O-GlcNAc Moieties Research Articles

Optimization of 2-DE and multiplexed detection of O-GlcNAcome, phosphoproteome and whole proteome protocol of synapse-associated proteins within the rat sensorimotor cortex

BackgroundSeveral studies have shown the importance of phosphorylation, O-GlcNAcylation and their interplay in neuronal processes. New methodTo get understanding about molecular mechanisms of synaptic plasticity, we performed a preparation of synaptic protein-enriched fraction on a small sample of rat sensorimotor cortex. We then optimized a multiplexed proteomic strategy to detect O-GlcNAcylated proteins, phosphoproteins, and the whole proteome within the same bidimensional gel. We compared different protocols (solubilisation buffer, reticulation and composition of the gel, migration buffer) to optimize separating conditions for 2D-gel electrophoresis of synaptic proteins. The O-GlcNAcome was revealed using Click chemistry and the azide–alkyne cycloaddition of a fluorophore on O-GlcNAc moieties. The phosphoproteome was detected by Phospho-Tag staining, while the whole proteome was visualized through SYPRORuby staining. ResultsThis method permitted, after sequential image acquisition, the direct in-gel detection of O-GlcNAcome, phosphoproteome, and whole proteome of synapse-associated proteins. ConclusionThis original method of differential proteomic analysis will permit to identify key markers of synaptic plasticity that are O-GlcNAcylated and/or phosphorylated, and their molecular regulations in neuronal processes.

MK-8719, a Novel and Selective O-GlcNAcase Inhibitor That Reduces the Formation of Pathological Tau and Ameliorates Neurodegeneration in a Mouse Model of Tauopathy.

Deposition of hyperphosphorylated and aggregated tau protein in the central nervous system is characteristic of Alzheimer disease and other tauopathies. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-GlcNAcylation of tau has been shown to influence tau phosphorylation and aggregation. Inhibition of O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc moieties, is a novel strategy to attenuate the formation of pathologic tau. Here we described the in vitro and in vivo pharmacological properties of a novel and selective OGA inhibitor, MK-8719. In vitro, this compound is a potent inhibitor of the human OGA enzyme with comparable activity against the corresponding enzymes from mouse, rat, and dog. In vivo, oral administration of MK-8719 elevates brain and peripheral blood mononuclear cell O-GlcNAc levels in a dose-dependent manner. In addition, positron emission tomography imaging studies demonstrate robust target engagement of MK-8719 in the brains of rats and rTg4510 mice. In the rTg4510 mouse model of human tauopathy, MK-8719 significantly increases brain O-GlcNAc levels and reduces pathologic tau. The reduction in tau pathology in rTg4510 mice is accompanied by attenuation of brain atrophy, including reduction of forebrain volume loss as revealed by volumetric magnetic resonance imaging analysis. These findings suggest that OGA inhibition may reduce tau pathology in tauopathies. However, since hundreds of O-GlcNAcylated proteins may be influenced by OGA inhibition, it will be critical to understand the physiologic and toxicological consequences of chronic O-GlcNAc elevation in vivo. SIGNIFICANCE STATEMENT: MK-8719 is a novel, selective, and potent O-linked N-acetylglucosamine (O-GlcNAc)-ase (OGA) inhibitor that inhibits OGA enzyme activity across multiple species with comparable in vitro potency. In vivo, MK-8719 elevates brain O-GlcNAc levels, reduces pathological tau, and ameliorates brain atrophy in the rTg4510 mouse model of tauopathy. These findings indicate that OGA inhibition may be a promising therapeutic strategy for the treatment of Alzheimer disease and other tauopathies.

O-GlcNAc Transferase Regulates Cancer Stem-like Potential of Breast Cancer Cells.

Breast tumors are heterogeneous and composed of different subpopulation of cells, each with dynamic roles that can change with stage, site, and microenvironment. Cellular heterogeneity is, in part, due to cancer stem-like cells (CSC) that share properties with stem cells and are associated with treatment resistance. CSCs rewire metabolism to meet energy demands of increased growth and biosynthesis. O-GlcNAc transferase enzyme (OGT) uses UDP-GlcNAc as a substrate for adding O-GlcNAc moieties to nuclear and cytoplasmic proteins. OGT/O-GlcNAc levels are elevated in multiple cancers and reducing OGT in cancer cells blocks tumor growth. Here, we report that breast CSCs enriched in mammosphere cultures contain elevated OGT/O-GlcNAcylation. Inhibition of OGT genetically or pharmacologically reduced mammosphere forming efficiency, the CD44H/CD24L, NANOG+, and ALDH+ CSC population in breast cancer cells. Conversely, breast cancer cells overexpressing OGT increased mammosphere formation, CSC populations in vitro, and also increased tumor initiation and CSC frequency in vivo. Furthermore, OGT regulates expression of a number of epithelial-to-mesenchymal transition and CSC markers including CD44, NANOG, and c-Myc. In addition, we identify Krüppel-like factor 8 (KLF8) as a novel regulator of breast cancer mammosphere formation and a critical target of OGT in regulating CSCs. IMPLICATIONS: These findings demonstrate that OGT plays a key role in the regulation of breast CSCs in vitro and tumor initiation in vivo, in part, via regulation of KLF8, and thus inhibition of OGT may serve as a therapeutic strategy to regulate tumor-initiating activity.

O-GlcNAc transferase inhibits visceral fat lipolysis and promotes diet-induced obesity

Excessive visceral fat accumulation is a primary risk factor for metabolically unhealthy obesity and related diseases. The visceral fat is highly susceptible to the availability of external nutrients. Nutrient flux into the hexosamine biosynthetic pathway leads to protein posttranslational modification by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties. O-GlcNAc transferase (OGT) is responsible for the addition of GlcNAc moieties to target proteins. Here, we report that inducible deletion of adipose OGT causes a rapid visceral fat loss by specifically promoting lipolysis in visceral fat. Mechanistically, visceral fat maintains a high level of O-GlcNAcylation during fasting. Loss of OGT decreases O-GlcNAcylation of lipid droplet-associated perilipin 1 (PLIN1), which leads to elevated PLIN1 phosphorylation and enhanced lipolysis. Moreover, adipose OGT overexpression inhibits lipolysis and promotes diet-induced obesity. These findings establish an essential role for OGT in adipose tissue homeostasis and indicate a unique potential for targeting O-GlcNAc signaling in the treatment of obesity.

Regulatory Effects of O-GlcNAcylation in Vascular Smooth Muscle Cells on Diabetic Vasculopathy

Vascular complications from uncontrolled hyperglycemia are the leading cause of death in patients with diabetes mellitus. Previous reports have shown a strong correlation between hyperglycemia and vascular calcification, which increases mortality and morbidity in individuals with diabetes. However, the precise underlying molecular mechanisms of hyperglycemia-induced vascular calcification remain largely unknown. Transdifferentiation of vascular smooth muscle cells (VSMC) into osteoblast-like cells is a known culprit underlying the development of vascular calcification in the diabetic vasculature. Pathological conditions such as high glucose levels and oxidative stress are linked to enhanced osteogenic differentiation of VSMC both in vivo and in vitro. It has been demonstrated that increased expression of runt-related transcription factor 2 (Runx2), a bone-related transcription factor, in VSMC is necessary and sufficient for the induction of VSMC calcification. Addition of a single O-linked β-N-acetylglucosamine (O-GlcNAc) moiety to the serine/threonine residues of target proteins (O-GlcNAcylation) has been observed in the arteries of diabetic patients, as well as in animal models in association with the enhanced expression of Runx2 and aggravated vascular calcification. O-GlcNAcylation is a dynamic and tightly regulated process, that is mediated by 2 enzymes, O-GlcNAc transferase and O-GlcNAcase. Glucose is metabolized into UDP-β-D-N-acetylglucosamine, an active sugar donor of O-GlcNAcylation via the hexosamine biosynthetic pathway. Overall increases in the O-GlcNAcylation of cellular proteins have been closely associated with cardiovascular complications of diabetes. In this review, the authors provide molecular insights into cardiovascular complications, including diabetic vasculopathy, that feature increased O-GlcNAcylation in people with diabetes.

O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry.

As a dynamic post-translational modification, O-linked β- N-acetylglucosamine ( O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-β-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.

A Novel Glycoproteomics Workflow Reveals Dynamic O-GlcNAcylation of COPγ1 as a Candidate Regulator of Protein Trafficking.

O-linked β-N-acetylglucosamine (O-GlcNAc) is an abundant and essential intracellular form of protein glycosylation in animals and plants. In humans, dysregulation of O-GlcNAcylation occurs in a wide range of diseases, including cancer, diabetes, and neurodegeneration. Since its discovery more than 30 years ago, great strides have been made in understanding central aspects of O-GlcNAc signaling, including identifying thousands of its substrates and characterizing the enzymes that govern it. However, while many O-GlcNAcylated proteins have been reported, only a small subset of these change their glycosylation status in response to a typical stimulus or stress. Identifying the functionally important O-GlcNAcylation changes in any given signaling context remains a significant challenge in the field. To address this need, we leveraged chemical biology and quantitative mass spectrometry methods to create a new glycoproteomics workflow for profiling stimulus-dependent changes in O-GlcNAcylated proteins. In proof-of-principle experiments, we used this new workflow to interrogate changes in O-GlcNAc substrates in mammalian protein trafficking pathways. Interestingly, our results revealed dynamic O-GlcNAcylation of COPγ1, an essential component of the coat protein I (COPI) complex that mediates Golgi protein trafficking. Moreover, we detected 11 O-GlcNAc moieties on COPγ1 and found that this modification is reduced by a model secretory stress that halts COPI trafficking. Our results suggest that O-GlcNAcylation may regulate the mammalian COPI system, analogous to its previously reported roles in other protein trafficking pathways. More broadly, our glycoproteomics workflow is applicable to myriad systems and stimuli, empowering future studies of O-GlcNAc in a host of biological contexts.

Chemical Reporters and Their Bioorthogonal Reactions for Labeling Protein O-GlcNAcylation.

Protein O-GlcNAcylation is a non-canonical glycosylation of nuclear, mitochondrial, and cytoplasmic proteins with the attachment of a single O-linked β-N-acetyl-glucosamine (O-GlcNAc) moiety. Advances in labeling and identifying O-GlcNAcylated proteins have helped improve the understanding of O-GlcNAcylation at levels that range from basic molecular biology to cell signaling and gene regulation to physiology and disease. This review describes these advances in chemistry involving chemical reporters and their bioorthogonal reactions utilized for detection and construction of O-GlcNAc proteomes in a molecular mechanistic view. This detailed view will help better understand the principles of the chemistries utilized for biology discovery and promote continued efforts in developing new molecular tools and new strategies to further explore protein O-GlcNAcylation.

Structural basis of O-GlcNAc recognition by mammalian 14-3-3 proteins

O-GlcNAc is an intracellular posttranslational modification that governs myriad cell biological processes and is dysregulated in human diseases. Despite this broad pathophysiological significance, the biochemical effects of most O-GlcNAcylation events remain uncharacterized. One prevalent hypothesis is that O-GlcNAc moieties may be recognized by "reader" proteins to effect downstream signaling. However, no general O-GlcNAc readers have been identified, leaving a considerable gap in the field. To elucidate O-GlcNAc signaling mechanisms, we devised a biochemical screen for candidate O-GlcNAc reader proteins. We identified several human proteins, including 14-3-3 isoforms, that bind O-GlcNAc directly and selectively. We demonstrate that 14-3-3 proteins bind O-GlcNAc moieties in human cells, and we present the structures of 14-3-3β/α and γ bound to glycopeptides, providing biophysical insights into O-GlcNAc-mediated protein-protein interactions. Because 14-3-3 proteins also bind to phospho-serine and phospho-threonine, they may integrate information from O-GlcNAc and O-phosphate signaling pathways to regulate numerous physiological functions.

O‐GlcNAc Transferase Regulates Phenotypic Transformation of Vascular Smooth Muscle Cells in Response to Hyperglycemia

Risks of vascular complications are enhanced two‐to‐four fold in diabetes, accounting for increased morbidity and mortality in these patients. Hyperglycemia is an important risk factor for macrovascular complications associated with diabetes. Diabetic patients exhibit an increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, a hallmark of VSMC phenotypic switching from the quiescent contractile state to a synthetic proliferative phenotype. Hyperglycemia increases glucose flux through the hexosamine biosynthetic pathway triggering an enhanced signaling via O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT), the key enzyme catalyzing addition of O‐GlcNAc moieties to proteins. Protein O‐GlcNAcylation is a unique post‐translational protein modification implicated in diabetes and related cardiovascular complications. Previous studies have suggested both cardio‐protective and cardio‐detrimental effects of protein O‐GlcNAcylation in diabetes. However, the precise role of OGT in the etiology of diabetic atherogenesis remains incompletely understood. The goal of the current study was to investigate whether OGT modulates VSMC transition to a de‐differentiated phenotype under hyperglycemic conditions. Immunoblotting experiments revealed that in primary cultures of human aortic smooth muscle cells (HASMC) in vitro, benzyl 2‐deoxy α‐D‐galactopyranoside (BG), a pharmacological inhibitor of OGT, decreased high glucose‐induced OGT and O‐GlcNAc protein expression, and this was accompanied with attenuated PCNA (proliferation marker), reduced vimentin (SM synthetic marker) and increased SM‐MHC (SM contractile marker) expression. Consistent with these findings, deletion of OGT using siRNA gene silencing significantly lowered protein O‐GlcNAc and PCNA expression in glucose‐stimulated HASMC compared with glucose‐treated cells transfected with control siRNA. Notably, under glucose‐stimulated conditions, OGT knockdown enhanced calponin (SM contractile marker) expression in HASMC transfected with OGT siRNA vs cells transfected with control siRNA. Finally, electrophoretic mobility shift assay (EMSA) revealed that specific OGT inhibitors (BG and ST045849) attenuated high glucose‐induced DNA‐binding activity of YY1 and Elk1 (transcriptional repressors of SM contractile phenotype) in HASMC compared to cells treated with glucose alone. Taken together, these data clearly demonstrate that loss of OGT inhibits VSMC phenotypic de‐differentiation in response to hyperglycemia, suggesting OGT as a potential therapeutic target in diabetic atherosclerosis.Support or Funding InformationAmerican Heart Association ‐ Grant‐in‐Aid 16GRNT31200034This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

O-GlcNAcylation affects β-catenin and E-cadherin expression, cell motility and tumorigenicity of colorectal cancer

O-GlcNAcylation, the addition of β-N-acetylglucosamine (O-GlcNAc) moiety to Ser/Thr residues, is a sensor of the cell metabolic state. Cancer diseases such as colon, lung and breast cancer, possess deregulated O-GlcNAcylation. Studies during the last decade revealed that O-GlcNAcylation is implicated in cancer tumorigenesis and proliferation. The Wnt/β-catenin signaling pathway and cadherin-mediated adhesion are also implicated in epithelial-mesenchymal transition (EMT), a key cellular process in invasion and cancer metastasis. Often, deregulation of the Wnt pathway is caused by altered phosphorylation of its components. Specifically, phosphorylation of Ser or Thr residues of β-catenin affects its location and interaction with E-cadherin, thus facilitating cell-cell adhesion.Consistent with previous studies, the current study indicates that β-catenin is O-GlcNAcylated. To test the effect of O-GlcNAcylation on cell motility and how O-GlcNAcylation might affect β-catenin and E-cadherin functions, the enzyme machinery of O-GlcNAcylation was modulated either with chemical inhibitors or by gene silencing. When O-GlcNAcase (OGA) was inhibited, a global elevation of protein O-GlcNAcylation and increase in the expression of E-cadherin and β-catenin were noted. Concomitantly with enhanced O-GlcNAcylation, β-catenin transcriptional activity were elevated. Additionally, fibroblast cell motility was enhanced. Stable silenced cell lines with adenoviral OGA or adenoviral O-GlcNAc transferase (OGT) were established. Consistent with the results obtained by OGA chemical inhibition by TMG, OGT-silencing led to a significant reduction in β-catenin level. In vivo, murine orthotropic colorectal cancer model indicates that elevated O-GlcNAcylation leads to increased mortality rate, tumor and metastasis development. However, reduction in O-GlcNAcylation promoted survival that could be attributed to attenuated tumor and metastasis development. The results described herein provide circumstantial clues that O-GlcNAcylation deregulates β-catenin and E-cadherin expression and activity in fibroblast cell lines and this might influence EMT and cell motility, which may further influence tumor development and metastasis.

Modulation of O-GlcNAc Levels in the Liver Impacts Acetaminophen-Induced Liver Injury by Affecting Protein Adduct Formation and Glutathione Synthesis.

Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single β-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.

Abstract 5457: OGT restrains the NRF2 antioxidant pathway via O-GlcNAcylation of KEAP1

Abstract O-GlcNAcylation is a reversible post-translational modification that adds an O-linked β-N-acetylglucosamine (O-GlcNAc) moiety onto serine/threonine residues of target proteins. This modification is regulated by only two enzymes: O-GlcNAc transferase (OGT, the writer) and O-GlcNAcase (OGA, the eraser) in mammals. Recent studies have revealed that OGT expression and O-GlcNAc modifications are elevated in several cancers, but specific O-GlcNAc targets are not well defined. We conducted a global transcriptome profiling in MDA-MB-231 breast cancer cells to search for signaling events that respond to O-GlcNAc fluctuation. We found significant up-regulation of genes involved in the NRF2-dependent stress response when OGT activity is inhibited in different tumor types. We also discovered a strong positive correlation of gene signatures between low OGT activity and NRF2 activation in multiple human tumor gene expression datasets. NRF2, the primary regulator of redox balance, is usually activated by oxidative stress and repressed under basal conditions by the KEAP1-CUL3 ubiquitin ligase complex. However, we found that OGT inhibition increases NRF2 protein level through reducing poly-ubiquitination of NRF2 in the absence of oxidative stress. By chemical sugar labeling and mass spectrometry assays, we identified that KEAP1 is directly O-GlcNAcylated by OGT especially within the BTB and Kelch motifs. Of all 11 putative O-GlcNAc sites on KEAP1, we found serine 104 is responsible for regulating NRF2 activity through affecting KEAP1-CUL3 interaction. Interestingly, we found the amount of intracellular glucose co-vary with KEAP1 O-GlcNAcylation and NRF2 protein, suggesting that glucose metabolites may utilize the nutrient sensing O-GlcNAcylation to fine-tune the antioxidant response. We propose that cancer cells could utilize O-GlcNAcylation, specifically on KEAP1, to regulate NRF2-mediated stress responses in response to the dynamics of intracellular glucose level. Since the NRF2 pathway is inappropriately activated in certain tumor types due to dysregulated function of KEAP1, such as non-small cell lung cancer, where it provides stress resistance and a growth advantage. Our study could provide new insight into redox cancer biology and provide novel strategy to modulate NRF2 activity during tumor development. Citation Format: Po-Han Chen, Timothy J. Smith, Jianli Wu, Michael Boyce, Jen-Tsan Ashley Chi. OGT restrains the NRF2 antioxidant pathway via O-GlcNAcylation of KEAP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5457. doi:10.1158/1538-7445.AM2017-5457

Direct Monitoring of Protein O-GlcNAcylation by High-Resolution Native Mass Spectrometry.

O-GlcNAcylation is one of the most abundant metazoan nuclear-cytoplasmic post-translational modifications. Proteins modified by O-GlcNAc play key cellular roles in signaling, transcription, metabolism, and cell division. Mechanistic studies on protein O-GlcNAcylation are hampered by the lack of methods that can simultaneously quantify O-GlcNAcylation, determine its stoichiometry, and monitor O-GlcNAcylation kinetics. Here, we demonstrate that high-resolution native mass spectrometry can be employed to monitor the small mass shifts induced by modification by O-GlcNAc on two known protein substrates, CK2α and TAB1, without the need for radioactive labeling or chemoenzymatic tagging using large mass tags. Limited proteolysis enabled further localization of the O-GlcNAc sites. In peptide-centric MS analysis, the O-GlcNAc moiety is known to be easily lost. In contrast, we demonstrate that the O-GlcNAc is retained under native MS conditions, enabling precise quantitative analysis of stoichiometry and O-GlcNAcylation kinetics. Together, the data highlight that high resolution native MS may provide an alternative tool to monitor kinetics on one of the most labile of protein post-translational modifications, in an efficient, reliable, and quantitative manner.

Protein O-GlcNAcylation: emerging mechanisms and functions.

O-GlcNAcylation - the attachment of O-linked N-acetylglucosamine (O-GlcNAc) moieties to cytoplasmic, nuclear and mitochondrial proteins - is a post-translational modification that regulates fundamental cellular processes in metazoans. A single pair of enzymes - O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) - controls the dynamic cycling of this protein modification in a nutrient- and stress-responsive manner. Recent years have seen remarkable advances in our understanding of O-GlcNAcylation at levels that range from structural and molecular biology to cell signalling and gene regulation to physiology and disease. New mechanisms and functions of O-GlcNAcylation that are emerging from these recent developments enable us to begin constructing a unified conceptual framework through which the significance of this modification in cellular and organismal physiology can be understood.

Yeast cells as an assay system for in vivo O-GlcNAc modification

BackgroundO-GlcNAcylation is a reversible protein post-translational modification, where O-GlcNAc moiety is attached to nucleocytoplasmic protein by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA). Although O-GlcNAc modification widely occurs in eukaryotic cells, the budding yeast Saccharomyces cerevisiae notably lacks this protein modification and the genes for the GlcNAc transferase and hydrolase. MethodsHuman OGT isoforms and OGA were ectopically expressed in S. cerevisiae, and the effects of their expressions on yeast growth and O-GlcNAc modification levels were assessed. ResultsExpression of sOGT, in S. cerevisiae catalyzes the O-GlcNAc modification of proteins in vivo; conversely, the expression of OGA mediates the hydrolysis of these sugars. sOGT expression causes a severe growth defect in yeast cells, which is remediated by the co-expression of OGA. The direct analysis of yeast proteins demonstrates protein O-GlcNAcylation is dependent on sOGT expression; conversely, the hydrolysis of these sugar modifications is induced by co-expression of OGA. Protein O-GlcNAcylation and the growth defects of yeast cells are caused by the O-GlcNAc transferase activity because catalytically inactive sOGT does not exhibit toxicity in yeast cells. Expression of another OGT isoform, ncOGT, also results in a growth defect in yeast cells. However, its toxicity is largely attributed to the TPR domain rather than the O-GlcNAc transferase activity. ConclusionsO-GlcNAc cycling can occur in yeast cells, and OGT and OGA activities can be monitored via yeast growth. General significanceYeast cells may be used to assess OGT and OGA.

The Role of Stress-Induced O-GlcNAc Protein Modification in the Regulation of Membrane Transport.

O-linked N-acetylglucosamine (O-GlcNAc) is a posttranslational modification that is increasingly recognized as a signal transduction mechanism. Unlike other glycans, O-GlcNAc is a highly dynamic and reversible process that involves the addition and removal of a single N-acetylglucosamine molecule to Ser/Thr residues of proteins. UDP-GlcNAc—the direct substrate for O-GlcNAc modification—is controlled by the rate of cellular metabolism, and thus O-GlcNAc is dependent on substrate availability. Serving as a feedback mechanism, O-GlcNAc influences the regulation of insulin signaling and glucose transport. Besides nutrient sensing, O-GlcNAc was also implicated in the regulation of various physiological and pathophysiological processes. Due to improvements of mass spectrometry techniques, more than one thousand proteins were detected to carry the O-GlcNAc moiety; many of them are known to participate in the regulation of metabolites, ions, or protein transport across biological membranes. Recent studies also indicated that O-GlcNAc is involved in stress adaptation; overwhelming evidences suggest that O-GlcNAc levels increase upon stress. O-GlcNAc elevation is generally considered to be beneficial during stress, although the exact nature of its protective effect is not understood. In this review, we summarize the current data regarding the oxidative stress-related changes of O-GlcNAc levels and discuss the implications related to membrane trafficking.

Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.

Nucleocytoplasmic human O-GlcNAc transferase is sufficient for O-GlcNAcylation of mitochondrial proteins.

O-linked N-acetylglucosamine modification (O-GlcNAcylation) is a nutrient-dependent protein post-translational modification (PTM), dynamically and reversibly driven by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) that catalyse the addition and the removal of the O-GlcNAc moieties to/from serine and threonine residues of target proteins respectively. Increasing evidence suggests involvement of O-GlcNAcylation in many biological processes, including transcription, signalling, neuronal development and mitochondrial function. The presence of a mitochondrial O-GlcNAc proteome and a mitochondrial OGT (mOGT) isoform has been reported. We explored the presence of mOGT in human cell lines and mouse tissues. Surprisingly, analysis of genomic sequences indicates that this isoform cannot be expressed in most of the species analysed, except some primates. In addition, we were not able to detect endogenous mOGT in a range of human cell lines. Knockdown experiments and Western blot analysis of all the predicted OGT isoforms suggested the expression of only a single OGT isoform. In agreement with this, we demonstrate that overexpression of the nucleocytoplasmic OGT (ncOGT) isoform leads to increased O-GlcNAcylation of mitochondrial proteins, suggesting that ncOGT is necessary and sufficient for the generation of the O-GlcNAc mitochondrial proteome.

O-GlcNAcylation is associated with the development and progression of gastric carcinoma

IntroductionO-GlcNAcylation occurs via an O-linked β-N-acetylglucosamine (O-GlcNAc) moiety linked to the side chain hydroxyl of a serine or threonine residue on nucleocytoplasmic proteins. This reaction, which is catalyzed by O-GlcNAc-transferase (OGT), is involved in a variety of human cancers; however, its clinical significance in gastric carcinomas (GC) has been poorly investigated in vivo. Materials and methodsImmunohistochemical staining for O-GlcNAcylation and OGT was performed in 64 primary GCs, 40 gastric adenomas and nonneoplastic tissues adjacent to GCs, including 31 tissues of intestinal metaplasia and 24 normal gastric tissues. Their expressions were also studied in 20 tissues of chronic gastritis according to Helicobacter pylori (H. pylori) infection. ResultsO-GlcNAcylation was expressed in the nucleus and both the nuclear rim and cytoplasm. OGT was strongly expressed in the nucleus and weakly expressed in the cytoplasm. O-GlcNAcylation expression levels were significantly correlated with those of OGT. Their expression levels were progressively increased during the carcinogenesis of GC. O-GlcNAcylation expression was higher in GC with intestinal type, higher pT stage and nodal metastasis, while OGT expression was higher in GC with nodal metastasis. Nuclear O-GlcNAcylation expression was more frequently observed in tumors including GC and adenoma than in nonneoplastic tissues including intestinal metaplasia and normal tissue. Nuclear O-GlcNAcylation expression in GC was closely associated with large size, moderate and poor differentiation, higher pT stage, nodal metastasis and higher clinical stage. In addition, the expression of O-GlcNAcylation and OGT was more elevated in H. pylori-infected chronic gastritis than in chronic gastritis without H. pylori infection. ConclusionsO-GlcNAcylation expression and its nuclear expression were associated with the carcinogenesis and progression of GC.