Sulfur dioxide inhibits mast cell degranulation by sulphenylation of galectin-9 at cysteine 74

Objectives Mast cell (MC) degranulation is a key process in allergic reactions and inflammatory responses. Aspartate aminotransferase 1 (AAT1)-derived endogenous sulfur dioxide (SO 2 ) 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 SO 2 controlled MC degranulation. Methods HMC-1 and Rat basophilic leukemia cell MC line (RBL-2H3) were used in the cell experiments. SO 2 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 SO 2 . Animal models of passive cutaneous anaphylaxis (PCA) and hypoxia-driven pulmonary vascular remodeling were used to investigate the effect of SO 2 on mast cell activation in vivo . Site-directed mutation of Gal-9 was conducted to confirm the exact site of SO 2 and support the significance of SO 2 /Gal-9 signal axis in the regulation of MC degranulation. Results Degranulation was increased in AAT1-knockdowned MCs, and SO 2 supplementation reversed the increase in MC degranulation. Furthermore, deficiency of endogenous SO 2 contributed to IgE-mediated degranulation in vitro. Besides, SO 2 inhibited IgE-mediated and hypoxia-driven MC degranulation in vivo . Mechanistically, LC-MS/MS analysis and site-directed mutation results showed that SO 2 sulfenylated Gal-9 at cysteine 74. Sulfenylation of the 74 th cysteine of Gal-9 protein was required in the SO 2 -inhibited MC degranulation under both physiological and pathophysiological conditions. Conclusion These findings elucidated that SO 2 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.