Search

O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a dynamic post-translational modification that regulates thousands of proteins and almost all cellular processes. Aberrant O-GlcNAcylation has been associated with numerous diseases, including cancer, neurodegenerative diseases, cardiovascular diseases, and type 2 diabetes. O-GlcNAcylation is highly nutrient-sensitive since it is dependent on UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). We previously observed daily rhythmicity of protein O-GlcNAcylation in a Drosophila model that is sensitive to the timing of food consumption. We showed that the circadian clock is pivotal in regulating daily O-GlcNAcylation rhythms given its control of the feeding-fasting cycle and hence nutrient availability. Interestingly, we reported that the circadian clock also modulates daily O-GlcNAcylation rhythm by regulating molecular mechanisms beyond the regulation of food consumption time. A large body of work now indicates that O-GlcNAcylation is likely a generalized cellular status effector as it responds to various cellular signals and conditions, such as ER stress, apoptosis, and infection. In this review, we summarize the metabolic regulation of protein O-GlcNAcylation through nutrient availability, HBP enzymes, and O-GlcNAc processing enzymes. We discuss the emerging roles of circadian clocks in regulating daily O-GlcNAcylation rhythm. Finally, we provide an overview of other cellular signals or conditions that impact O-GlcNAcylation. Many of these cellular pathways are themselves regulated by the clock and/or metabolism. Our review highlights the importance of maintaining optimal O-GlcNAc rhythm by restricting eating activity to the active period under physiological conditions and provides insights into potential therapeutic targets of O-GlcNAc homeostasis under pathological conditions.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

BACKGROUND: In the demyelinating disease multiple sclerosis (MS), chronic-active brain inflammation, remyelination failure and neurodegeneration remain major issues despite immunotherapy. While B cell depletion and blockade/sequestration of T and B cells potently reduces episodic relapses, they act peripherally to allow persistence of chronic-active brain inflammation and progressive neurological dysfunction. N-acetyglucosamine (GlcNAc) is a triple modulator of inflammation, myelination and neurodegeneration. GlcNAc promotes biosynthesis of Asn (N)-linked-glycans, which interact with galectins to co-regulate the clustering/signaling/endocytosis of multiple glycoproteins simultaneously. In mice, GlcNAc crosses the blood brain barrier to raise N-glycan branching, suppress inflammatory demyelination by T and B cells and trigger stem/progenitor cell mediated myelin repair. MS clinical severity, demyelination lesion size and neurodegeneration inversely associate with a marker of endogenous GlcNAc, while in healthy humans, age-associated increases in endogenous GlcNAc promote T cell senescence. OBJECTIVES AND METHODS: An open label dose-escalation mechanistic trial of oral GlcNAc at 6 g (n = 18) and 12 g (n = 16) for 4 weeks was performed in MS patients on glatiramer acetate and not in relapse from March 2016 to December 2019 to assess changes in serum GlcNAc, lymphocyte N-glycosylation and inflammatory markers. Post-hoc analysis examined changes in serum neurofilament light chain (sNfL) as well as neurological disability via the Expanded Disability Status Scale (EDSS). RESULTS: Prior to GlcNAc therapy, high serum levels of the inflammatory cytokines IFNγ, IL-17 and IL-6 associated with reduced baseline levels of a marker of endogenous serum GlcNAc. Oral GlcNAc therapy was safe, raised serum levels and modulated N-glycan branching in lymphocytes. Glatiramer acetate reduces TH1, TH17 and B cell activity as well as sNfL, yet the addition of oral GlcNAc dose-dependently lowered serum IFNγ, IL-17, IL-6 and NfL. Oral GlcANc also dose-dependently reduced serum levels of the anti-inflammatory cytokine IL-10, which is increased in the brain of MS patients. 30% of treated patients displayed confirmed improvement in neurological disability, with an average EDSS score decrease of 0.52 points. CONCLUSIONS: Oral GlcNAc inhibits inflammation and neurodegeneration markers in MS patients despite concurrent immunomodulation by glatiramer acetate. Blinded studies are required to investigate GlcNAcs potential to control residual brain inflammation, myelin repair and neurodegeneration in MS.

The rapid emergence and global dissemination of SARS-CoV-2 that causes COVID-19 continues to cause an unprecedented global health burden resulting in nearly 7 million deaths. While multiple vaccine countermeasures have been approved for emergency use, additional treatments are still needed due to sluggish vaccine rollout, vaccine hesitancy, and inefficient vaccine-mediated protection. Immunoadjuvant compounds delivered intranasally can guide non-specific innate immune responses during the critical early stages of viral replication, reducing morbidity and mortality. N-dihydrogalactochitosan (GC) is a novel mucoadhesive immunostimulatory polymer of β-0-4-linked N-acetylglucosamine that is solubilized by the conjugation of galactose glycans with current applications as a cancer immunotherapeutic. We tested GC as a potential countermeasure for COVID-19. GC was well-tolerated and did not produce histopathologic lesions in the mouse lung. GC administered intranasally before and after SARS-CoV-2 exposure diminished morbidity and mortality in humanized ACE2 receptor expressing mice by up to 75% and reduced infectious virus levels in the upper airway. Fluorescent labeling of GC shows that it is confined to the lumen or superficial mucosa of the nasal cavity, without involvement of adjacent or deeper tissues. Our findings demonstrate a new application for soluble immunoadjuvants such as GC for preventing disease associated with SARS-CoV-2 and may be particularly attractive to persons who are needle-averse.

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a nutrient-sensitive protein modification that alters the structure and function of a wide range of proteins involved in diverse cellular processes. Similar to phosphorylation, another protein modification that targets serine and threonine residues, O-GlcNAcylation occupancy on cellular proteins exhibits daily rhythmicity and has been shown to play critical roles in regulating daily rhythms in biology by modifying circadian clock proteins and downstream effectors. We recently reported that daily rhythm in global O-GlcNAcylation observed in Drosophila tissues is regulated via the integration of circadian and metabolic signals. Significantly, mistimed feeding, which disrupts coordination of these signals, is sufficient to dampen daily O-GlcNAcylation rhythm and is predicted to negatively impact animal biological rhythms and health span. In this review, we provide an overview of published and potential mechanisms by which metabolic and circadian signals regulate hexosamine biosynthetic pathway metabolites and enzymes, as well as O-GlcNAc processing enzymes to shape daily O-GlcNAcylation rhythms. We also discuss the significance of functional interactions between O-GlcNAcylation and other post-translational modifications in regulating biological rhythms. Finally, we highlight organ/tissue-specific cellular processes and molecular pathways that could be modulated by rhythmic O-GlcNAcylation to regulate time-of-day-specific biology.

All living organisms have the ability to sense nutrient levels to coordinate cellular metabolism. Despite the importance of nutrient-sensing pathways that detect the levels of amino acids and glucose, how the availability of these two types of nutrients is integrated is unclear. Here, we show that glucose availability regulates the central nutrient effector mTORC1 through intracellular leucine sensor leucyl-tRNA synthetase 1 (LARS1). Glucose starvation results in O-GlcNAcylation of LARS1 on residue S1042. This modification inhibits the interaction of LARS1 with RagD GTPase and reduces the affinity of LARS1 for leucine by promoting phosphorylation of its leucine-binding site by the autophagy-activating kinase ULK1, decreasing mTORC1 activity. The lack of LARS1 O-GlcNAcylation constitutively activates mTORC1, supporting its ability to sense leucine, and deregulates protein synthesis and leucine catabolism under glucose starvation. This work demonstrates that LARS1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.

A decline in skeletal muscle mass and low muscular strength are prognostic factors in advanced human cancers. Here we found that breast cancer suppressed O-linked N-acetylglucosamine (O-GlcNAc) protein modification in muscle through extracellular-vesicle-encapsulated miR-122, which targets O-GlcNAc transferase (OGT). Mechanistically, O-GlcNAcylation of ryanodine receptor 1 (RYR1) competed with NEK10-mediated phosphorylation and increased K48-linked ubiquitination and proteasomal degradation; the miR-122-mediated decrease in OGT resulted in increased RYR1 abundance. We further found that muscular protein O-GlcNAcylation was regulated by hypoxia and lactate through HIF1A-dependent OGT promoter activation and was elevated after exercise. Suppressed O-GlcNAcylation in the setting of cancer, through increasing RYR1, led to higher cytosolic Ca2+ and calpain protease activation, which triggered cleavage of desmin filaments and myofibrillar destruction. This was associated with reduced skeletal muscle mass and contractility in tumour-bearing mice. Our findings link O-GlcNAcylation to muscular protein homoeostasis and contractility and reveal a mechanism of cancer-associated muscle dysregulation.

[Figure: see text].

The integration of circadian and metabolic signals is essential for maintaining robust circadian rhythms and ensuring efficient metabolism and energy use. Using Drosophila as an animal model, we show that cellular protein O-GlcNAcylation exhibits robust 24-hour rhythm and represents a key post-translational mechanism that regulates circadian physiology. We observe strong correlation between protein O-GlcNAcylation rhythms and clock-controlled feeding-fasting cycles, suggesting that O-GlcNAcylation rhythms are primarily driven by nutrient input. Interestingly, daily O-GlcNAcylation rhythms are severely dampened when we subject flies to time-restricted feeding at unnatural feeding time. This suggests the presence of clock-regulated buffering mechanisms that prevent excessive O-GlcNAcylation at non-optimal times of the day-night cycle. We show that this buffering mechanism is mediated by the expression and activity of GFAT, OGT, and OGA, which are regulated through integration of circadian and metabolic signals. Finally, we generate a mathematical model to describe the key factors that regulate daily O-GlcNAcylation rhythm.

[Figure: see text].

O-GlcNAcylation, the addition of a single N-acetylglucosamine residue to serine and threonine residues of cytoplasmic, nuclear, or mitochondrial proteins, is a widespread regulatory posttranslational modification. It is involved in the response to nutritional status and stress, and its dysregulation is associated with diseases ranging from Alzheimer's to diabetes. Although the modification was first detected over 35 years ago, research into the function of O-GlcNAcylation has accelerated dramatically in the last 10 years owing to the development of new enrichment and mass spectrometry techniques that facilitate its analysis. This article summarizes methods for O-GlcNAc enrichment, key mass spectrometry instrumentation advancements, particularly those that allow modification site localization, and software tools that allow analysis of data from O-GlcNAc-modified peptides.

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.

Introduction: Melasma is a human melanogenesis dysfunction that results in localized, chronic acquired hypermelanosis of the skin difficult to treat. Methods: This prospective, randomized, single‐blind, study aimed to compare the efficacy and tolerability of a liposomal emulsion based on Politranexamide® (SAMPLE A) with that of a competitor based on acetylglucosamine, ethyl linoleate and phenyl ethyl resorcinol (SAMPLE B) in patients affected by facial melasma on 26 patients. Disease severity was assessed by the Melasma Area Severity Index (MASI) at baseline and after 6 and 12 weeks of therapy. All patients were subjected to photo documentation using DermaView camera and Antera 3D camera. Results: The mean MASI score at baseline was 10.93 ± 7 in the group A and 9.34 ± 6.29 in the group B, respectively. A significant decrease in MASI score from baseline was noted in both treatment groups as early as 6 weeks of follow‐up (p = 0.00096 for SAMPLE A and p = 0.0049 for SAMPLE B) and was confirmed at the end of the treatment (p = 0.0006 for SAMPLE A and p = 0.00039 for SAMPLE B). Intergroup comparison revealed a greater improvement of melasma among patients in group A compared to those in group B that was quite statistically significant at weeks 6 (p = 0.055009) and significant after 12 weeks of follow‐up (p = 0.032942). Both treatment groups experienced an improvement in Antera average level of melanin. Conclusion: Our results suggested Politranexamide® to be a useful and safe therapeutic option in treating melasma, more effective than competitor used in this study. [ABSTRACT FROM AUTHOR]

The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essentially all tumors when compared to healthy tissue, and lowering O-GlcNAcylation levels results in reduced tumorigenesis and increased cancer cell death. Therefore, the pharmacological reduction of O-GlcNAc may represent a therapeutic vulnerability. The most direct approach to this goal is the inhibition of O-GlcNAc transferase (OGT), the enzyme that directly adds the modification to proteins. However, despite some recent success, this enzyme has proven difficult to inhibit. An alternative strategy involves starving OGT of its sugar substrate UDP-GlcNAc by targeting enzymes of the hexosamine biosynthetic pathway (HBP). Here, we explore the potential of the rate-determining enzyme of this pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). We first show that CRISPR-mediated knockout of GFAT results in inhibition of cancer cell growth in vitro and a xenograft model that correlates with O-GlcNAcylation levels. We then demonstrate that pharmacological inhibition of GFAT sensitizes a small panel of cancer cells to undergo apoptosis in response to diamide-induced oxidative stress. Finally, we find that GFAT expression and O-GlcNAc levels are increased in a spontaneous mouse model of liver cancer. Together these experiments support the further development of inhibitors of the HBP as an indirect approach to lowering O-GlcNAcylation levels in cancer.

Mounting evidence in animal models indicates potential for rejuvenation of cellular and cognitive functions in the aging brain. However, the ability to utilize this potential is predicated on identifying molecular targets that reverse the effects of aging in vulnerable regions of the brain, such as the hippocampus. The dynamic post-translational modification O-linked N-Acetylglucosamine (O-GlcNAc) has emerged as an attractive target for regulating aging-specific synaptic alterations as well as neurodegeneration. While speculation exists about the role of O-GlcNAc in neurodegenerative conditions, such as Alzheimer's disease, its role in physiological brain aging remains largely unexplored. Here, we report that countering age-related decreased O-GlcNAc transferase (OGT) expression and O-GlcNAcylation ameliorates cognitive impairments in aged mice. Mimicking an aged condition in young adults by abrogating OGT, using a temporally controlled neuron-specific conditional knockout mouse model, recapitulated cellular and cognitive features of brain aging. Conversely, overexpressing OGT in mature hippocampal neurons using a viral-mediated approach enhanced associative fear memory in young adult mice. Excitingly, in aged mice overexpressing neuronal OGT in the aged hippocampus rescued in part age-related impairments in spatial learning and memory as well as associative fear memory. Our data identify O-GlcNAcylaton as a key molecular mediator promoting cognitive rejuvenation.

KRAS mutations are the earliest events found in approximately 90% of pancreatic ductal adenocarcinomas (PDACs). However, little is known as to why KRAS mutations preferentially occur in PDACs and what processes/factors generate these mutations. While abnormal carbohydrate metabolism is associated with a high risk of pancreatic cancer, it remains elusive whether a direct relationship between KRAS mutations and sugar metabolism exists. Here, we show that under high-glucose conditions, cellular O-GlcNAcylation is significantly elevated in pancreatic cells that exhibit lower phosphofructokinase (PFK) activity than other cell types. This post-translational modification specifically compromises the ribonucleotide reductase (RNR) activity, leading to deficiency in dNTP pools, genomic DNA alterations with KRAS mutations, and cellular transformation. These results establish a mechanistic link between a perturbed sugar metabolism and genomic instability that induces de novo oncogenic KRAS mutations preferentially in pancreatic cells.

Regulatory T (Treg) cells control self-tolerance, inflammatory responses and tissue homeostasis. In mature Treg cells, continued expression of FOXP3 maintains lineage identity, while T cell receptor (TCR) signaling and interleukin-2 (IL-2)/STAT5 activation support the suppressive effector function of Treg cells, but how these regulators synergize to control Treg cell homeostasis and function remains unclear. Here we show that TCR-activated posttranslational modification by O-linked N-Acetylglucosamine (O-GlcNAc) stabilizes FOXP3 and activates STAT5, thus integrating these critical signaling pathways. O-GlcNAc-deficient Treg cells develop normally but display modestly reduced FOXP3 expression, strongly impaired lineage stability and effector function, and ultimately fatal autoimmunity in mice. Moreover, deficiency in protein O-GlcNAcylation attenuates IL-2/STAT5 signaling, while overexpression of a constitutively active form of STAT5 partially ameliorates Treg cell dysfunction and systemic inflammation in O-GlcNAc deficient mice. Collectively, our data demonstrate that protein O-GlcNAcylation is essential for lineage stability and effector function in Treg cells.

An efficient streamlined chemoenzymatic approach has been developed for gram-scale synthesis of Lewis a angtigen (LeaβProN3) and a library of sialyl Lewis a antigens (sLeaβProN3) containing different sialic acid forms. Intially, commercially available inexpensive N-acetylglucosamine (GlcNAc) was converted to its N'-glycosyl p-toluenesulfonohydrazide in one step. Followed by chemical glycosylation, GlcNAcβProN3 was synthesized using this protecting group-free method in high yield (82%). Sequential one-pot multienzyme (OPME) β1-3-galactosylation of GlcNAcβProN3 followed by OPME α1-4-fucosylation reactions produced target LeaβProN3 in gram-scale. Structurally diverse sialic acid forms was successfully introduced using a OPME sialylation reation containing a CMP-sialic acid synthetase and Pasteurella multocida α2-3-sialyltransferase 1 (PmST1) mutant PmST1 M144D with or without a sialic acid aldolase to form sLeaβProN3 containing naturally occurring or non-natural sialic acid forms in preparative scales.

N-acetylglucosamine (GlcNAc) branching of Asn (N)-linked glycans inhibits pro-inflammatory T cell responses and models of autoimmune diseases such as Multiple Sclerosis (MS). Metabolism controls N-glycan branching in T cells by regulating de novo hexosamine pathway biosynthesis of UDP-GlcNAc, the donor substrate for the Golgi branching enzymes. Activated T cells switch metabolism from oxidative phosphorylation to aerobic glycolysis and glutaminolysis. This reduces flux of glucose and glutamine into the hexosamine pathway, thereby inhibiting de novo UDP-GlcNAc synthesis and N-glycan branching. Salvage of GlcNAc into the hexosamine pathway overcomes this metabolic suppression to restore UDP-GlcNAc synthesis and N-glycan branching, thereby promoting anti-inflammatory T regulatory (Treg) over pro-inflammatory T helper (TH) 17 and TH1 differentiation to suppress autoimmunity. However, GlcNAc activity is limited by the lack of a cell surface transporter and requires high doses to enter cells via macropinocytosis. Here we report that GlcNAc-6-acetate is a superior pro-drug form of GlcNAc. Acetylation of amino-sugars improves cell membrane permeability, with subsequent de-acetylation by cytoplasmic esterases allowing salvage into the hexosamine pathway. Per- and bi-acetylation of GlcNAc led to toxicity in T cells, whereas mono-acetylation at only the 6 > 3 position raised N-glycan branching greater than GlcNAc without inducing significant toxicity. GlcNAc-6-acetate inhibited T cell activation/proliferation, TH1/TH17 responses and disease progression in Experimental Autoimmune Encephalomyelitis (EAE), a mouse model of MS. Thus, GlcNAc-6-Acetate may provide an improved therapeutic approach to raise N-glycan branching, inhibit pro-inflammatory T cell responses and treat autoimmune diseases such as MS.

Understanding the cellular properties controlling neural stem and progenitor cell (NSPC) fate choice will improve their therapeutic potential. The electrophysiological measure whole-cell membrane capacitance reflects fate bias in the neural lineage but the cellular properties underlying membrane capacitance are poorly understood. We tested the hypothesis that cell surface carbohydrates contribute to NSPC membrane capacitance and fate. We found NSPCs differing in fate potential express distinct patterns of glycosylation enzymes. Screening several glycosylation pathways revealed that the one forming highly branched N-glycans differs between neurogenic and astrogenic populations of cells in vitro and in vivo. Enhancing highly branched N-glycans on NSPCs significantly increases membrane capacitance and leads to the generation of more astrocytes at the expense of neurons with no effect on cell size, viability, or proliferation. These data identify the N-glycan branching pathway as a significant regulator of membrane capacitance and fate choice in the neural lineage.

Glycosylinositol phosphorylceramides (GIPCs), which have a ceramide core linked to a glycan headgroup of varying structures, are the major sphingolipids in the plant plasma membrane. Recently, we identified the major biosynthetic genes for GIPC glycosylation in Arabidopsis (Arabidopsis thaliana) and demonstrated that the glycan headgroup is essential for plant viability. However, the function of GIPCs and the significance of their structural variation are poorly understood. Here, we characterized the Arabidopsis glycosyltransferase GLUCOSAMINE INOSITOLPHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) and showed that it is responsible for the glycosylation of a subgroup of GIPCs found in seeds and pollen that contain GlcNAc and GlcN [collectively GlcN(Ac)]. In Arabidopsis gint1 plants, loss of the GlcN(Ac) GIPCs did not affect vegetative growth, although seed germination was less sensitive to abiotic stress than in wild-type plants. However, in rice, where GlcN(Ac) containing GIPCs are the major GIPC subgroup in vegetative tissue, loss of GINT1 was seedling lethal. Furthermore, we could produce, de novo, "rice-like" GlcN(Ac) GIPCs in Arabidopsis leaves, which allowed us to test the function of different sugars in the GIPC headgroup. This study describes a monocot GIPC biosynthetic enzyme and shows that its Arabidopsis homolog has the same biochemical function. We also identify a possible role for GIPCs in maintaining cell-cell adhesion.

Bone loss and fractures are underrecognized complications of type 1 diabetes and are primarily due to impaired bone formation by osteoblasts. The mechanisms leading to osteoblast dysfunction in diabetes are incompletely understood, but insulin deficiency, poor glycemic control, and hyperglycemia-induced oxidative stress likely contribute. Here we show that insulin promotes osteoblast proliferation and survival via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signal transduction pathway and that PKG stimulation of Akt provides a positive feedback loop. In osteoblasts exposed to high glucose, NO/cGMP/PKG signaling was reduced due in part to the addition of O-linked N-acetylglucosamine to NO synthase-3, oxidative inhibition of guanylate cyclase activity, and suppression of PKG transcription. Cinaciguat-an NO-independent activator of oxidized guanylate cyclase-increased cGMP synthesis under diabetic conditions and restored proliferation, differentiation, and survival of osteoblasts. Cinaciguat increased trabecular and cortical bone in mice with type 1 diabetes by improving bone formation and osteocyte survival. In bones from diabetic mice and in osteoblasts exposed to high glucose, cinaciguat reduced oxidative stress via PKG-dependent induction of antioxidant genes and downregulation of excess NADPH oxidase-4-dependent H2O2 production. These results suggest that cGMP-elevating agents could be used as an adjunct treatment for diabetes-associated osteoporosis.

Many pathogenic bacteria, including Streptococcus gordonii, possess a pathway for the cellular export of a single serine-rich-repeat protein that mediates the adhesion of bacteria to host cells and the extracellular matrix. This adhesin protein is O-glycosylated by several cytosolic glycosyltransferases and requires three accessory Sec proteins (Asp1-3) for export, but how the adhesin protein is processed for export is not well understood. Here, we report that the S. gordonii adhesin GspB is sequentially O-glycosylated by three enzymes (GtfA/B, Nss, and Gly) that attach N-acetylglucosamine and glucose to Ser/Thr residues. We also found that modified GspB is transferred from the last glycosyltransferase to the Asp1/2/3 complex. Crystal structures revealed that both Asp1 and Asp3 are related to carbohydrate-binding proteins, suggesting that they interact with carbohydrates and bind glycosylated adhesin, a notion that was supported by further analyses. We further observed that Asp1 also has an affinity for phospholipids, which is attenuated by Asp2. In summary, our findings support a model in which the GspB adhesin is sequentially glycosylated by GtfA/B, Nss, and Gly and then transferred to the Asp1/2/3 complex in which Asp1 mediates the interaction of the Asp1/2/3 complex with the lipid bilayer for targeting of matured GspB to the export machinery.

Classification of streptococci is based upon expression of unique cell wall carbohydrate antigens. All serotypes of group A Streptococcus (GAS; Streptococcus pyogenes), a leading cause of infection-related mortality worldwide, express the group A carbohydrate (GAC). GAC, the classical Lancefield antigen, is comprised of a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side chains. The immunodominant GlcNAc epitope of GAC is the basis of all rapid diagnostic testing for GAS infection. We previously identified the 12-gene GAC biosynthesis gene cluster and determined that the glycosyltransferase GacI was required for addition of the GlcNAc side chain to the polyrhamnose core. Loss of the GAC GlcNAc epitope in serotype M1 GAS resulted in attenuated virulence in two animal infection models and increased GAS sensitivity to killing by whole human blood, serum, neutrophils, and antimicrobial peptides. Here, we report that the GAC biosynthesis gene cluster is ubiquitous among 520 GAS isolates from global sources, representing 105 GAS emm serotypes. Isogenic ΔgacI mutants were constructed in M2, M3, M4, M28, and M89 backgrounds and displayed an array of phenotypes in susceptibility to killing by whole human blood, baby rabbit serum, human platelet releasate, human neutrophils, and antimicrobial peptide LL-37. The contribution of the GlcNAc side chain to GAS survival in vivo also varied by strain, demonstrating that it is not a prerequisite for virulence in the murine infection model. Thus, the relative contribution of GAC to virulence in non-M1 serotypes appears to depend on the quorum of other virulence factors that each strain possesses.IMPORTANCE The Lancefield group A carbohydrate (GAC) is the species-defining antigen for group A Streptococcus (GAS), comprising ~50% of the cell wall of this major human pathogen. We previously showed that the GlcNAc side chain of GAC contributes to the innate immune resistance and animal virulence phenotypes of the globally disseminated strain of serotype M1 GAS. Here, we use isogenic mutagenesis to examine the role of GAC GlcNAc in five additional medically relevant GAS serotypes. Overall, the GlcNAc side chain of GAC contributes to the innate immune resistance of GAS, but the relative contribution varies among individual strains. Moreover, the GAC GlcNAc side chain is not a universal prerequisite for GAS virulence in the animal model.

Amino sugars are good sources of both ammonia and fructose-6-phosphate, produced by the glucosamine 6-phosphate deaminase, NagB. NagB is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the phosphocarrier protein of the bacterial phosphotransferase system, HPr, in Escherichia coli We provide evidence that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein (U-PII) also allosterically activate NagB by direct protein-protein interactions. NanE is essential for neuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and PII is known to be a central metabolic nitrogen regulator. We demonstrate that uridylylated PII (but not underivatized PII) activates NagB >10-fold at low concentrations of substrate, whereas NanE increases NagB activity >2-fold. NanE activates NagB in the absence or presence of GlcNAc-6P, but HPr and U-PII activation requires the presence of GlcNAc-6P. Activation of NagB by HPr and uridylylated PII, as well as by NanE and HPr (but not by NanE and U-PII), is synergistic, and the modeling, which suggests specific residues involved in complex formation, provides possible explanations. Specific physiological functions for the regulation of NagB by its three protein activators are proposed. Each regulatory agent is suggested to mediate signal transduction in response to a different stimulus.IMPORTANCE The regulation of amino sugar utilization is important for the survival of bacteria in a competitive environment. NagB, a glucosamine 6-phosphate deaminase in Escherichia coli, is essential for amino sugar utilization and is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the histidine-phosphorylatable phosphocarrier protein, HPr. We provide evidence here that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein allosterically activate NagB by direct protein-protein interactions. NanE is essential for N-acetylneuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and the PII protein is known to be a central metabolic nitrogen regulator. Regulatory links between carbon and nitrogen metabolism are important for adaptation of metabolism to different growth conditions.

The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multiuse mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition "click" reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1)-(3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation.

BackgroundProtein O-linked-β-N-acetyl glucosamine (O-GlcNAc) is a post-translational modification to Ser/Thr residues that integrates energy supply with demand. Abnormal O-GlcNAc patterning is evident in several neurological disease states including epilepsy, Alzheimer's disease and autism spectrum disorder (ASD). A potential treatment option for these disorders includes the high-fat, low-carbohydrate, ketogenic diet (KD). The goal of this study was to determine whether the KD induces changes in O-GlcNAc in the BTBRT+tf/j (BTBR) mouse model of ASD.MethodsJuvenile male (5weeks), age-matched C57 or BTBR mice consumed a chow diet (13% kcal fat) or KD (75% kcal fat) for 10-14days. Following these diets, brain (prefrontal cortex) and liver were examined for gene expression levels of key O-GlcNAc mediators, global and protein specific O-GlcNAc as well as indicators of energy status.ResultsThe KD reduced global O-GlcNAc in the livers of all animals (p0.05), yet OGT and OGA expression (mRNA) were elevated in both C57 and BTBR animals (p

The Nrf1 (Nuclear factor E2-related factor 1) transcription factor performs a critical role in regulating cellular homeostasis. Using a proteomic approach, we identified Host Cell Factor-1 (HCF1), a co-regulator of transcription, and O-GlcNAc transferase (OGT), the enzyme that mediates protein O-GlcNAcylation, as cellular partners of Nrf1a, an isoform of Nrf1. Nrf1a directly interacts with HCF1 through the HCF1 binding motif (HBM), while interaction with OGT is mediated through HCF1. Overexpression of HCF1 and OGT leads to increased Nrf1a protein stability. Addition of O-GlcNAc decreases ubiquitination and degradation of Nrf1a. Transcriptional activation by Nrf1a is increased by OGT overexpression and treatment with PUGNAc. Together, these data suggest that OGT can act as a regulator of Nrf1a.

Genetic studies have shown essential functions of O-linked N-acetylglucosamine (O-GlcNAc) modification in plants. However, the proteins and sites subject to this posttranslational modification are largely unknown. Here, we report a large-scale proteomic identification of O-GlcNAc-modified proteins and sites in the model plant Arabidopsis thaliana Using lectin weak affinity chromatography to enrich modified peptides, followed by mass spectrometry, we identified 971 O-GlcNAc-modified peptides belonging to 262 proteins. The modified proteins are involved in cellular regulatory processes, including transcription, translation, epigenetic gene regulation, and signal transduction. Many proteins have functions in developmental and physiological processes specific to plants, such as hormone responses and flower development. Mass spectrometric analysis of phosphopeptides from the same samples showed that a large number of peptides could be modified by either O-GlcNAcylation or phosphorylation, but cooccurrence of the two modifications in the same peptide molecule was rare. Our study generates a snapshot of the O-GlcNAc modification landscape in plants, indicating functions in many cellular regulation pathways and providing a powerful resource for further dissecting these functions at the molecular level.

O-GlcNAc glycosylation (or O-GlcNAcylation) is a dynamic, inducible posttranslational modification found on proteins associated with neurodegenerative diseases such as α-synuclein, amyloid precursor protein, and tau. Deletion of the O-GlcNAc transferase (ogt) gene responsible for the modification causes early postnatal lethality in mice, complicating efforts to study O-GlcNAcylation in mature neurons and to understand its roles in disease. Here, we report that forebrain-specific loss of OGT in adult mice leads to progressive neurodegeneration, including widespread neuronal cell death, neuroinflammation, increased production of hyperphosphorylated tau and amyloidogenic Aβ-peptides, and memory deficits. Furthermore, we show that human cortical brain tissue from Alzheimer's disease patients has significantly reduced levels of OGT protein expression compared with cortical tissue from control individuals. Together, these studies indicate that O-GlcNAcylation regulates pathways critical for the maintenance of neuronal health and suggest that dysfunctional O-GlcNAc signaling may be an important contributor to neurodegenerative diseases.

Intracellular GlcNAcylation of Ser and Thr residues is a well-known and widely investigated post-translational modification. This post-translational modification has been shown to play a significant role in cell signaling and in many regulatory processes within cells. O-GlcNAc transferase is the enzyme responsible for glycosylating cytosolic and nuclear proteins with a single GlcNAc residue on Ser and Thr side-chains. Here we report that the same enzyme may also be responsible for S-GlcNAcylation, i.e. for linking the GlcNAc unit to the peptide by modifying a cysteine side-chain. We also report that O-GlcNAcase, the enzyme responsible for removal of O-GlcNAcylation does not appear to remove the S-linked sugar. Such Cys modifications have been detected and identified in mouse and rat samples. This work has established the occurrence of 14 modification sites assigned to 11 proteins unambiguously. We have also identified S-GlcNAcylation from human Host Cell Factor 1 isolated from HEK-cells. Although these site assignments are primarily based on electron-transfer dissociation mass spectra, we also report that S-linked GlcNAc is more stable under collisional activation than O-linked GlcNAc derivatives.

N-acetylglucosamine (GlcNAc) exists ubiquitously as a component of the surface on a wide range of cells, from bacteria to humans. Many fungi are able to utilize environmental GlcNAc to support growth and induce cellular development, a property important for their survival in various host niches. However, how the GlcNAc signal is sensed and subsequently transduced is largely unknown. Here, we identify a gene that is essential for GlcNAc signalling (NGS1) in Candida albicans, a commensal and pathogenic yeast of humans. Ngs1 can bind GlcNAc through the N-terminal β-N-acetylglucosaminidase homology domain. This binding activates N-acetyltransferase activity in the C-terminal GCN5-related N-acetyltransferase domain, which is required for GlcNAc-induced promoter histone acetylation and transcription. Ngs1 is targeted to the promoters of GlcNAc-inducible genes constitutively by the transcription factor Rep1. Ngs1 is conserved in diverse fungi that have GlcNAc catabolic genes. Thus, fungi use Ngs1 as a GlcNAc-sensor and transducer for GlcNAc-induced transcription.

UnlabelledSchwann cells (SCs), ensheathing glia of the peripheral nervous system, support axonal survival and function. Abnormalities in SC metabolism affect their ability to provide this support and maintain axon integrity. To further interrogate this metabolic influence on axon-glial interactions, we generated OGT-SCKO mice with SC-specific deletion of the metabolic/nutrient sensing protein O-GlcNAc transferase that mediates the O-linked addition of N-acetylglucosamine (GlcNAc) moieties to Ser and Thr residues. The OGT-SCKO mice develop tomaculous demyelinating neuropathy characterized by focal thickenings of the myelin sheath (tomacula), progressive demyelination, axonal loss, and motor and sensory nerve dysfunction. Proteomic analysis identified more than 100 O-GlcNAcylated proteins in rat sciatic nerve, including Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans. PRX lacking O-GlcNAcylation is mislocalized within the myelin sheath of these mutant animals. Furthermore, phenotypes of OGT-SCKO and Prx-deficient mice are very similar, suggesting that metabolic control of PRX O-GlcNAcylation is crucial for myelin maintenance and axonal integrity.Significance statementThe nutrient sensing protein O-GlcNAc transferase (OGT) mediates post-translational O-linked N-acetylglucosamine (GlcNAc) modification. Here we find that OGT functions in Schwann cells (SCs) to maintain normal myelin and prevent axonal loss. SC-specific deletion of OGT (OGT-SCKO mice) causes a tomaculous demyelinating neuropathy accompanied with progressive axon degeneration and motor and sensory nerve dysfunction. We also found Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans, is O-GlcNAcylated. Importantly, phenotypes of OGT-SCKO and Prx mutant mice are very similar, implying that compromised PRX function contributes to the neuropathy of OGT-SCKO mice. This study will be useful in understanding how SC metabolism contributes to PNS function and in developing new strategies for treating peripheral neuropathy by targeting SC function.

O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked β-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma.

Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1β and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor. PAPERCLIP.

The post-translational modification of serine or threonine residues of proteins with a single N-acetylglucosamine monosaccharide (O-GlcNAcylation) is essential for cell survival and function. However, relatively few O-GlcNAc modification sites have been mapped due to the difficulty of enriching and detecting O-GlcNAcylated peptides from complex samples. Here we describe an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labelling followed by copper(i)-catalysed azide-alkyne cycloaddition (CuAAC) installs a new mass spectrometry (MS)-compatible linker designed for facile purification of O-GlcNAcylated proteins from cell lysates. The linker also allows subsequent quantitative release of O-GlcNAcylated proteins for downstream MS analysis. We validate the approach by unambiguously identifying several established O-GlcNAc sites on the proteins α-crystallin and O-GlcNAc transferase (OGT), as well as discovering new, previously unreported sites on OGT. Notably, these novel sites on OGT lie in key functional domains of the protein, underscoring how this site identification method may reveal important biological insights into protein activity and regulation.

The transcription factor SOX2 is central in establishing and maintaining pluripotency. The processes that modulate SOX2 activity to promote pluripotency are not well understood. Here, we show SOX2 is O-GlcNAc modified in its transactivation domain during reprogramming and in mouse embryonic stem cells (mESCs). Upon induction of differentiation SOX2 O-GlcNAcylation at serine 248 is decreased. Replacing wild type with an O-GlcNAc-deficient SOX2 (S248A) increases reprogramming efficiency. ESCs with O-GlcNAc-deficient SOX2 exhibit alterations in gene expression. This change correlates with altered protein-protein interactions and genomic occupancy of the O-GlcNAc-deficient SOX2 compared to wild type. In addition, SOX2 O-GlcNAcylation impairs the SOX2-PARP1 interaction, which has been shown to regulate ESC self-renewal. These findings show that SOX2 activity is modulated by O-GlcNAc, and provide a novel regulatory mechanism for this crucial pluripotency transcription factor.

BackgroundIn 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.ResultsDNA sequence analysis of these genes revealed a number of characteristic nucleotide substitutions including a unique and distinctive guanine insertion that shifts the reading frame and generates a premature stop codon (TGA) 154 bp downstream of the ATG start codon of the HXK1 gene encoding the GlcNAc-kinase, a key enzyme of the GlcNAc catabolic pathway. However, all examined genes produced transcripts even though different levels of expression were observed among the Candida isolates examined. In particular, we found an HXK1-idependent relationship of the NGT1 gene and a considerable influence of the GlcNAc-kinase functionality on the transcription of the DAC1 and NAG1 genes. Additional phenotypic analysis revealed that C. africana isolates are hyperfilamentous in the first 24-48h of growth on filament-inducing media and revert to the yeast morphological form after 72h of incubation on these media.ConclusionsOur results show that C. africana is a natural HXK1 mutant, displaying a number of phenotypic characteristics distinct from typical C. albicans isolates.

AimsTo evaluate the ability of N-acetylglucosamine (GlcNAc) decorated nanoparticles and their cargo to modulate calcium handling in failing cardiac myocytes (CMs).Materials & methodsPrimary CMs isolated from normal and failing hearts were treated with GlcNAc nanoparticles in order to assess the ability of the nanoparticles and their cargo to correct dysfunctional calcium handling in failing myocytes.Results & conclusionGlcNAc particles reduced aberrant calcium release in failing CMs and restored sarcomere function. Additionally, encapsulation of a small calcium-modulating protein, S100A1, in GlcNAc nanoparticles also showed improved calcium regulation. Thus, the development of our bioactive nanoparticle allows for a 'two-hit' treatment, by which the cargo and also the nanoparticle itself can modulate intracellular protein activity.

Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

UnlabelledSingle-celled organisms have different strategies to sense and utilize nutrients in their ever-changing environments. The opportunistic fungal pathogen Candida albicans is a common member of the human microbiota, especially that of the gastrointestinal (GI) tract. An important question concerns how C. albicans gained a competitive advantage over other microbes to become a successful commensal and opportunistic pathogen. Here, we report that C. albicans uses N-acetylglucosamine (GlcNAc), an abundant carbon source present in the GI tract, as a signal for nutrient availability. When placed in water, C. albicans cells normally enter the G0 phase and remain viable for weeks. However, they quickly lose viability when cultured in water containing only GlcNAc. We term this phenomenon GlcNAc-induced cell death (GICD). GlcNAc triggers the upregulation of ribosomal biogenesis genes, alterations of mitochondrial metabolism, and the accumulation of reactive oxygen species (ROS), followed by rapid cell death via both apoptotic and necrotic mechanisms. Multiple pathways, including the conserved cyclic AMP (cAMP) signaling and GlcNAc catabolic pathways, are involved in GICD. GlcNAc acts as a signaling molecule to regulate multiple cellular programs in a coordinated manner and therefore maximizes the efficiency of nutrient use. This adaptive behavior allows C. albicans' more efficient colonization of the gut.ImportanceThe ability to rapidly and appropriately respond to nutrients in the environment is crucial to free-living microorganisms. To maximize the use of available nutrients, microorganisms often use a limiting nutritional component as a signal to coordinate multiple biological processes. The human fungal pathogen Candida albicans uses N-acetylglucosamine (GlcNAc) as a signal for the availability of external nutrient resources. GlcNAc induces rapid cell death in C. albicans due to the constitutive activation of oxidative metabolism and accumulation of reactive oxygen species (ROS), and multiple pathways are involved in its regulation. This study sheds light on the mechanisms of niche specialization of pathogenic fungi and raises the possibility that this cell death pathway could be an unexplored therapeutic target.

Single-molecule techniques can monitor the kinetics of transitions between enzyme open and closed conformations, but such methods usually lack the resolution to observe the underlying transition pathway or intermediate conformational dynamics. We have used a 1 MHz bandwidth carbon nanotube transistor to electronically monitor single molecules of the enzyme T4 lysozyme as it processes substrate. An experimental resolution of 2 μs allowed the direct recording of lysozyme's opening and closing transitions. Unexpectedly, both motions required 37 μs, on average. The distribution of transition durations was also independent of the enzyme's state: either catalytic or nonproductive. The observation of smooth, continuous transitions suggests a concerted mechanism for glycoside hydrolysis with lysozyme's two domains closing upon the polysaccharide substrate in its active site. We distinguish these smooth motions from a nonconcerted mechanism, observed in approximately 10% of lysozyme openings and closings, in which the enzyme pauses for an additional 40-140 μs in an intermediate, partially closed conformation. During intermediate forming events, the number of rate-limiting steps observed increases to four, consistent with four steps required in the stepwise, arrow-pushing mechanism. The formation of such intermediate conformations was again independent of the enzyme's state. Taken together, the results suggest lysozyme operates as a Brownian motor. In this model, the enzyme traces a single pathway for closing and the reverse pathway for enzyme opening, regardless of its instantaneous catalytic productivity. The observed symmetry in enzyme opening and closing thus suggests that substrate translocation occurs while the enzyme is closed.

Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development.

Non-genetic phenotypic variations play a critical role in the adaption to environmental changes in microbial organisms. Candida albicans, a major human fungal pathogen, can switch between several morphological phenotypes. This ability is critical for its commensal lifestyle and for its ability to cause infections. Here, we report the discovery of a novel morphological form in C. albicans, referred to as the "gray" phenotype, which forms a tristable phenotypic switching system with the previously reported white and opaque phenotypes. White, gray, and opaque cell types differ in a number of aspects including cellular and colony appearances, mating competency, secreted aspartyl proteinase (Sap) activities, and virulence. Of the three cell types, gray cells exhibit the highest Sap activity and the highest ability to cause cutaneous infections. The three phenotypes form a tristable phenotypic switching system, which is independent of the regulation of the mating type locus (MTL). Gray cells mate over 1,000 times more efficiently than do white cells, but less efficiently than do opaque cells. We further demonstrate that the master regulator of white-opaque switching, Wor1, is essential for opaque cell formation, but is not required for white-gray transitions. The Efg1 regulator is required for maintenance of the white phenotype, but is not required for gray-opaque transitions. Interestingly, the wor1/wor1 efg1/efg1 double mutant is locked in the gray phenotype, suggesting that Wor1 and Efg1 could function coordinately and play a central role in the regulation of gray cell formation. Global transcriptional analysis indicates that white, gray, and opaque cells exhibit distinct gene expression profiles, which partly explain their differences in causing infections, adaptation ability to diverse host niches, metabolic profiles, and stress responses. Therefore, the white-gray-opaque tristable phenotypic switching system in C. albicans may play a significant role in a wide range of biological aspects in this common commensal and pathogenic fungus.

Studies of AMCase inhibition in mouse models of lung eosinophilic inflammation have produced conflicting results with some studies demonstrating inhibition of eosinophilic inflammation and others not. No studies have investigated the role of AMCase inhibition in eosinophilic esophagitis (EoE). We have used a mouse model of egg (OVA) induced EoE to determine whether pharmacologic inhibition of AMCase with allosamidin reduced eosinophilic inflammation and remodeling in the esophagus in EoE. Administration of intra-esophageal OVA for 6weeks to BALB/c mice induced increased levels of esophageal eosinophils, mast cells, and features of esophageal remodeling (fibrosis, basal zone hyperplasia, deposition of the extracellular matrix protein fibronectin). Administration of intraperitoneal (ip) allosamidin to BALB/c mice significantly inhibited AMCase enzymatic activity in the esophagus. Pharmacologic inhibition of AMCase with ip allosamidin inhibited both OVA induced increases in esophageal eosinophilic inflammation and OVA induced esophageal remodeling (fibrosis, epithelial basal zone hyperplasia, extracellular matrix deposition of fibronectin). This inhibition of eosinophilic inflammation in the esophagus by ip allosamidin was associated with reduced eotaxin-1 expression in the esophagus. Oral allosamidin inhibited eosinophilic inflammation in the epithelium but did not inhibit esophageal remodeling. These studies suggest that pharmacologic inhibition of AMCase results in inhibition of eosinophilic inflammation and remodeling in the esophagus in a mouse model of egg induced EoE partially through effects in the esophagus on reducing chemokines (i.e. eotaxin-1) implicated in the pathogenesis of EoE.

In vitro culture of Plasmodium falciparum gametocytes is essential for studying sexual development of the parasite. Here we describe a simple method for producing synchronous gametocyte culture without contamination of asexual stages. This method employs heparin's activity in blocking merozoite invasion of erythrocytes to eliminate asexual stage parasites from gametocyte culture. We show that following induction of gametocyte formation, addition of heparin in culture medium for four days effectively eliminates asexual stages and produces pure, synchronous cultures of gametocytes. Compared with the commonly used N-acetylglucosamine treatment method, heparin treatment requires shorter time to eliminate asexual stages and causes significantly less hemolysis in late stage gametocyte cultures.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca(2+) handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca(2+) concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca(2+) release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.

Posttranslational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3β-dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3β. Interestingly, OGT activity is regulated by GSK3β; hence, OGT and GSK3β exhibit reciprocal regulation. Modulating O-GlcNAcylation levels alter circadian period length in both mice and Drosophila; conversely, protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662-S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine-tune circadian clock.

Posttranslational modifications play central roles in myriad biological pathways including circadian regulation. We employed a circadian proteomic approach to demonstrate that circadian timing of phosphorylation is a critical factor in regulating complex GSK3β-dependent pathways and identified O-GlcNAc transferase (OGT) as a substrate of GSK3β. Interestingly, OGT activity is regulated by GSK3β; hence, OGT and GSK3β exhibit reciprocal regulation. Modulating O-GlcNAcylation levels alter circadian period length in both mice and Drosophila; conversely, protein O-GlcNAcylation is circadianly regulated. Central clock proteins, Clock and Period, are reversibly modified by O-GlcNAcylation to regulate their transcriptional activities. In addition, O-GlcNAcylation of a region in PER2 known to regulate human sleep phase (S662-S674) competes with phosphorylation of this region, and this interplay is at least partly mediated by glucose levels. Together, these results indicate that O-GlcNAcylation serves as a metabolic sensor for clock regulation and works coordinately with phosphorylation to fine-tune circadian clock.

O-Linked β-N-acetylglucosaminyl transferase (OGT) plays an important role in the glycosylation of proteins, which is involved in various cellular events. In human, three isoforms of OGT (short OGT [sOGT]; mitochondrial OGT [mOGT]; and nucleocytoplasmic OGT [ncOGT]) share the same catalytic domain, implying that they might adopt a similar catalytic mechanism, including sugar donor recognition. In this work, the sugar-nucleotide tolerance of sOGT was investigated. Among a series of uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) analogs tested using the casein kinase II (CKII) peptide as the sugar acceptor, four compounds could be used by sOGT, including UDP-6-deoxy-GlcNAc, UDP-GlcNPr, UDP-6-deoxy-GalNAc and UDP-4-deoxy-GlcNAc. Determined values of Km showed that the substitution of the N-acyl group, deoxy modification of C6/C4-OH or epimerization of C4-OH of the GlcNAc in UDP-GlcNAc decreased its affinity to sOGT. A molecular docking study combined with site-directed mutagenesis indicated that the backbone carbonyl oxygen of Leu653 and the hydroxyl group of Thr560 in sOGT contributed to the recognition of the sugar moiety via hydrogen bonds. The close vicinity between Met501 and the N-acyl group of GlcNPr, as well as the hydrophobic environment near Met501, were responsible for the selective binding of UDP-GlcNPr. These findings illustrate the interaction of OGT and sugar nucleotide donor, providing insights into the OGT catalytic mechanism.