Substrate specificity of the bovine and feline neutral α-mannosidases

Abstract

Neutral α-mannosidases were prepared from bovine and cat liver. The activities were distinguished from lysosomal and Golgi α-mannosidases by their neutral pH optima, relatively low K(m) for their synthetic substrate p-nitrophenyl α-D-mannoside, inhibition by Zn2+ and absence of inhibition by Co2+, EDTA, low concentrations of swainsonine, or deoxymannojirimycin. The cytosolic α-mannosidases were not retained by concanavalin A-Sepharose. They were able to degrade efficiently a variety of oligosaccharides with structures corresponding to certain high-mannose glycans or the oligomannosyl parts of hybrid and complex glycans. However, unlike lysosomal α-mannosidases from the same species these enzymes were not able to degrade Man9GlcNAc2 efficiently, and the bovine neutral α-mannosidase was not able to degrade a hexasaccharide with a structure analogous to Man5GlcNAc2-PP-dolichol. Sharp differences were noted for the bovine and cat enzymes with regard to the specificity of degradation. The bovine neutral α-mannosidase degraded the substrates by defined pathways, but the cat neutral α-mannosidase often produced complex mixtures of products, especially from the larger oligosaccharides. Therefore the bovine enzyme resembled the rat and human cytosolic α-mannosidases, but the cat enzyme did not. The bovine and cat neutral α-mannosidases, unlike the corresponding lysosomal activities, did not show specificity for the hydrolysis of the (1→3)- and (1→6)-linked mannose residues in the N-linked glycan pentasaccharide core.