Department Graduate School of Science and Technology, Gunma University, 1-5-1 Tenjin-cho, Kiryu, Gunma, 376-8515, Japan.
Glycometabolome Team, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center for Systems Chemical Biology, RIKEN Global Research Cluster, Wako, Saitama, 351-0198, Japan.
Chembiochem. 2018 Apr 4;19(7):660-663. doi: 10.1002/cbic.201700662. Epub 2018 Feb 16.
We developed a fluorescence-quenching-based assay system to determine the hydrolysis activity of endo-β-N-acetylglucosaminidases (ENGases). The pentasaccharide derivative 1 was labeled with an N-methylanthraniloyl group as a reporter dye at the non-reducing end and with a 2,4-dinitrophenyl group as a quencher molecule at the reducing end. This derivative is hydrolyzed by ENGase, resulting in an increase in fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of the tetrasaccharide derivative, hence allowing ENGase activity to be evaluated easily and quantitatively. Using this system, we succeeded in measuring the hydrolysis activities of ENGases and thus the inhibitory activities of known inhibitors. We confirmed that this assay system is suitable for high-throughput screening for potential inhibitors of human ENGase that might serve as therapeutic agents for the treatment of N-glycanase 1 (NGLY1) deficiency.
我们开发了一种基于荧光猝灭的测定体系，用于测定内-β-N-乙酰氨基葡萄糖苷酶（ENGases）的水解活性。五糖衍生物 1 在非还原端标记有 N-甲基邻氨基苯甲酸基团作为报告染料，在还原端标记有 2,4-二硝基苯基团作为猝灭分子。该衍生物可被 ENGase 水解，导致荧光强度增加。因此，荧光信号与四糖衍生物的量成正比，从而可以轻松且定量地评估 ENGase 活性。使用该系统，我们成功地测量了 ENGases 的水解活性，从而评估了已知抑制剂的抑制活性。我们证实，该测定系统适用于筛选人 ENGase 的潜在抑制剂，这些抑制剂可能成为治疗 N-糖基化酶 1（NGLY1）缺乏症的治疗剂。
