International Center for Biotechnology, Osaka University, Suita, Osaka, Japan.
Biochimie. 2011 Apr;93(4):766-71. doi: 10.1016/j.biochi.2011.01.005. Epub 2011 Jan 21.
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.
杰克豆α-甘露糖苷酶（JBM）是一种研究充分的植物液泡α-甘露糖苷酶，被广泛用作糖蛋白糖链酶分析的工具。在这项研究中，使用吡啶氨（PA-）糖链研究了 JBM 对杂合型 N-聚糖的消化图谱。PA 标记的杂合型 N-聚糖 Manα1,6(Manα1,3)Manα1,6(GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA（GNM5-PA）和 Manα1,6(Manα1,3)Manα1,6(Galβ1,4GlcNAcβ1,2Manα1,3)Manβ1,4GlcNAcβ1,4GlcNAc-PA（GalGNM5-PA）的消化效率明显低于寡甘露糖型 N-聚糖 Manα1,6(Manα1,3)Manα1,6Manβ1,4GlcNAcβ1,4GlcNAc-PA（M4-PA），并且 GNM5-PA 和 GalGNM5-PA 的修剪途径与 M4-PA 不同，这表明杂合型 N-聚糖中 GlcNAcβ1-2Man(α)残基对 JBM 活性存在空间位阻。我们还发现，JBM 对杂合型 N-聚糖末端 Manα1-6Man(α)和 Manα1-3Man(α)键的底物偏好性因反应 pH 的变化而改变，这表明酶-底物相互作用存在 pH 依赖性变化。
