Identification and initial characterizations of free, glycosylated, and phosphorylated ceramides of Paramecium

Abstract

Paremecium tetraurelia contains high concentrations of six ethanolamine sphingolipids in the cell surface membrane surrounding somatic cilia. Three have phosphoryl groups and the other three have phosphonyl groups, and each contains either dihydrosphingosine, sphingosine, or phytosphingosine. In the present study, free, phosphorylated, and three major glycosylated ceramides were identified in the neutral sphingolipid fraction of this organism when it was grown on a crude medium. Individual lipids were characterized by specific staining on thin-layer chromatographic plates and the fatty acids derived from them were identified by gas-liquid chromatography and mass spectrometry. Unlike the esterified fatty acid composition of the ciliate's phospholipids, which have only small amounts of fatty acids greater than 20 carbons in length, the neutral sphingolipids mainly contain fatty acids greater than 22 carbons in length as well as high concentrations of long chain hydroxy fatty acids. The sugars, glucose, galactose, and fructose, were identified in total neutral sphingolipid fraction. The long chain bases in the neutral sphingolipid fraction were mainly C18 compounds and were identified as C18 dihydrosphingosine, C20 sphingosine, and isomers of C18 sphingosine. Phytosphingosine was not detected in the neutral sphingolipid fraction obtained from whole cells. Because most of the cell's phytosphingosine-containing ethanolamine sphingolipids are in cilia, the inability to detect phytosphingosine as part of putative precursor ceramide compounds suggests that conversions of the long chain base moiety of ethanolamine phospho- and phosphonosphingolipids occur in situ in the ciliary membrane.