Enzymatic Release of Oligosaccharides from Glycolipids

Adriana E. Manzi1

1 University of California San Diego, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.17A
DOI:  10.1002/0471142727.mb1717as32
Online Posting Date:  May, 2001
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Selective cleavage of the oligosaccharide moiety of glycolipids for further structural analysis can be achieved by means of endoglycoceramidase (EGCase), an enzyme specific for the linkages between oligosaccharide and ceramide residues in glycolipids. The method described here can be used for analysis of glycolipids present in macroscopic amounts or for glycolipids that have been radiolabeled in their carbohydrate moiety.

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Table of Contents

  • Basic Protocol 1: Use of Endoglycoceramidase for Selective Cleavage of Glycolipids
  • Reagents and Solutions
  • Commentary
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Basic Protocol 1: Use of Endoglycoceramidase for Selective Cleavage of Glycolipids

  • Sample to be analyzed—neutral glycolipids (unit 17.2), gangliosides (unit 17.17), or desialylated gangliosides (see unit 17.12)—in organic solvent
  • 1 mg/ml disialoganglioside‐G D1a (Sigma) in 2:1 (v/v) chloroform/methanol
  • 0.5% (w/v) sodium taurocholate in 0.05 M sodium acetate buffer, pH 6.0
  • Endoglycoceramidase (EGCase; from Rhodococcus sp.; for purification see Ito and Yamagata, )
  • Toluene
  • Methanol
  • 60:40:9 (v/v/v) chloroform/methanol/0.2% (w/v; aqueous) calcium chloride
  • recipeOrcinol/sulfuric acid reagent (see recipe)
  • 2:1 (v/v) chloroform/methanol
  • Bath sonicator
  • Precoated high‐performance thin‐layer (HPTLC) silica gel 60 plates (10 × 10–cm; Merck)
  • Glass chromatography tank
  • 140°C oven
  • Tabletop centrifuge
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Literature Cited

Literature Cited
   Ito, M. and Yamagata, T. 1986. A novel glycosphingolipid‐degrading enzyme cleaves the linkage between the oligosaccharide and ceramide of neutral and acidic glycosphingolipids. J. Biol. Chem. 261:14278‐14282.
   Ito, M. and Yamagata, T. 1989a. Endoglycoceramidase from Rhodococcus species G‐74‐2. Methods Enzymol 179:488‐495.
   Ito, M. and Yamagata, T. 1989b. Purification and characterization of glycosphingolipid‐specific endoglycosidases (endoglycoceramidases) from a mutant strain of Rhodococcus sp. Evidence for three species of endoglycoceramidase with different specificities. J. Biol. Chem. 264:9510‐9519.
   Ito, M., Ikegami, Y., and Yamagata, T. 1991. Activator proteins for glycosphingolipid hydrolysis by endoglycoceramidases. Elucidation of biological functions of cell‐surface glycosphingolipids in situ by endoglycoceramidases made possible using these activator proteins. J. Biol. Chem. 266:7919‐7926.
   Li, S.‐C., Degasperi, R., Muldrey, J.E., and Li, Y.‐T. 1986. A unique glycosphingolipid‐splitting enzyme (ceramide‐glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide. Biochem. Biophys. Res. Commun. 141:346‐352.
   Li, Y.‐T., Ishikawa, Y., and Li, S.‐C. 1987. Occurrence of ceramide‐glycanase in the earthworm, Lumbricus terrestris. Biochem. Biophys. Res. Commun. 149:167‐172.
   Manzi, A.E., Sjoberg, E.R., Diaz, S., and Varki, A. 1990. Biosynthesis and turnover of O‐acetyl and N‐acetyl groups in the gangliosides of human melanoma cells. J. Biol. Chem. 265:13091‐13103.
   Rasilo, M.‐L., Ito, M., and Yamagata, T. 1989. Liberation of oligosaccharides from glycosphingolipids on PC12 cell surface with endoglycoceramidase. Biochem. Biophys. Res. Commun. 162:1093‐1099.
   Shimamura, M., Hayase, T., Ito, M., Rasilo, M.‐L., and Yamagata, T. 1988. Characterization of a major neutral glycolipid in PC12 cells as III3Galα‐globotriaosylceramide by the method for determining glycosphingolipid saccharide sequence with endoglycoceramidase. J. Biol. Chem. 263:12124‐12128.
Key References
   Ito and Yamagata, 1986. See above.
  Describes the purification of the enzyme, its specificity, and examples of its use.
   Ito and Yamagata, 1989a. See above.
  Describes the purification and properties of the enzyme and contains many references regarding its application.
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