Transient expression of type 2 chain in A-active hexaglycosylceramide of rat small intestine at weaning time. Demonstration by affinity chromatography and ceramide glycanase hydrolysis of A-active glycosphingolipids followed by gas chromatography and mass spectrometry of permethylated hexasaccharides

Abstract

The small intestine of 15- to 23-day-old rats was cut into four segments from the duodenum to the ileum. Neutral glycosphingolipids were purified from each segment and submitted to thin-layer chromatography and immunostaining with the A005 monoclonal anti-A antibody. This antibody detected an hexaglycosyleeramide located mainly in the duodenum during the postnatal development. In order to characterize hexaglycosylceramides, blood group A-active glycolipids were purified by affinity chromatography on immobilized Helix pomatia lectin in organic solvent. Hexaglycosylceramides (A-6) were subsequently isolated by preparative thin-layer chromatography and hydrolyzed with ceramide glycanase. The free hexasaccharides were permethylated and analyzed by gas chromatography. Two peaks were detected in varying ratios during development, corresponding to type 1 and type 2 chain A hexasaccharides. Gas chromatography clearly demonstrated that type 2 A-6 occurred in the duodenum of developing rats, and that a shift from type 2 to type 1 A-6 occurred with growing age. The change from type 2 to type 1 chain was also assessed by methylation analysis, and by the variation of the characteristic fragmentations of type 1 and type 2 chain hexasaccharides upon mass spectometry of the permethylated A-6 oligosaccharides from the duodenum of 19-day-old and adult rats.