O-GlcNAcase Activity Research Articles

Isoindoline-based fluorogenic probes bearing a self-immolative linker for the sensitive and selective detection of O-GlcNAcase activity.

O-GlcNAcase (OGA) is implicated in several important biological and disease-relevant processes. Here, we synthesized fluorogenic probes for OGA by grafting GlcNAc directly or using a self-immolative linker to the hydroxyl position of 4-hydroxylisoindoline (BHID), a typical excited-state intramolecular proton transfer (ESIPT) probe. The probe was used for a fluorogenic assay to determine the half maximal inhibitory concentration of a known OGA inhibitor and differentiate between OGA and hexosaminidase when GlcNAc is replaced by GlcNPr, where a propionyl group is used instead of an acetyl group.

Cardioprotective O-GlcNAc signaling is elevated in murine female hearts via enhanced O-GlcNAc transferase activity

The post-translational modification of intracellular proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has emerged as a critical regulator of cardiac function. Enhanced O-GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia reperfusion (I/R) injury; however, the mechanisms underpinning O-GlcNAc-cycling in response to I/R injury have not been comprehensively assessed. The cycling of O-GlcNAc is regulated by the collective efforts of two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which catalyze the addition and hydrolysis of O-GlcNAc respectively. It has previously been shown that baseline heart physiology and pathophysiology are impacted by sex. Here, we hypothesized that sex differences in molecular signaling may target protein O-GlcNAcylation both basally and in ischemic hearts. To address this question, we subjected male and female wild type murine hearts to ex vivo ischemia or ischemia-reperfusion injury. We assessed hearts for protein O-GlcNAcylation, abundance of OGT, OGA, and glutamine:fructose-6-phosphate aminotransferase (GFAT2), activity of OGT and OGA, and UDP-GlcNAc levels. Our data demonstrate elevated O-GlcNAcylation in female hearts both basally and during ischemia. We show OGT activity was enhanced in female hearts in all treatments, suggesting a mechanism for these observations. Furthermore, we found ischemia led to reduced O-GlcNAcylation and OGT specific activity. Our findings provide a foundation for understanding molecular mechanisms that regulate O-GlcNAcylation in the heart and highlight the importance of sex as a significant factor when assessing key regulatory events that control O-GlcNAc-cycling. These data suggest the intriguing possibility that elevated O-GlcNAcylation in females contributes to reduced ischemic susceptibility.

Protein O-GlcNAcylation homeostasis regulates facultative heterochromatin to fine-tune sog-Dpp signaling during Drosophila early embryogenesis

Protein O-GlcNAcylation is a monosaccharide post-translational modification maintained by two evolutionarily conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Mutations in human OGT have recently been associated with neurodevelopmental disorders, although the mechanisms linking O-GlcNAc homeostasis to neurodevelopment are not understood. Here, we investigate the effects of perturbing protein O-GlcNAcylation using transgenic Drosophila lines that overexpress a highly active OGA. We reveal that temporal reduction of protein O-GlcNAcylation in early embryos leads to reduced brain size and olfactory learning in adult Drosophila. Downregulation of O-GlcNAcylation induced by the exogenous OGA activity promotes nuclear foci formation of Polycomb-group protein Polyhomeotic and the accumulation of excess K27 trimethylation of histone H3 (H3K27me3) at the mid-blastula transition. These changes interfere with the zygotic expression of several neurodevelopmental genes, particularly shortgastrulation (sog), a component of an evolutionarily conserved sog-Decapentaplegic (Dpp) signaling system required for neuroectoderm specification. Our findings highlight the importance of early embryonic O-GlcNAcylation homeostasis for the fidelity of facultative heterochromatin redeployment and initial cell fate commitment of neuronal lineages, suggesting a possible mechanism underpinning OGT-associated intellectual disability.

An O-GlcNAcase responsive fluorogenic probe for biological applications

In the current study, we designed and prepared five fluorogenic probes (β-GlcNAc-FC, β-GlcNPr-FC, β-GlcNBu-FC, β-GlcNVa-FC and β-GlcNAc-Bn-FC) for the detection of O-GlcNAcase in live cells. Among these probes, β-GlcNAc-FC was found to be the best fluorogenic substrate for O-GlcNAcase. In addition, β-GlcNAc-FC was able to monitor cytosolic and nuclear O-GlcNAcase in cells. By using this probe, we found that O-GlcNAcase activity in AGS cells is higher than those in Capan-1 and HaCaT cells. Also, we observed that fluorescence signals arising from β-GlcNAc-FC are very low in cells cultured under glucose deprivation conditions and gradually increases when the concentration of glucose in culture media increases up to 10 mM. Furthermore, ER and mitochondrial stresses caused by respective treatment with tunicamycin and FCCP did not affect O-GlcNAcase activity, as judged from the observation that the fluorescence intensity arising from β-GlcNAc-FC in cells exposed to these stresses was quite similar to that in cells cultured under normal conditions. Taken together, the findings indicate that β-GlcNAc-FC is a useful fluorogenic probe to determine the level of O-GlcNAcase activity in live cells.

Structure-based discovery and development of novel O-GlcNAcase inhibitors for the treatment of Alzheimer's disease

The neurofibrillary tangles (NFTs) formed from hyperphosphorylation of tau protein are closely associated with Alzheimer's disease (AD). O-GlcNAcylation of tau can negatively regulate hyperphosphorylation and the O-GlcNAcase (OGA) catalyzes the removal of O-linked β-N-acetylglucosamine (O-GlcNAc) from tau protein. Therefore, preventing tau hyperphosphorylation by increasing the levels of tau O-GlcNAcylation via OGA inhibitors could be a promising approach. Based on Thiamet-G, a potent OGA inhibitor, and its binding mode to OGA, a novel OGA inhibitor scaffold bearing three parts was designed and hit compound 7j was successfully identified via extensive exploring. Further chemical optimization and diversification of the 7j structure resulted in compound 39 which possesses excellent OGA inhibition, no cytotoxicity, and has good pharmacokinetic properties. In acute AD model mice, 39 was more effective than Thiamet-G in inhibiting OGA activity attributable to its better blood-brain barrier permeability. In addition, 39 restored the cognitive function in mice and reduced amyloid-β (Aβ) concentrations to a greater extent than Thiamet-G. Molecular docking studies demonstrated that 39 was well associated with OGA through H-bonds and hydrophobic interaction. Together, these findings suggest that 39 was promising as a potent OGA inhibitor in the treatment of AD.

O‐GlcNAc characterization during Tribolium castaneumdevelopment

O‐GlcNAc (O‐linked β‐N‐acetylglucosamine) is a dynamic intracellular post‐translational modification (PTM) that regulates several cellular processes. This PTM is catalyzed by the covalent attachment of GlcNAc on residues of serine/threonine in proteins by the enzyme O‐GlcNAc transferase (OGT), while its removal is performed by O‐GlcNAcase (OGA). This PTM is limited by the concentration of UDP‐GlcNAc, the end‐product from the hexosamine biosynthetic pathway (HBP) that, together with OGT and OGA levels and activity, will regulate protein O‐GlcNAcylation. Although O‐GlcNAc has been reported in all studied metazoans, functional analysis during development in Hexapoda is restricted to the model organism Drosophila melanogaster. In this study, the role of O‐GlcNAc during development was investigated in an emerging model system, the beetle Tribolium castaneum. Comparison of the transcriptional profile of the HBP rate‐limiting step enzyme glutamine‐6‐phosphate amidotransferase (GFAT) during developmental stages showed higher levels during larval stages. Interestingly, OGT is modulated during embryonic development with an increase of mRNA levels during gastrulation while OGA remained unaltered in all stages. In situ hybridization analysis revealed an embryonic limited ubiquitous pattern of OGA during egg development, while GFAT is expressed in both embryonic and extra‐embryonic cells. Finally, parental knock down of OGT by RNA interference (RNAi) shows physiological impairment of oocyte maturation and a decrease in egg laying and embryonic survival, while OGA knockdown did not lead to physiologically and phenotypic significant changes. Altogehter, these results provide an important characterization of the O‐GlcNAc machinery in the most diverse order of insects, and suggest a critical role of O‐GlcNAcylation in a stage‐specific manner during Tribolium castaneum development.

Traditional herbal formula Wu-Mei-Wan alleviates TNBS-induced colitis in mice by inhibiting necroptosis through increasing RIPK3 O-GlcNAcylation

BackgroundAccumulating evidence indicated that necroptosis plays an essential role in the pathogenesis of inflammatory bowel disease (IBD). The O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) of necroptotic signal molecule receptor-interacting serine-threonine kinase 3 (RIPK3) was reported to exert a protective effect in gut inflammation. Our recent study suggested traditional Chinese herbal formula Wu-Mei-Wan (WMW) as an effective prescription in mouse colitis. However, the potential mechanisms are not fully understood. Considering the crucial role of necroptosis in the pathogenesis of IBD, therefore, this study was designed to explain whether the anti-colitis effect of WMW is mediated by modulating necroptosis and its related mechanisms.MethodsThe protective effects of WMW on colitis have been determined by detecting colitis mice body weight, disease activity index (DAI), survival rate and colon length. Colonic inflammation was examined by inflammatory cells infiltration and local cytokines levels. After then, we measured the levels of necroptosis and O-GlcNAcylation. C O-immunoprecipitation experiments were used to address whether elevated O-GlcNAcylation can inhibit necroptotic signal transduction in the treatment of WMW. Finally, the key enzymes in O-GlcNAcylation: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) were examined and molecular docking analysis was used to determine effective natural compounds in the regulation on OGT and OGA activities.ResultsOur results showed that WMW significantly improved mice body weight, survival rate and colon length, decreased DAI in TNBS-induced colitis. WMW obviously alleviated colonic inflammatory responses with reduced macrophages, neutrophils infiltration and local IL-1β, IL-6, TNF-α and IFN-γ levels. It was found that WMW increased colonic O-GlcNAcylation level and inhibited the activation of RIPK1, RIPK3 and MLKL. Then, further experiments revealed that WMW enhanced OGT activity and suppressed OGA activity, thereby increasing RIPK3 O-GlcNAcylation and inhibiting the binding of RIPK3 and MLKL, which led to the inhibition of necroptosis. Additionally, docking analysis demonstrated that hesperidin, coptisine and ginsenoside Rb1 may exert a major role in the regulation on OGT and OGA activities by WMW.ConclusionOur work demonstrated that WMW can alleviate TNBS-induced colitis in mice by inhibiting necroptosis through increasing RIPK3 O-GlcNAcylation.

Post-natal activity of cardiac O-GlcNAcase is regulated to maintain physiological levels of O-GlcNAc

During post-natal development, the organism is subjected to 2 metabolic transitions. The weaning transition is particularly interesting as it is associated with a change in glucose metabolism. O-GlcNAcylation is an ubiquitous post-translational modification, described as a nutrient sensor. The GlcNAc moiety is added and removed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Interestingly, cardiac O-GlcNAc levels decrease throughout post-natal development but its regulatory mechanism and impact on proteins have never been studied under this condition. Study the regulatory mechanism involved in O-GlcNAc regulation and its potential impact on proteins implied in cardiac metabolism. Hearts from 28 day-olds (D28) Wistar rats fed with a standard or low-carbohydrate (low-CHO) diet (D28F: 50% fat, 30% protein, 3% carbohydrates) during the weaning period (D12 to D28) were studied. O-GlcNAc levels and regulatory enzymes expression were evaluated by western blot. The OGA activity was evaluated with a spectrophotometric assay. The impact of selected protein expression in response to O-GlcNAc levels variations (Thiamet-G, OGA inhibitor) was evaluated on cell culture (HEK and COS). No change was reported on O-GlcNAc levels between the diets (D28: 1.00 ± 0.06; D28F: 0.99 ± 0.08). Cardiac OGA expression was 5 times lower in D28F however OGA activity remained stable (D28: 1.00 ± 0.03; D28F: 0.83 ± 0.16 AU). Protein expression, acetyl-CoA transferase 1 (ACAT1) and acetyl-CoA synthetase 1 (ACSS1), was reduced both in HEK and COS cells in responses to O-GlcNAc stimulation with Thiamet-G. While OGA expression is lower with the low-CHO diet, OGA activity and O-GlcNAc levels remain stable. This indicates a strong regulatory mechanism to control O-GlcNAc levels throughout post-natal development. This regulatory mechanism could help to modulate the expression of key metabolic enzymes such as ACAT1 or ACSS1.

Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress

The dynamic modification of specific serine and threonine residues of intracellular proteins by O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) mitigates injury and promotes cytoprotection in a variety of stress models. The O-GlcNAc transferase (OGT) and the O-GlcNAcase are the sole enzymes that add and remove O-GlcNAc, respectively, from thousands of substrates. It remains unclear how just two enzymes can be specifically controlled to affect glycosylation of target proteins and signaling pathways both basally and in response to stress. Several lines of evidence suggest that protein interactors regulate these responses by affecting OGT and O-GlcNAcase activity, localization, and substrate specificity. To provide insight into the mechanisms by which OGT function is controlled, we have used quantitative proteomics to define OGT’s basal and stress-induced interactomes. OGT and its interaction partners were immunoprecipitated from OGT WT, null, and hydrogen peroxide–treated cell lysates that had been isotopically labeled with light, medium, and heavy lysine and arginine (stable isotopic labeling of amino acids in cell culture). In total, more than 130 proteins were found to interact with OGT, many of which change their association upon hydrogen peroxide stress. These proteins include the major OGT cleavage and glycosylation substrate, host cell factor 1, which demonstrated a time-dependent dissociation after stress. To validate less well-characterized interactors, such as glyceraldehyde 3-phosphate dehydrogenase and histone deacetylase 1, we turned to parallel reaction monitoring, which recapitulated our discovery-based stable isotopic labeling of amino acids in cell culture approach. Although the majority of proteins identified are novel OGT interactors, 64% of them are previously characterized glycosylation targets that contain varied domain architecture and function. Together these data demonstrate that OGT interacts with unique and specific interactors in a stress-responsive manner.

Loss of O-GlcNAcase catalytic activity leads to defects in mouse embryogenesis

O-GlcNAcylation is an essential post-translational modification that has been implicated in neurodevelopmental and neurodegenerative disorders. O-GlcNAcase (OGA), the sole enzyme catalyzing the removal of O-GlcNAc from proteins, has emerged as a potential drug target. OGA consists of an N-terminal OGA catalytic domain and a C-terminal pseudo histone acetyltransferase (HAT) domain with unknown function. To investigate phenotypes specific to loss of OGA catalytic activity and dissect the role of the HAT domain, we generated a constitutive knock-in mouse line, carrying a mutation of a catalytic aspartic acid to alanine. These mice showed perinatal lethality and abnormal embryonic growth with skewed Mendelian ratios after day E18.5. We observed tissue-specific changes in O-GlcNAc homeostasis regulation to compensate for loss of OGA activity. Using X-ray microcomputed tomography on late gestation embryos, we identified defects in the kidney, brain, liver, and stomach. Taken together, our data suggest that developmental defects during gestation may arise upon prolonged OGA inhibition specifically because of loss of OGA catalytic activity and independent of the function of the HAT domain.

Pregnancy decreases O-GlcNAc-modified proteins in systemic arteries of normotensive and spontaneously hypertensive rats

AimWe determined the role played by O-linked N-acetylglucosamine (O-GlcNAc) of proteins in systemic arteries during late pregnancy in normotensive and hypertensive rats. Main methodsO-GlcNAc levels and O-GlcNAc modification of endothelial nitric oxide synthase (eNOS) were determined in aorta (conductance vessel) and mesenteric arteries (resistance vessels) of non-pregnant (NP) and pregnant (P) Wistar rats and spontaneously hypertensive rats (SHR). Vascular O-GlcNAc-modified proteins, O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT) expression, and OGA activity were analyzed. Concentration–response to phenylephrine (PE) curves were constructed for arteries with and without endothelium. Arteries were treated with vehicle or PugNAc (OGA inhibitor, 100 μmol/L) in the presence of L-NAME (NOS inhibitor, 100 μmol/L). Key findingsThe content of vascular O-GlcNAc-modified proteins was lower, OGT and OGA expression did not change, and OGA activity was higher in arteries of P-Wistar rats and P-SHR compared to arteries of NP-groups. Reactivity to PE increased in arteries of P-Wistar rats treated with PugNAc compared to vehicle. O-GlcNAcylation of eNOS decreased in P-SHR compared to NP-SHR. PugNAc partially inhibited the effects of endothelium removal and L-NAME on reactivity to PE in arteries of P-Wistar rats. However, PugNAc did not alter reactivity to PE in arteries of P-SHR. Our data showed that pregnancy decreased the content of vascular O-GlcNAc-modified proteins. SignificanceIncreased OGA activity and decreased O-GlcNAc modification of eNOS boosts eNOS activity in arteries of P-Wistar rats. In P-SHR, altered OGA activity may lower the content of O-GlcNAc-modified proteins, but decreased OGT activity seems a potential mechanism to reduce glycosylation.

Involvement of NDPK-B in Glucose Metabolism-Mediated Endothelial Damage via Activation of the Hexosamine Biosynthesis Pathway and Suppression of O-GlcNAcase Activity.

Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.

O-GlcNAcase contributes to cognitive function in Drosophila

O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, we generated catalytically inactive and precise knockout Oga alleles (OgaD133N and OgaKO, respectively) in Drosophila melanogaster. Adult OgaD133N and OgaKO flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionarily conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected a number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, we report behavioral and neurodevelopmental phenotypes associated with Oga alleles and show that Oga contributes to cognition and synaptic morphology in Drosophila.

Identifying Regulators of O‐GlcNAcylation in the Broken Heart

The post‐translational modification of intracellular proteins by monosaccharides of O‐linked b‐N‐acetylglucosamine (O‐GlcNAc) has emerged as a critical regulator of cellular function. Previous research has demonstrated that enhanced O‐GlcNAcylation activates cytoprotective pathways in cardiac models of ischemia reperfusion (I/R) injury. The cycling of O‐GlcNAc is regulated by the collective efforts of two enzymes: O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA), which catalyze the addition and hydrolysis of O‐GlcNAc respectively. The goal of these studies is to understand the mechanisms by which the heart regulates O‐GlcNAcylation in response to injury. Sex and gender have been linked to the pathophysiology of cardiovascular disease; however, the impact of sex on O‐GlcNAc cycling has not been studied. Thus, to address our goal, we harvested male and female hearts from mice and extracted the total heart proteome and assessed the levels of O‐GlcNAc, OGT, and OGA as well as key enzymes within the hexosamine biosynthetic pathway (HBP). We observed a modest elevation in O‐GlcNAc levels in female hearts. Counter‐intuitively, OGA levels appeared to be elevated in the female hearts. To assess the impact of sex on O‐GlcNAc cycling in response to injury, we used the Langendorff perfused mouse heart model to collect male and female hearts exposed to either control, ischemic or I/R conditions. Hearts were partitioned into a soluble fraction enriched in cytosolic and mitochondrial proteins, as well as a myofibrillar fraction enriched in contractile proteins. Our data demonstrate that the majority of the O‐GlcNAc‐modified proteins are detected in the soluble fraction. Ischemia results in a suppression of O‐GlcNAcylation; however, O‐GlcNAc levels were modestly elevated in hearts subjected to I/R injury. Future work will assess the impact of sex on the activity of OGT and OGA, as well as the nucleotide sugar UDP‐GlcNAc. Collectively, these findings will provide a foundation for understanding whether sex is an important factor to consider when assessing molecular mechanisms regulating O‐GlcNAc cycling in the heart.Support or Funding InformationK12 HL141952, U01 CA2300978, R01 HL129640, P01 HL107153

Modulation of O-GlcNAcylation Regulates Autophagy in Cortical Astrocytes.

The addition of O-linked β-N-acetylglucosamine (O-GlcNAcylation) to serine and threonine residues is a common posttranslational modification of intracellular proteins which modulates protein functions and neurodegenerative diseases, controlled by a single pair of enzymes, O-GlcNAcase (OGA), and O-GlcNAcylation transferase (OGT). Autophagy is a cellular recycling pathway activated by stress and nutrient signaling; however, the mechanism by which O-GlcNAcylation modification regulates autophagy in cortical astrocytes is poorly understood. Here, we report that increased O-GlcNAcylation by the suppression of OGA activity using thiamet-G and OGA siRNA did not affect autophagy, whereas decreased O-GlcNAcylation caused by OGT inhibition by alloxan and OGT siRNA increased autophagy. OGT inhibitor and siRNA accumulated LC3 puncta, and cotreatment with chloroquine (CQ), an autophagy inhibitor, significantly increased LC3 puncta and LC3-II protein, confirming that decreased O-GlcNAcylation promotes autophagic flux. In particular, we found that OGT knockdown increases the fusion between autophagosomes as well as lysosomes and stimulates autophagy to promote lysosomal-associated membrane protein 1 (LAMP-1). Additionally, decreasing O-GlcNAcylation by treatment with alloxan, OGT siRNA, and OGA overexpression significantly decreased the level of autophagy substrate SQSTM1/p62, indicating that autophagic degradation was activated. Together, our study reveals a mechanism by which the modulation of O-GlcNAcylation modification regulates autophagy in mouse cortical astrocytes.

New insights into mechanism of bisphenol analogue neurotoxicity: implications of inhibition of O-GlcNAcase activity in PC12 cells.

Bisphenol analogues including bisphenol A and its derivatives are ubiquitous environmental contaminants and have been linked to adverse neurodevelopment effects on animals and humans. Most toxicological research focused on estrogen receptor mediated pathways and did not comprehensively clarify the observed toxicity. O-GlcNAcase (OGA), the highest level in brain, plays a critical role in controlling neuronal functions at multi-levels from molecule to animal behaviors. In this work, we intend to investigate the underlying molecular mechanisms for the neurotoxicity of bisphenol analogues by identifying their cellular targets and the resultant effects. The inhibitory actions of seven bisphenol analogues on the OGA activity at molecular level were investigated by our developed electrochemical biosensor. We found that their potency varied with substituent groups, in which tetrabromo bisphenol A (TBBPA) was the strongest. The seven bisphenol analogues (0-100μM exposure) significantly inhibited OGA activity and up-regulated protein O-GlcNAcylation level in PC12 cells. Inhibition of OGA by bisphenol analogues further induced intracellular calcium, ROS, inflammation, repressed proliferation, interfered with cell cycle, induced apoptosis. And especially, 10μM tetrabromo bisphenol A (TBBPA) exposure could impair the growth and development of neurite in human neural stem cells (hNSCs). Molecular docking for OGA/bisphenol analogue complexes revealed the hydrophobicity-dominated inhibition potency. OGA, as a new cellular target of bisphenol analogues, would illuminate the molecular mechanism of bisphenol analogues neurotoxicity.

O-GlcNacylation Links TxNIP to Inflammasome Activation in Pancreatic β Cells.

Thioredoxin interacting protein (TxNIP), which strongly responds to glucose, has emerged as a central mediator of glucotoxicity in pancreatic β cells. TxNIP is a scaffold protein interacting with target proteins to inhibit or stimulate their activity. Recent studies reported that high glucose stimulates the interaction of TxNIP with the inflammasome protein NLRP3 (NLR family, pyrin domain containing 3) to increase interleukin-1 β (IL1β) secretion by pancreatic β cells. To better understand the regulation of TxNIP by glucose in pancreatic β cells, we investigated the implication of O-linked β-N-acetylglucosamine (O-GlcNAcylation) in regulating TxNIP at the posttranslational level. O-GlcNAcylation of proteins is controlled by two enzymes: the O-GlcNAc transferase (OGT), which transfers a monosaccharide to serine/threonine residues on target proteins, and the O-GlcNAcase (OGA), which removes it. Our study shows that TxNIP is subjected to O-GlcNAcylation in response to high glucose concentrations in β cell lines. Modification of the O-GlcNAcylation pathway through manipulation of OGT or OGA expression or activity significantly modulates TxNIP O-GlcNAcylation in INS1 832/13 cells. Interestingly, expression and O-GlcNAcylation of TxNIP appeared to be increased in islets of diabetic rodents. At the mechanistic level, the induction of the O-GlcNAcylation pathway in human and rat islets promotes inflammasome activation as evidenced by enhanced cleaved IL1β. Overexpression of OGT in HEK293 or INS1 832/13 cells stimulates TxNIP and NLRP3 interaction, while reducing TxNIP O-GlcNAcylation through OGA overexpression destabilizes this interaction. Altogether, our study reveals that O-GlcNAcylation represents an important regulatory mechanism for TxNIP activity in β cells.

F199. O-GLCNAC DYSREGULATION IN SCHIZOPHRENIA CORTEX

BackgroundO-linked β-linked N-acetylglucosamine (O-GlcNAc) is a post-translational glycosylation modification with ubiquitous functions in cell biology. The attachment of GlcNAc to serine or threonine (S/T) residues transiently adorns thousands of nuclear, cytosolic, and mitochondrial proteins and modulates protein function and localization via dynamic cooperation with kinase and phosphatase enzymes. O-GlcNAc transferase (OGT) exists in complex with S/T phosphatase subunits PP1β and PP1γ and, along with the activity of O-GlcNAcase (OGA), facilitates rapid cycling between O-GlcNAcylation and phosphorylation states to serve as an “on/off switch” for substrate activation. Altered levels of OGT, OGA, and/or O-GlcNAc have been shown to influence many pathways pertinent to schizophrenia (SZ) pathophysiology. Notably, elevated O-GlcNAc and enhanced O-GlcNAcylation mediate glucose tolerance and insulin resistance which can lead to diabetes, an illness often found comorbid with SZ. Elevated O-GlcNAcylation can also produce mitochondrial abnormalities consistent with those identified in SZ. In an exploratory study of glycosylation enzyme transcript expression, our lab found OGT mRNA levels 253% higher in SZ than non-psychiatrically ill comparison (COMP) subjects (p < 0.0001). Based on this evidence, we hypothesized that OGT protein levels or the ratio of OGT:OGA enzymes are elevated in SZ brain.MethodsExpression of OGT and OGA were measured using western blots of superior temporal gyrus (STG; Brodmann Area 22) homogenates from sex- and age-matched pairs of SZ and COMP subjects (N = 17). Standard immunoblotting methods and commercially available antibodies were used to detect the targets of interest and protein levels were normalized to intralane valosin containing protein (VCP) expression; VCP expression has previously been found to be unchanged in SZ STG.ResultsIn the current study, we found OGA protein levels reduced 18% in SZ (p < 0.01) and SZ subjects demonstrate a trend toward increased ratios of OGT:OGA (p = 0.05). OGT was not different between groups (p = 0.77).DiscussionOur current results partially support our original hypothesis that an altered ratio of OGT:OGA may contribute to abnormalities of O-GlcNAcylation and consequent cellular metabolic abnormalities in SZ. A trend toward increased OGT:OGA along with decreased expression of OGA would produce the same functional outcome as the originally predicted OGT increase: upregulation of protein O-GlcNAcylation. Given that the mRNA study used samples of dorsolateral prefrontal cortex (DLPFC) while our protein-level measures were from STG, it is not inconceivable that potential O-GlcNAc dysregulation could arise from upregulated OGT in one brain region, but downregulated OGA in another. To elaborate on these findings, we will investigate OGT and OGA expression in the DLPFC and will assess total O-GlcNAcylation in both brain regions to determine functional consequences of altered enzyme expression.

Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription.

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5’LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.

Carbohydrate Bis-acetal-Based Substrates as Tunable Fluorescence-Quenched Probes for Monitoring exo-Glycosidase Activity.

Tunable Förster resonance energy transfer (FRET)-quenched substrates are useful for monitoring the activity of various enzymes within their relevant physiological environments. Development of FRET-quenched substrates for exo-glycosidases, however, has been hindered by their constrained pocket-shaped active sites. Here we report the design of a new class of substrate that overcomes this problem. These Bis-Acetal-Based Substrates (BABS) bear a hemiacetal aglycon leaving group that tethers fluorochromes in close proximity, also positioning them distant from the active site pocket. Following cleavage of the glycosidic bond, the liberated hemiacetal spontaneously breaks down, leading to separation of the fluorophore and quencher. We detail the synthesis and characterization of GlcNAc-BABS, revealing a striking 99.9% quenching efficiency. These substrates are efficiently turned over by the human exo-glycosidase O-GlcNAcase (OGA). We find the hemiacetal leaving group rapidly breaks down, enabling quantitative monitoring of OGA activity. We expect this strategy to be broadly useful for the development of substrate probes for monitoring exo-glycosidases, as well as a range of other enzymes having constrained pocket-shaped active sites.