Metabolic Labeling of Glycoproteins with Radioactive Sugars

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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: [³⁵ S]sulfate, [³² P]orthophosphate, or sugar labeled with either ³ H or ¹⁴ C
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 H₂ O), ice cold
80% (v/v) aqueous acetone, ice cold
1 M NaBH₄ /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

Figure 7.8.1 Pathways of sugar metabolism in mammalian cells. Compounds in boxes are precursors that can be taken up and incorporated into glycoconjugates. Pathways for conversion into the nucleotide sugar donors for biosynthesis are shown. Sugars in open boxes compete with Glc for uptake and incorporation. These are incorporated efficiently and can be used in the labeling procedures (see and ). Sugars in shaded boxes do not compete with Glc. They are incorporated less efficiently and should be used only in long‐term labeling experiments (see ). Abbreviations used: Fru, fructose; Fuc, fucose; Gal, galactose; GalNAc, N‐acetylgalactosamine; Glc, glucose; GlcA, glucuronic acid; GlcNAc, N‐acetylglucosamine; GlcNH₂ , glucosamine; Man, mannose; ManNAc, N‐acetylmannosamine; NeuAc, N‐acetylneuraminic acid (a sialic acid); P, phosphate; UDP, uracil diphosphate; Xyl, xylose.

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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.