A role for N-glycosylation in active adenosine deaminase 2 production
A role for N-glycosylation in active adenosine deaminase 2 production
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N-糖基化在活性腺苷脱氨酶 2 生产中的作用
DOI：
10.1016/j.bbagen.2022.130237
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Sachiko Iwaki-Egawa
中科院分区：
文献类型：
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作者：
Moeko Ito;Yuko Maejima;Kazuki Nishimura;Yusei Nakae;Ayami Ono;Sachiko Iwaki-Egawa
背景腺苷脱氨酶2（Adenosine Deaminase 2，ADA 2）调节细胞外腺苷水平，其最佳表达对调节免疫系统至关重要。然而，调节活性ADA 2酶产生的机制尚未完全了解。本研究旨在探讨N-糖基化在ADA 2功能结构形成和分泌途径中的作用。方法通过定点突变和N-糖基化抑制剂的应用研究了N-糖基化在ADA 2活性、同源二聚体和分泌中的作用。共聚焦荧光显微镜下观察ADA 2沿着内质网（ER）葡萄糖苷酶抑制剂的亚细胞定位。结果通过定点突变或N-糖基化抑制剂处理抑制ER中ADA 2的初始N-糖基化，可降低细胞内ADA 2的活性和分泌。此时，在细胞中观察到ADA 2同源二聚体和ADA 2聚集的减少。用内质网中N-糖链编辑抑制剂栗精胺处理细胞，导致高尔基体定位率降低，并显著抑制ADA 2分泌。结论这些数据表明，内质网中的初始N-糖基化和N-糖链编辑对于活性ADA 2酶的产生和向细胞外空间的适当运输至关重要。ADA 2发挥其功能并分泌到细胞外。
BackgroundAdenosine deaminase 2 (ADA2) regulates extracellular levels of adenosine and the optimal expression of ADA2 is essential for modulating the immune system. However, the mechanisms regulating the production of active ADA2 enzyme are not fully understood. In this study, we examined the role ofN-glycosylation in the formation of functional structures and the secretory pathway of ADA2.MethodsWe investigated the roles ofN-glycosylation in the activity, homodimerization, and secretion of ADA2 via site-directed mutagenesis and the application of N-glycosylation inhibitors. Subcellular localization of ADA2 along with the endoplasmic reticulum (ER) glucosidase inhibitor was observed under confocal fluorescence microscope.ResultsInhibiting the initialN-glycosylation of ADA2 in the ER via site-directed mutagenesis or treatment withN-glycosylation inhibitors reduced the intracellular ADA2 activity and secretion. At this time, decreases in the ADA2 homodimers and ADA2 aggregation were observed in the cells. Treating the cells with castanospermine, an inhibitor ofN-glycan editing in the ER, resulted in a reduction of the localization rate to the Golgi and markedly suppressed the ADA2 secretion.ConclusionsThese data suggest that the initialN-glycosylation andN-glycan editing in the ER are essential for the production of an active ADA2 enzyme and proper trafficking to the extracellular space.General significanceWith sufficientN-glycosylation in the ER, ADA2 exerts its function and is secreted extracellularly.