Changes In O-GlcNAcylation Research Articles

Diet-inducing hypercholesterolemia show decreased O-GlcNAcylation of liver proteins through modulation of AMPK.

O-GlcNAcylation, a nutritionally driven, post-translational modification of proteins, is gaining importance because of its health implications. Changes in O-GlcNAcylation are observed in various disease conditions. Changes in O-GlcNAcylation by diet that causes hypercholesterolemia are not critically looked into in the liver. To address it, both in vitro and in vivo approaches were employed. Hypercholesterolemia was induced individually by feeding cholesterol (H)/high-fat (HF) diet. Global O-GlcNAcylation levels and modulation of AMPK activation in both preventive and curative approaches were looked into. Diet-induced hypercholesterolemia resulted in decreased O-GlcNAcylation of liver proteins which was associated with decreased O-linked N-acetylglucosaminyltransferase (OGT) and Glutamine fructose-6-phosphate amidotransferase-1 (GFAT1). Activation of AMPK by metformin in preventive mode restored the O-GlcNAcylation levels; however, metformin treatment of HepG2 cells in curative mode restored O-GlcNAcylation levels in HF but failed to in H condition (at 24 h). Further, maternal faulty diet resulted in decreased O-GlcNAcylation in pup liver despite feeding normal diet till adulthood. A faulty diet modulates global O-GlcNAcylation of liver proteins which is accompanied by decreased AMPK activation which could exacerbate metabolic syndromes through fat accumulation in the liver.

Best practices in assessing cardiac protein O-GlcNAcylation by immunoblot.

The modification of serine and threonine amino acids of proteins by O-linked N-acetylglucosamine (O-GlcNAc) regulates the activity, stability, function, and subcellular localization of proteins. Dysregulation of O-GlcNAc homeostasis is well established as a hallmark of various cardiac diseases, including cardiac hypertrophy, heart failure, complications associated with diabetes, and responses to acute injuries such as oxidative stress and ischemia-reperfusion. Given the limited availability of site-specific O-GlcNAc antibodies, studies of changes in O-GlcNAcylation in the heart frequently use pan-O-GlcNAc antibodies for semiquantitative evaluation of overall O-GlcNAc levels. However, there is a high degree of variability in many published cardiac O-GlcNAc blots. For example, many blots often have regions that lack O-GlcNAc positive staining of proteins either below 50 or above 100 kDa. In some O-GlcNAc blots, only a few protein bands are detected, while in others, intense bands around 75 kDa dominate the gel due to nonspecific IgM band staining, making it difficult to visualize less intense bands. Therefore, the goal of this study was to develop a modifiable protocol that optimizes O-GlcNAc positive banding of proteins in cardiac tissue extracts. We showed that O-GlcNAc blots using CTD110.6 antibody of proteins ranging from <30 to ∼450 kDa could be obtained while also limiting nonspecific staining. We also show that some myofilament proteins are recognized by the CTD110.6 antibody. Therefore, by protocol optimization using the widely available CTD110.6 antibody, we found that it is possible to obtain pan-O-GlcNAc blots of cardiac tissue, which minimizes common limitations associated with this technique.NEW & NOTEWORTHY The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is recognized as mediating cardiac pathophysiology. However, there is considerable variability in the quality of O-GlcNAc immunoblots used to evaluate changes in cardiac O-GlcNAc levels. Here we show that with relatively minor changes to a commonly used protocol it is possible to minimize the intensity of nonspecific bands while also reproducibly generating O-GlcNAc immunoblots covering a range of molecular weights from <30 to ∼450 kDa.

Differential Detection of O-GlcNAcylated proteins in the heart using antibodies

Thousands of mammalian intracellular proteins are dynamically modified by O-linked β−N-acetylglucosamine (O-GlcNAc). Global changes in O-GlcNAcylation have been associated with the development of cardiomyopathy, heart failure, hypertension, and neurodegenerative disease. Levels of O-GlcNAc in cells and tissues can be detected using numerous approaches; however, immunoblotting using GlcNAc-specific antibodies and lectins is commonplace. The goal of this study was to optimize the detection of O-GlcNAc in heart lysates by immunoblotting. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, as well as hearts from wild-type and O-GlcNAc transferase transgenic mice, we demonstrate that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, and as these proteins represent a major fraction of the heart proteome, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The data presented highlight tissue lysis approaches that should aid the assessment of the cardiac O-GlcNAcylation by immunoblotting.

IP-LC-MSMS Enables Identification of Three Tau O-GlcNAcylation Sites as O-GlcNAcase Inhibition Pharmacodynamic Readout in Transgenic Mice Overexpressing Human Tau.

O-β-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates tau phosphorylation and aggregation: the pharmacological increase of tau O-GlcNAcylation upon treatment with inhibitors of O-GlcNAc hydrolase (OGA) constitutes a potential strategy to tackle neurodegenerative diseases. Analysis of tau O-GlcNAcylation could potentially be used as a pharmacodynamic biomarker both in preclinical and clinical studies. The goal of the current study was to confirm tau O-GlcNAcylation at S400 as a pharmacodynamic readout of OGA inhibition in P301S transgenic mice overexpressing human tau and treated with the OGA inhibitor Thiamet G and to explore if additional O-GlcNAcylation sites on tau could be identified. As a first step, an immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) methodology was developed to monitor changes in O-GlcNAcylation around S400 of tau in mouse brain homogenate (BH) extracts. Second, additional O-GlcNAc sites were identified in in-house produced recombinant O-GlcNAcylated human tau at relatively high concentrations, thereby facilitating collection of informative LC-MS data for identification of low-concentration O-GlcNAc-tryptic tau peptides in human transgenic mouse BH extracts. This strategy enabled, for the first time, identification of three low abundant N-terminal and mid-domain O-GlcNAc sites of tau (at S208, S191, and S184 or S185) in human transgenic mouse BH. Data are openly available at data.mendeley.com (doi: 10.17632/jp57yk9469.1; doi: 10.17632/8n5j45dnd8.1; doi: 10.17632/h5vdrx4n3d.1).

Recent development of analytical methods for disease-specific protein O-GlcNAcylation.

The enzymatic modification of protein serine or threonine residues by N-acetylglucosamine, namely O-GlcNAcylation, is a ubiquitous post-translational modification that frequently occurs in the nucleus and cytoplasm. O-GlcNAcylation is dynamically regulated by two enzymes, O-GlcNAc transferase and O-GlcNAcase, and regulates nearly all cellular processes in epigenetics, transcription, translation, cell division, metabolism, signal transduction and stress. Aberrant O-GlcNAcylation has been shown in a variety of diseases, including diabetes, neurodegenerative diseases and cancers. Deciphering O-GlcNAcylation remains a challenge due to its low abundance, low stoichiometry and extreme lability in most tandem mass spectrometry. Separation or enrichment of O-GlcNAc proteins or peptides from complex mixtures has been of great interest because quantitative analysis of protein O-GlcNAcylation can elucidate their functions and regulatory mechanisms in disease. However, valid and specific analytical methods are still lacking, and efforts are needed to further advance this direction. Here, we provide an overview of recent advances in various analytical methods, focusing on chemical oxidation, affinity of antibodies and lectins, hydrophilic interaction, and enzymatic addition of monosaccharides in conjugation with these methods. O-GlcNAcylation quantification has been described in detail using mass-spectrometric or non-mass-spectrometric techniques. We briefly summarized dysregulated changes in O-GlcNAcylation in disease.

Integration of O-GlcNAc into Stress Response Pathways.

The modification of nuclear, mitochondrial, and cytosolic proteins by O-linked βN-acetylglucosamine (O-GlcNAc) has emerged as a dynamic and essential post-translational modification of mammalian proteins. O-GlcNAc is cycled on and off over 5000 proteins in response to diverse stimuli impacting protein function and, in turn, epigenetics and transcription, translation and proteostasis, metabolism, cell structure, and signal transduction. Environmental and physiological injury lead to complex changes in O-GlcNAcylation that impact cell and tissue survival in models of heat shock, osmotic stress, oxidative stress, and hypoxia/reoxygenation injury, as well as ischemic reperfusion injury. Numerous mechanisms that appear to underpin O-GlcNAc-mediated survival include changes in chaperone levels, impacts on the unfolded protein response and integrated stress response, improvements in mitochondrial function, and reduced protein aggregation. Here, we discuss the points at which O-GlcNAc is integrated into the cellular stress response, focusing on the roles it plays in the cardiovascular system and in neurodegeneration.

Dynamic changes in O-GlcNAcylation regulate osteoclast differentiation and bone loss via nucleoporin 153

Bone mass is maintained by the balance between osteoclast-induced bone resorption and osteoblast-triggered bone formation. In inflammatory arthritis such as rheumatoid arthritis (RA), however, increased osteoclast differentiation and activity skew this balance resulting in progressive bone loss. O-GlcNAcylation is a posttranslational modification with attachment of a single O-linked β-D-N-acetylglucosamine (O-GlcNAc) residue to serine or threonine residues of target proteins. Although O-GlcNAcylation is one of the most common protein modifications, its role in bone homeostasis has not been systematically investigated. We demonstrate that dynamic changes in O-GlcNAcylation are required for osteoclastogenesis. Increased O-GlcNAcylation promotes osteoclast differentiation during the early stages, whereas its downregulation is required for osteoclast maturation. At the molecular level, O-GlcNAcylation affects several pathways including oxidative phosphorylation and cell-cell fusion. TNFα fosters the dynamic regulation of O-GlcNAcylation to promote osteoclastogenesis in inflammatory arthritis. Targeted pharmaceutical or genetic inhibition of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) arrests osteoclast differentiation during early stages of differentiation and during later maturation, respectively, and ameliorates bone loss in experimental arthritis. Knockdown of NUP153, an O-GlcNAcylation target, has similar effects as OGT inhibition and inhibits osteoclastogenesis. These findings highlight an important role of O-GlcNAcylation in osteoclastogenesis and may offer the potential to therapeutically interfere with pathologic bone resorption.

Dynamic changes in O-GlcNAcylation regulate osteoclast differentiation and bone loss via nucleoporin 153

Dynamic changes in O-GlcNAcylation regulate osteoclast differentiation and bone loss via nucleoporin 153

Dynamic changes in O-GlcNAcylation regulate osteoclast differentiation and bone loss via nucleoporin 153

Dynamic changes in O-GlcNAcylation regulate osteoclast differentiation and bone loss via nucleoporin 153

Glycosylation Changes in Prostate Cancer Progression.

Prostate Cancer (PCa) is the most commonly diagnosed malignancy and second leading cause of cancer-related mortality in men. With the use of next generation sequencing and proteomic platforms, new biomarkers are constantly being developed to both improve diagnostic sensitivity and specificity and help stratify patients into different risk groups for optimal management. In recent years, it has become well accepted that altered glycosylation is a hallmark of cancer progression and that the glycan structures resulting from these mechanisms show tremendous promise as both diagnostic and prognostic biomarkers. In PCa, a wide range of structural alterations to glycans have been reported such as variations in sialylation and fucosylation, changes in branching, altered levels of Lewis and sialyl Lewis antigens, as well as the emergence of high mannose “cryptic” structures, which may be immunogenic and therapeutically relevant. Furthermore, aberrant expression of galectins, glycolipids, and proteoglycans have also been reported and associated with PCa cell survival and metastasis. In this review, we discuss the findings from various studies that have explored altered N- and O-linked glycosylation in PCa tissue and body fluids. We further discuss changes in O-GlcNAcylation as well as altered expression of galectins and glycoconjugates and their effects on PCa progression. Finally, we emphasize the clinical utility and potential impact of exploiting glycans as both biomarkers and therapeutic targets to improve our ability to diagnose clinically relevant tumors as well as expand treatment options for patients with advanced disease.

Changes in O‐GlcNAcylation Alter Mitochondrial Function

Mitochondrial dysfunction is a common characteristic of many diseases, such as Alzheimer's disease, diabetes, and cancer. Normal mitochondrial and cellular function is regulated by post-translational modifications (PTM) of proteins, which alter protein function to adapt to changes in the cellular environment. One PTM that plays a key role in protein function is the attachment of a single N-acetylglucosamine (O-GlcNAc) sugar to nuclear, mitochondrial, or cytoplasmic proteins at their serine or threonine residues. This process, known as O-GlcNAcylation, is catalyzed by only two enzymes: O-GlcNAc transferase (OGT), which attaches the sugar, and O-GlcNAcase (OGA), which removes the sugar. O-GlcNAcylation is sensitive to changes in various metabolic pathways, such as the metabolism of glucose, fatty acids, and amino acids. Thus, this modification is necessary for maintaining cellular homeostasis. Our previous data showed that changes in O-GlcNAcylation, such as loss of OGT or inhibition of OGA via thiamet-G (TMG) treatment in the liver, showed disruption of the electron transport chain (ETC); however, we still do not know which complexes are most affected by these changes. One potential explanation lies in the relationship between O-GlcNAcylation and acetylation, as acetylation is a highly abundant mitochondrial PTM responsible for the regulation of many components of the ETC. A variety of cell line knockouts and animal models were studied to more fully understand the role of O-GlcNAcylation in regulating mitochondrial function and acetylation. We generated a stable cell line in SH-SY5Y human neuroblastoma cells with shRNA mediated knockdown of OGT. These cells demonstrated an increase in acetylation in the mitochondria as a result of the loss of OGT. Further analysis also showed changes in the expressions of two proteins that are critical for mitochondrial acetylation: acetyl-coenzyme A synthetase 2-like (ACSS1) and NAD-dependent deacetylase sirtuin-3 (SIRT3). Taken together, these findings suggest that reduced O-GlcNAcylation alters the mitochondrial proteome including proteins that are regulated via acetylation. We also observed that oxidative phosphorylation rates were altered in the OGT knockdown cell lines, demonstrating alterations in mitochondrial function due to the reduction of OGT. To further elucidate the role of O-GlcNAcylation in the electron transport chain, ETC complex assays were run to assess changes in complex function due to different levels of O-GlcNAcylation. SH-SY5Y cells treated with and without TMG expressed different levels of complex activity, providing key insights about the importance of O-GlcNAcylation in the ETC. Based on these findings, more studies are needed to understand how O-GlcNAcylation and acetylation work in tandem to maintain electron transport chain function.

Exploring the Potential of β-Catenin O-GlcNAcylation by Using Fluorescence-Based Engineering and Imaging.

Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator β-catenin in its O-GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of β-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the O-GlcNAcylation status of β-catenin in HeLa cells. The changes in O-GlcNAcylation of β-catenin were varied by perturbing global cellular O-GlcNAc levels with the inhibitors of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.

O-GlcNAcylation and phosphorylation of β-actin Ser199 in diabetic nephropathy.

The function of actin is regulated by various posttranslational modifications. We have previously shown that in the kidneys of nonobese type 2 diabetes model Goto-Kakizaki rats, increased O-GlcNAcylation of β-actin protein is observed. It has also been reported that both O-GlcNAcylation and phosphorylation occur on Ser199 of β-actin. However, their roles are not known. To elucidate their roles in diabetic nephropathy, we examined the rat kidney for changes in O-GlcNAcylation of Ser199 (gS199)-actin and in the phosphorylation of Ser199 (pS199)-actin. Both gS199- and pS199-actin molecules had an apparent molecular weight of 40 kDa and were localized as nonfilamentous actin in both the cytoplasm and nucleus. Compared with the normal kidney, the immunostaining intensity of gS199-actin increased in podocytes of the glomeruli and in proximal tubules of the diabetic kidney, whereas that of pS199-actin did not change in podocytes but decreased in proximal tubules. We confirmed that the same results could be observed in the glomeruli of the human diabetic kidney. In podocytes of glomeruli cultured in the presence of the O-GlcNAcase inhibitor Thiamet G, increased O-GlcNAcylation was accompanied by a concomitant decrease in the amount of filamentous actin and in morphological changes. Our present results demonstrate that dysregulation of O-GlcNAcylation and phosphorylation of Ser199 occurred in diabetes, which may contribute partially to the causes of the morphological changes in the glomeruli and tubules. gS199- and pS199-actin will thus be useful for the pathological evaluation of diabetic nephropathy.

Acute increases in O-GlcNAc indirectly impair mitochondrial bioenergetics through dysregulation of LonP1-mediated mitochondrial protein complex turnover.

The attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) to the serine and threonine residues of proteins in distinct cellular compartments is increasingly recognized as an important mechanism regulating cellular function. Importantly, the O-GlcNAc modification of mitochondrial proteins has been identified as a potential mechanism to modulate metabolism under stress with both potentially beneficial and detrimental effects. This suggests that temporal and dose-dependent changes in O-GlcNAcylation may have different effects on mitochondrial function. In the current study, we found that acutely augmenting O-GlcNAc levels by inhibiting O-GlcNAcase with Thiamet-G for up to 6 h resulted in a time-dependent decrease in cellular bioenergetics and decreased mitochondrial complex I, II, and IV activities. Under these conditions, mitochondrial number was unchanged, whereas an increase in the protein levels of the subunits of several electron transport complex proteins was observed. However, the observed bioenergetic changes appeared not to be due to direct increased O-GlcNAc modification of complex subunit proteins. Increases in O-GlcNAc were also associated with an accumulation of mitochondrial ubiquitinated proteins; phosphatase and tensin homolog induced kinase 1 (PINK1) and p62 protein levels were also significantly increased. Interestingly, the increase in O-GlcNAc levels was associated with a decrease in the protein levels of the mitochondrial Lon protease homolog 1 (LonP1), which is known to target complex IV subunits and PINK1, in addition to other mitochondrial proteins. These data suggest that impaired bioenergetics associated with short-term increases in O-GlcNAc levels could be due to impaired, LonP1-dependent, mitochondrial complex protein turnover.

A genetic model to study O-GlcNAc cycling in immortalized mouse embryonic fibroblasts

O-GlcNAcylation is an abundant posttranslational protein modification in which the monosaccharide O-GlcNAc is added to Ser/Thr residues by O-GlcNAc transferase and removed by O-GlcNAcase. Analyses of O-GlcNAc-mediated signaling and metabolic phenomena are complicated by factors including unsatisfactory inhibitors and loss-of-function cell lines lacking identical genetic backgrounds. In this work, we generated immortalized WT, Oga knockout, and Ogt floxed allele (Ogt floxed) mouse embryonic fibroblast (MEF) cell lines with similar genetic backgrounds. These lines will facilitate experiments and serve as a platform to study O-GlcNAc cycling in mammals. As a test paradigm, we used the immortalized MEF lines to investigate how changes in O-GlcNAcylation affected pathological phosphorylation of the tau protein. The activity of glycogen synthase kinase 3β (GSK3β), a kinase that phosphorylates tau, decreases when expressed in Oga knockout MEFs compared with WT cells. Phosphorylation at Thr231 in recombinant, tauopathy-associated tau with a proline-to-leucine mutation at position 301 (P301L) was altered when expressed in MEFs with altered O-GlcNAc cycling. In aggregate, our data support that O-GlcNAc cycling indirectly affects tau phosphorylation at Thr231, but tau phosphorylation was highly variable, even in genetically stable, immortalized MEF cells. The variable nature of tau phosphorylation observed here supports the need to use cells akin to those generated here with genetically defined lesions and similar backgrounds to study complex biological processes.

O-GlcNAcylation of the Tumor Suppressor FOXO3 Triggers Aberrant Cancer Cell Growth.

Posttranslational modifications of tumor suppressors can induce abnormal cell growth. Here, we identify site-specific O-GlcNAcylation as a critical block of FOXO3 that may abrogate a part of the p53 pathway, resulting in aberrant cancer cell growth. Of seven O-GlcNAcylation sites identified within the FOXO3 transactivation domain, we found that changes in O-GlcNAcylation at Ser284 modulated p21-mediated cancer cell growth. Overexpression of either O-GlcNAcylated FOXO3 (FOX-OV) or a Ser-to-Ala mutant (S284A) in PANC-1 cells indicated that S284 O-GlcNAc acts as a critical block of the FOXO tumor suppressor and induces proliferation in PANC-1 cancer cells by stimulating the MDM2-p53-p21 axis. Furthermore, S284A mutant cells lacking S284 O-GlcNAc and FOX-OV cells exhibited opposing MDM2-p53-p21 axis expression patterns at both the mRNA and protein levels. Thus, our study provides evidence to support a role for S284 O-GlcNAc as a critical block of FOXO3 to induce subsequent cancer cell growth via abrogation of the p53 regulatory circuit.Significance: These findings highlight a posttranslational mechanism for indirect abrogation of the p53 pathway, one that may occur with some frequency in human cancer cells. Cancer Res; 78(5); 1214-24. ©2018 AACR.

Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells

O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked β-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma.

A mutant O-GlcNAcase as a probe to reveal global dynamics of protein O-GlcNAcylation during Drosophila embryonic development.

O-GlcNAcylation is a reversible type of serine/threonine glycosylation on nucleocytoplasmic proteins in metazoa. Various genetic approaches in several animal models have revealed that O-GlcNAcylation is essential for embryogenesis. However, the dynamic changes in global O-GlcNAcylation and the underlying mechanistic biology linking them to embryonic development is not understood. One of the limiting factors towards characterizing changes in O-GlcNAcylation has been the limited specificity of currently available tools to detect this modification. In the present study, harnessing the unusual properties of an O-GlcNAcase (OGA) mutant that binds O-GlcNAc (O-N-acetylglucosamine) sites with nanomolar affinity, we uncover changes in protein O-GlcNAcylation as a function of Drosophila development.

Disruption of O-GlcNAc Cycling in C. elegans Perturbs Nucleotide Sugar Pools and Complex Glycans.

The carbohydrate modification of serine and threonine residues with O-linked beta- N-acetylglucosamine (O-GlcNAc) is ubiquitous and governs cellular processes ranging from cell signaling to apoptosis. The O-GlcNAc modification along with other carbohydrate modifications, including N-linked and O-linked glycans, glycolipids, and sugar polymers, all require the use of the nucleotide sugar UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). In this paper, we describe the biochemical consequences resulting from perturbation of the O-GlcNAc pathway in C. elegans lacking O-GlcNAc transferase and O-GlcNAcase activities. In ogt-1 null animals, steady-state levels of UDP-GlcNAc/UDP-GalNAc and UDP-glucose were substantially elevated. Transcripts of genes encoding for key members in the HBP (gfat-2, gna-2, C36A4.4) and trehalose metabolism (tre-1, tre-2, tps-2) were elevated in ogt-1 null animals. While there is no evidence to suggest changes in the profile of N-linked glycans in the ogt-1 and oga-1 mutants, glycans insensitive to PNGase digestion (including O-linked glycans, glycolipids, and glycopolymers) were altered in these strains. Our data support that changes in O-GlcNAcylation alters nucleotide sugar production, overall glycan composition, and transcription of genes encoding glycan processing enzymes. These data along with our previous findings that disruption in O-GlcNAc cycling alters macronutrient storage underscores the noteworthy influence this posttranslational modification plays in nutrient sensing.

A role for O-GlcNAcylation in setting circadian clock speed

Post-translational modifications of one or more central "clock" proteins, most notably time-of-day-dependent changes in phosphorylation, are critical for setting the pace of circadian (≅24 h) clocks. In animals, PERIOD (PER) proteins are the key state variable regulating circadian clock speed and undergo daily changes in abundance and cytoplasmic-nuclear distribution that are partly driven by a complex phosphorylation program. Here, we identify O-GlcNAcylation (O-GlcNAc) as a critical post-translational modification in circadian regulation that also contributes to setting clock speed. Knockdown or overexpression of Drosophila O-GlcNAc transferase (ogt) in clock cells either shortens or lengthens circadian behavioral rhythms, respectively. The Drosophila PERIOD protein (dPER) is a direct target of OGT and undergoes daily changes in O-GlcNAcylation, a modification that is mainly observed during the first half of the night, when dPER is predominantly located in the cytoplasm. Intriguingly, the timing of when dPER translocates from the cytoplasm to the nucleus is advanced or delayed in flies, wherein ogt expression is reduced or increased, respectively. Our results suggest that O-GlcNAcylation of dPER contributes to setting the correct pace of the clock by delaying the timing of dPER nuclear entry. In addition, OGT stabilizes dPER, suggesting that O-GlcNAcylation has multiple roles in circadian timing systems.