The N-glycans of jack bean alpha-mannosidase. Structure, topology and function.
The acid hydrolase alpha-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 alpha-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 alpha-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 alpha-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 alpha-mannosidase activity. However, the activity could largely be recovered by renaturation in an appropriate renaturation buffer. In contrast, recovery of alpha-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.
Kimura
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Y
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Japan.
Hess
D
D
Sturm
A
A
D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D007887:Fabaceae / Q000201:enzymology*; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D009682:Magnetic Resonance Spectroscopy; D008361:Mannosidases / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D043323:alpha-Mannosidase
D006020:Glycopeptides; D011134:Polysaccharides; D011522:Protons; D008361:Mannosidases; D043323:alpha-Mannosidase