Peptide substrate-assisted study of O-GlcNAc transferase and O-GlcNAcylation

Abstract

O-GlcNAcylation is a post translational modification (PTM) that corresponds to the addition of a single β-linked N-Acetyl-D-glucosamine (GlcNAc) sugar moiety onto the hydroxyl group of serine and threonine residues in numerous proteins. The addition of O-GlcNAc to proteins is catalyzed by O-GlcNAc transferase (OGT), while its removal is catalyzed by O-GlcNAcase (OGA). In contrast to complex glycans that typically function on the cell surface by mediating numerous ligand-receptor interactions, O-GlcNAc modifications of proteins ubiquitously occur inside the cells where they regulate many basic cellular processes such as gene expression, cell cycle, metabolism, cellular stress response, insulin signaling, and proteostasis. Aberrant regulation of O-GlcNAcylation has been linked to chronic diseases such as type 2 diabetes, cancer and Alzheimer’s Disease (AD). Although advanced mass spectrometry and protein analysis techniques have been used for the study of O-GlcNAcylation of proteins, it is still a challenging task to study this modification at the protein level and in living systems. In this thesis, we studied O-GlcNAcylation at the peptide level, taking advantage of peptide chemistry and the power of peptide display techniques. We developed a dynamic peptide microarray approach to discover novel OGT substrates and study its catalytic properties. With this approach, the O-GlcNAcylation of hundreds of peptides immobilized on the microarray can be monitored in parallel by a fluorescent labeled anti O-GlcNAc antibody. Another important feature of this microarray-based assay is the kinetics readout of the O-GlcNAcylation process. This approach will likely prove valuable in diagnosis of GlcNAcylation-linked disease. The peptide microarray-based approach was further used to study the cross-talk between O-GlcNAcylation and tyrosine phosphorylation in a direct manner. From 256 known tyrosine kinase peptide substrates, we found that the phosphorylation of 6 peptides by Jak2 inhibited their subsequent O-GlcNAcylation while O-GlcNAcylation had no detectable effect on their subsequent phosphorylation. This type of crosstalk might prove to be more general with additional studies. These findings provide a glimpse of a possible new paradigm for cellular signaling control by cross-talk, although these results have to be validated on proteins and in living systems. Selective and potent inhibitors are valuable tools for deeper investigation of the biological functions of OGT. We found that two excellent OGT peptide substrates in which the O-GlcNAcylation site was replaced with an alanine, which can’t be O-GlcNAcylated, exhibited significant inhibitory effect on OGT activity. These data suggest that residues around the O-GlcNAc site maintain important interactions with the protein which could possibly be utilized to design selective OGT inhibitor. In addition, we used an mRNA display-based approach to discover short peptide substrates of OGT by taking advantage of its fast selection of peptides of interest from a library with a large diversity and complexity (5.12 x109 peptides). High-throughput sequencing of the selected libraries would yield information that can be used to compare the relative abundance of each sequence in the same selected population, which in our case might help identify the detailed substrate preference of OGT.