Glycolipids in Mouse Thymocytes Stimulated by Concanavalin A. Characterisation of Glycolipids and Rate of Lipid Biosynthesis during Stimulation

Abstract

Mouse thymocytes were cultivated in the presence of concanavalin A and the incorporation of label derived from d-[1-14C]galactose, d-[1-14C]glucosamine and l-[3-14C]serine into various (glyco)-lipid fractions was studied. About 80% of galactose-derived label was present in glycolipids. Labelled glycolipids were separated by down-scaled thin-layer chromatography. The major neutral glycolipid was characterised as glucosylceramide. A variety of Vibrio cholerae neuraminidase-labile gangliosides were found to be derived from two different asialotetraosylceramides. The migration behaviour of one of them is similar to that of gangliotetraosylceramide, the other one may contain a novel type of oligosaccharide side chain. The time course of the rates of labelling of phospholipids and glycolipids were compared in cultures of concanavalin-A-stimulated mouse thymocytes using [32P]phosphate as phospholipid-label and [14C]galactose as glycolipid label in identical cultures. The data obtained from these experiments indicate a different time dependency of biosynthesis and/or turnover of both lipid classes. The rate of incorporation into phospholipids has peak activity in the beginning of the cultivation, and only a small maximum after 30 h whereas incorporation into glycolipids has a pronounced maximum 30 h after concanavalin A stimulation. Following the rates of incorporation into individual glycolipid fractions i.e. gangliosides and neutral glycolipids, no differences in kinetics were observed, indicating no preferential synthesis of certain glycolipids. Since the rates of incorporation of galac-tose-label into the carbohydrate moiety and of serine-label into the ceramide moiety were similarly increased in stimulated cultures at 30 h, it is concluded that mainly biosynthesis de novo of glycolipids occurs at this time.