Characterization of a major neutral glycolipid in PC12 cells as III3Gal alpha-globotriaosylceramide by the method for determining glycosphingolipid saccharide sequence with endoglycoceramidase.

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

doi:10.1016/s0021-9258(18)37901-8

M Shimamura, T Hayase + Show 3 more

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https://doi.org/10.1016/s0021-9258(18)37901-8

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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 alpha-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 Gal alpha Gal alpha Gal beta GlcCer. 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 Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer, III3-Gal alpha-globotriaosylceramide. This is the first report indicating the presence of this glycosphingolipid in PC12 cells.

Highlights

Thisreportpresents the structure of a major glycolipid,l whose presence in PC12 cells has so far not been reported in previous papers
Be GalaGalaGalbGlcCerC. eramide 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 Galal-3Galal-4Gal~1-4Glc/31-1Ce1r1,1’

Summary

Present address

Dept. of General Chemistry, Faculty of Agricul- amide; HPTLC, high performance thin layer chromatography; HPLC, tureand Forestry, University of Helsinki, Viikki 00710 Helsinki, high performance liquid chromatography; CTH, ceramide trihexoside; Finland. Determination of the Saccharide Sequence of Gal-Gbs-Gal-Gb3 (50 nmol) was digested with endoglycoceramidase (total, 12 milliunits) prepared as described below in 0.1 M acetate buffer (pH 6.0, containing 1 mg/ml sodium taurodeoxycholate, 50 pl) for 3 days at 37 "C. The 2-aminopyridyl oligosaccharide was dissolved in 10 pl of 0.05 M acetate buffer (pH 5.5) and digested with a-galactosidase (3.3 milliunits, from green coffee, Sigma) for 1 day a t 37 "C. It was further digested with pgalactosidase (0.5 unit, from Escherichia coli, Sigma) for 1 day at 37 "C.

A Major Neutral Glycolipid in PC12Cells l l

RESULTS

DISCUSSION

A Major Neutral Glycolipid in PC12 Cells