SUBSTRATE-SPECIFICITY OF RAT-LIVER CYTOSOLIC ALPHA-D-MANNOSIDASE - NOVEL DEGRADATIVE PATHWAY FOR OLIGOMANNOSIDIC TYPE GLYCANS

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-1-2)Man(alpha-1-3)]Man(beta-1-4)GlcNAc; Man7GlcNAc, 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 H-1-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 Man5GlcNAc 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-1-2)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.