Studies on glycosphingolipid biosynthesis by lectin-stimulated human lymphocytes

Abstract

1. Human peripheral lymphocytes, normal and stimulated by phytohemagglutinin or concanavalin A. were investigated with respect to their ability to biosynthesize neutral and acidic glycosphingolipids from D-[U-14C]glucose, D-[U-14C]galactose and D-[U-14C]glucosamine as precursors. 2. Galactose and glucosamie are taken up selectively, in the presence of excessive glucose concentrations. Labeling of total neutral glycosphingolipids from D-[U-14]galactose in normal cells decreases after the first 6 h while in stimulated cells there is a fourfold and a sevenfold increase after 6 and 12 h of incubation respectively. Under similar conditions stimulation with concanavalin A gives a fourfold increase after 12 h of incubation. 3. Analysis of individual glycosphingolipids biosynthesized from [U-14C]galactose shows that lactosylceramide is the major radioactive neutral glycosphingolipid and that stimulation by phytohemagglutinin yields an almost sevenfold and 13-fold increase in the radioactivity incorporated within 6 and 12 h of incubation respectively. Glucosylceramide shows about an eightfold increase, globotetraosylceramide a threefold increase and globotriaosylceramide a fourfold increase. The rate of incorporation into glucosylceramide of stimulated lymphocytes declines after 6 h of incubation, accompanied by a concomitant increase of incorporation into lactosylceramide. 4. At 12 h of incubation the ratios of radioactivities incorporated into neutral glycosphingolipids of phytohemagglutinin-stimulated cells compared to normal cells were 1.0 for D-[U-14C]glucose 7.0 for D-[U-14C]galactose and 2.5 for D-[U-14C]glucosamine. Respective ratios for lactosylceramide are 1.0 for [U-14C]glucose and 13.0 for [U-14C]galactose. These differences did not arise from changes of the uptake of the glycosyl precursor by the cell due to stimulation. 5. Incorporation of D-[U-14C]galactose into II3-N-acetylneuraminosyllactosylceramide by cells stimulated by phytohemagglutinin and concanavalin A is enhanced 14-times and 15-times respectively. With [U-14C]glucosamine as precursor, this increase in the labeling is much more impressive, 80-fold after 12 h of incubation by the phytohemagglutinin-stimulated lymphocytes. Neuraminidase treatment and gas radiochromatographic analysis of the labeled compound derived from [U-14C]-galactose as precursor indicate that 89% of the radioactivity was incorporated into the glucosyl and galactosyl moieties, in a ratio 1:1. With [U-14C]glucosamine as precursor, a selective labeling of the sialyl moiety of the II3-N-acetylneuraminosyllactosylceramide was indicated. 6. The pattern of complex gangliosides (more complex than II3-N-acetylneuraminosyllactosylceramide) which are biosynthesized after phytohemagglutinin stimulation of the cells, show no significant differences when compared to the patterns obtained from normal cells.