title: Lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells.
abstract: Sandhoff and Tay-Sachs disease are autosomal recessive GM2 gangliosidoses where a deficiency of lysosomal beta-hexosaminidase results in storage of glycoconjugates. Imino sugar (2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol) inhibition of beta-hexosaminidase in murine RAW264.7 macrophage-like cells led to lysosomal storage of glycoconjugates that were characterised structurally using fluorescence labelling of the free or glycolipid-derived oligosaccharides followed by HPLC and mass spectrometry. Stored glycoconjugates were confirmed as containing non-reducing GlcNAc or GalNAc residues resulting from the incomplete degradation of N-linked glycoprotein oligosaccharide and glycolipids, respectively. When substrate reduction therapeutics N-butyl-deoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ) were applied to the storage phenotype cells, an increase in glucosylated and galactosylated oligosaccharide species was observed due to endoplasmic reticulum alpha-glucosidases and lysosomal beta-galactosidase inhibition, respectively. Hexosaminidase inhibition triggered a tightly regulated cytokine-mediated inflammatory response that was normalised using imino sugars NB-DNJ and NB-DGJ, which restored the GM2 ganglioside storage burden but failed to reduce the levels of GA2 glycolipid or glycoprotein-derived N-linked oligosaccharides. Using a chemically induced gangliosidosis phenotype that can be modulated with substrate lowering drugs, the critical role of GM2 ganglioside in the progression of inflammatory disease is also demonstrated.
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Stephanie D
authors.initials: SD
authors.lastname: Boomkamp
authors.forename: J S Shane
authors.initials: JS
authors.lastname: Rountree
authors.forename: David C A
authors.initials: DC
authors.lastname: Neville
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Dwek
authors.forename: George W J
authors.initials: GW
authors.lastname: Fleet
authors.forename: Terry D
authors.initials: TD
authors.lastname: Butters
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects; D005733:Gangliosidoses / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism*; D050111:Imino Sugars / Q000494:pharmacology*; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D013058:Mass Spectrometry; D051379:Mice; D009844:Oligosaccharides / Q000378:metabolism*; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
chemical_list: D006028:Glycosphingolipids; D050111:Imino Sugars; D009844:Oligosaccharides; D017485:1-Deoxynojirimycin; C090092:migalastat; D001619:beta-N-Acetylhexosaminidases