UDP-GlcNAc Research Articles

Cell-free expression and biochemical characterization of polysaccharide-synthesizing glycosyltransferases

Polysaccharides like cellulose and hyaluronan are synthesized by membrane-bound family-2 glycosyltransferases (GTs) that play critical structural, metabolic, or functional roles in cells. Though GT-2 family has the maximum number of deposited gene sequences, the biochemistry is poorly understood due to enzyme production challenges. Here, we developed a cell-free expression (CFE) protocol to produce two GT-2 family representative cellulose synthase (PttCesA8 from Populus tremula x tremuloides) and hyaluronan synthase (SeHAS from Streptococcus equisimilis). The CFE products were obtained as reconstituted proteoliposomes directly at high yields and short processing times compared to other approaches. Enzyme expression was confirmed using SDS-PAGE and immunoblotting, while integration of GTs into lipid layers was observed using cryogenic electron microscopy. Both GTs were catalytically active with Michalis-Menten kinetic constants, Km for PttCesA8, was 295.8 µM, and SeHAS was 321.51 µM (toward UDP N-Acetyl Glucosamine) and 207.88 µM (toward UDP Glucuronic Acid), respectively, and with UDP inhibiting both GTs. Mutation of conserved residues in SeHAS also confirmed the importance of lysine-139, glutamine-248, and threonine-283 residues in hyaluronan biosynthesis. In summary, CFE methods enable expression of polysaccharide-synthesizing enzymes as proteoliposomes at high yields with relative ease for rapid biochemical and structural characterization studies.

Functional overlap of inborn errors of immunity and metabolism genes defines T cell metabolic vulnerabilities.

Inborn errors of metabolism (IEMs) and immunity (IEIs) are Mendelian diseases in which complex phenotypes and patient rarity have limited clinical understanding. Whereas few genes have been annotated as contributing to both IEMs and IEIs, immunometabolic demands suggested greater functional overlap. Here, CRISPR screens tested IEM genes for immunologic roles and IEI genes for metabolic effects and found considerable previously unappreciated crossover. Analysis of IEMs showed that N-linked glycosylation and the hexosamine pathway enzyme Gfpt1 are critical for T cell expansion and function. Further, T helper (TH1) cells synthesized uridine diphosphate N-acetylglucosamine more rapidly and were more impaired by Gfpt1 deficiency than TH17 cells. Screening IEI genes found that Bcl11b promotes the CD4 T cell mitochondrial activity and Mcl1 expression necessary to prevent metabolic stress. Thus, a high degree of functional overlap exists between IEM and IEI genes, and immunometabolic mechanisms may underlie a previously underappreciated intersection of these disorders.

Exploring marine-derived bioactive compounds for dual inhibition of Pseudomonas aeruginosa LpxA and LpxD: integrated bioinformatics and cheminformatics approaches.

Pseudomonas aeruginosa can cause serious nosocomial infections. Targeting the biosynthesis of Lipid A, a major structural domain of lipopolysaccharide (LPS) in P. aeruginosa has emerged as a valuable strategy for developing novel therapeutic agents. The biosynthesis of Lipid A involves the activation of homolog enzymes including LpxA and LpxD. LpxA enzyme facilitates the transfer of R-3-hydroxydecanoic fatty acid to uridine diphosphate N-acetylglucosamine in the first step. While LPxD is accountable in third step, wherein R-3-hydroxydodecanoate is transferred to the 2' amine of UDP-3-O-(3-hydroxydecanoyl) utilizing an ACP donor. The exploration of LpxA and LpxD has been largely neglected, as no specific small-molecule inhibitors have been identified, thus far, except for peptide inhibitors. Here, we report the identification of potential dual inhibitors of the lipid A biosynthesis pathway that target both the LpxA and LpxD enzymes as novel antibiotic agents. Among the virtually screened 32,000 marine bioactive compounds Oscillatoxin A, NCI60_041046, and LTS0192263 exhibited optimal docking interactions with LpxA and LpxD, respectively. MD simulation and MMPBSA data showcased stable interactions between selected marine products and LpxA/LpxD. FMO analysis showed that Oscillatoxin A and NCI60_041046 are the most chemically active molecules. MEP analysis data highlighted the possible electrophilic and nucleophilic distribution zones present in the structure. In addition, these bioactive molecules showed acceptable ADMET profiles. These data confirmed that Oscillatoxin A, NCI60_041046, and LTS0192263 could serve as seeds for the development of potential therapeutics to combat P. aeruginosa infection.

Identification and in vitro characterization of UDP-GlcNAc-RNA cap-modifying and decapping enzymes.

In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurBand MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs. Additionally, we provide useful enzymatic tools that could be utilized in the development and application of UDP-GlcNAc capture and sequencing protocols. Such protocols are essential for deepening our understanding of the widespread yet enigmatic GlcNAc modification of RNA and its physiological significance.

The GFPT2-O-GlcNAcylation-YBX1 axis promotes IL-18 secretion to regulate the tumor immune microenvironment in pancreatic cancer

The immunosuppressive microenvironment caused by several intrinsic and extrinsic mechanism has brought great challenges to the immunotherapy of pancreatic cancer. We identified GFPT2, the key enzyme in hexosamine biosynthesis pathway (HBP), as an immune-related prognostic gene in pancreatic cancer using transcriptome sequencing and further confirmed that GFPT2 promoted macrophage M2 polarization and malignant phenotype of pancreatic cancer. HBP is a glucose metabolism pathway leading to the generation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which is further utilized for protein O-GlcNAcylation. We confirmed GFPT2-mediated O-GlcNAcylation played an important role in regulating immune microenvironment. Through cellular proteomics, we identified IL-18 as a key downstream of GFPT2 in regulating the immune microenvironment. Through CO-IP and protein mass spectrum, we confirmed that YBX1 was O-GlcNAcylated and nuclear translocated by GFPT2-mediated O-GlcNAcylation. Then, YBX1 functioned as a transcription factor to promote IL-18 transcription. Our study elucidated the relationship between the metabolic pathway of HBP in cancer cells and the immune microenvironment, which might provide some insights into the combination therapy of HBP vulnerability and immunotherapy in pancreatic cancer.

Role of UDP-N-acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

In vitro and in silico studies of enterobactin-inspired Ciprofloxacin and Fosfomycin first generation conjugates on the antibiotic resistant E. coli OQ866153

BackgroundThe emergence of antimicrobial resistance in bacterial pathogens is a growing concern worldwide due to its impact on the treatment of bacterial infections. The "Trojan Horse" strategy has been proposed as a potential solution to overcome drug resistance caused by permeability issues.ObjectiveThe objective of our research was to investigate the bactericidal activity and mechanism of action of the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin against the antibiotic-resistant Escherichia coli strain OQ866153.MethodologyEnterobactin, a mixed ligand of E. coli OQ866153, was conjugated with Ciprofloxacin and Fosfomycin individually to aid active absorption via specific enterobactin binding proteins (FepABCDG). The effectiveness of the conjugates was assessed by measuring their bactericidal activity against E. coli OQ866153, as well as their ability to inhibit DNA gyrase enzyme and biofilm formation.ResultsThe Fe+3-enterobactin-Ciprofloxacin conjugate effectively inhibited the DNA gyrase enzyme (Docking score = -8.597 kcal/mol) and resulted in a lower concentration (25 μg/ml) required to eliminate supercoiled DNA plasmids compared to the parent drug (35 μg/ml; Docking score = -6.264 kcal/mol). The Fe+3-Enterobactin-Fosfomycin conjugate showed a higher inhibition percentage (100%) of biofilm formation compared to Fosfomycin (21.58%) at a concentration of 2 mg/ml, with docking scores of -5.481 and -3.756 kcal/mol against UDP-N acetylglucosamine 1-carboxyvinyltransferase MurA.ConclusionThe findings of this study suggest that the "Trojan Horse" strategy using enterobactin conjugated with Ciprofloxacin and Fosfomycin can effectively overcome permeability issues caused by efflux proteins and enhance the bactericidal activity of these drugs against antibiotic-resistant strains of E. coli.

Hypothetical protein CuvA (Rv1422) from Mycobacterium tuberculosis H37Rv interacts with uridine diphosphate N-acetylglucosamine as a key precursor of cell wall

Mycobacterium tuberculosis CuvA (Rv1422, MtCuvA) has previously been suggested that it may play a critical role in nutrient utilization and cell wall synthesis required for physiological adaptation in a host cell, but its biochemical details remain unclear. Our previous studies showed that MtCuvA can bind to uridine diphosphate (UDP) sugars as a cell wall precursor component. To verify its functional roles, we report here the biochemical properties of MtCuvA for the binding of UDP-N-acetylglucosamine (GlcNAc) using site-directed mutagenesis and docking simulation. The KD values for UDP-sugars indicate that MtCuvA prefers to bind UDP-GlcNAc as a physiological ligand compared to UDP-glucose. Mutational studies of MtCuvA showed that H12A, T33A, D36A, Q154A, S196, T199A, N226A, and H298A mutants significantly affected the binding to UDP-GlcNAc. We also observed that UDP, but not GlcNAc, could bind to MtCuvA. These results imply that the presence of UDP moiety in the ligand is necessary for interaction with MtCuvA. Moreover, mutational studies of MtCuvA with UDP showed that residues H12, S196, T199, N226, and H298 may be involved in its binding to the UDP moiety, almost consistent with the docking simulation results. Our results provide an insight into the interaction of MtCuvA with UDP-GlcNAc as a key precursor of peptidoglycan.

Inhibition of glycolysis-driven immunosuppression with a nano-assembly enhances response to immune checkpoint blockade therapy in triple negative breast cancer

Immune-checkpoint inhibitors (ICI) are promising modalities for treating triple negative breast cancer (TNBC). However, hyperglycolysis, a hallmark of TNBC cells, may drive tumor-intrinsic PD-L1 glycosylation and boost regulatory T cell function to impair ICI efficacy. Herein, we report a tumor microenvironment-activatable nanoassembly based on self-assembled aptamer-polymer conjugates for the targeted delivery of glucose transporter 1 inhibitor BAY-876 (DNA-PAE@BAY-876), which remodels the immunosuppressive TME to enhance ICI response. Poly β-amino ester (PAE)-modified PD-L1 and CTLA-4-antagonizing aptamers (aptPD-L1 and aptCTLA-4) are synthesized and co-assembled into supramolecular nanoassemblies for carrying BAY-876. The acidic tumor microenvironment causes PAE protonation and triggers nanoassembly dissociation to initiate BAY-876 and aptamer release. BAY-876 selectively inhibits TNBC glycolysis to deprive uridine diphosphate N-acetylglucosamine and downregulate PD-L1 N-linked glycosylation, thus facilitating PD-L1 recognition of aptPD-L1 to boost anti-PD-L1 therapy. Meanwhile, BAY-876 treatment also elevates glucose supply to tumor-residing regulatory T cells (Tregs) for metabolically rewiring them into an immunostimulatory state, thus cooperating with aptCTLA-4-mediated immune-checkpoint inhibition to abolish Treg-mediated immunosuppression. DNA-PAE@BAY-876 effectively reprograms the immunosuppressive microenvironment in preclinical models of TNBC in female mice and provides a distinct approach for TNBC immunotherapy in the clinics.

Small Molecules Targeting Human UDP-GlcNAc 2-Epimerase.

Uridine diphosphate N-acetylglucosamine 2-epimerase (GNE) is a key enzyme in the sialic acid biosynthesis pathway. Sialic acids are primarily terminal carbohydrates on glycans and play fundamental roles in health and disease. In search of effective GNE inhibitors not based on a carbohydrate scaffold, we performed a high-throughput screening campaign of 68,640 drug-like small molecules against recombinant GNE using a UDP detection assay. We validated nine of the primary actives with an orthogonal real-time NMR assay and verified their IC50 values in the low micromolar to nanomolar range manually. Stability and solubility studies revealed three compounds for further evaluation. Thermal shift assays, analytical size exclusion, and interferometric scattering microscopy demonstrated that the GNE inhibitors acted on the oligomeric state of the protein. Finally, hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed which sections of GNE were shifted upon the addition of the inhibitors. In summary, we have identified three small molecules as GNE inhibitors with high potency in vitro, which serve as promising candidates to modulate sialic acid biosynthesis in more complex systems.

Nanopore Discrimination of Nucleotide Sugars.

Nucleotide sugars, the glycosyl donors in the biosynthesis of carbohydrates, are critical ingredients in the growth and development of all living organisms. A variety of nucleotide sugars simultaneously exist in biological samples. They, however, have only minor structural differences, which make them extremely difficult to discriminate. In this work, a phenylboronic acid (PBA)-modified Mycobacterium smegmatis porin A (MspA) hetero-octamer was applied to sense nucleotide sugars. Five representative nucleotide sugars, including guanosine diphosphate mannose (GDP-Man), adenosine diphosphate glucose (ADP-Glc), uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), uridine diphosphate glucose (UDP-Glc), and uridine diphosphate glucoronic acid (UDP-GlcA), were successfully distinguished. A custom machine learning algorithm was also employed to automatically identify events, reporting a general accuracy of 99.4%. This sensing strategy provides a rapid, direct, and accurate method for identifying different nucleotide sugars. However, single-molecule identification of nucleotide sugars has never been previously reported, to the best of our knowledge.

Genetically Encoded Boronolectin as a Specific Red Fluorescent UDP-GlcNAc Biosensor.

There is great interest in developing boronolectins that are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid- and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.

Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic non-canonical glycosylation of intracellular proteins. Several branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation, the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of UDP-GlcNAc is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it are restricted to specialized chromatographic procedures. Here, we introduce an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by O-linked N-acetylglucosamine transferase (OGT) and subsequent immunodetection of the modification. The assay can be performed in dot-blot or microplate format. We apply it to quantify UDP-GlcNAc concentrations in several mouse tissues and cell lines. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).

Conserved active site architecture between bacterial cellulose and chitin synthases.

Glycosyltransferases (GTs) are a large and diverse group of enzymes responsible for catalyzing the formation of a glycosidic bond between a donor molecule, usually a monosaccharide, and a wide range of acceptor molecules, thus, playing critical roles in various essential biological processes. Chitin and cellulose synthases are two inverting processive integral membrane GTs, belonging to the type-2 family involved in the biosynthesis of chitin and cellulose, respectively. Herein, we report that bacterial cellulose and chitin synthases share an E-D-D-ED-QRW-TK common motif spatially co-localized among distant bacterial evolutionary species despite their low amino acid sequence and structural similarities. This theoretical framework offers a new perspective to the current view that bacterial cellulose and chitin synthases are substrate specific and that chitin and cellulose are organism specific. It lays the ground for future in vivo and in silico experimental assessment of cellulose synthase catalytic promiscuity against uridine diphosphate N-acetylglucosamine and chitin synthase against uridine diphosphate glucose, respectively.

Tisp40 prevents cardiac ischemia/reperfusion injury through the hexosamine biosynthetic pathway in male mice

The hexosamine biosynthetic pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to facilitate O-linked GlcNAc (O-GlcNAc) protein modifications, and subsequently enhance cell survival under lethal stresses. Transcript induced in spermiogenesis 40 (Tisp40) is an endoplasmic reticulum membrane-resident transcription factor and plays critical roles in cell homeostasis. Here, we show that Tisp40 expression, cleavage and nuclear accumulation are increased by cardiac ischemia/reperfusion (I/R) injury. Global Tisp40 deficiency exacerbates, whereas cardiomyocyte-restricted Tisp40 overexpression ameliorates I/R-induced oxidative stress, apoptosis and acute cardiac injury, and modulates cardiac remodeling and dysfunction following long-term observations in male mice. In addition, overexpression of nuclear Tisp40 is sufficient to attenuate cardiac I/R injury in vivo and in vitro. Mechanistic studies indicate that Tisp40 directly binds to a conserved unfolded protein response element (UPRE) of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) promoter, and subsequently potentiates HBP flux and O-GlcNAc protein modifications. Moreover, we find that I/R-induced upregulation, cleavage and nuclear accumulation of Tisp40 in the heart are mediated by endoplasmic reticulum stress. Our findings identify Tisp40 as a cardiomyocyte-enriched UPR-associated transcription factor, and targeting Tisp40 may develop effective approaches to mitigate cardiac I/R injury.

Identification and functional characterisation of N-acetylglucosamine kinase from Helicoverpa armigera divulge its potential role in growth and development via UDP-GlcNAc salvage pathway

N-acetylglucosamine kinase (NAGK), a major enzyme of sugar-kinase/Hsp70/actin superfamily, catalyses the conversion of N-acetylglucosamine to N-acetylglucosamine-6-phosphate, the first step leading to the salvage synthesis of uridine diphosphate N-acetylglucosamine. Here, we present the first report on identification, cloning, recombinant expression and functional characterisation of NAGK from Helicoverpa armigera (HaNAGK). The purified soluble HaNAGK exhibited a molecular mass of ∼39 kDa with monomeric conformation. It catalysed the sequential transformation of GlcNAc into UDP-GlcNAc, indicating its role as the initiator of UDP-GlcNAc salvage pathway. HaNAGK exhibited ubiquitous expressions across all the developmental stages and major tissues of H. armigera. The gene was significantly upregulated (80 %; p < 0.01) by the moulting hormone 20-hydroxyecdysone and significantly downregulated (89 %; p < 0.001) by the chitin synthesis inhibitor novaluron, indicating its involvement in ecdysis and chitin metabolism. Furthermore, RNAi of HaNAGK caused poor weight gain, deformed insect bodies, aberrant metamorphosis and pronounced wing abnormalities in >55 % of surviving adults, while recording 7.79 ± 1.52 % and 24.25 ± 7.21 % mortality during larval and pupal stages, respectively. Altogether, the present findings suggest that HaNAGK plays a crucial role in the growth and development of H. armigera and thus, could be considered as a compelling gene of interest while formulating novel pest management strategies.

Development of simultaneous quantitative analytical method for metabolites of hexosamine biosynthesis pathway in lung cancer cells using UPLC-MS/MS.

The hexosamine biosynthesis pathway (HBP) is a glucose metabolism pathway that produces uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc). Substantial changes in HBP, including elevated HBP flux and UDP-GlcNAc levels, are associated with cancer pathogenesis. Particularly, cancer cells expressing oncogenic Kirsten rat sarcoma virus (KRAS) are highly dependent on HBP for growth and survival. To differentiate between HBP metabolites in KRAS wild-type (WT) and mutant (MT) lung cancer cells, a simultaneous quantitative method for analyzing seven HPB metabolites was developed using ultra-high-performance liquid chromatography-tandem mass spectrometry. A simple method without complicated preparation steps, such as derivatization or isotope labeling, was optimized for the simultaneous analysis of highly hydrophilic HBP metabolites, and the developed method was successfully verified. The intra- and inter-day coefficients of variation were less than 15% for all HBP metabolites, and the recovery was 89.67-114.5%. All results of the validation list were in accordance with ICM M10 guidelines. Through this method, accurate quantification of HBP metabolites in lung cancer cells was performed, and it was confirmed that all HBP metabolites were upregulated in KRAS MT cells compared with KRAS WT lung cancer cells. We expect that this will be a useful tool for metabolic research on cancer and for the development of new drugs for cancer treatment.

Mice lacking nucleotide sugar transporter SLC35A3 exhibit lethal chondrodysplasia with vertebral anomalies and impaired glycosaminoglycan biosynthesis.

SLC35A3 is considered an uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) transporter in mammals and regulates the branching of N-glycans. A missense mutation in SLC35A3 causes complex vertebral malformation (CVM) in cattle. However, the biological functions of SLC35A3 have not been fully clarified. To address these issues, we have established Slc35a3-/-mice using CRISPR/Cas9 genome editing system. The generated mutant mice were perinatal lethal and exhibited chondrodysplasia recapitulating CVM-like vertebral anomalies. During embryogenesis, Slc35a3 mRNA was expressed in the presomitic mesoderm of wild-type mice, suggesting that SLC35A3 transports UDP-GlcNAc used for the sugar modification that is essential for somite formation. In the growth plate cartilage of Slc35a3-/-embryos, extracellular space was drastically reduced, and many flat proliferative chondrocytes were reshaped. Proliferation, apoptosis and differentiation were not affected in the chondrocytes of Slc35a3-/-mice, suggesting that the chondrodysplasia phenotypes were mainly caused by the abnormal extracellular matrix quality. Because these histological abnormalities were similar to those observed in several mutant mice accompanying the impaired glycosaminoglycan (GAG) biosynthesis, GAG levels were measured in the spine and limbs of Slc35a3-/-mice using disaccharide composition analysis. Compared with control mice, the amounts of heparan sulfate, keratan sulfate, and chondroitin sulfate/dermatan sulfate, were significantly decreased in Slc35a3-/-mice. These findings suggest that SLC35A3 regulates GAG biosynthesis and the chondrodysplasia phenotypes were partially caused by the decreased GAG synthesis. Hence, Slc35a3-/- mice would be a useful model for investigating the in vivo roles of SLC35A3 and the pathological mechanisms of SLC35A3-associated diseases.

O-GlcNAc transferase in astrocytes modulates depression-related stress susceptibility through glutamatergic synaptic transmission.

Major depressive disorder is a common and devastating psychiatric disease, the prevalence and burden are substantially increasing worldwide. Multiple studies of depression patients have implicated glucose metabolic dysfunction in the pathophysiology of depression. However, the molecular mechanisms by which glucose and related metabolic pathways modulate depressive-like behaviors are largely uncharacterized. UDP-GlcNAc is a glucose metabolite with pivotal functions as a donor molecule for O-GlcNAcylation. O-GlcNAc transferase (OGT), a key enzyme in protein O-GlcNAcylation, catalyzes protein posttranslational modification by O-GlcNAc and acts as a stress sensor. Here, we show that Ogt mRNA was increased in depression patients and that astroglial OGT expression was specifically upregulated in the medial prefrontal cortex of susceptible mice after chronic social defeat stress. The selective deletion of astrocytic OGT resulted in antidepressant-like behaviors, moreover, astrocytic OGT in the mPFC bidirectionally regulated vulnerability to social stress. Furthermore, OGT modulated glutamatergic synaptic transmission through O-GlcNAcylation of glutamate transporter-1 (GLT-1) in astrocytes. OGT astrocyte-specific knockout preserved the neuronal morphology atrophy and Ca2+ activity deficits caused by chronic stress and resulted in antidepressant effects. Altogether, our study reveals that astrocytic OGT in the mPFC regulates depressive-like behaviors through the O-GlcNAcylation of GLT-1 and could be a potential target for antidepressants.

Listeria monocytogenes GlmR Is an Accessory Uridyltransferase Essential for Cytosolic Survival and Virulence.

The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions against these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell-wall stress, cytosolic survival, inflammasome avoidance, and, ultimately, virulence in vivo. In this study, a genetic suppressor screen revealed that blocking utilization of UDP N-acetylglucosamine (UDP-GlcNAc) by a nonessential wall teichoic acid decoration pathway restored resistance to lysozyme and partially restored virulence of ΔglmR mutants. In parallel, metabolomic analysis revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggests that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell-wall stress responses and virulence in vivo. Taken together, these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes. IMPORTANCE Bacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK), to survive in the host cytosol. GlmR is important for resistance to some cell-wall stresses and is essential for virulence. The ΔglmR mutant is deficient in production of an essential cell-wall metabolite, UDP-GlcNAc, and suppressors that increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc, and this enzymatic activity is conserved in both Bacillus subtilis and Staphylococcus aureus. These results highlight the importance of accessory cell wall metabolism enzymes in responding to cell-wall stress in a variety of Gram-positive bacteria.