Brefeldin A induced inhibition of de novo globo- and neolacto-series glycolipid core chain biosynthesis in human cells. Evidence for an effect on beta 1-->4galactosyltransferase activity.
De novo synthesis of neolacto-series glycolipids has been studied in human cell lines via metabolic labeling of ceramide with [3H]serine. Intense labeling of ceramide mono- and dihexoside glycolipids occurred with labeling of progressively longer chain derivatives with increasing time. Most of the label was recovered in neutral glycolipids with about 5% of the total labeling in the ganglioside fraction. Experiments done using cell treatment with 2.5 micrograms/ml brefeldin A resulted in a stimulation in the total amount of labeling, accumulation of a neutral glycolipid identified as Lc3 due to inhibited transfer of the neolacto-series core chain terminal beta-Gal residue, and a corresponding inhibition of labeling of longer chain neutral glycolipids in all cell lines. Brefeldin A also blocked synthesis of the globo-series precursor, Gb3, longer chain sialylated structures such as IV3NeuAcnLc4, but not de novo GM3 synthesis. Brefeldin A treatment had no effect on cellular beta 1-->3N-acetylglucosaminyl-, beta 1-->4galactosyl-, or alpha 1-->3fucosyltransferase specific activities, nor was it inhibitory in beta 1-->4galactosyltransferase assays in vitro. The results describe brefeldin A-induced blocks in globo- and neolacto-series glycolipid biosynthesis, consistent with differential localization of enzymes in intracellular membranes. In particular, the results suggest that the beta 1-->4galactosyltransferase in these cells is either not redistributed by brefeldin A or is otherwise rendered nonfunctional.
Sherwood
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Pacific Northwest Research Foundation, Seattle, Washington 98122.
Holmes
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EH
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D020126:Brefeldin A; D002240:Carbohydrate Sequence; D002518:Ceramides / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003517:Cyclopentanes / Q000494:pharmacology*; D005915:Globosides / Q000096:biosynthesis*; D006017:Glycolipids / Q000096:biosynthesis* / Q000302:isolation & purification; D006056:Golgi Apparatus / Q000378:metabolism; D006602:Hexosyltransferases / Q000037:antagonists & inhibitors*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009237:N-Acetyllactosamine Synthase / Q000037:antagonists & inhibitors*; D011863:Radioimmunoassay; D012694:Serine / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000900:Anti-Bacterial Agents; D002518:Ceramides; D003517:Cyclopentanes; D005915:Globosides; D006017:Glycolipids; D020126:Brefeldin A; D012694:Serine; D006602:Hexosyltransferases; D009237:N-Acetyllactosamine Synthase