Metabolic Radiolabeling of Animal Cell Glycoconjugates

Sandra Diaz1, Ajit Varki1

1 University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 12.2
DOI:  10.1002/0471140864.ps1202s57
Online Posting Date:  August, 2009
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Abstract

Useful information about glycoconjugates can be obtained by labeling their aglycone (noncarbohydrate) portions—e.g., labeling proteins with radioactive amino acids—and then using techniques described elsewhere in this chapter to infer the presence, type, and nature of glycan chains. This unit describes metabolic labeling techniques that provide more specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. Following metabolic labeling, the radioactive glycoconjugate of interest is isolated, individual glycosylation sites are identified and separated if necessary, and the labeled glycans are subjected to structural analysis. Curr. Protoc. Protein Sci. 57:12.2.1‐12.2.15. © 2009 by John Wiley & Sons, Inc.

Keywords: glycoconjugates; glycan; carbohydrate; metabolic labeling

     
 
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Table of Contents

  • Introduction
  • 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, [3H]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; appendix 2E), pH 7.2, ice cold
  • Disposable sterile 50‐ml vacuum‐suction filter device: filter flask fitted with 0.22‐µm filter
  • Disposable 0.22‐µm sterile filter attached to sterile plastic syringe, both with Luer‐Lok fittings
  • Sterile pipet tips
  • Tissue culture plates or flasks
  • Screw‐cap centrifuge tubes
  • Tabletop centrifuges, at room temperature and 4°C
  • Rubberman policeman or disposable cell scraper
  • Scintillation counter
  • Additional reagents and equipment for splitting monolayer cells using trypsinization ( appendix 3C)

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

  • Multiply deficient medium (MDM; see protocol 4 and Table 12.2.1) supplemented as appropriate
  • Fetal bovine serum (FBS; Invitrogen), dialyzed ( appendix 3B) against sterile 0.15 M NaCl (glucose concentration ∼250 µM)
    Table 2.2.1   Additional Materials (also see protocol 1)   Additional MaterialsComposition of Multiply Deficient Medium (MDM)

    Component Final concentration in complete medium Stock
    CaCl 2 200 mg/liter Salt, reagent grade
    KCl 400 mg/liter Salt, reagent grade
    MgCl 2 a 75 mg/liter Salt, reagent grade
    NaCl 6800 mg/liter Salt, reagent grade
    NaH 2PO⋅H 2O 140 mg/liter Salt, reagent grade
    Phenol red 10 mg/liter 10×
    Sodium pyruvate 110 mg/liter 100×
    L‐Alanine 25 mg/liter 100×
    L‐Arginine 126 mg/liter 100×
    L‐Asparagine 50 mg/liter 100×
    L‐Aspartic acid 30 mg/liter 100×
    L‐Cysteine NONE b
    L‐Glutamic acid 75 mg/liter 100×
    L‐Glutamine NONE b
    L‐Glycine 50 mg/liter 100×
    L‐Histidine 42 mg/liter 100×
    L‐Isoleucine 52 mg/liter 100×
    L‐Leucine 52 mg/liter 100×
    L‐Lysine 72 mg/liter 100×
    L‐Methionine NONE b
    L‐Phenylalanine 32 mg/liter 100×
    L‐Proline 40 mg/liter 100×
    L‐Serine NONEb
    L‐Threonine 48 mg/liter 100×
    L‐Tryptophan 10 mg/liter 100×
    L‐Tyrosine 36 mg/liter 100×
    L‐Valine 46 mg/liter 100×
    MEM vitamins (mixture) 100×

     aDo not use MgSO 4 in place of MgCl 2.
     bNot present in MDM; add as needed for specific experiments.

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

  • Multiply deficient medium (MDM; see protocol 4 and Table 12.2.1), supplemented as appropriate
  • Fetal bovine serum (FBS; Invitrogen), dialyzed ( appendix 3B) against sterile 0.15 M NaCl (glucose concentration ∼250 µM)

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

  • Stock solutions for multiply deficient medium (MDM; Table 12.2.1)
  • 100× stock solutions for reconstituting MDM (Table 12.2.2)
  • 50‐ml tubes
    Table 2.2.2   Additional Materials (also see protocol 1)   Additional Materials   Stock Solutions for Reconstitution of MDM c   Stock Solutions for Reconstitution of MDM

    Component Final concentration in complete medium Stock
    NaHCO 3 d 1× (2200 mg/liter) 100×
    HEPES⋅HCl d 20 mM (4.76 g/liter) 2 M, pH 7.3
    Na 2SO 4 0.81 mM (115 mg/liter) 100 mM, sterile
    D‐Glucose 1× (1000 mg/ml) 100×
    L‐Cysteine 1× (100 mg/liter) 100×
    L‐Glutamine 1× (292 mg/liter) 100×
    L‐Methionine 1× (15 mg/liter) 100×
    L‐Serine 1× (25 mg/liter) 100×

     cIndividual components are added to MDM at full strength, lower concentration, or left out altogether, depending upon the experiment planned.
     dUse either NaHCO 3/CO 2or HEPES⋅HCl (not both); these control the pH of the final medium.
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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.
   Fitch, F.W., Gajewski, T.F., and Yokoyama, W.M. 1997. Diagnosis and treatment of mycoplasma‐contaminated cell cultures. Curr. Protoc. Immunol. A.3E.1‐A.3E.4.
   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‐glycolylneuraminic 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., and Atkinson, P.H. 1978. Labeling complex carbohydrates of animal cells with monosaccharides. Methods Enzymol. 50:175‐204.
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