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Metabolic Radiolabeling of Animal Cell Glycoconjugates

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Abstract
Table of Contents
Materials
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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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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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Basic Protocol 1: Steady‐State Labeling with Radioactive Precursors Materials

Radioactive precursor: 3 H‐ or 14 C‐labeled monosaccharide, [35 S]sulfate, [3 H]acetate, or [32 P]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 2 PO⋅H 2 O	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)	1×	100×

a Do not use MgSO 4 in place of MgCl 2 . b Not 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 2 SO 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×

c Individual components are added to MDM at full strength, lower concentration, or left out altogether, depending upon the experiment planned. d Use either NaHCO 3 /CO 2 or HEPES⋅HCl (not both); these control the pH of the final medium.

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Figure 12.2.1 Incorporation of radiolabeled sulfates into macromolecules in cells labeled in sulfate‐free medium and the effect of adding increasing amounts of unlabeled sulfate. The arrow indicates the point at which the sulfate is no longer limiting.

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Figure 12.2.2 Scheme for sequential pulse labeling of cells reutilizing radioactive medium.

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Figure 12.2.3 Strategy for planning metabolic labeling of animal cell glycoconjugates.

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Figure 12.2.4 Cytosolic pathways for the interconversion of monosaccharides and their nucleotide sugar forms.

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