The N‐glycans of jack bean α‐mannosidase

The acid hydrolase α‐mannosidase, which accumulates in plant vacuoles and probably is involved in the catabolism and turnover of N‐linked glycoproteins, is itself a glycoprotein with at least one high‐mannose‐type and one complex‐type N ‐glycan. The puzzling finding that α‐mannosidase stably carries its own substrate suggests that the N ‐glycans have unique topologies, and important functions in protein folding, oligomerization or enzyme activity. As a first step towards the elucidation of this enigma, we purified the N ‐glycans of jack bean α‐mannosidase and determined their structures by sugar composition analysis, mass spectrometry and 1 H‐NMR. The structures of two N ‐glycans were identified in an approximate ratio of one‐to‐one: a glucose‐containing high‐mannose‐type glycan (Glc1Man9GlcNAc2) and a small xylose‐ and fucose‐containing complex‐type glycan (Xyl1Man1Fuc1GlcNAc2). Isolation and sequencing of glycopeptides strongly suggests that one high‐mannose‐type and one complex‐type glycan are linked to specific glycosylation sites of the large α‐mannosidase subunit. The high‐mannose‐type glycan, which is a good substrate of the endoglycosidase (endo‐H), can only be removed from the enzyme after denaturation and cleavage of disulfide bonds by a reducing agent, suggesting that this glycan is buried within the folded polypeptide and, thus, protected from its hydrolytic activity. Denaturation and reduction of the native enzyme led to a marked decrease in α‐mannosidase activity. However, the activity could largely be recovered by renaturation in an appropriate renaturation buffer. In contrast, recovery of α‐mannosidase activity failed when the high‐mannose‐type glycan was removed by endo‐H prior to renaturation, indicating that this glycan appears to be important for enzyme activity.