Effect of substrate structure on the activity of Man9-mannosidase from pig liver involved in N-linked oligosaccharide processing.

Man9-mannosidase, an alpha 1,2-specific enzyme located in the endoplasmic reticulum and involved in N-linked-oligosaccharide processing, has been isolated from crude pig-liver microsomes and its substrate specificity studied using a variety of free and peptide-bound high-mannose oligosaccharide derivatives. The purified enzyme displays no activity towards synthetic alpha-mannosides, but removes three alpha 1,2-mannose residues from the natural Man9-(GlcNAc)2 substrate (M9). The alpha 1,2-mannosidic linkage remaining in the M6 intermediate is cleaved about 40-fold more slowly. Similar kinetics of hydrolysis were determined with Man9-(GlcNAc)2 N-glycosidically attached to the hexapeptide Tyr-Asn-Lys-Thr-Ser-Val (GP-M9), indicating that the specificity of the enzyme is not influenced by the peptide moiety of the substrate. The alpha 1,2-mannose residue which is largely resistant to hydrolysis, was found to be attached in both the M6 and GP-M6 intermediate to the alpha 1,3-mannose of the peripheral alpha 1,3/alpha 1,6-branch of the glycan chain. Studies with glycopeptides varying in the size and branching pattern of the sugar chains, revealed that the relative rates at which the various alpha 1,2-mannosidic linkages were cleaved, differed depending on their structural complexity. This suggests that distinct sugar residues in the aglycon moiety may be functional in substrate recognition and binding. Reduction or removal of the terminal GlcNAc residue of the chitobiose unit in M9 increased the hydrolytic susceptibility of the fourth (previously resistant) alpha 1,2-mannosidic linkage significantly. We conclude from this observation that, in addition to peripheral mannose residues, the intact chitobiose core represents a structural element affecting Man9-mannosidase specificity. A possible biological role of the enzyme during N-linked-oligosaccharide processing is discussed.