Identification of a specific inhibitor of nOGA — a caspase-3 cleaved O-GlcNAcase variant during apoptosis

O-Linked N-acetylglucosamine (O-GlcNAc) modification of serines/threonines on cytoplasmic proteins is a significant signal regulating cellular processes such as cell cycle, cell development, and cell apoptosis. O-GlcNAcase (OGA) is responsible for the removal of O-GlcNAc, and it thus plays a critical role in O-GlcNAc metabolism. Interestingly, OGA can be cleaved by caspase-3 into two fragments during apoptosis, producing an N-terminal fragment (1–413 a.a.), termed nOGA. Here, using 4-MU-GlcNAc (4-methylumbelliferyl 2-acetamido-2-deoxy-β-D-glucopyranoside) as substrate, we found that the nOGA fragment retains high glycosidase activity. To probe the role of nOGA in apoptosis, it is essential to develop a potent and specific nOGA inhibitor. However, many reported inhibitors active at nanomolar concentrations (including PUGNAc, STZ, GlcNAc-statin, and NAG-thiazoline) against full-length OGA were not potent for nOGA. Next, we screened a small triazole-linked carbohydrate library and first identified compound 4 (4-pyridyl-1-(2′-deoxy-2′-acetamido-β-D-glucopyranosyl)-1,2,3-triazole) as a potent and competitive inhibitor for nOGA. This compound shows 15-fold selectivity for nOGA (K _i = 48 μM) over the full-length OGA (K _i = 725 μM) and 10-fold selectivity over human lysosomal β-hexosaminidase A&B (Hex A&B) (K _i = 502 μM). These results reveal that compound 4 can be used as a potent and selective inhibitor for probing the role of nOGA in biological systems.     

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.     

Screening-based discovery of drug-like O-GlcNAcase inhibitor scaffolds

O-GlcNAcylation is an essential posttranslational modification in metazoa. Modulation of O-GlcNAc levels with small molecule inhibitors of O-GlcNAc hydrolase (OGA) is a useful strategy to probe the role of this modification in a range of cellular processes. Here we report the discovery of novel, low molecular weight and drug-like O-GlcNAcase inhibitor scaffolds by high-throughput screening. Kinetic and X-ray crystallographic analyses of the binding modes with human/bacterial O-GlcNAcases identify some of these as competitive inhibitors. Comparative kinetic experiments with the mechanistically related human lysosomal hexosaminidases reveal that three of the inhibitor scaffolds show selectivity towards human OGA. These scaffolds provide attractive starting points for the development of non-carbohydrate, drug-like OGA inhibitors.     

Isoforms of human O-GlcNAcase show distinct catalytic efficiencies

O-GlcNAcase (OGA) is a family 84 glycoside hydrolase catalyzing the hydrolytic cleavage of O-linked β-N-acetylglucosamine (O-GlcNAc) from serine and threonine residues of proteins. Thus far, three forms of OGA have been identified in humans. Here we optimized the expression of these isoforms in E. coli and characterized their kinetic properties. Using Geno 3D, we predicted that N-terminal amino acids 63–342 form the catalytic site for O-GlcNAc removal and characterized it. Large differences are observed in the K _m value and catalytic efficiency (k _cat/K _m) for the three OGA variants, though all of them displayed O-GlcNAc hydrolase activity. The full-length OGA had the lowest K _m value of 0.26 mM and the highest catalytic efficiency of 3.51·10³. These results reveal that the N-terminal region (a.a. 1–350) of OGA contains the catalytic site for glycoside hydrolase and the C-terminal region of the coding sequence has the ability to stabilize the native three-dimensional structure and further affect substrate affinity.     

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.     

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.     

Selective decrease of membrane-associated PKC-α and PKC-ε in response to elevated intracellular O-GlcNAc levels in transformed human glial cells

Increased flux through the hexosamine biosynthetic pathway (HBP) has been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. It has been suggested that this effect is due to increases in the β-O-linked N-acetylglucosamine (O-GlcNAc) modification. Herein, we demonstrate the effect of increasing the O-GlcNAc modification on the translocation of select PKC isozymes in a human astroglial cell line. Treating cells with either 8 mM d-glucosamine (GlcN), 5 mM streptozotocin (STZ), or 80 μM O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) produced a significant increase in the O-GlcNAc modification on both cytosolic and membrane proteins; however, both the level and rate of O-GlcNAc increase varied with the compound. GlcN treatment resulted in a rapid, transient translocation of PKC-βII that was maximal after 3 h (73±8%) and also produced a 48±15% decrease in membrane-associated PKC-ε after 9 h of treatment. Similar to GlcN treatment, STZ and PUGNAc treatment also resulted in decreased levels of PKC-ε in the membrane fraction. Significant decreases were seen as early as 5 h and, by 9 h of treatment, had decreased by 87±6% with STZ and 73±7% with PUGNAc. Unlike GlcN, both STZ and PUGNAc produced a decrease in PKC-α membrane levels by 9 h posttreatment (78±10% with STZ and 66±8% with PUGNAc) while neither compound produced any changes in PKC-βII translocation. In addition, none of the three compounds affected membrane levels of PKC-ι. Altogether, these results demonstrate a novel link between increased levels of the O-GlcNAc modification and the regulation of specific PKC isoforms.     

Insight into a strategy for attenuating AmpC‐mediated β‐lactam resistance: Structural basis for selective inhibition of the glycoside hydrolase NagZ

NagZ is an exo‐N‐acetyl‐β‐glucosaminidase, found within Gram‐negative bacteria, that acts in the peptidoglycan recycling pathway to cleave N‐acetylglucosamine residues off peptidoglycan fragments. This activity is required for resistance to cephalosporins mediated by inducible AmpC β‐lactamase. NagZ uses a catalytic mechanism involving a covalent glycosyl enzyme intermediate, unlike that of the human exo‐N‐acetyl‐β‐glucosaminidases: O‐GlcNAcase and the β‐hexosaminidase isoenzymes. These latter enzymes, which remove GlcNAc from glycoconjugates, use a neighboring‐group catalytic mechanism that proceeds through an oxazoline intermediate. Exploiting these mechanistic differences we previously developed 2‐N‐acyl derivatives of O‐(2‐acetamido‐2‐deoxy‐D ‐glucopyranosylidene)amino‐N‐phenylcarbamate (PUGNAc), which selectively inhibits NagZ over the functionally related human enzymes and attenuate antibiotic resistance in Gram‐negatives that harbor inducible AmpC. To understand the structural basis for the selectivity of these inhibitors for NagZ, we have determined its crystallographic structure in complex with N‐valeryl‐PUGNAc, the most selective known inhibitor of NagZ over both the human β‐hexosaminidases and O‐GlcNAcase. The selectivity stems from the five‐carbon acyl chain of N‐valeryl‐PUGNAc, which we found ordered within the enzyme active site. In contrast, a structure determination of a human O‐GlcNAcase homologue bound to a related inhibitor N‐butyryl‐PUGNAc, which bears a four‐carbon chain and is selective for both NagZ and O‐GlcNAcase over the human β‐hexosamnidases, reveals that this inhibitor induces several conformational changes in the active site of this O‐GlcNAcase homologue. A comparison of these complexes, and with the human β‐hexosaminidases, reveals how selectivity for NagZ can be engineered by altering the 2‐N‐acyl substituent of PUGNAc to develop inhibitors that repress AmpC mediated β‐lactam resistance.     

O-GlcNAcase: Promiscuous Hexosaminidase or Key Regulator of O-GlcNAc Signaling?

O-GlcNAc signaling is regulated by an opposing pair of enzymes: O-GlcNAc transferase installs and O-GlcNAcase (OGA) removes the modification from proteins. The dynamics and regulation of this process are only beginning to be understood as the physiological functions of both enzymes are being probed using genetic and pharmacological approaches. This minireview charts the discovery and functional and structural analysis of OGA and summarizes the insights gained from recent studies using OGA inhibition, gene knock-out, and overexpression. We identify several areas of “known unknowns” that would benefit from future research, such as the enigmatic C-terminal domain of OGA.     

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.     

Elevation of cellular O-GlcNAcylation level by a potent and selective O-GlcNAcase inhibitor based on tetrahydroimidazopyridine scaffold

Protein O-GlcNAc glycosylation is a ubiquitous post-translational modification in metazoans. O-GlcNAcase (OGA), which is responsible for removing O-GlcNAc from serine or threonine residues, plays a key role in O-GlcNAc metabolism. Potent and selective O-GlcNAcase (OGA) inhibitors are useful tools for investigating the role of this modification in a broad range of cellular processes, and may also serve as drug candidates for treatment of neurodegenerative diseases. Biological screening of the gluco-configured tetrahydroimidazopyridine derivatives identified a compound as a potent and competitive inhibitor of human O-GlcNAcase (OGA) with a K_i of 5.9 μM, and it also displayed 28-fold selectivity for human OGA over human lysosomal β-hexosaminidase A (Hex A, K_i = 163 μM). In addition, cell-based assay revealed that this compound was cell-permeant and effectively induced cellular hyper-O-GlcNAcylation at 10 μM concentration.     

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.     

Glucosamine protects neonatal cardiomyocytes from ischemia-reperfusion injury via increased protein-associated O-GlcNAc

Increased levels of protein O-linked N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to determine whether in isolated neonatal rat ventricular myocytes (NRVMs) an increase in protein O-GlcNAcylation resulted in improved survival and viability following ischemia-reperfusion (I/R). NRVMs were exposed to 4 h of ischemia and 16 h of reperfusion, and cell viability, necrosis, apoptosis, and O-GlcNAc levels were assessed. Treatment of cells with glucosamine, hyperglycemia, or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate(PUGNAc), an inhibitor of O-GlcNAcase, significantly increased O-GlcNAc levels and improved cell viability, as well as reducing both necrosis and apoptosis compared with untreated cells following I/R. Alloxan, an inhibitor of O-GlcNAc transferase, markedly reduced O-GlcNAc levels and exacerbated I/R injury. The improved survival with hyperglycemia was attenuated by azaserine, which inhibits glucose metabolism via the hexosamine biosynthesis pathway. Reperfusion in the absence of glucose reduced O-GlcNAc levels on reperfusion compared with normal glucose conditions and decreased cell viability. O-GlcNAc levels significantly correlated with cell viability during reperfusion. The effects of glucosamine and PUGNAc on cellular viability were associated with reduced calcineurin activation as measured by translocation of nuclear factor of activated T cells, suggesting that increased O-GlcNAc levels may attenuate I/R induced increase in cytosolic Ca²⁺. These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection. hexosamine biosynthesis; calcium; protein O-glycosylation     

Conformational flexibility of the glycosidase NagZ allows it to bind structurally diverse inhibitors to suppress β‐lactam antibiotic resistance

NagZ is an N‐acetyl‐β‐d ‐glucosaminidase that participates in the peptidoglycan (PG) recycling pathway of Gram‐negative bacteria by removing N‐acetyl‐glucosamine (GlcNAc) from PG fragments that have been excised from the cell wall during growth. The 1,6‐anhydromuramoyl‐peptide products generated by NagZ activate β‐lactam resistance in many Gram‐negative bacteria by inducing the expression of AmpC β‐lactamase. Blocking NagZ activity can thereby suppress β‐lactam antibiotic resistance in these bacteria. The NagZ active site is dynamic and it accommodates distortion of the glycan substrate during catalysis using a mobile catalytic loop that carries a histidine residue which serves as the active site general acid/base catalyst. Here, we show that flexibility of this catalytic loop also accommodates structural differences in small molecule inhibitors of NagZ, which could be exploited to improve inhibitor specificity. X‐ray structures of NagZ bound to the potent yet non‐selective N‐acetyl‐β‐glucosaminidase inhibitor PUGNAc (O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidene) amino‐N‐phenylcarbamate), and two NagZ‐selective inhibitors – EtBuPUG, a PUGNAc derivative bearing a 2‐N‐ethylbutyryl group, and MM‐156, a 3‐N‐butyryl trihydroxyazepane, revealed that the phenylcarbamate moiety of PUGNAc and EtBuPUG completely displaces the catalytic loop from the NagZ active site to yield a catalytically incompetent form of the enzyme. In contrast, the catalytic loop was found positioned in the catalytically active conformation within the NagZ active site when bound to MM‐156, which lacks the phenylcarbamate extension. Displacement of the catalytic loop by PUGNAc and its N‐acyl derivative EtBuPUG alters the active site conformation of NagZ, which presents an additional strategy to improve the potency and specificity of NagZ inhibitors.     

Targeting O-Glycosyltransferase (OGT) to Promote Healing of Diabetic Skin Wounds

Non-healing wounds are a significant source of morbidity. This is particularly true for diabetic patients, who tend to develop chronic skin wounds. O-GlcNAc modification of serine and threonine residues is a common regulatory post-translational modification analogous to protein phosphorylation; increased intracellular protein O-GlcNAc modification has been observed in diabetic and hyperglycemic states. Two intracellular enzymes, UDP-N-acetylglucosamine-polypeptide β-N-acetylglucosaminyl transferase (OGT) and O-GlcNAc-selective N-acetyl-β-d-glucosaminidase (OGA), mediate addition and removal, respectively, of N-acetylglucosamine (GlcNAc) from intracellular protein substrates. Alterations in O-GlcNAc modification of intracellular proteins is linked to diabetes, and the increased levels of protein O-GlcNAc modification observed in diabetic tissues may in part explain some of the observed underlying pathophysiology that contributes to delayed wound healing. We have previously shown that increasing protein O-GlcNAc modification by overexpression of OGT in murine keratinocytes results in elevated protein O-GlcNAc modification and a hyperadhesive phenotype. This study was undertaken to explore the hypothesis that increased O-GlcNAc modification of cellular proteins in diabetic skin could contribute to the delayed wound healing observed in patients with diabetic skin ulcers. In the present study, we show that human keratinocytes cultured under hyperglycemic conditions display increased levels of O-GlcNAc modification as well as a delay in the rate of wound closure in vitro. We further show that specific knockdown of OGT by RNA interference (RNAi) reverses this effect, thereby opening up the opportunity for OGT-targeted therapies to promote wound healing in diabetic patients.     

<Emphasis Type="Italic">O</Emphasis>-GlcNAcylation of the <Emphasis Type="Italic">Plum pox virus</Emphasis> capsid protein catalyzed by SECRET AGENT: characterization of <Emphasis Type="Italic">O</Emphasis>-GlcNAc sites by electron transfer dissociation mass spectrometry

The capsid protein of Plum pox virus (PPV-CP) is modified with O-linked β-N-acetylglucosamine (O-GlcNAc). In Arabidopsis thaliana this modification is made by an O-GlcNAc transferase named SECRET AGENT (SEC). Modification of PPV-CP by SEC is hypothesized to have a direct role in the infection process, because virus titer and rate of spread are reduced in SEC mutants. Previous studies used deletion mapping and site-directed mutagenesis to identify four O-GlcNAc sites on the capsid protein that are modified by Escherichia coli-expressed SEC. The infection process was not affected when two of these sites were mutated suggesting that O-GlcNAcylation of these sites does not have a significant role in the infection process or that a subset of the modifications is sufficient. Since it is possible that the mutational mapping approach missed or incorrectly identified O-GlcNAc sites, the modifications produced by E. coli-expressed SEC were characterized using mass spectrometry. O-GlcNAcylated peptides were enzymatically tagged with galactose, the products were enriched on immobilized Ricinus communis agglutinin I and sequenced by electron transfer dissociation (ETD) mass spectrometry. Five O-GlcNAc sites on PPV-CP were identified. Two of these sites were not identified in by the previous mutational mapping. In addition, one site previously predicted by mutation mapping was not detected, but modification of this site was not supported when the mutation mapping was repeated. This study suggests that mapping modification sites by ETD mass spectrometry is more comprehensive and accurate than mutational mapping.