title: Substrate specificity of rat liver cytosolic alpha-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans.
abstract: The substrate specificity of rat liver cytosolic neutral alpha-D-mannosidase was investigated by in vitro incubation with a crude cytosolic fraction of oligomannosyl oligosaccharides Man9GlcNAc, Man7GlcNAc, Man5GlcNAc I and II isomers and Man4GlcNAc having the following structures: Man9GlcNAc, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-2)Man(alpha 1-6)]Man(alpha 1-6) [Man(alpha 1-2)Man(alpha 1-3)]Man(beta 1-4)GlcNAc; Man5GlcNAc I, Man(alpha 1-3)[Man(alpha 1-6)]-Man(alpha 1-6)Man(alpha 1-3)] Man(beta 1-4)GlcNAc; Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3) [Man(alpha 1-6)]Man(beta 1-4)GlcNAc; Man4GlcNAc, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc. The different oligosaccharide isomers resulting from alpha-D-mannosidase hydrolysis were analyzed by 1H-NMR spectroscopy after HPLC separation. The cytosolic alpha-D-mannosidase activity is able to hydrolyse all types of alpha-mannosidic linkages found in the glycans of the oligomannosidic type, i.e. alpha-1,2, alpha-1,3 and alpha-1,6. Nevertheless the enzyme is highly active on branched Man9GlcNAc or Man5GlcNAc I oligosaccharides and rather inactive towards the linear Man4GlcNAc oligosaccharide. Structural analysis of the reaction products of the soluble alpha-D-mannosidase acting on Man5-GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, represents the 'construction' Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The cytosolic alpha-D-mannosidase is activated by Co2+, insensitive to 1-deoxymannojirimycin but strongly inhibited by swainsonine in the presence of Co2+ ions. The enzyme shows a highly specific action different from that previously described for the lysosomal alpha-D-mannosidases [Michalski, J.C., Haeuw, J.F., Wieruszeski, J.M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369-379]. A possible complementarity between cytosolic and lysosomal alpha-D-mannosidase activities in the catabolism of N-glycosylprotein is proposed.
authors.affiliation: Laboratoire de Chimie Biologique, UMR du CNRSn. 111, Université des Sciences et Techniques de Lille Flandres-Artois, Villeneuve d'Ascq, France.
authors.forename: J F
authors.initials: JF
authors.lastname: Haeuw
authors.forename: G
authors.initials: G
authors.lastname: Strecker
authors.forename: J M
authors.initials: JM
authors.lastname: Wieruszeski
authors.forename: J
authors.initials: J
authors.lastname: Montreuil
authors.forename: J C
authors.initials: JC
authors.lastname: Michalski
mesh_terms: D017485:1-Deoxynojirimycin; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003600:Cytosol / Q000201:enzymology; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D007536:Isomerism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008361:Mannosidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides; D051381:Rats; D013379:Substrate Specificity; D017026:Swainsonine / Q000494:pharmacology; D043323:alpha-Mannosidase
chemical_list: D009844:Oligosaccharides; C028569:oligomannoside; D017485:1-Deoxynojirimycin; D008361:Mannosidases; D043323:alpha-Mannosidase; D005944:Glucosamine; D017026:Swainsonine