O-GlcNAc modification of GSDMD attenuates LPS-induced endothelial cells pyroptosis

Abstract Objective Increased O -linked β- N -acetylglucosamine ( O -GlcNAc) stimulation has been reported to protect against sepsis associated mortality and cardiovascular derangement. Previous studies, including our own research, have indicated that gasdermin-D(GSDMD)-mediated endothelial cells pyroptosis contributes to sepsis-associated endothelial injury. This study explored the functions and mechanisms of O -GlcNAc modification on lipopolysaccharide (LPS)-induced pyroptosis and its effects on the function of GSDMD. Methods A LPS-induced septic mouse model administrated with O -GlcNAcase (OGA) inhibitor thiamet-G (TMG) was used to assess the effects of O -GlcNAcylation on sepsis-associated vascular dysfunction and pyroptosis. We conducted experiments on human umbilical vein endothelial cells (HUVECs) by challenging them with LPS and TMG to investigate the impact of O -GlcNAcylation on endothelial cell pyroptosis and implications of GSDMD. Additionally, we identified potential O -GlcNAcylation sites in GSDMD by utilizing four public O -GlcNAcylation site prediction database, and these sites were ultimately established through gene mutation. Results Septic mice with increased O -GlcNAc stimulation exhibited reduced endothelial injury, GSDMD cleavage (a marker of pyroptosis). O -GlcNAc modification of GSDMD mitigates LPS-induced pyroptosis in endothelial cells by preventing its interaction with caspase-11 (a human homologous of caspases-4/5). We also identified GSDMD Serine 338 (S338) as a novel site of O -GlcNAc modification, leading to decreased association with caspases-4 in HEK293T cells. Conclusions Our findings identified a novel post-translational modification of GSDMD and elucidated the O -GlcNAcylation of GSDMD inhibits LPS-induced endothelial injury, suggesting that O -GlcNAc modification-based treatments could serve as potential interventions for sepsis-associated vascular endothelial injury.