Like phosphorylation, O-GlcNAc-glycosylation (O-GlcNAcylation) controls the activity, stability and/or localization of proteins. This post-translational modification, which depends on the metabolic environment of the cell, plays a crucial role in many cellular processes and has been implicated in several human pathologies such as diabetes, cancer and neurodegenerative diseases.
Due to alternative splicing, 3 isoforms of OGT have been described: two nucleocytoplasmic isoforms (ncOGT and sOGT) and one mitochondrial isoform (mOGT). Although OGA activity had been demonstrated in mitochondria, characterization of mitochondrial OGA had not been performed yet. Two isoforms of OGA, long (L-OGA) and short (S-OGA), had been described as resulting from alternative splicing. While L-OGA was shown to be predominantly nucleocytoplasmic, S-OGA had been very little studied to date, and contradictory results were obtained regarding its subcellular localization (first described as nuclear, it was then found associated with lipid droplets in a second publication).
By studying the expression of mRNAs of the different isoforms of OGA and OGT in human leukocytes, the authors observed a strong correlation between mOGT and S-OGA, suggesting a potential role for S-OGA in the mitochondria.
Cell fractionation experiments then showed that S-OGA was preferentially found in mitochondria-enriched fractions of HEK-293T cells, RAW 264.7 macrophages and mouse embryonic fibroblasts. In addition, high-resolution confocal microscopy imaging confirmed that S-OGA was addressed to mitochondria. Using a BRET biosensor, the de-glycosylation activity of S-OGA in mitochondria was demonstrated in living cells. Finally, using fluorescent mitochondrial probes, the authors showed that overexpression of S-OGA increased the levels of reactive oxygen species (ROS) in mitochondria.
A fraction (2-5%) of the glucose entering the cell is used in the hexosamine biosynthetic pathway to produce UDP-GlcNAc, the substrate used by ncOGT or sOGT to O-GlcNAcylate cytosolic and nuclear proteins. UDP-GlcNAc can also enter the mitochondria via a mitochondrial nucleotide transporter, where it is used by mOGT to O-GlcNAcylate mitochondrial proteins. The authors identified S-OGA as the enzyme preferentially addressed to mitochondria to de-GlcNAcylate mitochondrial proteins. Overexpression of S-OGA leads to an increase in the production of ROS, suggesting an important role of this enzyme in the regulation of oxidative stress.
Taken together, this work reveals that the S-OGA isoform is addressed to the mitochondria where it regulates ROS homeostasis. This work could therefore suggest new therapeutic approaches to reduce oxidative stress in pathological situations associated with excessive production of ROS (metabolic diseases, neurodegenerative diseases, chronic inflammation...).
Pagesy P, Bouaboud A, Feng Z, Hulin P, Issad T. Short O-GlcNAcase Is Targeted to the Mitochondria and Regulates Mitochondrial Reactive Oxygen Species Level. Cells. (2022) Jun 2;11(11):1827. doi: 10.3390/cells11111827