肥大细胞（MC）脱颗粒是过敏反应和炎症反应的关键过程。天冬氨酸转氨酶 1 (AAT1) 衍生的内源性二氧化硫 (SO2) 是 MC 功能的重要调节剂。然而，其在 MC 脱颗粒中的作用机制仍不清楚。本研究旨在探讨内源性SO2控制MC脱粒的机制。细胞实验采用HMC-1和大鼠嗜碱性白血病细胞MC系(RBL-2H3)。采用原位荧光探针检测SO2含量。使用比色测定法测定以 MC β-己糖胺酶的释放速率表示的 MC 脱颗粒。使用生物素开关测定检测 MC 和纯化蛋白中半乳糖凝集素 9 (Gal-9) 的磺酰化。采用液相色谱-串联质谱(LC-MS/MS) 确定了Gal-9 被SO2 准确磺化的位点。使用被动皮肤过敏反应（PCA）和缺氧驱动的肺血管重塑的动物模型来研究SO2对体内肥大细胞活化的影响。对 Gal-9 进行定点突变，以确认 SO2 的确切位点，并支持 SO2/Gal-9 信号轴在 MC 脱颗粒调节中的重要性。AAT1 敲低的 MC 中脱颗粒增加，补充 SO2 逆转了MC 脱粒增加。此外，内源性 SO2 的缺乏导致 IgE 介导的体外脱颗粒。此外，SO2 还能抑制体内 IgE 介导和缺氧驱动的 MC 脱颗粒。从机制上来说，LC-MS/MS分析和定点突变结果表明，SO2在半胱氨酸74处磺酰化Gal-9。在生理和病理生理条件下，SO2抑制MC脱颗粒需要Gal-9蛋白的第74个半胱氨酸磺酰化。 .这些研究结果阐明SO2在生理和病理生理条件下通过磺化Gal-9抑制MC脱颗粒，这可能为MC激活相关疾病提供一种新的治疗方法。版权所有©2024宋，郑，李，付，杨，李，于、吕、杜、黄、金。

Mast cell (MC) degranulation is a key process in allergic reactions and inflammatory responses. Aspartate aminotransferase 1 (AAT1)-derived endogenous sulfur dioxide (SO2) is an important regulator of MC function. However, the mechanism underlying its role in MC degranulation remains unclear. This study aimed to investigate the mechanism by which endogenous SO2 controlled MC degranulation.HMC-1 and Rat basophilic leukemia cell MC line (RBL-2H3) were used in the cell experiments. SO2 content was detected by in situ fluorescent probe. MC degranulation represented by the release rate of MC β-hexosaminidase was determined using a colorimetric assay. Sulfenylation of galectin-9 (Gal-9) in MCs and purified protein was detected using a biotin switch assay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the exact sulfenylation sites of Gal-9 by SO2. Animal models of passive cutaneous anaphylaxis (PCA) and hypoxia-driven pulmonary vascular remodeling were used to investigate the effect of SO2 on mast cell activation in vivo. Site-directed mutation of Gal-9 was conducted to confirm the exact site of SO2 and support the significance of SO2/Gal-9 signal axis in the regulation of MC degranulation.Degranulation was increased in AAT1-knockdowned MCs, and SO2 supplementation reversed the increase in MC degranulation. Furthermore, deficiency of endogenous SO2 contributed to IgE-mediated degranulation in vitro. Besides, SO2 inhibited IgE-mediated and hypoxia-driven MC degranulation in vivo. Mechanistically, LC-MS/MS analysis and site-directed mutation results showed that SO2 sulfenylated Gal-9 at cysteine 74. Sulfenylation of the 74th cysteine of Gal-9 protein was required in the SO2-inhibited MC degranulation under both physiological and pathophysiological conditions.These findings elucidated that SO2 inhibited MC degranulation via sulfenylating Gal-9 under both physiological and pathophysiological conditions, which might provide a novel treatment approach for MC activation-related diseases.Copyright © 2024 Song, Zheng, Li, Fu, Yang, Li, Yu, Lv, Du, Huang and Jin.