The Regulative Mechanism Of Core Fucosylation In T-B Cell Interaction

Core fucosyltransferase?Fut8?is the only enzyme that catalyzes the core fucosylation of N-glycans and regulates the action of the protein in the body via post-translational modification of the protein.It has been reported that Fut8 is abnormally expressed in various cancers and plays an important role in immune response.The activation of CD4⁺T cells is regulated by glycosylation,and most of the molecules are glycoproteins involved in the activation of CD4⁺T cells.Fut8 affects protein conformation,localization and expression through core fucosylation.T cell receptors?TCRs?are highly core fucosylated glycoproteins.Glycosylation affects T cell activation by affecting TCR signaling,cytokine secretion and its receptor expression.Appropriate levels of glycosylation contribute to the maintenance of T cell function,however,the disorder of glycosylation can cause abnormal activation of T cells and further promote the pathogenesis of autoimmune diseases such as systemic lupus erythematosus?SLE?.Related research has proved that glycosylation and CD4⁺T cell activation play an important role in the pathogenesis of autoimmune diseases.However,the role of core fucosylation in T cell activation and autoimmune diseases has not yet been fully studied.Objective:In this study,we will establish Fut8 knockout mouse and OT-II transgenic mouse model for CD4⁺T cell activation related research.To clarify the regulatory effect and mechanism of core fucosylation on CD4⁺T cell activation,and provide a basis for the diagnosis and treatment of autoimmune diseases.Methods:?1?To establish Fut8 wild type(Fut8^+/+)and knockout(Fut8^-/-)mouse model,and establish OT-II transgenic Fut8 wild type(Fut8^+/+OT-II)and knockout(Fut8^-/-OT-II)mouse model.?2?To detect the core fucosylation level of serum protein and peripheral CD4⁺T cell ratio in SLE patients via Lectin Blot?LB?and flow cytometry?FACS?.?3?Related studies of CD4⁺T cell activation were performed by the Fut8^+/+and Fut8^-/-mouse models.The role of core fucosylation in IgG class-switching,TCR signaling,T cell activation and proliferation was detected via ELISA,Western blot,Lectin Blot and FACS et al.?4?Based on the Fut8^+/+OT-II and Fut8^-/-OT-II mouse models,we detected the regulatory function of core fucosylation in TCR signaling,T cell activation and T-B cell conjugates formation by ELISA,Western blot,Lectin Blot and FACS et al.Results:?1?The level of core fucosylation in the serum protein of SLE patients was increased.Though the proportion of peripheral CD4⁺T cells did not change,the number of activated cells was increased significantly.?2?Core fucosylation was attenuated in the spleen cells?SPLs?of Fut8^-/-mice and the ability of inducing experimental allergic encephalomyelitis?EAE?model was decreased.After OVA immunization,the proportion of CD4⁺T cells was reduced and IgG class-switching was also affected in the spleen of Fut8^-/-mice.The phosphorylation of ZAP70 was reduced.Moreover,the activation and proliferation of CD4⁺T cells were weakened in Fut8^-/-mice.?3?The phosphorylation of ZAP70 was decreased in Fut8^-/-OT-II CD4⁺T cells.The phosphorylation of ZAP-70 was significantly reduced in Fut8^+/+OT-II CD4⁺T cells by the treatment of fucosidase.The lack of core fucosylation reduced T-B cells conjugates formation.Moreover,the activation and proliferation of CD4⁺T cells were weakened in Fut8^-/-OT-II mice.Conlusion:Loss of core fucosyltransferase?Fut8?,the sole enzyme for catalyzing the core fucosylation of N-glycan,significantly reduced CD4⁺T cell activation and ameliorated the EAE-induced syndrome in Fut8^-/-mice.T cell activation with OVA_323-339 loaded major histocompatibility complex II?p MHC-II?on B cell was dramatically attenuated in Fut8^-/-OT-II CD4⁺T cells compared with Fut8^+/+OT-II CD4⁺T cells.Moreover,the TCR signaling was significantly reduced in Fut8^+/+OT-II CD4⁺T cells by the treatment of fucosidase.Our results suggest that core fucosylation is required for efficient TCR-p MHC-II contacts in CD4⁺T cell activation,and hyper core fucosylation in the sera may serve as a potential novel biomarker for the clinical diagnosis and treatment of SLE patients.