The Study Of Recognition Between OGT And Sugar Donor&the Function Of OGT Self-Glycosylation

O-GlcNAc modification, namely O-GlcNAcylation, is an essential post-translational modification with a single β-N-acetylglucosamine linked to Ser or Thr residues of various nucleocytoplasmic proteins. It contributes to various cellular cascades, including signal transduction, gene expression and protein trafficking. Dysregulation in O-GlcNAcylation is assumed to be tightly linked to chronic diseases, such as cancers, diabetes and neurodegenerative diseases.O-Linked β-N-acetylglucosaminyl transferase (OGT) plays an important role in the glycosylation of proteins, which is involved in various cellular events. In human, three isoforms of OGT (short OGT [sOGT]; mitochondrial OGT [mOGT]; and nucleocytoplasmic OGT [ncOGT]) share the same catalytic domain, implying that they might adopt a similar catalytic mechanism, including sugar donor recognition. In this work, the sugar-nucleotide tolerance of sOGT was investigated. Among a series of uridine5’-diphosphate-N-acetylglucosamine (UDP-GlcNAc) analogs tested using the casein kinase Ⅱ(CKⅡ) peptide as the sugar acceptor, four compounds could be used by sOGT, including UDP-6-deoxy-GlcNAc, UDP-GlcNPr, UDP-6-deoxy-GalNAc and UDP-4-deoxy-GlcNAc. Determined values of Km showed that the substitution of the N-acyl group, deoxy modification of C6/C4-OH or epimerization of C4-OH of the GlcNAc in UDP-GlcNAc decreased its affinity to sOGT. A molecular docking study combined with site-directed mutagenesis indicated that the backbone carbonyl oxygen of Leu653and the hydroxyl group of Thr560in sOGT contributed to the recognition of the sugar moiety via hydrogen bonds. The close vicinity between Met501and the N-acyl group of GlcNPr, as well as the hydrophobic environment near Met501, were responsible for the selective binding of UDP-GlcNPr. These findings illustrate the interaction of OGT and sugar nucleotide donor, providing insights into the OGT catalytic mechanism.Self-glycosylation of OGT was reported by several research groups. Based on the function of O-GlcNAcylation, we proposed that self-glycosylation was involved in the regulation of OGT. A variety of biological and chemical approaches were applied in the study of self-glycosylation. Six potential glycosylation sites were found, and three of them had significant influence on the level of glycosylation. The glycosylation level of sOGT was higher in the cytoplasm than that in the nuclear, when it was overexpressed in the HEK293T cells. In the analysis of subcellular localization, the mount of T449A and T662A mutants was much less compared with the wild type, and the mount of other mutants was almost equal to the wild type. It was indicated that T449and T662might be involved in the translocation of sOGT. According to the results obtained with pull down and mass spectrometry, self-glycosylation was supposed to play an essential role in the interaction between sOGT and other proteins, which is associated with various cellular events. As a part of regulation of OGT, the function and mechanism of self-glycosylation’effect on OGT were not fully understood. We provided several clues for the further study of OGT self-glycosylation, as well as for the pathogenesis of chronic diseases associated with O-GlcNAcylation dysregulation.