Cellular effects of deoxynojirimycin analogues: inhibition of N-linked oligosaccharide processing and generation of free glucosylated oligosaccharides.
In the accompanying paper [Mellor, Neville, Harvey, Platt, Dwek and Butters (2004) Biochem. J. 381, 861-866] we treated HL60 cells with N-alk(en)yl-deoxynojirimycin (DNJ) compounds to inhibit glucosphingolipid (GSL) biosynthesis and identified a number of non-GSL-derived, small, free oligosaccharides (FOS) most likely produced due to inhibition of the oligosaccharide-processing enzymes a-glucosidases I and II. When HL60 cells were treated with concentrations of N-alk(en)ylated DNJ analogues that inhibited GSL biosynthesis completely, N-butyl- and N-nonyl-DNJ inhibited endoplasmic reticulum (ER) glucosidases I and II, but octadecyl-DNJ did not, probably due to the lack of ER lumen access for this novel, long-chain derivative. Glucosidase inhibition resulted in the appearance of free Glc1-3Man structures, which is evidence of Golgi glycoprotein endomannosidase processing of oligosaccharides with retained glucose residues. Additional large FOS was also detected in cells following a 16 h treatment with N-butyl- and N-nonyl-DNJ. When these FOS structures (>30, including >20 species not present in control cells) were characterized by enzyme digests and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, all were found to be polymannose-type oligosaccharides, of which the majority were glucosylated and had only one reducing terminal GlcNAc (N-acetylglucosamine) residue (FOS-GlcNAc1), demonstrating a cytosolic location. These results support the proposal that the increase in glucosylated FOS results from enzyme-mediated cytosolic cleavage of oligosaccharides from glycoproteins exported from the ER because of misfolding or excessive retention. Importantly, the present study characterizes the cellular properties of DNJs further and demonstrates that side-chain modifications allow selective inhibition of protein and lipid glycosylation pathways. This represents the most detailed characterization of the FOS structures arising from ER a-glucosidase inhibition to date.
Mellor
Howard R
HR
Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Neville
David C A
DC
Harvey
David J
DJ
Platt
Frances M
FM
Dwek
Raymond A
RA
Butters
Terry D
TD
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000117:Acetylglucosamine / Q000737:chemistry; D000606:Amino Sugars / Q000494:pharmacology; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005947:Glucose / Q000737:chemistry; D006031:Glycosylation / Q000187:drug effects; D018922:HL-60 Cells / Q000737:chemistry / Q000187:drug effects*; D006801:Humans; D008358:Mannose / Q000737:chemistry; D009844:Oligosaccharides / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D000520:alpha-Glucosidases / Q000378:metabolism; D043323:alpha-Mannosidase / Q000378:metabolism
D000606:Amino Sugars; D009844:Oligosaccharides; D017485:1-Deoxynojirimycin; D000520:alpha-Glucosidases; D043323:alpha-Mannosidase; D005947:Glucose; D008358:Mannose; D000117:Acetylglucosamine