The N-glycans of jack bean α-mannosidase: Structure, topology and function

Yoshinobu Kimura, Daniel Hess, Arnd Sturm

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60 Citations (Scopus)


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 1H-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.

Original languageEnglish
Pages (from-to)168-175
Number of pages8
JournalEuropean Journal of Biochemistry
Issue number1
Publication statusPublished - Aug 15 1999
Externally publishedYes


  • Jack bean α-mannosidase
  • N-glycan function
  • N-glycan structure
  • N-glycan topology
  • Plant glycoprotein

ASJC Scopus subject areas

  • Biochemistry


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