β‐Elimination for Release of O‐Linked Glycosaminoglycans from Proteoglycans

H. Edward Conrad1

1 University of Illinois, Urbana, Illinois
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.15A
DOI:  10.1002/0471142727.mb1715as31
Online Posting Date:  May, 2001
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Abstract

O‐linked glycosaminoglycan (GAG) chains in proteoglycans are readily released from their core proteins by treatment with alkali at room temperature. This β‐elimination is the same type of reaction as that for releasing O‐linked oligosaccharides from their core proteins. Under the reaction conditions described here, N‐linked oligosaccharides remain attached to the core protein, but any O‐linked oligosaccharides will be released along with the GAG chains. The procedure can be used to isolate the free GAG chains, the free O‐linked oligosaccharides, and the core protein (which will still have any N‐linked oligosaccharides that were originally present).

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1:

  Materials
  • Proteoglycan solution to be analyzed
  • recipeAlkaline borohydride reagent (see recipe)
  • 2 M HCl
  • 1 M NaOH
  • Additional reagents and equipment for dialysis ( appendix 3C)
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Figures

Videos

Literature Cited

Literature Cited
   Bienkowski, M.J. and Conrad, H.E. 1984. Kinetics of proteoheparan sulfate synthesis, secretion, endocytosis, and catabolism by a hepatocyte cell line. J. Biol. Chem. 259:12989‐12996.
   Conrad, H.E., James, M.E., and Varboncoeur, E. 1973. Qualitative and quantitative analysis of reducing carbohydrates by radiochromatography on ion exchange papers. Anal. Biochem. 51:486‐500.
   Fedarko, N.F. and Conrad, H.E. 1986. A unique heparan sulfate in the nuclei of hepatocytes. Structural changes with the growth state of the cells. J. Cell Biol. 102:587‐599.
   Mayo, J.W. and Carlson, D.M. 1970. Effect of alkali and sodium borohydride at alkaline pH on N‐acetylchondrosine: Reduction versus cleavage. Carbohydr. Res. 15:300‐303.
   Ogata, S.‐I. and Lloyd, K.O. 1982. Mild alkaline borohydride treatment of glycoproteins—A method for liberating both N‐ and O‐linked carbohydrate chains. Anal. Biochem. 119:351‐359.
   Seno, N. and Sekizuka, E. 1978. Quantitative β‐elimination of O‐glycosyl linkages in chondroitin sulfates. Carbohydr. Res. 62:271‐299.
Key Reference
   Seno and Sekizuka, 1978. See above.
  Describes the basic procedure used here and includes the use of PdCl2 to reduce the dehydroalanine residues to alanine residues.
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