title: Formation of lipid-linked oligosaccharides by MOPC 315 plasmacytoma cells. Decreased synthesis by a nonsecretory variant.
abstract: MOPC 315 is a BALB/c plasmacytoma which secretes a trinitrophenol-binding IgA lambda 2 paraprotein. We have investigated the incorporation of [3H]mannose into lipid-linked oligosaccharide precursors in wild-type MOPC 315/J and variant nonsecretory 315/P cells. In pulse labeling experiments, no differences could be detected in the ability of the two cell types to incorporate [3H]mannose into lipid-linked oligosaccharides containing 5 or less mannose residues. In contrast, quantitation of the incorporation of [3H]mannose into larger lipid-linked oligosaccharides and proteins revealed a 49 and 40% decrease, respectively, in the 315/P cells compared to wild-type cells. Further characterization of the lipid-linked structures documented a marked decrease in glucosylated oligosaccharides isolated from 315/P cells. When membranes from the two cell lines were analyzed for their ability to transfer [3H]glucose from UDP-[3H]glucose to [3H]glucosylphosphoryldolichol, an apparent deficiency was noted in the 315/P preparations. However, if assay conditions were adjusted to include AMP in the reaction mixtures, no differences in the in vitro synthesis of [3H]glucosylphosphoryldolichol or [3H]glucose-labeled oligosaccharide-lipid could be detected. In these reactions AMP was found to prevent hydrolysis of UDP-[3H]glucose by inhibiting nucleotide pyrophosphatase (EC 3.6.1.9), the specific activity of which was determined to be more than 100 times greater in variant 315/P compared to wild-type MOPC 315/J cells. This large difference in specific activity was not accompanied by similar differences in the activity of several other enzymes analyzed. A decrease in whole cell UDP-glucose pool size was not detected in 315/P cells. Therefore, if nucleotide pyrophosphatase is important for the control of substrates for glycosylation, it must regulate nucleotide sugar levels at a site other than the cytoplasm of cells, perhaps at the location of synthesis of the larger lipid-linked oligosaccharides.
authors.forename: S
authors.initials: S
authors.lastname: Hickman
authors.forename: Y P
authors.initials: YP
authors.lastname: Wong-Yip
authors.forename: N F
authors.initials: NF
authors.lastname: Rebbe
authors.forename: J M
authors.initials: JM
authors.lastname: Greco
mesh_terms: D000818:Animals; D002460:Cell Line; D005947:Glucose / Q000378:metabolism; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008358:Mannose / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D051379:Mice; D009154:Mutation; D010954:Plasmacytoma / Q000235:genetics / Q000378:metabolism*; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000096:biosynthesis; D011755:Pyrophosphatases / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D011103:Polyisoprenyl Phosphate Monosaccharides; C023023:lipid-linked oligosaccharides; C002968:dolichol-D-glucosylmonophosphate; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase; D005947:Glucose; D008358:Mannose; D014532:Uridine Diphosphate Glucose