Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) donates GlcNAc for various glycans and glycoconjugates. We previously found that GlcNAc supplementation increases the UDP-GlcNAc content in Arabidopsis; however, the metabolic pathway was undefined. Here, we show that the homolog of human GlcNAc kinase (GNK) is conserved in land plants. Enzymatic assays of the Arabidopsis homologous protein (AtGNK) revealed kinase activity that was highly specific for GlcNAc. We also demonstrate the role of AtGNK in plants by using its knockout mutant, which presents lower UDP-GlcNAc contents and is insensitive to GlcNAc supplementation. Moreover, our results demonstrate the presence of a GlcNAc salvage pathway in plants.

In this study we report the genetic characterization, including expression analysis, of the genes involved in the uptake (NGT1) and catabolism (HXK1/NAG5, DAC1/NAG2, NAG1) of the aminosugar N-acetylglucosamine (GlcNAc) in Candida africana, a pathogenic biovariant of Candida albicans that is naturally unable to assimilate the GlcNAc.

There is increasing evidence that O-linked N-acetylglucosamine (O-GlcNAc) plays an important role in cell signaling pathways. It has also been reported that increases in O-GlcNAc contribute to the development of diabetes and diabetic complications; however, little is known about O-GlcNAc levels in diabetic nephropathy (DNP). Therefore the goal of this study was to determine whether O-GlcNAc could be detected in human kidney biopsy specimens, and if so to examine whether O-GlcNAc levels were increased in the kidneys of patients with DNP compared to the non-diabetic individuals.

Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events.

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The non-conventional yeast Yarrowia lipolytica possesses an ORF, YALI0E20207g, which encodes a protein with an amino acid sequence similar to hexokinases from different organisms. We have cloned that gene and determined several enzymatic properties of its encoded protein showing that it is an N-acetylglucosamine (NAGA) kinase. This conclusion was supported by the lack of growth in NAGA of a strain carrying a YALI0E20207g deletion. We named this gene YlNAG5. Expression of YlNAG5 as well as that of the genes encoding the enzymes of the NAGA catabolic pathway-identified by a BLAST search-was induced by this sugar. Deletion of YlNAG5 rendered that expression independent of the presence of NAGA in the medium and reintroduction of the gene restored the inducibility, indicating that YlNag5 participates in the transcriptional regulation of the NAGA assimilatory pathway genes. Expression of YlNAG5 was increased during sporulation and homozygous Ylnag5/Ylnag5 diploid strains sporulated very poorly as compared with a wild type isogenic control strain pointing to a participation of the protein in the process. Overexpression of YlNAG5 allowed growth in glucose of an Ylhxk1glk1 double mutant and produced, in a wild type background, aberrant morphologies in different media. Expression of the gene in a Saccharomyces cerevisiae hxk1 hxk2 glk1 triple mutant restored ability to grow in glucose.

Myelination plays an important role in cognitive development and in demyelinating diseases like multiple sclerosis (MS), where failure of remyelination promotes permanent neuro-axonal damage. Modification of cell surface receptors with branched N-glycans coordinates cell growth and differentiation by controlling glycoprotein clustering, signaling, and endocytosis. GlcNAc is a rate-limiting metabolite for N-glycan branching. Here we report that GlcNAc and N-glycan branching trigger oligodendrogenesis from precursor cells by inhibiting platelet-derived growth factor receptor-α cell endocytosis. Supplying oral GlcNAc to lactating mice drives primary myelination in newborn pups via secretion in breast milk, whereas genetically blocking N-glycan branching markedly inhibits primary myelination. In adult mice with toxin (cuprizone)-induced demyelination, oral GlcNAc prevents neuro-axonal damage by driving myelin repair. In MS patients, endogenous serum GlcNAc levels inversely correlated with imaging measures of demyelination and microstructural damage. Our data identify N-glycan branching and GlcNAc as critical regulators of primary myelination and myelin repair and suggest that oral GlcNAc may be neuroprotective in demyelinating diseases like MS.

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification of serine and threonine residues on nuclear and cytoplasmic proteins. O-GlcNAc modification influences many cellular mechanisms, including carbohydrate metabolism, signal transduction and protein degradation. Multiple studies also showed that cell cycle might be modulated by O-GlcNAc. Although the role of O-GlcNAc in the regulation of some cell cycle processes such as mitotic spindle organization or histone phosphorylation is well established, the general behaviour of O-GlcNAc regulation during cell cycle is still controversial. In this study, we analysed the dynamic changes of overall O-GlcNAc levels in HeLa cells using double thymidine block. O-GlcNAc levels in G₁, S, G₂ and M phase were measured. We observed that O-GlcNAc levels are significantly increased during mitosis in comparison to the other cell cycle phases. However, this change could only be detected when mitotic cells were enriched by harvesting round shaped cells from the G₂/M fraction of the synchronized cells. Our data verify that O-GlcNAc is elevated during mitosis, but also emphasize that O-GlcNAc levels can significantly change in a short period of time. Thus, selection and collection of cells at specific cell-cycle checkpoints is a challenging, but necessary requirement for O-GlcNAc studies.

In Yarrowia lipolytica, expression of the genes encoding the enzymes of the N-acetylglucosamine (NAGA) utilization pathway (NAG genes) becomes independent of the presence of NAGA in a Ylnag5 mutant lacking NAGA kinase. We addressed the question of whether the altered transcription was due to a lack of kinase activity or to a moonlighting role of this protein. Glucosamine-6-phosphate deaminase (Nag1) activity was measured as a reporter of NAG genes expression. The NGT1 gene encoding the NAGA transporter was deleted, creating a Ylnag5 ngt1 strain. In glucose cultures of this strain, Nag1 activity was similar to that of the Ylnag5 strain, ruling out the possibility that NAGA derived from cell wall turnover could trigger the derepression. Heterologous NAGA kinases were expressed in a Ylnag5 strain. Among them, the protein from Arabidopsis thaliana did not restore kinase activity but lowered Nag1 activity 4-fold with respect to a control. Expression in the Ylnag5 strain of YlNag5 variants F320S or D214V with low kinase activity caused a repression similar to that of the wild-type protein. Together, these results indicate that YlNag5 behaves as a moonlighting protein. An RNA-seq analysis revealed that the Ylnag5 mutation had a limited transcriptomic effect besides derepression of the NAG genes.

Phagocytosis by innate immune cells is one of the most effective barriers against the multiplication and dissemination of microbes within the mammalian host. Candida albicans, a pathogenic yeast, has robust mechanisms that allow survival upon macrophage phagocytosis. C. albicans survives in part because it can utilize the alternative carbon sources available in the phagosome, including carboxylic acids and amino acids. Furthermore, metabolism of these compounds raises the pH of the extracellular environment, which combats the acidification and maturation of the phagolysosome. In this study, we demonstrate that metabolism by C. albicans of an additional carbon source, N-acetylglucosamine (GlcNAc), facilitates neutralization of the phagosome by a novel mechanism. Catabolism of GlcNAc raised the ambient pH through release of ammonia, which is distinct from growth on carboxylic acids but similar to growth on amino acids. However, the effect of GlcNAc metabolism on pH was genetically distinct from the neutralization induced by catabolism of amino acids, as mutation of STP2 or ATO5 did not impair the effects of GlcNAc. In contrast, mutants lacking the dedicated GlcNAc transporter gene NGT1 or the enzymes responsible for catabolism of GlcNAc were defective in altering the pH of the phagosome. This correlated with reduced survival following phagocytosis and decreased ability to damage macrophages. Thus, GlcNAc metabolism represents the third genetically independent mechanism that C. albicans utilizes to combat the rapid acidification of the phagolysosome, allowing for cells to escape and propagate infection. IMPORTANCECandida albicans is the most important medically relevant fungal pathogen, with disseminated candidiasis being the fourth most common hospital-associated bloodstream infection. Macrophages and neutrophils are innate immune cells that play a key role in host defense by phagocytosing and destroying C. albicans cells. To survive this attack by macrophages, C. albicans generates energy by utilizing alternative carbon sources that are available in the phagosome. Interestingly, metabolism of amino acids and carboxylic acids by C. albicans raises the pH of the phagosome and thereby blocks the acidification of the phagosome, which is needed to initiate antimicrobial attack. In this work, we demonstrate that metabolism of a third type of carbon source, the amino sugar GlcNAc, also induces pH neutralization and survival of C. albicans upon phagocytosis. This mechanism is genetically and physiologically distinct from the previously described mechanisms of pH neutralization, indicating that the robust metabolic plasticity of C. albicans ensures survival upon macrophage phagocytosis.

Osteoclasts are the only cells in an organism capable of resorbing bone. These cells differentiate from monocyte/macrophage lineage cells upon stimulation by receptor activator of NF-κB ligand (RANKL). On the other hand, osteoclastogenesis is reportedly suppressed by glucose via the downregulation of NF-κB activity through suppression of reactive oxygen species generation. To examine whether other sugars might also affect osteoclast development, we compared the effects of monomeric sugars (glucose, galactose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc)) on the osteoclastogenesis of murine RAW264 cells. Our results demonstrated that, in addition to glucose, both GlcNAc and GalNAc, which each have little effect on the generation of reactive oxygen species, suppress osteoclastogenesis. We hypothesized that GlcNAc might affect osteoclastogenesis through the upregulation of O-GlcNAcylation and showed that GlcNAc increases global O-GlcNAcylation, thereby suppressing the RANKL-dependent phosphorylation of NF-κB p65. Furthermore, an inhibitor of N-acetyl-β-D-glucosaminidase, O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenylcarbamate (PUGNAc), which also increases O-GlcNAcylation, suppressed the osteoclastogenesis of RAW264 cells and that of human peripheral blood mononuclear cells. Together, these data suggest that GlcNAc suppresses osteoclast differentiation in part through the promotion of O-GlcNAcylation.

We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), inhibits the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase) which is responsible for the removal of O-GlcNAc from proteins. Alloxan, another beta-cell toxin is a uracil analog. Since the O-GlcNAc transferase (OGT) uses UDP-GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in beta-cells. In isolated pancreatic islets, alloxan almost completely blocked both glucosamine-induced and STZ-induced protein O-GlcNAcylation, suggesting that alloxan indeed was inhibiting (OGT). In order to show definitively that alloxan was inhibiting OGT activity, recombinant OGT was incubated with 0-10 mM alloxan, and OGT activity was measured directly by quantitating UDP-[(3)H]-GlcNAc incorporation into the recombinant protein substrate, nucleoporin p62. Under these conditions, OGT activity was completely inhibited by 1 mM alloxan with half-maximal inhibition achieved at a concentration of 0.1 mM alloxan. Together, these data demonstrate that alloxan is an inhibitor of OGT, and as such, is the first OGT inhibitor described.

O-linked N-acetylglucosamine transferase (OGT) is an essential enzyme that catalyzes the covalent bonding of N-acetylglucosamine (GlcNAc) to the hydroxyl group of a serine or threonine in the target protein. It plays an important role in many important cellular physiological catalytic reactions. Here, we determine the binding mode and the binding free energy of the OGT product (uridine diphosphate, UDP) as well as the hydrogen-bond-dependent release mechanism using extensive molecular dynamic simulations. The Lys634, Asn838, Gln839, Lys842, His901, and Asp925 residues were identified to play a major role in the UDP stabilization in the active site of OGT, where hydrogen bonding and π-π interactions mainly occur. The calculations on the mutant forms support our results. Sixteen possible release channels were identified while the two most favorable channels were determined using random acceleration molecular dynamics (RAMD) simulations combined with the constant velocity pulling (PCV) method. The thermodynamic and dynamic properties as along with the corresponding mechanism were determined and discussed according to the umbrella sampling technique. For the most optimal channel, the main free energy barrier is 13 kcal/mol, which probably originates from the hydrogen bonds between UDP and the Ala896 and Asp925 residues. Moreover, the unstable hydrogen bonds and the rollback of the ligand likely cause the other two small obstacles. This work clarifies the ligand transport mechanism in the OGT enzymatic process and is a great resource for designing inhibitors based on UDP or UDP-GlcNAc.

Bisecting N-acetylglucosamine (GlcNAc), a GlcNAc linked to the core β-mannose residue via a β1,4 linkage, is a special type of N-glycosylation that has been reported to be involved in various biological processes, such as cell adhesion and fetal development. This N-glycan structure is abundant in human trophoblasts, which is postulated to be resistant to natural killer cell-mediated cytotoxicity, enabling a mother to nourish a fetus without rejection. In this study, we hypothesized that the human amniotic membrane, which serves as the last barrier for the fetus, may also express bisected-type glycans. To test this hypothesis, glycomic analysis of the human amniotic membrane was performed, and bisected N-glycans were detected. Furthermore, our proteomic data, which have been previously employed to explore human missing proteins, were analyzed and the presence of bisecting GlcNAc-modified peptides was confirmed. A total of 41 glycoproteins with 43 glycopeptides were found to possess a bisecting GlcNAc, and 25 of these glycoproteins were reported to exhibit this type of modification for the first time. These results provide insights into the potential roles of bisecting GlcNAc modification in the human amniotic membrane, and can be beneficial to functional studies on glycoproteins with bisecting GlcNAc modifications and functional studies on immune suppression in human placenta.

A gene encoding Trypanosoma brucei UDP-N-acetylglucosamine pyrophosphorylase was identified, and the recombinant protein was shown to have enzymatic activity. The parasite enzyme is unusual in having a strict substrate specificity for N-acetylglucosamine 1-phosphate and in being located inside a peroxisome-like microbody, the glycosome. A bloodstream form T. brucei conditional null mutant was constructed and shown to be unable to sustain growth in vitro or in vivo under nonpermissive conditions, demonstrating that there are no alternative metabolic or nutritional routes to UDP-N-acetylglucosamine and providing a genetic validation for the enzyme as a potential drug target. The conditional null mutant was also used to investigate the effects of N-acetylglucosamine starvation in the parasite. After 48 h under nonpermissive conditions, about 24 h before cell lysis, the status of parasite glycoprotein glycosylation was assessed. Under these conditions, UDP-N-acetylglucosamine levels were less than 5% of wild type. Lectin blotting and fluorescence microscopy with tomato lectin revealed that poly-N-acetyllactosamine structures were greatly reduced in the parasite. The principal parasite surface coat component, the variant surface glycoprotein, was also analyzed. Endoglycosidase digestions and mass spectrometry showed that, under UDP-N-acetylglucosamine starvation, the variant surface glycoprotein was specifically underglycosylated at its C-terminal Asn-428 N-glycosylation site. The significance of this finding, with respect to the hierarchy of site-specific N-glycosylation in T. brucei, is discussed.

Taking advantage of the known planarity of the N-acetyl group of N-acetylglucosamine, an analysis of the quality of carbohydrate structures found in the protein databank was performed. Few obvious defects of the local geometry of the carbonyl group were observed. However, the N-acetyl group was often found in the less favorable cis conformation (12% of the cases). It was also found severely twisted in numerous instances, especially in structures with a resolution poorer than 1.9 Å determined between 2000 and 2015. Though the automated PDB-REDO procedure has proved able to improve nearly 85% of the structural models deposited to the PDB, and does prove able to cure most severely twisted conformations of the N-acetyl group, it fails to correct its high rate of cis conformations. More generally, for structures with a resolution poorer than 1.6 Å, it produces N-acetylglucosamine models in slightly poorer agreement with experimental data, as measured using real-space correlation coefficients. Significant improvements are thus still needed, at least as far as this carbohydrate structure is concerned.

The first step of lipid A biosynthesis in Escherichia coli (E. coli) is catalyzed by LpxA (EcLpxA), an acyltransferase selective for UDP-N-acetylglucosamine (UDP-GlcNAc) and R-3-hydroxymyristoyl-acyl carrier protein (3-OH-C14-ACP), and is an essential step in majority of Gram-negative bacteria. Since the majority of lipid A species isolated from F. novicida contains 3-OH-C16 or 3-OH-C18 at its C3 and C3' positions, FnLpxA was thought to be selective for longer acyl chain (3-OH-C16 and 3-OH-C18) over short acyl chain (3-OH-C14, 3-OH-C12, and 3-OH-C10). Here we demonstrate that Francisella novicida (F. novicida) lpxA functionally complements an E. coli lpxA knockout mutant and efficiently transfers 3-OH-C14 as well as 3-OH-C16 in E. coli. Our results implicate that the acyl chain length of lipid A is determined by several factors including acyl chain selectivity of LpxA and downstream enzymes, as well as the composition of the acyl-ACP pool in vivo. We also report the crystal structure of F. novicida LpxA (FnLpxA) at 2.06 Å. The N-terminal parallel beta-helix (LβH) and C-terminal alpha-helical domain are similar to other reported structures of LpxAs. However, our structure indicates that the supposed ruler residues for hydrocarbon length, 171L in one monomer and 168H in the adjacent monomer in a functional trimer of FnLpxA, are located just 3.8 Å apart that renders not enough space for binding of 3-OH-C12 or longer acyl chains. This implicates that FnLpxA may have an alternative hydrophobic pocket, or the acyl chain may bend while binding to FnLpxA. In addition, the FnLpxA structure suggests a potential inhibitor binding site for development of antibiotics.

The balance between bone formation and bone resorption is maintained by osteoblasts and osteoclasts, and an imbalance in this bone metabolism leads to osteoporosis. Here, we found that osteoblast differentiation in MC3T3-E1 cells is promoted by the inactivation of O-linked β-N-acetylglucosaminidase (O-GlcNAcase) and suppressed by the inactivation of O-GlcNAc transferase, as indicated by extracellular matrix calcification. The expression of osteogenic genes such as alp, ocn, and bsp during osteoblast differentiation was positively regulated in a O-GlcNAc glycosylation-dependent manner. Because it was confirmed that Ets1 and Runx2 are the two key transcription factors responsible for the expression of these osteogenic genes, their transcriptional activity might therefore be regulated by O-GlcNAc glycosylation. However, osteoclast differentiation of RAW264 cells, as indicated by the expression and activity of tartrate-resistant acid phosphatase, was unaffected by the inactivation of either O-GlcNAcase or O-GlcNAc transferase. Our findings suggest that an approach to manipulate O-GlcNAc glycosylation could be useful for developing the therapeutics for osteoporosis.

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is an acetylated amino sugar nucleotide that naturally serves as precursor in bacterial cell wall synthesis and is involved in prokaryotic and eukaryotic glycosylation reactions. UDP-GlcNAc finds application in various fields including the production of oligosaccharides and glycoproteins with therapeutic benefits. At present, nucleotide sugars are produced either chemically or in vitro by enzyme cascades. However, chemical synthesis is complex and non-economical, and in vitro synthesis requires costly substrates and often purified enzymes. A promising alternative is the microbial production of nucleotide sugars from cheap substrates. In this study, we aimed to engineer the non-pathogenic, Gram-positive soil bacterium Corynebacterium glutamicum as a host for UDP-GlcNAc production. The native glmS, glmU, and glmM genes and glmM of Escherichia coli, encoding the enzymes for UDP-GlcNAc synthesis from fructose-6-phosphate, were over-expressed in different combinations and from different plasmids in C. glutamicum GRS43, which lacks the glucosamine-6-phosphate deaminase gene (nagB) for glucosamine degradation. Over-expression of glmS, glmU and glmM, encoding glucosamine-6-phosphate synthase, the bifunctional glucosamine-1-phosphate acetyltransferase/N-acetyl glucosamine-1-phosphate uridyltransferase and phosphoglucosamine mutase, respectively, was confirmed using activity assays or immunoblot analysis. While the reference strain C. glutamicum GlcNCg1 with an empty plasmid in the exponential growth phase contained intracellularly only about 0.25 mM UDP-GlcNAc, the best engineered strain GlcNCg4 accumulated about 14 mM UDP-GlcNAc. The extracellular UDP-GlcNAc concentrations in the exponential growth phase did not exceed 2 mg/L. In the stationary phase, about 60 mg UDP-GlcNAc/L was observed extracellularly with strain GlcNCg4, indicating the potential of C. glutamicum to produce and to release the activated sugar into the culture medium. To our knowledge, the observed UDP-GlcNAc levels are the highest obtained with microbial hosts, emphasizing the potential of C. glutamicum as a suitable platform for activated sugar production.

The ever increasing number of antibiotic resistant bacteria has fuelled interest in the development of new antibiotics and other antibacterial agents. The major structural element of the bacterial cell wall is the heteropolymer peptidoglycan and the enzymes of peptidoglycan biosynthesis are potential targets for antibacterial agents. One such enzyme is UDP-N-acetylglucosamine enolpyruvyltransferase (EPT) which catalyzes the first committed step in peptidoglycan biosynthesis: the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 3-hydroxyl of UDP-N-acetylglucosamine (UDPGlcNAc). EPT is of potential pharmaceutical interest because it is inhibited by the broad spectrum antibiotic fosfomycin.