Effects of brefeldin A on oligosaccharide processing. Evidence for decreased branching of complex-type glycans and increased formation of hybrid-type glycans.
Brefeldin A (BFA), a drug that induces redistribution of Golgi-apparatus proteins into the endoplasmic reticulum, was used to determine the role of subcellular compartmentalization in the processing of asparagine-linked oligosaccharides. Baby-hamster kidney cells were pulse-labelled with [3H]mannose for 30-60 min and chased for up to several hours in the presence or in the absence of BFA or labelled continuously for several hours with and without the drug. Cellular glycoproteins were digested to glycopeptides with Pronase and either fractionated into glycan classes by lectin affinity chromatography or digested further by endoglycosidase H and endoglycosidase D. Released oligosaccharides obtained in the latter procedure were then separated from each other and from endoglycosidase-resistant glycopeptides by paper chromatography. The results show that BFA induces a very fast processing of protein-linked Glc3Man9GlcNAc2 oligosaccharide down to man5GlcNAc2 and conversion into complex-type and hybrid-type glycans. The major difference between untreated and BFA-treated cells is a large increase in bi-antennary and hybrid-type glycans in the latter cells. These results indicate that galactosylation of a mono-antennary GlcNAcMan5GlcNAc2 hybrid blocks subsequent action by mannosidase II and N-acetylglucosaminyl transferase II, producing galactosylated hybrid-type glycans. Similarly, galactosylation of the product of N-acetylglucosaminyltransferases I and II, i.e. a Man3GlcNAc2 core substituted with GlcNAc beta 1----2 on both alpha 1----3- and alpha 1----6-linked mannose residues, blocks branching N-acetylglucosaminyltransferases IV and V, thereby causing an increase in bi-antennary glycans and a decrease in tri- and tetra-antennary glycans.
National Institute for Medical Research, The Ridgeway, Mill Hill, London, U.K.
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D000818:Animals; D020126:Brefeldin A; D002460:Cell Line; D002853:Chromatography, Liquid; D002854:Chromatography, Paper; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology*; D006020:Glycopeptides / Q000378:metabolism; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009844:Oligosaccharides / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*
D003517:Cyclopentanes; D006020:Glycopeptides; D009844:Oligosaccharides; D011134:Polysaccharides; D020126:Brefeldin A; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase