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Effect of substrate structure on the activity of Man9-mannosidase from pig liver involved in N-linked oligosaccharide
E Bause1, W Breuer, J Schweden
1Institut für Physiologische Chemie, Bonn, Federal Republic of Germany.
Abstract:
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.
Insights
Man9-mannosidase, an enzyme in N-linked-oligosaccharide processing, specifically removes mannose residues from substrates. Its specificity is influenced by the chitobiose core, not the peptide moiety.
Area of Science:
- Biochemistry
- Glycobiology
- Enzymology
Background:
- N-linked-oligosaccharide processing is crucial for protein function.
- Man9-mannosidase is an alpha 1,2-specific enzyme in the endoplasmic reticulum.
- Understanding enzyme specificity is key to elucidating biological pathways.
Purpose of the Study:
- To isolate and characterize pig-liver Man9-mannosidase.
- To determine the substrate specificity of Man9-mannosidase.
- To investigate factors influencing Man9-mannosidase activity and its biological role.
Main Methods:
- Isolation of Man9-mannosidase from pig-liver microsomes.
- Substrate specificity studies using free and peptide-bound high-mannose oligosaccharides.
- Kinetic analysis of enzyme activity on various glycopeptide substrates.
Main Results:
- Purified Man9-mannosidase removed three alpha 1,2-mannose residues from Man9-(GlcNAc)2 (M9).
- Enzyme specificity was not affected by the peptide moiety of glycopeptide substrates.
- The chitobiose core, along with peripheral mannose residues, influences Man9-mannosidase specificity.
Conclusions:
- Man9-mannosidase exhibits specific cleavage patterns based on oligosaccharide structure.
- The enzyme's activity is modulated by both peripheral mannose and the chitobiose core.
- Further studies are needed to fully understand the biological role of Man9-mannosidase in N-linked-oligosaccharide processing.