Cellular O-GlcNAc Levels Research Articles

Enzymatic characterization and inhibition of the nuclear variant of human O-GlcNAcase

Increasing cellular O-GlcNAc levels through pharmacological inhibition of O-GlcNAcase, the enzyme responsible for removal of the O-GlcNAc post-translational modification, is being increasingly used to aid in discerning the roles played by this form of intracellular glycosylation. Interestingly, two forms of O-GlcNAcase have been studied; a full-length isoform that is better characterized, and a shorter nuclear-localized variant, arising from failure to splice out one intron, which has not been as well characterized. Given the increasing use of O-GlcNAcase inhibitors as research tools, we felt that a clear understanding of how these inhibitors affect both isoforms of O-GlcNAcase is important for proper interpretation of studies making use of these inhibitors in cell culture and in vivo. Here we describe an enzymatic characterization of the nuclear variant of human O-GlcNAcase. We find that this short nuclear variant of O-GlcNAcase, which has the identical catalytic domain as the full-length enzyme, has similar trends in a pH-rate profile and Taft linear free energy analysis as the full-length enzyme. These findings strongly suggest that both enzymes use broadly similar transition states. Consistent with this interpretation, the short isoform is potently inhibited by several previously described inhibitors of full-length O-GlcNAcase including PUGNAc, NAG-thiazoline, and the selective O-GlcNAcase inhibitor NButGT. These findings contrast with earlier studies and suggest that studies using O-GlcNAcase inhibitors in cultured cells or in vivo can be interpreted with the knowledge that both these forms of O-GlcNAcase are inhibited when present.

Elevation of Global O-GlcNAc Levels in 3T3-L1 Adipocytes by Selective Inhibition of O-GlcNAcase Does Not Induce Insulin Resistance

The O-GlcNAc post-translational modification is considered to act as a sensor of nutrient flux through the hexosamine biosynthetic pathway. A cornerstone of this hypothesis is that global elevation of protein O-GlcNAc levels, typically induced with the non-selective O-GlcNAcase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-D-glycopyranosylidene) amino-N-phenylcarbamate), causes insulin resistance in adipocytes. Here we address the potential link between elevated O-GlcNAc and insulin resistance by using a potent and selective inhibitor of O-GlcNAcase (NButGT (1,2-dideoxy-2'-propyl-alpha-D-glucopyranoso-[2,1-D]-Delta 2'-thiazoline), 1200-fold selectivity). A comparison of the structures of a bacterial homologue of O-GlcNAcase in complex with PUGNAc or NButGT reveals that these inhibitors bind to the same region of the active site, underscoring the competitive nature of their inhibition of O-GlcNAcase and the molecular basis of selectivity. Treating 3T3-L1 adipocytes with NButGT induces rapid increases in global O-GlcNAc levels, but strikingly, NButGT treatment does not replicate the insulin desensitizing effects of the non-selective O-GlcNAcase inhibitor PUGNAc. Consistent with these observations, NButGT also does not recapitulate the impaired insulin-mediated phosphorylation of Akt that is induced by treatment with PUGNAc. Collectively, these results suggest that increases in global levels of O-GlcNAc-modified proteins of cultured adipocytes do not, on their own, cause insulin resistance.

Direct in-gel fluorescence detection and cellular imaging of O-GlcNAc-modified proteins.

We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3 + 2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct in-gel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of O-GlcNAc.

Cardioprotection by N -Acetylglucosamine Linkage to Cellular Proteins

The modification of proteins with O-linked beta-N-acetylglucosamine (O-GlcNAc) represents a key posttranslational modification that modulates cellular function. Previous data suggest that O-GlcNAc may act as an intracellular metabolic or stress sensor, linking glucose metabolism to cellular function. Considering this, we hypothesized that augmentation of O-GlcNAc levels represents an endogenously recruitable mechanism of cardioprotection. In mouse hearts subjected to in vivo ischemic preconditioning, O-GlcNAc levels were significantly elevated. Pharmacological augmentation of O-GlcNAc levels in vivo was sufficient to reduce myocardial infarct size. We investigated the influence of O-GlcNAc levels on cardiac injury at the cellular level. Lethal oxidant stress of cardiac myocytes produced a time-dependent loss of cellular O-GlcNAc levels. This pathological response was largely reversible by pharmacological augmentation of O-GlcNAc levels and was associated with improved cardiac myocyte survival. The diminution of O-GlcNAc levels occurred synchronously with the loss of mitochondrial membrane potential in isolated cardiac myocytes. Pharmacological enhancement of O-GlcNAc levels attenuated the loss of mitochondrial membrane potential. Proteomic analysis identified voltage-dependent anion channel as a potential target of O-GlcNAc modification. Mitochondria isolated from adult mouse hearts with elevated O-GlcNAc levels had more O-GlcNAc-modified voltage-dependent anion channel and were more resistant to calcium-induced swelling than cardiac mitochondria from vehicle mice. O-GlcNAc signaling represents a unique endogenously recruitable mechanism of cardioprotection that may involve direct modification of mitochondrial proteins critical for survival such as voltage-dependent anion channel.