Jack bean α-mannosidase digestion profile of hybrid-type N-glycans: effect of reaction pH on substrate preference.
Jack bean α-mannosidase (JBM) is a well-studied plant vacuolar α-mannosidase, and is widely used as a tool for the enzymatic analysis of sugar chains of glycoproteins. In this study, the JBM digestion profile of hybrid-type N-glycans was examined using pyridylamino (PA-) sugar chains. The digestion efficiencies of the PA-labeled hybrid-type N-glycans Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GNM5-PA) and Manα1,6(Manα1,3)Manα1,6(Galβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA (GalGNM5-PA) were significantly lower than that of the oligomannose-type N-glycan Manα1,6(Manα1,3)Manα1,6Manβ1,4GlcNAcβ1,4GlcNAc-PA (M4-PA), and the trimming pathways of GNM5-PA and GalGNM5-PA were different from that of M4-PA, suggesting a steric hindrance to the JBM activity caused by GlcNAcβ1-2Man(α) residues of the hybrid-type N-glycans. We also found that the substrate preference of JBM for the terminal Manα1-6Man(α) and Manα1-3Man(α) linkages in the hybrid-type N-glycans was altered by the change in reaction pH, suggesting a pH-dependent change in the enzyme-substrate interaction.
International Center for Biotechnology, Osaka University, Suita, Osaka, Japan.
Koji
K
Dohi
Junko
J
Isoyama-Tanaka
Ryo
R
Misaki
Kazuhito
K
Fujiyama
D007887:Fabaceae / Q000201:enzymology*; D006023:Glycoproteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D043323:alpha-Mannosidase / Q000737:chemistry* / Q000378:metabolism
D006023:Glycoproteins; D011134:Polysaccharides; D043323:alpha-Mannosidase