Graduate School of Science and Technology, Gunma University, 1-5-1, Tenjin-cho, Kiryu, Gunma, 376-8515, Japan.
Graduate School of Science and Technology, Gunma University, 1-5-1, Tenjin-cho, Kiryu, Gunma, 376-8515, Japan; Biologics Technology Research Laboratories, Daiichi Sankyo Co., Ltd., 3-5-1, Nihonbashi-honcho, Tokyo, 103-8426, Japan.
Carbohydr Res. 2023 Jan;523:108724. doi: 10.1016/j.carres.2022.108724. Epub 2022 Nov 17.
A fluorescence-quenching-based assay system to determine the hydrolytic activity of endo-β-N-acetylglucosaminidases (ENGases), which act on the innermost N-acetylglucosamine (GlcNAc) residue of the chitobiose segment of core-fucosylated N-glycans, was constructed using a dual-labeled fluorescent probe with a hexasaccharide structure. The fluorogenic probe was evaluated using a variety of ENGases, including Endo-M W251N mutant, Endo-F3, and Endo-S, which recognize core fucosylated N-glycans. The occurrence of a hydrolysis reaction was detected by observing an increased fluorescence intensity, ultimately allowing the ENGase activities to be easily and quantitatively evaluated, with the exception of Endo-S. The obtained results clearly indicated the substrate specificities of the examined ENGases.
构建了一种基于荧光猝灭的分析系统，用于测定内切-β-N-乙酰氨基葡萄糖苷酶（ENGases）的水解活性，这些酶作用于核心岩藻糖基化 N-聚糖中壳二糖片段的最内部 N-乙酰氨基葡萄糖（GlcNAc）残基。该分析系统使用带有六糖结构的双标记荧光探针。使用多种 ENGases（包括识别核心岩藻糖基化 N-聚糖的 Endo-M W251N 突变体、Endo-F3 和 Endo-S）对荧光发生探针进行了评估。通过观察荧光强度的增加来检测水解反应的发生，最终可以轻松且定量地评估 ENGase 活性，但 Endo-S 除外。获得的结果清楚地表明了所研究的 ENGases 的底物特异性。
