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‐[U14C]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‐14C]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 indicated 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.