The spectrum of incomplete N-linked oligosaccharides synthesized by endothelial cells in the presence of brefeldin A.
Previous studies in many cell lines have shown that Brefeldin A (BFA) inhibits the forward movement of newly synthesized glycoconjugates by fusing the cis-, medial-, and trans-Golgi compartments with the rough endoplasmic reticulum. Studies on the oligosaccharide processing of individual glycoproteins have yielded confusing and incomplete results regarding the location of the block. Assuming that all glycoproteins with N-linked oligosaccharides follow the same endoplasmic reticulum to the Golgi pathway, a more complete picture on the location and nature of the block can be determined by analyzing N-linked oligosaccharides synthesized in the presence of BFA. In bovine pulmonary artery endothelial cells, BFA (0.1 microgram/ml) reversibly inhibits the secretion of greater than 95% of Tran35S and [3H]Man-labeled glycoproteins without affecting protein synthesis or N-linked glycosylation. In addition, BFA inhibits the synthesis and secretion of 35SO4-labeled oligosaccharides. Initial oligosaccharide trimming is uninhibited, but further processing is affected since the majority (65%) of the chains terminate only in beta-GlcNAc residues. Concomitantly, the proportion of [3H]Man-labeled N-linked anionic oligosaccharides is reduced from 60 to 20%, and the great majority of the charge is due to one sialic acid. The rate-limiting step for sialylation appears to be the branch selective addition of beta-Gal residues. The remaining charge is due to sulfate esters (0.6%) which normally account for greater than 10% of the anionic substituents. BFA also reduces the amount of phosphorylated chains by 80% and greatly diminishes further phosphodiester processing since the majority of these oligosaccharides (60%) contain a Man-6-PO4 residue in an acid-sensitive diester linkage. The addition of all polylactosamine chains, outer-branch fucose and terminal alpha-Gal residues are completely inhibited by BFA. Secretion, fucosylation, and sialylation are completely restored when BFA is removed, but the other modification steps are only partially restored. Our results indicate that addition of sulfate esters, terminal alpha-Gal residues, polylactosamine chains, outer-branch fucose residues, some initial phosphorylation, and most phosphodiester processing may occur beyond a compartment where some beta-Gal and sialic acid residues can be added. Essentially, all of the effects on oligosaccharide processing are partially or completely reversible.
Sampath
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Glycobiology/Chemistry Program, La Jolla Cancer Research Foundation, California 92037.
Varki
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A
Freeze
H H
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D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D020126:Brefeldin A; D002240:Carbohydrate Sequence; D002417:Cattle; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003517:Cyclopentanes / Q000494:pharmacology*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
D003517:Cyclopentanes; D009844:Oligosaccharides; D020126:Brefeldin A; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase