The Protective Effect And Mechanism Of FUT2 On Intestinal Stem Cells Against Inflammatory Injury

Objective: Dysregulation of intestinal homeostasis is a crucial factor in the progression of a variety of gastrointestinal and systemic diseases.Enhancement of intestinal epithelial repair after injury is the core process to restore homeostasis,and epithelial regeneration is predominently coordinated by intestinal stem cells.As a crucial dietary monosaccharide,fucose protects the intestinal mucosal barrier by activating fucosyltransferase 2(FUT2).However,the specific effects and mechanisms of fucose and FUT2 on intestinal stem cells are still poorly defined.Methods: 1.Acute intestinal inflammation model of mice treated with dextran sulfate sodium(DSS),and lipopolysaccharide(LPS)-induced injury model of intestinal organoids were established and fucose was continuously given to mice by oral gavage.The destruction of epithelial inflammatory injury and the impairment of barrier function were evaluated.The alternations in the number,stemness genes,and proliferation capacity of intestinal stem cells were detected.Alternations in the potential of organoid budding,oxidative stress,mitochondrial dysfunction,and apoptosis were examined.2.Nglycoproteomics was applied for sorted intestinal stem cells derived from intestinal stem cell-specific Fut2 knockout(Fut2ΔISC)and wild type mice.Changes in the critical biological process mediated by FUT2 in intestinal stem cells were clarified,and the fucosylated proteins and their modification sites that regulating the survival and function of intestinal stem cells were identified.3.UEA-1 chromatography was performed to identify the potential key fucosylated protein.Site-directed mutagenesis was used to evaluted the effects of the fucosylation levels on the self-renewal and regenerative capacities of intestinal stem cells.Results: 1.Fucose could retard the DSS-induced decrease in the number and proliferation capacity of intestinal stem cells via upregulating the fucosylation level of intestinal stem cells,thereby alleviating intestinal inflammatory injury,restoring mucosal barrier function,and maintaining the integrity of intestinal epithelial.2.Fucose could ameliorate LPSinduced reactive oxygen species accumulation,mitochondrial dysfunction,and apoptosis.Moreover,fucose restored the potential of organoid budding,the expression of stemness genes,and the proliferation capacity.3.N-glycoproteomics revealed that endoplasmic reticulum was the notably differentially expressed organelle mediated by FUT2,and endoplasmic reticulum chaperone protein was a key component in control of FUT2.4.Fucose could mitigated the activation of inflammation-induced endoplasmic reticulum stress.While in the FUT2-specific-knockout intestinal stem cells,the protective effects of fucose on the potential of organoid budding and proliferation capacity were suppressed.FUT2 depletion in intestinal stem cells triggered the aggravation of endoplasmic reticulum stress and apoptosis through the pro-apoptotic IRE1/TRAF2/ASK1/JNK signaling branch of unfolded protein response.5.UEA-1 affinity chromatography showed that HYOU1 was a vital fucosylated protein mediated by FUT2 in intestinal stem cells.6.Fucosylation of the chaperone protein HYOU1 at the N-glycosylation site of asparagine 862 mediated by FUT2.Site-directed mutagensis of the regulated fucosylation modification site inhibited the potential of organoid budding and the proliferative capacity,and diminished the resistance of intestinal stem cells to endoplasmic reticulum stress and apoptosis.Conclusion: FUT2 promotes survival and self-renewal of intestinal stem cells upon intestinal injury via regulating UPR and the fucosylated modification of the ER chaperone protein HYOU1.