Hypertonic stress modulates eNOS function through O-GlcNAc modification at Thr-866.

O-GlcNAcylation, an energy-sensitive posttranslational modification, can regulate the activity of endothelial nitric oxide synthase (eNOS). Previous studies found that Thr866 is the key site for low-glucose-mediated regulation of eNOS O-GlcNAc. However, it is not known whether this activity functions through the Thr866 site concomitant with other physical and chemical factors. Therefore, we first explored the effects of physical and chemical factors on eNOS O-GlcNAc and its Thr866 site. In this study, hypertonic stress, hyperthermia and hydrogen peroxide all increased the expression levels of eNOS O-GlcNAc, whereas hypoxia and high levels of alcohol had no effect. on the expression levels of eNOS O-GlcNAc; by contrast, low pH led to a decrease in eNOS O-GlcNAc levels. Notably, eNOS O-GlcNAc protein levels were unchanged after Thr866 site mutation only under hypertonic conditions, suggesting that hypertonic stress may act through the Thr866 site. Upon exploring the mechanism of hypertonic stress on eNOS O-GlcNAc activity and function, we found that hypertonic stress can upregulate the expression of O-linked N-acetylglucosamine (GlcNAc) transferase (OGT), which is dependent on AMPK. When AMPK was knocked out, the upregulation of OGT expression and increased O-GlcNAc modifications induced by hypertonic stress were reversed.

Hyperglycemia-Induced Aberrant Cell Proliferation; A Metabolic Challenge Mediated by Protein O-GlcNAc Modification.

Chronic hyperglycemia has been associated with an increased prevalence of pathological conditions including cardiovascular disease, cancer, or various disorders of the immune system. In some cases, these associations may be traced back to a common underlying cause, but more often, hyperglycemia and the disturbance in metabolic balance directly facilitate pathological changes in the regular cellular functions. One such cellular function crucial for every living organism is cell cycle regulation/mitotic activity. Although metabolic challenges have long been recognized to influence cell proliferation, the direct impact of diabetes on cell cycle regulatory elements is a relatively uncharted territory. Among other "nutrient sensing" mechanisms, protein O-linked ß-N-acetylglucosamine (O-GlcNAc) modification emerged in recent years as a major contributor to the deleterious effects of hyperglycemia. An increasing amount of evidence suggest that O-GlcNAc may significantly influence the cell cycle and cellular proliferation. In our present review, we summarize the current data available on the direct impact of metabolic changes caused by hyperglycemia in pathological conditions associated with cell cycle disorders. We also review published experimental evidence supporting the hypothesis that O-GlcNAc modification may be one of the missing links between metabolic regulation and cellular proliferation.

Functional significance of O-GlcNAc modification in regulating neuronal properties.

Post-translational modifications (PTMs) covalently modify proteins and diversify protein functions. Along with protein phosphorylation, another common PTM is the addition of O-linked ß-N-acetylglucosamine (O-GlcNAc) to serine and/or threonine residues. O-GlcNAc modification is similar to phosphorylation in that it occurs to serine and threonine residues and cycles on and off with a similar time scale. However, a striking difference is that the addition and removal of the O-GlcNAc moiety on all substrates are mediated by the two enzymes regardless of proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. O-GlcNAcylation can interact or potentially compete with phosphorylation on serine and threonine residues, and thus serves as an important molecular mechanism to modulate protein functions and activation. However, it has been challenging to address the role of O-GlcNAc modification in regulating protein functions at the molecular level due to the lack of convenient tools to determine the sites and degrees of O-GlcNAcylation. Studies in this field have only begun to expand significantly thanks to the recent advances in detection and manipulation methods such as quantitative proteomics and highly selective small-molecule inhibitors for OGT and OGA. Interestingly, multiple brain regions, especially hippocampus, express high levels of both OGT and OGA, and a number of neuron-specific proteins have been reported to undergo O-GlcNAcylation. This review aims to discuss the recent updates concerning the impacts of O-GlcNAc modification on neuronal functions at multiple levels ranging from intrinsic neuronal properties to synaptic plasticity and animal behaviors.

Dexamethasone provoked mitochondrial perturbations in thymus: Possible role of N-acetylglucosamine in restoration of mitochondrial function.

Thymus mitochondria play a crucial role in immune function. This study identifies the novel protective role of N-Acetylglucosamine (NAG) in dexamethasone (DEX) induced mitochondrial perturbations in mice thymus. Mice were induced with DEX (5mg/kg) and treated with NAG i.p. (266µg/kg, 400µg/kg and 800µg/kg) for 14 days, Withanolide A (800µg/kg) has been used as positive control. Dose dependent treatment of NAG against DEX significantly restored the mitochondrial enzyme levels (ICDH, KDH, SDH and MDH) and elevated the mitochondrial glutathione antioxidants defense (GSH, SOD, GPX and GST) thus improving the ATP status which was confirmed by ultrastructural alterations in mitochondria and nucleus using TEM studies. Further histopathological studies also revealed that NAG attenuate DEX induced thymotoxicity. Finally, the study concludes that dose dependent treatment of NAG supports a potential role in preventing DEX induced thymotoxicity and NAG acts as a beneficial pharmacological intervention in the DEX induced thymic repercussions.

Identification of Poly-N-acetylglucosamine as a Major Polysaccharide Component of the Bacillus subtilis Biofilm Matrix.

Bacillus subtilis is intensively studied as a model organism for the development of bacterial biofilms or pellicles. A key component is currently undefined exopolysaccharides produced from proteins encoded by genes within the eps locus. Within this locus are four genes, epsHIJK, known to be essential for pellicle formation. We show they encode proteins synthesizing the broadly expressed microbial carbohydrate poly-N-acetylglucosamine (PNAG). PNAG was present in both pellicle and planktonic wild-type B. subtilis cells and in strains with deletions in the epsA-G and -L-O genes but not in strains deleted for epsH-K. Cloning of the B. subtilis epsH-K genes into Escherichia coli with in-frame deletions in the PNAG biosynthetic genes pgaA-D, respectively, restored PNAG production in E. coli. Cloning the entire B. subtilis epsHIJK locus into pga-deleted E. coli, Klebsiella pneumoniae, or alginate-negative Pseudomonas aeruginosa restored or conferred PNAG production. Bioinformatic and structural predictions of the EpsHIJK proteins suggest EpsH and EpsJ are glycosyltransferases (GT) with a GT-A fold; EpsI is a GT with a GT-B fold, and EpsK is an α-helical membrane transporter. B. subtilis, E. coli, and pga-deleted E. coli carrying the epsHIJK genes on a plasmid were all susceptible to opsonic killing by antibodies to PNAG. The immunochemical and genetic data identify the genes and proteins used by B. subtilis to produce PNAG as a significant carbohydrate factor essential for pellicle formation.

Reduced gland mucin-specific O-glycan in gastric atrophy: A possible risk factor for differentiated-type adenocarcinoma of the stomach.

BACKGROUND AND AIMS: O-glycans exhibiting terminal α1,4-linked N-acetylglucosamine (αGlcNAc) are attached to MUC6 in gastric gland mucins and serve as a tumor suppressor for gastric adenocarcinoma. Gastric atrophy is associated with risk for gastric cancer. However, the significance of αGlcNAc expression in pyloric glands of chronic atrophic gastritis remains unknown. Here, we asked whether reduced αGlcNAc expression in chronic atrophic gastritis is associated with risk for gastric cancer. METHODS: We quantitatively analyzed expression of αGlcNAc relative to MUC6 in pyloric glands by immunohistochemistry in 67 patients with normal mucosa, 70 with chronic atrophic gastritis, 68 with intramucosal differentiated-type adenocarcinoma, and 11 with intramucosal undifferentiated-type adenocarcinoma. We also compared the Ki-67 labeling index in gastric epithelial cells between chronic atrophic gastritis and normal gastric mucosa with respect to αGlcNAc reduction. RESULTS: In normal pyloric mucosa, αGlcNAc was co-expressed with MUC6. By contrast, in chronic atrophic gastritis, pyloric gland αGlcNAc expression was significantly reduced relative to MUC6. In intramucosal gastric cancer, αGlcNAc expression in pyloric glands found just beneath differentiated-type adenocarcinoma was also reduced relative to MUC6. However, pyloric glands present beneath undifferentiated-type adenocarcinoma exhibited no αGlcNAc decrease. The Ki-67 labeling index in chronic atrophic gastritis showing αGlcNAc reduction was significantly increased relative to that in normal gastric mucosa. CONCLUSIONS: Because αGlcNAc prevents the gastric cancer development, reduced αGlcNAc expression in chronic atrophic gastritis is a possible risk factor for differentiated-type adenocarcinoma of the stomach.

Effect of sypQ gene on poly-N-acetylglucosamine biosynthesis in Vibrio parahaemolyticus and its role in infection process.

The syp locus includes four genes encoding putative regulators, six genes encoding glycosyltransferases, two encoding export proteins, and six other genes encoding unidentified functional proteins associated with biofilm formation and symbiotic colonization. However, the individual functions of the respective genes remain unclear. Amino acid alignment indicates that sypQ is presumably involved in biosynthesizing poly-N-acetylglucosamine (PNAG), which is proposed to be a critical virulence factor in pathogen infection and is regarded as a target for protective immunity against a variety of Gram-negative/positive pathogens. However, no evidence showing that Vibrio parahaemolyticus also produces PNAG has been reported. Herein, the V. parahaemolyticus is confirmed to possess potential for producing PNAG for the first time. Our results indicated that gene sypQ is associated with PNAG biosynthesis and PNAG is involved in pathogen colonization. We propose that the function of pgaC in Escherichia coli could be taken over by sypQ from V. parahaemolyticus. We also tested whether PNAG can be used as a target against V. parahaemolyticus when it infects Pseudosciaena crocea. Our results showed that PNAG isolated from V. parahaemolyticus is an effective agent for decreasing V. parahaemolyticus invasion, implying that PNAG could be used to develop an effective vaccine against V. parahaemolyticus infection.

Glucose starvation induces cell death in K-ras-transformed cells by interfering with the hexosamine biosynthesis pathway and activating the unfolded protein response.

Cancer cells, which use more glucose than normal cells and accumulate extracellular lactate even under normoxic conditions (Warburg effect), have been reported to undergo cell death under glucose deprivation, whereas normal cells remain viable. As it may be relevant to exploit the molecular mechanisms underlying this biological response to achieve new cancer therapies, in this paper we sought to identify them by using transcriptome and proteome analysis applied to an established glucose-addicted cellular model of transformation, namely, murine NIH-3T3 fibroblasts harboring an oncogenic K-RAS gene, compared with parental cells. Noteworthy is that the analyses performed in high- and low-glucose cultures indicate that reduction of glucose availability induces, especially in transformed cells, a significant increase in the expression of several unfolded protein response (UPR) hallmark genes. We show that this response is strictly associated with transformed cell death, given that its attenuation, by reducing protein translation or by increasing cell protein folding capacity, preserves the survival of transformed cells. Such an effect is also observed by inhibiting c-Jun NH2-terminal kinase, a pro-apoptotic signaling mediator set downstream of UPR. Strikingly, addition of N-acetyl-D-glucosamine, a specific substrate for the hexosamine biosynthesis pathway (HBP), to glucose-depleted cells completely prevents transformed cell death, stressing the important role of glucose in HBP fuelling to ensure UPR attenuation and increased cell survival. Interestingly, these results have been fully recognized in a human model of breast cancer, MDA-MB-231 cells. In conclusion, we show that glucose deprivation, leading to harmful accumulation of unfolded proteins in consequence of a reduction of protein glycosylation, induces a UPR-dependent cell death mechanism. These findings may open the way for new therapeutic strategies to specifically kill glycolytic cancer cells.

Impact of O-GlcNAc on cardioprotection by remote ischaemic preconditioning in non-diabetic and diabetic patients.

AIMS: Post-translational modification of proteins by O-linked ß-N-acetylglucosamine (O-GlcNAc) is cardioprotective but its role in cardioprotection by remote ischaemic preconditioning (rIPC) and the reduced efficacy of rIPC in type 2 diabetes mellitus is unknown. In this study we achieved mechanistic insight into the remote stimulus mediating and the target organ response eliciting the cardioprotective effect by rIPC in non-diabetic and diabetic myocardium and the influence of O-GlcNAcylation. METHODS AND RESULTS: The cardioprotective capacity and the influence on myocardial O-GlcNAc levels of plasma dialysate from eight healthy volunteers and eight type 2 diabetic patients drawn before and after subjection to an rIPC stimulus were tested on human isolated atrial trabeculae subjected to ischaemia/reperfusion injury. Dialysate from healthy volunteers exposed to rIPC improved post-ischaemic haemodynamic recovery (40 ± 6 vs. 16 ± 2%; P < 0.01) and increased myocardial O-GlcNAc levels. Similar observations were made with dialysate from diabetic patients before exposure to rIPC (43 ± 3 vs. 16 ± 2%; P < 0.001) but no additional cardioprotection or further increase in O-GlcNAc levels was achieved by perfusion with dialysate after exposure to rIPC (44 ± 4 and 42 ± 5 vs. 43 ± 3%; P = 0.7). The glutamine:fructose-6-phosphate amidotransferase (GFAT) inhibitor azaserine abolished the cardioprotective effects and the increment in myocardial O-GlcNAc levels afforded by plasma from diabetic patients and healthy volunteers treated with rIPC. CONCLUSIONS: rIPC and diabetes mellitus per se influence myocardial O-GlcNAc levels through circulating humoral factors. O-GlcNAc signalling participates in mediating rIPC-induced cardioprotection and maintaining a state of inherent chronic activation of cardioprotection in diabetic myocardium, restricting it from further protection by rIPC.

O-GlcNAc modification affects the ATM-mediated DNA damage response.

BACKGROUND: O-Linked ß-N-acetylglucosamine (O-GlcNAc) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. The role of O-GlcNAcylation in the ataxia-telangiectasia mutated (ATM)-mediated DNA damage response is unknown. It is unclear whether ATM, which is an early acting and central component of the signal transduction system activated by DNA double strand breaks, is an O-GlcNAc-modified protein. METHODS: The effect of O-GlcNAc modification on ATM activation was examined using two inhibitors, PUGNAc and DON that increase and decrease, respectively, levels of protein O-GlcNAcylation. To assess O-GlcNAcylation of ATM, immunoprecipitation and immunoblot analyses using anti-ATM or anti-O-GlcNAc antibody were performed in HeLa cells and primary cultured neurons. Interaction of ATM with O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to target proteins, was examined by immunoprecipitation and immunoblot analyses using anti-ATM. RESULTS: Enhancement of protein O-GlcNAcylation increased levels of X-irradiation-induced ATM activation. However, decreases in protein O-GlcNAcylation did not affect levels of ATM activation, but these decreases did delay ATM activation and ATM recovery processes based on assessment of de-phosphorylation of phospho-ATM. Thus, activation and recovery of ATM were affected by O-GlcNAcylation. ATM was subjected to O-GlcNAcylation, and ATM interacted with OGT. The steady-state O-GlcNAc level of ATM was not significantly responsive to X-irradiation or oxidative stress. GENERAL SIGNIFICANCE: ATM is an O-GlcNAc modified protein, and dynamic O-GlcNAc modification affects the ATM-mediated DNA damage response.

ß-N-Acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3.

O-Linked ß-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.

The roles of O-linked ß-N-acetylglucosamine in cardiovascular physiology and disease.

More than 1,000 proteins of the nucleus, cytoplasm, and mitochondria are dynamically modified by O-linked ß-N-acetylglucosamine (O-GlcNAc), an essential post-translational modification of metazoans. O-GlcNAc, which modifies Ser/Thr residues, is thought to regulate protein function in a manner analogous to protein phosphorylation and, on a subset of proteins, appears to have a reciprocal relationship with phosphorylation. Like phosphorylation, O-GlcNAc levels change dynamically in response to numerous signals including hyperglycemia and cellular injury. Recent data suggests that O-GlcNAc appears to be a key regulator of the cellular stress response, the augmentation of which is protective in models of acute vascular injury, trauma hemorrhage, and ischemia-reperfusion injury. In contrast to these studies, O-GlcNAc has also been implicated in the development of hypertension and type II diabetes, leading to vascular and cardiac dysfunction. Here we summarize the current understanding of the roles of O-GlcNAc in the heart and vasculature.

Cardiac O-GlcNAc signaling is increased in hypertrophy and heart failure.

Reversible protein O-GlcNAc modification has emerged as an essential intracellular signaling system in several tissues, including cardiovascular pathophysiology related to diabetes and acute ischemic stress. We tested the hypothesis that cardiac O-GlcNAc signaling is altered in chronic cardiac hypertrophy and failure of different etiologies. Global protein O-GlcNAcylation and the main enzymes regulating O-GlcNAc, O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine-fructose-6-phosphate amidotransferase (GFAT) were measured by immunoblot and/or real-time RT-PCR analyses of left ventricular tissue from aortic stenosis (AS) patients and rat models of hypertension, myocardial infarction (MI), and aortic banding (AB), with and without failure. We show here that global O-GlcNAcylation was increased by 65% in AS patients, by 47% in hypertensive rats, by 81 and 58% post-AB, and 37 and 60% post-MI in hypertrophic and failing hearts, respectively (P < 0.05). Noticeably, protein O-GlcNAcylation patterns varied in hypertrophic vs. failing hearts, and the most extensive O-GlcNAcylation was observed on proteins of 20-100 kDa in size. OGT, OGA, and GFAT2 protein and/or mRNA levels were increased by pressure overload, while neither was regulated by myocardial infarction. Pharmacological inhibition of OGA decreased cardiac contractility in post-MI failing hearts, demonstrating a possible role of O-GlcNAcylation in development of chronic cardiac dysfunction. Our data support the novel concept that O-GlcNAc signaling is altered in various etiologies of cardiac hypertrophy and failure, including human aortic stenosis. This not only provides an exciting basis for discovery of new mechanisms underlying pathological cardiac remodeling but also implies protein O-GlcNAcylation as a possible new therapeutic target in heart failure.

Role of the O-linked ß-N-acetylglucosamine in the cardioprotection induced by isoflurane.

BACKGROUND: Cardiac protection by volatile anesthetic-induced preconditioning and ischemic preconditioning have similar signaling pathways. Recently, it was reported that augmentation of protein modified with O-linked ß-N-acetylglucosamine (O-GlcNAc) contributes to cardiac protection. This study investigated the role of O-GlcNAc in cardiac protection induced by anesthetic-induced preconditioning. METHODS: O-GlcNAc-modified proteins were visualized by immunoblotting. Tolerance against ischemia or reperfusion was tested in vivo (n = 8) and in vitro (n = 6). The opening of the mitochondrial permeability transition pore (mPTP) upon oxidative stress was examined in myocytes treated with calcein AM (n = 5). Coimmunoprecipitation and enzymatic labeling were performed to detect the mitochondrial protein responsible for the mPTP opening. RESULTS: Isoflurane treatment and the consequent augmentation of O-GlcNAc concentrations reduced the infarct size (26 ± 5% [mean ± SD], P < 0.001) compared with the control. The protective effect of O-GlcNAc was eliminated in the group pretreated with the O-GlcNAc transferase inhibitor alloxan (39 ± 5%, P < 0.001). Myocyte survival also showed the same result in vitro. Formation of the mPTP was abrogated in the isoflurane-treated cells (86 ± 4%, P < 0.001) compared with the control and alloxan-plus-isoflurane-treated cells (57 ± 7%, P < 0.001). Coimmunoprecipitation and enzymatic labeling studies revealed that the O-GlcNAc-modified, voltage-dependent anion channel restained the mPTP opening. CONCLUSIONS: Isoflurane induced O-GlcNAc modification of mitochondrial voltage-dependent anion channel. This modification inhibited the opening of the mPTP and conferred resistance to ischemia-reperfusion stress.

Functional roles of the bisecting GlcNAc in integrin-mediated cell adhesion.

N-acetylglucosaminyltransferase III (GnT-III) transfers N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to core mannose with a beta1,4 linkage, so-called bisecting GlcNAc, in N-glycans. The bisecting GlcNAc is found in various hybrid and complex N-glycans. GnT-III is generally regarded as a key glycosyltransferase in N-glycan biosynthetic pathways. Introduction of a bisecting GlcNAc suppresses further processing and elongation of N-glycans catalyzed by other GlcNAc transferases to form branching structures, such as N-acetylglucosaminyltransferase V (GnT-V), since GnT-V cannot utilize the bisected oligosaccharide as a substrate. Considering that expression of the enzyme leads to a remarkable structural alteration of the N-glycans on cell surface, it has been postulated that the enzyme is associated with various biological events such as cell adhesion, migration, cell growth, cell differentiation, and tumor invasion. Integrin is a major carrier of N-glycans. In fact, overexpression of GnT-III reduced the beta1,6 GlcNAc branching structures, in conjunction with the increase in the bisected N-glycans on integrins, and resulted in an inhibition of integrin-mediated cell spreading and migration, and the cellular phosphorylation levels. Conversely, knockdown of endogenous GnT-III expression resulted in increased cell migration, concomitant with an increase in beta1,6 GlcNAc-branched N-glycans on integrins. Thus, N-glycan could be considered as either a positive or negative regulator for biological functions of integrin.

The role of protein O-linked beta-N-acetylglucosamine in mediating cardiac stress responses.

The modification of serine and threonine residues of nuclear and cytoplasmic proteins by O-linked beta-N-acetylglucosamine (O-GlcNAc) has emerged as a highly dynamic post-translational modification that plays a critical role in regulating numerous biological processes. Much of our understanding of the mechanisms underlying the role of O-GlcNAc on cellular function has been in the context of its adverse effects in mediating a range of chronic disease processes, including diabetes, cancer and neurodegenerative diseases. However, at the cellular level it has been shown that O-GlcNAc levels are increased in response to stress; augmentation of this response improved cell survival while attenuation decreased cell viability. Thus, it has become apparent that strategies that augment O-GlcNAc levels are pro-survival, whereas those that reduce O-GlcNAc levels decrease cell survival. There is a long history demonstrating the effectiveness of acute glucose-insulin-potassium (GIK) treatment and to a lesser extent glutamine in protecting against a range of stresses, including myocardial ischemia. A common feature of these approaches for metabolic cardioprotection is that they both have the potential to stimulate O-GlcNAc synthesis. Consequently, here we examine the links between metabolic cardioprotection with the ischemic cardioprotection associated with acute increases in O-GlcNAc levels. Some of the protective mechanisms associated with activation of O-GlcNAcylation appear to be transcriptionally mediated; however, there is also strong evidence to suggest that transcriptionally independent mechanisms also play a critical role. In this context we discuss the potential link between O-GlcNAcylation and cardiomyocyte calcium homeostasis including the role of non-voltage gated, capacitative calcium entry as a potential mechanism contributing to this protection.

The hexosamine signaling pathway: O-GlcNAc cycling in feast or famine.

The enzymes of O-GlcNAc cycling couple the nutrient-dependent synthesis of UDP-GlcNAc to O-GlcNAc modification of Ser/Thr residues of key nuclear and cytoplasmic targets. This series of reactions culminating in O-GlcNAcylation of targets has been termed the hexosamine signaling pathway (HSP). The evolutionarily ancient enzymes of O-GlcNAc cycling have co-evolved with other signaling effecter molecules; they are recruited to their targets by many of the same mechanisms used to organize canonic kinase-dependent signaling pathways. This co-recruitment of the enzymes of O-GlcNAc cycling drives a binary switch impacting pathways of anabolism and growth (nutrient uptake) and catabolic pathways (nutrient sparing and salvage). The hexosamine signaling pathway (HSP) has thus emerged as a versatile cellular regulator modulating numerous cellular signaling cascades influencing growth, metabolism, cellular stress, circadian rhythm, and host-pathogen interactions. In mammals, the nutrient-sensing HSP has been harnessed to regulate such cell-specific functions as neutrophil migration, and activation of B-cells and T-cells. This review summarizes the diverse approaches being used to examine O-GlcNAc cycling. It will emphasize the impact O-GlcNAcylation has upon signaling pathways that may be become deregulated in diseases of the immune system, diabetes mellitus, cancer, cardiovascular disease, and neurodegenerative diseases.

Effect of N-acetyl-D-glucosamine on bovine sperm-oocyte interactions.

N-Acetyl-D-glucosamine (GlcNAc) is a major component of glycosaminoglycan, which is involved in sperm-oocyte interactions. We examined the effect of adding GlcNAc and other monosaccharides, D-mannose and D-fucose, to the in vitro fertilization (IVF) medium on bovine sperm-oocyte interactions. In medium in which sperm and a zona pellucida (ZP) were co-incubated with monosaccharides for 5 min, addition of GlcNAc (5 or 25 mM) significantly reduced the number of sperm that attached to the ZP. Pretreatment of gametes with GlcNAc (5 mM) prior to co-incubation also suppressed sperm-ZP attachment. Addition of GlcNAc (5 or 25 mM) to the medium in which sperm and a ZP were co-incubated for 5 h, however, significantly increased the number of sperm binding to and penetrating the ZP in a concentration-related manner. The other monosaccharides, D-fucose and D-mannose, did not have this effect. Supplementation of the sperm-oocyte co-incubation medium with 5 mM GlcNAc also enhanced the rate of polyspermic fertilization. When the ZPs were removed from the oocytes, GlcNAc did not affect the fertilization rate. Furthermore, incubation of sperm with 5 mM GlcNAc induced sperm membrane destabilization and an acrosome reaction, as evidenced by the hypo-osmotic swelling test and fluorescein isothiocyanate-labeled peanut agglutinin/propidium iodide (FITC-PNA/PI) staining. Finally, GlcNAc suppressed ZP hardening following fertilization, as determined by measuring the time required for pronase to dissolve the ZP. In conclusion, supplementation of IVF medium with GlcNAc has various effects on sperm-oocyte interactions including suppression of initial attachment, induction of sperm membrane destabilization and acrosome reaction, increase in the number of sperm secondarily bound to and penetrating the ZP, suppression of ZP hardening following sperm-oocyte co-incubation and increase in the rate of polyspermic fertilization.

Relevant role of fibronectin-binding proteins in Staphylococcus aureus biofilm-associated foreign-body infections.

Staphylococcus aureus can establish chronic infections on implanted medical devices due to its capacity to form biofilms. Analysis of the factors that assemble cells into a biofilm has revealed the occurrence of strains that produce either a polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide- or a protein-dependent biofilm. Examination of the influence of matrix nature on the biofilm capacities of embedded bacteria has remained elusive, because a natural strain that readily converts between a polysaccharide- and a protein-based biofilm has not been studied. Here, we have investigated the clinical methicillin (meticillin)-resistant Staphylococcus aureus strain 132, which is able to alternate between a proteinaceous and an exopolysaccharidic biofilm matrix, depending on environmental conditions. Systematic disruption of each member of the LPXTG surface protein family identified fibronectin-binding proteins (FnBPs) as components of a proteinaceous biofilm formed in Trypticase soy broth-glucose, whereas a PIA/PNAG-dependent biofilm was produced under osmotic stress conditions. The induction of FnBP levels due to a spontaneous agr deficiency present in strain 132 and the activation of a LexA-dependent SOS response or FnBP overexpression from a multicopy plasmid enhanced biofilm development, suggesting a direct relationship between the FnBP levels and the strength of the multicellular phenotype. Scanning electron microscopy revealed that cells growing in the FnBP-mediated biofilm formed highly dense aggregates without any detectable extracellular matrix, whereas cells in a PIA/PNAG-dependent biofilm were embedded in an abundant extracellular material. Finally, studies of the contribution of each type of biofilm matrix to subcutaneous catheter colonization revealed that an FnBP mutant displayed a significantly lower capacity to develop biofilm on implanted catheters than the isogenic PIA/PNAG-deficient mutant.