Allosteric regulation by c-di-AMP modulates a complete N-acetylglucosamine signaling cascade in Saccharopolyspora erythraea.

c-di-AMP is an essential and widespread nucleotide second messenger in bacterial signaling. For most c-di-AMP synthesizing organisms, c-di-AMP homeostasis and the molecular mechanisms pertaining to its signal transduction are of great concern. Here we show that c-di-AMP binds the N-acetylglucosamine (GlcNAc)-sensing regulator DasR, indicating a direct link between c-di-AMP and GlcNAc signaling. Beyond its foundational role in cell-surface structure, GlcNAc is attractive as a major nutrient and messenger molecule regulating multiple cellular processes from bacteria to humans. We show that increased c-di-AMP levels allosterically activate DasR as a master repressor of GlcNAc utilization, causing the shutdown of the DasR-mediated GlcNAc signaling cascade and leading to a consistent enhancement in the developmental transition and antibiotic production in Saccharopolyspora erythraea. The expression of disA, encoding diadenylate cyclase, is directly repressed by the regulator DasR in response to GlcNAc signaling, thus forming a self-sustaining transcriptional feedback loop for c-di-AMP synthesis. These findings shed light on the allosteric regulation by c-di-AMP, which appears to play a prominent role in global signal integration and c-di-AMP homeostasis in bacteria and is likely widespread in streptomycetes that produce c-di-AMP.

RAD18 O-GlcNAcylation promotes translesion DNA synthesis and homologous recombination repair.

RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.

O-GlcNAcylation levels remain stable regardless of the anaesthesia in healthy rats.

Anaesthetics are used daily in human and veterinary medicine as well as in scientific research. Anaesthetics have an impact on cell homeostasis especially through modulation of protein post-translational modifications. O-GlcNAcylation, a ubiquitous post-translational modification, plays a role in many biological processes. The aims of this study were to evaluate whether (1) anaesthesia influences O-GlcNAcylation and (2) its stimulation affects physiological parameters. Male Wistar rats (n = 38) were anaesthetized with ketamine-xylazine or isoflurane. They randomly received either an intravenous injection of Ringer's lactate or NButGT (10mg/kg) in order to increase O-GlcNAcylation levels. One hour after induction of anaesthesia, haemodynamic parameters and plasmatic markers were evaluated. Heart, brain and lungs were harvested and O-GlcNAcylation levels and O-GlcNAc-related enzymes were evaluated by western blot. Cardiac and pulmonary O-GlcNAcylation levels and cardiac, cerebral and pulmonary O-GlcNAc associated enzyme expression were not impacted with anaesthesia. Compared with ketamine-xylazine, isoflurane had a lower impact on blood pressure, heart rate and glycaemia. Pharmacological stimulation of O-GlcNAcylation by NButGT did not affect the physiological parameters. This study offers unprecedented insights into the regulation of O-GlcNAcylation and O-GlcNAc related enzymes during anaesthesia. Pharmacological stimulation of O-GlcNAcylation over a 1-h period did not disrupt the physiological balance in healthy anaesthetized rats.

Cyanovirin-N Binding to N-Acetyl-d-glucosamine Requires Carbohydrate-Binding Sites on Two Different Protomers.

Cyanovirin-N (CV-N) binds high-mannose oligosaccharides on enveloped viruses with two carbohydrate-binding sites, one bearing high affinity and one low affinity to Manα(1-2)Man moieties. A tandem repeat of two CV-N molecules (CVN2) was tested for antiviral activity against human immunodeficiency virus type I (HIV-1) by using a domain-swapped dimer. CV-N was shown to bind N-acetylmannosamine (ManNAc) and N-acetyl-d-glucosamine (GlcNAc) when the carbohydrate-binding sites in CV-N were free to interact with these monosaccharides independently. CVN2 recognized ManNAc at a Kd of 1.4 µM and bound this sugar in solution, regardless of the lectin making amino acid side chain contacts on the targeted viral glycoproteins. An interdomain cross-contacting residue Glu41, which has been shown to be hydrogen bonding with dimannose, was substituted in the monomeric CV-N. The amide derivative of glucose, GlcNAc, achieved similar high affinity to the new variant CVN-E41T as high-mannose N-glycans, but binding to CVN2 in the nanomolar range with four binding sites involved or binding to the monomeric CVN-E41A. A stable dimer was engineered and expressed from the alanine-to-threonine-substituted monomer to confirm binding to GlcNAc. In summary, low-affinity binding was achieved by CVN2 to dimannosylated peptide or GlcNAc with two carbohydrate-binding sites of differing affinities, mimicking biological interactions with the respective N-linked glycans of interest and cross-linking of carbohydrates on human T cells for lymphocyte activation.

Biological Functions and Potential Therapeutic Significance of O-GlcNAcylation in Hepatic Cellular Stress and Liver Diseases.

O-linked-ß-D-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation), which is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), is a post-translational modification involved in multiple cellular processes. O-GlcNAcylation of proteins can regulate their biological functions via crosstalk with other post-translational modifications, such as phosphorylation, ubiquitination, acetylation, and methylation. Liver diseases are a major cause of death worldwide; yet, key pathological features of the disease, such as inflammation, fibrosis, steatosis, and tumorigenesis, are not fully understood. The dysregulation of O-GlcNAcylation has been shown to be involved in some severe hepatic cellular stress, viral hepatitis, liver fibrosis, nonalcoholic fatty acid liver disease (NAFLD), malignant progression, and drug resistance of hepatocellular carcinoma (HCC) through multiple molecular signaling pathways. Here, we summarize the emerging link between O-GlcNAcylation and hepatic pathological processes and provide information about the development of therapeutic strategies for liver diseases.

GlycoID Proximity Labeling to Identify O-GlcNAcylated Protein Interactomes in Live Cells.

Cells continuously remodel their intracellular proteins with the monosaccharide O-linked N-acetylglucosamine (O-GlcNAc) to regulate metabolism, signaling, and stress. This protocol describes the use of GlycoID tools to capture O-GlcNAc dynamics in live cells. GlycoID constructs contain an O-GlcNAc binding domain linked to a proximity labeling domain and a subcellular localization sequence. When expressed in mammalian cells, GlycoID tracks changes in O-GlcNAc-modified proteins and their interactomes in response to chemical induction with biotin over time. Pairing the subcellular localization of GlycoID with the chemical induction of activity enables spatiotemporal studies of O-GlcNAc biology during cellular events such as insulin signaling. However, optimizing intracellular labeling experiments requires attention to several variables. Here, we describe two protocols to adapt GlycoID methods to a cell line and biological process of interest. Next, we describe how to conduct a semiquantitative proteomic analysis of O-GlcNAcylated proteins and their interactomes using insulin versus glucagon signaling as a sample application. This articles aims to establish baseline GlycoID protocols for new users and set the stage for widespread use over diverse cellular applications for the functional study of O-GlcNAc glycobiology. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Expression of targeted GlycoID constructs to verify subcellular location and labeling activity in mammalian cells Basic Protocol 2: GlycoID labeling in live HeLa cells for O-GlcNAc proteomic comparisons.

Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Synthesis and anti-inflammatory activities of two new N-acetyl glucosamine derivatives.

N-acetyl glucosamine (NAG) is a natural amino sugar found in various human tissues with previously described anti-inflammatory effects. Various chemical modifications of NAG have been made to promote its biomedical applications. In this study, we synthesized two bi-deoxygenated NAG, BNAG1 and BNAG2 and investigated their anti-inflammatory properties, using an in vivo and in vitro inflammation mouse model induced by lipopolysaccharide (LPS). Among the parent molecule NAG, BNAG1 and BNAG2, BNAG1 showed the highest inhibition against serum levels of IL-6 and TNF α and the leukocyte migration to lungs and peritoneal cavity in LPS challenged mice, as well as IL-6 and TNF α production in LPS-stimulated primary peritoneal macrophages. BNAG2 displayed an anti-inflammatory effect which was comparable to NAG. These findings implied potential application of these novel NAG derivatives, especially BNAG1, in treatment of certain inflammation-related diseases.

A novel N-acetylglucosamine-6-phosphate deacetylase that is essential for chitin utilization in the chitinolytic bacterium, Chitiniphilus shinanonensis.

AIMS: We aimed to investigate the function of an unidentified gene annotated as a PIG-L domain deacetylase (cspld) in Chitiniphilus shinanonensis SAY3. cspld was identified using transposon mutagenesis, followed by negatively selecting a mutant incapable of growing on chitin, a polysaccharide consisting of N-acetyl-d-glucosamine (GlcNAc). We focused on the physiological role of CsPLD protein in chitin utilization. METHODS AND RESULTS: Recombinant CsPLD expressed in Escherichia coli exhibited GlcNAc-6-phosphate deacetylase (GPD) activity, which is involved in the metabolism of amino sugars. However, SAY3 possesses two genes (csnagA1 and csnagA2) in its genome that code for proteins whose primary sequences are homologous to those of typical GPDs. Recombinant CsNagA1 and CsNagA2 also exhibited GPD activity with 23 and 1.6% of catalytic efficiency (kcat/Km), respectively, compared to CsPLD. The gene-disrupted mutant, Δcspld was unable to grow on chitin or GlcNAc, whereas the three mutants, ΔcsnagA1, ΔcsnagA2, and ΔcsnagA1ΔcsnagA2 grew similarly to SAY3. The determination of GPD activity in the crude extracts of each mutant revealed that CsPLD is a major enzyme that accounts for almost all cellular activities. CONCLUSIONS: Deacetylation of GlcNAc-6P catalyzed by CsPLD (but not by typical GPDs) is essential for the assimilation of chitin and its constituent monosaccharide, GlcNAc, as a carbon and energy source in C. shinanonensis.

O-GlcNAcylation: roles and potential therapeutic target for bone pathophysiology.

O-linked N-acetylglucosamine (O-GlcNAc) protein modification (O-GlcNAcylation) is a critical post-translational modification (PTM) of cytoplasmic and nuclear proteins. O-GlcNAcylation levels are regulated by the activity of two enzymes, O-GlcNAc transferase (OGT) and O­GlcNAcase (OGA). While OGT attaches O-GlcNAc to proteins, OGA removes O-GlcNAc from proteins. Since its discovery, researchers have demonstrated O-GlcNAcylation on thousands of proteins implicated in numerous different biological processes. Moreover, dysregulation of O-GlcNAcylation has been associated with several pathologies, including cancers, ischemia-reperfusion injury, and neurodegenerative diseases. In this review, we focus on progress in our understanding of the role of O-GlcNAcylation in bone pathophysiology, and we discuss the potential molecular mechanisms of O-GlcNAcylation modulation of bone-related diseases. In addition, we explore significant advances in the identification of O-GlcNAcylation-related regulators as potential therapeutic targets, providing novel therapeutic strategies for the treatment of bone-related disorders.

Expression of the O-Linked N-Acetylglucosamine-containing Epitope H (O-GlcNAcH) in Human Uterine Cervical Mucosa.

BACKGROUND/AIM: Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation or environment, recognized by a site-specific monoclonal mouse IgM antibody H. O-GlcNAcH occurs in several normal and pathological cells and in several polypeptides, including keratin-8 and vimentin, on the latter in cells under stress. MATERIALS AND METHODS: In this work, we studied the distribution of O-GlcNAcH on cells of endocervical mucosa in 60 specimens of endocervical curettings, 10 of which contained 15 inflamed polyps. RESULTS: In our results, expression of O-GlcNAcH was weak in the mucosa with <5% mucin-secreting cells and up to 30% of the polyps staining positively. All non-ciliated, non-mucin-secreting cells, normal and hyperplastic 'reserve' cells, as well as the cells of immature squamous metaplasia, showed strong diffuse cytoplasmic staining for O-GlcNAcH. In mature squamous epithelium, fewer than 5% of basal cells and all the intermediate and superficial cells showed cytoplasmic staining for O-GlcNAcH, whereas parabasal cells were negative. All ciliated cells showed patchy or diffuse cytoplasmic staining. Nuclear staining for O-GlcNAcH was weak with fewer than 5% of hyperplastic 'reserve' and ciliated cells staining positively. Moreover, mucosal fibroblasts were negative, whereas all stromal cells of the polyps showed strong cytoplasmic staining for O-GlcNAcH. CONCLUSION: O-GlcNAcH is: a) differentially expressed among the cellular elements of mucosa and polyps, b) upregulated in mucin-secreting cells of polyps, c) induced in stromal cells of inflamed polyps, and d) can be used as a marker to differentiate between 'reserve' (positive) and parabasal (negative) cells, which have similar morphology using conventional cytological stains.

Malnutrition enteropathy in Zambian and Zimbabwean children with severe acute malnutrition: A multi-arm randomized phase II trial.

Malnutrition underlies almost half of all child deaths globally. Severe Acute Malnutrition (SAM) carries unacceptable mortality, particularly if accompanied by infection or medical complications, including enteropathy. We evaluated four interventions for malnutrition enteropathy in a multi-centre phase II multi-arm trial in Zambia and Zimbabwe and completed in 2021. The purpose of this trial was to identify therapies which could be taken forward into phase III trials. Children of either sex were eligible for inclusion if aged 6-59 months and hospitalised with SAM (using WHO definitions: WLZ <-3, and/or MUAC <11.5 cm, and/or bilateral pedal oedema), with written, informed consent from the primary caregiver. We randomised 125 children hospitalised with complicated SAM to 14 days treatment with (i) bovine colostrum (n = 25), (ii) N-acetyl glucosamine (n = 24), (iii) subcutaneous teduglutide (n = 26), (iv) budesonide (n = 25) or (v) standard care only (n = 25). The primary endpoint was a composite of faecal biomarkers (myeloperoxidase, neopterin, α1-antitrypsin). Laboratory assessments, but not treatments, were blinded. Per-protocol analysis used ANCOVA, adjusted for baseline biomarker value, sex, oedema, HIV status, diarrhoea, weight-for-length Z-score, and study site, with pre-specified significance of P < 0.10. Of 143 children screened, 125 were randomised. Teduglutide reduced the primary endpoint of biomarkers of mucosal damage (effect size -0.89 (90% CI: -1.69,-0.10) P = 0.07), while colostrum (-0.58 (-1.4, 0.23) P = 0.24), N-acetyl glucosamine (-0.20 (-1.01, 0.60) P = 0.67), and budesonide (-0.50 (-1.33, 0.33) P = 0.32) had no significant effect. All interventions proved safe. This work suggests that treatment of enteropathy may be beneficial in children with complicated malnutrition. The trial was registered at ClinicalTrials.gov with the identifier NCT03716115.

Targeted Protein O-GlcNAcylation Using Bifunctional Small Molecules.

Protein O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of the homeostasis of O-GlcNAcylation has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of the O-GlcNAcylation challenging. To address this, we have developed heterobifunctional small molecules, named O-GlcNAcylation TArgeting Chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in living cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2α, and EZH2 in cellulo by recruiting FKBP12F36V-fused O-GlcNAc transferase (OGT), with temporal, magnitude, and reversible control. Overall, the OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.

Protective effect of increased O-GlcNAc cycling against 6-OHDA induced Parkinson's disease pathology.

This study aimed to elucidate the role of O-GlcNAc cycling in 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease (PD)-like neurodegeneration and the underlying mechanisms. We observed dose-dependent downregulation of O-GlcNAcylation, accompanied by an increase in O-GlcNAcase following 6-OHDA treatment in both mouse brain and Neuro2a cells. Interestingly, elevating O-GlcNAcylation through glucosamine (GlcN) injection provided protection against PD pathogenesis induced by 6-OHDA. At the behavioral level, GlcN mitigated motor deficits induced by 6-OHDA, as determined using the pole, cylinder, and apomorphine rotation tests. Furthermore, GlcN attenuated 6-OHDA-induced neuroinflammation and mitochondrial dysfunction. Notably, augmented O-GlcNAcylation, achieved through O-GlcNAc transferase (OGT) overexpression in mouse brain, conferred protection against 6-OHDA-induced PD pathology, encompassing neuronal cell death, motor deficits, neuroinflammation, and mitochondrial dysfunction. These collective findings suggest that O-GlcNAcylation plays a crucial role in the normal functioning of dopamine neurons. Moreover, enhancing O-GlcNAcylation through genetic and pharmacological means could effectively ameliorate neurodegeneration and motor impairment in an animal model of PD. These results propose a potential strategy for safeguarding against the deterioration of dopamine neurons implicated in PD pathogenesis.

A comprehensive synthetic library of poly-N-acetyl glucosamines enabled vaccine against lethal challenges of Staphylococcus aureus.

Poly-ß-(1-6)-N-acetylglucosamine (PNAG) is an important vaccine target, expressed on many pathogens. A critical hurdle in developing PNAG based vaccine is that the impacts of the number and the position of free amine vs N-acetylation on its antigenicity are not well understood. In this work, a divergent strategy is developed to synthesize a comprehensive library of 32 PNAG pentasaccharides. This library enables the identification of PNAG sequences with specific patterns of free amines as epitopes for vaccines against Staphylococcus aureus (S. aureus), an important human pathogen. Active vaccination with the conjugate of discovered PNAG epitope with mutant bacteriophage Qß as a vaccine carrier as well as passive vaccination with diluted rabbit antisera provides mice with near complete protection against infections by S. aureus including methicillin-resistant S. aureus (MRSA). Thus, the comprehensive PNAG pentasaccharide library is an exciting tool to empower the design of next generation vaccines.

The Immunometabolic Gene N-Acetylglucosamine Kinase Is Uniquely Involved in the Heritability of Multiple Sclerosis Severity.

The clinical severity of multiple sclerosis (MS), an autoimmune disorder of the central nervous system, is thought to be determined by environmental and genetic factors that have not yet been identified. In a recent genome-wide association study (GWAS), a single nucleotide polymorphism (SNP), rs10191329, has been associated with MS severity in two large independent cohorts of patients. Different approaches were followed by the authors to prioritize the genes that are transcriptionally regulated by such an SNP. It was concluded that the identified SNP regulates a group of proximal genes involved in brain resilience and cognitive abilities rather than immunity. Here, by conducting an alternative strategy for gene prioritization, we reached the opposite conclusion. According to our re-analysis, the main target of rs10191329 is N-Acetylglucosamine Kinase (NAGK), a metabolic gene recently shown to exert major immune functions via the regulation of the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) pathway. To gain more insights into the immunometabolic functions of NAGK, we analyzed the currently known list of NAGK protein partners. We observed that NAGK integrates a dense network of human proteins that are involved in glucose metabolism and are highly expressed by classical monocytes. Our findings hold potentially major implications for the understanding of MS pathophysiology.

Lysinibacillus piscis sp. nov. isolated from the gut of mottled spinefoot Siganus fuscescens.

A novel Lysinibacillus strain, designated KH24T, was isolated from the gut of Siganus fuscescens, a herbivorous fish, which was captured off the coast of Okinawa, Japan. Strain KH24T is a rod-shaped, Gram-stain-positive, spore-forming, and motile bacterium that forms off-white colonies. The 16S rRNA gene sequence of strain KH24T showed the highest similarity (97.4%) with Lysinibacillus pakistanensis JCM 18776T and L. irui IRB4-01T. Genomic similarities between strain KH24T and Lysinibacillus type strains, based on average nucleotide identity, digital DNA-DNA hybridization (genome-to-genome distance calculation), and average amino acid identity were 70.4-77.7%, 17.1-24.4%, and 69.2-81.2%, respectively, which were lower than species delineation thresholds. Strain KH24T growth occurred at pH values of 5.5-8.5, temperatures of 20-40 °C, and NaCl concentrations of 0-4.0%, and optimally at pH 7.0, 30 °C, and 0%, respectively. Unlike related Lysinibacillus type strains, strain KH24T could assimilate D-glucose, D-fructose, N-acetyl-glucosamine, amygdalin, arbutin, esculin, ferric citrate, salicin, D-cellobiose, D-maltose, D-sucrose, and gentiobiose. Major fatty acids included iso-C15:0 (45.8%), anteiso-C15:0 (15.1%), iso-C17:0 (12.6%), and anteiso-C17:0 (10.9%). Menaquinone-7 was the predominant quinone, and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and lysophosphatidylethanolamine. Based on its genetic and phenotypic properties, strain KH24T represents a novel species of the genus Lysinibacillus, for which the name Lysinibacillus piscis sp. nov. is proposed. The type strain is KH24T (= JCM 36611 T = KCTC 43676 T).

O-GlcNAcylation of RBM14 contributes to elevated cellular O-GlcNAc through regulation of OGA protein stability.

Dysregulation of O-GlcNAcylation has emerged as a potential biomarker for several diseases, particularly cancer. The role of OGT (O-GlcNAc transferase) in maintaining O-GlcNAc homeostasis has been extensively studied; nevertheless, the regulation of OGA (O-GlcNAcase) in cancer remains elusive. Here, we demonstrated that the multifunctional protein RBM14 is a regulator of cellular O-GlcNAcylation. By investigating the correlation between elevated O-GlcNAcylation and increased RBM14 expression in lung cancer cells, we discovered that RBM14 promotes ubiquitin-dependent proteasomal degradation of OGA, ultimately mediating cellular O-GlcNAcylation levels. In addition, RBM14 itself is O-GlcNAcylated at serine 521, regulating its interaction with the E3 ligase TRIM33, consequently affecting OGA protein stability. Moreover, we demonstrated that mutation of serine 521 to alanine abrogated the oncogenic properties of RBM14. Collectively, our findings reveal a previously unknown mechanism for the regulation of OGA and suggest a potential therapeutic target for the treatment of cancers with dysregulated O-GlcNAcylation.

Next-Generation Metabolic Glycosylation Reporters Enable Detection of Protein O-GlcNAcylation in Living Cells without S-Glyco Modification.

Protein O-GlcNAcylation is a ubiquitous posttranslational modification of cytosolic and nuclear proteins involved in numerous fundamental regulation processes. Investigation of O-GlcNAcylation by metabolic glycoengineering (MGE) has been carried out for two decades with peracetylated N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine derivatives modified with varying reporter groups. Recently, it has been shown that these derivatives can result in non-specific protein labeling termed S-glyco modification. Here, we report norbornene-modified GlcNAc derivatives with a protected phosphate at the anomeric position and their application in MGE. These derivatives overcome two limitations of previously used O-GlcNAc reporters. They do not lead to detectable S-glyco modification, and they efficiently react in the inverse-electron-demand Diels-Alder (IEDDA) reaction, which can be carried out even within living cells. Using a derivative with an S-acetyl-2-thioethyl-protected phosphate, we demonstrate the protein-specific detection of O-GlcNAcylation of several proteins and the protein-specific imaging of O-GlcNAcylation inside living cells by Förster resonance energy transfer (FRET) visualized by confocal fluorescence lifetime imaging microscopy (FLIM).

The O-GlcNAc Modification of Recombinant Tau Protein and Characterization of the O-GlcNAc Pattern for Functional Study.

The neuronal microtubule-associated tau protein is characterized in vivo by a large number of post-translational modifications along the entire primary sequence that modulates its function. The primary modification of tau is phosphorylation of serine/threonine or tyrosine residues that is involved in the regulation of microtubule binding and polymerization. In neurodegenerative disorders referred to as tauopathies including Alzheimer's disease, tau is abnormally hyperphosphorylated and forms fibrillar inclusions in neurons progressing throughout different brain area during the course of the disease. The O-ß-linked N-acetylglucosamine (O-GlcNAc) is another reversible post-translational modification of serine/threonine residues that is installed and removed by the unique O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA), respectively. This modification was described as a potential modulator of tau phosphorylation and functions in the physiopathology. Moreover, reducing protein O-GlcNAc levels in the brain upon treatment of tauopathy mouse models with an OGA inhibitor reveals a beneficial effect on tau pathology and neurodegeneration. However, whether the role of tau O-GlcNAcylation is responsible of the protective effect against tau toxicity remains to be determined. The production of O-GlcNAc modified recombinant tau protein is a valuable tool for the investigations of the impact of O-GlcNAcylation on tau functions, modulation of interactions with partners and crosstalk with other post-translational modifications, including but not restricted to phosphorylation. We describe here the in vitro O-GlcNAcylation of tau with recombinant OGT for which we provide an expression and purification protocol. The use of the O-GlcNAc tau protein in functional studies requires the analytical characterization of the O-GlcNAc pattern. Here, we describe a method for the O-GlcNAc modification of tau protein with recombinant OGT and the analytical characterization of the resulting O-GlcNAc pattern by a combination of methods for the overall characterization of tau O-GlcNAcylation by chemoenzymatic labeling and mass spectrometry, as well as the quantitative, site-specific pattern by NMR spectroscopy.