Characterization of a major neutral glycolipid in PC12 cells as III3Galα-globotriaosylceramide by the method for determining glycosphingolipid saccharide sequence with endoglycoceramidase

M. Shimamura, T. Hayase, Makoto Ito, M. L. Rasilo, T. Yamagata

Research output: Contribution to journalArticle

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Abstract

Neutral glycolipids in PC12 cells were examined. A major neutral glycosphingolipid, isolated from a chloroform/methanol extract of the cells, was found to contain only galactose and glucose at a ratio of 3:1 and identified as ceramide tetrahexoside by fast atom bombardment (FAB) mass spectrometry. Its saccharide sequence was determined by a new method developed here using endoglycoceramidase (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). The glycosphingolipid was digested with endoglycoceramidase to produce oligosaccharide which was subsequently pyridylaminated. The fluorescence-labeled oligosaccharide was digested with a series of specific exoglycosidases and fractionated by high performance liquid chromatography. The 2-aminopyridyl oligosaccharide was hydrolyzed by α-galactosidase to give a 2-aminopyridyl oligosaccharide which was identified as 2-aminopyridyl lactose by high performance liquid chromatography, indicating the glycolipid structure to be GalαGalαGalβGlcCer. Ceramide trihexoside obtained by limited digestion of the intact glycolipid was clearly identical with ceramide trihexoside obtained from human erythrocytes, according to NMR spectroscopy and methylation analysis. From these and other data on the intact glycolipid, obtained by methylation analysis and NMR spectroscopy, its structure was confirmed as Galα1-3Galα1-4Galβ1-4Glcβ1-1Cer, III3-Galα-globotriaosylceramide. This is the first report indicating the presence of this glycosphingolipid in PC12 cells.

Original languageEnglish
Pages (from-to)12124-12128
Number of pages5
JournalJournal of Biological Chemistry
Volume263
Issue number24
Publication statusPublished - Jan 1 1988
Externally publishedYes

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endoglycoceramidase
Glycosphingolipids
PC12 Cells
Glycolipids
Oligosaccharides
Methylation
High performance liquid chromatography
Nuclear magnetic resonance spectroscopy
Magnetic Resonance Spectroscopy
Neutral Glycosphingolipids
High Pressure Liquid Chromatography
Galactosidases
Fast Atom Bombardment Mass Spectrometry
Ceramides
Glycoside Hydrolases
Lactose
Chloroform
Cell Extracts
Galactose
Mass spectrometry

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Characterization of a major neutral glycolipid in PC12 cells as III3Galα-globotriaosylceramide by the method for determining glycosphingolipid saccharide sequence with endoglycoceramidase. / Shimamura, M.; Hayase, T.; Ito, Makoto; Rasilo, M. L.; Yamagata, T.

In: Journal of Biological Chemistry, Vol. 263, No. 24, 01.01.1988, p. 12124-12128.

Research output: Contribution to journalArticle

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abstract = "Neutral glycolipids in PC12 cells were examined. A major neutral glycosphingolipid, isolated from a chloroform/methanol extract of the cells, was found to contain only galactose and glucose at a ratio of 3:1 and identified as ceramide tetrahexoside by fast atom bombardment (FAB) mass spectrometry. Its saccharide sequence was determined by a new method developed here using endoglycoceramidase (Ito, M., and Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282). The glycosphingolipid was digested with endoglycoceramidase to produce oligosaccharide which was subsequently pyridylaminated. The fluorescence-labeled oligosaccharide was digested with a series of specific exoglycosidases and fractionated by high performance liquid chromatography. The 2-aminopyridyl oligosaccharide was hydrolyzed by α-galactosidase to give a 2-aminopyridyl oligosaccharide which was identified as 2-aminopyridyl lactose by high performance liquid chromatography, indicating the glycolipid structure to be GalαGalαGalβGlcCer. Ceramide trihexoside obtained by limited digestion of the intact glycolipid was clearly identical with ceramide trihexoside obtained from human erythrocytes, according to NMR spectroscopy and methylation analysis. From these and other data on the intact glycolipid, obtained by methylation analysis and NMR spectroscopy, its structure was confirmed as Galα1-3Galα1-4Galβ1-4Glcβ1-1Cer, III3-Galα-globotriaosylceramide. This is the first report indicating the presence of this glycosphingolipid in PC12 cells.",
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