Expression of dolichol-linked saccharide intermediate synthesis during the development of B lymphocytes

There are large developmental increases in the rates of dolichol-linked oligosaccharide synthesis and protein N-glycosylation when resting murine splenic B lymphocytes are activated by bacterial lipopolysaccharide (LPS). These in vivo and in vitro studies were carried out to investigate the underlying biochemical mechanisms involved in the dramatic increase in the rate of oligosaccharide-lipid biosynthesis in LPS-stimulated B cells. Metabolic labelling experiments showed that the rate of synthesis of N-acetyl-glucosaminylpyrophosphoryldolichol (GlcNAc-P-P-Dol), mannosylphosphoryldolichol (Man-P-Dol) and glucosylphos-phoryldolichol (Glc-P-Dol) increased 4- to 15-fold between 20 and 40 h after exposure to LPS. When the glyco-syltransferase activities catalysing the formation of the three dolichol-bound monosaccharides were assayed in vitro with endoplasmic reticulum (ER)-enriched fractions, the initial rates were found to be elevated 4-fold prior to the major increases in oligosaccharide-lipid intermediate biosynthesis observed in vivo. Based on kinetic analyses, the higher enzyme activities were due to an increase in the amount of the three glycosyltransferases in activated cells. The time courses for elevated cellular content and rate of synthesis of guanosine-diphosphomannose (GDP)-Man corresponded to the developmental increase in oligosaccharide-lipid synthesis. The kinetics and magnitude of the induction of oligosaccharide-lipid synthesis were similar whether the initial rates were calculated on the basis of [2-³H]mannose-labelling or the specific activity of the GDP-[2-³H]mannose pool. This study shows that the induction of the glycosyltransferases forming GlcNAc-P-P-Dol, Man-P-Dol and Glc-P-Dol slightly precedes the synthesis of the dolichol-bound monosaccharides in vivo and the full expression of the lipid intermediate pathway. The results also indicate that elevated levels of the enzymes synthesizing the three monosaccharide-lipid intermediates and an expansion of the GDP-Man poo contribute to, but do not fully account for, the substantial increase in the rate of synthesis of dolichol-linked oligosaccharides in LPS-stimulated B cells.