AUTHOR=Song Jiaru , Zheng Jie , Li Zongmin , Fu Ling , Yang Jing , Li Kun , Yu Xiaoqi , Lv Boyang , Du Junbao , Huang Yaqian , Jin Hongfang 

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

JOURNAL=Frontiers in Immunology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1369326

DOI=10.3389/fimmu.2024.1369326

ISSN=1664-3224

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