 251:6210‐6217.
   Muchmore, E.A., Milewski, M., Varki, A., and Diaz, S. 1989. Biosynthesis of N‐glycolyl‐neuraminic acid: The primary site of hydroxylation of N‐acetylneuraminic acid is the cytosolic sugar nucleotide pool. J. Biol. Chem. 264:20216‐20223.
   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.
   Roux, L., Holoyda, S., Sunblad, G., Freeze, H.H., and Varki, A. 1988. Sulfated N‐linked oligosaccharides in mammalian cells I: Complex‐type chains with sialic acids and O‐sulfate esters. J. Biol. Chem. 236:8879‐8889.
   Tabas, I. and Kornfeld, S. 1980. Biosynthetic intermediates of beta‐glucuronidase contain high mannose oligosaccharides with blocked phosphate residues. J. Biol. Chem. 255:6633‐6639.
   Varki, A. 1991. Radioactive tracer techniques in the sequencing of glycoprotein oligosaccharides. FASEB J. 5:226‐235.
   Varki, A. and Kornfeld, S. 1982. The spectrum of anionic oligosaccharides released by endo‐β‐N‐acetylglucosaminidase H from glycoproteins. J. Biol. Chem. 258:2808‐2818.
   Yanagishita, M., Salustri, A. and Hascall, V.C. 1989. Specific activity of radiolabeled hexosamines in metabolic labeling experiments. Methods Enzymol. 179:435‐445.
   Yurchenco, P.D., Ceccarini, C., Atkinson, P.H. 1978. Labeling complex carbohydrates of animal cells with monosaccharides. Methods Enzymol. 50:175‐204.
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