Substrate specificity of rat liver cytosolic α‐d‐mannosidase Novel degradative pathway for oligomannosidic type glycans

Abstract

The substrate specificity of rat liver cytosolic neutral α‐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(α‐2)Man(α‐3)[Man(α1–2)Man(α1–6)]Man(α1–2)Manα1–2)Man(α1–3)]Man(β‐4)GlcNAc; Man7GlcNac, Man(α1–3)[Man(α1–2)Man(α1–6)]Man(α1–6)[Man(α1–2)Man(α1–3]Man(β1–4)GlcNAc; Man5GlcNAc I, Man(α1–3)[Man(α1–6)]Man(α1–6)Man(α1–3)]Man(β1–4)GlcNAc; Man5GlcNAc II, Man(α1–2)Man(α1–2)Man(α1–3)[Man(α1–6)]Man(β1–4)GlcNAc; Man4GlcNAc, Man(α1–2)Man(α1–2)Man(α1–3)Man(β1–4)GlcNAc. The different oligosaccharide isomers resulting from α‐d‐mannosidase hydrolysis were analyzed by 1H‐NMR spectroscopy after HPLC separation. The cytosolic α‐d‐mannosidase activity is able to hydrolyse all types of α‐mannosidic linkages found in the glycans of the oligomannosidic type, i.e. α‐1,2, α‐1,3 and α‐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 α‐d‐mannosidase acting on Man5GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(α1–6)[Man(α1–3)]Man(β1–4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(α1–2)Man(α1–2)Man(α1–3)[Man(α1–6)]Man(β1–4)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 α‐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 α‐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 cytoslic and lysosomal α‐d‐mannosidase activities in the catabolism of N‐glycosylprotein is proposed. Copyright © 1991, Wiley Blackwell. All rights reserved