title: Novel mannosidase inhibitors probe glycoprotein degradation pathways in cells.
abstract: Multiple isoforms of mammalian alpha-mannosidases are active in the pathways of N-linked glycoprotein synthesis and catabolism. They differ in specificity, function and location within the cell and can be selectively inhibited by imino sugar monosaccharide mimics. Previously, a series of structurally related novel 7-membered iminocyclitols were synthesised and found to be inhibitors of alpha-mannosidase using in vitro assays. The present study aimed to delineate alpha-mannosidases hydrolytic pathways in azepane inhibitor treated cells by the analysis of free oligosaccharides (FOS) as markers of endoplasmic reticulum (ER), Golgi, lysosomal and cytosolic alpha-mannosidase activities. Two compounds were identified as potent and selective cytosolic alpha-mannosidase inhibitors. Two related compounds were shown to be potent inhibitors of lysosomal alpha-mannosidase with different potencies towards alpha1,6 mannosidase. The specificities of these novel 7-membered imino sugars are related to differences in their structure and D: -mannose-like stereochemistry. Specific ER-mannosidase inhibition by kifunensine also reveals significant non-proteasomal degradation following FOS analysis and appears to be cell line dependent. The availability of more selective inhibitors allows the pathways of N-linked oligosaccharide metabolism to be dissected.
authors.affiliation: Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK. terry.butters@bioch.ox.ac.uk
authors.forename: Terry D
authors.initials: TD
authors.lastname: Butters
authors.forename: Dominic S
authors.initials: DS
authors.lastname: Alonzi
authors.forename: Nikolay V
authors.initials: NV
authors.lastname: Kukushkin
authors.forename: Yuan
authors.initials: Y
authors.lastname: Ren
authors.forename: Yves
authors.initials: Y
authors.lastname: Blériot
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002240:Carbohydrate Sequence; D002477:Cells / Q000187:drug effects* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006023:Glycoproteins / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D008361:Mannosidases / Q000037:antagonists & inhibitors*; D053858:Metabolic Networks and Pathways / Q000187:drug effects*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013329:Structure-Activity Relationship
chemical_list: D000470:Alkaloids; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D009844:Oligosaccharides; C065629:kifunensine; D008361:Mannosidases