Synthetic trisaccharides reveal discrimination of endo-glycosidic linkages by exo-acting α-1,2-mannosidases in the endoplasmic reticulum

A tri-antennary Man(9)GlcNAc(2) glycan on the surface of endoplasmic reticulum (ER) glycoproteins functions as a glycoprotein secretion or degradation signal after regioselective cleavage of the terminal alpha-1,2-mannose residue of each branch. Four alpha-1,2-mannosidases-ER mannosidase I, ER degradation-enhancing alpha-mannosidase-like protein 1 (EDEM1), EDEM2, and EDEM3-are involved in the production of these signal glycans. Although selective production of signal glycans is important in determining the fate of glycoproteins, the branch-discrimination abilities of the alpha-1,2-mannosidases are not well understood. A structural feature of the Man(9)GlcNAc(2) glycan is that all terminal glycosidic linkages of the three branches are of the alpha-1,2 type, while the adjacent inner glycosidic linkages are different. In this study, we examined whether the alpha-1,2-mannosidases showed branch specificity by discriminating between different inner glycosides. Four trisaccharides with different glycosidic linkages [Man alpha 1-2Man alpha 1-2Man (natural A-branch), Man alpha 1-2Man alpha 1-3Man (natural B-branch), Man alpha 1-2Man alpha 1-6Man (natural C-branch), and Man alpha 1-2Man alpha 1-4Man (unnatural D-branch)] were synthesized and used to evaluate the hypothesis. When synthesizing these oligosaccharides, highly stereoselective glycosylation was achieved with a high yield in each case by adding a weak base or tuning the polarity of the mixed solvent. Enzymatic hydrolysis of the synthetic trisaccharides by a mouse liver ER fraction containing the target enzymes showed that the ER alpha-1,2-mannosidases had clear specificity for the trisaccharides in the order of A-branch > B-branch > C-branch approximate to D-branch. Various competitive experiments have revealed for the first time that alpha-1,2-mannosidase with inner glycoside specificity is present in the ER. Our findings suggest that exo-acting ER alpha-1,2-mannosidases can discriminate between endo-glycosidic linkages.

Automated summary

This study revealed the branch specificity of ER alpha-1,2-mannosidases by discriminating different inner glycosides, which can differentiate between the glycoproteins' branches.