Fluorescence quenching-based assay for measuring Golgi endo-α-mannosidase
Fluorescence quenching-based assay for measuring Golgi endo-α-mannosidase
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基于荧光猝灭的测定高尔基体内切 α-甘露糖苷酶的方法
DOI：
10.1002/asia.201900240
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发表时间：
2019
影响因子：
4.1
通讯作者：
Ichiro Matsuo
中科院分区：
文献类型：
--
作者：
Kanae Sano;Taiki Kuribara;Nozomi Ishii;Ayumi Kurokawa;Toshitada Yoshihara;Seiji Tobita;Kiichiro Totani;Ichiro Matsuo
高尔金多-α-甘露糖苷酶 (G-EM) 催化 N-聚糖的替代脱糖基过程，并在内质网后 (ER) 质量控制途径中发挥重要作用。为了了解 ER 后质量控制机制，我们合成了一种四糖探针，用于基于荧光猝灭测定检测 G-EM 的水解活性。该探针在非还原端用 N-甲基邻氨基苯甲酰基作为报告染料，在还原端用 2,4-二硝基苯基作为猝灭剂标记。该探针被 G-EM 水解成二糖衍生物，导致荧光强度增加。因此，荧光信号与存在的二糖衍生物的量成正比，从而可以轻松定量地评估 G-EM 活性。
Golgiendo‐α‐mannosidase (G‐EM) catalyzes an alternative deglucosylation process for N‐glycans and plays important roles in the post‐endoplasmic reticulum (ER) quality control pathway. To understand the post‐ER quality control mechanism, we synthesized a tetrasaccharide probe for the detection of the hydrolytic activity of G‐EM based on a fluorescence quenching assay. The probe was labeled with anN‐methylanthraniloyl group as a reporter dye at the non‐reducing end and a 2,4‐dinitrophenyl group as a quencher at the reducing end. This probe is hydrolyzed to disaccharide derivatives by G‐EM, resulting in increased fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of disaccharide derivative present, allowing the G‐EM activity to be evaluated easily and quantitatively.