Structural identification of two ten-sugar branched chain glycosphingolipids of blood group H type present in epithelial cells of rat small intestine.

Abstract

Two novel blood group H-type decaglycosylceramides with a branched core saccharide have been identified in mixture in a fraction isolated from rat small intestine. They were present exclusively in the epithelial cells. The number and sequence of sugars were established by direct inlet mass spectrometry of the permethylated and LiAlH4-reduced permethylated derivatives. Gas-liquid chromatography of the products after degradation of the native and permethylated glycolipids gave the type of sugars and the binding positions. A di- and a trisaccharide were identified by mass spectrometry after degradation of the permethylated-reduced derivative. One trisaccharide had the structure (formula see text) and was therefore additional evidence for a branched structure. Treatment of the decaglycosylceramide fraction with alpha-L-fucosidase gave free fucose and an octaglycosylceramide identified by mass spectrometry. Proton NMR spectra of the permethylated and permethylated-reduced octa- and decaglycosylceramides provided evidence for the binding configurations and the localization of type 1 and type 2 sequences in the two branches. The 3-linked branch was homogeneous with a type 1 saccharide (Gal beta 1 leads to 3GlcNAc) but the 6-linked branch had both type 1 and type 2 (Gal beta 1 leads to 4GlcNAc) sequences. Two glycolipids with the following probable structures were therefore present, making up 60 and 40% of the mixture, respectively: (formula see text) The lipophilic part contained mainly trihydroxy 18:0 long chain base (phytosphingosine) and 16:0 to 24:0 nonhydroxy fatty acids.