■肥大细胞(MC)脱颗粒是过敏反应和炎症反应中的关键过程。天冬氨酸氨基转移酶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尚不清楚.本研究旨在探讨内源性SO2控制MC脱粒的机理。
■HMC-1和大鼠嗜碱性白血病细胞MC系(RBL-2H3)用于细胞实验。用原位荧光探针检测SO2含量。使用比色法测定由MCβ-己糖胺酶的释放速率表示的MC脱粒。使用生物素开关测定法检测MC和纯化蛋白中半乳糖凝集素-9(Gal-9)的磺酰化。使用液相色谱-串联质谱(LC-MS/MS)确定SO2对Gal-9的确切磺酰化位点。采用被动皮肤过敏反应(PCA)和低氧驱动的肺血管重塑动物模型,研究SO2对体内肥大细胞活化的影响。进行Gal-9的定点突变以确认SO2的确切位点并支持SO2/Gal-9信号轴在MC脱粒调节中的重要性。
■在AAT1敲除的MC中脱粒增加,和SO2的补充逆转了MC脱粒的增加。此外,内源性SO2的缺乏导致IgE介导的体外脱颗粒。此外,SO2在体内抑制IgE介导和缺氧驱动的MC脱颗粒。机械上,LC-MS/MS分析和定点突变结果显示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抑制的MC脱粒都需要Gal-9蛋白的第74个半胱氨酸的磺酰化。
■这些发现阐明了在生理和病理生理条件下,SO2通过磺酰化Gal-9抑制MC脱粒,这可能为MC激活相关疾病提供新的治疗方法。
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.