Identification and functional characterization of O-GlcNAcylation and phosphorylation on O-GlcNAcase

• Main Authors: Pin-Jong Lee, 李品融
• Other Authors: Teh-Ying Chou
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/44288079310933338630

碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 101 === Protein O-GlcNAcylation is increasingly recognized as a key regulatory mechanism of diverse cellular processes. This post-translational modification involves the attachment of the N-acetylglucosamine (GlcNAc) to serine and threonine residues of nuclear and cytoplasmic proteins by the cellular O-GlcNAc transferase (OGT), and the modification can be removed by the O-GlcNAcase (OGA). It has been reported that protein O-GlcNAcylation can regulate essential cellular processes such as signal transduction, transcription, translation, and protein degradation. Studies have also showed that O-GlcNAcylation is involved in many human diseases, such as Alzheimer disease, diabetes, cardiovascular diseases and cancers. Cellular O-GlcNAc levels are regulated by two key enzymes: OGT and OGA. However, mechanisms involved in regulating the activity of these two enzymes are still not clear. Otherwise, OGA has been shown to be modified by O-GlcNAcylation and O-phosphorylation. However, how these modifications affect OGA activity or its protein functions remains unknown. This thesis research aims to characterize the functional role of O-GlcNAcylation and O-phosphorylation on OGA. First, by literature search and mass spectrometry, we identified Ser405 as an O-GlcNAcylation site and Ser364 and Thr709 as O-phosphorylation sites on OGA. We constructed plasmids to express OGA with amino acid substitution at these modification sites. Using cells transfect by these plasmids, we compared the wild-type and mutant OGA in several functional assays. We found that OGA-S405A displayed decreased extent of O-GlcNAcylation compared to wild-type OGA, suggesting that Ser405 of OGA is a major O-GlcNAcylation site. Furthermore, we found that OGA-S405A exhibited decreased OGA protein half-life in comparison with wild-type. We also found that phosphorylation of Ser364 decreased OGA protein stability and slightly decreased O-GlcNAcase activity. These finding can point out the direction of our further investigation into the role of post-translational modifications on OGA.