Identification and characterization of an intracellular lectin, calnexin, from Aspergillus oryzae using N-glycan-conjugated beads.
Recently, asparagine-linked oligosaccharides (N-glycans) have been found to play a pivotal role in glycoprotein quality control in the endoplasmic reticulum (ER). In order to screen proteins interacting with N-glycans, we developed affinity chromatography by conjugating synthetic N-glycans on sepharose beads. Using the affinity beads with the dodecasaccharide Glc(1)Man(9)GlcNAc(2), one structure of the N-glycans, a 75-kDa protein, was isolated from the membranous fraction including the ER in Aspergillus oryzae. By LC-MS/MS analysis using the A. oryzae genome database, the protein was identified as one (AO090009000313) sharing similarities with calnexin. Further affinity chromatographic experiments suggested that the protein specifically bound to Glc(1)Man(9)GlcNAc(2), similarly to mammalian calnexins. We designated the gene AoclxA and expressed it as a fusion gene with egfp, revealing the ER localization of the AoClxA protein. Our results suggest that our affinity chromatography with synthetic N-glycans might help in biological analysis of glycoprotein quality control in the ER.
Watanabe
Taisuke
T
RIKEN (The Institute of Physical and Chemical Research), Saitama 351-0198, Japan.
Matsuo
Ichiro
I
Maruyama
Jun-Ichi
J
Kitamoto
Katsuhiko
K
Ito
Yukishige
Y
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000235:genetics / Q000378:metabolism*; D037281:Calnexin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D005800:Genes, Fungal; D016681:Genome, Fungal / Q000235:genetics; D037102:Lectins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008863:Microspheres; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D012685:Sepharose / Q000737:chemistry; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D005656:Fungal Proteins; C092383:Glc(1)Man(9)GlcNAc(2) oligosaccharide; D037102:Lectins; D009844:Oligosaccharides; D011134:Polysaccharides; D037281:Calnexin; D012685:Sepharose