| Summary: | 博士 === 國立陽明大學 === 微生物及免疫學研究所 === 104 === O-linked β-N-acetylglucosamine (O-GlcNAc) modification of proteins is a post-translational modification (PTM). The modification is catalyzed by cellular enzyme, OGT, to add a GlcNAc residue on protein serine and threonine sites. This reaction could be reversed by O-GlcNAcase, OGA. Because O-GlcNAcylation occurs on serine and threonine sites, it is implicated in many cellular events through affecting phosphorylation-mediated function. B cell activation and apoptosis induced by B cell receptor (BCR) ligation involves plenty of phosphorylation signaling cascades. The aim of this thesis is to investigate whether and how O-GlcNAcylation regulate B cell activation and apoptosis. We found that O-GlcNAcylation accumulation by treating mouse splenic B cells with OGA inhibitor, thiamet G, enhances BCR crosslinking-induced B cell activation and apoptosis. Besides, comparative phosphoproteomics analysis identified phosphopeptides and proteins sensitive to O-GlcNAcylation accumulation. Lymphocyte-specific protein 1 (Lsp1), one of the proteins responsive to O-GlcNAcylation accumulation, is demonstrated to be O-GlcNAcylated on serine 209 site. Further study examining the level of Lsp1 O-GlcNAcylation and phosphorylation during B cell activation shows that the amount of serine 243 phosphorylation is increased following the increment of Lsp1 O-GlcNAcylation and that O-GlcNAcylation on S209 site is required for the phosphorylation of serine 243 site. Functional analysis using different Lsp1 mutants demonstrates that serine 209 site O-GlcNAcylation and serine 243 site phosphorylation are both critical for BCR cross-linking induced apoptosis. The mechanism linking serine 209 site O-GlcNAcylation and serine 243 site phosphorylation is mediated by the enhanced recruitment of the kinase, PKCβ1, to O-GlcNAcylated Lsp1, which leads to higher activation of downstream apoptosis related signalsome and the reduced expression of anti-apoptosis proteins, BCL-2 and BCL-xL. In conclusion, our finding demonstrates that the interplay of O-GlcNAcylation and phosphorylation on Lsp1 determines the anti-IgM-induced B cell apoptosis.
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