O-GlcNAc glycosylation stoichiometry of the FET protein family: only EWS is glycosylated with a high stoichiometry

Of the FET (fused in sarcoma [FUS]/Ewing sarcoma protein [EWS]/TATA binding protein-associated factor 15 [TAF15]) family of heterogeneous nuclear ribonucleoprotein particle proteins, FUS and TAF15 are consistently and EWS variably found in inclusion bodies in neurodegenerative diseases such as frontotemporal lobar degeneration associated with FUS. It is speculated that dysregulation of FET proteins at the post-translational level is involved in their cytoplasmic deposition. Here, the O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation stoichiometry of the FET proteins was chemoenzymatically analyzed, and it was found that only EWS is dynamically glycosylated with a high stoichiometry in the neural cell lines tested and in mouse brain. It was also confirmed that EWS, but not FUS and TAF15, is glycosylated with a high stoichiometry not only in the neural cells but also in the non-neural cell lines tested. These results indicate that O-GlcNAc glycosylation imparts a physicochemical property on EWS that is distinct from that of the other FET proteins in most of cell lineages or tissues.     

Adipogenesis stimulates the nuclear localization of EWS with an increase in its O-GlcNAc glycosylation in 3T3-L1 cells

Although the Ewing sarcoma (EWS) proto-oncoprotein is found in the nucleus and cytosol and is associated with the cell membrane, the regulatory mechanisms of its subcellular localization are still unclear. Here we found that adipogenic stimuli induce the nuclear localization of EWS in 3T3-L1 cells. Tyrosine phosphorylation in the C-terminal PY-nuclear localization signal of EWS was negative throughout adipogenesis. Instead, an adipogenesis-dependent increase in O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation of EWS was observed. Pharmacological inactivation of O-GlcNAcase in preadipocytes promoted perinuclear localization of EWS. Our findings suggest that the nuclear localization of EWS is partly regulated by the glycosylation.     

Does O-GlcNAc play a role in neurodegenerative diseases?

There are several lines of evidence that the modification of proteins by cytosolic- and nuclear-specific O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is closely related to neuropathologies, particularly Alzheimer’s disease. Several neuronal proteins have been identified as being modified with O-GlcNAc; these proteins could form part of the inclusion bodies found, for example, in the most frequently observed neurologic disorder (i.e., Alzheimer’s disease; Tau protein and β-amyloid peptide are the well known aggregated proteins). O-GlcNAc proteins are also implicated in synaptosomal transport (e.g., synapsins and clathrin-assembly proteins). Inclusion bodies are partly characterized by a deficiency in the ubiquitin–proteasome system, avoiding the degradation of aggregated proteins. From this perspective, it appears interesting that substrate proteins could be protected against proteasomal degradation by being covalently modified with single N-acetylglucosamine on serine or threonine, and that the proteasome itself is modified and regulated by O-GlcNAc (in this case the turnover of neuronal proteins correlates with extracellular glucose). Interestingly, glucose uptake and metabolism are impaired in neuronal disorders, and this phenomenon is linked to increased phosphorylation. In view of the existence of the dynamic interplay between O-GlcNAc and phosphorylation, it is tempting to draw a parallel between the use of glucose, O-GlcNAc glycosylation and phosphorylation. Lastly, the two enzymes responsible for O-GlcNAc dynamism (i.e., O-GlcNAc transferase and glucosaminidase) are both enriched in the brain and genes that encode the two enzymes are located in two regions that are found to be frequently mutated in neurologic disorders. The data presented in this review strongly suggest that O-GlcNAc could play an active role in neurodegenerative diseases.     

Survey of <Emphasis Type="Italic">O</Emphasis>-GlcNAc level variations in <Emphasis Type="Italic">Xenopus laevis</Emphasis> from oogenesis to early development

Little is known about the impact of O-linked-N-acetylglucosaminylation (O-GlcNAc) in gametes production and developmental processes. Here we investigated changes in O-GlcNAc, UDP-GlcNAc and O-GlcNAc transferase (OGT) levels in Xenopus laevis from oogenesis to embryo hatching. We showed that in comparison to stage VI, stages I–V oocytes expressed higher levels of O-GlcNAc correlating changes in OGT expression, but not in UDP-GlcNAc pools. Upon progesterone stimulation, an O-GlcNAc level burst occurred during meiotic resumption long before MPF and Mos-Erk2 pathways activations. Finally, we observed high levels of O-GlcNAc, UDP-GlcNAc and OGT during segmentation that decreased concomitantly at the onset of gastrulation. Nevertheless, no correlation between the glycosylation, the nucleotide-sugar and the glycosyltransferase was observed after neurulation. Our results show that O-GlcNAc is regulated throughout oogenesis and development within a complex pattern and suggest that dysfunctions in the dynamics of this glycosylation could lead to developmental abnormalities.     

Chemical tools to explore nutrient-driven O-GlcNAc cycling

Abstract

Posttranslational modifications (PTM) including glycosylation, phosphorylation, acetylation, methylation and ubiquitination dynamically alter the proteome. The evolutionarily conserved enzymes O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and O-GlcNAcase are responsible for the addition and removal, respectively, of the nutrient-sensitive PTM of protein serine and threonine residues with O-GlcNAc. Indeed, the O-GlcNAc modification acts at every step in the “central dogma” of molecular biology and alters signaling pathways leading to amplified or blunted biological responses. The cellular roles of OGT and the dynamic PTM O-GlcNAc have been clarified with recently developed chemical tools including high-throughput assays, structural and mechanistic studies and potent enzyme inhibitors. These evolving chemical tools complement genetic and biochemical approaches for exposing the underlying biological information conferred by O-GlcNAc cycling.     

Substrate and product analogues as human <Emphasis Type="Italic">O</Emphasis>-GlcNAc transferase inhibitors

Protein glycosylation on serine/threonine residues with N-acetylglucosamine (O-GlcNAc) is a dynamic, inducible and abundant post-translational modification. It is thought to regulate many cellular processes and there are examples of interplay between O-GlcNAc and protein phosphorylation. In metazoa, a single, highly conserved and essential gene encodes the O-GlcNAc transferase (OGT) that transfers GlcNAc onto substrate proteins using UDP–GlcNAc as the sugar donor. Specific inhibitors of human OGT would be useful tools to probe the role of this post-translational modification in regulating processes in the living cell. Here, we describe the synthesis of novel UDP–GlcNAc/UDP analogues and evaluate their inhibitory properties and structural binding modes in vitro alongside alloxan, a previously reported weak OGT inhibitor. While the novel analogues are not active on living cells, they inhibit the enzyme in the micromolar range and together with the structural data provide useful templates for further optimisation.     

Human Alzheimer’s disease synaptic <Emphasis Type="Italic">O</Emphasis>-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry

Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer’s disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.     

Increasing O-GlcNAc levels: An overview of small-molecule inhibitors of O-GlcNAcase

The O-GlcNAc modification is found on many nucleocytoplasmic proteins. The dynamic nature of O-GlcNAc, which in some ways is reminiscent of phosphorylation, has enabled investigators to modulate the stoichiometry of O-GlcNAc on proteins in order to study its function. Although several genetic and pharmacological methods for manipulating O-GlcNAc levels have been described, one of the most direct approaches of increasing global O-GlcNAc levels is by using small-molecule inhibitors of O-GlcNAcase (OGA). As the interest in increasing O-GlcNAc levels has grown, so too has the number of OGA inhibitors. This review provides an overview of the available methods of increasing O-GlcNAc levels, with a special emphasis on inhibition of OGA by small molecules. Known inhibitors of OGA are discussed with particular attention on those most suitable for cell-based biological studies. Several examples in which OGA inhibitors have been used to study the functional role of the O-GlcNAc modification in biological systems are discussed, highlighting the pros and cons of different inhibitors.     

Ac4GlcNAcF3, an OGT-tolerated but OGA-resistant regulator for O-GlcNAcylation

O-Linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational monosaccaride-modification found on Ser or Thr residues of intracellular proteins in most eukaryotes. The dynamic nature of O-GlcNAc has enabled researchers to modulate the stoichiometry of O-GlcNAc on proteins in order to investigate its function. Cell permeable small moleculars have proven invaluable tools to increase O-GlcNAc levels. Herein, using in vitro substrate screening, we identified GlcNAcF₃ as an OGT-accepted but OGA-resistant sugar mimic. Cellular experiments with cell-permeable peracetylated-GlcNAcF₃ (Ac₄GlcNAcF₃) displayed that Ac₄GlcNAcF₃ was a potent tool to increase O-GlcNAc levels in several cell lines. Further, NIH3T3 cells interfered with OGT (siOGT) showed significant decreasing of O-GlcNAc levels with Ac₄GlcNAcF₃ treatment, indicating O-GlcNAcF₃ was an OGT-dependent modification. In addition, cellular toxic assay confirmed O-GlcNAcF₃ production has no significant effect on cell proliferation or viability. Thus, Ac₄GlcNAcF₃ represents a safe and dual regulator for both OGT and OGA, which will benefit the study of O-GlcNAc.     

Monitoring protein O-linked β-N-acetylglucosamine status via metabolic labeling and copper-free click chemistry

O-Linked β-N-acetylglucosamine (O-GlcNAc) modification found on the serine and threonine residues of intracellular proteins is an inducible post-translational modification that regulates numerous biological processes. In combination with other cell biological and biochemical approaches, a robust and streamlined strategy for detecting the number and stoichiometry of O-GlcNAc modification can provide valuable insights for decoding the functions of O-GlcNAc at the molecular level. Here, we report an optimized workflow for evaluating the O-GlcNAc status of proteins using a combination of metabolic labeling and click chemistry-based mass tagging. This method is strategically complementary to the chemoenzymatic-based mass-tagging method.     

Mapping <Emphasis Type="Italic">O</Emphasis>-GlcNAc modification sites on tau and generation of a site-specific <Emphasis Type="Italic">O</Emphasis>-GlcNAc tau antibody

The microtubule-associated protein tau is known to be post-translationally modified by the addition of N-acetyl-d-glucosamine monosaccharides to certain serine and threonine residues. These O-GlcNAc modification sites on tau have been challenging to identify due to the inherent complexity of tau from mammalian brains and the fact that the O-GlcNAc modification typically has substoichiometric occupancy. Here, we describe a method for the production of recombinant O-GlcNAc modified tau and, using this tau, we have mapped sites of O-GlcNAc on tau at Thr-123 and Ser-400 using mass spectrometry. We have also detected the presence of a third O-GlcNAc site on either Ser-409, Ser-412, or Ser-413. Using this information we have raised a rabbit polyclonal IgG antibody (3925) that detects tau O-GlcNAc modified at Ser-400. Further, using this antibody we have detected the Ser-400 tau O-GlcNAc modification in rat brain, which confirms the validity of this in vitro mapping approach. The identification of these O-GlcNAc sites on tau and this antibody will enable both in vivo and in vitro experiments designed to understand the possible functional roles of O-GlcNAc on tau.     

The O-GlcNAc Modification of CDK5 Involved in Neuronal Apoptosis Following In Vitro Intracerebral Hemorrhage

Contrary to cell cycle-associated cyclin-dependent kinases, CDK5 is best known for its regulation of signaling processes in regulating mammalian CNS development. Studies of CDK5 have focused on its phosphorylation, although the diversity of CDK5 functions in the brain suggests additional forms of regulation. Here we expanded on the functional roles of CDK5 glycosylation in neurons. We showed that CDK5 was dynamically modified with O-GlcNAc in response to neuronal activity and that glycosylation represses CDK5-dependent apoptosis by impairing its association with p53 pathway. Blocking glycosylation of CDK5 alters cellular function and increases neuronal apoptosis in the cell model of the ICH. Our findings demonstrated a new role for O-glycosylation in neuronal apoptosis and provided a mechanistic understanding of how glycosylation contributes to critical neuronal functions. Moreover, we identified a previously unknown mechanism for the regulation of activity-dependent gene expression, neural development, and apoptosis.     

The dynamic stress-induced “<Emphasis Type="Italic">O</Emphasis>-GlcNAc-ome” highlights functions for <Emphasis Type="Italic">O</Emphasis>-GlcNAc in regulating DNA damage/repair and other cellular pathways

The modification of nuclear, mitochondrial, and cytoplasmic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic and essential post-translational modification of metazoans. Numerous forms of cellular injury lead to elevated levels of O-GlcNAc in both in vivo and in vitro models, and elevation of O-GlcNAc levels before, or immediately after, the induction of cellular injury is protective in models of heat stress, oxidative stress, endoplasmic reticulum (ER) stress, hypoxia, ischemia reperfusion injury, and trauma hemorrhage. Together, these data suggest that O-GlcNAc is a regulator of the cellular stress response. However, the molecular mechanism(s) by which O-GlcNAc regulates protein function leading to enhanced cell survival have not been identified. In order to determine how O-GlcNAc modulates stress tolerance in these models we have used stable isotope labeling with amino acids in cell culture to determine the identity of proteins that undergo O-GlcNAcylation in response to heat shock. Numerous proteins with diverse functions were identified, including NF-90, RuvB-like 1 (Tip49α), RuvB-like 2 (Tip49β), and several COPII vesicle transport proteins. Many of these proteins bind double-stranded DNA-dependent protein kinase (PK), or double-stranded DNA breaks, suggesting a role for O-GlcNAc in regulating DNA damage signaling or repair. Supporting this hypothesis, we have shown that DNA-PK is O-GlcNAc modified in response to numerous forms of cellular stress.     

<Emphasis Type="Italic">O</Emphasis>-GlcNAc modification of the anti-malarial vaccine candidate <Emphasis Type="Italic">Pf</Emphasis>AMA1: <Emphasis Type="Italic">in silico</Emphasis>-defined structural changes and potential to generate a better vaccine

The complex life cycle of plasmodial parasites makes the selection of a single subunit protein a less than optimal strategy to generate an efficient vaccinal protection against malaria. Moreover, the full protection afforded by malarial proteins carried by intact parasites implies that immune responses against different antigens expressed in different phases of the cycle are required, but also suggests that native malarial antigens are presented to the host immune system in a manner that recombinant proteins do not achieve. The malarial apical membrane antigen 1 (AMA1) represents a suitable vaccine candidate because AMA1 is expressed on sporozoites and merozoites and allows them to invade hepatocytes and erythrocytes, respectively. Anti-AMA1 antibodies and cytotoxic T-cells are therefore expected to interfere both with the primary invasion of hepatocytes by sporozoites and with the later propagation of merozoites in erythrocytes, and thus efficiently counteract parasite development in its human host. AMA1 bears potential glycosylation sites and the human erythrocytic O-linked N-acetylglucosamine transferase (OGT) could glycosylate AMA1 through combinatorial metabolism. This hypothesis was tested in silico by developing binding models of AMA1 with human OGT complexed with UDP-GlcNc, and followed by the binding of O-GlcNAc with the hydroxyl group of AMA1 serine and threonine residues. Our results suggests that AMA1 shows potential for glycosylation at Thr517 and Ser498 and that O-GlcNAc AMA1 may constitute a conformationally more appropriate antigen for developing a protective anti-malarial immune response.     

Nutrient-responsive O-GlcNAcylation dynamically modulates the secretion of glycan-binding protein galectin 3

Endomembrane glycosylation and cytoplasmic O-GlcNAcylation each play essential roles in nutrient sensing, and characteristic changes in glycan patterns have been described in disease states such as diabetes and cancer. These changes in glycosylation have important functional roles and can drive disease progression. However, little is known about the molecular mechanisms underlying how these signals are integrated and transduced into biological effects. Galectins are proteins that bind glycans and that are secreted by a poorly characterized nonclassical secretory mechanism. Once outside the cell, galectins bind to the terminal galactose residues of cell surface glycans and modulate numerous extracellular functions, such as clathrin-independent endocytosis (CIE). Originating in the cytoplasm, galectins are predicted substrates for O-GlcNAc addition and removal; and as we have shown, galectin 3 is a substrate for O-GlcNAc transferase. In this study, we also show that galectin 3 secretion is sensitive to changes in O-GlcNAc levels. We determined using immunoprecipitation and Western blotting that there is a significant difference in O-GlcNAcylation status between cytoplasmic and secreted galectin 3. We observed dramatic alterations in galectin 3 secretion in response to nutrient conditions, which were dependent on dynamic O-GlcNAcylation. Importantly, we showed that these O-GlcNAc-driven alterations in galectin 3 secretion also facilitated changes in CIE. These results indicate that dynamic O-GlcNAcylation of galectin 3 plays a role in modulating its secretion and can tune its function in transducing nutrient-sensing information coded in cell surface glycosylation into biological effects.     

Nutrient-driven O-GlcNAc cycling influences autophagic flux and neurodegenerative proteotoxicity

O-GlcNAcylation is an abundant post-translational modification implicated in human neurodegenerative diseases. We showed that loss-of-function of OGT (O-linked GlcNAc transferase) alleviated, while loss of OGA (O-GlcNAc selective β-N-acetyl-D-glucosaminidase) enhanced, the proteotoxicity of C. elegans neurodegenerative disease models including tauopathy, β-amyloid peptide and polyglutamine expansion. The O-GlcNAc cycling mutants act, in part, by altering insulin signaling, proteasome activity and autophagy. In mutants lacking either of these enzymes of O-GlcNAc cycling, there is a striking accumulation of GFP::LGG-1 (C. elegans homolog of Atg8 and LC3) and increased phosphatidylethanolamine (PE)-modified GFP::LGG-1 upon starvation. We speculate that O-GlcNAc cycling is a key nutrient-responsive regulator of autophagic flux acting at multiple levels including direct modification of BECN1 and BCL2.