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 glucosylphosphoryldolichol (Glc-P-Dol) increased 4- to 15-fold between 20 and 40 h after exposure to LPS. When the glycosyltransferase 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Rush
J S
JS
Department of Biochemistry, A.B. Chandler Medical Center, University of Kentucky College of Medicine, Lexington 40536.
Waechter
C J
CJ
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism*; D005260:Female; D005690:Galactose / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D016695:Glycosyltransferases / Q000378:metabolism*; D006155:Guanosine Diphosphate Mannose / Q000032:analysis / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D051379:Mice; D008811:Mice, Inbred DBA; D008861:Microsomes / Q000378:metabolism; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism*; D013154:Spleen / Q000378:metabolism; D014316:Tritium
D006023:Glycoproteins; D011103:Polyisoprenyl Phosphate Monosaccharides; D014316:Tritium; D006155:Guanosine Diphosphate Mannose; D004287:Dolichol Monophosphate Mannose; C002968:dolichol-D-glucosylmonophosphate; C018439:N-acetylglucosaminylpyrophosphoryldolichol; D016695:Glycosyltransferases; D005944:Glucosamine; D008358:Mannose; D005690:Galactose