O-GlcNAc transferase regulates transcriptional activity of human Oct4.

PubMed

Constable, Sandii; Lim, Jae-Min; Vaidyanathan, Krithika; Wells, Lance

2017-10-01

O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells*

PubMed Central

de Queiroz, Rafaela Muniz; Madan, Rashna; Chien, Jeremy; Dias, Wagner Barbosa; Slawson, Chad

2016-01-01

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. PMID:27402830

O-GlcNAc Transferase/Host Cell Factor C1 Complex Regulates Gluconeogenesis by Modulating PGC-1α Stability

PubMed Central

Ruan, Hai-Bin; Han, Xuemei; Li, Min-Dian; Singh, Jay Prakash; Qian, Kevin; Azarhoush, Sascha; Zhao, Lin; Bennett, Anton M.; Samuel, Varman T.; Wu, Jing; Yates, John R.; Yang, Xiaoyong

2012-01-01

SUMMARY A major cause of hyperglycemia in diabetic patients is inappropriate hepatic gluconeogenesis. PGC-1α is a master regulator of gluconeogenesis, and its activity is controlled by various post-translational modifications. A small portion of glucose metabolizes through the hexosamine biosynthetic pathway, which leads to O-linked β-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins. Using a proteomic approach, we identified a broad variety of proteins associated with O-GlcNAc transferase (OGT), among which host cell factor C1 (HCF-1) is highly abundant. HCF-1 recruits OGT to O-GlcNAcylate PGC-1α and O-GlcNAcylation facilitates the binding of the deubiquitinase BAP1, thus protecting PGC-1α from degradation and promoting gluconeogenesis. Glucose availability modulates gluconeogenesis through the regulation of PGC-1α O-GlcNAcylation and stability by the OGT/HCF1 complex. Hepatic knockdown of OGT and HCF-1 improves glucose homeostasis in diabetic mice. These findings define the OGT/HCF-1 complex as a glucose sensor and key regulator of gluconeogenesis, shedding light on new strategies for treating diabetes. PMID:22883232

Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes

PubMed Central

Kapuria, Vaibhav; Röhrig, Ute F.; Bhuiyan, Tanja; Borodkin, Vladimir S.; van Aalten, Daan M.F.; Zoete, Vincent; Herr, Winship

2016-01-01

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc), O-linked-GlcNAc transferase (OGT) catalyzes Ser/Thr O-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase–protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase–protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT. PMID:27056667

O-GlcNAc transferase enables AgRP neurons to suppress browning of white fat

PubMed Central

Ruan, Hai-Bin; Dietrich, Marcelo O.; Liu, Zhong-Wu; Zimmer, Marcelo R.; Li, Min-Dian; Singh, Jay Prakash; Zhang, Kaisi; Yin, Ruonan; Wu, Jing; Horvath, Tamas L.; Yang, Xiaoyong

2014-01-01

SUMMARY Induction of beige cells causes the browning of white fat and improves energy metabolism. However, the central mechanism that controls adipose tissue browning and its physiological relevance are largely unknown. Here we demonstrate that fasting and chemical-genetic activation of orexigenic AgRP neurons in the hypothalamus suppress the browning of white fat. O-linked β-N-acetylglucosamine (O-GlcNAc) modification of cytoplasmic and nuclear proteins regulates fundamental cellular processes. The levels of O-GlcNAc transferase (OGT) and O-GlcNAc modification are enriched in AgRP neurons and are elevated by fasting. Genetic ablation of OGT in AgRP neurons inhibits neuronal excitability through the voltage-dependent potassium channel, promotes white adipose tissue browning, and protects mice against diet-induced obesity and insulin resistance. These data reveal adipose tissue browning as a highly dynamic physiological process under central control, in which O-GlcNAc signaling in AgRP neurons is essential for suppressing thermogenesis to conserve energy in response to fasting. PMID:25303527

O-linked N-acetylglucosamine (O-GlcNAc) protein modification is increased in the cartilage of patients with knee osteoarthritis.

PubMed

Tardio, L; Andrés-Bergós, J; Zachara, N E; Larrañaga-Vera, A; Rodriguez-Villar, C; Herrero-Beaumont, G; Largo, R

2014-02-01

There is increasing evidence that the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins plays an important role in cell signaling pathways. In chondrocytes, accumulation of O-GlcNAc-modified proteins induces hypertrophic differentiation. Osteoarthritis (OA) is characterized by cartilage degradation, and hypertrophic-like changes in hyaline chondrocytes. However, the mechanisms responsible for these changes have not been described. Our aim was to study whether O-GlcNAcylation and the enzymes responsible for this modification are dysregulated in the cartilage of patients with knee OA and whether interleukin-1 could induce these modifications in cultured human OA chondrocytes (HOC). Human cartilage was obtained from patients with knee OA and from age and sex-matched healthy donors. HOC were cultured and stimulated with the catabolic cytokine IL-1α. Global protein O-GlcNAcylation and the synthesis of the key enzymes responsible for this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), were assessed by western blot. OA was associated with a 4-fold increase in the global O-GlcNAcylation in the cartilage. OA cartilage showed a re-distribution of the OGT and OGA isoforms, with a net increase in the presence of both enzymes, in comparison to healthy cartilage. In HOC, IL-1α stimulation rapidly increased O-GlcNAcylation and OGT and OGA synthesis. Our results indicate that a proinflammatory milieu could favor the accumulation of O-GlcNAcylated proteins in OA cartilage, together with the dysregulation of the enzymes responsible for this modification. The increase in O-GlcNAcylation could be responsible, at least partially, for the re-expression of hypertrophic differentiation markers that have been observed in OA. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

O-linked-N-acetylglucosamine modification of mammalian Notch receptors by an atypical O-GlcNAc transferase Eogt1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakaidani, Yuta; Ichiyanagi, Naoki; Saito, Chika

2012-03-02

Highlights: Black-Right-Pointing-Pointer We characterized A130022J15Rik (Eogt1)-a mouse gene homologous to Drosophila Eogt. Black-Right-Pointing-Pointer Eogt1 encodes EGF domain O-GlcNAc transferase. Black-Right-Pointing-Pointer Expression of Eogt1 in Drosophila rescued the cell-adhesion defect in the Eogt mutant. Black-Right-Pointing-Pointer O-GlcNAcylation reaction in the secretory pathway is conserved through evolution. -- Abstract: O-linked-{beta}-N-acetylglucosamine (O-GlcNAc) modification is a unique cytoplasmic and nuclear protein modification that is common in nearly all eukaryotes, including filamentous fungi, plants, and animals. We had recently reported that epidermal growth factor (EGF) repeats of Notch and Dumpy are O-GlcNAcylated by an atypical O-GlcNAc transferase, EOGT, in Drosophila. However, no study has yet shownmore » whether O-GlcNAcylation of extracellular proteins is limited to insects such as Drosophila or whether it occurs in other organisms, including mammals. Here, we report the characterization of A130022J15Rik, a mouse gene homolog of Drosophila Eogt (Eogt 1). Enzymatic analysis revealed that Eogt1 has a substrate specificity similar to that of Drosophila EOGT, wherein the Thr residue located between the fifth and sixth conserved cysteines of the folded EGF-like domains is modified. This observation is supported by the fact that the expression of Eogt1 in Drosophila rescued the cell-adhesion defect caused by Eogt downregulation. In HEK293T cells, Eogt1 expression promoted modification of Notch1 EGF repeats by O-GlcNAc, which was further modified, at least in part, by galactose to generate a novel O-linked-N-acetyllactosamine structure. These results suggest that Eogt1 encodes EGF domain O-GlcNAc transferase and that O-GlcNAcylation reaction in the secretory pathway is a fundamental biochemical process conserved through evolution.« less

Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dehennaut, Vanessa; EA 4020, Laboratoire de Regulation des Signaux de Division, USTL, IFR147, Villeneuve d'Ascq; Hanoulle, Xavier

2008-05-02

In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads tomore » a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.« less

Cross regulation between mTOR signaling and O-GlcNAcylation.

PubMed

Very, Ninon; Steenackers, Agata; Dubuquoy, Caroline; Vermuse, Jeanne; Dubuquoy, Laurent; Lefebvre, Tony; El Yazidi-Belkoura, Ikram

2018-06-01

The hexosamine biosynthetic pathway (HBP) integrates glucose, amino acids, fatty acids and nucleotides metabolisms for uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) synthesis. UDP-GlcNAc is the nucleotide sugar donor for O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) processes. O-GlcNAc transferase (OGT) is the enzyme which transfers the N-acetylglucosamine (O-GlcNAc) residue onto target proteins. Several studies previously showed that glucose metabolism dysregulations associated with obesity, diabetes or cancer correlated with an increase of OGT expression and global O-GlcNAcylation levels. Moreover, these diseases present an increased activation of the nutrient sensing mammalian target of rapamycin (mTOR) pathway. Other works demonstrate that mTOR regulates protein O-GlcNAcylation in cancer cells through stabilization of OGT. In this context, we studied the cross-talk between these two metabolic sensors in vivo in obese mice predisposed to diabetes and in vitro in normal and colon cancer cells. We report that levels of OGT and O-GlcNAcylation are increased in obese mice colon tissues and colon cancer cells and are associated with a higher activation of mTOR signaling. In parallel, treatments with mTOR regulators modulate OGT and O-GlcNAcylation levels in both normal and colon cancer cells. However, deregulation of O-GlcNAcylation affects mTOR signaling activation only in cancer cells. Thus, a crosstalk exists between O-GlcNAcylation and mTOR signaling in contexts of metabolism dysregulation associated to obesity or cancer.

The O-GlcNAc Transferase Intellectual Disability Mutation L254F Distorts the TPR Helix.

PubMed

Gundogdu, Mehmet; Llabrés, Salomé; Gorelik, Andrii; Ferenbach, Andrew T; Zachariae, Ulrich; van Aalten, Daan M F

2018-05-17

O-linked β-N-acetyl- D -glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cloning, Expression and Characterization of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) from Wolbachia Endosymbiont of Human Lymphatic Filarial Parasite Brugia malayi

PubMed Central

Shahab, Mohd; Verma, Meenakshi; Pathak, Manisha; Mitra, Kalyan; Misra-Bhattacharya, Shailja

2014-01-01

Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA) was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (K m) for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA) suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds. PMID:24941309

Exploring reaction pathways for O-GlcNAc transferase catalysis. A string method study.

PubMed

Kumari, Manju; Kozmon, Stanislav; Kulhánek, Petr; Štepán, Jakub; Tvaroška, Igor; Koča, Jaroslav

2015-03-26

The inverting O-GlcNAc glycosyltransferase (OGT) is an important post-translation enzyme, which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine (UDP-GlcNAc) to the hydroxyl group of the Ser/Thr of cytoplasmic, nuclear, and mitochondrial proteins. In the past, three different catalytic bases were proposed for the reaction: His498, α-phosphate, and Asp554. In this study, we used hybrid quantum mechanics/molecular mechanics (QM/MM) Car-Parrinello molecular dynamics to investigate reaction paths using α-phosphate and Asp554 as the catalytic bases. The string method was used to calculate the free-energy reaction profiles of the tested mechanisms. During the investigations, an additional mechanism was observed. In this mechanism, a proton is transferred to α-phosphate via a water molecule. Our calculations show that the mechanism with α-phosphate acting as the base is favorable. This reaction has a rate-limiting free-energy barrier of 23.5 kcal/mol, whereas reactions utilizing Asp554 and water-assisted α-phosphate have barriers of 41.7 and 40.9 kcal/mol, respectively. Our simulations provide a new insight into the catalysis of OGT and may thus guide rational drug design of transition-state analogue inhibitors with potential therapeutic use.

Glucose-induced expression of MIP-1 genes requires O-GlcNAc transferase in monocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chikanishi, Toshihiro; ERATO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012; Fujiki, Ryoji

2010-04-16

O-glycosylation has emerged as an important modification of nuclear proteins, and it appears to be involved in gene regulation. Recently, we have shown that one of the histone methyl transferases (MLL5) is activated through O-glycosylation by O-GlcNAc transferase (OGT). Addition of this monosaccharide is essential for forming a functional complex. However, in spite of the abundance of OGT in the nucleus, the impact of nuclear O-glycosylation by OGT remains largely unclear. To address this issue, the present study was undertaken to test the impact of nuclear O-glycosylation in a monocytic cell line, THP-1. Using a cytokine array, MIP-1{alpha} and -1{beta}more » genes were found to be regulated by nuclear O-glycosylation. Biochemical purification of the OGT interactants from THP-1 revealed that OGT is an associating partner for distinct co-regulatory complexes. OGT recruitment and protein O-glycosylation were observed at the MIP-1{alpha} gene promoter; however, the known OGT partner (HCF-1) was absent when the MIP-1{alpha} gene promoter was not activated. From these findings, we suggest that OGT could be a co-regulatory subunit shared by functionally distinct complexes supporting epigenetic regulation.« less

O-GlcNAcase Expression is Sensitive to Changes in O-GlcNAc Homeostasis.

PubMed

Zhang, Zhen; Tan, Ee Phie; VandenHull, Nicole J; Peterson, Kenneth R; Slawson, Chad

2014-01-01

O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification involving an attachment of a single β-N-acetylglucosamine moiety to serine or threonine residues in nuclear and cytoplasmic proteins. Cellular O-GlcNAc levels are regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove the modification, respectively. The levels of O-GlcNAc can rapidly change in response to fluctuations in the extracellular environment; however, O-GlcNAcylation returns to a baseline level quickly after stimulus removal. This process termed O-GlcNAc homeostasis appears to be critical to the regulation of many cellular functions including cell cycle progress, stress response, and gene transcription. Disruptions in O-GlcNAc homeostasis are proposed to lead to the development of diseases, such as cancer, diabetes, and Alzheimer's disease. O-GlcNAc homeostasis is correlated with the expression of OGT and OGA. We reason that alterations in O-GlcNAc levels affect OGA and OGT transcription. We treated several human cell lines with Thiamet-G (TMG, an OGA inhibitor) to increase overall O-GlcNAc levels resulting in decreased OGT protein expression and increased OGA protein expression. OGT transcript levels slightly declined with TMG treatment, but OGA transcript levels were significantly increased. Pretreating cells with protein translation inhibitor cycloheximide did not stabilize OGT or OGA protein expression in the presence of TMG; nor did TMG stabilize OGT and OGA mRNA levels when cells were treated with RNA transcription inhibitor actinomycin D. Finally, we performed RNA Polymerase II chromatin immunoprecipitation at the OGA promoter and found that RNA Pol II occupancy at the transcription start site was lower after prolonged TMG treatment. Together, these data suggest that OGA transcription was sensitive to changes in O-GlcNAc homeostasis and was potentially regulated by O-GlcNAc.

O-GlcNAcase Expression is Sensitive to Changes in O-GlcNAc Homeostasis

PubMed Central

Zhang, Zhen; Tan, Ee Phie; VandenHull, Nicole J.; Peterson, Kenneth R.; Slawson, Chad

2014-01-01

O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification involving an attachment of a single β-N-acetylglucosamine moiety to serine or threonine residues in nuclear and cytoplasmic proteins. Cellular O-GlcNAc levels are regulated by two enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which add and remove the modification, respectively. The levels of O-GlcNAc can rapidly change in response to fluctuations in the extracellular environment; however, O-GlcNAcylation returns to a baseline level quickly after stimulus removal. This process termed O-GlcNAc homeostasis appears to be critical to the regulation of many cellular functions including cell cycle progress, stress response, and gene transcription. Disruptions in O-GlcNAc homeostasis are proposed to lead to the development of diseases, such as cancer, diabetes, and Alzheimer’s disease. O-GlcNAc homeostasis is correlated with the expression of OGT and OGA. We reason that alterations in O-GlcNAc levels affect OGA and OGT transcription. We treated several human cell lines with Thiamet-G (TMG, an OGA inhibitor) to increase overall O-GlcNAc levels resulting in decreased OGT protein expression and increased OGA protein expression. OGT transcript levels slightly declined with TMG treatment, but OGA transcript levels were significantly increased. Pretreating cells with protein translation inhibitor cycloheximide did not stabilize OGT or OGA protein expression in the presence of TMG; nor did TMG stabilize OGT and OGA mRNA levels when cells were treated with RNA transcription inhibitor actinomycin D. Finally, we performed RNA Polymerase II chromatin immunoprecipitation at the OGA promoter and found that RNA Pol II occupancy at the transcription start site was lower after prolonged TMG treatment. Together, these data suggest that OGA transcription was sensitive to changes in O-GlcNAc homeostasis and was potentially regulated by O-GlcNAc. PMID:25520704

O-Linked N-Acetylglucosamine Cycling Regulates Mitotic Spindle Organization*

PubMed Central

Tan, Ee Phie; Caro, Sarah; Potnis, Anish; Lanza, Christopher; Slawson, Chad

2013-01-01

Any defects in the correct formation of the mitotic spindle will lead to chromosomal segregation errors, mitotic arrest, or aneuploidy. We demonstrate that O-linked N-acetylglucosamine (O-GlcNAc), a post-translational modification of serine and threonine residues in nuclear and cytoplasmic proteins, regulates spindle function. In O-GlcNAc transferase or O-GlcNAcase gain of function cells, the mitotic spindle is incorrectly assembled. Chromosome condensation and centrosome assembly is impaired in these cells. The disruption in spindle architecture is due to a reduction in histone H3 phosphorylation by Aurora kinase B. However, gain of function cells treated with the O-GlcNAcase inhibitor Thiamet-G restored the assembly of the spindle and partially rescued histone phosphorylation. Together, these data suggest that the coordinated addition and removal of O-GlcNAc, termed O-GlcNAc cycling, regulates mitotic spindle organization and provides a potential new perspective on how O-GlcNAc regulates cellular events. PMID:23946484

Thio-Linked UDP–Peptide Conjugates as O-GlcNAc Transferase Inhibitors

PubMed Central

2018-01-01

O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP–peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol–ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA (Ki = 1.3 μM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay. PMID:29723473

Structure of human O-GlcNAc transferase and its complex with a peptide substrate

PubMed Central

Lazarus, Michael B.; Nam, Yunsun; Jiang, Jiaoyang; Sliz, Piotr; Walker, Suzanne

2010-01-01

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that couples metabolic status to the regulation of a wide variety of cellular signaling pathways by acting as a nutrient sensor1. OGT catalyzes the transfer of N-acetyl-glucosamine from UDP-GlcNAc to serines and threonines of cytoplasmic, nuclear and mitochondrial proteins2,3, including numerous transcription factors4, tumor suppressors, kinases5, phosphatases1, and histone-modifying proteins6. Aberrant O-GlcNAcylation by OGT has been linked to insulin resistance7, diabetic complications8, cancer9 and neurodegenerative diseases including Alzheimer’s10. Despite the importance of OGT, the details of how it recognizes and glycosylates its protein substrates are largely unknown. We report here two crystal structures of human OGT, as a binary complex with UDP (2.8 A) and a ternary complex with UDP and a peptide substrate (1.95 A). The structures provide clues to the enzyme mechanism, show how OGT recognizes target peptide sequences, and reveal the fold of the unique domain between the two halves of the catalytic region. This information will accelerate the rational design of biological experiments to investigate OGT’s functions and the design of inhibitors for use as cellular probes and to assess its potential as a therapeutic target. PMID:21240259

The omniscient placenta: Metabolic and epigenetic regulation of fetal programming

PubMed Central

Nugent, Bridget M.; Bale, Tracy L.

2015-01-01

Fetal development could be considered a sensitive period wherein exogenous insults and changes to the maternal milieu can have long-term impacts on developmental programming. The placenta provides the fetus with protection and necessary nutrients for growth, and responds to maternal cues and changes in nutrient signaling through multiple epigenetic mechanisms. The X-linked enzyme O-linked-N-acetylglucosamine transferase (OGT) acts as a nutrient sensor that modifies numerous proteins to alter various cellular signals, including major epigenetic processes. This review describes epigenetic alterations in the placenta in response to insults during pregnancy, the potential links of OGT as a nutrient sensor to placental epigenetics, and the implications of placental epigenetics in long-term neurodevelopmental programming. We describe the role of placental OGT in the sex-specific programming of hypothalamic-pituitary-adrenal (HPA) axis programming deficits by early prenatal stress as an example of how placental signaling can have long-term effects on neurodevelopment. PMID:26368654

O-GlcNAc Transferase Is Essential for Sensory Neuron Survival and Maintenance

PubMed Central

Su, Cathy

2017-01-01

O-GlcNAc transferase (OGT) regulates a wide range of cellular processes through the addition of the O-GlcNAc sugar moiety to thousands of protein substrates. Because nutrient availability affects the activity of OGT, its role has been broadly studied in metabolic tissues. OGT is enriched in the nervous system, but little is known about its importance in basic neuronal processes in vivo. Here, we show that OGT is essential for sensory neuron survival and maintenance in mice. Sensory neuron-specific knock-out of OGT results in behavioral hyposensitivity to thermal and mechanical stimuli accompanied by decreased epidermal innervation and cell-body loss in the dorsal root ganglia. These effects are observed early in postnatal development and progress as animals age. Cultured sensory neurons lacking OGT also exhibit decreased axonal outgrowth. The effects on neuronal health in vivo are not solely due to disruption of developmental processes, because inducing OGT knock-out in the sensory neurons of adult mice results in a similar decrease in nerve fiber endings and cell bodies. Significant nerve-ending loss occurs before a decrease in cell bodies; this phenotype is indicative of axonal dieback that progresses to neuronal death. Our findings demonstrate that OGT is important in regulating axonal maintenance in the periphery and the overall health and survival of sensory neurons. SIGNIFICANCE STATEMENT We show the importance of O-GlcNAc transferase (OGT) for sensory neuron health and survival in vivo. This study is the first to find that loss of OGT results in neuronal cell death. Moreover, it suggests that aberrant O-GlcNAc signaling can contribute to the development of neuropathy. The sensory neurons lie outside of the blood–brain barrier and therefore, compared to central neurons, may have a greater need for mechanisms of metabolic sensing and compensation. Peripheral sensory neurons in particular are subject to degeneration in diabetes. Our findings provide a

Synthesis of Fucosyl-N-Acetylglucosamine Disaccharides by Transfucosylation Using α-l-Fucosidases from Lactobacillus casei

PubMed Central

Rodríguez-Díaz, Jesús; Carbajo, Rodrigo J.; Pineda-Lucena, Antonio; Monedero, Vicente

2013-01-01

AlfB and AlfC α-l-fucosidases from Lactobacillus casei were used in transglycosylation reactions, and they showed high efficiency in synthesizing fucosyldisaccharides. AlfB and AlfC activities exclusively produced fucosyl-α-1,3-N-acetylglucosamine and fucosyl-α-1,6-N-acetylglucosamine, respectively. The reaction kinetics showed that AlfB can convert 23% p-nitrophenyl-α-l-fucopyranoside into fucosyl-α-1,3-N-acetylglucosamine and AlfC at up to 56% into fucosyl-α-1,6-N-acetylglucosamine. PMID:23542622

Aβ-affected pathogenic induction of S-nitrosylation of OGT and identification of Cys-NO linkage triplet.

PubMed

Ryu, In-Hyun; Lee, Ki-Young; Do, Su-Il

2016-05-01

Mechanistic link of protein hypo-O-GlcNAcylation to the pathogenesis of Alzheimer's disease (AD) remains unclear. Here, we found that S-nitrosylation of O-linked N-acetylglucosaminyltransferase (SNO-OGT) was induced by β-amyloid peptide (Aβ) exposure to SK-N-MC and SK-N-SH human neuroblastoma cells. Subsequently, Aβ-induced SNO-OGT led to protein hypo-O-GlcNAcylation globally including tau hypo-O-GlcNAcylation. Our results support that underlying mechanism for induction of SNO-OGT comprises the concerted action of Aβ-triggered Ca2+ entry into cells and nNOS-catalyzed NO production. Intriguingly, OGT was found to be associated with nNOS and its association was enhanced during Aβ treatment. In parallel with SNO-OGT-mediated tau hypo-O-GlcNAcylation, Aβ led to SNO-Akt-mediated GSK3β activation for tau phosphorylation, suggesting that tau hyperphosphorylation is established by synergistic connection between SNO-OGT and GSK3β activation. We also observed that Aβ-neurotoxicity including both reactive oxygen species (ROS) production and cell death was amplified with DON treatment, whereas it was restored by PUGNAc treatment, GlcNH2 treatment or OGT overexpression. Early time-course Aβ-monitoring assay revealed that premaintained hyper-O-GlcNAcylation inside cells blocked not only Aβ-triggered Ca2+ entry into cells but also induction of SNO-OGT and SNO-Akt. Together, these findings suggest that induction of SNO-OGT by Aβ exposure is a pathogenic mechanism to cause cellular hypo-O-GlcNAcylation by which Aβ neurotoxicity is executed, and conversely, hyper-O-GlcNAcylation within cells can defend against Aβ neurotoxicity. Furthermore, our Cys mapping demonstrates that cysteine-nitric oxide (Cys-NO) linkages in SNO-OGT occur at triple Cys845, Cys921, and Cys965 residues in C-terminal catalytic domain (C-CAT), suggesting that Cys-NO linkage triplet in SNO-OGT is associated with null OGT activity. Copyright © 2016 Elsevier B.V. All rights reserved.

Conditions Inducing Excessive O-GlcNAcylation Inhibit BMP2-Induced Osteogenic Differentiation of C2C12 Cells.

PubMed

Gu, Hanna; Song, Mina; Boonanantanasarn, Kanitsak; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

2018-01-09

Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O -linked-β- N -acetylglucosamine glycosylation ( O -GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O -GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N -acetylglucosamine concentrations or by O -GlcNAc transferase (OGT) overexpression. The effect of O -GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N -acetylglucosamine increased O -GlcNAcylation of Runx2 and the total levels of O -GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O -GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing β- N -acetylglucosaminidase. Our findings suggest that excessive protein O -GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation.

Lack of O-GlcNAcylation enhances exercise-dependent glucose utilization potentially through AMP-activated protein kinase activation in skeletal muscle.

PubMed

Murata, Koichiro; Morino, Katsutaro; Ida, Shogo; Ohashi, Natsuko; Lemecha, Mengistu; Park, Shi-Young; Ishikado, Atsushi; Kume, Shinji; Choi, Cheol Soo; Sekine, Osamu; Ugi, Satoshi; Maegawa, Hiroshi

2018-01-08

O-GlcNAcylation is a post-translational modification that is characterized by the addition of N-acetylglucosamine (GlcNAc) to proteins by O-GlcNAc transferase (Ogt). The degree of O-GlcNAcylation is thought to be associated with glucotoxicity and diabetic complications, because GlcNAc is produced by a branch of the glycolytic pathway. However, its role in skeletal muscle has not been fully elucidated. In this study, we created skeletal muscle-specific Ogt knockout (Ogt-MKO) mice and analyzed their glucose metabolism. During an intraperitoneal glucose tolerance test, blood glucose was slightly lower in Ogt-MKO mice than in control Ogt-flox mice. High fat diet-induced obesity and insulin resistance were reversed in Ogt-MKO mice. In addition, 12-month-old Ogt-MKO mice had lower adipose and body mass. A single bout of exercise significantly reduced blood glucose in Ogt-MKO mice, probably because of higher AMP-activated protein kinase α (AMPKα) protein expression. Furthermore, intraperitoneal injection of 5-aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, resulted in a more marked decrease in blood glucose levels in Ogt-MKO mice than in controls. Finally, Ogt knockdown by siRNA in C2C12 myotubes significantly increased protein expression of AMPKα, glucose uptake and oxidation. In conclusion, loss of O-GlcNAcylation facilitates glucose utilization in skeletal muscle, potentially through AMPK activation. The inhibition of O-GlcNAcylation in skeletal muscle may have an anti-diabetic effect, through an enhancement of glucose utilization during exercise. Copyright © 2017 Elsevier Inc. All rights reserved.

O-GlcNAc site-mapping of liver X receptor-α and O-GlcNAc transferase.

PubMed

Fan, Qiong; Moen, Anders; Anonsen, Jan Haug; Bindesbøll, Christian; Sæther, Thomas; Carlson, Cathrine Rein; Grønning-Wang, Line M

2018-05-05

The Liver X Receptor α (LXRα) belongs to the nuclear receptor superfamily and plays an essential role in regulating cholesterol, lipid and glucose metabolism and inflammatory responses. We have previously shown that LXRα is post-translationally modified by O-linked β-N-acetyl-glucosamine (O-GlcNAc) with increased transcriptional activity. Moreover, we showed that LXRα associates with O-GlcNAc transferase (OGT) in vitro and in vivo in mouse liver. In this study, we report that human LXRα is O-GlcNAc modified in its N-terminal domain (NTD) by identifying a specific O-GlcNAc site S49 and a novel O-GlcNAc modified peptide 20 LWKPGAQDASSQAQGGSSCILRE 42 . However, O-GlcNAc site-mutations did not modulate LXRα transactivation of selected target gene promoters in vitro. Peptide array and co-immunoprecipitation assays demonstrate that LXRα interacts with OGT in its NTD and ligand-binding domain (LBD) in a ligand-independent fashion. Moreover, we map two new O-GlcNAc sites in the longest OGT isoform (ncOGT): S437 in the tetratricopeptide repeat (TPR) 13 domain and T1043 in the far C-terminus, and a new O-GlcNAc modified peptide (amino acids 826-832) in the intervening region (Int-D) within the catalytic domain. We also map four new O-GlcNAc sites in the short isoform sOGT: S391, T393, S399 and S437 in the TPRs 11-13 domain. Future studies will reveal the biological role of identified O-GlcNAc sites in LXRα and OGT. Copyright © 2018 Elsevier Inc. All rights reserved.

Both UDP N-acetylglucosamine pyrophosphorylases of Tribolium castaneum are critical for molting, survival, and fecundity

USDA-ARS?s Scientific Manuscript database

A bioinformatics search of the genome of the red flour beetle, Tribolium castaneum, resulted in the identification of two genes encoding proteins closely related to UDP-N-acetylglucosamine pyrophosphorylases (UAP), which provide the activated precursor, UDP-N-acetylglucosamine, for the synthesis of ...

The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

PubMed Central

López-Fernández, Loida; Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; Prieto, Alicia; Khraiwesh, Husam; Roncero, M. Isabel G.

2013-01-01

With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity. PMID:24416097

Structure of a Novel O-Linked N-Acetyl-d-glucosamine (O-GlcNAc) Transferase, GtfA, Reveals Insights into the Glycosylation of Pneumococcal Serine-rich Repeat Adhesins*

PubMed Central

Shi, Wei-Wei; Jiang, Yong-Liang; Zhu, Fan; Yang, Yi-Hu; Shao, Qiu-Yan; Yang, Hong-Bo; Ren, Yan-Min; Wu, Hui; Chen, Yuxing; Zhou, Cong-Zhao

2014-01-01

Protein glycosylation catalyzed by the O-GlcNAc transferase (OGT) plays a critical role in various biological processes. In Streptococcus pneumoniae, the core enzyme GtfA and co-activator GtfB form an OGT complex to glycosylate the serine-rich repeat (SRR) of adhesin PsrP (pneumococcal serine-rich repeat protein), which is involved in the infection and pathogenesis. Here we report the 2.0 Å crystal structure of GtfA, revealing a β-meander add-on domain beyond the catalytic domain. It represents a novel add-on domain, which is distinct from the all-α-tetratricopeptide repeats in the only two structure-known OGTs. Structural analyses combined with binding assays indicate that this add-on domain contributes to forming an active GtfA-GtfB complex and recognizing the acceptor protein. In addition, the in vitro glycosylation system enables us to map the O-linkages to the serine residues within the first SRR of PsrP. These findings suggest that fusion with an add-on domain might be a universal mechanism for diverse OGTs that recognize varying acceptor proteins/peptides. PMID:24936067

The involvement of beta-1,4-Galactosyltransferase and N-Acetylglucosamine residues in fertilization has been lost in the horse.

PubMed

Mugnier, Sylvie; Boittin, Stéphane; Douet, Cécile; Monget, Philippe; Magistrini, Michèle; Goudet, Ghylène

2008-11-14

In human and rodents, sperm-zona pellucida binding is mediated by a sperm surface Galactosyltransferase that recognizes N-Acetylglucosamine residues on a glycoprotein ZPC. In large domestic mammals, the role of these molecules remains unclear: in bovine, they are involved in sperm-zona pellucida binding, whereas in porcine, they are not necessary. Our aim was to clarify the role of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding in ungulates. For this purpose, we analyzed the mechanism of sperm-zona pellucida interaction in a third ungulate: the horse, since the Galactosyltransferase and N-Acetylglucosamine residues have been localized on equine gametes. We masked the Galactosyltransferase and N-Acetylglucosamine residues before the co-incubation of gametes. Galactosyltransferase was masked either with an anti-Galactosyltransferase antibody or with the enzyme substrate, UDP Galactose. N-Acetylglucosamine residues were masked either with a purified Galactosyltransferase or with an anti-ZPC antibody. The number of spermatozoa bound to the zona pellucida did not decrease after the masking of Galactosyltransferase or N-Acetylglucosamine. So, these two molecules may not be necessary in the mechanism of in vitro sperm-zona pellucida interaction in the horse. The involvement of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding may have been lost during evolution in some ungulates, such as porcine and equine species.

Purification, crystallization and preliminary X-ray diffraction studies of N-acetylglucosamine-phosphate mutase from Candida albicans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishitani, Yuichi; Maruyama, Daisuke; Nonaka, Tsuyoshi

2006-04-01

Preliminary X-ray diffraction studies on N-acetylglucosamine-phosphate mutase from C. albicans are reported. N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) in eukaryotes and belongs to the α-d-phosphohexomutase superfamily. AGM1 from Candida albicans (CaAGM1) was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals obtained belong to the primitive monoclinic space group P2{sub 1}, with unit-cell parameters a = 60.2, b = 130.2, c = 78.0 Å, β = 106.7°. The crystals diffract X-rays to beyond 1.8 Å resolution using synchrotron radiation.

Label-free electrochemical biosensing of small-molecule inhibition on O-GlcNAc glycosylation.

PubMed

Yang, Yu; Gu, Yuxin; Wan, Bin; Ren, Xiaomin; Guo, Liang-Hong

2017-09-15

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) plays a critical role in modulating protein function in many cellular processes and human diseases such as Alzheimer's disease and type II diabetes, and has emerged as a promising new target. Specific inhibitors of OGT could be valuable tools to probe the biological functions of O-GlcNAcylation, but a lack of robust nonradiometric assay strategies to detect glycosylation, has impeded efforts to identify such compounds. Here we have developed a novel label-free electrochemical biosensor for the detection of peptide O-GlcNAcylation using protease-protection strategy and electrocatalytic oxidation of tyrosine mediated by osmium bipyridine as a signal reporter. There is a large difference in the abilities of proteolysis of the glycosylated and the unglycosylated peptides by protease, thus providing a sensing mechanism for OGT activity. When the O-GlcNAcylation is achieved, the glycosylated peptides cannot be cleaved by proteinase K and result in a high current response on indium tin oxide (ITO) electrode. However, when the O-GlcNAcylation is successfully inhibited using a small molecule, the unglycosylated peptides can be cleaved easily and lead to low current signal. Peptide O-GlcNAcylation reaction was performed in the presence of a well-defined small-molecule OGT inhibitor. The results indicated that the biosensor could be used to screen the OGT inhibitors effectively. Our label-free electrochemical method is a promising candidate for protein glycosylation pathway research in screening small-molecule inhibitors of OGT. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas.

PubMed Central

Harris, L. C.; Margison, G. P.

1993-01-01

V79 Chinese hamster cells expressing either the O6-alkylguanine-DNA-alkyltransferase (ATase) encoded by the E. coli ogt gene or a truncated version of the E. coli ada gene have been exposed to various alkylnitrosoureas to investigate the contribution of ATase repairable lesions to the toxicity of these compounds. Both ATases are able to repair O6-alkylguanine (O6-AlkG) and O4-alkylthymine (O4-AlkT) but the ogt ATase is more efficient in the repair of O4-methylthymine (O4-MeT) and higher alkyl derivatives of O6-AlkG than is the ada ATase. Expression of the ogt ATase provided greater protection against the toxic effects of the alkylating agents then the ada ATase particularly with N-ethyl-N-nitrosourea (ENU) and N-butyl-N-nitrosourea (BNU) to which the ada ATase expressing cells were as sensitive as parent vector transfected cells. Although ogt was expressed at slightly higher levels than the truncated ada in the transfected cells, this could not account for the differential protection observed. For-N-methyl-N-nitrosourea (MNU) the increased protection in ogt-transfected cells is consistent with O4-MeT acting as a toxic lesion. For the longer chain alkylating agents and chloroethylating agents, the protection afforded by the ogt protein may be a consequence of the more efficient repair of O6-AlkG, O4-AlkT or both of these lesions in comparison with the ada-encoded ATase. Images Figure 2 Figure 3 PMID:8512805

Modulation of O-GlcNAc Levels in the Liver Impacts Acetaminophen-Induced Liver Injury by Affecting Protein Adduct Formation and Glutathione Synthesis.

PubMed

McGreal, Steven R; Bhushan, Bharat; Walesky, Chad; McGill, Mitchell R; Lebofsky, Margitta; Kandel, Sylvie E; Winefield, Robert D; Jaeschke, Hartmut; Zachara, Natasha E; Zhang, Zhen; Tan, Ee Phie; Slawson, Chad; Apte, Udayan

2018-04-01

Overdose of acetaminophen (APAP) results in acute liver failure. We have investigated the role of a posttranslational modification of proteins called O-GlcNAcylation, where the O-GlcNAc transferase (OGT) adds and O-GlcNAcase (OGA) removes a single β-D-N-acetylglucosamine (O-GlcNAc) moiety, in the pathogenesis of APAP-induced liver injury. Hepatocyte-specific OGT knockout mice (OGT KO), which have reduced O-GlcNAcylation, and wild-type (WT) controls were treated with 300 mg/kg APAP and the development of injury was studied over a time course from 0 to 24 h. OGT KO mice developed significantly lower liver injury as compared with WT mice. Hepatic CYP2E1 activity and glutathione (GSH) depletion following APAP treatment were not different between WT and OGT KO mice. However, replenishment of GSH and induction of GSH biosynthesis genes were significantly faster in the OGT KO mice. Next, male C57BL/6 J mice were treated Thiamet-G (TMG), a specific inhibitor of OGA to induce O-GlcNAcylation, 1.5 h after APAP administration and the development of liver injury was studied over a time course of 0-24 h. TMG-treated mice exhibited significantly higher APAP-induced liver injury. Treatment with TMG did not affect hepatic CYP2E1 levels, GSH depletion, APAP-protein adducts, and APAP-induced mitochondrial damage. However, GSH replenishment and GSH biosynthesis genes were lower in TMG-treated mice after APAP overdose. Taken together, these data indicate that induction in cellular O-GlcNAcylation exacerbates APAP-induced liver injury via dysregulation of hepatic GSH replenishment response.

Determination of N-acetylglucosamine in cosmetic formulations and skin test samples by hydrophilic interaction liquid chromatography and UV detection.

PubMed

Pedrali, Alice; Bleve, Mariella; Capra, Priscilla; Jonsson, Tobias; Massolini, Gabriella; Perugini, Paola; Marrubini, Giorgio

2015-03-25

N-Acetylglucosamine is an ingredient in pharmaceuticals, nutritional supplements and in cosmetics. N-Acetylglucosamine in cosmetics is expected to improve skin hydration, reparation, and to contribute as anti-wrinkle agent. This study reports on the validation and application of an HPLC method based on HILIC and UV detection for determining N-acetylglucosamine in cosmetics and in samples obtained after testing the skin exposed to cosmetics formulations. The chromatographic column used is a ZIC(®)-pHILIC (150 mm × 4.6 mm, 5 μm particle size) on which a mobile phase containing acetonitrile-aqueous KH2PO4 (70:30, v/v) 15 mM was applied in isocratic elution mode injecting 20 μl of sample at 0.5 ml/min constant flow-rate and 10±1°C column temperature. Under these conditions the total run time was 10 min and N-acetylglucosamine eluted baseline separated from all other compounds in the samples. Calibration in the range from 40 to 80 μg/ml allowed to assess the method linearity (R(2)>0.999) in a concentration range corresponding to about 50% to 120% of the expected levels of N-acetylglucosamine in the formulations. Precision expressed by RSD% was always better than 2% in intra-day and inter-day assays of authentic samples. Accuracy was in all cases within 95-105% of the expected concentration value in formulations containing N-acetylglucosamine. The sensitivity of the method was at the level of 10 μg/ml as limit of detection, and at 40 μg/ml as limit of quantitation. The application of the method to formulations containing solid lipid nanoparticles documents its usefulness in cosmetic quality control. The results witness that the method is also suitable for the determination of N-acetylglucosamine in samples obtained from skin test strips. Copyright © 2014 Elsevier B.V. All rights reserved.

Purification, crystallization and preliminary X-ray diffraction studies of UDP-N-acetylglucosamine pyrophosphorylase from Candida albicans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maruyama, Daisuke; Nishitani, Yuichi; Nonaka, Tsuyoshi

2006-12-01

UDP-N-acetylglucosamine pyrophosphorylase was purified and crystallized and X-ray diffraction data were collected to 2.3 Å resolution. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP-N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.3 Å resolution using synchrotron radiation. The crystals of the substrate complex belong to the triclinic space group P1, with unit-cell parameters a = 47.77, b = 62.89, c = 90.60 Å, α = 90.01, β = 97.72, γ = 92.88°, whereas those of the productmore » complex belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 61.95, b = 90.87, c = 94.88 Å.« less

Cardiomyocyte Ogt limits ventricular dysfunction in mice following pressure overload without affecting hypertrophy.

PubMed

Dassanayaka, Sujith; Brainard, Robert E; Watson, Lewis J; Long, Bethany W; Brittian, Kenneth R; DeMartino, Angelica M; Aird, Allison L; Gumpert, Anna M; Audam, Timothy N; Kilfoil, Peter J; Muthusamy, Senthilkumar; Hamid, Tariq; Prabhu, Sumanth D; Jones, Steven P

2017-05-01

The myocardial response to pressure overload involves coordination of multiple transcriptional, posttranscriptional, and metabolic cues. The previous studies show that one such metabolic cue, O-GlcNAc, is elevated in the pressure-overloaded heart, and the increase in O-GlcNAcylation is required for cardiomyocyte hypertrophy in vitro. Yet, it is not clear whether and how O-GlcNAcylation participates in the hypertrophic response in vivo. Here, we addressed this question using patient samples and a preclinical model of heart failure. Protein O-GlcNAcylation levels were increased in myocardial tissue from heart failure patients compared with normal patients. To test the role of OGT in the heart, we subjected cardiomyocyte-specific, inducibly deficient Ogt (i-cmOgt -/- ) mice and Ogt competent littermate wild-type (WT) mice to transverse aortic constriction. Deletion of cardiomyocyte Ogt significantly decreased O-GlcNAcylation and exacerbated ventricular dysfunction, without producing widespread changes in metabolic transcripts. Although some changes in hypertrophic and fibrotic signaling were noted, there were no histological differences in hypertrophy or fibrosis. We next determined whether significant differences were present in i-cmOgt -/- cardiomyocytes from surgically naïve mice. Interestingly, markers of cardiomyocyte dedifferentiation were elevated in Ogt-deficient cardiomyocytes. Although no significant differences in cardiac dysfunction were apparent after recombination, it is possible that such changes in dedifferentiation markers could reflect a larger phenotypic shift within the Ogt-deficient cardiomyocytes. We conclude that cardiomyocyte Ogt is not required for cardiomyocyte hypertrophy in vivo; however, loss of Ogt may exert subtle phenotypic differences in cardiomyocytes that sensitize the heart to pressure overload-induced ventricular dysfunction.

BLT1-mediated O-GlcNAcylation is required for NOX2-dependent migration, exocytotic degranulation and IL-8 release of human mast cell induced by Trichomonas vaginalis-secreted LTB4.

PubMed

Min, Arim; Lee, Young Ah; Kim, Kyeong Ah; Shin, Myeong Heon

2018-05-31

Trichomonas vaginalis is a sexually-transmitted protozoan parasite that causes vaginitis and cervicitis. Although mast cell activation is important for provoking tissue inflammation during infection with parasites, information regarding the signaling mechanisms in mast cell activation and T. vaginalis infection is limited. O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serine and threonine residues that functions as a critical regulator of intracellular signaling, regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). We investigated if O-GlcNAcylation was associated with mast cell activation induced by T. vaginalis-derived secretory products (TvSP). Modified TvSP collected from live trichomonads treated with the 5-lipooxygenase inhibitor AA861 inhibited migration of mast cells. This result suggested that mast cell migration was caused by stimulation of T. vaginalis-secreted leukotrienes. Using the BLT1 antagonist U75302 or BLT1 siRNA, we found that migration of mast cells was evoked via LTB 4 receptor (BLT1). Furthermore, TvSP induced protein O-GlcNAcylation and OGT expression in HMC-1 cells, which was prevented by transfection with BLT1 siRNA. TvSP-induced migration, ROS generation, CD63 expression and IL-8 release were significantly suppressed by pretreatmemnt with OGT inhibitor ST045849 or OGT siRNA. These results suggested that BLT1-mediated OGlcNAcylation was important for mast cell activation during trichomoniasis. Copyright © 2018. Published by Elsevier Masson SAS.

Swim-exercised mice show a decreased level of protein O-GlcNAcylation and expression of O-GlcNAc transferase in heart.

PubMed

Belke, Darrell D

2011-07-01

Swim-training exercise in mice leads to cardiac remodeling associated with an improvement in contractile function. Protein O-linked N-acetylglucosamine (O-GlcNAcylation) is a posttranslational modification of serine and threonine residues capable of altering protein-protein interactions affecting gene transcription, cell signaling pathways, and general cell physiology. Increased levels of protein O-GlcNAcylation in the heart have been associated with pathological conditions such as diabetes, ischemia, and hypertrophic heart failure. In contrast, the impact of physiological exercise on protein O-GlcNAcylation in the heart is currently unknown. Swim-training exercise in mice was associated with the development of a physiological hypertrophy characterized by an improvement in contractile function relative to sedentary mice. General protein O-GlcNAcylation was significantly decreased in swim-exercised mice. This effect was mirrored in the level of O-GlcNAcylation of individual proteins such as SP1. The decrease in protein O-GlcNAcylation was associated with a decrease in the expression of O-GlcNAc transferase (OGT) and glutamine-fructose amidotransferase (GFAT) 2 mRNA. O-GlcNAcase (OGA) activity was actually lower in swim-trained than sedentary hearts, suggesting that it did not contribute to the decreased protein O-GlcNAcylation. Thus it appears that exercise-induced physiological hypertrophy is associated with a decrease in protein O-GlcNAcylation, which could potentially contribute to changes in gene expression and other physiological changes associated with exercise.

N-Acetylglucosamine: Production and Applications

PubMed Central

Chen, Jeen-Kuan; Shen, Chia-Rui; Liu, Chao-Lin

2010-01-01

N-Acetylglucosamine (GlcNAc) is a monosaccharide that usually polymerizes linearly through (1,4)-β-linkages. GlcNAc is the monomeric unit of the polymer chitin, the second most abundant carbohydrate after cellulose. In addition to serving as a component of this homogeneous polysaccharide, GlcNAc is also a basic component of hyaluronic acid and keratin sulfate on the cell surface. In this review, we discuss the industrial production of GlcNAc, using chitin as a substrate, by chemical, enzymatic and biotransformation methods. Also, newly developed methods to obtain GlcNAc using glucose as a substrate in genetically modified microorganisms are introduced. Moreover, GlcNAc has generated interest not only as an underutilized resource but also as a new functional material with high potential in various fields. Here we also take a closer look at the current applications of GlcNAc, and several new and cutting edge approaches in this fascinating area are thoroughly discussed. PMID:20948902

O-GlcNAcylation enhances anaplastic thyroid carcinoma malignancy.

PubMed

Cheng, Y U; Li, Honglun; Li, Jianlin; Li, Jisheng; Gao, Yan; Liu, Baodong

2016-07-01

O-linked N -acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation), a dynamic post-translational modification of nuclear and cytoplasmic proteins, may have a critical role in the regulation of biological cell processes and human cancer. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Accumulating evidence suggests that O-GlcNAcylation is involved in a variety of types of human cancer. However, the exact role of O-GlcNAcylation in tumor pathogenesis or progression remains to be established. Computed tomography scans of patients with anaplastic thyroid carcinoma (ATC) reveal a rapid growth rate and invasion. The present study demonstrated that O-GlcNAcylation accelerates the progression of ATC. The global O-GlcNAc level of intracellular proteins was increased by overexpression of OGT or downregulation of OGA activity with the specific inhibitor Thiamet-G. By contrast, the global O-GlcNAc level was decreased by silencing of OGT. MTT assay indicated that O-GlcNAcylation significantly promotes cell proliferation. Furthermore, O-GlcNAcylation enhanced cellular biological functions, such as colony formation ability, migration and invasion, of ATC cells in vitro . The findings of the present study suggest that O-GlcNAcylation is associated with malignant properties of thyroid cancer, and may be a potential target for the diagnosis and treatment of thyroid cancer.

Protein O-GlcNAcylation: emerging mechanisms and functions

PubMed Central

Yang, Xiaoyong; Qian, Kevin

2017-01-01

O-GlcNAcylation—the attachment of O-linked N-acetylglucosamine (O-GlcNAc) moieties to cytoplasmic, nuclear and mitochondrial proteins—is a post-translational modification that regulates fundamental cellular processes in metazoans. A single pair of enzymes—O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA)—controls the dynamic cycling of this post-translational modification in a nutrient- and stress-responsive manner. Recent years have seen remarkable advances in our understanding of O-GlcNAcylation at levels ranging from structural and molecular biology to cell signalling and gene regulation to physiology and disease. Emerging from these recent developments are new mechanisms and functions of O-GlcNAcylation that enable us to begin constructing a unified conceptual framework through which to understand the significance of this modification in cellular and organismal physiology. PMID:28488703

Extensive Crosstalk Between O-GlcNAcylation and Phosphorylation Regulates Cytokinesis

PubMed Central

Wang, Zihao; Udeshi, Namrata D.; Slawson, Chad; Compton, Philip D.; Sakabe, Kaoru; Cheung, Win D.; Shabanowitz, Jeffrey; Hunt, Donald F.; Hart, Gerald W.

2010-01-01

Like phosphorylation, the addition of O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways, but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division. PMID:20068230

Potential coordination role between O-GlcNAcylation and epigenetics.

PubMed

Wu, Donglu; Cai, Yong; Jin, Jingji

2017-10-01

Dynamic changes of the post-translational O-GlcNAc modification (O-GlcNAcylation) are controlled by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) and the glycoside hydrolase O-GlcNAcase (OGA) in cells. O-GlcNAcylation often occurs on serine (Ser) and threonine (Thr) residues of the specific substrate proteins via the addition of O-GlcNAc group by OGT. It has been known that O-GlcNAcylation is not only involved in many fundamental cellular processes, but also plays an important role in cancer development through various mechanisms. Recently, accumulating data reveal that O-GlcNAcylation at histones or non-histone proteins can lead to the start of the subsequent biological processes, suggesting that O-GlcNAcylation as 'protein code' or 'histone code' may provide recognition platforms or executive instructions for subsequent recruitment of proteins to carry out the specific functions. In this review, we summarize the interaction of O-GlcNAcylation and epigenetic changes, introduce recent research findings that link crosstalk between O-GlcNAcylation and epigenetic changes, and speculate on the potential coordination role of O-GlcNAcylation with epigenetic changes in intracellular biological processes.

Lactobacillus casei Ferments the N-Acetylglucosamine Moiety of Fucosyl-α-1,3-N-Acetylglucosamine and Excretes l-Fucose

PubMed Central

Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio

2012-01-01

We have previously characterized from Lactobacillus casei BL23 three α-l-fucosidases, AlfA, AlfB, and AlfC, which hydrolyze in vitro natural fucosyl-oligosaccharides. In this work, we have shown that L. casei is able to grow in the presence of fucosyl-α-1,3-N-acetylglucosamine (Fuc-α-1,3-GlcNAc) as a carbon source. Interestingly, L. casei excretes the l-fucose moiety during growth on Fuc-α-1,3-GlcNAc, indicating that only the N-acetylglucosamine moiety is being metabolized. Analysis of the genomic sequence of L. casei BL23 shows that downstream from alfB, which encodes the α-l-fucosidase AlfB, a gene, alfR, that encodes a transcriptional regulator is present. Divergently from alfB, three genes, alfEFG, that encode proteins with homology to the enzyme IIAB (EIIAB), EIIC, and EIID components of a mannose-class phosphoenolpyruvate:sugar phosphotransferase system (PTS) are present. Inactivation of either alfB or alfF abolishes the growth of L. casei on Fuc-α-1,3-GlcNAc. This proves that AlfB is involved in Fuc-α-1,3-GlcNAc metabolism and that the transporter encoded by alfEFG participates in the uptake of this disaccharide. A mutation in the PTS general component enzyme I does not eliminate the utilization of Fuc-α-1,3-GlcNAc, suggesting that the transport via the PTS encoded by alfEFG is not coupled to phosphorylation of the disaccharide. Transcriptional analysis with alfR and ccpA mutants shows that the two gene clusters alfBR and alfEFG are regulated by substrate-specific induction mediated by the inactivation of the transcriptional repressor AlfR and by carbon catabolite repression mediated by the catabolite control protein A (CcpA). This work reports for the first time the characterization of the physiological role of an α-l-fucosidase in lactic acid bacteria and the utilization of Fuc-α-1,3-GlcNAc as a carbon source for bacteria. PMID:22544237

OGT-related mitochondrial motility is associated with sex differences and exercise effects in depression induced by prenatal exposure to glucocorticoids.

PubMed

Liu, Weina; Wang, Hongmei; Xue, Xiangli; Xia, Jie; Liu, Jiatong; Qi, Zhengtang; Ji, Liu

2018-01-15

Prenatal exposure to glucocorticoids (GCs) has been found to trigger abnormal behaviors and deleterious neurological effects on offspring both in animals and in humans. The sex differences in depression have been replicated in numerous studies across cultures, persisting throughout the reproductive years. As an X-linked gene in rodents and in humans, O-GlcNAc transferase (OGT) may provide a novel perspective for the sex differences in depression. In the last third of pregnancy (gestational day 14-21), rats were subcutaneously administered either 0.13mg/kg dexamethasone-21-phosphate disodium salt (0.1mg/kg DEX) or vehicle (0.9% saline) once a day for 7 days. Adolescent (4 weeks) offspring were then trained in a swimming program or not. Here we found that adult offspring rats exposed to DEX prenatally exhibited sex-specific depression-like behaviors, males being more vulnerable than females. Swimming exercise ameliorated the above-mentioned depressive syndromes, which may be a compensatory effect for male disadvantage suffering from prenatal stress. Furthermore, the effects of prenatal DEX exposure and swimming exercise on depression were associated with OGT-related mitochondrial motility, including PINK1/Parkin pathway and AKT/GSK3β pathway. Representative kymographs of mitochondrial motility were not detected and no causal effects were obtained by OGT gene overexpression or gene knockout in this study. Our results provide a new perspective for better understanding sex differences and exercise effects in depression and may offer new mechanism-based therapeutic targets for depression. Copyright © 2017 Elsevier B.V. All rights reserved.

Immediate effects of a single exercise bout on protein O-GlcNAcylation and chromatin regulation of cardiac hypertrophy

PubMed Central

Medford, Heidi M.; Porter, Karen

2013-01-01

Cardiac hypertrophy induced by pathological stimuli is regulated by a complex formed by the repressor element 1-silencing transcription factor (REST) and its corepressor mSin3A. We previously reported that hypertrophic signaling is blunted by O-linked attachment of β-N-acetylglucosamine (O-GlcNAc) to proteins. Regular exercise induces a physiological hypertrophic phenotype in the heart that is associated with decreased O-GlcNAc levels, but a link between O-GlcNAc, the REST complex, and initiation of exercise-induced cardiac hypertrophy is not known. Therefore, mice underwent a single 15- or 30-min bout of moderate- to high-intensity treadmill running, and hearts were harvested immediately and compared with sedentary controls. Cytosolic O-GlcNAc was lower (P < 0.05) following 15 min exercise with no differences in nuclear levels (P > 0.05). There were no differences in cytosolic or nuclear O-GlcNAc levels in hearts after 30 min exercise (P > 0.05). Cellular compartment levels of O-GlcNAc transferase (OGT, the enzyme that removes the O-GlcNAc moiety from proteins), REST, mSin3A, and histone deacetylases (HDACs) 1, 2, 3, 4, and 5 were not changed with exercise. Immunoprecipitation revealed O-GlcNAcylation of OGT and HDACs 1, 2, 4, and 5 that was not changed with acute exercise; however, exercised hearts did exhibit lower interactions between OGT and REST (P < 0.05) but not between OGT and mSin3A. These data suggest that hypertrophic signaling in the heart may be initiated by as little as 15 min of exercise via intracellular changes in protein O-GlcNAcylation distribution and reduced interactions between OGT and the REST chromatin repressor. PMID:23624624

Regulatory insights into the production of UDP-N-acetylglucosamine by Lactobacillus casei

PubMed Central

Rodríguez-Díaz, Jesús; Rubio-del-Campo, Antonio; Yebra, María J.

2012-01-01

UDP-N-acetylglucosamine (UDP-GlcNAc) is an important sugar nucleotide used as a precursor of cell wall components in bacteria, and as a substrate in the synthesis of oligosaccharides in eukaryotes. In bacteria UDP-GlcNAc is synthesized from the glycolytic intermediate D-fructose-6-phosphate (fructose-6P) by four successive reactions catalyzed by three enzymes: glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM) and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). We have previously reported a metabolic engineering strategy in Lactobacillus casei directed to increase the intracellular levels of UDP-GlcNAc by homologous overexpression of the genes glmS, glmM and glmU. One of the most remarkable features regarding the production of UDP-GlcNAc in L. casei was to find multiple regulation points on its biosynthetic pathway: (1) regulation by the NagB enzyme, (2) glmS RNA specific degradation through the possible participation of a glmS riboswitch mechanism, (3) regulation of the GlmU activity probably by end product inhibition and (4) transcription of glmU. PMID:22825354

Elevated O-GlcNAcylation promotes gastric cancer cells proliferation by modulating cell cycle related proteins and ERK 1/2 signaling.

PubMed

Jiang, Mingzuo; Qiu, Zhaoyan; Zhang, Song; Fan, Xing; Cai, Xiqiang; Xu, Bing; Li, Xiaowei; Zhou, Jinfeng; Zhang, Xiangyuan; Chu, Yi; Wang, Weijie; Liang, Jie; Horvath, Tamas; Yang, Xiaoyong; Wu, Kaichun; Nie, Yongzhan; Fan, Daiming

2016-09-20

O-GlcNAc transferase (OGT) is the only enzyme in mammals that catalyzes the attachment of β-D-N-acetylglucosamine (GlcNAc) to serine or threonine residues of target proteins. Hyper-O-GlcNAcylation is becoming increasingly realized as a general feature of cancer and contributes to rapid proliferation of cancer cells. In this study, we demonstrated that O-GlcNAc and OGT levels were increased in all six gastric cancer (GC) cell lines as compared with immortal gastric epithelial cells. Downregulation of the O-GlcNAcylation level by silencing OGT inhibited cell viability and growth rate via the cdk-2, cyclin D1 and ERK 1/2 pathways. In vivo xenograft assays also demonstrated that the hyper-O-GlcNAc level markedly promoted the proliferation of tumors. Moreover, compared with noncancerous tissues, the O-GlcNAcylation level was increased in cancerous tissues. GC patients with higher levels of O-GlcNAcylation exhibited large tumor sizes (≥5 cm), deep tumor invasion (T3 and T4), high AJCC stages (stage III and IV), more lymph node metastases and lower overall survival. Notably, the phosphorylation level of ERK 1/2 was increased progressively with the increase of O-GlcNAcylation in both SGC 7901 and AGS cells. Consistently, human GC tissue arrays also revealed that ERK 1/2 signaling was positively correlated to O-GlcNAcylation (r = 0.348; P = 0.015). Taken together, here we reported that hyper-O-GlcNAcylation significantly promotes GC cells proliferation by modulating cell cycle related proteins and ERK 1/2 signaling, suggesting that inhibition of OGT may be a potential novel therapeutic target of GC.

Identification and phylogenetic relationship of Iranian strains of various Leishmania species isolated from cutaneous and visceral cases of leishmaniasis based on N-acetylglucosamine-1-phosphate transferase gene.

PubMed

Hajjaran, Homa; Mohebali, Mehdi; Teimouri, Aref; Oshaghi, Mohammad Ali; Mirjalali, Hamed; Kazemi-Rad, Elham; Shiee, Mohammad Reza; Naddaf, Saied Reza

2014-08-01

The identity of Iranian Leishmania species has been resolved to some extent by some genetic markers. In this study, based on N-acetylglucosamine-1-phosphate transferase (nagt) gene, we further elucidated the identity and phylogeny of the prevalent species in this country. DNAs of 121 isolates belonging to cutaneous leishmaniasis (CL) patients, canine visceral leishmaniasis (CVL) cases, and Rhombomys opimus rodents were amplified by targeting a partial sequence of nagt gene. All the amplicons were analyzed with restriction fragment length polymorphism (RFLP) using Acc1 enzyme, and 49 amplicons representing different reservoir hosts were sequenced and aligned with similar sequences from GenBank database. The RFLP analysis revealed that 41 CL patients were infected Leishmania tropica and 36 with Leishmania major. Among 10 CVL isolates, 6 were identified as Leishmania infantum and 4 as L. tropica. Amongst 34 rodents' isolates, 11 and 23 isolates exhibited patterns similar to those of L. major, and L. tropica/Leishmania turanica, respectively. The sequencing results from all CL patients, CVL cases, and 4 reservoir rodents were in agreement with RFLP analysis and showed 99-100% homologies with the registered species of L. major, L. tropica, and L. infantum from Turkey, Tunisia, Iraq and Israel. Of the 7 rodent isolates exhibiting RFLP patterns similar to L. tropica/L. turanica, 3 exhibited the highest homologies (99-100%) with L. turanica and 4 with Leishmania gerbilli. The 49 nagt DNA sequences were grouped into five clusters representing L. major, L. tropica, L. infantum, L. turanica and L. gerbilli species, encompassing 19 haplotypes. No correlation was observed between intraspecies divergence and geographic distribution of haplotypes. The L. tropica haplotypes exhibited more homologies with those of L. infantum than L. major (97.2% vs. 96.9%), a probable indication to the potential ability of L. tropica to visceralize. Characterization of Iranian Leishmania isolates

O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis

PubMed Central

Zentella, Rodolfo; Hu, Jianhong; Hsieh, Wen-Ping; Matsumoto, Peter A.; Dawdy, Andrew; Barnhill, Benjamin; Oldenhof, Harriëtte; Hartweck, Lynn M.; Maitra, Sushmit; Thomas, Stephen G.; Cockrell, Shelley; Boyce, Michael; Shabanowitz, Jeffrey; Hunt, Donald F.; Olszewski, Neil E.; Sun, Tai-ping

2016-01-01

The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein–protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors—PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)—that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development. PMID:26773002

Identification of a negative regulator of gibberellin action, HvSPY, in barley.

PubMed Central

Robertson, M; Swain, S M; Chandler, P M; Olszewski, N E

1998-01-01

To broaden our understanding of the molecular mechanisms of gibberellin (GA) action, we isolated a spindly clone (HvSPY) from barley cultivar Himalaya and tested whether the HvSPY protein would modulate GA action in barley aleurone. The HvSPY cDNA showed high sequence identity to Arabidopsis SPY along its entire length, and the barley protein functionally complemented the spy-3 mutation. HvSPY and SPY proteins showed sequence relatedness with animal O-linked N-acetylglucosamine transferases (OGTs), suggesting that they may also have OGT activity. HvSPY has a locus distinct from that of Sln, a mutation that causes the constitutive GA responses of slender barley, which phenotypically resembles Arabidopsis spy mutants. The possibility that the HvSPY gene encodes a negative regulator of GA action was tested by expressing HvSPY in a barley aleurone transient assay system. HvSPY coexpression largely abolished GA3-induced activity of an alpha-amylase promoter. Surprisingly, HvSPY coexpression increased reporter gene activity from an abscisic acid (ABA)-inducible gene promoter (dehydrin), even in the absence of exogenous ABA. These results show that HvSPY modulates the transcriptional activities of two hormonally regulated promoters: negatively for a GA-induced promoter and positively for an ABA-induced promoter. PMID:9634587

O-GlcNAcylation of master growth repressor DELLA by SECRET AGENT modulates multiple signaling pathways in Arabidopsis.

PubMed

Zentella, Rodolfo; Hu, Jianhong; Hsieh, Wen-Ping; Matsumoto, Peter A; Dawdy, Andrew; Barnhill, Benjamin; Oldenhof, Harriëtte; Hartweck, Lynn M; Maitra, Sushmit; Thomas, Stephen G; Cockrell, Shelley; Boyce, Michael; Shabanowitz, Jeffrey; Hunt, Donald F; Olszewski, Neil E; Sun, Tai-Ping

2016-01-15

The DELLA family of transcription regulators functions as master growth repressors in plants by inhibiting phytohormone gibberellin (GA) signaling in response to developmental and environmental cues. DELLAs also play a central role in mediating cross-talk between GA and other signaling pathways via antagonistic direct interactions with key transcription factors. However, how these crucial protein-protein interactions can be dynamically regulated during plant development remains unclear. Here, we show that DELLAs are modified by the O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) SECRET AGENT (SEC) in Arabidopsis. O-GlcNAcylation of the DELLA protein REPRESSOR OF ga1-3 (RGA) inhibits RGA binding to four of its interactors-PHYTOCHROME-INTERACTING FACTOR3 (PIF3), PIF4, JASMONATE-ZIM DOMAIN1, and BRASSINAZOLE-RESISTANT1 (BZR1)-that are key regulators in light, jasmonate, and brassinosteroid signaling pathways, respectively. Consistent with this, the sec-null mutant displayed reduced responses to GA and brassinosteroid and showed decreased expression of several common target genes of DELLAs, BZR1, and PIFs. Our results reveal a direct role of OGT in repressing DELLA activity and indicate that O-GlcNAcylation of DELLAs provides a fine-tuning mechanism in coordinating multiple signaling activities during plant development. © 2016 Zentella et al.; Published by Cold Spring Harbor Laboratory Press.

Structural Basis of Specific Recognition of Non-Reducing Terminal N-Acetylglucosamine by an Agrocybe aegerita Lectin

PubMed Central

Ren, Xiao-Ming; Li, De-Feng; Jiang, Shuai; Lan, Xian-Qing; Hu, Yonglin; Sun, Hui; Wang, Da-Cheng

2015-01-01

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification that plays essential roles in many cellular pathways. Research in this field, however, is hampered by the lack of suitable probes to identify, accumulate, and purify the O-GlcNAcylated proteins. We have previously reported the identification of a lectin from the mushroom Agrocybe aegerita, i.e., Agrocybe aegerita lectin 2, or AAL2, that could bind terminal N-acetylglucosamine with higher affinities and specificity than other currently used probes. In this paper, we report the crystal structures of AAL2 and its complexes with GlcNAc and GlcNAcβ1-3Galβ1-4GlcNAc and reveal the structural basis of GlcNAc recognition by AAL2 and residues essential for the binding of terminal N-acetylglucosamine. Study on AAL2 may enable us to design a protein probe that can be used to identify and purify O-GlcNAcylated proteins more efficiently. PMID:26114302

Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

PubMed

Rancourt, Ann; Dufresne, Sébastien S; St-Pierre, Guillaume; Lévesque, Julie-Christine; Nakamura, Haruka; Kikuchi, Yodai; Satoh, Masahiko S; Frenette, Jérôme; Sato, Sachiko

2018-06-12

The muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), in which mutations in the dystrophin gene disrupts the firm adhesion. In patients with DMD, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in patients with DMD, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina and increases the efficiency of the myogenesis. Galectin-3 is an oligosaccharide-binding protein and is known to be involved in cell-cell interactions and cell-matrix interactions. Galectin-3 is expressed in myoblasts and skeletal muscle, although its function in muscle remains elusive. In this study, we found evidence that galectin-3 and the monosaccharide N-acetylglucosamine, which increases the synthesis of binding partners (oligosaccharides) of galectin-3, promote myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N-acetylglucosamine increased muscle-force production. The results suggest that treatment with N-acetylglucosamine might mitigate the burden of DMD.-Rancourt, A., Dufresne, S. S., St-Pierre, G., Lévesque, J.-C., Nakamura, H., Kikuchi, Y., Satoh, M. S., Frenette, J., Sato, S. Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy.

In vitro evidence for the participation of Drosophila melanogaster sperm β-N-acetylglucosaminidases in the interactions with glycans carrying terminal N-acetylglucosamine residues on the egg's envelopes.

PubMed

Intra, Jari; Veltri, Concetta; De Caro, Daniela; Perotti, Maria Elisa; Pasini, Maria Enrica

2017-09-01

Fertilization is a complex and multiphasic process, consisting of several steps, where egg-coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm-associated β-N-acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid-phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well-defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β-N-acetylglucosamine, but not core β-N-acetylglucosamine residues. The capacity of sperm β-N-acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β-N-acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β-N-acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm-egg interactions in Drosophila. © 2017 Wiley Periodicals, Inc.

Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity.

PubMed

Shi, Hao; Munk, Alexander; Nielsen, Thomas S; Daughtry, Morgan R; Larsson, Louise; Li, Shize; Høyer, Kasper F; Geisler, Hannah W; Sulek, Karolina; Kjøbsted, Rasmus; Fisher, Taylor; Andersen, Marianne M; Shen, Zhengxing; Hansen, Ulrik K; England, Eric M; Cheng, Zhiyong; Højlund, Kurt; Wojtaszewski, Jørgen F P; Yang, Xiaoyong; Hulver, Matthew W; Helm, Richard F; Treebak, Jonas T; Gerrard, David E

2018-05-01

Given that cellular O-GlcNAcylation levels are thought to be real-time measures of cellular nutrient status and dysregulated O-GlcNAc signaling is associated with insulin resistance, we evaluated the role of O-GlcNAc transferase (OGT), the enzyme that mediates O-GlcNAcylation, in skeletal muscle. We assessed O-GlcNAcylation levels in skeletal muscle from obese, type 2 diabetic people, and we characterized muscle-specific OGT knockout (mKO) mice in metabolic cages and measured energy expenditure and substrate utilization pattern using indirect calorimetry. Whole body insulin sensitivity was assessed using the hyperinsulinemic euglycemic clamp technique and tissue-specific glucose uptake was subsequently evaluated. Tissues were used for histology, qPCR, Western blot, co-immunoprecipitation, and chromatin immunoprecipitation analyses. We found elevated levels of O-GlcNAc-modified proteins in obese, type 2 diabetic people compared with well-matched obese and lean controls. Muscle-specific OGT knockout mice were lean, and whole body energy expenditure and insulin sensitivity were increased in these mice, consistent with enhanced glucose uptake and elevated glycolytic enzyme activities in skeletal muscle. Moreover, enhanced glucose uptake was also observed in white adipose tissue that was browner than that of WT mice. Interestingly, mKO mice had elevated mRNA levels of Il15 in skeletal muscle and increased circulating IL-15 levels. We found that OGT in muscle mediates transcriptional repression of Il15 by O-GlcNAcylating Enhancer of Zeste Homolog 2 (EZH2). Elevated muscle O-GlcNAc levels paralleled insulin resistance and type 2 diabetes in humans. Moreover, OGT-mediated signaling is necessary for proper skeletal muscle metabolism and whole-body energy homeostasis, and our data highlight O-GlcNAcylation as a potential target for ameliorating metabolic disorders. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

Regulation of Dauer formation by O-GlcNAcylation in Caenorhabditis elegans.

PubMed

Lee, Jeeyong; Kim, Kwang-Youl; Lee, Jihyun; Paik, Young-Ki

2010-01-29

Modification of proteins at serine or threonine residues with N-acetylglucosamine, termed O-GlcNAcylation, plays an important role in most eukaryotic cells. To understand the molecular mechanism by which O-GlcNAcylation regulates the entry of Caenorhabditis elegans into the non-aging dauer state, we performed proteomic studies using two mutant strains: the O-GlcNAc transferase-deficient ogt-1(ok430) strain and the O-GlcNAcase-defective oga-1(ok1207) strain. In the presence of the dauer pheromone daumone, ogt-1 showed suppression of dauer formation, whereas oga-1 exhibited enhancement of dauer formation. Consistent with these findings, treatment of wild-type N2 worms with low concentrations of daumone and the O-GlcNAcase inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) enhanced dauer formation, which was dependent on intact O-GlcNAcylation metabolism. We also found that the treatment of daumone enhanced O-GlcNAcylation in vivo. Seven proteins, identified by coupled two-dimensional electrophoresis/liquid chromatography-mass spectroscopy (LC-MS) analysis, were differentially expressed in oga-1(ok1207) worms compared with wild-type N2 worms. The identities of these proteins suggest that O- GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. Using O-GlcNAc labeling with fluorescent dye combined with two-dimensional electrophoresis/LC-MS analysis, we also identified five proteins that were differentially O-GlcNAcylated during dauer formation. Analysis of these candidate O-GlcNAcylated proteins suggests that O-GlcNAcylation may regulate cytoskeleton modifications and protein turnover during dauer formation.

Hyper-O-GlcNAcylation inhibits the induction of heat shock protein 70 (Hsp 70) by sodium arsenite in HeLa cells.

PubMed

Miura, Yuri; Sato, Takatoshi; Sakurai, Yoko; Sakai, Ryo; Hiraoka, Wakako; Endo, Tamao

2014-01-01

O-Linked β-N-acetylglucosamine-modification (O-GlcNAcylation) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. However, the role of O-GlcNAcylation in the induction of heat shock proteins (Hsps) by arsenite remains unclear. We used O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate (PUGNAc), an inhibitor of O-GlcNAcase, and glucosamine (GlcN), an enhancer of the hexosamine biosynthesis pathway, or O-GlcNAc transferase (OGT) short interfering RNA (siRNA) to enhance or suppress cellular O-GlcNAcylation levels, respectively, in HeLa cells. The exposure to arsenite increased O-GlcNAcylation and Hsp 70 levels in HeLa cells. However, the pre-treatment with PUGNAc or GlcN, which enhanced O-GlcNAcylation levels, decreased the arsenite-induced expression of Hsp 70. The pre-treatment with OGT siRNA, which suppressed O-GlcNAcylation levels, did not affect the induction of Hsp 70. We then examined the effects of O-GlcNAcylation on the nuclear translocation and phosphorylation of heat shock factor 1 (HSF1), and found that neither the nuclear translocation nor phosphorylation of HSF1 was regulated by O-GlcNAcylation. Finally, Hsp 70 mRNA expression was induced by arsenite, whereas the addition of PUGNAc slightly suppressed its induction. These results indicate that O-GlcNAcylation is related to arsenite-induced Hsp 70 expression, and demonstrated that hyper-O-GlcNAcylation inhibited the induction of Hsp 70 via transcriptional factors instead of HSF1.

Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription

PubMed Central

Waast, Laetitia; Kuo, Mei-Shiue; Mangeney, Marianne; Martella, Christophe; Souidi, Mouloud; Issad, Tarik

2017-01-01

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5’LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O

Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription.

PubMed

Groussaud, Damien; Khair, Mostafa; Tollenaere, Armelle I; Waast, Laetitia; Kuo, Mei-Shiue; Mangeney, Marianne; Martella, Christophe; Fardini, Yann; Coste, Solène; Souidi, Mouloud; Benit, Laurence; Pique, Claudine; Issad, Tarik

2017-07-01

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5'LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway

2-acylamido analogues of N-acetylglucosamine prime formation of chitin oligosaccharides by yeast chitin synthase 2

USDA-ARS?s Scientific Manuscript database

Chitin, a polymer of beta-1,4-linked N-acetylglucosamine (GlcNAc), is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases (CSs) transfer GlcNAc from UDP-GlcNAc to pre-existing chitin chains in reactions that are typically stimulated by free GlcNAc. The eff...

Functional Analysis of SPINDLY in Gibberellin Signaling in Arabidopsis1[C][W][OA

PubMed Central

Silverstone, Aron L.; Tseng, Tong-Seung; Swain, Stephen M.; Dill, Alyssa; Jeong, Sun Yong; Olszewski, Neil E.; Sun, Tai-ping

2007-01-01

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-Δ17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-Δ17 phenotype but does not reduce rga-Δ17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification. PMID:17142481

O-GlcNAc cycling: Emerging Roles in Development and Epigenetics

PubMed Central

Love, Dona C.; Krause, Michael W.; Hanover, John A.

2010-01-01

The nutrient-sensing hexosamine signaling pathway modulates the levels of O-linked N-acetylglucosamine (O-GlcNAc) on key targets impacting cellular signaling, protein turnover and gene expression. O-GlcNAc cycling may be deregulated in neurodegenerative disease, cancer, and diabetes. Studies in model organisms demonstrate that the O-GlcNAc transferase (OGT/Sxc) is essential for Polycomb group (PcG) repression of the homeotic genes, clusters of genes responsible for the adult body plan. Surprisingly, from flies to man, the O-GlcNAcase (OGA, MGEA5) gene is embedded within the NK cluster, the most evolutionarily ancient of three homeobox gene clusters regulated by PcG repression. PcG repression also plays a key role in maintaining stem cell identity, recruiting the DNA methyltransferase machinery for imprinting, and in X-chromosome inactivation. Intriguingly, the Ogt gene resides near the Xist locus in vertebrates and is subject to regulation by PcG-dependent X-inactivation. OGT is also an enzymatic component of the human dosage compensation complex. These ‘evo-devo’ relationships linking O-GlcNAc cycling to higher order chromatin structure provide insights into how nutrient availability may influence the epigenetic regulation of gene expression. O-GlcNAc cycling at promoters and PcG repression represent concrete mechanisms by which nutritional information may be transmitted across generations in the intra-uterine environment. Thus, the nutrient-sensing hexosamine signaling pathway may be a key contributor to the metabolic deregulation resulting from prenatal exposure to famine, or the ‘vicious cycle’ observed in children of mothers with type-2 diabetes and metabolic disease. PMID:20488252

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

PubMed

Hwang, Hongik; Rhim, Hyewhon

2018-03-01

Post-translational modifications (PTMs) covalently modify proteins and diversify protein functions. Along with protein phosphorylation, another common PTM is the addition of O-linked β-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. Copyright © 2017 Elsevier Ltd. All rights reserved.

N-acetylglucosamine 6-Phosphate Deacetylase (nagA) Is Required for N-acetyl Glucosamine Assimilation in Gluconacetobacter xylinus

PubMed Central

Yadav, Vikas; Panilaitis, Bruce; Shi, Hai; Numuta, Keiji; Lee, Kyongbum; Kaplan, David L.

2011-01-01

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tetr; named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species. PMID:21655093

N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus.

PubMed

Yadav, Vikas; Panilaitis, Bruce; Shi, Hai; Numuta, Keiji; Lee, Kyongbum; Kaplan, David L

2011-01-01

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.

O-GlcNAcylation in oral squamous cell carcinoma.

PubMed

Kongkaew, Tassaporn; Aung, Win Pa Pa; Supanchart, Chayarop; Makeudom, Anupong; Langsa-Ard, Sarawat; Sastraruji, Thanapat; Chaiyarit, Ponlatham; Krisanaprakornkit, Suttichai

2018-03-01

Two post-translational mechanisms commonly demonstrated in various cancers are protein phosphorylation and glycosylation by O-linked β-N-acetylglucosamine (O-GlcNAc). However, only phosphorylation of the epidermal growth factor receptor (EGFR)/Akt pathway has been reported in oral squamous cell carcinoma (OSCC). Therefore, we aimed to determine both post-translational modifications in OSCC tissues and in oral cancer cells compared to normal tissues and oral keratinocytes and to find correlations of these modifications with histological grading. Thirty-two OSCC and ten normal formalin-fixed and paraffin-embedded sections were probed with the anti-O-GlcNAc, anti-O-GlcNAc transferase (OGT), anti-phosphorylated-EGFR tyr1173 , and anti-phosphorylated-Akt ser473 antibodies following standard immunohistochemistry. The immunohistochemical (IHC) score was determined using the Fromowitz standard. Whole cell lysates of oral cancer cells and normal oral keratinocytes were immunoblotted with the anti-O-GlcNAc antibody. The median IHC scores of O-GlcNAc or OGT between OSCC and normal tissues were not different, whereas those of phosphorylated-EGFR tyr1173 and phosphorylated-Akt ser473 were significantly higher in OSCC than normal tissues (P < .001 and P < .01, respectively). Similarly, expression of O-GlcNAcylated proteins in oral cancer cells and normal oral keratinocytes did not differ. In the OSCC group, the median IHC scores of O-GlcNAc and OGT were significantly lower than those of phosphorylated-EGFR tyr1173 and phosphorylated-Akt ser473 (P < .01 and P < .001, respectively). The IHC scores of O-GlcNAc or OGT were not determined to correlate with histological grading. Unlike other types of cancers, our findings demonstrate that the levels of O-GlcNAcylation are not significantly increased in OSCC tissues or in oral cancer cells and are not associated with the histological grading of OSCC. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Quantitative assessment of the dose-response of alkylating agents in DNA repair proficient and deficient ames tester strains.

PubMed

Tang, Leilei; Guérard, Melanie; Zeller, Andreas

2014-01-01

Mutagenic and clastogenic effects of some DNA damaging agents such as methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) have been demonstrated to exhibit a nonlinear or even "thresholded" dose-response in vitro and in vivo. DNA repair seems to be mainly responsible for these thresholds. To this end, we assessed several mutagenic alkylators in the Ames test with four different strains of Salmonella typhimurium: the alkyl transferases proficient strain TA1535 (Ogt+/Ada+), as well as the alkyl transferases deficient strains YG7100 (Ogt+/Ada-), YG7104 (Ogt-/Ada+) and YG7108 (Ogt-/Ada-). The known genotoxins EMS, MMS, temozolomide (TMZ), ethylnitrosourea (ENU) and methylnitrosourea (MNU) were tested in as many as 22 concentration levels. Dose-response curves were statistically fitted by the PROAST benchmark dose model and the Lutz-Lutz "hockeystick" model. These dose-response curves suggest efficient DNA-repair for lesions inflicted by all agents in strain TA1535. In the absence of Ogt, Ada is predominantly repairing methylations but not ethylations. It is concluded that the capacity of alkyl-transferases to successfully repair DNA lesions up to certain dose levels contributes to genotoxicity thresholds. Copyright © 2013 Wiley Periodicals, Inc.

The molecular mechanism of N-acetylglucosamine side-chain attachment to the Lancefield group A carbohydrate in Streptococcus pyogenes.

PubMed

Rush, Jeffrey S; Edgar, Rebecca J; Deng, Pan; Chen, Jing; Zhu, Haining; van Sorge, Nina M; Morris, Andrew J; Korotkov, Konstantin V; Korotkova, Natalia

2017-11-24

In many Lactobacillales species ( i.e. lactic acid bacteria), peptidoglycan is decorated by polyrhamnose polysaccharides that are critical for cell envelope integrity and cell shape and also represent key antigenic determinants. Despite the biological importance of these polysaccharides, their biosynthetic pathways have received limited attention. The important human pathogen, Streptococcus pyogenes , synthesizes a key antigenic surface polymer, the Lancefield group A carbohydrate (GAC). GAC is covalently attached to peptidoglycan and consists of a polyrhamnose polymer, with N -acetylglucosamine (GlcNAc) side chains, which is an essential virulence determinant. The molecular details of the mechanism of polyrhamnose modification with GlcNAc are currently unknown. In this report, using molecular genetics, analytical chemistry, and mass spectrometry analysis, we demonstrated that GAC biosynthesis requires two distinct undecaprenol-linked GlcNAc-lipid intermediates: GlcNAc-pyrophosphoryl-undecaprenol (GlcNAc-P-P-Und) produced by the GlcNAc-phosphate transferase GacO and GlcNAc-phosphate-undecaprenol (GlcNAc-P-Und) produced by the glycosyltransferase GacI. Further investigations revealed that the GAC polyrhamnose backbone is assembled on GlcNAc-P-P-Und. Our results also suggested that a GT-C glycosyltransferase, GacL, transfers GlcNAc from GlcNAc-P-Und to polyrhamnose. Moreover, GacJ, a small membrane-associated protein, formed a complex with GacI and significantly stimulated its catalytic activity. Of note, we observed that GacI homologs perform a similar function in Streptococcus agalactiae and Enterococcus faecalis In conclusion, the elucidation of GAC biosynthesis in S. pyogenes reported here enhances our understanding of how other Gram-positive bacteria produce essential components of their cell wall. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeting Protein O-GlcNAc Modifications In Breast Cancer

DTIC Science & Technology

2010-09-30

O-GlcNAcation and elevated expression of O-GlcNAc transferase (OGT), the enzyme catalyzing addition of O-GlcNAc to proteins. Reduction of O...regulatory switch mechanism analogous to phosphorylation (28). Cytosolic and nuclear enzymes dynamically catalyze addition (O-GlcNAc transferase or OGT) and...levels, through pharmacological inhibition or genetic knock-down of enzymes that add or remove O-GlcNAc, can inhibit ErbB2-mediated oncogenic

Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity[S

PubMed Central

Bindesbøll, Christian; Fan, Qiong; Nørgaard, Rikke C.; MacPherson, Laura; Ruan, Hai-Bin; Wu, Jing; Pedersen, Thomas Å.; Steffensen, Knut R.; Yang, Xiaoyong; Matthews, Jason; Mandrup, Susanne; Nebb, Hilde I.; Grønning-Wang, Line M.

2015-01-01

Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/β+/+ and LXRα/β−/− mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPβ. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity. PMID:25724563

N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi.

PubMed

Su, Chang; Lu, Yang; Liu, Haoping

2016-10-03

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.

N-acetylglucosamine sensing by a GCN5-related N-acetyltransferase induces transcription via chromatin histone acetylation in fungi

PubMed Central

Su, Chang; Lu, Yang; Liu, Haoping

2016-01-01

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. PMID:27694804

Quinovosamycins: New tunicamycin-type antibiotics in which the alpha, beta-1", 11'-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine.

USDA-ARS?s Scientific Manuscript database

Tunicamycins (TUN) are potent inhibitors of polyprenyl phosphate N-acetylhexosamine 1-phosphate transferases (PPHP), including essential eukaryotic GPT enzymes and bacterial HexNAc 1-P translocases. Hence, TUN blocks the formation of eukaryotic N-glycoproteins and the assembly of bacterial call wall...

Functional Analysis of the N-Acetylglucosamine Metabolic Genes of Streptomyces coelicolor and Role in Control of Development and Antibiotic Production

PubMed Central

Świątek, Magdalena A.; Tenconi, Elodie; Rigali, Sébastien

2012-01-01

N-Acetylglucosamine, the monomer of chitin, is a favored carbon and nitrogen source for streptomycetes. Its intracellular catabolism requires the combined actions of the N-acetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase NagA and the glucosamine-6-phosphate (GlcN-6P) deaminase/isomerase NagB. GlcNAc acts as a signaling molecule in the DasR-mediated nutrient sensing system, activating development and antibiotic production under poor growth conditions (famine) and blocking these processes under rich conditions (feast). In order to understand how a single nutrient can deliver opposite information according to the nutritional context, we carried out a mutational analysis of the nag metabolic genes nagA, nagB, and nagK. Here we show that the nag genes are part of the DasR regulon in Streptomyces coelicolor, which explains their transcriptional induction by GlcNAc. Most likely as the result of the intracellular accumulation of GlcN-6P, nagB deletion mutants fail to grow in the presence of GlcNAc. This toxicity can be alleviated by the additional deletion of nagA. We recently showed that in S. coelicolor, GlcNAc is internalized as GlcNAc-6P via the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS). Considering the relevance of GlcNAc for the control of antibiotic production, improved insight into GlcNAc metabolism in Streptomyces may provide new leads toward biotechnological applications. PMID:22194457

Retrospective analysis of insulin responses to standard dosed oral glucose tests (OGTs) via naso-gastric tubing towards definition of an objective cut-off value.

PubMed

Warnken, Tobias; Delarocque, Julien; Schumacher, Svenja; Huber, Korinna; Feige, Karsten

2018-01-19

Insulin dysregulation (ID) with basal or postprandial hyperinsulinemia is one of the key findings in horses and ponies suffering from the equine metabolic syndrome (EMS). Assessment of ID can easily be performed in clinical settings by the use of oral glucose challenge tests. Oral glucose test (OGT) performed with 1 g/kg bodyweight (BW) glucose administered via naso-gastric tube allows the exact administration of a defined glucose dosage in a short time. However, reliable cut-off values have not been available so far. Therefore, the aim of the study was to describe variations in insulin response to OGT via naso-gastric tubing and to provide a clinical useful cut-off value for ID when using the insulin quantification performed with an equine-optimized insulin enzyme-linked immunosorbent assay. Data visualization revealed no clear separation in the serum insulin concentration of insulin sensitive and insulin dysregulated horses during OGT. Therefore, a model based clustering method was used to circumvent the use of an arbitrary limit for categorization. This method considered all data-points for the classification, taking into account the individual insulin trajectory during the OGT. With this method two clusters were differentiated, one with low and one with high insulin responses during OGT. The cluster of individuals with low insulin response was consistently detected, independently of the initialization parameters of the algorithm. In this cluster the 97.5% quantile of insulin is 110 µLU/mL at 120 min. We suggest using this insulin concentration of 110 µLU/mL as a cut-off value for samples obtained at 120 min in OGT. OGT performed with 1 g/kg BW glucose and administration via naso-gastric tubing can easily be performed under clinical settings. Application of the cut-off value of 110 µLU/mL at 120 min allows assessment of ID in horses.

O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.

PubMed

Makino, Ayako; Dai, Anzhi; Han, Ying; Youssef, Katia D; Wang, Weihua; Donthamsetty, Reshma; Scott, Brian T; Wang, Hong; Dillmann, Wolfgang H

2015-11-01

Cardiovascular disease is the primary cause of morbidity and mortality in diabetes, and endothelial dysfunction is commonly seen in these patients. Increased O-linked N-acetylglucosamine (O-GlcNAc) protein modification is one of the central pathogenic features of diabetes. Modification of proteins by O-GlcNAc (O-GlcNAcylation) is regulated by two key enzymes: β-N-acetylglucosaminidase [O-GlcNAcase (OGA)], which catalyzes the reduction of protein O-GlcNAcylation, and O-GlcNAc transferase (OGT), which induces O-GlcNAcylation. However, it is not known whether reducing O-GlcNAcylation can improve endothelial dysfunction in diabetes. To examine the effect of endothelium-specific OGA overexpression on protein O-GlcNAcylation and coronary endothelial function in diabetic mice, we generated tetracycline-inducible, endothelium-specific OGA transgenic mice, and induced OGA by doxycycline administration in streptozotocin-induced type 1 diabetic mice. OGA protein expression was significantly decreased in mouse coronary endothelial cells (MCECs) isolated from diabetic mice compared with control MCECs, whereas OGT protein level was markedly increased. The level of protein O-GlcNAcylation was increased in diabetic compared with control mice, and OGA overexpression significantly decreased the level of protein O-GlcNAcylation in MCECs from diabetic mice. Capillary density in the left ventricle and endothelium-dependent relaxation in coronary arteries were significantly decreased in diabetes, while OGA overexpression increased capillary density to the control level and restored endothelium-dependent relaxation without changing endothelium-independent relaxation. We found that connexin 40 could be the potential target of O-GlcNAcylation that regulates the endothelial functions in diabetes. These data suggest that OGA overexpression in endothelial cells improves endothelial function and may have a beneficial effect on coronary vascular complications in diabetes. Copyright © 2015 the

The NEXT-A (N-terminal EXtension with Transferase and ARS) reaction.

PubMed

Taki, Masumi; Kuroiwa, Hiroyuki; Sisido, Masahiko

2009-01-01

L/F-transferase is known to catalyze transfer of hydrophobic amino acids from aminoacyl tRNA to the N-terminus of a protein possessing lysine or arginine as the N-terminus. Combining L/F-transferase with E. coli phenylalanyl-tRNA synthetase (ARS), we achieved non-ribosomal N-terminal-specific introduction of various kinds of nonnatural amino acids to a protein. A nonnatural amino acid is once charged onto an E. coli tRNA(Phe) by a mutant ARS in situ, and successively transferred from the tRNA to a target protein, namely the NEXT-A reaction. Besides alphaA294G mutation on the ARS, alphaT251A, betaG318W, or betaA356W double-mutation were effective to increase the introduction efficiency through the NEXT-A reaction. Protein specific fluorescence labelling via the NEXT-A reaction followed by Huisgen cycloaddition was also demonstrated.

Meningococcal X polysaccharide quantification by high-performance anion-exchange chromatography using synthetic N-acetylglucosamine-4-phosphate as standard.

PubMed

Micoli, F; Adamo, R; Proietti, D; Gavini, M; Romano, M R; MacLennan, C A; Costantino, P; Berti, F

2013-11-15

A method for meningococcal X (MenX) polysaccharide quantification by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is described. The polysaccharide is hydrolyzed by strong acidic treatment, and the peak of glucosamine-4-phosphate (4P-GlcN) is detected and measured after chromatography. In the selected conditions of hydrolysis, 4P-GlcN is the prevalent species formed, with GlcN detected for less than 5% in moles. As standard for the analysis, the monomeric unit of MenX polysaccharide, N-acetylglucosamine-4-phosphate (4P-GlcNAc), was used. This method for MenX quantification is highly selective and sensitive, and it constitutes an important analytical tool for the development of a conjugate vaccine against MenX. Copyright © 2013 Elsevier Inc. All rights reserved.

Structural and functional determination of homologs of the Mycobacterium tuberculosisN-acetylglucosamine-6-phosphate deacetylase (NagA).

PubMed

Ahangar, Mohd Syed; Furze, Christopher M; Guy, Collette S; Cooper, Charlotte; Maskew, Kathryn S; Graham, Ben; Cameron, Alexander D; Fullam, Elizabeth

2018-05-04

The Mycobacterium tuberculosis (Mtb) pathogen encodes an N -acetylglucosamine-6-phosphate deacetylase enzyme, NagA (Rv3332), that belongs to the amidohydrolase superfamily. NagA enzymes catalyze the deacetylation of N -acetylglucosamine-6-phosphate (GlcNAc6P) to glucosamine-6-phosphate (GlcN6P). NagA is a potential anti-tubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments. Here, we report the structural and functional characterization of NagA from Mycobacterium smegmatis (MSNagA) and Mycobacterium marinum (MMNagA), close relatives of Mtb Using a combination of X-ray crystallography, site-directed mutagenesis, and biochemical and biophysical assays, we show that these mycobacterial NagA enzymes are selective for GlcNAc6P. Site-directed mutagenesis studies revealed crucial roles of conserved residues in the active site that underpin stereo-selective recognition, binding, and catalysis of substrates. Moreover, we report the crystal structure of MSNagA in both ligand-free form and in complex with the GlcNAc6P substrate at 2.6 Å and 2.0 Å resolutions, respectively. The GlcNAc6P-complex structure disclosed the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the α/β binuclear site. Furthermore, we observed a cysteine residue located on a flexible loop region that occludes the active site. This cysteine is unique to mycobacteria and may represent a unique subsite for targeting mycobacterial NagA enzymes. Our results provide critical insights into the structural and mechanistic properties of mycobacterial NagA enzymes having an essential role in amino-sugar and nucleotide metabolism in mycobacteria. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

New use for CETSA: monitoring innate immune receptor stability via post-translational modification by OGT.

PubMed

Drake, Walter R; Hou, Ching-Wen; Zachara, Natasha E; Grimes, Catherine Leimkuhler

2018-06-01

O-GlcNAcylation is a dynamic and functionally diverse post-translational modification shown to affect thousands of proteins, including the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mutations of Nod2 (R702W, G908R and 1007 fs) are associated with Crohn's disease and have lower stabilities compared to wild type. Cycloheximide (CHX)-chase half-life assays have been used to show that O-GlcNAcylation increases the stability and response of both wild type and Crohn's variant Nod2, R702W. A more rapid method to assess stability afforded by post-translational modifications is necessary to fully comprehend the correlation between NLR stability and O-GlcNAcylation. Here, a recently developed cellular thermal shift assay (CETSA) that is typically used to demonstrate protein-ligand binding was adapted to detect shifts in protein stabilization upon increasing O-GlcNAcylation levels in Nod2. This assay was used as a method to predict if other Crohn's associated Nod2 variants were O-GlcNAcylated, and also identified the modification on another NLR, Nod1. Classical immunoprecipitations and NF-κB transcriptional assays were used to confirm the presence and effect of this modification on these proteins. The results presented here demonstrate that CETSA is a convenient method that can be used to detect the stability effect of O-GlcNAcylation on O-GlcNAc-transferase (OGT) client proteins and will be a powerful tool in studying post-translational modification.

Candida albicans Adheres to Chitin by Recognizing N-acetylglucosamine (GlcNAc).

PubMed

Ishijima, Sanae A; Yamada, Tsuyoshi; Maruyama, Naho; Abe, Shigeru

2017-01-01

The binding of Candida albicans cells to chitin was examined in a cell-binding assay. Microscopic observations indicated that both living and heat-killed Candida cells bound to chitin-coated substrates. C. albicans preferentially bound to chitin-coated plastic plates over chitosan-coated and uncoated plates. We prepared 125 I-labeled Candida cells for quantitative analysis of their binding to chitin. Heat-killed 125 I-labeled Candida cells bound to chitin-coated plates in a time-dependent manner until 1.5 hours after start of incubation at 4℃. The binding of 125 I-labeled Candida cells to chitin-coated plates was inhibited by adding unlabeled living or unlabeled heat-killed Candida cells. The binding of Candida to chitin was also reduced by addition of 25 mg/ml chitin or chitosan up to 10%. N-acetylglucosamine (GlcNAc), which is a constituent of chitin, inhibited binding of Candida to chitin in a dose-dependent manner between 12.5 and 200 mM. Glucosamine, which is a constituent of chitosan, showed no such inhibitory effect. These findings suggest that the binding of Candida to chitin may be mediated by recognition of GlcNAc.

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

PubMed Central

2012-01-01

More than 1,000 proteins of the nucleus, cytoplasm, and mitochondria are dynamically modified by O-linked β-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. PMID:22287582

Thermodynamic parameters of the interaction of Urtica dioica agglutinin with N-acetylglucosamine and its oligomers.

PubMed

Lee, R T; Gabius, H J; Lee, Y C

1998-07-01

The interaction between Urtica dioica agglutinin (UDA) and N-acetylglucosamine (GlcNAc) and its beta(1-4)-linked oligomers was studied by fluorescence titration and isothermal titration microcalorimetry. UDA possesses one significant binding site that can be measured calorimetrically. This site is composed of three subsites, each subsite accommodating one GlcNAc residue. The interaction is enthalpically driven, and the binding area of UDA is characterized by a deltaH of interaction for a given oligosaccharide considerably smaller than that of wheat germ agglutinin (WGA), despite the fact that they both belong to a family of proteins composed entirely of hevein domains. Relatively high deltaCp values of the UDA-carbohydrate interactions and more favorable entropy term compared to WGA suggest that binding of the carbohydrate ligands by UDA has a higher hydrophobic component than that of WGA.

Functional O-GlcNAc modifications: Implications in molecular regulation and pathophysiology

PubMed Central

Wells, Lance

2016-01-01

O-linked β-N-acetylglucosamine (O-GlcNAc) is a regulatory post-translational modification of intracellular proteins. The dynamic and inducible cycling of the modification is governed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in response to UDP-GlcNAc levels in the hexosamine biosynthetic pathway (HBP). Due to its reliance on glucose flux and substrate availability, a major focus in the field has been on how O-GlcNAc contributes to metabolic disease. For years this post-translational modification has been known to modify thousands of proteins implicated in various disorders, but direct functional connections have until recently remained elusive. New research is beginning to reveal the specific mechanisms through which O-GlcNAc influences cell dynamics and disease pathology including clear examples of O-GlcNAc modification at a specific site on a given protein altering its biological functions. The following review intends to focus primarily on studies in the last half decade linking O-GlcNAc modification of proteins with chromatin-directed gene regulation, developmental processes, and several metabolically related disorders including Alzheimer’s, heart disease and cancer. These studies illustrate the emerging importance of this post-translational modification in biological processes and multiple pathophysiologies. PMID:24524620

N-acetylglucosamine, the building block of chitin, inhibits growth of Neurospora crassa.

PubMed

Gaderer, Romana; Seidl-Seiboth, Verena; de Vries, Ronald P; Seiboth, Bernhard; Kappel, Lisa

2017-10-01

N-acetylglucosamine (GlcNAc) is the monomer of the polysaccharide chitin, an essential structural component of the fungal cell wall and the arthropod exoskeleton. We recently showed that the genes encoding the enzymes for GlcNAc catabolism are clustered in several ascomycetes. In the present study we tested these fungi for growth on GlcNAc and chitin. All fungi, containing the GlcNAc gene cluster, could grow on GlcNAc with the exception of four independent Neurospora crassa wild-type isolates, which were however able to grow on chitin. GlcNAc even inhibited their growth in the presence of other carbon sources. Genes involved in GlcNAc catabolism were strongly upregulated in the presence of GlcNAc, but during growth on chitin their expression was not increased. Deletion of hxk-3 (encoding the first catabolic enzyme, GlcNAc-hexokinase) and ngt-1 (encoding the GlcNAc transporter) improved growth of N. crassa on GlcNAc in the presence of glycerol. A crucial step in GlcNAc catabolism is enzymatic conversion from glucosamine-6-phosphate to fructose-6-phosphate, catalyzed by the glucosamine-6-phosphate deaminase, DAM-1. To assess, if DAM-1 is compromised in N. crassa, the orthologue from Trichoderma reesei, Trdam1, was expressed in N. crassa. Trdam1 expression partially alleviated the negative effects of GlcNAc in the presence of a second carbon source, but did not fully restore growth on GlcNAc. Our results indicate that the GlcNAc-catabolism pathway is bypassed during growth of N. crassa on chitin by use of an alternative pathway, emphasizing the different strategies that have evolved in the fungal kingdom for chitin utilization. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Glucose Alters Per2 Rhythmicity Independent of AMPK, Whereas AMPK Inhibitor Compound C Causes Profound Repression of Clock Genes and AgRP in mHypoE-37 Hypothalamic Neurons.

PubMed

Oosterman, Johanneke E; Belsham, Denise D

2016-01-01

Specific neurons in the hypothalamus are regulated by peripheral hormones and nutrients to maintain proper metabolic control. It is unclear if nutrients can directly control clock gene expression. We have therefore utilized the immortalized, hypothalamic cell line mHypoE-37, which exhibits robust circadian rhythms of core clock genes. mHypoE-37 neurons were exposed to 0.5 or 5.5 mM glucose, comparable to physiological levels in the brain. Per2 and Bmal1 mRNAs were assessed every 3 hours over 36 hours. Incubation with 5.5 mM glucose significantly shortened the period and delayed the phase of Per2 mRNA levels, but had no effect on Bmal1. Glucose had no significant effect on phospho-GSK3β, whereas AMPK phosphorylation was altered. Thus, the AMPK inhibitor Compound C was utilized, and mRNA levels of Per2, Bmal1, Cryptochrome1 (Cry1), agouti-related peptide (AgRP), carnitine palmitoyltransferase 1C (Cpt1c), and O-linked N-acetylglucosamine transferase (Ogt) were measured. Remarkably, Compound C dramatically reduced transcript levels of Per2, Bmal1, Cry1, and AgRP, but not Cpt1c or Ogt. Because AMPK was not inhibited at the same time or concentrations as the clock genes, we suggest that the effect of Compound C on gene expression occurs through an AMPK-independent mechanism. The consequences of inhibition of the rhythmic expression of clock genes, and in turn downstream metabolic mediators, such as AgRP, could have detrimental effects on overall metabolic processes. Importantly, the effects of the most commonly used AMPK inhibitor Compound C should be interpreted with caution, considering its role in AMPK-independent repression of specific genes, and especially clock gene rhythm dysregulation.

Glucose Alters Per2 Rhythmicity Independent of AMPK, Whereas AMPK Inhibitor Compound C Causes Profound Repression of Clock Genes and AgRP in mHypoE-37 Hypothalamic Neurons

PubMed Central

Oosterman, Johanneke E.; Belsham, Denise D.

2016-01-01

Specific neurons in the hypothalamus are regulated by peripheral hormones and nutrients to maintain proper metabolic control. It is unclear if nutrients can directly control clock gene expression. We have therefore utilized the immortalized, hypothalamic cell line mHypoE-37, which exhibits robust circadian rhythms of core clock genes. mHypoE-37 neurons were exposed to 0.5 or 5.5 mM glucose, comparable to physiological levels in the brain. Per2 and Bmal1 mRNAs were assessed every 3 hours over 36 hours. Incubation with 5.5 mM glucose significantly shortened the period and delayed the phase of Per2 mRNA levels, but had no effect on Bmal1. Glucose had no significant effect on phospho-GSK3β, whereas AMPK phosphorylation was altered. Thus, the AMPK inhibitor Compound C was utilized, and mRNA levels of Per2, Bmal1, Cryptochrome1 (Cry1), agouti-related peptide (AgRP), carnitine palmitoyltransferase 1C (Cpt1c), and O-linked N-acetylglucosamine transferase (Ogt) were measured. Remarkably, Compound C dramatically reduced transcript levels of Per2, Bmal1, Cry1, and AgRP, but not Cpt1c or Ogt. Because AMPK was not inhibited at the same time or concentrations as the clock genes, we suggest that the effect of Compound C on gene expression occurs through an AMPK-independent mechanism. The consequences of inhibition of the rhythmic expression of clock genes, and in turn downstream metabolic mediators, such as AgRP, could have detrimental effects on overall metabolic processes. Importantly, the effects of the most commonly used AMPK inhibitor Compound C should be interpreted with caution, considering its role in AMPK-independent repression of specific genes, and especially clock gene rhythm dysregulation. PMID:26784927

Glutamine-induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels.

PubMed

Liu, Jia; Marchase, Richard B; Chatham, John C

2007-01-01

It has been shown that glutamine protects the heart from ischemia/reperfusion (I/R) injury; however, the mechanisms underlying this protection have not been identified. Glutamine:fructose-6-phosphate amidotransferase (GFAT) regulates the entry of glucose into the hexosamine biosynthesis pathway (HBP), and activation of this pathway has been shown to be cardioprotective. Glutamine is required for metabolism of glucose via GFAT; therefore, the goal of this study was to determine whether glutamine cardioprotection could be attributed to increased flux through the HBP and elevated levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. Hearts from male rats were isolated and perfused with Krebs-Henseliet buffer containing 5 mM glucose, and global, no-flow ischemia was induced for 20 min followed by 60 min of reperfusion. Thirty-minute pre-treatment with 2.5 mM glutamine significantly improved functional recovery (RPP: 15.6+/-5.7% vs. 59.4+/-6.1%; p<0.05) and decreased cardiac troponin I release (25.4+/-3.0 vs. 4.7+/-1.9 ng/ml; p<0.05) during reperfusion. This protection was associated with a significant increase in the levels of protein O-GlcNAc and ATP. Pre-treatment with 80 muM azaserine, an inhibitor of GFAT, completely reversed the protection seen with glutamine and prevented the increase in protein O-GlcNAc. O-GlcNAc transferase (OGT) catalyzes the formation of O-GlcNAc, and inhibition of OGT with 5 mM alloxan also reversed the protection associated with glutamine. These data support the hypothesis that in the ex vivo perfused heart glutamine cardioprotection is due, at least in part, to enhanced flux through the HBP and increased protein O-GlcNAc levels.

Structure-based design of diverse inhibitors of Mycobacterium tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase: combined molecular docking, dynamic simulation, and biological activity.

PubMed

Soni, Vijay; Suryadevara, Priyanka; Sriram, Dharmarajan; Kumar, Santhosh; Nandicoori, Vinay Kumar; Yogeeswari, Perumal

2015-07-01

Persistent nature of Mycobacterium tuberculosis is one of the major factors which make the drug development process monotonous against this organism. The highly lipophilic cell wall, which constituting outer mycolic acid and inner peptidoglycan layers, acts as a barrier for the drugs to enter the bacteria. The rigidity of the cell wall is imparted by the peptidoglycan layer, which is covalently linked to mycolic acid by arabinogalactan. Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) serves as the starting material in the biosynthesis of this peptidoglycan layers. This UDP-GlcNAc is synthesized by N-acetylglucosamine-1-phosphate uridyltransferase (GlmU(Mtb)), a bi-functional enzyme with two functional sites, acetyltransferase site and uridyltransferase site. Here, we report design and screening of nine inhibitors against UTP and NAcGlc-1-P of uridyltransferase active site of glmU(Mtb). Compound 4 was showing good inhibition and was selected for further analysis. The isothermal titration calorimetry (ITC) experiments showed the binding energy pattern of compound 4 to the uridyltransferase active site is similar to that of substrate UTP. In silico molecular dynamics (MD) simulation studies, for compound 4, carried out for 10 ns showed the protein-compound complex to be stable throughout the simulation with relative rmsd in acceptable range. Hence, these compounds can serve as a starting point in the drug discovery processes against Mycobacterium tuberculosis.

Antinutritive effects of wheat-germ agglutinin and other N-acetylglucosamine-specific lectins.

PubMed

Pusztai, A; Ewen, S W; Grant, G; Brown, D S; Stewart, J C; Peumans, W J; Van Damme, E J; Bardocz, S

1993-07-01

Incorporation of N-acetylglucosamine-specific agglutinins from wheat germ (Triticum aestivum; WGA), thorn apple (Datura stramonium) or nettle (Urtica dioica) rhizomes in the diet at the level of 7 g/kg reduced the apparent digestibility and utilization of dietary proteins and the growth of rats, with WGA being the most damaging. As a result of their binding and endocytosis by the epithelial cells of the small intestine, all three lectins were growth factors for the gut and interfered with its metabolism and function to varying degrees. WGA was particularly effective; it induced extensive polyamine-dependent hyperplastic and hypertrophic growth of the small bowel by increasing its content of proteins, RNA and DNA. Furthermore, an appreciable portion of the endocytosed WGA was transported across the gut wall into the systemic circulation, where it was deposited in the walls of the blood and lymphatic vessels. WGA also induced the hypertrophic growth of the pancreas and caused thymus atrophy. Although the transfer of the gene of WGA into crop plants has been advocated to increase their insect resistance, as the presence of this lectin in the diet may harm higher animals at the concentrations required to be effective against most pests, its use in plants as natural insecticide is not without health risks for man.

Understanding the Role of O-GlcNAc Modifications in Plant Development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olszewski, Neil, E.

2011-06-16

This project has contributed towards understanding the role of O-GlcNAc (O-linked N-acetylglucosamine) transferases (OGTs) in plants. Through analyses of single and double mutants, we have investigated the unique and overlapping functions of SECRET AGENT (SEC) and SPINDLY (SPY), the arabidopsis OGTs. This work showed that SEC functions as negative regulators of the long-day flowering pathway. SEC also has a positive role in regulation of rosette. An E. coli co-expression system that allows potential substrates to be co-expressed with and O-GlcNAc modified by SEC was developed. We showed that SEC is a bona fide OGT that modifies itself with single O-linkedmore » GlcNAc(s). Using this system, we tested a number of proteins that were hypothesized to be substrates of SEC and identified a number of substrates include GIGANTEA (GI), a component of the long day flowering pathway. The hypothesis that O-GlcNAc modification controls GI activity was tested by first mapping where E. coli-expressed SEC modifies GI and then assessing the activity of a non-modifiable mutant form of GI. The activity of the mutant form of GI was indistinguishable from that of wild type suggesting that either O-GlcNAc does not regulate GI activity or that additional modification sites exist on GI. In collaboration with Dr. Juan Antonio Garcia at Universidad Autónoma de Madrid the role of O-GlcNAc modification of the plum pox virus coat protein (PPV-CP) was investigated. SEC was shown to O-GlcNAc modify PPV-CP and the modification was shown to facilitate the infection process. E. coli-expressed SEC was shown to modify the same PPV-CP sites that are modified in plants. SEC has a large protein interaction domain called the TPR domain that has been hypothesized to have a role in determining the substrate specificity of the enzyme and/or to regulate its activity. A mutational analysis of the TPR domain did not find evidence for a role in substrate specificity but did obtain evidence that the domain

Poly-N-Acetylglucosamine Production by Staphylococcus epidermidis Cells Increases Their In Vivo Proinflammatory Effect

PubMed Central

Ferreirinha, Pedro; Pérez-Cabezas, Begoña; Correia, Alexandra; Miyazawa, Bruna; França, Angela; Carvalhais, Virgínia; Faustino, Augusto; Cordeiro-da-Silva, Anabela; Teixeira, Luzia; Pier, Gerald B.

2016-01-01

Poly-N-acetylglucosamine (PNAG) is a major component of the Staphylococcus epidermidis biofilm extracellular matrix. However, it is not yet clear how this polysaccharide impacts the host immune response and infection-associated pathology. Faster neutrophil recruitment and bacterial clearance were observed in mice challenged intraperitoneally with S. epidermidis biofilm cells of the PNAG-producing 9142 strain than in mice similarly challenged with the isogenic PNAG-defective M10 mutant. Moreover, intraperitoneal priming with 9142 cells exacerbated liver inflammatory pathology induced by a subsequent intravenous S. epidermidis challenge, compared to priming with M10 cells. The 9142-primed mice had elevated splenic CD4+ T cells producing gamma interferon and interleukin-17A, indicating that PNAG promoted cell-mediated immunity. Curiously, despite having more marked liver tissue pathology, 9142-primed mice also had splenic T regulatory cells with greater suppressive activity than those of their M10-primed counterparts. By showing that PNAG production by S. epidermidis biofilm cells exacerbates host inflammatory pathology, these results together suggest that this polysaccharide contributes to the clinical features associated with biofilm-derived infections. PMID:27481237

The EGF Repeat-Specific O-GlcNAc-Transferase Eogt Interacts with Notch Signaling and Pyrimidine Metabolism Pathways in Drosophila

PubMed Central

Müller, Reto; Jenny, Andreas; Stanley, Pamela

2013-01-01

The O-GlcNAc transferase Eogt modifies EGF repeats in proteins that transit the secretory pathway, including Dumpy and Notch. In this paper, we show that the Notch ligands Delta and Serrate are also substrates of Eogt, that mutation of a putative UDP-GlcNAc binding DXD motif greatly reduces enzyme activity, and that Eogt and the cytoplasmic O-GlcNAc transferase Ogt have distinct substrates in Drosophila larvae. Loss of Eogt is larval lethal and disrupts Dumpy functions, but does not obviously perturb Notch signaling. To identify novel genetic interactions with eogt, we investigated dominant modification of wing blister formation caused by knock-down of eogt. Unexpectedly, heterozygosity for several members of the canonical Notch signaling pathway suppressed wing blister formation. And importantly, extensive genetic interactions with mutants in pyrimidine metabolism were identified. Removal of pyrimidine synthesis alleles suppressed wing blister formation, while removal of uracil catabolism alleles was synthetic lethal with eogt knock-down. Therefore, Eogt may regulate protein functions by O-GlcNAc modification of their EGF repeats, and cellular metabolism by affecting pyrimidine synthesis and catabolism. We propose that eogt knock-down in the wing leads to metabolic and signaling perturbations that increase cytosolic uracil levels, thereby causing wing blister formation. PMID:23671640

Role of UDP-N-Acetylglucosamine (GlcNAc) and O-GlcNAcylation of Hyaluronan Synthase 2 in the Control of Chondroitin Sulfate and Hyaluronan Synthesis*

PubMed Central

Vigetti, Davide; Deleonibus, Sara; Moretto, Paola; Karousou, Eugenia; Viola, Manuela; Bartolini, Barbara; Hascall, Vincent C.; Tammi, Markku; De Luca, Giancarlo; Passi, Alberto

2012-01-01

Hyaluronan (HA) is a glycosaminoglycan present in most tissue microenvironments that can modulate many cell behaviors, including proliferation, migration, and adhesive proprieties. In contrast with other glycosaminoglycans, which are synthesized in the Golgi, HA is synthesized at the plasma membrane by one or more of the three HA synthases (HAS1–3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Previous studies revealed the importance of UDP-sugars for regulating HA synthesis. Therefore, we analyzed the effect of UDP-GlcNAc availability and protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAcylation) on HA and chondroitin sulfate synthesis in primary human aortic smooth muscle cells. Glucosamine treatment, which increases UDP-GlcNAc availability and protein O-GlcNAcylation, increased synthesis of both HA and chondroitin sulfate. However, increasing O-GlcNAcylation by stimulation with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate without a concomitant increase of UDP-GlcNAc increased only HA synthesis. We found that HAS2, the main synthase in aortic smooth muscle cells, can be O-GlcNAcylated on serine 221, which strongly increased its activity and its stability (t½ >5 h versus ∼17 min without O-GlcNAcylation). S221A mutation prevented HAS2 O-GlcNAcylation, which maintained the rapid turnover rate even in the presence of GlcN and increased UDP-GlcNAc. These findings could explain the elevated matrix HA observed in diabetic vessels that, in turn, could mediate cell dedifferentiation processes critical in vascular pathologies. PMID:22887999

Construction integrity assessment report (ETN-98-0005) S-Farm overground transfer (OGT) system valve pit 241-S-B to valve pit 241-S-D

DOE Office of Scientific and Technical Information (OSTI.GOV)

HICKS, D.F.

1999-08-12

The S-Farm overground transfer (OGT) line will bypass the existing line(s), between valve pits 241-S-B and 241-S-D that no longer meet system requirements. The new OGT line will provide a waste transfer pipeline between these valve pits in support of saltwell pumping activities. The length of the OGT line is approximately 180 ft from pit to pit. The primary pipe is nominal 1-in. diameter stainless steel (SST) braided Ethylene-propylene Diene Monomer (EPDM) hose. The encasement pipe is a nominal 3-in., flanged, SST pipe made up of several different length pipe spool pieces (drawing H-2-829564, sh. 1 and sh. 2). Themore » OGT line slopes from valve pit 241-S-B toward valve pit 241-S-D. At each end, the primary and encasement pipe connect to a pit entry spool piece. The pit entry spool pieces are constructed of prefabricated SST materials. These spool pieces allow for the separation of the primary and encasement pipelines after the pipes have entered the valve pits (drawing H-2-818280, sh. 2). The pit entry spool pieces also allow for leak detection of the encasement pipe at each end (drawing H-2-829564, sh. 2). The OGT encasement pipeline is supported above ground by adjustable height unistrut brackets and precast concrete bases (drawing H-2-829654, sh. 1). The pipeline is heat-traced and insulated. The heat tracing and insulation supply and retain latent heat that prevents waste solidification during transfers and provides freeze protection. The total length of the pipeline is above ground, thereby negating the need for cathodic corrosion protection. This Construction Integrity Assessment Report (CIAR) is prepared by Fluor Daniel Northwest for Numatec Hanford Corporation/Lockheed Martin Hanford Corporation, the operations contractor, and the U. S. Department of Energy, the system owner. The CIAR is intended to verify that construction was performed in accordance with the provisions of Washington Administrative Code, WAC-173-303-640 (3) (c), (e), (f) and (h).« less

N-acetylglucosamine affects Cryptococcus neoformans cell-wall composition and melanin architecture.

PubMed

Camacho, Emma; Chrissian, Christine; Cordero, Radames J B; Liporagi-Lopes, Livia; Stark, Ruth E; Casadevall, Arturo

2017-11-01

Cryptococcus neoformans is an environmental fungus that belongs to the phylum Basidiomycetes and is a major pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin pigments represents its second most important virulence factor, after the presence of a polysaccharide capsule. Both the capsule and melanin are closely associated with the fungal cell wall, a complex structure that is essential for maintaining cell morphology and viability under conditions of stress. The amino sugar N-acetylglucosamine (GlcNAc) is a key constituent of the cell-wall chitin and is used for both N-linked glycosylation and GPI anchor synthesis. Recent studies have suggested additional roles for GlcNAc as an activator and mediator of cellular signalling in fungal and plant cells. Furthermore, chitin and chitosan polysaccharides interact with melanin pigments in the cell wall and have been found to be essential for melanization. Despite the importance of melanin, its molecular structure remains unresolved; however, we previously obtained critical insights using advanced nuclear magnetic resonance (NMR) and imaging techniques. In this study, we investigated the effect of GlcNAc supplementation on cryptococcal cell-wall composition and melanization. C. neoformans was able to metabolize GlcNAc as a sole source of carbon and nitrogen, indicating a capacity to use a component of a highly abundant polymer in the biospherenutritionally. C. neoformans cells grown with GlcNAc manifested changes in the chitosan cell-wall content, cell-wall thickness and capsule size. Supplementing cultures with isotopically 15 N-labelled GlcNAc demonstrated that the exogenous monomer serves as a building block for chitin/chitosan and is incorporated into the cell wall. The altered chitin-to-chitosan ratio had no negative effects on the mother-daughter cell separation; growth with GlcNAc affected the fungal cell-wall scaffold, resulting in increased melanin deposition and assembly. In

N-acetylglucosamine affects Cryptococcus neoformans cell-wall composition and melanin architecture

PubMed Central

Camacho, Emma; Chrissian, Christine; Cordero, Radames J. B.; Liporagi-Lopes, Livia; Stark, Ruth E.; Casadevall, Arturo

2017-01-01

Cryptococcus neoformans is an environmental fungus that belongs to the phylum Basidiomycetes and is a major pathogen in immunocompromised patients. The ability of C. neoformans to produce melanin pigments represents its second most important virulence factor, after the presence of a polysaccharide capsule. Both the capsule and melanin are closely associated with the fungal cell wall, a complex structure that is essential for maintaining cell morphology and viability under conditions of stress. The amino sugar N-acetylglucosamine (GlcNAc) is a key constituent of the cell-wall chitin and is used for both N-linked glycosylation and GPI anchor synthesis. Recent studies have suggested additional roles for GlcNAc as an activator and mediator of cellular signalling in fungal and plant cells. Furthermore, chitin and chitosan polysaccharides interact with melanin pigments in the cell wall and have been found to be essential for melanization. Despite the importance of melanin, its molecular structure remains unresolved; however, we previously obtained critical insights using advanced nuclear magnetic resonance (NMR) and imaging techniques. In this study, we investigated the effect of GlcNAc supplementation on cryptococcal cell-wall composition and melanization. C. neoformans was able to metabolize GlcNAc as a sole source of carbon and nitrogen, indicating a capacity to use a component of a highly abundant polymer in the biospherenutritionally. C. neoformans cells grown with GlcNAc manifested changes in the chitosan cell-wall content, cell-wall thickness and capsule size. Supplementing cultures with isotopically 15N-labelled GlcNAc demonstrated that the exogenous monomer serves as a building block for chitin/chitosan and is incorporated into the cell wall. The altered chitin-to-chitosan ratio had no negative effects on the mother–daughter cell separation; growth with GlcNAc affected the fungal cell-wall scaffold, resulting in increased melanin deposition and assembly. In

Detection of glycosylation abnormality in rheumatoid IgG using N-acetylglucosamine-specific Psathyrella velutina lectin.

PubMed

Tsuchiya, N; Endo, T; Matsuta, K; Yoshinoya, S; Takeuchi, F; Nagano, Y; Shiota, M; Furukawa, K; Kochibe, N; Ito, K

1993-07-15

Although the galactose deficiency in the Asn297-linked sugar chains of serum IgG from patients with rheumatoid arthritis (RA) has been established, structural analysis of sugar chains has not been readily available. Psathyrella velutina lectin (PVL) preferentially interacts with the N-acetylglucosamine beta 1-->2Man group, exposed at the termini of sugar chains in agalacto IgG. Biotinylated PVL reacted strongly in Western blotting with H chains of IgG derived from patients with RA. An ELISA-based assay for the detection of agalacto IgG was developed using recombinant protein G and biotinylated PVL in combination, and the screening of patients' sera was performed. PVL binding of serum IgG significantly correlated with percentage of galactose-deficient IgG determined by the structural analysis. Age-related slight increase in PVL binding was observed among normal controls. Patients with RA showed significantly higher PVL binding (37.90 +/- 42.25 U/ml, n = 93) as compared with normal controls (5.75 +/- 2.92 U/ml, n = 112) (p = 0.0001). Patients with SLE showed lower but still significant PVL binding (17.86 +/- 5.18 U/ml, n = 10, p = 0.0001). PVL binding correlated with C-reactive protein level in serial analysis of individual RA patients, and was significantly higher in the synovial fluid compared with paired serum samples. PVL binding assay may provide an ideal tool for the simple and sensitive detection of agalacto IgG.

Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom.

PubMed

Kochibe, N; Matta, K L

1989-01-05

A lectin in the fruiting bodies of Psathyrella velutina was purified by affinity chromatography on a chitin column and subsequent ion-exchange chromatography. P. velutina lectin (PVL) tends to aggregate irreversibly in buffered saline, but the addition of glycerol (10%, v/v) to lectin solutions was found to prevent aggregate formation. PVL is assumed to occur as a monomer of a polypeptide of Mr = 40,000 as determined by gel filtration and by gel electrophoresis in the presence of sodium dodecyl sulfate. PVL is specific for N-acetylglucosamine (GlcNAc). It was determined by equilibrium dialysis to have four binding sites/polypeptide molecule showing an average intrinsic association constant of K0 = 6.4 x 10(3) M-1 toward this sugar. The binding specificity of the lectin was studied by hemagglutination inhibition assays and by avidin-biotin-mediated enzyme immunoassays using various GlcNAc-containing saccharides. The results indicate that methyl N-acetyl beta-glucosaminide was a slightly better inhibitor than the corresponding alpha-anomer. PVL binds well to oligosaccharides bearing nonreducing terminal beta-GlcNAc linked 1----6 or 1----3 but poorly to those having a 1----4 linkage, such as N-acetylated chito-oligosaccharides. It also binds to the subterminal GlcNAc moiety when it is substituted at the C-6 position but does not interact with the moiety when substituted either at C-3 or C-4. Thus, these results show that PVL is quite different in its binding specificity from other GlcNAc-binding lectins of higher plants since they bind preferentially to beta-GlcNAc in 1----4 linkage and they have a high affinity for chitin oligosaccharides.

Plant nuclear pore complex proteins are modified by novel oligosaccharides with terminal N-acetylglucosamine.

PubMed Central

Heese-Peck, A; Cole, R N; Borkhsenious, O N; Hart, G W; Raikhel, N V

1995-01-01

Only a few nuclear pore complex (NPC) proteins, mainly in vertebrates and yeast but none in plants, have been well characterized. As an initial step to identify plant NPC proteins, we examined whether NPC proteins from tobacco are modified by N-acetylglucosamine (GlcNAc). Using wheat germ agglutinin, a lectin that binds specifically to GlcNAc in plants, specific labeling was often found associated with or adjacent to NPCs. Nuclear proteins containing GlcNAc can be partially extracted by 0.5 M salt, as shown by a wheat germ agglutinin blot assay, and at least eight extracted proteins were modified by terminal GlcNAc, as determined by in vitro galactosyltransferase assays. Sugar analysis indicated that the plant glycans with terminal GlcNAc differ from the single O-linked GlcNAc of vertebrate NPC proteins in that they consist of oligosaccharides that are larger in size than five GlcNAc residues. Most of these appear to be bound to proteins via a hydroxyl group. This novel oligosaccharide modification may convey properties to the plant NPC that are different from those of vertebrate NPCs. PMID:8589629

Characterization of Streptococcus pneumoniae N-acetylglucosamine-6-phosphate deacetylase as a novel diagnostic marker.

PubMed

Choi, Chi-Won; An, Hee-Young; Lee, Yong Ju; Lee, Yeol Gyun; Yun, Sung Ho; Park, Edmond Changkyun; Hong, Yeonhee; Kim, Gun-Hwa; Park, Jae-Eun; Baek, Sun Jong; Kim, Hyun Sik; Kim, Seung Il

2013-10-01

The identification of novel diagnostic markers of pathogenic bacteria is essential for improving the accuracy of diagnoses and for developing targeted vaccines. Streptococcus pneumoniae is a significant human pathogenic bacterium that causes pneumonia. N-acetylglucosamine-6-phosphate deacetylase (NagA) was identified in a protein mixture secreted by S. pneumoniae and its strong immunogenicity was confirmed in an immuno-proteomic assay against the anti-serum of the secreted protein mixture. In this study, recombinant S. pneumoniae NagA protein was expressed and purified to analyze its protein characteristics, immunospecificity, and immunogenicity, thereby facilitating its evaluation as a novel diagnostic marker for S. pneumoniae. Mass spectrometry analysis showed that S. pneumoniae NagA contains four internal disulfide bonds and that it does not undergo post-translational modification. S. pneumoniae NagA antibodies successfully detected NagA from different S. pneumoniae strains, whereas NagA from other pathogenic bacteria species was not detected. In addition, mice infected with S. pneumoniae generated NagA antibodies in an effective manner. These results suggest that NagA has potential as a novel diagnostic marker for S. pneumoniae because of its high immunogenicity and immunospecificity.

Increased Bisecting N-Acetylglucosamine and Decreased Branched Chain Glycans of N-linked Glycoproteins in Expressed Prostatic Secretions Associated with Prostate Cancer Progression

PubMed Central

Nyalwidhe, Julius O.; Betesh, Lucy R.; Powers, Thomas W.; Jones, E. Ellen; White, Krista Y.; Burch, Tanya C.; Brooks, Jasmin; Watson, Megan T.; Lance, Raymond S.; Troyer, Dean A.; Semmes, O. John; Mehta, Anand; Drake, Richard R.

2013-01-01

Purpose Using prostatic fluids rich in glycoproteins like prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) , the goal of this study was to identify the structural types and relative abundance of glycans associated with prostate cancer status for subsequent use in emerging mass spectrometry-based glycopeptide analysis platforms. Experimental Design A series of pooled samples of expressed prostatic secretions (EPS) and exosomes reflecting different stages of prostate cancer disease were used for N-linked glycan profiling by three complementary methods, MALDI-TOF profiling, normal-phase HPLC separation, and triple quadropole MS analysis of PAP glycopeptides. Results Glycan profiling of N-linked glycans from different EPS fluids indicated a global decrease in larger branched tri- and tetra-antennary glycans. Differential exoglycosidase treatments indicated a substantial increase in bisecting N-acetylglucosamines correlated with disease severity. A triple quadrupole MS analysis of the N-linked glycopeptides sites from PAP in aggressive prostate cancer pools was done to cross-reference with the glycan profiling data. Conclusion and clinical relevance Changes in glycosylation as detected in EPS fluids reflect the clinical status of prostate cancer. Defining these molecular signatures at the glycopeptide level in individual samples could improve current approaches of diagnosis and prognosis. PMID:23775902

The insect repellent DEET (N,N-diethyl-3-methylbenzamide) increases the synthesis of glutathione S-transferase in cultured mosquito cells.

PubMed

Hellestad, Vanessa J; Witthuhn, Bruce A; Fallon, Ann M

2011-04-01

DEET (N,N-diethyl-3-methylbenzamide) is the active ingredient used in many commonly used insect repellents, but its mode of action remains poorly understood. Efforts to identify properties that could lead to the development of more effective active ingredients have distinguished among DEET's repellent, deterrent, and insecticidal activities. We used an Aedes albopictus mosquito cell line to evaluate DEET's toxicological properties in the absence of sensory input mediated by the olfactory system. When cells were treated with DEET and labeled with [(35)S]methionine/cysteine, a single 25-kDa protein was induced, relative to other proteins, on SDS-polyacrylamide gels. The 25-kDa band from DEET-treated cells was enriched in peptides corresponding to glutathione S-transferase D10 and/or theta in the Aedes aegypti genome. Consistent with the increased expression of the labeled protein, DEET-treated cells had increased glutathione S-transferase activity, and the radiolabeled band bound to Sepharose 4B containing reduced glutathione. By analyzing partial tryptic digests, we established that DEET induces the homolog of A. aegypti glutathione S-transferase, class theta, corresponding to protein XP_001658009.1 in the NCBI database. This specific effect of DEET at the subcellular level suggests that DEET induces physiological responses that extend beyond recognition by the peripheral olfactory system.

The Gene YALI0E20207g from Yarrowia lipolytica Encodes an N-Acetylglucosamine Kinase Implicated in the Regulated Expression of the Genes from the N-Acetylglucosamine Assimilatory Pathway

PubMed Central

Flores, Carmen-Lisset; Gancedo, Carlos

2015-01-01

The non-conventional yeast Yarrowia lipolytica possesses an ORF, YALI0E20207g, which encodes a protein with an amino acid sequence similar to hexokinases from different organisms. We have cloned that gene and determined several enzymatic properties of its encoded protein showing that it is an N-acetylglucosamine (NAGA) kinase. This conclusion was supported by the lack of growth in NAGA of a strain carrying a YALI0E20207g deletion. We named this gene YlNAG5. Expression of YlNAG5 as well as that of the genes encoding the enzymes of the NAGA catabolic pathway—identified by a BLAST search—was induced by this sugar. Deletion of YlNAG5 rendered that expression independent of the presence of NAGA in the medium and reintroduction of the gene restored the inducibility, indicating that YlNag5 participates in the transcriptional regulation of the NAGA assimilatory pathway genes. Expression of YlNAG5 was increased during sporulation and homozygous Ylnag5/Ylnag5 diploid strains sporulated very poorly as compared with a wild type isogenic control strain pointing to a participation of the protein in the process. Overexpression of YlNAG5 allowed growth in glucose of an Ylhxk1glk1 double mutant and produced, in a wild type background, aberrant morphologies in different media. Expression of the gene in a Saccharomyces cerevisiae hxk1 hxk2 glk1 triple mutant restored ability to grow in glucose. PMID:25816199

N-acetylglucosamine-Mediated Expression of nagA and nagB in Streptococcus pneumoniae.

PubMed

Afzal, Muhammad; Shafeeq, Sulman; Manzoor, Irfan; Henriques-Normark, Birgitta; Kuipers, Oscar P

2016-01-01

In this study, we have explored the transcriptomic response of Streptococcus pneumoniae D39 to N-acetylglucosamine (NAG). Transcriptome comparison of S. pneumoniae D39 wild-type grown in chemically defined medium (CDM) in the presence of 0.5% NAG to that grown in the presence of 0.5% glucose revealed elevated expression of many genes/operons, including nagA, nagB, manLMN , and nanP . We have further confirmed the NAG-dependent expression of nagA, nagB, manLMN , and nanP by β-galactosidase assays. nagA, nagB and glmS are putatively regulated by a transcriptional regulator NagR. We predicted the operator site of NagR ( dre site) in P nagA , P nagB , and P glmS , which was further confirmed by mutating the predicted dre site in the respective promoters ( nagA, nagB , and glmS ). Growth comparison of Δ nagA , Δ nagB , and Δ glmS with the D39 wild-type demonstrates that nagA and nagB are essential for S. pneumoniae D39 to grow in the presence of NAG as a sole carbon source. Furthermore, deletion of ccpA shows that CcpA has no effect on the expression of nagA, nagB , and glmS in the presence of NAG in S . pneumoniae .

N-acetylglucosamine-Mediated Expression of nagA and nagB in Streptococcus pneumoniae

PubMed Central

Afzal, Muhammad; Shafeeq, Sulman; Manzoor, Irfan; Henriques-Normark, Birgitta; Kuipers, Oscar P.

2016-01-01

In this study, we have explored the transcriptomic response of Streptococcus pneumoniae D39 to N-acetylglucosamine (NAG). Transcriptome comparison of S. pneumoniae D39 wild-type grown in chemically defined medium (CDM) in the presence of 0.5% NAG to that grown in the presence of 0.5% glucose revealed elevated expression of many genes/operons, including nagA, nagB, manLMN, and nanP. We have further confirmed the NAG-dependent expression of nagA, nagB, manLMN, and nanP by β-galactosidase assays. nagA, nagB and glmS are putatively regulated by a transcriptional regulator NagR. We predicted the operator site of NagR (dre site) in PnagA, PnagB, and PglmS, which was further confirmed by mutating the predicted dre site in the respective promoters (nagA, nagB, and glmS). Growth comparison of ΔnagA, ΔnagB, and ΔglmS with the D39 wild-type demonstrates that nagA and nagB are essential for S. pneumoniae D39 to grow in the presence of NAG as a sole carbon source. Furthermore, deletion of ccpA shows that CcpA has no effect on the expression of nagA, nagB, and glmS in the presence of NAG in S. pneumoniae. PMID:27900287

N-acetylglucosamine increases symptoms and fungal burden in a murine model of oral candidiasis.

PubMed

Ishijima, Sanae A; Hayama, Kazumi; Takahashi, Miki; Holmes, Ann R; Cannon, Richard D; Abe, Shigeru

2012-04-01

The amino sugar N-acetylglucosamine (GlcNAc) is an in vitro inducer of the hyphal mode of growth of the opportunistic pathogen Candida albicans. The development of hyphae by C. albicans is considered to contribute to the pathogenesis of mucosal oral candidiasis. GlcNAc is also a commonly used nutritional supplement for the self-treatment of conditions such as arthritis. To date, no study has investigated whether ingestion of GlcNAc has an effect on the in vivo growth of C. albicans or the pathogenesis of a C. albicans infection. Using a murine model of oral candidiasis, we have found that administration of GlcNAc, but not glucose, increased oral symptoms of candidiasis and fungal burden. Groups of mice were given GlcNAc in either water or in a viscous carrier, i.e., 1% methylcellulose. There was a dose-dependent relationship between GlcNAc concentration and the severity of oral symptoms. Mice given the highest dose of GlcNAc, 45.2 mM, also showed a significant increase in fungal burden, and increased histological evidence of infection compared to controls given water alone. We propose that ingestion of GlcNAc, as a nutritional supplement, may have an impact on oral health in people susceptible to oral candidiasis.

Survey of O-GlcNAc level variations in Xenopus laevis from oogenesis to early development.

PubMed

Dehennaut, Vanessa; Lefebvre, Tony; Leroy, Yves; Vilain, Jean-Pierre; Michalski, Jean-Claude; Bodart, Jean-François

2009-04-01

Little is known about the impact of O-linked-N-acetylglucosaminylation (O-GlcNAc) in gametes production and developmental processes. Here we investigated changes in O-GlcNAc, UDP-GlcNAc and O-GlcNAc transferase (OGT) levels in Xenopus laevis from oogenesis to embryo hatching. We showed that in comparison to stage VI, stages I-V oocytes expressed higher levels of O-GlcNAc correlating changes in OGT expression, but not in UDP-GlcNAc pools. Upon progesterone stimulation, an O-GlcNAc level burst occurred during meiotic resumption long before MPF and Mos-Erk2 pathways activations. Finally, we observed high levels of O-GlcNAc, UDP-GlcNAc and OGT during segmentation that decreased concomitantly at the onset of gastrulation. Nevertheless, no correlation between the glycosylation, the nucleotide-sugar and the glycosyltransferase was observed after neurulation. Our results show that O-GlcNAc is regulated throughout oogenesis and development within a complex pattern and suggest that dysfunctions in the dynamics of this glycosylation could lead to developmental abnormalities.

Synthesis of FUDP-N-acetylglucosamine and FUDP-glucose in Saccharomyces cerevisiae cells treated with 5-fluorouracil.

PubMed

Günther Sillero, María A; Pérez-Zúñiga, Francisco; Gomes, Joana; de Carvalho, Ana Isabel; Martins, Susana; Silles, Eduardo; Sillero, Antonio

2008-03-01

Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1% glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism.

A little sugar goes a long way: The cell biology of O-GlcNAc

PubMed Central

2015-01-01

Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate–GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport. PMID:25825515

The antifungal caspofungin increases fluoroquinolone activity against Staphylococcus aureus biofilms by inhibiting N-acetylglucosamine transferase.

PubMed

Siala, Wafi; Kucharíková, Soňa; Braem, Annabel; Vleugels, Jef; Tulkens, Paul M; Mingeot-Leclercq, Marie-Paule; Van Dijck, Patrick; Van Bambeke, Françoise

2016-11-03

Biofilms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against S. aureus biofilms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with β-1-3-glucan synthase (caspofungin's pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biofilms, and increases fluoroquinolone penetration inside biofilms. Our study identifies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biofilm-related infections.

The antifungal caspofungin increases fluoroquinolone activity against Staphylococcus aureus biofilms by inhibiting N-acetylglucosamine transferase

PubMed Central

Siala, Wafi; Kucharíková, Soňa; Braem, Annabel; Vleugels, Jef; Tulkens, Paul M; Mingeot-Leclercq, Marie-Paule; Van Dijck, Patrick; Van Bambeke, Françoise

2016-01-01

Biofilms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against S. aureus biofilms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with β-1-3-glucan synthase (caspofungin's pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biofilms, and increases fluoroquinolone penetration inside biofilms. Our study identifies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biofilm-related infections. PMID:27808087

The participation of ribosomes in protein glycosylation. Interaction of the ribosome-UDP-N-acetyl-glucosamine complex with dolichol phosphate.

PubMed

Paszkiewicz-Gadek, A; Porowska, H; Gałasiński, W

1992-01-01

UDP-N-acetylglucosamine can be bound by pure ribosomes. The part of N-acetylglucosamine-1-P can be transferred from the complex ribosome-UDP-N-acetylglucosamine onto dolichol phosphate. Evidence is presented that N-acetylglucosamine bound to dolichol phosphate can be transferred to the nascent peptide synthesized on the ribosome.

O-Linked-N-Acetylglucosamine Cycling and Insulin Signaling Are Required for the Glucose Stress Response in Caenorhabditis elegans

PubMed Central

Mondoux, Michelle A.; Love, Dona C.; Ghosh, Salil K.; Fukushige, Tetsunari; Bond, Michelle; Weerasinghe, Gayani R.; Hanover, John A.; Krause, Michael W.

2011-01-01

In a variety of organisms, including worms, flies, and mammals, glucose homeostasis is maintained by insulin-like signaling in a robust network of opposing and complementary signaling pathways. The hexosamine signaling pathway, terminating in O-linked-N-acetylglucosamine (O-GlcNAc) cycling, is a key sensor of nutrient status and has been genetically linked to the regulation of insulin signaling in Caenorhabditis elegans. Here we demonstrate that O-GlcNAc cycling and insulin signaling are both essential components of the C. elegans response to glucose stress. A number of insulin-dependent processes were found to be sensitive to glucose stress, including fertility, reproductive timing, and dauer formation, yet each of these differed in their threshold of sensitivity to glucose excess. Our findings suggest that O-GlcNAc cycling and insulin signaling are both required for a robust and adaptable response to glucose stress, but these two pathways show complex and interdependent roles in the maintenance of glucose–insulin homeostasis. PMID:21441213

New insights into metabolic signaling and cell survival: the role of beta-O-linkage of N-acetylglucosamine.

PubMed

Ngoh, Gladys A; Jones, Steven P

2008-12-01

The involvement of glucose in fundamental metabolic pathways represents a core element of biology. Late in the 20th century, a unique glucose-derived signal was discovered, which appeared to be involved in a variety of cellular processes, including mitosis, transcription, insulin signaling, stress responses, and potentially, Alzheimer's disease, and diabetes. By definition, this glucose-fed signaling system was a post-translational modification to proteins. However, unlike classical cotranslational N-glycosylation occurring in the endoplasmic reticulum and Golgi apparatus, this process occurs elsewhere throughout the cell in a highly dynamic fashion, similar to the quintessential post-translational modification, phosphorylation. This more recently described post-translational modification, the beta-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to nucleocytoplasmic proteins, represents an under-investigated area of biology. This signaling system operates in all of the tissues examined and seems to have persisted throughout all multicellular eukaryotes. Thus, it comes with little surprise that O-GlcNAc signaling is an integral system and viable target for biomedical investigation. This system may be a boundless source for insight into a variety of diseases and yield numerous opportunities for drug design. This Perspective will address recent insights into O-GlcNAc signaling in the cardiovascular system as a paradigm for its involvement in other biological systems.

Biosynthesis of truncated N-linked oligosaccharides results from non-orthologous hexosaminidase-mediated mechanisms in nematodes, plants, and insects.

PubMed

Gutternigg, Martin; Kretschmer-Lubich, Dorothea; Paschinger, Katharina; Rendić, Dubravko; Hader, Josef; Geier, Petra; Ranftl, Ramona; Jantsch, Verena; Lochnit, Günter; Wilson, Iain B H

2007-09-21

In many invertebrates and plants, the N-glycosylation profile is dominated by truncated paucimannosidic N-glycans, i.e. glycans consisting of a simple trimannosylchitobiosyl core often modified by core fucose residues. Even though they lack antennal N-acetylglucosamine residues, the biosynthesis of these glycans requires the sequential action of GlcNAc transferase I, Golgi mannosidase II, and, finally, beta-N-acetylglucosaminidases. In Drosophila, the recently characterized enzyme encoded by the fused lobes (fdl) gene specifically removes the non-reducing N-acetylglucosamine residue from the alpha1,3-antenna of N-glycans. In the present study, we examined the products of five beta-N-acetylhexosaminidase genes from Caenorhabditis elegans (hex-1 to hex-5, corresponding to reading frames T14F9.3, C14C11.3, Y39A1C.4, Y51F10.5, and Y70D2A.2) in addition to three from Arabidopsis thaliana (AtHEX1, AtHEX2, and AtHEX3, corresponding to reading frames At1g65590, At3g55260, and At1g05590). Based on homology, the Caenorhabditis HEX-1 and all three Arabidopsis enzymes are members of the same sub-family as the aforementioned Drosophila fused lobes enzyme but either act as chitotriosidases or non-specifically remove N-acetylglucosamine from both N-glycan antennae. The other four Caenorhabditis enzymes are members of a distinct sub-family; nevertheless, two of these enzymes displayed the same alpha1,3-antennal specificity as the fused lobes enzyme. Furthermore, a deletion of part of the Caenorhabditis hex-2 gene drastically reduces the native N-glycan-specific hexosaminidase activity in mutant worm extracts and results in a shift in the N-glycan profile, which is a demonstration of its in vivo enzymatic relevance. Based on these data, it is hypothesized that the genetic origin of paucimannosidic glycans in nematodes, plants, and insects involves highly divergent members of the same hexosaminidase gene family.

Functional characterization of Gne (UDP-N-acetylglucosamine-4-epimerase), Wzz (chain length determinant), and Wzy (O-antigen polymerase) of Yersinia enterocolitica serotype O:8.

PubMed

Bengoechea, José Antonio; Pinta, Elise; Salminen, Tiina; Oertelt, Clemens; Holst, Otto; Radziejewska-Lebrecht, Joanna; Piotrowska-Seget, Zofia; Venho, Reija; Skurnik, Mikael

2002-08-01

The lipopolysaccharide (LPS) O-antigen of Yersinia enterocolitica serotype O:8 is formed by branched pentasaccharide repeat units that contain N-acetylgalactosamine (GalNAc), L-fucose (Fuc), D-galactose (Gal), D-mannose (Man), and 6-deoxy-D-gulose (6d-Gul). Its biosynthesis requires at least enzymes for the synthesis of each nucleoside diphosphate-activated sugar precursor; five glycosyltransferases, one for each sugar residue; a flippase (Wzx); and an O-antigen polymerase (Wzy). As this LPS shows a characteristic preferred O-antigen chain length, the presence of a chain length determinant protein (Wzz) is also expected. By targeted mutagenesis, we identify within the O-antigen gene cluster the genes encoding Wzy and Wzz. We also present genetic and biochemical evidence showing that the gene previously called galE encodes a UDP-N-acetylglucosamine-4-epimerase (EC 5.1.3.7) required for the biosynthesis of the first sugar of the O-unit. Accordingly, the gene was renamed gne. Gne also has some UDP-glucose-4-epimerase (EC 5.1.3.2) activity, as it restores the core production of an Escherichia coli K-12 galE mutant. The three-dimensional structure of Gne was modeled based on the crystal structure of E. coli GalE. Detailed structural comparison of the active sites of Gne and GalE revealed that additional space is required to accommodate the N-acetyl group in Gne and that this space is occupied by two Tyr residues in GalE whereas the corresponding residues present in Gne are Leu136 and Cys297. The Gne Leu136Tyr and Cys297Tyr variants completely lost the UDP-N-acetylglucosamine-4-epimerase activity while retaining the ability to complement the LPS phenotype of the E. coli galE mutant. Finally, we report that Yersinia Wzx has relaxed specificity for the translocated oligosaccharide, contrary to Wzy, which is strictly specific for the O-unit to be polymerized.

Interaction of a lectin from Psathyrella velutina mushroom with N-acetylneuraminic acid.

PubMed

Ueda, H; Kojima, K; Saitoh, T; Ogawa, H

1999-04-01

A lectin from the fruiting body of Psathyrella velutina has been used as a specific probe for non-reducing terminal N-acetylglucosamine residues. We reveal in this report that P. velutina lectin recognizes a non-reducing terminal N-acetylneuraminic acid residue in glycoproteins and oligosaccharides. Binding of biotinyl P. velutina lectin to N-acetylneuraminic acid residues was prevented by desialylation of glycoconjugates and was distinguished from the binding to N-acetylglucosamine. Sialooligosaccharides were retarded or bound and eluted with N-acetylglucosamine on a P. velutina lectin column, being differentiated from each other and also from the oligosaccharides with non-reducing terminal N-acetylglucosamine which bound more strongly to the column.

O-GlcNAcylation of NF-κB Promotes Lung Metastasis of Cervical Cancer Cells via Upregulation of CXCR4 Expression.

PubMed

Ali, Akhtar; Kim, Sung Hwan; Kim, Min Jun; Choi, Mee Young; Kang, Sang Soo; Cho, Gyeong Jae; Kim, Yoon Sook; Choi, Jun-Young; Choi, Wan Sung

2017-07-31

C-X-C chemokine receptor 4 (CXCR4) stimulates cancer metastasis. NF-κB regulates CXCR4 expression in cancer cells, and O-GlcNAc modification of NF-κB promotes its transcriptional activity. Here, we determined whether CXCR4 expression is affected by O-GlcNAcylation of NF-κB in lung metastasis of cervical cancer. We found elevated levels of O-linked-N-actylglucosamine transferase (OGT) and O-GlcNAcylation in cervical cancer cells compared to those in non-malignant epithelial cells and detected increased expression of NF-κB p65 (p65) and CXCR4 in cervical cancer cells. Knockdown of OGT inhibited the O-GlcNAcylation of p65 and decreased CXCR4 expression levels in HeLa cells. Thiamet G treatment increased O-GlcNAcylated p65, which subsequently enhanced CXCR4 expression levels. Inhibition of O-GlcNAcylation by 6-Diazo-5-oxo-L-norleucine (DON) treatment decreased p65 activation, eventually inhibiting CXCR4 expression in HeLa cells. Lung tissues from mice engrafted with OGT-knockdown HeLa cells (shOGT) exhibited lower expression of Ki-67 and HPV E6 and E7 oncogenes compared to lung tissues from mice engrafted with control HeLa cells (shCTL). In addition, lung tissues from mice engrafted with shOGT cells exhibited lower p65 and CXCR4 immunoreactivity compared to tissues from mice engrafted with shCTL cells. Taken together, our data suggest that p65 O-GlcNAcylation promotes lung metastasis of cervical cancer cells by activating CXCR4 expression.

Increased OGA Expression and Activity in Leukocytes from Patients with Diabetes: Correlation with Inflammation Markers.

PubMed

Pagesy, Patrick; Tachet, Caroline; Mostefa-Kara, Ali; Larger, Etienne; Issad, Tarik

2018-06-11

O-linked-β-N-Acetylglucosaminylation (O-GlcNAcylation), a reversible post-translational modification involved in diabetic complications, is regulated by only two enzymes, O-linked N-acetylglucosamine transferase (OGT) and β-N-Acetylglucosaminidase (OGA). Increased OGA expression has been described previously in blood cells from patients with diabetes and was interpreted as an adaptative response to hyperglycemia-induced O-GlcNAcylation. OGA expression was thus proposed to have potential utility as a diagnostic marker. The present work was undertaken to determine whether determination of OGA enzymatic activity in blood cells could constitute a more rapidly accessible marker than OGA expression level measurements.Blood samples were obtained from patients with type 2 diabetes from the Department of Diabetology of the Cochin Hospital and healthy volunteers from the French blood Agency. OGA enzymatic activity and OGA mRNA expression levels were evaluated in leucocytes from patients with type 2 diabetes and from healthy donors.OGA activity was higher in leucocytes from patients with diabetes compared to control individuals. Surprisingly, OGA activity was not correlated hyperglycaemia markers (blood glucose, fructosamine, HbA 1c ) but was positively correlated with the inflammatory marker C-reactive protein. OGA mRNA levels were also increased in leucocytes from patients with diabetes and were correlated with mRNA coding for two pro-inflammatory proteins, TNFα and TxNIP.Therefore, OGA activity in leucocytes might be a more easily accessible biomarker than OGA expression levels. However, changes in OGA activity observed in patients with type 2 diabetes may reflect the inflammatory rather than the glycaemic status of these patients. © Georg Thieme Verlag KG Stuttgart · New York.

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase in nuclei and rimmed vacuoles of muscle fibers in DMRV (distal myopathy with rimmed vacuoles).

PubMed

Ishihara, Shoichiro; Tomimitsu, Hiroyuki; Fujigasaki, Hiroto; Saito, Fumiaki; Mizusawa, Hidehiro

2008-03-01

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key molecule in the pathogenesis of distal myopathy with rimmed vacuoles (DMRV) and hereditary inclusion body myopathy (HIBM) and almost all such patients have some mutations in GNE. However, subcellular localization of GNE and the mechanism of muscular damage have not been clarified. A rabbit polyclonal antibody for GNE was prepared. Immunohistochemistry was performed using anti-GNE and anti-nuclear protein antibodies. Western blotting with subcellular fractionated proteins was performed to determine subcellular localization of GNE. The sizes of myonuclei were quantified in muscle biopsies from patients with DMRV and amyotrophic lateral sclerosis (ALS). In DMRV muscles, immunohistochemistry identified GNE in sarcoplasm and specifically in myonuclei and rimmed vacuoles (RV). Nuclear proteins were also found in RVs. Immunohistochemistry showed colocalization of GNE and emerin in C2C12 cells. Western blotting revealed the presence of GNE in nuclear fractions of human embryonic kidney (HEK) 293T cells. The mean size of myonuclei of DMRV was significantly larger than that of ALS. GNE is present in myonuclei near nuclear membrane. Our results suggest that myonuclei are involved in RV formation in DMRV, and that mutant GNE in myonuclei seems to play some role in this process.

A novel lectin from Agrocybe aegerita shows high binding selectivity for terminal N-acetylglucosamine

PubMed Central

Jiang, Shuai; Chen, Yijie; Wang, Man; Yin, Yalin; Pan, Yongfu; Gu, Bianli; Yu, Guojun; Li, Yamu; Wong, Barry Hon Cheung; Liang, Yi; Sun, Hui

2012-01-01

A novel lectin was isolated from the mushroom Agrocybe aegerita (designated AAL-2) by affinity chromatography with GlcNAc (N-acetylglucosamine)-coupled Sepharose 6B after ammonium sulfate precipitation. The AAL-2 coding sequence (1224 bp) was identified by performing a homologous search of the five tryptic peptides identified by MS against the translated transcriptome of A. aegerita. The molecular mass of AAL-2 was calculated to be 43.175 kDa from MS, which was consistent with the data calculated from the amino acid sequence. To analyse the carbohydrate-binding properties of AAL-2, a glycan array composed of 465 glycan candidates was employed, and the result showed that AAL-2 bound with high selectivity to terminal non-reducing GlcNAc residues, and further analysis revealed that AAL-2 bound to terminal non-reducing GlcNAc residues with higher affinity than previously well-known GlcNAc-binding lectins such as WGA (wheatgerm agglutinin) and GSL-II (Griffonia simplicifolia lectin-II). ITC (isothermal titration calorimetry) showed further that GlcNAc bound to AAL-2 in a sequential manner with moderate affinity. In the present study, we also evaluated the anti-tumour activity of AAL-2. The results showed that AAL-2 could bind to the surface of hepatoma cells, leading to induced cell apoptosis in vitro. Furthermore, AAL-2 exerted an anti-hepatoma effect via inhibition of tumour growth and prolongation of survival time of tumour-bearing mice in vivo. PMID:22268569

Multi-specificity of a Psathyrella velutina mushroom lectin: heparin/pectin binding occurs at a site different from the N-acetylglucosamine/N-acetylneuraminic acid-specific site.

PubMed

Ueda, H; Saitoh, T; Kojima, K; Ogawa, H

1999-09-01

An N-acetylglucosamine (GlcNAc)/N-acetylneuraminic acid-specific lectin from the fruiting body of Psathyrella velutina (PVL) is a useful probe for the detection and fractionation of specific carbohydrates. In this study, PVL was found to exhibit multispecificity to acidic polysaccharides and sulfatides. Purified PVL and a counterpart lectin to PVL in the mycelium interact with heparin neoproteoglycans, as detected by both membrane analysis and solid phase assay. The pH-dependencies of the binding to heparin and GlcNAc5-6 differ. The heparin binding of PVL is inhibited best by pectin, polygalacturonic acid, and highly sulfated polysaccharides, but not by GlcNAc, colominic acid, or other glycosaminoglycans. Sandwich affinity chromatography indicated that PVL can simultaneously interact with heparin- and GlcNAc-containing macromolecules. Extensive biotinylation was found to suppress the binding activity to heparin while the GlcNAc binding activity is retained. On the other hand, biotinyl PVL binds to sulfatide and the binding is not inhibited by GlcNAc, N-acetylneuraminic acid, or heparin. These results indicate that PVL is a multi-ligand adhesive lectin that can interact with various glycoconjugates. This multispecificity needs to be recognized when using PVL as a sugar-specific probe to avoid misleading information about the nature of glycoforms.

Both Leukotoxin and Poly-N-Acetylglucosamine Surface Polysaccharide Protect Aggregatibacter actinomycetemcomitans Cells from Macrophage Killing

PubMed Central

Venketaraman, Vishwanath; Lin, Albert K.; Le, Amy; Kachlany, Scott C.; Connell, Nancy D.; Kaplan, Jeffrey B.

2008-01-01

Two virulence factors produced by the periodontopathogen Aggregatibacter actinomycetemcomitans are leukotoxin, a secreted lipoprotein that kills human polymorphonuclear leukocytes and macrophages, and poly-N-acetylglucosamine (PGA), a surface polysaccharide that mediates intercellular adhesion, biofilm formation and detergent resistance. In this study we examined the roles of leukotoxin and PGA in protecting A. actinomycetemcomitans cells from killing by the human macrophage cell line THP-1. Monolayers of THP-1 cells were infected with single-cell suspensions of a wild-type A. actinomycetemcomitans strain, or of isogenic leukotoxin or PGA mutant strains. After 48 h, viable bacteria were enumerated by dilution plating, macrophage morphology was evaluated microscopically, and macrophage viability was measured by a Trypan blue dye exclusion assay. The number of A. actinomycetemcomitans CFUs increased approximately 2-fold in wells infected with the wild-type strain, but decreased by approximately 70–90% in wells infected with the leukotoxin and PGA mutant strains. Infection with the wild-type or leukotoxin mutant strain caused a significant decrease in THP-1 cell viability, whereas infection with the PGA mutant strain did not result in any detectable changes in THP-1 viability. Pre-treatment of wild-type A. actinomycetemcomitans cells with the PGA-hydrolyzing enzyme dispersin B rendered them sensitive to killing by THP-1 cells. We concluded that both leukotoxin and PGA are necessary for evasion of macrophage killing by A. actinomycetemcomitans. PMID:18573331

Poly-N-acetylglucosamine mediates biofilm formation and antibiotic resistance in Actinobacillus pleuropneumoniae

PubMed Central

Izano, Era A.; Sadovskaya, Irina; Vinogradov, Evgeny; Mulks, Martha H.; Velliyagounder, Kabilan; Ragunath, Chandran; Kher, William B.; Ramasubbu, Narayanan; Jabbouri, Saïd; Perry, Malcolm B.; Kaplan, Jeffrey B.

2007-01-01

Most field isolates of the swine pathogen Actinobacillus pleuropneumoniae form tenacious biofilms on abiotic surfaces in vitro. We purified matrix polysaccharides from biofilms produced by A. pleuropneumoniae field isolates IA1 and IA5 (serotypes 1 and 5, respectively), and determined their chemical structures by using NMR spectroscopy. Both strains produced matrix polysaccharides consisting of linear chains of N-acetyl-D-glucosamine (GlcNAc) residues in β(1,6) linkage (poly-β-1,6-GlcNAc or PGA). A small percentage of the GlcNAc residues in each polysaccharide were N-deacetylated. These structures were nearly identical to those of biofilm matrix polysaccharides produced by Escherichia coli, Staphylococcus aureus and S. epidermidis. PCR analyses indicated that a gene encoding the PGA-specific glycoside transferase enzyme PgaC was present on the chromosome of 15 out of 15 A. pleuropneumoniae reference strains (serotypes 1-12) and 76 out of 77 A. pleuropneumoniae field isolates (serotypes 1, 5 and 7). A pgaC mutant of strain IA5 failed to form biofilms in vitro, as did wild-type strains IA1 and IA5 when grown in broth supplemented with the PGA-hydrolyzing enzyme dispersin B. Treatment of IA5 biofilms with dispersin B rendered them more sensitive to killing by ampicillin. Our findings suggest that PGA functions as a major biofilm adhesin in A. pleuropneumoniae. Biofilm formation may have relevance to the colonization and pathogenesis of A. pleuropneumoniae in pigs. PMID:17412552

Inhibitors of acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). Part 1: Hit to lead evaluation of a novel arylsulfonamide series.

PubMed

Green, Oluyinka M; McKenzie, Andrew R; Shapiro, Adam B; Otterbein, Ludovic; Ni, Haihong; Patten, Arthur; Stokes, Suzanne; Albert, Robert; Kawatkar, Sameer; Breed, Jason

2012-02-15

A novel arylsulfonamide-containing series of compounds represented by 1, discovered by highthroughput screening, inhibit the acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). X-ray structure determination confirmed that inhibitor binds at the site occupied by acetyl-CoA, indicating that series is competitive with this substrate. This letter documents our early hit-to-lead evaluation of the chemical series and some of the findings that led to improvement in in-vitro potency against Gram-negative and Gram-positive bacterial isozymes, exemplified by compound 40. Copyright © 2012 Elsevier Ltd. All rights reserved.

Exposure of Trypanosoma brucei to an N-acetylglucosamine-Binding Lectin Induces VSG Switching and Glycosylation Defects Resulting in Reduced Infectivity

PubMed Central

Castillo-Acosta, Víctor M.; Ruiz-Pérez, Luis M.; Van Damme, Els J. M.; Balzarini, Jan; González-Pacanowska, Dolores

2015-01-01

Trypanosoma brucei variant surface glycoproteins (VSG) are glycosylated by both paucimannose and oligomannose structures which are involved in the formation of a protective barrier against the immune system. Here, we report that the stinging nettle lectin (UDA), with predominant N-acetylglucosamine-binding specificity, interacts with glycosylated VSGs and kills parasites by provoking defects in endocytosis together with impaired cytokinesis. Prolonged exposure to UDA induced parasite resistance based on a diminished capacity to bind the lectin due to an enrichment of biantennary paucimannose and a reduction of triantennary oligomannose structures. Two molecular mechanisms involved in resistance were identified: VSG switching and modifications in N-glycan composition. Glycosylation defects were correlated with the down-regulation of the TbSTT3A and/or TbSTT3B genes (coding for oligosaccharyltransferases A and B, respectively) responsible for glycan specificity. Furthermore, UDA-resistant trypanosomes exhibited severely impaired infectivity indicating that the resistant phenotype entails a substantial fitness cost. The results obtained further support the modification of surface glycan composition resulting from down-regulation of the genes coding for oligosaccharyltransferases as a general resistance mechanism in response to prolonged exposure to carbohydrate-binding agents. PMID:25746926

Identification of the S-transferase like superfamily bacillithiol transferases encoded by Bacillus subtilis

PubMed Central

Perera, Varahenage R.; Lapek, John D.; Newton, Gerald L.; Gonzalez, David J.; Pogliano, Kit

2018-01-01

Bacillithiol is a low molecular weight thiol found in Firmicutes that is analogous to glutathione, which is absent in these bacteria. Bacillithiol transferases catalyze the transfer of bacillithiol to various substrates. The S-transferase-like (STL) superfamily contains over 30,000 putative members, including bacillithiol transferases. Proteins in this family are extremely divergent and are related by structural rather than sequence similarity, leaving it unclear if all share the same biochemical activity. Bacillus subtilis encodes eight predicted STL superfamily members, only one of which has been shown to be a bacillithiol transferase. Here we find that the seven remaining proteins show varying levels of metal dependent bacillithiol transferase activity. We have renamed the eight enzymes BstA-H. Mass spectrometry and gene expression studies revealed that all of the enzymes are produced to varying levels during growth and sporulation, with BstB and BstE being the most abundant and BstF and BstH being the least abundant. Interestingly, several bacillithiol transferases are induced in the mother cell during sporulation. A strain lacking all eight bacillithiol transferases showed normal growth in the presence of stressors that adversely affect growth of bacillithiol-deficient strains, such as paraquat and CdCl2. Thus, the STL bacillithiol transferases represent a new group of proteins that play currently unknown, but potentially significant roles in bacillithiol-dependent reactions. We conclude that these enzymes are highly divergent, perhaps to cope with an equally diverse array of endogenous or exogenous toxic metabolites and oxidants. PMID:29451913

Identification of human phosphoglucomutase 3 (PGM3) as N-acetylglucosamine-phosphate mutase (AGM1).

PubMed

Pang, H; Koda, Y; Soejima, M; Kimura, H

2002-03-01

We performed phenotyping of human phosphoglucomutase 3 (PGM(3)) and screening for mutations in the human N-acetylglucosamine-phosphate mutase gene (AGM(1)) to identify PGM(3) as AGM(1). By sequencing the coding region of AGM(1), two alleles containing a G or A base at nucleotide 1396, that can respectively encode aspartic acid or asparagine at codon 466, were identified. Cell extracts of COS7 cells after transfection with the pcDNA 3.1(+) plasmid containing an AGM(1) allele with 1396G or 1396A showed similar electrophoretic patterns to the PGM(3) 1 or PGM(3) 2 protein, respectively, with the isozyme detection method used for PGM(3) phenotyping. The genotypes determined by the two alleles of AGM(1) coincided exactly with the PGM(3) phenotypes in 20 individuals. We also investigated the allele frequency of the AGM(1) nucleotide polymorphism in a Japanese population by DNA sequencing and found that the frequencies of alleles 1396G and 1396A were similar to previously reported PGM(3) *1 and PGM(3) *2 frequencies. Overall, the facts that the AGM(1) gene product shows PGM activity, AGM(1) is polymorphic, the electrophoretic mobility is similar between AGM(1) allele-specific products and PGM(3) 1 and 2 proteins, PGM(3) phenotypes and AGM(1) genotypes completely coincide in 20 individuals, and AGM(1) allele frequencies are similar to those of PGM(3) *1 and PGM(3) *2 in Japanese populations, suggest that PGM(3) is identical to AGM(1).

A two-stage process facilitating microbial lipid production from N-acetylglucosamine by Cryptococcus curvatus cultured under non-sterile conditions.

PubMed

Tang, Mou; Zhou, Wenting; Liu, Yi; Yan, Jiabao; Gong, Zhiwei

2018-06-01

N-acetylglucosamine (GlcNAc), the monomeric constituent of chitin, is rarely studied for lipid production by oleaginous species. This study demonstrated that Cryptococcus curvatus had a great capacity to convert GlcNAc into lipid with high yield using a two-stage production process. Optimal inoculum age and inoculation size strongly improved the two-stage lipid production efficiency. More interestingly, this process rendered superior lipid production under non-sterile condition. The acetate liberated from GlcNAc was consumed timely, while the NH 4 + released was rarely assimilated. Lipid titre, lipid content and lipid yield reached 9.9 g/L, 56.9% and 0.23 g/g, respectively, which were significantly higher than those from the conventional process where cell growth and lipid accumulation were coupled. The resulting lipid samples had similar fatty acid compositional profiles to those of vegetable oil, suggesting their potential for biodiesel production. These findings strongly supported the two-stage process as an attractive strategy for better techno-economics of the chitin-to-biodiesel routes. Copyright © 2018 Elsevier Ltd. All rights reserved.

O-GlcNAc modification is essential for the regulation of autophagy in Drosophila melanogaster.

PubMed

Park, Sujin; Lee, Yangsin; Pak, Jin Won; Kim, Hanbyeol; Choi, Hyeonjin; Kim, Jae-woo; Roth, Jürgen; Cho, Jin Won

2015-08-01

O-GlcNAcylation is a dynamic post-translational modification that takes place on ser/thr residues of nucleocytoplasmic proteins. O-GlcNAcylation regulates almost all cellular events as a nutrient sensor, a transcriptional and translational regulator, and a disease-related factor. Although the role of O-GlcNAcylation in insulin signaling and metabolism are well established, the relationship between O-GlcNAcylation and autophagy is largely unknown. Here, we manipulated O-GlcNAcylation in Drosophila and found that it regulates autophagy through Akt/dFOXO signaling. We demonstrate that O-GlcNAcylation and the levels of O-GlcNAc transferase (OGT) are increased during starvation. Furthermore, Atg proteins and autolysosomes are increased in OGT-reduced flies without fasting. Atg proteins and autophagosomes are reduced in OGT-overexpressing flies. Our results suggest that not only autophagy gene expression but also autophagic structures are regulated by OGT through Akt and dFOXO. These data imply that O-GlcNAcylation is important in modulating autophagy as well as insulin signaling in Drosophila.

High-Level Fosfomycin Resistance in Vancomycin-Resistant Enterococcus faecium

PubMed Central

Guo, Yan; Tomich, Adam D.; McElheny, Christi L.; Cooper, Vaughn S.; Tait-Kamradt, Amelia; Wang, Minggui; Hu, Fupin; Rice, Louis B.; Sluis-Cremer, Nicolas

2017-01-01

Of 890 vancomycin-resistant Enterococcus faecium isolates obtained by rectal screening from patients in Pittsburgh, Pennsylvania, USA, 4 had MICs >1,024 μg/mL for fosfomycin. These isolates had a Cys119Asp substitution in the active site of UDP-N-acetylglucosamine enolpyruvyl transferase. This substitution increased the fosfomycin MIC >4-fold and rendered this drug inactive in biochemical assays. PMID:29048285

O-Linked N-Acetylglucosaminylation of Sp1 Inhibits the Human Immunodeficiency Virus Type 1 Promoter▿

PubMed Central

Jochmann, Ramona; Thurau, Mathias; Jung, Susan; Hofmann, Christian; Naschberger, Elisabeth; Kremmer, Elisabeth; Harrer, Thomas; Miller, Matthew; Schaft, Niels; Stürzl, Michael

2009-01-01

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5′ long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5′ LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked N-acetyl-d-glucosamine (O-GlcNAc), we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the O-GlcNAcylation-enhancing agent glucosamine (GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of O-GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of O-GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4+ T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (>95%) when OGT was recombinantly overexpressed prior to infection. O-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that O-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS. PMID:19193796

O-GlcNAcylation affects β-catenin and E-cadherin expression, cell motility and tumorigenicity of colorectal cancer.

PubMed

Harosh-Davidovich, Shani Ben; Khalaila, Isam

2018-03-01

O-GlcNAcylation, the addition of β-N-acetylglucosamine (O-GlcNAc) moiety to Ser/Thr residues, is a sensor of the cell metabolic state. Cancer diseases such as colon, lung and breast cancer, possess deregulated O-GlcNAcylation. Studies during the last decade revealed that O-GlcNAcylation is implicated in cancer tumorigenesis and proliferation. The Wnt/β-catenin signaling pathway and cadherin-mediated adhesion are also implicated in epithelial-mesenchymal transition (EMT), a key cellular process in invasion and cancer metastasis. Often, deregulation of the Wnt pathway is caused by altered phosphorylation of its components. Specifically, phosphorylation of Ser or Thr residues of β-catenin affects its location and interaction with E-cadherin, thus facilitating cell-cell adhesion. Consistent with previous studies, the current study indicates that β-catenin is O-GlcNAcylated. To test the effect of O-GlcNAcylation on cell motility and how O-GlcNAcylation might affect β-catenin and E-cadherin functions, the enzyme machinery of O-GlcNAcylation was modulated either with chemical inhibitors or by gene silencing. When O-GlcNAcase (OGA) was inhibited, a global elevation of protein O-GlcNAcylation and increase in the expression of E-cadherin and β-catenin were noted. Concomitantly with enhanced O-GlcNAcylation, β-catenin transcriptional activity were elevated. Additionally, fibroblast cell motility was enhanced. Stable silenced cell lines with adenoviral OGA or adenoviral O-GlcNAc transferase (OGT) were established. Consistent with the results obtained by OGA chemical inhibition by TMG, OGT-silencing led to a significant reduction in β-catenin level. In vivo, murine orthotropic colorectal cancer model indicates that elevated O-GlcNAcylation leads to increased mortality rate, tumor and metastasis development. However, reduction in O-GlcNAcylation promoted survival that could be attributed to attenuated tumor and metastasis development. The results described herein provide

Structural studies on molecular interactions between camel peptidoglycan recognition protein, CPGRP-S, and peptidoglycan moieties N-acetylglucosamine and N-acetylmuramic acid.

PubMed

Sharma, Pradeep; Yamini, Shavait; Dube, Divya; Singh, Amar; Sinha, Mau; Dey, Sharmistha; Mitra, Dipendra K; Kaur, Punit; Sharma, Sujata; Singh, Tej P

2012-06-22

Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoo, Jiho; Mashalidis, Ellene H.; Kuk, Alvin C. Y.

N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structuralmore » information. Here we present crystal structures of human GPT in complex with tunicamycin. In conclusion, structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.« less

Quinovosamycins: new tunicamycin-type antibiotics in which the α, β-1″,11'-linked N-acetylglucosamine residue is replaced by N-acetylquinovosamine.

PubMed

Price, Neil Pj; Labeda, David P; Naumann, Todd A; Vermillion, Karl E; Bowman, Michael J; Berhow, Mark A; Metcalf, William W; Bischoff, Kenneth M

2016-08-01

Tunicamycins (TUN) are potent inhibitors of polyprenyl phosphate N-acetylhexosamine 1-phosphate transferases (PPHP), including essential eukaryotic GPT enzymes and bacterial HexNAc 1-P translocases. Hence, TUN blocks the formation of eukaryotic N-glycoproteins and the assembly of bacterial call wall polysaccharides. The genetic requirement for TUN production is well-established. Using two genes unique to the TUN pathway (tunB and tunD) as probes we identified four new prospective TUN-producing strains. Chemical analysis showed that one strain, Streptomyces niger NRRL B-3857, produces TUN plus new compounds, named quinovosamycins (QVMs). QVMs are structurally akin to TUN, but uniquely in the 1″,11'-HexNAc sugar head group, which is invariably d-GlcNAc for the known TUN, but is d-QuiNAc for the QVM. Surprisingly, this modification has only a minor effect on either the inhibitory or antimicrobial properties of QVM and TUN. These findings have unexpected consequences for TUN/QVM biosynthesis, and for the specificity of the PPHP enzyme family.

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

PubMed

Chatham, John C; Marchase, Richard B

2010-02-01

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. Copyright 2009 Elsevier B.V. All rights reserved.

Characterization of N-Acetylglucosamine Biosynthesis in Pneumocystis species. A New Potential Target for Therapy

PubMed Central

Kottom, Theodore J.; Hebrink, Deanne M.; Jenson, Paige E.; Ramirez-Prado, Jorge H.

2017-01-01

N-acetylglucosamine (GlcNAc) serves as an essential structural sugar on the cell surface of organisms. For example, GlcNAc is a major component of bacterial peptidoglycan, it is an important building block of fungal cell walls, including a major constituent of chitin and mannoproteins, and it is also required for extracellular matrix generation by animal cells. Herein, we provide evidence for a uridine diphospho (UDP)–GlcNAc pathway in Pneumocystis species. Using an in silico search of the Pneumocystis jirovecii and P. murina (Pm) genomic databases, we determined the presence of at least four proteins implicated in the Saccharomyces cerevisiae UDP-GlcNAc biosynthetic pathway. These genes, termed GFA1, GNA1, AGM1, and UDP-GlcNAc pyrophosphorylase (UAP1), were either confirmed to be present in the Pneumocystis genomes by PCR, or, in the case of Pm uap1 (Pmuap1), functionally confirmed by direct enzymatic activity assay. Expression analysis using quantitative PCR of Pneumocystis pneumonia in mice demonstrated abundant expression of the Pm uap1 transcript. A GlcNAc-binding recombinant protein and a novel GlcNAc-binding immune detection method both verified the presence of GlcNAc in P. carinii (Pc) lysates. Studies of Pc cell wall fractions using high-performance gas chromatography/mass spectrometry documented the presence of GlcNAc glycosyl residues. Pc was shown to synthesize GlcNAc in vitro. The competitive UDP-GlcNAc substrate synthetic inhibitor, nikkomycin Z, suppressed incorporation of GlcNAc by Pc preparations. Finally, treatment of rats with Pneumocystis pneumonia using nikkomycin Z significantly reduced organism burdens. Taken together, these data support an important role for GlcNAc generation in the cell surface of Pneumocystis organisms. PMID:27632412

Hibiscus cannabinus feruloyl-coa:monolignol transferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilkerson, Curtis; Ralph, John; Withers, Saunia

The invention relates to isolated nucleic acids encoding a feruloyl-CoA:monolignol transferase and feruloyl-CoA:monolignol transferase enzymes. The isolated nucleic acids and/or the enzymes enable incorporation of monolignol ferulates into the lignin of plants, where such monolignol ferulates include, for example, p-coumaryl ferulate, coniferyl ferulate, and/or sinapyl ferulate. The invention also includes methods and plants that include nucleic acids encoding a feruloyl-CoA:monolignol transferase enzyme and/or feruloyl-CoA:monolignol transferase enzymes.

Engineering of N-acetylglucosamine metabolism for improved antibiotic production in Streptomyces coelicolor A3(2) and an unsuspected role of NagA in glucosamine metabolism.

PubMed

Świątek, Magdalena A; Urem, Mia; Tenconi, Elodie; Rigali, Sébastien; van Wezel, Gilles P

2012-01-01

N-acetylglucosamine (GlcNAc), the monomer of chitin and constituent of bacterial peptidoglycan, is a preferred carbon and nitrogen source for streptomycetes. Recent studies have revealed new functions of GlcNAc in nutrient signaling of bacteria. Exposure to GlcNAc activates development and antibiotic production of Streptomyces coelicolor under poor growth conditions (famine) and blocks these processes under rich conditions (feast). Glucosamine-6-phosphate (GlcN-6P) is a key molecule in this signaling pathway and acts as an allosteric effector of a pleiotropic transcriptional repressor DasR, the regulon of which includes the GlcNAc metabolic enzymes N-actetylglucosamine-6-phosphate (GlcNAc-6P) deacetylase (NagA) and GlcN-6P deaminase (NagB). Intracellular accumulation of GlcNAc-6P and GlcN-6P enhanced production of the pigmented antibiotic actinorhodin. When the nagB mutant was challenged with GlcNAc or GlcN, spontaneous second-site mutations that relieved the toxicity of the accumulated sugar phosphates were obtained. Surprisingly, deletion of nagA also relieved toxicity of GlcN, indicating novel linkage between the GlcN and GlcNAc utilization pathways. The strongly enhanced antibiotic production observed for many suppressor mutants shows the potential of the modulation of GlcNAc and GlcN metabolism as a metabolic engineering tool toward the improvement of antibiotic productivity or even the discovery of novel compounds.

Enzymatic Glycosylation by Transferases

NASA Astrophysics Data System (ADS)

Blixt, Ola; Razi, Nahid

Glycosyltransferases are important biological catalysts in cellular systems generating complex cell surface glycans involved in adhesion and signaling processes. Recent advances in glycoscience have increased the demands to access significant amount of glycans representing the glycome. Glycosyltransferases are now playing a key role for in vitro synthesis of oligosaccharides and the bacterial genome are increasingly utilized for cloning and over expression of active transferases in glycosylation reactions. This chapter highlights the recent progress towards preparative synthesis of oligosaccharides representing terminal sequences of glycoproteins and glycolipids using recombinant transferases. Transferases are also being explored in the context of solid-phase synthesis, immobilized on resins and over expression in vivo by engineered bacteria.

Structures of the Peptidoglycan N-Acetylglucosamine Deacetylase Bc1974 and Its Complexes with Zinc Metalloenzyme Inhibitors.

PubMed

Giastas, Petros; Andreou, Athena; Papakyriakou, Athanasios; Koutsioulis, Dimitris; Balomenou, Stavroula; Tzartos, Socrates J; Bouriotis, Vassilis; Eliopoulos, Elias E

2018-02-06

The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme. These metalloenzymes belong to the carbohydrate esterase family 4 (CE4) and are attractive targets for the development of new anti-infective agents. Herein we report the first X-ray crystal structures of the NodB domain of Bc1974, the conserved catalytic core of CE4s, in the unliganded form and in complex with four known metalloenzyme inhibitors and two amino acid hydroxamates that target the active site metal. These structures revealed the presence of two conformational states of a catalytic loop known as motif-4 (MT4), which were not observed previously for peptidoglycan deacetylases, but were recently shown in the structure of a Vibrio clolerae chitin deacetylase. By employing molecular docking of a substrate model, we describe a catalytic mechanism that probably involves initial binding of the substrate in a receptive, more open state of MT4 and optimal catalytic activity in the closed state of MT4, consistent with the previous observations. The ligand-bound structures presented here, in addition to the five Bc1974 inhibitors identified, provide a valuable basis for the design of antibacterial agents that target the peptidoglycan deacetylase Ba1977.

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

PubMed

Ye, Libin; Zheng, Xiaolin; Zheng, Hongjian

2014-04-01

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.

Feruloyl-CoA:monolignol transferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wilkerson, Curtis; Ralph, John; Withers, Saunia

The invention relates to nucleic acids encoding a feruloyl-CoA:monolignol transferase and the feruloyl-CoA:monolignol transferase enzyme that enables incorporation of monolignol ferulates, for example, including p-coumaryl ferulate, coniferyl ferulate, and sinapyl ferulate, into the lignin of plants.

"Nutrient-sensing" and self-renewal: O-GlcNAc in a new role.

PubMed

Sharma, Nikita S; Saluja, Ashok K; Banerjee, Sulagna

2018-06-01

Whether embryonic, hematopoietic or cancer stem cells, this metabolic reprogramming is dependent on the nutrient-status and bioenergetic pathways that is influenced by the micro-environmental niches like hypoxia. Thus, the microenvironment plays a vital role in determining the stem cell fate by inducing metabolic reprogramming. Under the influence of the microenvironment, like hypoxia, the stem cells have increased glucose and glutamine uptake which result in activation of hexosamine biosynthesis pathway (HBP) and increased O-GlcNAc Transferase (OGT). The current review is focused on understanding how HBP, a nutrient-sensing pathway (that leads to increased OGT activity) is instrumental in regulating self-renewal not only in embryonic and hematopoietic stem cells (ESC/HSC) but also in cancer stem cells.

The three-dimensional structure of the N-acetylglucosamine-6-phosphate deacetylase, NagA, from Bacillus subtilis: a member of the urease superfamily.

PubMed

Vincent, Florence; Yates, David; Garman, Elspeth; Davies, Gideon J; Brannigan, James A

2004-01-23

The enzyme N-acetylglucosamine-6-phosphate deacetylase, NagA, catalyzes the hydrolysis of the N-acetyl group of GlcNAc-6-P to yield glucosamine 6-phosphate and acetate, the first committed step in the biosynthetic pathway to amino-sugar-nucleotides. It is classified into carbohydrate esterase family CE-9 (see afmb.cnrs-mrs.fr/CAZY/). Here we report the cloning, expression, and three-dimensional structure (Protein Data Bank code 1un7) determination by x-ray crystallography of the Bacillus subtilis NagA at a resolution of 2.0 A. The structure presents two domains, a (beta/alpha)(8) barrel enclosing the active center and a small beta barrel domain. The structure is dimeric, and the substrate phosphate coordination at the active center is provided by an Arg/His pair contributed from the second molecule of the dimer. Both the overall structure and the active center bear a striking similarity to the urease superfamily with two metals involved in substrate binding and catalysis. PIXE (Proton-Induced x-ray Emission) data show that iron is the predominant metal in the purified protein. We propose a catalytic mechanism involving proton donation to the leaving group by aspartate, nucleophilic attack by an Fe-bridged hydroxide, and stabilization of the carbonyl oxygen by one of the two Fe atoms of the pair. We believe that this is the first sugar deacetylase to utilize this fold and catalytic mechanism.

Evolved osmotolerant Escherichia coli mutants frequently exhibit defective N-acetylglucosamine catabolism and point mutations in cell shape-regulating protein MreB.

PubMed

Winkler, James D; Garcia, Carlos; Olson, Michelle; Callaway, Emily; Kao, Katy C

2014-06-01

Biocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable of in situ DNA exchange) and asexual Escherichia coli strains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controlling N-acetylglucosamine catabolism (nagC, nagA), cell shape (mrdA, mreB), osmoprotectant uptake (proV), and motility (fimA). Possible epistatic interactions between nagC, nagA, fimA, and proV deletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes in ompACGL porin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Rewiring the Glucose Transportation and Central Metabolic Pathways for Overproduction of N-Acetylglucosamine in Bacillus subtilis.

PubMed

Gu, Yang; Deng, Jieying; Liu, Yanfeng; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Chen, Jian; Liu, Long

2017-10-01

N-acetylglucosamine (GlcNAc) is an important amino sugar extensively used in the healthcare field. In a previous study, the recombinant Bacillus subtilis strain BSGN6-P xylA -glmS-pP43NMK-GNA1 (BN0-GNA1) had been constructed for microbial production of GlcNAc by pathway design and modular optimization. Here, the production of GlcNAc is further improved by rewiring both the glucose transportation and central metabolic pathways. First, the phosphotransferase system (PTS) is blocked by deletion of three genes, yyzE (encoding the PTS system transporter subunit IIA YyzE), ypqE (encoding the PTS system transporter subunit IIA YpqE), and ptsG (encoding the PTS system glucose-specific EIICBA component), resulting in 47.6% increase in the GlcNAc titer (from 6.5 ± 0.25 to 9.6 ± 0.16 g L -1 ) in shake flasks. Then, reinforcement of the expression of the glcP and glcK genes and optimization of glucose facilitator proteins are performed to promote glucose import and phosphorylation. Next, the competitive pathways for GlcNAc synthesis, namely glycolysis, peptidoglycan synthesis pathway, pentose phosphate pathway, and tricarboxylic acid cycle, are repressed by initiation codon-optimization strategies, and the GlcNAc titer in shake flasks is improved from 10.8 ± 0.25 to 13.2 ± 0.31 g L -1 . Finally, the GlcNAc titer is further increased to 42.1 ± 1.1 g L -1 in a 3-L fed-batch bioreactor, which is 1.72-fold that of the original strain, BN0-GNA1. This study shows considerably enhanced GlcNAc production, and the metabolic engineering strategy described here will be useful for engineering other prokaryotic microorganisms for the production of GlcNAc and related molecules. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

O-GlcNAcomic Profiling Identifies Widespread O-Linked β-N-Acetylglucosamine Modification (O-GlcNAcylation) in Oxidative Phosphorylation System Regulating Cardiac Mitochondrial Function*♦

PubMed Central

Ma, Junfeng; Liu, Ting; Wei, An-Chi; Banerjee, Partha; O'Rourke, Brian; Hart, Gerald W.

2015-01-01

Dynamic cycling of O-linked β-N-acetylglucosamine (O-GlcNAc) on nucleocytoplasmic proteins serves as a nutrient sensor to regulate numerous biological processes. However, mitochondrial protein O-GlcNAcylation and its effects on function are largely unexplored. In this study, we performed a comparative analysis of the proteome and O-GlcNAcome of cardiac mitochondria from rats acutely (12 h) treated without or with thiamet-G (TMG), a potent and specific inhibitor of O-GlcNAcase. We then determined the functional consequences in mitochondria isolated from the two groups. O-GlcNAcomic profiling finds that over 88 mitochondrial proteins are O-GlcNAcylated, with the oxidative phosphorylation system as a major target. Moreover, in comparison with controls, cardiac mitochondria from TMG-treated rats did not exhibit altered protein abundance but showed overall elevated O-GlcNAcylation of many proteins. However, O-GlcNAc was unexpectedly down-regulated at certain sites of specific proteins. Concomitantly, TMG treatment resulted in significantly increased mitochondrial oxygen consumption rates, ATP production rates, and enhanced threshold for permeability transition pore opening by Ca2+. Our data reveal widespread and dynamic mitochondrial protein O-GlcNAcylation, serving as a regulator to their function. PMID:26446791

Dynamic O-linked N-acetylglucosamine modification of proteins affects stress responses and survival of mesothelial cells exposed to peritoneal dialysis fluids.

PubMed

Herzog, Rebecca; Bender, Thorsten O; Vychytil, Andreas; Bialas, Katarzyna; Aufricht, Christoph; Kratochwill, Klaus

2014-12-01

The ability of cells to respond and survive stressful conditions is determined, in part, by the attachment of O-linked N-acetylglucosamine (O-GlcNAc) to proteins (O-GlcNAcylation), a post-translational modification dependent on glucose and glutamine. This study investigates the role of dynamic O-GlcNAcylation of mesothelial cell proteins in cell survival during exposure to glucose-based peritoneal dialysis fluid (PDF). Immortalized human mesothelial cells and primary mesothelial cells, cultured from human omentum or clinical effluent of PD patients, were assessed for O-GlcNAcylation under normal conditions or after exposure to PDF. The dynamic status of O-GlcNAcylation and effects on cellular survival were investigated by chemical modulation with 6-diazo-5-oxo-L-norleucine (DON) to decrease or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc) to increase O-GlcNAc levels. Viability was decreased by reducing O-GlcNAc levels by DON, which also led to suppressed expression of the cytoprotective heat shock protein 72. In contrast, increasing O-GlcNAc levels by PUGNAc or alanyl-glutamine led to significantly improved cell survival paralleled by higher heat shock protein 72 levels during PDF treatment. Addition of alanyl-glutamine increased O-GlcNAcylation and partly counteracted its inhibition by DON, also leading to improved cell survival. Immunofluorescent analysis of clinical samples showed that the O-GlcNAc signal primarily originates from mesothelial cells. In conclusion, this study identified O-GlcNAcylation in mesothelial cells as a potentially important molecular mechanism after exposure to PDF. Modulating O-GlcNAc levels by clinically feasible interventions might evolve as a novel therapeutic target for the preservation of peritoneal membrane integrity in PD. Copyright © 2014 by the American Society of Nephrology.

Inhibitors incorporating zinc-binding groups target the GlcNAc-PI de-N-acetylase in Trypanosoma brucei, the causative agent of African sleeping sickness.

PubMed

Abdelwahab, Nuha Z; Crossman, Arthur T; Sullivan, Lauren; Ferguson, Michael A J; Urbaniak, Michael D

2012-03-01

Disruption of glycosylphosphatidylinositol biosynthesis is genetically and chemically validated as a drug target against the protozoan parasite Trypanosoma brucei, the causative agent of African sleeping sickness. The N-acetylglucosamine-phosphatidylinositol de-N-acetylase (deNAc) is a zinc metalloenzyme responsible for the second step of glycosylphosphatidylinositol biosynthesis. We recently reported the synthesis of eight deoxy-2-C-branched monosaccharides containing carboxylic acid, hydroxamic acid, or N-hydroxyurea substituents at the C2 position that may act as zinc-binding groups. Here, we describe the synthesis of a glucocyclitol-phospholipid incorporating a hydroxamic acid moiety and report the biochemical evaluation of the monosaccharides and the glucocyclitol-phospholipid as inhibitors of the trypanosome deNAc in the cell-free system and against recombinant enzyme. Monosaccharides with carboxylic acid or hydroxamic acid substituents were found to be the inhibitors of the trypanosome deNAc with IC(50) values 0.1-1.5mM and the glucocyclitol-phospholipid was found to be a dual inhibitor of the deNAc and the α1-4-mannose transferase with an apparent IC(50)= 19±0.5μm. © 2011 John Wiley & Sons A/S.

Hexose rearrangements upon fragmentation of N-glycopeptides and reductively aminated N-glycans.

PubMed

Wuhrer, Manfred; Koeleman, Carolien A M; Deelder, André M

2009-06-01

Tandem mass spectrometry of glycans and glycoconjugates in protonated form is known to result in rearrangement reactions leading to internal residue loss. Here we studied the occurrence of hexose rearrangements in tandem mass spectrometry of N-glycopeptides and reductively aminated N-glycans by MALDI-TOF/TOF-MS/MS and ESI-ion trap-MS/MS. Fragmentation of proton adducts of oligomannosidic N-glycans of ribonuclease B that were labeled with 2-aminobenzamide and 2-aminobenzoic acid resulted in transfer of one to five hexose residues to the fluorescently tagged innermost N-acetylglucosamine. Glycopeptides from various biological sources with oligomannosidic glycans were likewise shown to undergo hexose rearrangement reactions, resulting in chitobiose cleavage products that have acquired one or two hexose moieties. Tryptic immunoglobulin G Fc-glycopeptides with biantennary N-glycans likewise showed hexose rearrangements resulting in hexose transfer to the peptide moiety retaining the innermost N-acetylglucosamine. Thus, as a general phenomenon, tandem mass spectrometry of reductively aminated glycans as well as glycopeptides may result in hexose rearrangements. This characteristic of glycopeptide MS/MS has to be considered when developing tools for de novo glycopeptide structural analysis.

Accumulation of N-Acetylglucosamine Oligomers in the Plant Cell Wall Affects Plant Architecture in a Dose-Dependent and Conditional Manner1[W][OPEN

PubMed Central

Vanholme, Bartel; Vanholme, Ruben; Turumtay, Halbay; Goeminne, Geert; Cesarino, Igor; Goubet, Florence; Morreel, Kris; Rencoret, Jorge; Bulone, Vincent; Hooijmaijers, Cortwa; De Rycke, Riet; Gheysen, Godelieve; Ralph, John; De Block, Marc; Meulewaeter, Frank; Boerjan, Wout

2014-01-01

To study the effect of short N-acetylglucosamine (GlcNAc) oligosaccharides on the physiology of plants, N-ACETYLGLUCOSAMINYLTRANSFERASE (NodC) of Azorhizobium caulinodans was expressed in Arabidopsis (Arabidopsis thaliana). The corresponding enzyme catalyzes the polymerization of GlcNAc and, accordingly, β-1,4-GlcNAc oligomers accumulated in the plant. A phenotype characterized by difficulties in developing an inflorescence stem was visible when plants were grown for several weeks under short-day conditions before transfer to long-day conditions. In addition, a positive correlation between the oligomer concentration and the penetrance of the phenotype was demonstrated. Although NodC overexpression lines produced less cell wall compared with wild-type plants under nonpermissive conditions, no indications were found for changes in the amount of the major cell wall polymers. The effect on the cell wall was reflected at the transcriptome level. In addition to genes encoding cell wall-modifying enzymes, a whole set of genes encoding membrane-coupled receptor-like kinases were differentially expressed upon GlcNAc accumulation, many of which encoded proteins with an extracellular Domain of Unknown Function26. Although stress-related genes were also differentially expressed, the observed response differed from that of a classical chitin response. This is in line with the fact that the produced chitin oligomers were too small to activate the chitin receptor-mediated signal cascade. Based on our observations, we propose a model in which the oligosaccharides modify the architecture of the cell wall by acting as competitors in carbohydrate-carbohydrate or carbohydrate-protein interactions, thereby affecting noncovalent interactions in the cell wall or at the interface between the cell wall and the plasma membrane. PMID:24664205

21 CFR 862.1535 - Ornithine carbamyl transferase test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Test Systems § 862.1535 Ornithine carbamyl transferase test system. (a) Identification. An ornithine carbamyl transferase test system is a device intended to measure the activity of the enzyme ornithine... and treatment of liver diseases, such as infectious hepatitis, acute cholecystitis (inflammation of...

Aralia elata inhibits neurodegeneration by downregulating O-GlcNAcylation of NF-κB in diabetic mice.

PubMed

Kim, Seong-Jae; Kim, Min-Jun; Choi, Mee-Young; Kim, Yoon-Sook; Yoo, Ji-Myong; Hong, Eun-Kyung; Ju, Sunmi; Choi, Wan-Sung

2017-01-01

To investigate the role of O-GlcNAcylation of nuclear factor-kappa B (NF-κB) in retinal ganglion cell (RGC) death and analysedthe effect of Aralia elata (AE) on neurodegeneration in diabetic mice. C57BL/6mice with streptozotocin-induced diabetes were fed daily with AE extract or control (CTL) diet at the onset of diabetes mellitus (DM). Two months after injection of streptozotocin or saline, the degree of cell death and the expression of O-GlcNAc transferase (OGT), N-acetyl-b-D-glucosaminidase (OGA), O-GlcNAcylated proteins, and O-GlcNAcylation of NF-κB were examined. AE did not affect the metabolic status of diabetic mice. The decrease in the inner retinal thickness ( P <0.001 vs CTL, P <0.01 vs DM) and increases in RGCs with terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling ( P <0.001 vs CTL, P <0.0001 vs DM), glial activation, and active caspase-3 ( P <0.0001 vs CTL, P <0.0001 vs DM) were blocked in diabetic retinas of AE extract-fed mice. Expression levels of protein O-GlcNAcylation and OGT were increased in diabetic retinas ( P <0.0001 vs CTL), and the level of O-GlcNAcylation of the NF-κB p65 subunit was higher in diabetic retinas than in controls ( P <0.0001 vs CTL). AE extract downregulated O-GlcNAcylation of NF-κB and prevented neurodegeneration induced by hyperglycemia ( P <0.0001 vs DM). O-GlcNAcylation of NF-κB is concerned in neuronal degeneration and that AE prevents diabetes-induced RGC apoptosis via downregulation of NF-κB O-GlcNAcylation. Hence, O-GlcNAcylation may be a new object for the treatment of DR, and AE may have therapeutic possibility to prevent diabetes-induced neurodegeneration.

Retinal O-linked N-acetylglucosamine protein modifications: implications for postnatal retinal vascularization and the pathogenesis of diabetic retinopathy

PubMed Central

Sieg, Kelsey M.; Shallow, Keegan D.; Sorenson, Christine M.; Sheibani, Nader

2013-01-01

Purpose Hyperglycemia activates several metabolic pathways, including the hexosamine biosynthetic pathway. Uridine diphosphate N-acetylglucosamine (GlcNAc) is the product of the hexosamine biosynthetic pathway and the substrate for O-linked GlcNAc (O-GlcNAc) modification. This modification affects a wide range of proteins by altering their activity, cellular localization, and/or protein interactions. However, the role O-GlcNAcylation may play in normal postnatal retinal vascular development and in the ocular complications of diabetes, including diabetic retinopathy, requires further investigation. Methods The total levels of O-GlcNAc-modified proteins were evaluated by western blot analysis of lysates prepared from retinas obtained at different days during postnatal retinal vascularization and oxygen-induced ischemic retinopathy. Similar experiments were performed with retinal lysate prepared from diabetic Ins2Akita/+ mice with different durations of diabetes and retinal vascular cells cultured under various glucose conditions. The localization of O-GlcNAc-modified proteins in the retinal vasculature was confirmed by immunofluorescence staining. The impact of altered O-GlcNAcylation on the migration of retinal vascular cells was determined using scratch wound and transwell migration assays. Results We detected an increase in protein O-GlcNAcylation during mouse postnatal retinal vascularization and aging, in part through the regulation of the enzymes that control this modification. The study of the diabetic Ins2Akita/+ mouse retina showed an increase in the O-GlcNAc modification of retinal proteins. We also observed an increase in retinal O-GlcNAcylated protein levels during the neovascularization phase of oxygen-induced ischemic retinopathy. Our fluorescence microscopy data confirmed that the alterations in retinal O-GlcNAcylation are similarly represented in the retinal vasculature and in retinal pericytes and endothelial cells. Particularly, the migration of

21 CFR 862.1535 - Ornithine carbamyl transferase test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Test Systems § 862.1535 Ornithine carbamyl transferase test system. (a) Identification. An ornithine...

21 CFR 862.1535 - Ornithine carbamyl transferase test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... Test Systems § 862.1535 Ornithine carbamyl transferase test system. (a) Identification. An ornithine...

Polymorphisms of glutathione S-transferase Mu 1, glutathione S-transferase theta 1 and glutathione S-transferase Pi 1 genes in Hodgkin's lymphoma susceptibility and progression.

PubMed

Lourenço, Gustavo J; Néri, Iramaia A; Sforni, Vitor C S; Kameo, Rodolfo; Lorand-Metze, Irene; Lima, Carmen S P

2009-06-01

We tested in this study whether the polymorphisms of the glutathione S-transferase Mu1 (GSTM1), glutathione S-transferase Theta 1 (GSTT1) and glutathione S-transferase Pi 1 (GSTP1), involved in metabolism of chemical agents, cell proliferation and cell survival, alter the risk for Hodgkin lymphoma (HL). Genomic DNA from 110 consecutive patients with HL and 226 controls was analysed by polymerase chain reaction and restriction digestion for the polymorphism analyses. Similar frequencies of the GSTM1 and GSTT1 genotypes were seen in patients and controls. In contrast, the frequency of the GSTP1 wild genotype (59.1%versus 36.3%, P = 0.004) was higher in patients than in controls. Individuals with the wild genotype had a 2.68 (95%CI: 1.38-5.21)-fold increased risk for the disease than others. An excess of the GSTP1 wild genotype was also observed in patients with tumors of stages III + IV when compared with those with tumors of stages I + II (39.1%versus 20.0%, P = 0.03). These results suggest that the wild allele of the GSTP1 gene is linked to an increased risk and high aggressiveness of the HL in our cases but they should be confirmed by further studies with larger cohorts of patients and controls.

Moderate mammalian target of rapamycin inhibition induces autophagy in HTR8/SVneo cells via O-linked β-N-acetylglucosamine signaling.

PubMed

Zhang, Qiuxia; Na, Quan; Song, Weiwei

2017-10-01

Autophagy, a highly regulated process with a dual role (pro-survival or pro-death), has been implicated in adverse pregnancy outcomes. The aim of this study was to explore the mechanism whereby mammalian target of rapamycin (mTOR) signaling regulates autophagy by modulating protein O-GlcNAcylation in human trophoblasts. HTR8/SVneo cells were incubated in serum-free medium for different time intervals or treated with varying doses of Torin1. Protein expression and cell apoptosis were detected by immunoblotting and flow cytometry, respectively. Short-term serum starvation or slight suppression of mTOR signaling promoted autophagy and decreased apoptosis in HTR8/SVneo cells. Conversely, prolonged serum starvation or excessive inhibition of mTOR reduced autophagy and enhanced cell apoptosis. Both serum starvation and mTOR signaling suppression reduced protein O-GlcNAcylation. Upregulation and downregulation of O-linked β-N-acetylglucosamine (O-GlcNAc) levels attenuated and augmented autophagy, respectively. Moderate mTOR inhibition-induced autophagy was blocked by upregulation of protein O-GlcNAcylation. Furthermore, immunoprecipitation studies revealed that Beclin1 and synaptosome associated protein 29 (SNAP29) could be O-GlcNAcylated, and that slight mTOR inhibition resulted in decreased O-GlcNAc modification of Beclin1 and SNAP29. Notably, we observed an inverse correlation between phosphorylation (Ser15) and O-GlcNAcylation of Beclin1. mTOR signaling inhibition played dual roles in regulating autophagy and apoptosis in HTR8/SVneo cells. Moderate mTOR suppression might induce autophagy via modulating O-GlcNAcylation of Beclin1 and SNAP29. Moreover, the negative interplay between Beclin1 O-GlcNAcylation and phosphorylation (Ser15) may be involved in autophagy regulation by mTOR signaling. © 2017 Japan Society of Obstetrics and Gynecology.

O-GlcNAcylation: A New Cancer Hallmark?

PubMed

Fardini, Yann; Dehennaut, Vanessa; Lefebvre, Tony; Issad, Tarik

2013-01-01

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification consisting in the addition of a sugar moiety to serine/threonine residues of cytosolic or nuclear proteins. Catalyzed by O-GlcNAc-transferase (OGT) and removed by O-GlcNAcase, this dynamic modification is dependent on environmental glucose concentration. O-GlcNAcylation regulates the activities of a wide panel of proteins involved in almost all aspects of cell biology. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake, an important cancer risk factor, on protein activities and cellular functions. Indeed, O-GlcNAcylation has been shown to play a significant role in cancer development through different mechanisms. O-GlcNAcylation and OGT levels are increased in different cancers (breast, prostate, colon…) and vary during cell cycle progression. Modulating their expression or activity can alter cancer cell proliferation and/or invasion. Interestingly, major oncogenic factors have been shown to be directly O-GlcNAcylated (p53, MYC, NFκB, β-catenin…). Furthermore, chromatin dynamics is modulated by O-GlcNAc. DNA methylation enzymes of the Tet family, involved epigenetic alterations associated with cancer, were recently found to interact with and target OGT to multi-molecular chromatin-remodeling complexes. Consistently, histones are subjected to O-GlcNAc modifications which regulate their function. Increasing number of evidences point out the central involvement of O-GlcNAcylation in tumorigenesis, justifying the attention received as a potential new approach for cancer treatment. However, comprehension of the underlying mechanism remains at its beginnings. Future challenge will be to address directly the role of O-GlcNAc-modified residues in oncogenic-related proteins to eventually propose novel strategies to alter cancer development and/or progression.

O-GlcNAcylation: A New Cancer Hallmark?

PubMed Central

Fardini, Yann; Dehennaut, Vanessa; Lefebvre, Tony; Issad, Tarik

2013-01-01

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a reversible post-translational modification consisting in the addition of a sugar moiety to serine/threonine residues of cytosolic or nuclear proteins. Catalyzed by O-GlcNAc-transferase (OGT) and removed by O-GlcNAcase, this dynamic modification is dependent on environmental glucose concentration. O-GlcNAcylation regulates the activities of a wide panel of proteins involved in almost all aspects of cell biology. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake, an important cancer risk factor, on protein activities and cellular functions. Indeed, O-GlcNAcylation has been shown to play a significant role in cancer development through different mechanisms. O-GlcNAcylation and OGT levels are increased in different cancers (breast, prostate, colon…) and vary during cell cycle progression. Modulating their expression or activity can alter cancer cell proliferation and/or invasion. Interestingly, major oncogenic factors have been shown to be directly O-GlcNAcylated (p53, MYC, NFκB, β-catenin…). Furthermore, chromatin dynamics is modulated by O-GlcNAc. DNA methylation enzymes of the Tet family, involved epigenetic alterations associated with cancer, were recently found to interact with and target OGT to multi-molecular chromatin-remodeling complexes. Consistently, histones are subjected to O-GlcNAc modifications which regulate their function. Increasing number of evidences point out the central involvement of O-GlcNAcylation in tumorigenesis, justifying the attention received as a potential new approach for cancer treatment. However, comprehension of the underlying mechanism remains at its beginnings. Future challenge will be to address directly the role of O-GlcNAc-modified residues in oncogenic-related proteins to eventually propose novel strategies to alter cancer development and/or progression. PMID:23964270

O-GlcNAcylation of histone deacetylases 1 in hepatocellular carcinoma promotes cancer progression.

PubMed

Zhu, Guizhou; Tao, Tao; Zhang, Dongmei; Liu, Xiaojuan; Qiu, Huiyuan; Han, LiJian; Xu, Zhiwei; Xiao, Ying; Cheng, Chun; Shen, Aiguo

2016-08-01

Hepatocellular carcinoma (HCC) is a malignant tumor originating in the liver. Previous studies have indicated that O-GlcNAc transferase (OGT) and histone deacetylase-1 (HDAC1) play important roles in the pathogenesis of HCC. In the present study, we investigated the physical link between OGT and HDAC1. The O-GlcNAcylation of HDAC1 is overexpressed in HCC. We found that HDAC1 has two major sites of O-GlcNAcylation in its histone deacetylase domain. HDAC1 O-GlcNAcylation increases the activated phosphorylation of HDAC1, which enhances its enzyme activity. HDAC1 O-GlcNAc mutants promote the p21 transcription regulation through affecting the acetylation levels of histones from chromosome, and then influence the proliferation of HCC cells. We also found that mutants of O-GlcNAcylation site of HDAC1 affect invasion and migration of HepG2 cells. E-cadherin level is highly up-regulated in HDAC1 O-GlcNAc mutant-treated liver cancer cells, which inhibit the occurrence and development of HCC. Our findings suggest that OGT promotes the O-GlcNAc modification of HDAC1in the development of HCC. Therefore, inhibiting O-GlcNAcylation of HDAC1 may repress the progression of HCC. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Differential membranous E-cadherin expression, cell proliferation and O-GlcNAcylation between primary and metastatic nodal lesion in colorectal cancer.

PubMed

Jang, Tae Jung

2016-02-01

O-GlcNAcylation is an O-linked β-N-acetylglucosamine (O-GlcNAc) moiety linked to the side chain hydroxyl of a serine or threonine residue. The E-cadherin/β-catenin system, an integral component of epithelial to mesenchymal transition (EMT)/mesenchymal to epithelial transition (MET), is affected through O-GlcNAcylation. The current study examined the status of EMT/MET in both the tumor center and invasive front of the primary colorectal carcinoma (CRC) and metastatic nodal lesions, which were compared to O-GlcNAcylation expression levels in those areas. In addition, the cliniopathological significance of O-GlcNAcylation was studied Immunohistochemical staining for E-cadherin, β-catenin, Snail, O-GlcNAc and Ki67 was performed in 40 primary CRC tissues, 40 nonneoplastic colons, and 17 nodal metastatic lesions. Western blot was also conducted in primary CRC tissue Membranous E-cadherin expression was lowest in the invasive front, but showed greater increases in metastatic nodal lesions. Moreover, its expression level was negatively correlated with that of nuclear β-catenin and Snail. The Ki67 labeling index (LI) was lowest in the invasive front, and increased in metastatic nodal lesions. Primary CRC showed higher expression of O-GlcNAcylation and O-GlcNAc-transferase (OGT) than nonneoplastic colons. O-GlcNAcylation expression decreased in metastatic nodal lesions compared to the invasive front and tumor center, and was inversely correlated with Ki67 LI. However, O-GlcNAcylation expression was only slightly changed between tumor center and invasive front. In addition, there was no correlation between its expression and the level of nuclear β-catenin, membranous E-cadherin and Snail. No significant relationship was observed between O-GlcNAcylation level and cliniopathological parameters. Differential membranous E-cadherin expression, cell proliferation and O-GlcNAcylation in metastatic nodal lesion compared to primary CRC may play role in establishing its lesions

Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei.

PubMed

Pettersson, Eva U; Ljunggren, Erland L; Morrison, David A; Mattsson, Jens G

2005-01-01

The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide. Our interest in S. scabiei led us to further characterise a glutathione S-transferase. This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases. The S. scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26 kDa. Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects. The recombinant S. scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis. The S. scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme. Polyclonal antibodies raised against S. scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites. However, some minor staining of parasite intestines was observed. No staining was seen in host tissues, nor could we detect any antibody response against S. scabiei glutathione S-transferase in sera from naturally S. scabiei infected dogs or pigs. Additionally, the polyclonal sera raised against recombinant S. scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.

Structure-activity relationships of 4-hydroxyalkenals in the conjugation catalysed by mammalian glutathione transferases.

PubMed Central

Danielson, U H; Esterbauer, H; Mannervik, B

1987-01-01

The substrate specificities of 15 cytosolic glutathione transferases from rat, mouse and man have been explored by use of a homologous series of 4-hydroxyalkenals, extending from 4-hydroxypentenal to 4-hydroxypentadecenal. Rat glutathione transferase 8-8 is exceptionally active with the whole range of 4-hydroxyalkenals, from C5 to C15. Rat transferase 1-1, although more than 10-fold less efficient than transferase 8-8, is the second most active transferase with the longest chain length substrates. Other enzyme forms showing high activities with these substrates are rat transferase 4-4 and human transferase mu. The specificity constants, kcat./Km, for the various enzymes have been determined with the 4-hydroxyalkenals. From these constants the incremental Gibbs free energy of binding to the enzyme has been calculated for the homologous substrates. The enzymes responded differently to changes in the length of the hydrocarbon side chain and could be divided into three groups. All glutathione transferases displayed increased binding energy in response to increased hydrophobicity of the substrate. For some of the enzymes, steric limitations of the active site appear to counteract the increase in binding strength afforded by increased chain length of the substrate. Comparison of the activities with 4-hydroxyalkenals and other activated alkenes provides information about the active-site properties of certain glutathione transferases. The results show that the ensemble of glutathione transferases in a given species may serve an important physiological role in the conjugation of the whole range of 4-hydroxyalkenals. In view of its high catalytic efficiency with all the homologues, rat glutathione transferase 8-8 appears to have evolved specifically to serve in the detoxication of these reactive compounds of oxidative metabolism. PMID:3426557

Development of the sigma-1 receptor in C-terminals of motoneurons and colocalization with the N,N'-dimethyltryptamine forming enzyme, indole-N-methyl transferase.

PubMed

Mavlyutov, T A; Epstein, M L; Liu, P; Verbny, Y I; Ziskind-Conhaim, L; Ruoho, A E

2012-03-29

The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein-coupled receptors (GPCR). In the CNS, the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and S1Rs suggest that DMT is synthesized locally to effectively activate S1R in MN. Published by Elsevier Ltd.

Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase.

PubMed

Murphy, Jesse R; Mullins, Elwood A; Kappock, T Joseph

2016-01-01

Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.

Functional Dissection of the Bipartite Active Site of the Class I Coenzyme A (CoA)-Transferase Succinyl-CoA:Acetate CoA-Transferase

PubMed Central

Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

2016-01-01

Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. In this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes and orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. The ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA. PMID:27242998

Functional dissection of the bipartite active site of the class I coenzyme A (CoA)-transferase succinyl-CoA:acetate CoA-transferase

DOE PAGES

Murphy, Jesse R.; Mullins, Elwood A.; Kappock, T. Joseph

2016-05-23

Coenzyme A (CoA)-transferases catalyze the reversible transfer of CoA from acyl-CoA thioesters to free carboxylates. Class I CoA-transferases produce acylglutamyl anhydride intermediates that undergo attack by CoA thiolate on either the internal or external carbonyl carbon atoms, forming distinct tetrahedral intermediates <3 Å apart. Here in this study, crystal structures of succinyl-CoA:acetate CoA-transferase (AarC) from Acetobacter aceti are used to examine how the Asn347 carboxamide stabilizes the internal oxyanion intermediate. A structure of the active mutant AarC-N347A bound to CoA revealed both solvent replacement of the missing contact and displacement of the adjacent Glu294, indicating that Asn347 both polarizes andmore » orients the essential glutamate. AarC was crystallized with the nonhydrolyzable acetyl-CoA (AcCoA) analog dethiaacetyl-CoA (1a) in an attempt to trap a closed enzyme complex containing a stable analog of the external oxyanion intermediate. One active site contained an acetylglutamyl anhydride adduct and truncated 1a, an unexpected result hinting at an unprecedented cleavage of the ketone moiety in 1a. Solution studies confirmed that 1a decomposition is accompanied by production of near-stoichiometric acetate, in a process that seems to depend on microbial contamination but not AarC. A crystal structure of AarC bound to the postulated 1a truncation product (2a) showed complete closure of one active site per dimer but no acetylglutamyl anhydride, even with acetate added. These findings suggest that an activated acetyl donor forms during 1a decomposition; a working hypothesis involving ketone oxidation is offered. Finally, the ability of 2a to induce full active site closure furthermore suggests that it subverts a system used to impede inappropriate active site closure on unacylated CoA.« less

Glutathione transferases, regulators of cellular metabolism and physiology.

PubMed

Board, Philip G; Menon, Deepthi

2013-05-01

The cytosolic glutathione transferases (GSTs) comprise a super family of proteins that can be categorized into multiple classes with a mixture of highly specific and overlapping functions. The review covers the genetics, structure and function of the human cytosolic GSTs with particular attention to their emerging roles in cellular metabolism. All the catalytically active GSTs contribute to the glutathione conjugation or glutathione dependant-biotransformation of xenobiotics and many catalyze glutathione peroxidase or thiol transferase reactions. GSTs also catalyze glutathione dependent isomerization reactions required for the synthesis of several prostaglandins and steroid hormones and the catabolism of tyrosine. An increasing body of work has implicated several GSTs in the regulation of cell signaling pathways mediated by stress-activated kinases like Jun N-terminal kinase. In addition, some members of the cytosolic GST family have been shown to form ion channels in intracellular membranes and to modulate ryanodine receptor Ca(2+) channels in skeletal and cardiac muscle. In addition to their well established roles in the conjugation and biotransformation of xenobiotics, GSTs have emerged as significant regulators of pathways determining cell proliferation and survival and as regulators of ryanodine receptors that are essential for muscle function. This article is part of a Special Issue entitled Cellular functions of glutathione. Copyright © 2012 Elsevier B.V. All rights reserved.

Spectrofluorimetric assay method for glutathione and glutathione transferase using monobromobimane.

PubMed

Yakubu, S I; Yakasai, I A; Musa, A

2011-06-01

The primary role of glutathione transferase is to defend an organism from toxicities through catalyzing the reaction of glutathione (GSH) with potentially toxic compounds or metabolites to their chemically and biologically inert conjugates. The objective of the study was to develop a simple and sensitive spectrofluorimetric assay method for glutathione transferase using monobromobimane (MBB), a non fluorescent compound with electrophilic site. MBB slowly reacted with glutathione to form fluorescent glutathione conjugate and that the reaction was catalysed by glutathione transferase. Both non-enzymatic and enzymatic reaction products of MBB, in presence of GSH in phosphate buffer (pH 6.5), were measured by following increase of fluorescence at wavelength of 475nm. For validation of the assay method, the kinetic parameters such as the apparent Michaelis-Mente constants and maximum rates of conjugate formation as well as the specific activity of rat hepatic glutathione transferase were determined. The method was found to be sensitive, thus, applied to measure glutathione contents of crude preparation of rat hepatic cytosol fraction.

Rat lung glutathione S-transferases. Evidence for two distinct types of 22000-Mr subunits.

PubMed Central

Singh, S V; Partridge, C A; Awasthi, Y C

1984-01-01

Two immunologically distinct types of 22000-Mr subunits are present in rat lung glutathione S-transferases. One of these subunits is probably similar to Ya subunits of rat liver glutathione S-transferases, whereas the other subunit Ya' is immunologically distinct. Glutathione S-transferase II (pI7.2) of rat lung is a heterodimer (YaYa') of these subunits, and glutathione S-transferase VI (pI4.8) of rat lung is a homodimer of Ya' subunits. On hybridization in vitro of the subunits of glutathione S-transferase II of rat lung three active dimers having pI values 9.4, 7.2 and 4.8 are obtained. Immunological properties and substrate specificities indicate that the hybridized enzymes having pI7.2 and 4.8 correspond to glutathione S-transferases II and VI of rat lung respectively. Images Fig. 1. Fig. 5. PMID:6433888

AglH, a thermophilic UDP-N-acetylglucosamine-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase initiating protein N-glycosylation pathway in Sulfolobus acidocaldarius, is capable of complementing the eukaryal Alg7.

PubMed

Meyer, Benjamin H; Shams-Eldin, Hosam; Albers, Sonja-Verena

2017-01-01

AglH, a predicted UDP-GlcNAc-1-phosphate:dolichyl phosphate GlcNAc-1-phosphotransferase, is initiating the protein N-glycosylation pathway in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. AglH successfully replaced the endogenous GlcNAc-1-phosphotransferase activity of Alg7 in a conditional lethal Saccharomyces cerevisiae strain, in which the first step of the eukaryal protein N-glycosylation process was repressed. This study is one of the few examples of cross-domain complementation demonstrating a conserved polyprenyl phosphate transferase reaction within the eukaryal and archaeal domain like it was demonstrated for Methanococcus voltae (Shams-Eldin et al. 2008). The topology prediction and the alignment of the AglH membrane protein with GlcNAc-1-phosphotransferases from the three domains of life show significant conservation of amino acids within the different proposed cytoplasmic loops. Alanine mutations of selected conserved amino acids in the putative cytoplasmic loops II (D 100 ), IV (F 220 ) and V (F 264 ) demonstrated the importance of these amino acids for cross-domain AlgH activity in in vitro complementation assays in S. cerevisiae. Furthermore, antibiotic treatment interfering directly with the activity of dolichyl phosphate GlcNAc-1-phosphotransferases confirmed the essentiality of N-glycosylation for cell survival.

Effects of hypo-O-GlcNAcylation on Drosophila development.

PubMed

Mariappa, Daniel; Ferenbach, Andrew T; van Aalten, Daan M F

2018-05-11

Post-translational modification of serine/threonine residues in nucleocytoplasmic proteins with GlcNAc ( O -GlcNAcylation) is an essential regulatory mechanism in many cellular processes. In Drosophila , null mutants of the Polycomb gene O -GlcNAc transferase ( OGT ; also known as super sex combs ( sxc )) display homeotic phenotypes. To dissect the requirement for O -GlcNAc signaling in Drosophila development, we used CRISPR/Cas9 gene editing to generate rationally designed sxc catalytically hypomorphic or null point mutants. Of the fertile males derived from embryos injected with the CRISPR/Cas9 reagents, 25% produced progeny carrying precise point mutations with no detectable off-target effects. One of these mutants, the catalytically inactive sxc K872M , was recessive lethal, whereas a second mutant, the hypomorphic sxc H537A , was homozygous viable. We observed that reduced total protein O -GlcNAcylation in the sxc H537A mutant is associated with a wing vein phenotype and temperature-dependent lethality. Genetic interaction between sxc H537A and a null allele of Drosophila host cell factor ( dHcf ), encoding an extensively O -GlcNAcylated transcriptional coactivator, resulted in abnormal scutellar bristle numbers. A similar phenotype was also observed in sxc H537A flies lacking a copy of skuld ( skd ), a Mediator complex gene known to affect scutellar bristle formation. Interestingly, this phenotype was independent of OGT Polycomb function or dHcf downstream targets. In conclusion, the generation of the endogenous OGT hypomorphic mutant sxc H537A enabled us to identify pleiotropic effects of globally reduced protein O -GlcNAc during Drosophila development. The mutants generated and phenotypes observed in this study provide a platform for discovery of OGT substrates that are critical for Drosophila development. © 2018 Mariappa et al.

Alteration of O-GlcNAcylation affects serine phosphorylation and regulates gene expression and activity of pyruvate kinase M2 in colorectal cancer cells.

PubMed

Chaiyawat, Parunya; Chokchaichamnankit, Daranee; Lirdprapamongkol, Kriengsak; Srisomsap, Chantragan; Svasti, Jisnuson; Champattanachai, Voraratt

2015-10-01

O-GlcNAcylation is a dynamic post-translational modification that has extensive crosstalk with phosphorylation either at the same or adjacent sites of various proteins. We have previously reported that O-GlcNAcylation level was increased in primary breast and colorectal cancer, but the interplay of the two modifications remains unclear. Therefore, we explored crosstalk of the modifications by RNA interference against O-GlcNAc transferase (OGT) in colorectal cancer cells. Two-dimensional immunoblotting and mass spectrometric analysis showed that the levels of O-GlcNAc and serine phosphorylation of many proteins including serine hydroxymethyltransferase, cytokeratin-8, pyruvate kinase M2 (PKM2), heterogeneous nuclear ribonucleoprotein L, and lamin-B1, were reduced in siOGT cells compared to siScramble cells. In HT29 cells, immunoprecipitated PKM2 revealed decreased O-GlcNAc and serine phosphorylation levels after siOGT knockdown, but increased levels after treatment with Thiamet-G, an inhibitor of O-GlcNAcase (OGA). In addition, when global O-GlcNAcylation was enhanced by treating cells with Thiamet-G, PKM2 expression level was upregulated, but PKM2-specific activity was decreased. On the other hand, in OGT knockdown cells, PKM2 expression level was downregulated, but PKM2-specific activity was increased. Moreover, the metastatic colorectal cancer cells, SW620, had more O-GlcNAc-PKM2 and showed lower PKM2-specific activity compared to the non-metastatic colorectal cancer SW480 cells. These results suggested roles of O-GlcNAcylation in modulating serine phosphorylation, as well as in regulating PKM2 activity and expression. Interfering levels of O-GlcNAcylation of PKM2 may be a novel target in controlling cancer metabolism and tumorigenesis of colorectal cancer.

Regulation of the Hippo-YAP Pathway by Glucose Sensor O-GlcNAcylation.

PubMed

Peng, Changmin; Zhu, Yue; Zhang, Wanjun; Liao, Qinchao; Chen, Yali; Zhao, Xinyuan; Guo, Qiang; Shen, Pan; Zhen, Bei; Qian, Xiaohong; Yang, Dong; Zhang, Jin-San; Xiao, Dongguang; Qin, Weijie; Pei, Huadong

2017-11-02

The Hippo pathway is crucial in organ size control and tissue homeostasis, with deregulation leading to cancer. An extracellular nutrition signal, such as glucose, regulates the Hippo pathway activation. However, the mechanisms are still not clear. Here, we found that the Hippo pathway is directly regulated by the hexosamine biosynthesis pathway (HBP) in response to metabolic nutrients. Mechanistically, the core component of Hippo pathway (YAP) is O-GlcNAcylated by O-GlcNAc transferase (OGT) at serine 109. YAP O-GlcNAcylation disrupts its interaction with upstream kinase LATS1, prevents its phosphorylation, and activates its transcriptional activity. And this activation is not dependent on AMPK. We also identified OGT as a YAP-regulated gene that forms a feedback loop. Finally, we confirmed that glucose-induced YAP O-GlcNAcylation and activation promoted tumorigenesis. Together, our data establish a molecular mechanism and functional significance of the HBP in directly linking extracellular glucose signal to the Hippo-YAP pathway and tumorigenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular and biochemical characterization of tomato farnesyl-protein transferase.

PubMed

Schmitt, D; Callan, K; Gruissem, W

1996-10-01

The prenylation of membrane-associated proteins involved in the regulation of eukaryotic cell growth and signal transduction is critically important for their subcellular localization and biological activity. In contrast to mammalian cells and yeast, however, the function of protein prenylation in plants is not well understood and only a few prenylated proteins have been identified. We partially purified and characterized farnesyl-protein transferase from tomato (Lycopersicon esculentum, LeFTase) to analyze its biochemical and molecular properties. Using Ras- and G gamma-specific peptide substrates and competition assays we showed that tomato protein extracts have both farnesyl-protein transferase and geranylgeranyl-protein transferase 1 activities. Compared with the heterologous synthetic peptide substrates, the plant-specific CaaX sequence of the ANJ1 protein is a less efficient substrate for LeFTase in vitro. LeFTase activity profiles and LeFTase beta-subunit protein (LeFTB) levels differ significantly in various tissues and are regulated during fruit development. Partially purified LeFTase requires Zn2+ and Mg2+ for enzymatic activity and has an apparent molecular mass of 100 kD Immunoprecipitation experiments using anti-alpha LeFTB antibodies confirmed that LeFTB is a component of LeFTase but not of tomato geranylgeranyl-protein transferase 1. Based on their conserved bio-chemical activities, we expect that prenyltransferases are likely integrated with the sterol biosynthesis pathway in the control of plant cell growth.

Analysis of N-acetylaminosugars by CE: a comparative derivatization study.

PubMed

Rustighi, Isabella; Campa, Cristiana; Rossi, Marco; Semeraro, Sabrina; Vetere, Amedeo; Gamini, Amelia

2009-08-01

N-linked or O-linked glycans derived from glycoprotein processing carry, an N-acetylglucosamine or an N-acetylgalactosamine respectively, at their reducing termini. The presence of the N-acetylamino group on C-2 of reducing sugar residues has been reported to hamper the derivatization reaction with a chromophore at the anomeric centre. In this paper N-acetyllactosamine, N-acetylglucosamine, N-acetylgalactosamine and several other neutral monosaccharides are coupled to three different dyes (4-aminobenzonitrile, 2-aminopyridine, 2-aminobenzoic acid (2-AA)) by reductive amination and analysed by CE with UV detection. The 2-AA derivatives showed the lowest concentration detection limits, varying approximately in the 2-3 muM range for the saccharides tested including the N-acetamido ones. The possibility to separate and detect with the same sensitivity ten 2-AA-labelled monosaccharides mainly found in mammalian or plant glycoproteins in a single CE run is highlighted. The analysis has been carried out in less than 25 min using the borate-complexation method in CZE mode. The influence of the strength of the acid used as catalyst in the chemical modification of the sugars with 2-AA is also shortly addressed.

DEVELOPMENT OF THE SIGMA-1 RECEPTOR IN C-TERMINALS OF MOTONEURONS AND COLOCALIZATION WITH THE N,N’-DIMETHYLTRYPTAMINE FORMING ENZYME, INDOLE-N-METHYL TRANSFERASE

PubMed Central

Mavlyutov, Timur A.; Epstein, Miles L.; Liu, Patricia; Verbny, Yakov I.; Ziskind-Conhaim, Lea; Ruoho, Arnold E.

2012-01-01

The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein coupled receptors (GPCR). In the CNS the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that Indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and SIRs suggest that DMT is synthesized locally to effectively activate S1R in MN. PMID:22265729

O-GlcNAc regulates NEDD4-1 stability via caspase-mediated pathway.

PubMed

Jiang, Kuan; Bai, Bingyang; Ta, Yajie; Zhang, Tingling; Xiao, Zikang; Wang, Peng George; Zhang, Lianwen

2016-03-18

O-GlcNAc modification of cytosolic and nuclear proteins regulates essential cellular processes such as stress responses, transcription, translation, and protein degradation. Emerging evidence indicates O-GlcNAcylation has a dynamic interplay with ubiquitination in cellular regulation. Here, we report that O-GlcNAc indirectly targets a vital E3 ubiquitin ligase enzyme of NEDD4-1. The protein level of NEDD4-1 is accordingly decreased following an increase of overall O-GlcNAc level upon PUGNAc or glucosamine stimulation. O-GlcNAc transferase (OGT) knockdown, overexpression and mutation results confirm that the stability of NEDD4-1 is negatively regulated by cellular O-GlcNAc. Moreover, the NEDD4-1 degradation induced by PUGNAc or GlcN is significantly inhibited by the caspase inhibitor. Our study reveals a regulation mechanism of NEDD4-1 stability by O-GlcNAcylation. Copyright © 2016 Elsevier Inc. All rights reserved.

Cysteine S-linked N-acetylglucosamine (S-GlcNAcylation), A New Post-translational Modification in Mammals.

PubMed

Maynard, Jason C; Burlingame, Alma L; Medzihradszky, Katalin F

2016-11-01

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

GLUTATHIONE S-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE

EPA Science Inventory

GLUTATHIONE s-TRANSFERASE-MEDIATED METABOLISM OF BROMODICHLOROMETHANE. M K Ross1 and R A Pegram2. 1Curriculum in Toxicology, University of North Carolina at Chapel Hill; 2Experimental Toxicology Division, NHEERL/ORD, United States Environmental Protection Agency, Research Triangl...

Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors

PubMed Central

Kosa, Nicolas M.; Foley, Timothy L.; Burkart, Michael D.

2016-01-01

Phosphopantetheinyl transferase (E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low molecular weight coenzyme A substrate to higher molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of phosphopantetheinyl transferase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human phosphopantetheinyl transferase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity. PMID:24192555

Genomic organization of plant aminopropyl transferases.

PubMed

Rodríguez-Kessler, Margarita; Delgado-Sánchez, Pablo; Rodríguez-Kessler, Gabriela Theresia; Moriguchi, Takaya; Jiménez-Bremont, Juan Francisco

2010-07-01

Aminopropyl transferases like spermidine synthase (SPDS; EC 2.5.1.16), spermine synthase and thermospermine synthase (SPMS, tSPMS; EC 2.5.1.22) belong to a class of widely distributed enzymes that use decarboxylated S-adenosylmethionine as an aminopropyl donor and putrescine or spermidine as an amino acceptor to form in that order spermidine, spermine or thermospermine. We describe the analysis of plant genomic sequences encoding SPDS, SPMS, tSPMS and PMT (putrescine N-methyltransferase; EC 2.1.1.53). Genome organization (including exon size, gain and loss, as well as intron number, size, loss, retention, placement and phase, and the presence of transposons) of plant aminopropyl transferase genes were compared between the genomic sequences of SPDS, SPMS and tSPMS from Zea mays, Oryza sativa, Malus x domestica, Populus trichocarpa, Arabidopsis thaliana and Physcomitrella patens. In addition, the genomic organization of plant PMT genes, proposed to be derived from SPDS during the evolution of alkaloid metabolism, is illustrated. Herein, a particular conservation and arrangement of exon and intron sequences between plant SPDS, SPMS and PMT genes that clearly differs with that of ACL5 genes, is shown. The possible acquisition of the plant SPMS exon II and, in particular exon XI in the monocot SPMS genes, is a remarkable feature that allows their differentiation from SPDS genes. In accordance with our in silico analysis, functional complementation experiments of the maize ZmSPMS1 enzyme (previously considered to be SPDS) in yeast demonstrated its spermine synthase activity. Another significant aspect is the conservation of intron sequences among SPDS and PMT paralogs. In addition the existence of microsynteny among some SPDS paralogs, especially in P. trichocarpa and A. thaliana, supports duplication events of plant SPDS genes. Based in our analysis, we hypothesize that SPMS genes appeared with the divergence of vascular plants by a processes of gene duplication and the

A Novel Murine Candidiasis Model with Severe Colonization in the Stomach Induced by N-acetylglucosamine-treatment and Its Scoring System Based on Local Characteristic Stomach Symptoms.

PubMed

Ishijima, Sanae A; Abe, Shigeru

2015-01-01

We developed a novel murine candidiasis model of the gastrointestinal tract using N-acetylglucosamine ( GlcNAc ) as a tool to aggravate symptoms. Forty-eight hours after intragastrically inoculating Candida albicans cells to immunosuppressed and GlcNAc-treated mice, vigorously accumulating patchy whitish plaques were observed on their inner stomach surface. Candida cells colonizing the plaques consisted of both yeast and mycelia, and were directly stained with Calcofluor White M2R. Aggravation of the candidiasis symptoms was dependent on GlcNAc concentration in drinking water, wherein administration of 50 mM GlcNAc not only severely worsened stomach symptoms, but also significantly increased Candida cell number in the stomach and small intestine. The aggravation effect of GlcNAc was enhanced by addition of sedative chemical chlorpromazine chloride after inoculation. In order to semi-quantitatively assess colonization by Candida in the stomach, we devised a new symptom scoring system that represents the extent of the patchy whitish plaques on the mucosal epithelium of the stomach. Histochemical analysis of Candida-infected tissues revealed not only a large amount of thick Candida mycelia invading mucosal epithelial stomach tissues but also infiltrating inflammatory cells. These results suggest that this murine gastrointestinal candidiasis model could serve as a useful tool for evaluating the protective activity of antifungal agents, probiotics, or functional foods against gastrointestinal candidiasis. Furthermore, from another point of view, this novel murine model could also be used to analyze the pathological mechanisms behind the translocation of C. albicans across intestinal barriers, which results in systemic Candida dissemination and infection.

Molecular Cloning of Adenosinediphosphoribosyl Transferase.

DTIC Science & Technology

1987-09-08

nature of the blocking group is unknown, except its identity with pyroglutamic acid was ruled out by its insensitivity to pyroglutaminase (not shown...AdenosinediphosphoribOSyl Transferase (ADPRT) is: 1) the complete amino acid sequence of this large protein is best determined -from the DNA !equence of the gene, 2...enzyme (I), determination of its peptide structure (II) and application of synthetic DNA probes (III) derived from amino acid sequences, resulting in the

O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis.

PubMed

Zeng, Qinghua; Zhao, Rui-Xun; Chen, Jianfeng; Li, Yining; Li, Xiang-Dong; Liu, Xiao-Long; Zhang, Wei-Ming; Quan, Cheng-Shi; Wang, Yi-Shu; Zhai, Ying-Xian; Wang, Jian-Wei; Youssef, Mariam; Cui, Rutao; Liang, Jiyong; Genovese, Nicholas; Chow, Louise T; Li, Yu-Lin; Xu, Zhi-Xiang

2016-08-16

High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18-transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc.

O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis

PubMed Central

Zeng, Qinghua; Zhao, Rui-Xun; Chen, Jianfeng; Li, Yining; Li, Xiang-Dong; Liu, Xiao-Long; Zhang, Wei-Ming; Quan, Cheng-Shi; Wang, Yi-Shu; Zhai, Ying-Xian; Wang, Jian-Wei; Youssef, Mariam; Cui, Rutao; Liang, Jiyong; Genovese, Nicholas; Chow, Louise T.; Li, Yu-Lin; Xu, Zhi-Xiang

2016-01-01

High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18–transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc. PMID:27482104

Too sweet to resist: Control of immune cell function by O-GlcNAcylation.

PubMed

de Jesus, Tristan; Shukla, Sudhanshu; Ramakrishnan, Parameswaran

2018-06-02

O-linked β-N-acetyl glucosamine modification (O-GlcNAcylation) is a dynamic, reversible posttranslational modification of cytoplasmic and nuclear proteins. O-GlcNAcylation depends on nutrient availability and the hexosamine biosynthetic pathway (HBP), which produces the donor substrate UDP-GlcNAc. O-GlcNAcylation is mediated by a single enzyme, O-GlcNAc transferase (OGT), which adds GlcNAc and another enzyme, O-GlcNAcase (OGA), which removes O-GlcNAc from proteins. O-GlcNAcylation controls vital cellular processes including transcription, translation, the cell cycle, metabolism, and cellular stress. Aberrant O-GlcNAcylation has been implicated in various pathologies including Alzheimer's disease, diabetes, obesity, and cancer. Growing evidences indicate that O-GlcNAcylation plays crucial roles in regulating immunity and inflammatory responses, especially under hyperglycemic conditions. This review will highlight the emerging functions of O-GlcNAcylation in mammalian immunity under physiological and various pathological conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

Biosynthesis of 2-Hydroxyacid-Containing Polyhydroxyalkanoates by Employing butyryl-CoA Transferases in Metabolically Engineered Escherichia coli.

PubMed

David, Yokimiko; Joo, Jeong Chan; Yang, Jung Eun; Oh, Young Hoon; Lee, Sang Yup; Park, Si Jae

2017-11-01

The authors previously reported the production of polyhydroxyalkanoates (PHAs) containing 2-hydroxyacid monomers by expressing evolved Pseudomonas sp. 6-19 PHA synthase and Clostridium propionicum propionyl-CoA transferase in engineered microorganisms. Here, the authors examined four butyryl-CoA transferases from Roseburia sp., Eubacterium hallii, Faecalibacterium prausnitzii, and Anaerostipes caccae as potential CoA-transferases to support synthesis of polymers having 2HA monomer. In vitro activity analyses of the four butyryl-CoA transferases suggested that each butyryl-CoA transferase has different activities towards 2-hydroxybutyrate (2HB), 3-hydroxybutyrate (3HB), and lactate (LA). When Escherichia coli XL1-Blue expressing Pseudomonas sp. 6-19 PhaC1437 along with one butyryl-CoA transferase is cultured in chemically defined MR medium containing 20 g L -1 of glucose, 2 g L -1 of sodium 3-hydroxybutyrate, and various concentrations of sodium 2-hydroxybutyrate, PHAs consisting of 3HB, 2HB, and LA are produced. The monomer composition of PHAs agreed well with the substrate specificities of butyryl-CoA transferases from E. hallii, F. prausnitzii, and A. caccae, but not Roseburia sp. When E. coli XL1-Blue expressing PhaC1437 and E. hallii butyryl-CoA transferase is cultured in MR medium containing 20 g L -1 of glucose and 2 g L -1 of sodium 2-hydroxybutyrate, P(65.7 mol% 2HB-co-34.3 mol% LA) is produced with the highest PHA content of 30 wt%. Butyryl-CoA transferases also supported the production of P(3HB-co-2HB-co-LA) from glucose as the sole carbon source in E. coli XL1-Blue strains when one of these bct genes is expressed with phaC1437, cimA3.7, leuBCD, panE, and phaAB genes. Butyryl-CoA transferases characterized in this study can be used for engineering of microorganisms that produce PHAs containing novel 2-hydroxyacid monomers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The mechanism of cell death in human cultured colon adenocarcinoma cell line COLO 201 induced by beta-D-N-acetylglucosaminyl-p-nitrophenol.

PubMed

Kukidome, J; Kakizaki, I; Takagaki, K; Matsuki, A; Munakata, A; Endo, M

2001-05-01

COLO 201, human colon adenocarcinoma cells were incubated with artificial primers, p-nitrophenyl-glycoside derivatives at 1.0 mmol (mM) in the medium containing 10% fetal bovine serum to detect sugar chain elongation. However, when p-nitrophenyl-beta-N-acetylglucosamine (beta-GlcNAc-PNP) was added, the medium changed color to yellow and the cells were dead. To explain this finding, the cells were incubated with 1.0 mM each of beta-GlcNAc-PNP and 4-methylumbelliferyl-beta-N-acetylglucosamine, then the number of living cells was measured in a time course. In beta-GlcNAc-PNP, the living cells were decreased at 24 hours. The cells were survived with N-acetylglucosamine, whereas in the presence of p-nitrophenol (PNP) the living cells were decreased. It was suggested that PNP released from beta-GlcNAc-PNP induced the cell death. Activity of beta-D-N-acetylglucosaminidase was detected in fetal bovine serum. It was shown that PNP induced the cell death in time-and-dose dependent manner. Genomic DNA from COLO 201 analyzed by agarose gel electrophoresis was fragmentated. PNP analogues were tested for toxicity, and the results suggested that the phenolic OH-group linked to benzene ring and nitro-group linked to the structure in para-form (PNP) was the most effective.

Glutathione S-transferases in neonatal liver disease.

PubMed Central

Mathew, J.; Cattan, A. R.; Hall, A. G.; Hines, J. E.; Nelson, R.; Eastham, E.; Burt, A. D.

1992-01-01

AIMS: To investigate the distribution of alpha and pi class glutathione S-transferases (GST) in normal fetal, neonatal, and adult liver; and to examine changes in GST expression in neonatal liver disease. METHODS: alpha and pi class GST were immunolocalised in sections of formalin fixed liver tissue obtained from human fetuses (n = 21), neonates (n = 8), young children (n = 9) and adults (n = 10), and from neonates with extrahepatic biliary atresia (n = 15) and neonatal hepatitis (n = 12). Monospecific rabbit polyclonal antibodies were used with a peroxidase-antiperoxidase method. RESULTS: Expression of pi GST was localised predominantly within biliary epithelial cells of developing and mature bile ducts of all sizes from 16 weeks' gestation until term and in neonatal and adult liver. Coexpression of pi and alpha GST was seen in hepatocytes of developing fetal liver between 16 and 34 weeks' gestation. Although pi GST was seen in occasional hepatocytes up to six months of life, this isoenzyme was not expressed by hepatocytes in adult liver. By contrast, alpha GST continued to be expressed by hepatocytes in adult liver; this isoenzyme was also seen in some epithelial cells of large bile ducts in adult liver. No change was observed in the distribution of alpha GST in either neonatal hepatitis or extrahepatic biliary atresia. However, aberrant expression of pi GST was identified in hepatocytes of all but one case of extrahepatic biliary atresia but in only two cases of neonatal hepatitis. CONCLUSIONS: The phenotypic alterations noted in extrahepatic biliary atresia may result from the effect of cholate stasis. Evaluation of the pattern of pi and alpha GST distribution by immunohistochemical staining may provide valuable information in distinguishing between these two forms of neonatal liver disease. Images PMID:1401176

Cloning and expression of clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cary, J.W.; Petersen, D.J.; Bennett, G.N.

1990-06-01

Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase (butyrate-acetoacetate CoA-transferase) (EC 2.8.3.9)) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The genes encoding the two subunits of this enzyme have been cloned and subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defectmore » in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of M{sub r} of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E.coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli. In the plasmid, however, transcription appears to be primarily from the lac promoter of the vector.« less

O-Linked β-N-Acetylglucosaminylation (O-GlcNAcylation) in Primary and Metastatic Colorectal Cancer Clones and Effect of N-Acetyl-β-d-glucosaminidase Silencing on Cell Phenotype and Transcriptome*

PubMed Central

Yehezkel, Galit; Cohen, Liz; Kliger, Adi; Manor, Esther; Khalaila, Isam

2012-01-01

O-Linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is a regulatory post-translational modification occurring on the serine or threonine residues of nucleocytoplasmic proteins. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), which are responsible for O-GlcNAc addition and removal, respectively. Although O-GlcNAcylation was found to play a significant role in several pathologies such as type II diabetes and neurodegenerative diseases, the role of O-GlcNAcylation in the etiology and progression of cancer remains vague. Here, we followed O-GlcNAcylation and its catalytic machinery in metastatic clones of human colorectal cancer and the effect of OGA knockdown on cellular phenotype and on the transcriptome. The colorectal cancer SW620 metastatic clone exhibited increased O-GlcNAcylation and decreased OGA expression compared with its primary clone, SW480. O-GlcNAcylation elevation in SW620 cells, through RNA interference of OGA, resulted in phenotypic alterations that included acquisition of a fibroblast-like morphology, which coincides with epithelial metastatic progression and growth retardation. Microarray analysis revealed that OGA silencing altered the expression of about 1300 genes, mostly involved in cell movement and growth, and specifically affected metabolic pathways of lipids and carbohydrates. These findings support the involvement of O-GlcNAcylation in various aspects of tumor cell physiology and suggest that this modification may serve as a link between metabolic changes and cancer. PMID:22730328

Engineering N-Glycosylation Pathway in Insect Cells: Suppression of β-N-Acetylglucosaminidase and Expression of β-1,4-Galactosyltransferase.

PubMed

Kim, Yeon Kyu; Cha, Hyung Joon

2015-01-01

Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. It has been proposed that β-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation profile in several insect cells including Drosophila S2. Here, we describe GlcNAcase suppression strategy using RNA interference (RNAi) to avoid the formation of paucimannosidic glycans in insect S2 cells. In addition, we describe coexpression of β(1,4)-galactosyltransferase (GalT) as a strategy to improve N-glycosylation pattern and enable recombinant therapeutic proteins to be produced in S2 cells with more complex N-glycans.

Reaction of rat liver glutathione S-transferases and bacterial dichloromethane dehalogenase with dihalomethanes.

PubMed

Blocki, F A; Logan, M S; Baoli, C; Wackett, L P

1994-03-25

Dichloromethane dehalogenase from Methylophilus sp. DM11 is a glutathione S-transferase homolog that is specifically active with dihalomethane substrates. This bacterial enzyme and rat liver glutathione S-transferases were purified to investigate their relative reactivity with CH2Cl2 and related substrates. Rat liver alpha class glutathione transferases were inactive and mu class enzymes showed low activity (7-23 nmol/min/mg of protein) with CH2Cl2. theta class glutathione transferase 5-5 from rat liver and Methylophilus sp. dichloromethane dehalogenase showed specific activities of > or = 1 mumol/min/mg of protein. Apparent Kcat/Km were determined to be 3.3 x 10(4) and 6.0 x 10(4) L M-1 S-1 for the two enzymes, respectively. Dideutero-dichloromethane was processed to dideutereo-formaldehyde, consistent with a nucleophilic halide displacement mechanism. The possibility of a GSCH2X reaction intermediate (GS, glutathione; X, halide) was probed using CH2ClF to generate a more stable halomethylglutathione species (GSCH2F). The reaction of CH2ClF with dichloromethane dehalogenase produced a kinetically identifiable intermediate that decomposed to formaldehyde at a similar rate to synthetic HOCH2CH2SCH2F. 19F-NMR revealed the transient formation of an intermediate identified as GSCH2F by its chemical shift, its triplet resonance, and H-F coupling constant consistent with a fluoromethylthioether. Its decomposition was matched by a stoichiometric formation of fluoride. These studies indicated that the bacterial dichloromethane dehalogenase directs a nucleophilic attack of glutathione on CH2Cl2 to produce a halomethylthioether intermediate. This focuses attention on the mechanism used by theta class glutathione transferases to generate a halomethylthioeter from relatively unreactive dihalomethanes.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

PubMed Central

Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

2014-01-01

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

Glutathione S - transferases class Pi and Mi and their significance in oncology.

PubMed

Marchewka, Zofia; Piwowar, Agnieszka; Ruzik, Sylwia; Długosz, Anna

2017-06-19

In this article the current data, which shows that glutathione S-transferases (GST) class Pi and Mi are interesting and promising biomarkers in acute and chronic inflammatory processes as well as in the oncology, were presented based on the review of the latest experimental and clinical studies. The article shows their characteristics, functions and participation (direct - GST Pi, indirect - GST Mi) in the regulation of signaling pathways of JNK kinases, which are involved in cell differentiation. Overexpression of glutathione S-transferases class Pi and Mi in many cancer cells plays a key role in cancer treatment, making them resistant to chemotherapy. GST isoenzymes are involved in the metabolism of various types of xenobiotics and endogenous substrates, so their altered expression in cancer tissues as well as in serum and urine could be an important potential marker of the cancer and an indicator of oxidative stress. The study shows the role of glutathione S-transferases in redox homeostasis of tumor cells and in the mechanism of resistance to anticancer drugs.

Novel derivatives of aclacinomycin A block cancer cell migration through inhibition of farnesyl transferase.

PubMed

Magi, Shigeyuki; Shitara, Tetsuo; Takemoto, Yasushi; Sawada, Masato; Kitagawa, Mitsuhiro; Tashiro, Etsu; Takahashi, Yoshikazu; Imoto, Masaya

2013-03-01

In the course of screening for an inhibitor of farnesyl transferase (FTase), we identified two compounds, N-benzyl-aclacinomycin A (ACM) and N-allyl-ACM, which are new derivatives of ACM. N-benzyl-ACM and N-allyl-ACM inhibited FTase activity with IC50 values of 0.86 and 2.93 μM, respectively. Not only ACM but also C-10 epimers of each ACM derivative failed to inhibit FTase. The inhibition of FTase by N-benzyl-ACM and N-allyl-ACM seems to be specific, because these two compounds did not inhibit geranylgeranyltransferase or geranylgeranyl pyrophosphate (GGPP) synthase up to 100 μM. In cultured A431 cells, N-benzyl-ACM and N-allyl-ACM also blocked both the membrane localization of H-Ras and activation of the H-Ras-dependent PI3K/Akt pathway. In addition, they inhibited epidermal growth factor (EGF)-induced migration of A431 cells. Thus, N-benzyl-ACM and N-allyl-ACM inhibited EGF-induced migration of A431 cells by inhibiting the farnesylation of H-Ras and subsequent H-Ras-dependent activation of the PI3K/Akt pathway.

21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... of the enzyme galactose-1-phosphate uridyl transferase in erythrocytes (red blood cells... hereditary disease galactosemia (disorder of galactose metabolism) in infants. (b) Classification. Class II. ...

21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a) Identification...

21 CFR 862.1315 - Galactose-1-phosphate uridyl transferase test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry Test Systems § 862.1315 Galactose-1-phosphate uridyl transferase test system. (a) Identification...

21 CFR 862.1535 - Ornithine carbamyl transferase test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Ornithine carbamyl transferase test system. 862.1535 Section 862.1535 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

Rod outer segment-associated N-acetylgalactosaminylphosphotransferase.

PubMed

Sweatt, A J; Balsamo, J; Lilien, J

1995-01-01

To determine the exact location of a cell surface glycosyltransferase (N-acetylgalactosaminylphosphotransferase, (GalNAcPTase) immunochemically identified in mammalian rod outer segments (ROS), to determine whether anti-GalNAcPTase antibody recognizes retinal molecules that possess transferase activity and to characterize ROS transferase enzyme activity and acceptors. The GalNAcPTase is known to be associated with the adhesion molecule N-cadherin in embryonic avian retinas and with E-cadherin in mammalian pancreatic islet cells. Purified, fixed ROS were reacted with anti-chick GalNAcPTase antibody followed by secondary antibody conjugated to colloidal gold and were examined by electron microscopy. Fractions of retinal and ROS proteins enriched in the transferase were obtained through batch adsorption on Sepharose, separated by gel electrophoresis, transferred to nitrocellulose, and either reacted with anti-GalNAcPTase antibody or assayed for transferase activity. Interphotoreceptor matrix (IPM) was examined for the presence of immunoreactive GalNAcPTase by gel electrophoresis and immunoblot. The kinetics and endogenous acceptors of the cow ROS transferase were characterized. ROS are specifically labeled by anti-GalNAcPTase antibody at the cell surface. The immunogold label was associated with the cell surface and with flocculent material adherent to the cell surface. In addition, soluble and particulate fractions of the IPM showed GalNAcPTase-like immunoreactivity. The transferase appears as single immunoreactive band at or near 220 kd. Transferase enzyme activity was present at this position on Western transfers of retinal and ROS proteins. In whole ROS, transferase activity was directed toward endogenous acceptors of very high molecular mass. The GalNAcPTase is localized on ROS in association with the cell surface and with components of the IPM. The molecule recognized by the anti-GalNAcPTase antibody possesses transferase activity toward itself and a few other

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage.

PubMed

Goth, Christoffer K; Tuhkanen, Hanna E; Khan, Hamayun; Lackman, Jarkko J; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H; Overall, Christopher M; Clausen, Henrik; Schjoldager, Katrine T; Petäjä-Repo, Ulla E

2017-03-17

The β 1 -adrenergic receptor (β 1 AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β 1 AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O -glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O -glycosylates β 1 AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O -glycosylation and proteolytic cleavage assays, a cell line deficient in O -glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β 1 AR. Furthermore, we demonstrate that impaired O -glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O -glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β 1 AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage*

PubMed Central

Goth, Christoffer K.; Tuhkanen, Hanna E.; Khan, Hamayun; Lackman, Jarkko J.; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H.; Overall, Christopher M.; Clausen, Henrik; Schjoldager, Katrine T.; Petäjä-Repo, Ulla E.

2017-01-01

The β1-adrenergic receptor (β1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates β1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. PMID:28167537

Removal of O-GlcNAcylation is important for pig preimplantation development

PubMed Central

SHIBUTANI, Mihiro; MORI, Takeshi; MIYANO, Takashi; MIYAKE, Masashi

2015-01-01

Glucose has been recognized as an energy source for a long time, but it has recently been suggested that the hexosamine biosynthesis pathway (HBP) and downstream protein O-GlcNAcylation have important functions in mouse preimplantation development. Thus, whether or not O-GlcNAcylation was present and what functions O-GlcNAcylation has in pig preimplantation development were investigated in the present study. The expressions of mRNA of glutaminefructose-6-phosphate aminotransferase (Gfpt), O-GlcNAc transferase (Ogt) and O-GlcNAcase (Oga), which are involved in the HBP and O-GlcNAc cycling, were examined in pig parthenogenetic diploids at each preimplantation developmental stage. Gfpt and Ogt were detected in diploids at all stages. Though Oga was detected at all stages except the 4-cell stage, OGA proteins were detected in diploids from the 2-cell to blastocyst stage. Furthermore, O-GlcNAcylated proteins in MII oocytes and diploids were also detected by immunofluorescence at every stage. Inhibition of OGT by 4.0 mM BADGP did not affect development up to the blastocyst stage, while inhibition of OGA by 300 µM PUGNAc decreased the proportion of diploids beyond the 4-cell stage. Four-cell diploids cultured with PUGNAc until 48 h developed to the blastocyst stage after culture in a PUGNAc-free medium until 144 h after electrostimulation. RNA polymerase II (Pol II) phosphorylation, which indicates the onset of mRNA transcription, was detected in nuclei of diploids in the control group at 48 h but not in the PUGNAc-treated group. These results indicate that HBP and O-GlcNAcylation have important functions in pig preimplantation development and that inhibition of OGA is fatal for development. It is also suggested that OGA inhibition disrupts normal Pol II regulation and may cause a zygotic gene activation error. PMID:26004176

Designing Ligands for Leishmania, Plasmodium, and Aspergillus N-Myristoyl Transferase with Specificity and Anti-Target-Safe Virtual Libraries.

PubMed

Garcia-Sosa, Alfonso T

2018-01-01

Leishmaniasis, malaria, and fungal diseases are burdens on individuals and populations and can present severe complications. Easily accessible chemical treatments for these diseases are increasingly sought-after. Targeting the parasite N-myristoyl transferase while avoiding the human enzyme and other anti-targets may allow the prospect of compounds with pan-activity against these diseases, which would simplify treatments and costs. Developing chemical libraries, both virtual and physical, that have been filtered and flagged early on in the drug discovery process (before virtual screening) could reduce attrition rates of compounds being developed and failing late in development stages due to problems of side-effects or toxicity. Chemical libraries have been screened against the anti-targets pregnane-X-receptor, sulfotransferase, cytochrome P450 2a6, 2c9, and 3a4 with three different docking programs. Statistically significant differences are observed in their interactions with these enzymes as compared to small molecule drugs and bioactive non-drug datasets. A series of compounds are proposed with the best predicted profiles for inhibition of all parasite targets while sparing the human form and anti-targets. Some of the topranked compounds have confirmed experimental activity against Leishmania, and highlighted are those compounds with best properties for further development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Homogentisate solanesyl transferase (HST) cDNA’s in maize

USDA-ARS?s Scientific Manuscript database

Maize white seedling 3 (w3) has served as a model albino-seedling mutant since its discovery in 1923. We show that the w3 phenotype is caused by disruptions in homogentisate solanesyl transferase (HST), an enzyme that catalyzes the committed step in plastoquinone-9 (PQ9) biosynthesis. This reaction ...

Reversal of hypermethylation and reactivation of glutathione S-transferase pi 1 gene by curcumin in breast cancer cell line.

PubMed

Kumar, Umesh; Sharma, Ujjawal; Rathi, Garima

2017-02-01

One of the mechanisms for epigenetic silencing of tumor suppressor genes is hypermethylation of cytosine residue at CpG islands at their promoter region that contributes to malignant progression of tumor. Therefore, activation of tumor suppressor genes that have been silenced by promoter methylation is considered to be very attractive molecular target for cancer therapy. Epigenetic silencing of glutathione S-transferase pi 1, a tumor suppressor gene, is involved in various types of cancers including breast cancer. Epigenetic silencing of tumor suppressor genes can be reversed by several molecules including natural compounds such as polyphenols that can act as a hypomethylating agent. Curcumin has been found to specifically target various tumor suppressor genes and alter their expression. To check the effect of curcumin on the methylation pattern of glutathione S-transferase pi 1 gene in MCF-7 breast cancer cell line in dose-dependent manner. To check the reversal of methylation pattern of hypermethylated glutathione S-transferase pi 1, MCF-7 breast cancer cell line was treated with different concentrations of curcumin for different time periods. DNA and proteins of treated and untreated cell lines were isolated, and methylation status of the promoter region of glutathione S-transferase pi 1 was analyzed using methylation-specific polymerase chain reaction assay, and expression of this gene was analyzed by immunoblotting using specific antibodies against glutathione S-transferase pi 1. A very low and a nontoxic concentration (10 µM) of curcumin treatment was able to reverse the hypermethylation and led to reactivation of glutathione S-transferase pi 1 protein expression in MCF-7 cells after 72 h of treatment, although the IC 50 value of curcumin was found to be at 20 µM. However, curcumin less than 3 µM of curcumin could not alter the promoter methylation pattern of glutathione S-transferase pi 1. Treatment of breast cancer MCF-7 cells with curcumin

Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin.

PubMed

Nakamura, Toshio; Tonozuka, Takashi; Ide, Azusa; Yuzawa, Takayuki; Oguma, Keiji; Nishikawa, Atsushi

2008-02-22

Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal beta-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

PubMed

Endo, T; Ohbayashi, H; Kanazawa, K; Kochibe, N; Kobata, A

1992-01-15

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.

Production of N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) fused with secretory signal Igκ in insect cells.

PubMed

Horynová, Milada; Takahashi, Kazuo; Hall, Stacy; Renfrow, Matthew B; Novak, Jan; Raška, Milan

2012-02-01

The human UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyl-transferase 2 (GalNAc-T2) is one of the key enzymes that initiate synthesis of hinge-region O-linked glycans of human immunoglobulin A1 (IgA1). We designed secreted soluble form of human GalNAc-T2 as a fusion protein containing mouse immunoglobulin light chain kappa secretory signal and expressed it using baculovirus and mammalian expression vectors. The recombinant protein was secreted by insect cells Sf9 and human HEK 293T cells in the culture medium. The protein was purified from the media using affinity Ni-NTA chromatography followed by stabilization of purified protein in 50mM Tris-HCl buffer at pH 7.4. Although the purity of recombinant GalNAc-T2 was comparable in both expression systems, the yield was higher in Sf9 insect expression system (2.5mg of GalNAc-T2 protein per 1L culture medium). The purified soluble recombinant GalNAc-T2 had an estimated molecular mass of 65.8kDa and its amino-acid sequence was confirmed by mass-spectrometric analysis. The enzymatic activity of Sf9-produced recombinant GalNAc-T2 was determined by the quantification of enzyme-mediated attachment of GalNAc to synthetic IgA1 hinge-region peptide as the acceptor and UDP-GalNAc as the donor. In conclusion, murine immunoglobulin kappa secretory signal was used for production of secreted enzymatically active GalNAc-T2 in insect baculovirus expression system. Copyright © 2011 Elsevier Inc. All rights reserved.

21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

Code of Federal Regulations, 2012 CFR

2012-04-01

... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

Code of Federal Regulations, 2013 CFR

2013-04-01

... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

Code of Federal Regulations, 2011 CFR

2011-04-01

... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

21 CFR 573.130 - Aminoglycoside 3′-phospho- transferase II.

Code of Federal Regulations, 2014 CFR

2014-04-01

... SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.130 Aminoglycoside 3′-phospho- transferase II. The food additive aminoglycoside 3′-phosphotransferase II may be safely used in the development of...

Succinyl-CoA:(R)-Benzylsuccinate CoA-Transferase: an Enzyme of the Anaerobic Toluene Catabolic Pathway in Denitrifying Bacteria†

PubMed Central

Leutwein, Christina; Heider, Johann

2001-01-01

Anaerobic microbial toluene catabolism is initiated by addition of fumarate to the methyl group of toluene, yielding (R)-benzylsuccinate as first intermediate, which is further metabolized via β-oxidation to benzoyl-coenzyme A (CoA) and succinyl-CoA. A specific succinyl-CoA:(R)-benzylsuccinate CoA-transferase activating (R)-benzylsuccinate to the CoA-thioester was purified and characterized from Thauera aromatica. The enzyme is fully reversible and forms exclusively the 2-(R)-benzylsuccinyl-CoA isomer. Only some close chemical analogs of the substrates are accepted by the enzyme: succinate was partially replaced by maleate or methylsuccinate, and (R)-benzylsuccinate was replaced by methylsuccinate, benzylmalonate, or phenylsuccinate. In contrast to all other known CoA-transferases, the enzyme consists of two subunits of similar amino acid sequences and similar sizes (44 and 45 kDa) in an α2β2 conformation. Identity of the subunits with the products of the previously identified toluene-induced bbsEF genes was confirmed by determination of the exact masses via electrospray-mass spectrometry. The deduced amino acid sequences resemble those of only two other characterized CoA-transferases, oxalyl-CoA:formate CoA-transferase and (E)-cinnamoyl-CoA:(R)-phenyllactate CoA-transferase, which represent a new family of CoA-transferases. As suggested by kinetic analysis, the reaction mechanism of enzymes of this family apparently involves formation of a ternary complex between the enzyme and the two substrates. PMID:11418570

Recombinant human dihydroxyacetonephosphate acyl-transferase characterization as an integral monotopic membrane protein.

PubMed

Piano, Valentina; Nenci, Simone; Magnani, Francesca; Aliverti, Alessandro; Mattevi, Andrea

2016-12-02

Although the precise functions of ether phospholipids are still poorly understood, significant alterations in their physiological levels are associated either to inherited disorders or to aggressive metastatic cancer. The essential precursor, alkyl-dihydroxyacetone phosphate (DHAP), for all ether phospholipids species is synthetized in two consecutive reactions performed by two enzymes sitting on the inner side of the peroxisomal membrane. Here, we report the characterization of the recombinant human DHAP acyl-transferase, which performs the first step in alkyl-DHAP synthesis. By exploring several expression systems and designing a number of constructs, we were able to purify the enzyme in its active form and we found that it is tightly bound to the membrane through the N-terminal residues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Association of N-acetyltransferase-2 and glutathione S-transferase polymorphisms with idiopathic male infertility in Vietnam male subjects.

PubMed

Trang, Nguyen Thi; Huyen, Vu Thi; Tuan, Nguyen Thanh; Phan, Tran Duc

2018-04-25

N-acetyltransferase-2 (NAT2) and Glutathione S-transferases (GSTs) are phase-II xenobiotic metabolizing enzymes participating in detoxification of toxic arylamines, aromatic amines, hydrazines and reactive oxygen species (ROS), which are produced under oxidative and electrophile stresses. The purpose of this research was to investigate whether two common single-nucleotide polymorphisms (SNP) of NAT2 (rs1799929, rs1799930) and GSTP1 (rs1138272, rs1695) associated with susceptibility to idiopathic male infertility. A total 300 DNA samples (150 infertile patients and 150 healthy control) were genotyped for the polymorphisms by ARMS - PCR. We revealed a significant association between the NAT2 variant genotypes (CT + TT (rs1799929), (OR: 3.74; p < 0.001)) and (GA + AA (rs1799930), (OR: 3.75; p < 0.001)) or GSTP1 variant genotypes (GA + AA (rs1695), (OR: 5.11; p < 0,001)) and (CT + TT (rs1138272), (OR: 7.42; p < 0,001) with idiopathic infertility risk. Our findings rate the effect of single-nucleotide polymorphisms of GSTP1 and/or NAT2 in modulation of the risk of male infertility in subjects from Vietnam. This pilot study is the first (as far as we know) to reveal that polymorphisms of NAT2 (rs1799929, rs1799930) and GSTP1 (rs1138272, rs1695) are some novel genetic markers for susceptibility to idiopathic male infertility. Copyright © 2018 Elsevier B.V. All rights reserved.

Suppression of proliferation and neurite extension of human neuroblastoma SH-SY5Y cells on immobilized Psathyrella velutina lectin.

PubMed

Kitamura, Noriaki; Ikekita, Masahiko; Hayakawa, Satoru; Funahashi, Hisayuki; Furukawa, Kiyoshi

2004-02-01

Glycoproteins from mammalian brain tissues contain unique N-linked oligosaccharides terminating with beta-N-acetylglucosamine residues. Lectin blot analysis of membrane glycoprotein samples from human neuroblastoma SH-SY5Y cells showed that several protein bands bind to Psathylera velutina lectin (PVL), which interacts with beta-N-acetylglucosamine-terminating oligosaccharides. No lectin positive bands were detected by digestion with jack bean beta-N-acetyl-hexosaminidase or N-glycanase before incubation with the lectin, indicating that the cells contain beta-N-acetylglucosamine-terminating N-linked oligosaccharides. When cells were cultured in dishes with different concentrations of PVL, the cell proliferation was inhibited in a dose-dependent manner. Similarly, the neurite extension, which was stimulated with nerve growth factor, was also inhibited in a manner dependent on the lectin dose. Cell proliferation and neurite extension were recovered by the addition of 10 mM N-acetylglucosamine into the medium. Immunoblot analysis of the activation of mitogen-activated protein (MAP) kinases and protein kinase C revealed that phosphorylation of 42-kDa and 44-kDa MAP kinases and 80-kDa protein kinase C are inhibited when SH-SY5Y cells are cultured in PVL-coated dishes, but are restored by the addition of the haptenic sugar into the medium, indicating that MAP kinase and protein kinase C pathways are inhibited by interaction with immobilized PVL. These results indicate that beta-N-acetylglucosamine-terminating N-linked oligosaccharides expressed on neural cells can induce intracellular signals upon binding to extracellular receptors, and are important for growth regulation of neural cells. Copyright 2003 Wiley-Liss, Inc.

Transmutation of human glutathione transferase A2-2 with peroxidase activity into an efficient steroid isomerase.

PubMed

Pettersson, Par L; Johansson, Ann-Sofie; Mannervik, Bengt

2002-08-16

A major goal in protein engineering is the tailor-making of enzymes for specified chemical reactions. Successful attempts have frequently been based on directed molecular evolution involving libraries of random mutants in which variants with desired properties were identified. For the engineering of enzymes with novel functions, it would be of great value if the necessary changes of the active site could be predicted and implemented. Such attempts based on the comparison of similar structures with different substrate selectivities have previously met with limited success. However, the present work shows that the knowledge-based redesign restricted to substrate-binding residues in human glutathione transferase A2-2 can introduce high steroid double-bond isomerase activity into the enzyme originally characterized by glutathione peroxidase activity. Both the catalytic center activity (k(cat)) and catalytic efficiency (k(cat)/K(m)) match the values of the naturally evolved glutathione transferase A3-3, the most active steroid isomerase known in human tissues. The substrate selectivity of the mutated glutathione transferase was changed 7000-fold by five point mutations. This example demonstrates the functional plasticity of the glutathione transferase scaffold as well as the potential of rational active-site directed mutagenesis as a complement to DNA shuffling and other stochastic methods for the redesign of proteins with novel functions.

Purification and Biochemical Characterization of Glutathione S-Transferase from Down Syndrome and Normal Children Erythrocytes: A Comparative Study

ERIC Educational Resources Information Center

Hamed, Ragaa R.; Maharem, Tahany M.; Abdel-Meguid, Nagwa; Sabry, Gilane M.; Abdalla, Abdel-Monem; Guneidy, Rasha A.

2011-01-01

Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was…

Association of presenile cataract with galactose-1-phosphate uridyl transferase gene mutations.

PubMed

Nema, Nitin; Kumar, Ravindra; Verma, Abha; Verma, Sonam; Chaturvedi, Kiran

2017-01-01

Presenile cataract is commonly idiopathic in origin. However, patients with presenile cataract could have an underlying genetic abnormality of galactose metabolism. We studied the association, if any, between idiopathic presenile cataract and galactose-1 -phosphate uridyl transferase (GALT) gene mutation. We selected 50 patients with idiopathic presenile cataract, <45 years of age, and 50 age- and sex-matched controls for the study. Mutations in the GALT gene were determined by polymerase chain reaction restriction fragment length polymorphism. The classical galactosaemia was characterized by Q188R and K285N mutations, whereas Duarte galactosaemia by N314D mutations (Duarte-2: N314D with IVS5-24G >A and Duarte-1: N314D without IVS5- 24G>A). The most common mutation observed was the N314D (Duarte) mutation. The frequencies of classical and N31 4D alleles in patients with presenile cataract (16%) and controls (26%) were not statistically different (p=0.32, OR 0.54, 95% CI 0.20-1.45). Similarly, there was no statistically significant difference in the frequency distribution of Duarte-1 (p=0.77, OR 0.77, 95% CI 0.23-0.24) and Duarte-2 (p=0.44, OR 0.38, 95% CI 0.07-2.03) galactosaemia mutations in patients and controls. Duarte galactosaemia, a milder form of the disease, is more common than classical galactosaemia in the Indian population. Duarte galactosaemia is unlikely to be a causative factor in presenile cataract.

GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system

PubMed Central

Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D.; Baba, Hiroko; Ikenaka, Kazuhiro

2017-01-01

Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy. PMID:28186137

GlcNAc6ST-1 regulates sulfation of N-glycans and myelination in the peripheral nervous system.

PubMed

Yoshimura, Takeshi; Hayashi, Akiko; Handa-Narumi, Mai; Yagi, Hirokazu; Ohno, Nobuhiko; Koike, Takako; Yamaguchi, Yoshihide; Uchimura, Kenji; Kadomatsu, Kenji; Sedzik, Jan; Kitamura, Kunio; Kato, Koichi; Trapp, Bruce D; Baba, Hiroko; Ikenaka, Kazuhiro

2017-02-10

Highly specialized glial cells wrap axons with a multilayered myelin membrane in vertebrates. Myelin serves essential roles in the functioning of the nervous system. Axonal degeneration is the major cause of permanent neurological disability in primary myelin diseases. Many glycoproteins have been identified in myelin, and a lack of one myelin glycoprotein results in abnormal myelin structures in many cases. However, the roles of glycans on myelin glycoproteins remain poorly understood. Here, we report that sulfated N-glycans are involved in peripheral nervous system (PNS) myelination. PNS myelin glycoproteins contain highly abundant sulfated N-glycans. Major sulfated N-glycans were identified in both porcine and mouse PNS myelin, demonstrating that the 6-O-sulfation of N-acetylglucosamine (GlcNAc-6-O-sulfation) is highly conserved in PNS myelin between these species. P 0 protein, the most abundant glycoprotein in PNS myelin and mutations in which at the glycosylation site cause Charcot-Marie-Tooth neuropathy, has abundant GlcNAc-6-O-sulfated N-glycans. Mice deficient in N-acetylglucosamine-6-O-sulfotransferase-1 (GlcNAc6ST-1) failed to synthesize sulfated N-glycans and exhibited abnormal myelination and axonal degeneration in the PNS. Taken together, this study demonstrates that GlcNAc6ST-1 modulates PNS myelination and myelinated axonal survival through the GlcNAc-6-O-sulfation of N-glycans on glycoproteins. These findings may provide novel insights into the pathogenesis of peripheral neuropathy.

The identification and characterisation of a functional interaction between arginyl-tRNA-protein transferase and topoisomerase II.

PubMed

Barker, Catherine R; Mouchel, Nathalie A P; Jenkins, John R

2006-04-07

Topoisomerase II is required for the viability of all eukaryotic cells. It plays important roles in DNA replication, recombination, chromosome segregation, and the maintenance of the nuclear scaffold. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between arginyl-tRNA-protein transferase (Ate1) and the N-terminal domain of the S. cerevisiae topoisomerase II, including the potential site of interaction. Ate1 is a component of the N-end rule protein degradation pathway which targets proteins for degradation. We also propose a previously unidentified role for Ate1 in modulating the level of topoisomerase II through the cell cycle.

Glutathione S-transferase pi mediates MPTP-induced c-Jun N-terminal kinase activation in the nigrostriatal pathway.

PubMed

Castro-Caldas, Margarida; Carvalho, Andreia Neves; Rodrigues, Elsa; Henderson, Colin; Wolf, C Roland; Gama, Maria João

2012-06-01

Parkinson's disease (PD) is a progressive movement disorder resulting from the death of dopaminergic neurons in the substantia nigra. Neurotoxin-based models of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) recapitulate the neurological features of the disease, triggering a cascade of deleterious events through the activation of the c-Jun N-terminal kinase (JNK). The molecular mechanisms underlying the regulation of JNK activity under cellular stress conditions involve the activation of several upstream kinases along with the fine-tuning of different endogenous JNK repressors. Glutathione S-transferase pi (GSTP), a phase II detoxifying enzyme, has been shown to inhibit JNK-activated signaling by protein-protein interactions, preventing c-Jun phosphorylation and the subsequent trigger of the cell death cascade. Here, we use C57BL/6 wild-type and GSTP knockout mice treated with MPTP to evaluate the regulation of JNK signaling by GSTP in both the substantia nigra and the striatum. The results presented herein show that GSTP knockout mice are more susceptible to the neurotoxic effects of MPTP than their wild-type counterparts. Indeed, the administration of MPTP induces a progressive demise of nigral dopaminergic neurons together with the degeneration of striatal fibers at an earlier time-point in the GSTP knockout mice when compared to the wild-type mice. Also, MPTP treatment leads to increased p-JNK levels and JNK catalytic activity in both wild-type and GSTP knockout mice midbrain and striatum. Moreover, our results demonstrate that in vivo GSTP acts as an endogenous regulator of the MPTP-induced cellular stress response by controlling JNK activity through protein-protein interactions.

Regulation of Endothelial Permeability by Glutathione S-Transferase Pi Against Actin Polymerization.

PubMed

Yang, Yang; Yin, Fangyuan; Hang, Qiyun; Dong, Xiaoliang; Chen, Jiao; Li, Ling; Cao, Peng; Yin, Zhimin; Luo, Lan

2018-01-01

Inflammation-induced injury of the endothelial barrier occurs in several pathological conditions, including atherosclerosis, ischemia, and sepsis. Endothelial cytoskeleton rearrangement is an important pathological mechanism by which inflammatory stimulation triggers an increase of vascular endothelial permeability. However, the mechanism maintaining endothelial cell barrier function against inflammatory stress is not fully understood. Glutathione S-transferase pi (GSTpi) exists in various types of cells and protects them against different stresses. In our previous study, GSTpi was found to act as a negative regulator of inflammatory responses. We used a Transwell permeability assay to test the influence of GSTpi and its transferase activity on the increase of endothelial permeability induced by tumor necrosis factor alpha (TNF-α). TNF-α-induced actin remodeling and the influence of GSTpi were observed by using laser confocal microscopy. Western blotting was used to test the influence of GSTpi on TNF-α-activated p38 mitogen-activated protein kinase (MAPK)/MK2/heat shock protein 27 (HSP27). GSTpi reduced TNF-α-induced stress fiber formation and endothelial permeability increase by restraining actin cytoskeleton rearrangement, and this reduction was unrelated to its transferase activity. We found that GSTpi inhibited p38MAPK phosphorylation by directly binding p38 and influenced downstream substrate HSP27-induced actin remodeling. GSTpi inhibited TNF-α-induced actin remodeling, stress fiber formation and endothelial permeability increase by inhibiting the p38MAPK/HSP27 signaling pathway. © 2018 The Author(s). Published by S. Karger AG, Basel.

21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1100 - Aspartate amino transferase (AST/SGOT) test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Aspartate amino transferase (AST/SGOT) test system. 862.1100 Section 862.1100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

21 CFR 862.1030 - Alanine amino transferase (ALT/SGPT) test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Alanine amino transferase (ALT/SGPT) test system. 862.1030 Section 862.1030 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CLINICAL CHEMISTRY AND CLINICAL TOXICOLOGY DEVICES Clinical Chemistry...

O-Linked β-N-Acetylglucosamine Modification of A20 Enhances the Inhibition of NF-κB (Nuclear Factor-κB) Activation and Elicits Vascular Protection After Acute Endoluminal Arterial Injury.

PubMed

Yao, Dan; Xu, Lijuan; Xu, Oufan; Li, Rujun; Chen, Mingxing; Shen, Hui; Zhu, Huajiang; Zhang, Fengyi; Yao, Deshang; Chen, Yiu-Fai; Oparil, Suzanne; Zhang, Zhengang; Gong, Kaizheng

2018-06-01

Recently, we have demonstrated that acute glucosamine-induced augmentation of protein O-linked β-N-acetylglucosamine (O-GlcNAc) levels inhibits inflammation in isolated vascular smooth muscle cells and neointimal formation in a rat model of carotid injury by interfering with NF-κB (nuclear factor-κB) signaling. However, the specific molecular target for O-GlcNAcylation that is responsible for glucosamine-induced vascular protection remains unclear. In this study, we test the hypothesis that increased A20 (also known as TNFAIP3 [tumor necrosis factor α-induced protein 3]) O-GlcNAcylation is required for glucosamine-mediated inhibition of inflammation and vascular protection. In cultured rat vascular smooth muscle cells, both glucosamine and the selective O-linked N-acetylglucosaminidase inhibitor thiamet G significantly increased A20 O-GlcNAcylation. Thiamet G treatment did not increase A20 protein expression but did significantly enhance binding to TAX1BP1 (Tax1-binding protein 1), a key regulatory protein for A20 activity. Adenovirus-mediated A20 overexpression further enhanced the effects of thiamet G on prevention of TNF-α (tumor necrosis factor-α)-induced IκB (inhibitor of κB) degradation, p65 phosphorylation, and increases in DNA-binding activity. A20 overexpression enhanced the inhibitory effects of thiamet G on TNF-α-induced proinflammatory cytokine expression and vascular smooth muscle cell migration and proliferation, whereas silencing endogenous A20 by transfection of specific A20 shRNA significantly attenuated these inhibitory effects. In balloon-injured rat carotid arteries, glucosamine treatment markedly inhibited neointimal formation and p65 activation compared with vehicle treatment. Adenoviral delivery of A20 shRNA to the injured arteries dramatically reduced balloon injury-induced A20 expression and inflammatory response compared with scramble shRNA and completely abolished the vascular protection of glucosamine. These results suggest that

Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vetting,M.; Frantom, P.; Blanchard, J.

2008-01-01

The glycosyltransferase termed MshA catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-l-myo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis. The structure of MshA from Corynebacterium glutamicum was determined both in the absence of substrates and in a complex with UDP and 1-l-myo-inositol-1-phosphate. MshA belongs to the GT-B structural family whose members have a two-domain structure with both domains exhibiting a Rossman-type fold. Binding of the donor sugar to the C-terminal domain produces a 97 rotational reorientation of the N-terminal domain relative to the C-terminal domain, clamping down on UDP and generating the binding site for 1-l-myo-inositol-1-phosphate. The structuremore » highlights the residues important in binding of UDP-N-acetylglucosamine and 1-l-myo-inositol-1-phosphate. Molecular models of the ternary complex suggest a mechanism in which the {beta}-phosphate of the substrate, UDP-N-acetylglucosamine, promotes the nucleophilic attack of the 3-hydroxyl group of 1-l-myo-inositol-1-phosphate while at the same time promoting the cleavage of the sugar nucleotide bond.« less

A 4'-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus.

PubMed

Neville, Claire; Murphy, Alan; Kavanagh, Kevin; Doyle, Sean

2005-04-01

Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

Structural snapshots along the reaction pathway of Yersinia pestis RipA, a putative butyryl-CoA transferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Torres, Rodrigo; Lan, Benson; Latif, Yama

2014-04-01

The crystal structures of Y. pestis RipA mutants were determined to provide insights into the CoA transferase reaction pathway. Yersinia pestis, the causative agent of bubonic plague, is able to survive in both extracellular and intracellular environments within the human host, although its intracellular survival within macrophages is poorly understood. A novel Y. pestis three-gene rip (required for intracellular proliferation) operon, and in particular ripA, has been shown to be essential for survival and replication in interferon γ-induced macrophages. RipA was previously characterized as a putative butyryl-CoA transferase proposed to yield butyrate, a known anti-inflammatory shown to lower macrophage-produced NOmore » levels. RipA belongs to the family I CoA transferases, which share structural homology, a conserved catalytic glutamate which forms a covalent CoA-thioester intermediate and a flexible loop adjacent to the active site known as the G(V/I)G loop. Here, functional and structural analyses of several RipA mutants are presented in an effort to dissect the CoA transferase mechanism of RipA. In particular, E61V, M31G and F60M RipA mutants show increased butyryl-CoA transferase activities when compared with wild-type RipA. Furthermore, the X-ray crystal structures of E61V, M31G and F60M RipA mutants, when compared with the wild-type RipA structure, reveal important conformational changes orchestrated by a conserved acyl-group binding-pocket phenylalanine, Phe85, and the G(V/I)G loop. Binary structures of M31G RipA and F60M RipA with two distinct CoA substrate conformations are also presented. Taken together, these data provide CoA transferase reaction snapshots of an open apo RipA, a closed glutamyl-anhydride intermediate and an open CoA-thioester intermediate. Furthermore, biochemical analyses support essential roles for both the catalytic glutamate and the flexible G(V/I)G loop along the reaction pathway, although further research is required to fully

The O-β-linked N-acetylglucosaminylation of the Lamin B receptor and its impact on DNA binding and phosphorylation.

PubMed

Smet-Nocca, Caroline; Page, Adeline; Cantrelle, François-Xavier; Nikolakaki, Eleni; Landrieu, Isabelle; Giannakouros, Thomas

2018-04-01

Lamin B Receptor (LBR) is an integral protein of the interphase inner nuclear membrane that is implicated in chromatin anchorage to the nuclear envelope. Phosphorylation of a stretch of arginine-serine (RS) dipeptides in the amino-terminal nucleoplasmic domain of LBR regulates the interactions of the receptor with other nuclear proteins, DNA and RNA and thus modulates tethering of heterochromatin to the nuclear envelope. While phosphorylation has been extensively studied, very little is known about other post-translational modifications of the protein. There is only one report on the O-β-linked N-acetyl-glucosaminylation (O-GlcNAcylation) of a serine residue downstream of the RS domain of rat LBR. In the present study we identify additional O-GlcNAcylation sites by using as substrates of O-β-N-acetylglucosaminyltransferase (OGT) a set of peptides containing the entire LBR RS domain or parts of it as well as flanking sequences. The in vitro activity of OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Furthermore, we provide evidence that O-GlcNAcylation hampers DNA binding while it marginally affects RS domain phosphorylation mediated by SRPK1, Akt2 and cdk1 kinases. Our methodology providing a quantitative description of O-GlcNAc patterns based on a combination of mass spectrometry and high resolution NMR spectroscopy on short peptide substrates allows subsequent functional analyses. Hence, our approach is of general interest to a wide audience of biologists aiming at deciphering the functional role of O-GlcNAc glycosylation and its crosstalk with phosphorylation. Copyright © 2018 Elsevier B.V. All rights reserved.

A new method for determining the mycelial weight of the koji-mold Aspergillus oryzae by measuring its glycosylceramide content.

PubMed

Ferdouse, Jannatul; Miyagawa, Miyuki; Hirano, Mikako; Kitajima, Yuka; Inaba, Shigeki; Kitagaki, Hiroshi

2018-06-21

At present, the quantitation of the mycelial weight of the industrially important non-pathogenic fungus Aspergillus oryzae, which is used for manufacturing koji, is performed by quantitating N-acetylglucosamine. However, since N-acetylglucosamine is a cell wall component, the extraction procedure is costly and tedious, and its quantitative performance is poor. Here, we report a novel method for the quantitation of A. oryzae mycelial weight. The amount of glycosylceramide significantly correlated with both the mycelial weight of A. oryzae and the amount of N-acetylglucosamine, an established index of the mycelial weight of A. oryzae in koji. This new method is simple and efficient and can be used in the brewing and food industries to determine the mycelial weight of A. oryzae.

Characterization of glutathione transferases involved in the pathogenicity of Alternaria brassicicola.

PubMed

Calmes, Benoit; Morel-Rouhier, Mélanie; Bataillé-Simoneau, Nelly; Gelhaye, Eric; Guillemette, Thomas; Simoneau, Philippe

2015-06-18

Glutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection. Mining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were 'orphans'. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues. Among the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs.

Global deletion of glutathione S-Transferase A4 exacerbates developmental nonalcoholic steatohepatitis

USDA-ARS?s Scientific Manuscript database

We established a mouse model of developmental nonalcoholic steatohepatitis (NASH) by feeding a high polyunsaturated fat liquid diet to female glutathione-S-transferase 4-4 (Gsta4-/-)/peroxisome proliferator activated receptor a (Ppara-/-) double knockout 129/SvJ mice for 12 weeks from weaning. We us...

Functional Characterization of Lpt3 and Lpt6, the Inner-Core Lipooligosaccharide Phosphoethanolamine Transferases from Neisseria meningitidis▿

PubMed Central

Wenzel, Cory Q.; St. Michael, Frank; Stupak, Jacek; Li, Jianjun; Cox, Andrew D.; Richards, James C.

2010-01-01

The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner membrane, based on sucrose density gradient centrifugation. Lpt3-His6 and Lpt6-His6 were purified from Triton X-100-solubilized membranes by nickel chelation chromatography, and dot blot analysis of enzymatic reactions with 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully acylated LOS and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-mass spectrometry (MS) demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6 and 3 positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro. PMID:19854897

Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines.

PubMed

Sawers, L; Ferguson, M J; Ihrig, B R; Young, H C; Chakravarty, P; Wolf, C R; Smith, G

2014-09-09

Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity. Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT-PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays. Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers. Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients.

Inherited glutathione-S-transferase deficiency is a risk factor for pulmonary asbestosis.

PubMed

Smith, C M; Kelsey, K T; Wiencke, J K; Leyden, K; Levin, S; Christiani, D C

1994-09-01

Pulmonary diseases attributable to asbestos exposure constitute a significant public health burden, yet few studies have investigated potential genetic determinants of susceptibility to asbestos-related diseases. The glutathione-S-transferases are a family of conjugating enzymes that both catalyze the detoxification of a variety of potentially cytotoxic electrophilic agents and act in the generation of sulfadipeptide leukotriene inflammatory mediators. The gene encoding glutathione-S-transferase class mu (GSTM-1) is polymorphic; approximately 50% of Caucasian individuals have a homozygous deletion of this gene and do not produce functional enzyme. Glutathione-S-transferase mu (GST-mu) deficiency has been previously reported to be associated with smoking-induced lung cancer. We conducted a cross-sectional study to examine the prevalence of the homozygous deletion for the GSTM-1 gene in members of the carpentry trade occupationally exposed to asbestos. Members of the United Brotherhood of Carpenters and Joiners of America attending their 1991 National Union conference were invited to participate. Each participant was offered a chest X-ray and was asked to complete a comprehensive questionnaire and have their blood drawn. All radiographs were assessed for the presence of pneumoconiosis in a blinded fashion by a National Institute for Occupational Safety and Health-certified International Labor Office "B" reader. Individual GSTM-1 status was determined using polymerase chain reaction methods. Six hundred fifty-eight workers were studied. Of these, 80 (12.2%) had X-ray abnormalities associated with asbestos exposure. Individuals genetically deficient in GST-mu were significantly more likely to have radiographic evidence of nonmalignant asbestos-related disease than those who were not deficient (chi 2 = 5.0; P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

GLUTATHIONE S-TRANSFERASE THETA 1-1-DEPENDENT METABOLISM OF THE DISINFECTION BYPRODUCT BROMODICHLOROMETHANE

EPA Science Inventory

ABSTRACT
Bromodichloromethane (BDCM), a prevalent drinking water disinfection by-product, was previously shown to be mutagenic in Salmonella expressing glutathione S-transferase (GST) theta 1-1 (GST T1-1). In the present study, in vitro experiments were performed to study the...

Substrate specificities and intracellular distributions of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway.

PubMed

Geisler, Christoph; Jarvis, Donald L

2012-03-02

Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2) is a key branch point intermediate in the insect N-glycosylation pathway because it can be either trimmed by a processing β-N-acetylglucosaminidase (FDL) to produce paucimannosidic N-glycans or elongated by N-acetylglucosaminyltransferase II (GNT-II) to produce complex N-glycans. N-acetylglucosaminyltransferase I (GNT-I) contributes to branch point intermediate production and can potentially reverse the FDL trimming reaction. However, there has been no concerted effort to evaluate the relationships among these three enzymes in any single insect system. Hence, we extended our previous studies on Spodoptera frugiperda (Sf) FDL to include GNT-I and -II. Sf-GNT-I and -II cDNAs were isolated, the predicted protein sequences were analyzed, and both gene products were expressed and their acceptor substrate specificities and intracellular localizations were determined. Sf-GNT-I transferred N-acetylglucosamine to Man(5)GlcNAc(2), Man(3)GlcNAc(2), and GlcNAc(β1-2)Man(α1-6)[Man(α1-3)]ManGlcNAc(2), demonstrating its role in branch point intermediate production and its ability to reverse FDL trimming. Sf-GNT-II only transferred N-acetylglucosamine to Man(α1-6)[GlcNAc(β1-2)Man(α1-3)]ManGlcNAc(2), demonstrating that it initiates complex N-glycan production, but cannot use Man(3)GlcNAc(2) to produce hybrid or complex structures. Fluorescently tagged Sf-GNT-I and -II co-localized with an endogenous Sf Golgi marker and Sf-FDL co-localized with Sf-GNT-I and -II, indicating that all three enzymes are Golgi resident proteins. Unexpectedly, fluorescently tagged Drosophila melanogaster FDL also co-localized with Sf-GNT-I and an endogenous Drosophila Golgi marker, indicating that it is a Golgi resident enzyme in insect cells. Thus, the substrate specificities and physical juxtapositioning of GNT-I, GNT-II, and FDL support the idea that these enzymes function at the N-glycan processing branch point and are major factors determining the

DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1

EPA Science Inventory

DNA BINDING POTENTIAL OF BROMODICHLOROMETHANE MEDIATED BY GLUTATHIONE S-TRANSFERASE THETA 1-1. R A Pegram1 and M K Ross2. 2Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC; 1Pharmacokinetics Branch, NHEERL, ORD, United States Environmental Protection Ag...

Glutathione S-transferase P1 (GSTP1) directly influences platinum drug chemosensitivity in ovarian tumour cell lines

PubMed Central

Sawers, L; Ferguson, M J; Ihrig, B R; Young, H C; Chakravarty, P; Wolf, C R; Smith, G

2014-01-01

Background: Chemotherapy response in ovarian cancer patients is frequently compromised by drug resistance, possibly due to altered drug metabolism. Platinum drugs are metabolised by glutathione S-transferase P1 (GSTP1), which is abundantly, but variably expressed in ovarian tumours. We have created novel ovarian tumour cell line models to investigate the extent to which differential GSTP1 expression influences chemosensitivity. Methods: Glutathione S-transferase P1 was stably deleted in A2780 and expression significantly reduced in cisplatin-resistant A2780DPP cells using Mission shRNA constructs, and MTT assays used to compare chemosensitivity to chemotherapy drugs used to treat ovarian cancer. Differentially expressed genes in GSTP1 knockdown cells were identified by Illumina HT-12 expression arrays and qRT–PCR analysis, and altered pathways predicted by MetaCore (GeneGo) analysis. Cell cycle changes were assessed by FACS analysis of PI-labelled cells and invasion and migration compared in quantitative Boyden chamber-based assays. Results: Glutathione S-transferase P1 knockdown selectively influenced cisplatin and carboplatin chemosensitivity (2.3- and 4.83-fold change in IC50, respectively). Cell cycle progression was unaffected, but cell invasion and migration was significantly reduced. We identified several novel GSTP1 target genes and candidate platinum chemotherapy response biomarkers. Conclusions: Glutathione S-transferase P1 has an important role in cisplatin and carboplatin metabolism in ovarian cancer cells. Inter-tumour differences in GSTP1 expression may therefore influence response to platinum-based chemotherapy in ovarian cancer patients. PMID:25010864

Oncogenic BRAFV600E drives expression of MGL ligands in the colorectal cancer cell line HT29 through N-acetylgalactosamine-transferase 3.

PubMed

Sahasrabudhe, Neha M; Lenos, Kristiaan; van der Horst, Joost C; Rodríguez, Ernesto; van Vliet, Sandra J

2018-06-27

Colorectal cancer is the third most common cancer type worldwide. It is characterized by a high expression of aberrantly glycosylated ligands, such as the Tn antigen (GalNAcα1-Ser/Thr), which is a major ligand for the C-type lectin macrophage galactose-type lectin (MGL). We have previously determined that a high level of MGL ligands in colorectal tumors is associated with lower disease-free survival in patients with late stage disease, which we could attribute to the presence of oncogenic BRAFV600E mutations. Here we aimed to elucidate the downstream pathway of BRAFV600E governing high MGL ligand and Tn antigen expression. We focused on glycosylation-related enzymes involved in the synthesis or elongation of Tn antigen, N-acetylgalactosamine-transferases (GALNTs) and C1GalT1/COSMC, respectively. Both the activity and expression of C1GalT1 and COSMC were unrelated to the BRAF mutational status. In contrast, GALNT3, GALNT7 and GALNT12 were increased in colorectal cancer cells harboring the BRAFV600E mutation. Through CRISPR-Cas9 gene knockouts we could establish that GALNT3 increased MGL ligand synthesis in the HT29 cell line, while GALNT7 and GALNT12 appeared to have redundant roles. Together our results highlight a novel mechanistic pathway connecting BRAFV600E to aberrant glycosylation in colorectal cancer through GALNT3.

Characterization of a Bvg-regulated fatty acid methyl-transferase in Bordetella pertussis.

PubMed

Rivera-Millot, Alex; Lesne, Elodie; Solans, Luis; Coutte, Loic; Bertrand-Michel, Justine; Froguel, Philippe; Dhennin, Véronique; Hot, David; Locht, Camille; Antoine, Rudy; Jacob-Dubuisson, Françoise

2017-01-01

The whooping cough agent Bordetella pertussis controls the expression of its large virulence regulon in a coordinated manner through the two-component signal transduction system BvgAS. In addition to the genes coding for bona fide virulence factors, the Bvg regulon comprises genes of unknown function. In this work, we characterized a new Bvg-activated gene called BP2936. Homologs of BP2936 are found in other pathogenic Bordetellae and in several other species, including plant pathogens and environmental bacteria. We showed that the gene product of BP2936 is a membrane-associated methyl-transferase of free fatty acids. We thus propose to name it FmtB, for fatty acid methyl-transferase of Bordetella. The role of this protein was tested in cellular and animal models of infection, but the loss of BP2936 did not appear to affect host-pathogen interactions in those assays. The high level of conservation of BP2936 among B. pertussis isolates nevertheless argues that it probably plays a role in the life cycle of this pathogen.

The role of endoxyloglucan transferase in the organization of plant cell walls.

PubMed

Nishitani, K

1997-01-01

The plant cell wall plays a central role in morphogenesis as well as responsiveness to environmental signals. Xyloglucans are the principal component of the plant cell wall matrix and serve as cross-links between cellulose microfibrils to form the cellulose-xyloglucan framework. Endoxyloglucan transferase (EXGT), which was isolated and characterized in 1992, is an enzyme that mediates molecular grafting reaction between xyloglucan molecules. Structural studies on cDNAs encoding EXGT and its related proteins have disclosed the ubiquitous presence in the plant kingdom of a large multigene family of xyloglucan-related proteins (XRPs). Each XRP functions as either hydrolase or transferase acting on xyloglucans and is considered to be responsible for rearrangement of the cellulose-xyloglucan framework, the processes essential for the construction, modification, and degradation of plant cell walls. Different XRP genes exhibit potentially different expression profiles with respect to tissue specificity and responsiveness to hormonal and mechanical signals. The molecular approach to individual XRP genes will open a new path for exploring the controlling mechanisms by which the plant cell wall is constructed and reformed during plant growth and development.

Characterization of a Bvg-regulated fatty acid methyl-transferase in Bordetella pertussis

PubMed Central

Rivera-Millot, Alex; Lesne, Elodie; Solans, Luis; Coutte, Loic; Bertrand-Michel, Justine; Froguel, Philippe; Dhennin, Véronique; Hot, David; Locht, Camille; Antoine, Rudy

2017-01-01

The whooping cough agent Bordetella pertussis controls the expression of its large virulence regulon in a coordinated manner through the two-component signal transduction system BvgAS. In addition to the genes coding for bona fide virulence factors, the Bvg regulon comprises genes of unknown function. In this work, we characterized a new Bvg-activated gene called BP2936. Homologs of BP2936 are found in other pathogenic Bordetellae and in several other species, including plant pathogens and environmental bacteria. We showed that the gene product of BP2936 is a membrane-associated methyl-transferase of free fatty acids. We thus propose to name it FmtB, for fatty acid methyl-transferase of Bordetella. The role of this protein was tested in cellular and animal models of infection, but the loss of BP2936 did not appear to affect host-pathogen interactions in those assays. The high level of conservation of BP2936 among B. pertussis isolates nevertheless argues that it probably plays a role in the life cycle of this pathogen. PMID:28493897

A Tyrosine-Reactive Irreversible Inhibitor for Glutathione S-Transferase Pi (GSTP1)

PubMed Central

Crawford, L. A.; Weerapana, E.

2016-01-01

Glutathione S-Transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, we report LAS17, a dichlorotriazine-containing compound that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition. PMID:27113843

A tyrosine-reactive irreversible inhibitor for glutathione S-transferase Pi (GSTP1).

PubMed

Crawford, L A; Weerapana, E

2016-05-24

Glutathione S-transferase Pi (GSTP1) mediates cellular defense against reactive electrophiles. Here, we report LAS17, a dichlorotriazine-containing compound that irreversibly inhibits GSTP1 and is selective for GSTP1 within cellular proteomes. Mass spectrometry and mutational studies identified Y108 as the site of modification, providing a unique mode of GSTP1 inhibition.

Functional characterisation of ganglioside-induced differentiation-associated protein 1 as a glutathione transferase.

PubMed

Shield, Alison J; Murray, Tracy P; Board, Philip G

2006-09-08

Mutations in the ganglioside-induced differentiation-associated protein 1 (GDAP1) gene have been linked with Charcot-Marie-Tooth (CMT) disease. This protein, and its paralogue GDAP1L1, appear to be structurally related to the cytosolic glutathione S-transferases (GST) including an N-terminal thioredoxin fold domain with conserved active site residues. The specific function, of GDAP1 remains unknown. To further characterise their structure and function we purified recombinant human GDAP1 and GDAP1L1 proteins using bacterial expression and immobilised metal affinity chromatography. Like other cytosolic GSTs, GDAP1 protein has a dimeric structure. Although the full-length proteins were largely insoluble, the deletion of a proposed C-terminal transmembrane domain allowed the preparation of soluble protein. The purified proteins were assayed for glutathione-dependent activity against a library of 'prototypic' GST substrates. No evidence of glutathione-dependent activity or an ability to bind glutathione immobilised on agarose was found.

The Use of Amino Sugars by Bacillus subtilis: Presence of a Unique Operon for the Catabolism of Glucosamine

PubMed Central

Gaugué, Isabelle; Oberto, Jacques; Putzer, Harald; Plumbridge, Jacqueline

2013-01-01

B. subtilis grows more rapidly using the amino sugar glucosamine as carbon source, than with N-acetylglucosamine. Genes for the transport and metabolism of N-acetylglucosamine (nagP and nagAB) are found in all the sequenced Bacilli (except Anoxybacillus flavithermus). In B. subtilis there is an additional operon (gamAP) encoding second copies of genes for the transport and catabolism of glucosamine. We have developed a method to make multiple deletion mutations in B. subtilis employing an excisable spectinomycin resistance cassette. Using this method we have analysed the contribution of the different genes of the nag and gam operons for their role in utilization of glucosamine and N-acetylglucosamine. Faster growth on glucosamine is due to the presence of the gamAP operon, which is strongly induced by glucosamine. Although the gamA and nagB genes encode isozymes of GlcN6P deaminase, catabolism of N-acetylglucosamine relies mostly upon the gamA gene product. The genes for use of N-acetylglucosamine, nagAB and nagP, are repressed by YvoA (NagR), a GntR family regulator, whose gene is part of the nagAB yvoA(nagR) operon. The gamAP operon is repressed by YbgA, another GntR family repressor, whose gene is expressed divergently from gamAP. The nagAB yvoA synton is found throughout the Bacilli and most firmicutes. On the other hand the ybgA-gamAP synton, which includes the ybgB gene for a small protein of unknown provenance, is only found in B. subtilis (and a few very close relatives). The origin of ybgBA-gamAP grouping is unknown but synteny analysis suggests lateral transfer from an unidentified donor. The presence of gamAP has enabled B. subtilis to efficiently use glucosamine as carbon source. PMID:23667565

Identification of Bacillus anthracis by Using Monoclonal Antibody to Cell Wall Galactose-N-Acetylglucosamine Polysaccharide

DTIC Science & Technology

1990-02-01

which appear to be directed to an epitope associated with the galactose-N-acetyl-D- glucosamine polysaccharide. Both demonstrated specificity in their...liquid composed primarily of D-galactose and N-acetyl-D-glu - R medium (28) buffered with 50 mM Tris hydrochloride , pH cosamine (12, 13) (Gal-NAG...Ascites fluid (5 ml) was dialyzed (Cel-Line Associates, Inc., Newfield, N.J.). Suspensions against 20 mM Tris hydrochloride (pH 8.0) for 18 to 20 h, were

Increased N7-methyldeoxyguanosine DNA adducts after occupational exposure to pesticides and influence of genetic polymorphisms of paraoxonase-1 and glutathione S-transferase M1 and T1.

PubMed

Gómez-Martín, Antonio; Altakroni, Bashar; Lozano-Paniagua, David; Margison, Geoffrey P; de Vocht, Frank; Povey, Andrew C; Hernández, Antonio F

2015-06-01

There are concerns about genetic risks associated with long-term exposure to pesticides as these compounds may damage DNA, resulting in mutations that eventually lead to cancer, neurological, and reproductive adverse health effects. This study assessed DNA damage in intensive agricultural workers exposed to pesticides by determining the levels of N7-methyldeoxyguanosine (N7-MedG), an adduct known to be a robust biomarker of recent exposure to chemical methylating agents. A cohort of 39 plastic greenhouse workers was assessed for changes in lymphocyte DNA N7-MedG levels between low level and high level exposures during the course of a spraying season. The contributions of genetic polymorphisms of the pesticide-metabolizing enzymes paraoxonase-1 (PON1) and the glutathione S-transferases, GSTM1 and GSTT1, on N7-MedG levels and other potential confounders were also assessed. N7-MedG increased in the period of high pesticide exposure as compared to the low exposure period (0.23 and 0.18 µmol N7-MedG/mol dG for the unadjusted and adjusted linear mixed models, P = 0.02 and 0.08, respectively). Significant decreased levels of erythrocyte acetylcholinesterase and plasma cholinesterase were observed in the high versus low exposure period in both the unadjusted (2.85 U/g hemoglobin and 213.13 U/L, respectively) and adjusted linear mixed models (2.99 U/g hemoglobin and 230.77 U/L, respectively), indicating pesticide intake. In intensive agriculture workers, higher pesticide exposure increased DNA alkylation levels, further demonstrating the genotoxicity of pesticides in man. In addition, pesticide-exposed individuals with inherited susceptible metabolic genotypes (particularly, null genotype for GSTM1 and the PON1 192R allele) appear to have an increased risk of genotoxic DNA damage. Environ. Mol. Mutagen. 56:437-445, 2015. © 2014 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

Structural analysis of N-linked glycans in Caenorhabditis elegans.

PubMed

Natsuka, Shunji; Adachi, Jiro; Kawaguchi, Masahumi; Nakakita, Shin-ichi; Hase, Sumihiro; Ichikawa, Akira; Ikura, Koji

2002-06-01

Caenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Man(alpha)1-6(Man(alpha)1-3)Man(beta)1-4GlcNAc(beta)1-4GlcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.

Drug resistance in epithelial ovarian cancer: P-glycoprotein and glutation S-transferase. Can they play an important role in detecting response to platinum-based chemotherapy as a first-line therapy.

PubMed

Simşek, T; Ozbilim, G; Gülkesen, H; Kaya, H; Sargin, F; Karaveli, S

2001-01-01

Drug resistance is important for the treatment of ovarian cancer. P-glycoprotein and glutation S-transferase as resistance markers play an important role in the effectivity of chemotherapeutical agents. The role of P-glycoprotein and glutation S-transferase in the treatment of epithelial ovarian cancer is not well understood. We investigated the relation between P-glycoprotein and glutation S-transferase level for response to platinum-based chemotherapy in epithelial ovarian cancer. We reviewed 30 cases diagnosed as epithelial ovarian cancer and treated with platinum-based chemotherapy in the Department of Obstetrics and Gynecology, Akdeniz University School of Medicine. The material was attained from initial parafin-embeded blocks stained for P-glycoprotein and glutation S-transferase. The cases that were diagnosed and treated before attending our clinic were not enrolled in the study. Mean age was 58.2 (25-70) and mean gravida 4.1 (0-10). Twenty-four patients (80%) were glutation S-transferase positive. Three cases (10%) out of 30 had positive reaction for P-glycoprotein. No difference was revealed regarding chemotherapy response rate among the cases showing glutation S-transferase positivity and P-glycoprotein negativity. Detection of glutation S-transferase and P-glycoprotein levels in epithelial ovarian cancer tissue is not important for response to platinum-based chemotherapy as a first line.

Expression of spermidine/spermine N(1) -acetyl transferase (SSAT) in human prostate tissues is related to prostate cancer progression and metastasis.

PubMed

Huang, Wei; Eickhoff, Jens C; Mehraein-Ghomi, Farideh; Church, Dawn R; Wilding, George; Basu, Hirak S

2015-08-01

Prostate cancer (PCa) in many patients remains indolent for the rest of their lives, but in some patients, it progresses to lethal metastatic disease. Gleason score is the current clinical method for PCa prognosis. It cannot reliably identify aggressive PCa, when GS is ≤ 7. It is shown that oxidative stress plays a key role in PCa progression. We have shown that in cultured human PCa cells, an activation of spermidine/spermine N(1) -acetyl transferase (SSAT; EC 2.3.1.57) enzyme initiates a polyamine oxidation pathway and generates copious amounts of reactive oxygen species in polyamine-rich PCa cells. We used RNA in situ hybridization and immunohistochemistry methods to detect SSAT mRNA and protein expression in two tissue microarrays (TMA) created from patient's prostate tissues. We analyzed 423 patient's prostate tissues in the two TMAs. Our data show that there is a significant increase in both SSAT mRNA and the enzyme protein in the PCa cells as compared to their benign counterpart. This increase is even more pronounced in metastatic PCa tissues as compared to the PCa localized in the prostate. In the prostatectomy tissues from early-stage patients, the SSAT protein level is also high in the tissues obtained from the patients who ultimately progress to advanced metastatic disease. Based on these results combined with published data from our and other laboratories, we propose an activation of an autocrine feed-forward loop of PCa cell proliferation in the absence of androgen as a possible mechanism of castrate-resistant prostate cancer growth. © 2015 Wiley Periodicals, Inc.

β(1,3)-Glucanosyl-Transferase Activity Is Essential for Cell Wall Integrity and Viability of Schizosaccharomyces pombe

PubMed Central

de Medina-Redondo, María; Arnáiz-Pita, Yolanda; Clavaud, Cécile; Fontaine, Thierry; del Rey, Francisco; Latgé, Jean Paul; Vázquez de Aldana, Carlos R.

2010-01-01

Background The formation of the cell wall in Schizosaccharomyces pombe requires the coordinated activity of enzymes involved in the biosynthesis and modification of β-glucans. The β(1,3)-glucan synthase complex synthesizes linear β(1,3)-glucans, which remain unorganized until they are cross-linked to other β(1,3)-glucans and other cell wall components. Transferases of the GH72 family play important roles in cell wall assembly and its rearrangement in Saccharomyces cerevisiae and Aspergillus fumigatus. Four genes encoding β(1,3)-glucanosyl-transferases -gas1+, gas2+, gas4+ and gas5+- are present in S. pombe, although their function has not been analyzed. Methodology/Principal Findings Here, we report the characterization of the catalytic activity of gas1p, gas2p and gas5p together with studies directed to understand their function during vegetative growth. From the functional point of view, gas1p is essential for cell integrity and viability during vegetative growth, since gas1Δ mutants can only grow in osmotically supported media, while gas2p and gas5p play a minor role in cell wall construction. From the biochemical point of view, all of them display β(1,3)-glucanosyl-transferase activity, although they differ in their specificity for substrate length, cleavage point and product size. In light of all the above, together with the differences in expression profiles during the life cycle, the S. pombe GH72 proteins may accomplish complementary, non-overlapping functions in fission yeast. Conclusions/Significance We conclude that β(1,3)-glucanosyl-transferase activity is essential for viability in fission yeast, being required to maintain cell integrity during vegetative growth. PMID:21124977

Bisubstrate Kinetics of Glutathione S-Transferase: A Colorimetric Experiment for the Introductory Biochemistry Laboratory

ERIC Educational Resources Information Center

Stefanidis, Lazaros; Scinto, Krystal V.; Strada, Monica I.; Alper, Benjamin J.

2018-01-01

Most biochemical transformations involve more than one substrate. Bisubstrate enzymes catalyze multiple chemical reactions in living systems and include members of the transferase, oxidoreductase, and ligase enzyme classes. Working knowledge of bisubstrate enzyme kinetic models is thus of clear importance to the practicing biochemist. However,…

Identification of Glutathione S-Transferase Pi as a Protein Involved in Parkinson Disease Progression

PubMed Central

Shi, Min; Bradner, Joshua; Bammler, Theo K.; Eaton, David L.; Zhang, JianPeng; Ye, ZuCheng; Wilson, Angela M.; Montine, Thomas J.; Pan, Catherine; Zhang, Jing

2009-01-01

Parkinson disease (PD) typically affects the cortical regions during the later stages of disease, with neuronal loss, gliosis, and formation of diffuse cortical Lewy bodies in a significant portion of patients with dementia. To identify novel proteins involved in PD progression, we prepared synaptosomal fractions from the frontal cortices of pathologically verified PD patients at different stages along with age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique. Approximately 100 proteins displayed significant differences in their relative abundances between PD patients at various stages and controls; three of these proteins were validated using independent techniques. One of the confirmed proteins, glutathione S-transferase Pi, was further investigated in cellular models of PD, demonstrating that its level was intimately associated with several critical cellular processes that are directly related to neurodegeneration in PD. These results have, for the first time, suggested that the levels of glutathione S-transferase Pi may play an important role in modulating the progression of PD. PMID:19498008

The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism.

PubMed

Thom, R; Dixon, D P; Edwards, R; Cole, D J; Lapthorn, A J

2001-05-18

The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 A to 1.65 A. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed. Copyright 2001 Academic Press.

Imidazopyridine and Pyrazolopiperidine Derivatives as Novel Inhibitors of Serine Palmitoyl Transferase.

PubMed

Genin, Michael J; Gonzalez Valcarcel, Isabel C; Holloway, William G; Lamar, Jason; Mosior, Marian; Hawkins, Eric; Estridge, Thomas; Weidner, Jeffrey; Seng, Thomas; Yurek, David; Adams, Lisa A; Weller, Jennifer; Reynolds, Vincent L; Brozinick, Joseph T

2016-06-23

To develop novel treatments for type 2 diabetes and dyslipidemia, we pursued inhibitors of serine palmitoyl transferase (SPT). To this end compounds 1 and 2 were developed as potent SPT inhibitors in vitro. 1 and 2 reduce plasma ceramides in rodents, have a slight trend toward enhanced insulin sensitization in DIO mice, and reduce triglycerides and raise HDL in cholesterol/cholic acid fed rats. Unfortunately these molecules cause a gastric enteropathy after chronic dosing in rats.

Protein arginine (N)-methyl transferase 7 (PRMT7) as a potential target for the sensitization of tumor cells to camptothecins.

PubMed

Verbiest, Vincent; Montaudon, Danièle; Tautu, Michel T; Moukarzel, Joyce; Portail, Jean-Pierre; Markovits, Judith; Robert, Jacques; Ichas, François; Pourquier, Philippe

2008-04-30

PRMT7 belongs to the protein arginine methyl-transferases family. We show that downregulation of PRMT7alpha and beta isoforms in DC-3F hamster cells was associated with increased sensitivity to the Top1 inhibitor camptothecin (CPT). This effect was not due to a change in Top1 contents or catalytic activity, or to a difference in the reversal of DNA breaks. Overexpression of PRMT7alpha and beta in DC-3F cells had no effect on CPT sensitivity, whereas it conferred a resistance to DC-3F/9-OH-E cells for which both isoforms are reduced by two- to three-fold as compared to DC-3F parental cells. Finally, downregulation of the human PRMT7 could also sensitize HeLa cells to CPT, suggesting that it could be used as a target to potentiate CPT derivatives.

Structural analysis of N-linked carbohydrate chains of funnel web spider (Agelenopsis aperta) venom peptide isomerase.

PubMed

Shikata, Y; Ohe, H; Mano, N; Kuwada, M; Asakawa, N

1998-06-01

The structure of the N-linked carbohydrate chains of peptide isomerase from the venom of the funnel web spider (Agelenopsis aperta) has been analyzed. Carbohydrates were released from peptide isomerase by hydrazinolysis and reductively aminated with 2-aminopyridine. The fluorescent derivatives were purified by phenol/chloroform extraction, followed by size-exclusion HPLC. The structure of the purified pyridylamino (PA-) carbohydrate chains were analyzed by a combination of two-dimensional HPLC mapping, sugar composition analysis, sequential exoglycosidase digestions, and mass spectrometry. The peptide isomerase contains six kinds of N-linked carbohydrate chains of truncated high-mannose type, with a fucose alpha 1-6 linked to the reducing N-acetylglucosamine in approximately 80% of them.

Molecular mimicry between cockroach and helminth glutathione S-transferases promotes cross-reactivity and cross-sensitization

USDA-ARS?s Scientific Manuscript database

The extensive similarities between helminth proteins and allergens are thought to contribute to helminth-driven allergic sensitization. We investigated the molecular and structural similarities between Bla g 5, a major glutathione-S transferase (GST) allergen of cockroaches, and the GST of Wucherer...

Specificities of the enzymes of N-alkyltropane biosynthesis in Brugmansia and Datura.

PubMed

Boswell, H D; Dräger, B; McLauchlan, W R; Portsteffen, A; Robins, D J; Robins, R J; Walton, N J

1999-11-01

The enzymes N-methylputrescine oxidase (MPO), the tropine-forming tropinone reductase (TRI), the pseudotropine-forming tropinone reductase (TRII), the tropine:acyl-CoA transferase (TAT) and the pseudotropine:acyl-CoA transferase (PAT) extracted from transformed root cultures of Datura stramonium and a Brugmansia candida x aurea hybrid were tested for their ability to accept a range of alternative substrates. MPO activity was tested with N-alkylputrescines and N-alkylcadaverines as substrates. TRI and TRII reduction was tested against a series of N-alkylnortropinones, N-alkylnorpelletierines and structurally related ketones as substrates. TAT and PAT esterification tests used a series of N-substituted tropines, pseudotropines, pelletierinols and pseudopelletierinols as substrates to assess the formation of their respective acetyl and tigloyl esters. The results generally show that these enzymes will accept alien substrates to varying degrees. Such studies may shed some light on the overall topology of the active sites of the enzymes concerned.

Glutathione S-transferase M1 and glutathione S-transferase T1 genotype in chronic pancreatitis: a meta-analysis.

PubMed

Zhong, Yanjun; Zou, Runmei; Cao, Jie; Peng, Mou

2015-02-01

A meta-analysis to determine the association between chronic pancreatitis and glutathione-S transferase (GST) mu 1 (GSTM1) and theta 1 (GSTT1) deletions. Case-control studies concerning the relationship between chronic pancreatitis and GSTM1 or GSTT1 deletions were identified (up to October 2013). Meta-analyses of the association between GSTM1 and GSTT1 genotype and chronic pancreatitis or alcoholic chronic pancreatitis (ACP) were performed. Seven studies were included in the meta-analysis (650 patients/1382 controls for GSTM1 and 536 patients/1304 controls for GSTT1). There were no significant relationships between GSTM1/GSTT1 and chronic pancreatitis or GSTT1 and ACP. There was a significant association between GSTM1 null genotype and ACP (odds ratio 1.16, 95% confidence intervals 1.03, 1.30). The GSTM1 null genotype was significantly associated with ACP risk. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Pleiotropic Functions of Glutathione S-Transferase P

PubMed Central

Zhang, Jie; Grek, Christina; Ye, Zhi-Wei; Manevich, Yefim; Tew, Kenneth D.; Townsend, Danyelle M.

2016-01-01

Glutathione S-transferase P (GSTP) is one member of the GST superfamily that is prevalently expressed in mammals. Known to possess catalytic activity through deprotonating glutathione allowing formation of thioether bonds with electrophilic substrates, more recent discoveries have broadened our understanding of the biological roles of this protein. In addition to catalytic detoxification, other properties so far ascribed to GSTP include chaperone functions, regulation of nitric oxide pathways, regulation of a variety of kinase signaling pathways, and participation in the forward reaction of protein S-glutathionylation. The expression of GSTP has been linked with cancer and other human pathologies and more recently even with drug addiction. With respect to human health, polymorphic variants of GSTP may determine individual susceptibility to oxidative stress and/or be critical in the design and development of drugs that have used redox pathways as a discovery platform. PMID:24974181

The glutathione-S-transferase Mu 1 null genotype modulates ozone-induced airway inflammation in humans*

EPA Science Inventory

Background: The Glutathione-S-Transferase Mu 1 null genotype has been reported to be a risk factor for acute respiratory disease associated with increases in ambient air ozone. Ozone is known to cause an immediate decrease in lung function and increased airway inflammation. Howev...

Mannosyl- and Xylosyl-Containing Glycans Promote Tomato (Lycopersicon esculentum Mill.) Fruit Ripening

PubMed Central

Priem, Bernard; Gross, Kenneth C.

1992-01-01

The oligosaccharide glycans mannosylα1-6(mannosylα1-3)mannosylα1-6(mannosylα1-3) mannosylβ1-4-N-acetylglucosamine and mannosylα1-6(mannosylα1-3)(xylosylβ1-2) mannosylβ1-4-N-acetylglucosaminyl(fucosylα1-3) N-acetylglucosamine were infiltrated into mature green tomato fruit (Lycopersicon esculentum Mill., cv Rutgers). Coinfiltration of 1 nanogram per gram fresh weight of the glycans with 40 micrograms per gram fresh weight galactose, a level of galactose insufficient to promote ripening, stimulated ripening as measured by red coloration and ethylene production. PMID:16668644

Thermal- and urea-induced unfolding processes of glutathione S-transferase by molecular dynamics simulation.

PubMed

Li, Jiahuang; Chen, Yuan; Yang, Jie; Hua, Zichun

2015-05-01

The Schistosoma juponicum 26 kDa glutathione S-transferase (sj26GST) consists of the N-terminal domain (N-domain), containing three alpha-helices (named H1-H3) and four anti-parallel beta-strands (S1-S4), and the C-terminal domain (C-domain), comprising five alpha-helices (named H4-H8). In present work, molecular dynamics simulations and fluorescence spectroscopic were used to gain insights into the unfolding process of sj26GST. The molecular dynamics simulations on sj26GST subunit both in water and in 8 M urea were carried out at 300 K, 400 K and 500 K, respectively. Spectroscopic measurements were employed to monitor structural changes. Molecular dynamics simulations of sj26GST subunit induced by urea and temperature showed that the initial unfolding step of sj26GST both in water and urea occurred on N-domain, involving the disruption of helices H2, H3 and strands S3 and S4, whereas H6 was the last region exposed to solution and was the last helix to unfold. Moreover, simulations analyses combining with fluorescence and circular dichroism spectra indicated that N-domain could not fold independent, suggesting that correct folding of N-domain depended on its interactions with C-domain. We further proposed that the folding of GSTs could begin with the hydrophobic collapse of C-domain whose H4, H5, H6 and H7 could move close to each other and form a hydrophobic core, especially H6 wrapped in the hydrophobic center and beginning spontaneous formation of the helix. S3, S4, H3, and H2 could form in the wake of the interaction between C-domain and N-domain. The paper can offer insights into the molecular mechanism of GSTs unfolding. © 2014 Wiley Periodicals, Inc.

Dishevelled3 is a novel arginine methyl transferase substrate.

PubMed

Bikkavilli, Rama Kamesh; Avasarala, Sreedevi; Vanscoyk, Michelle; Sechler, Marybeth; Kelley, Nicole; Malbon, Craig C; Winn, Robert A

2012-01-01

Dishevelled, a phosphoprotein scaffold, is a central component in all the Wnt-sensitive signaling pathways. In the present study, we report that Dishevelled is post-translationally modified, both in vitro and in vivo, via arginine methylation. We also show protein arginine methyl transferases 1 and 7 as the key enzymes catalyzing Dishevelled methylation. Interestingly, Wnt3a stimulation of F9 teratocarcinoma cells results in reduced Dishevelled methylation. Similarly, the methylation-deficient mutant of Dishevelled, R271K, displayed spontaneous membrane localization and robust activation of Wnt signaling; suggesting that differential methylation of Dishevelled plays an important role in Wnt signaling. Thus arginine methylation is shown to be an important switch in regulation of Dishevelled function and Wnt signaling.

Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.

PubMed

Axarli, Irine; Muleta, Abdi W; Chronopoulou, Evangelia G; Papageorgiou, Anastassios C; Labrou, Nikolaos E

2017-01-01

Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher k cat /K m and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Šand 2.3Šresolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

Glutathione S-Transferases Interact with AMP-Activated Protein Kinase: Evidence for S-Glutathionylation and Activation In Vitro

PubMed Central

Polge, Cécile; Ramirez, Sacnicte; Michelland, Sylvie; Sève, Michel; Vertommen, Didier; Rider, Mark; Lentze, Nicolas; Auerbach, Daniel; Schlattner, Uwe

2013-01-01

AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions. PMID:23741294

Preliminary X-ray crystallographic analysis of glutathione transferase zeta 1 (GSTZ1a-1a)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boone, Christopher D.; Zhong, Guo; Smeltz, Marci

2014-01-21

Crystals of glutathione transferase zeta 1 were grown and shown to diffract X-rays to 3.1 Å resolution. They belonged to space group P1, with unit-cell parameters a = 42.0, b = 49.6, c = 54.6 Å, α = 82.9, β = 69.9, γ = 73.4°.

PABA/NO lead optimization: Improved targeting of cytotoxicity to glutathione S-transferase P1-overexpressing cancer cells.

PubMed

Kim, Youseung; Maciag, Anna E; Cao, Zhao; Deschamps, Jeffrey R; Saavedra, Joseph E; Keefer, Larry K; Holland, Ryan J

2015-08-01

PABA/NO [O(2)-{2,4-dinitro-5-[4-(N-methylamino)benzoyloxy]phenyl} 1-(N,N-dimethylamino) diazen-1-ium-1,2-diolate] is a nitric oxide (NO)-releasing arylating agent designed to be selectively activated by reaction with glutathione (GSH) on catalysis by glutathione S-transferase P1 (GSTP1), an enzyme frequently overexpressed in cancer cells. PABA/NO has proven active in several cancer models in vitro and in vivo, but its tendency to be metabolized via a variety of pathways, some that generate inactive metabolites and hydrolysis products, limits its potential as a drug. Here we show that a simple replacement of cyano for nitro at the 4 position to give compound 4b ('p-cyano-PABA/NO') has the dual effect of slowing the undesired side reactions while enhancing the proportion of NO release and arylating activity on catalysis by GSTP1. Compound 4b showed increased resistance to hydrolysis and uncatalyzed reaction with GSH, along with a more favorable product distribution in the presence of GSTP1. It also showed significant proapoptotic activity. The data suggest p-cyano-PABA/NO to be a more promising prodrug than PABA/NO, with better selectivity toward cancer cells. Published by Elsevier Ltd.

Detection of Glutathione by Glutathione-S-Transferase-Nanoconjugate Ensemble Electrochemical Device.

PubMed

Barman, Ujjwol; Mukhopadhyay, Gargi; Goswami, Namami; Ghosh, Siddhartha Sankar; Paily, Roy P

2017-06-01

This paper reports a novel electrochemical method for detection of Glutathione (GSH) using Glutathione-S-Transferase (GST) - ZnO composite nanoparticles to investigate the prospects of the method for detection of cancer at an early stage. The purified GST enzyme was bound with ZnO nanoparticles by electrostatic interactions and the nanocomposite was dropcast on a silicon dioxide wafer. The GST functionalized deposited layer was then used as a chemiresistive channel to detect conjugation reaction between GSH and 1-Chloro-2, 4-Dinitrobenzene (CDNB). The zeta potential values of the ZnO nanoparticles and the GST were found to be 13.4 mV and-6.21 mV, respectively. Around 73.8% binding was observed between the enzyme and ZnO nanoparticles. I - V analysis of the chemiresistive channel showed an increase in conductivity of the channel due to conjugation reaction between GSH and CDNB as compared with that of GSH or CDNB alone. I - V characterization of the GST functionalized layer was performed at various concentrations of GSH and a sensitivity and limit of detection of 5.68 nA/ [Formula: see text] and 41.9 nM were obtained, respectively. Thus from I - V analysis of the chemiresistivechannel, the detectionand quantification of GSH could be obtained. The kinetic parameters of both GST and nanoconjugate of ZnO nanoparticles andGSTwere determinedwith respect to its substrates, GSH and CDNB, using Michaelis-Mentenmodel. This novel approach of detection of GSH bymeans of ZnO nanoparticle and GST enzyme composite can be further analyzed for in vitro experiments, which will lead us to a new and efficient way of detecting certain types of cancers at an early stage.

Poly-N-acetylglucosamine mediates biofilm formation and detergent resistance in Aggregatibacter actinomycetemcomitans

PubMed Central

Izano, Era A.; Sadovskaya, Irina; Wang, Hailin; Vinogradov, Evgeny; Ragunath, Chandran; Ramasubbu, Narayanan; Jabbouri, Saïd; Perry, Malcolm B.; Kaplan, Jeffrey B.

2008-01-01

Clinical isolates of the periodontopathogen Aggregatibacter actinomycetemcomitans form matrix-encased biofilms on abiotic surfaces in vitro. A major component of the A. actinomycetemcomitans biofilm matrix is PGA, a hexosamine-containing polysaccharide that mediates intercellular adhesion. In this report we describe studies on the purification, structure, genetics and function of A. actinomycetemcomitans PGA. We found that PGA was very tightly attached to A. actinomycetemcomitans biofilm cells and could be efficiently separated from the cells only by phenol extraction. A. actinomycetemcomitans PGA copurified with LPS on a gel filtration column. 1H-NMR spectra of purified A. actinomycetemcomitans PGA were consistent with a structure containing a linear chain of N-acetyl-D-glucosamine residues in β(1,6) linkage. Genetic analyses indicated that all four genes of the pgaABCD locus were required for PGA production in A. actinomycetemcomitans. PGA mutant strains still formed biofilms in vitro. Unlike wild-type biofilms, however, PGA mutant biofilms were sensitive to detachment by DNase I and proteinase K. Treatment of A. actinomycetemcomitans biofilms with the PGA-hydrolyzing enzyme dispersin B made them 3 log units more sensitive to killing by the cationic detergent cetylpyridinium chloride. Our findings suggest that PGA, extracellular DNA and proteinaceous adhesins all contribute to the structural integrity of the A. actinomycetemcomitans biofilm matrix. PMID:17851029

Site-specific O-glycosylation of N-terminal serine residues by polypeptide GalNAc-transferase 2 modulates human δ-opioid receptor turnover at the plasma membrane.

PubMed

Lackman, Jarkko J; Goth, Christoffer K; Halim, Adnan; Vakhrushev, Sergey Y; Clausen, Henrik; Petäjä-Repo, Ulla E

2018-01-01

G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors

Partial purification and characterization of a mannosyl transferase involved in O -linked mannosylation of glycoproteins in Candida albicans.

PubMed

Arroyo-Flores, Blanca L; Calvo-Méndez, Carlos; Flores-Carreón, Arturo; López-Romero, Everardo

2004-04-01

Incubation of a mixed membrane fraction of C. albicans with the nonionic detergents Nonidet P-40 or Lubrol solubilized a fraction that catalyzed the transfer of mannose either from endogenously generated or exogenously added dolichol-P-[14C]Man onto endogenous protein acceptors. The protein mannosyl transferase solubilized with Nonidet P-40 was partially purified by a single step of preparative nondenaturing electrophoresis and some of its properties were investigated. Although transfer activity occurred in the absence of exogenous mannose acceptors and thus depended on acceptor proteins isolated along with the enzyme, addition of the protein fraction obtained after chemical de-mannosylation of glycoproteins synthesized in vitro stimulated mannoprotein labeling in a concentration-dependent manner. Other de-mannosylated glycoproteins, such as yeast invertase or glycoproteins extracted from C. albicans, failed to increase the amount of labeled mannoproteins. Mannosyl transfer activity was not influenced by common metal ions such as Mg(2+), Mn(2+) and Ca(2+), but it was stimulated up to 3-fold by EDTA. Common phosphoglycerides such as phosphatidylglycerol and, to a lower extent, phosphatidylinositol and phosphatidylcholine enhanced transfer activity. Interestingly, coupled transfer activity between dolichol phosphate mannose synthase, i.e., the enzyme responsible for Dol-P-Man synthesis, and protein mannosyl transferase could be reconstituted in vitro from the partially purified transferases, indicating that this process can occur in the absence of cell membranes.

Quantification of butyryl CoA:acetate CoA-transferase genes reveals different butyrate production capacity in individuals according to diet and age.

PubMed

Hippe, Berit; Zwielehner, Jutta; Liszt, Kathrin; Lassl, Cornelia; Unger, Frank; Haslberger, Alexander G

2011-03-01

The gastrointestinal microbiota produces short-chain fatty acids, especially butyrate, which affect colonic health, immune function and epigenetic regulation. To assess the effects of nutrition and aging on the production of butyrate, the butyryl-CoA:acetate CoA-transferase gene and population shifts of Clostridium clusters lV and XlVa, the main butyrate producers, were analysed. Faecal samples of young healthy omnivores (24 ± 2.5 years), vegetarians (26 ± 5 years) and elderly (86 ± 8 years) omnivores were evaluated. Diet and lifestyle were assessed in questionnaire-based interviews. The elderly had significantly fewer copies of the butyryl-CoA:acetate CoA-transferase gene than young omnivores (P=0.014), while vegetarians showed the highest number of copies (P=0.048). The thermal denaturation of the butyryl-CoA:acetate CoA-transferase gene variant melting curve related to Roseburia/Eubacterium rectale spp. was significantly more variable in the vegetarians than in the elderly. The Clostridium cluster XIVa was more abundant in vegetarians (P=0.049) and in omnivores (P<0.01) than in the elderly group. Gastrointestinal microbiota of the elderly is characterized by decreased butyrate production capacity, reflecting increased risk of degenerative diseases. These results suggest that the butyryl-CoA:acetate CoA-transferase gene is a valuable marker for gastrointestinal microbiota function. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Use of urinary γ-glutamyl transferase (GGT) to monitor the pattern of proteinuria in dogs with leishmaniasis treated with N-methylglucamine antimoniate.

PubMed

Paltrinieri, Saverio; Mangiagalli, Giulia; Ibba, Fabrizio

2018-05-25

The aim of this study was to assess if the coupled analysis of the urinary protein to creatinine (UPC) ratio and of the GGT/UC ratio (the ratio between urinary γ-glutamyl transferase activity and urinary creatinine) may be used in treated leishmaniotic dogs to differentiate dogs with transient impairment of tubular function from dogs with persistent tubular damage. To this aim, 40 urine from 10 proteinuric and leishmaniotic dogs that at the first visit had high GGT/UC ratio, consistent with tubular damage, were collected and analyzed before treatments and 2, 4 and 6 weeks after treatment with N-methylglucamine antimoniate and allopurinol. Compared with pre-treatment values, at the end of the study period the UPC ratio decreased only in 5/10 dogs, which, however, were still proteinuric or borderline proteinuric. Conversely, the GGT/CU ratio decreased in 8/10 dogs and in 3 of them the values at the end of the study period were below the threshold consistent with tubular proteinuria. The GGT/UC values at 6 weeks was significantly lower than before treatment. However, transient increases were frequent for both the analytes. These results indicate that in most of the dogs that remain proteinuric after treatment, likely due to the persistent glomerular damage, the GGT/UC ratio tends to normalize. This suggests that in these dogs tubular proteinuria at admission depends on functional impairment of tubular cells likely due to the overflow of proteins from damaged glomeruli. However, tubular proteinuria occasionally persists, suggesting that tubulointerstitial damages persist even in dogs responsive to treatments. Copyright © 2018 Elsevier Ltd. All rights reserved.

A model to environmental monitoring based on glutathione-S-transferase activity and branchial lesions in catfish

NASA Astrophysics Data System (ADS)

Neta, Raimunda Nonata Fortes Carvalho; Torres, Audalio Rebelo

2017-11-01

In this work, we validate the glutathione-S-transferase and branchial lesions as biomarkers in catfish Sciades herzbergii to obtain a predictive model of the environmental impact effects in a harbor of Brazil. The catfish were sampled from a port known to be contaminated with heavy metals and organic compounds and from a natural reserve in São Marcos Bay, Maranhão. Two biomarkers, hepatic glutathione S-transferase (GST) activity and branchial lesions were analyzed. The values for GST activity were modeled with the occurrence of branchial lesions by fitting a third order polynomial. Results from the mathematical model indicate that GST activity has a strong polynomial relationship with the occurrence of branchial lesions in both the wet and the dry seasons, but only at the polluted port site. Our mathematic model indicates that when the GST ceases to act, serious branchial lesions are observed in the catfish of the contaminated port area.

Characterization of spermidine hydroxycinnamoyl transferases from eggplant (Solanum melongena L.) and its wild relative Solanum richardii Dunal

USDA-ARS?s Scientific Manuscript database

Eggplant produces a variety of hydroxycinnamic acid amides (HCAAs) that play an important role in plant development and adaptation to environmental changes. However, the HCAA pathway remains largely uncharacterized in Solanaceae. In this study, a spermidine hydroxycinnamoyl transferase (SHT) from eg...

Identification and expression profiles of nine glutathione S-transferase genes from the important rice phloem sap-sucker and virus vector Laodelphax striatellus (Fallén) (Hemiptera: Delphacidae).

PubMed

Zhou, Wen-Wu; Li, Xi-Wang; Quan, Yin-Hua; Cheng, Jiaan; Zhang, Chuan-Xi; Gurr, Geoff; Zhu, Zeng-Rong

2012-09-01

Glutathione S-transferases (GSTs) have received considerable attention in insects for their roles in insecticide resistance. Laodelphax striatellus (Fallén) is a serious rice pest. L. striatellus outbreaks occur frequently throughout eastern Asia. A key problem in controlling this pest is its rapid adaptation to numerous insecticides. In this research, nine cDNAs encoding GSTs in L. striatellus were cloned and characterised. The cloned GSTs of L. striatellus belonged to six cytosolic classes and a microsomal subgroup. Exposure to sublethal concentrations of each of the six insecticides, DDT, chlorpyrifos, fipronil, imidacloprid, buprofezin and beta-cypermethrin, quickly induced (6 h) up-expression of LsGSTe1. The expression of LsGSTs2 was increased by chlorpyrifos, fipronil and beta-cypermethrin. Furthermore, exposure of L. striatellus to fipronil, imidacloprid, buprofezin and beta-cypermethrin increased the expression of the LsGSTm gene after 24 or 48 h. This work is the first identification of GST genes from different GST groups in Auchenorrhyncha species and their induction characteristics with insecticide types and time. The elevated expression of GST genes induced by insecticides might be related to the enhanced tolerance of this insect to insecticides and xenobiotics. Copyright © 2012 Society of Chemical Industry.

Borate-aided anion exchange high-performance liquid chromatography of uridine diphosphate-sugars in brain, heart, adipose and liver tissues.

PubMed

Oikari, Sanna; Venäläinen, Tuula; Tammi, Markku

2014-01-03

In this paper we describe a method optimized for the purification of uridine diphosphate (UDP)-sugars from liver, adipose tissue, brain, and heart, with highly reproducible up to 85% recoveries. Rapid tissue homogenization in cold ethanol, lipid removal by butanol extraction, and purification with a graphitized carbon column resulted in isolation of picomolar quantities of the UDP-sugars from 10 to 30mg of tissue. The UDP-sugars were baseline separated from each other, and from all major nucleotides using a CarboPac PA1 anion exchange column eluted with a gradient of acetate and borate buffers. The extraction and purification protocol produced samples with few unidentified peaks. UDP-N-acetylglucosamine was a dominant UDP-sugar in all the rat tissues studied. However, brain and adipose tissue showed high UDP-glucose levels, equal to that of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine showed 2.3-2.7 times higher levels than UDP-N-acetylgalactosamine in all tissues, and about the same ratio was found between UDP-glucose and UDP-galactose in adipose tissue and brain (2.6 and 2.8, respectively). Interestingly, the UDP-glucose/UDP-galactose ratio was markedly lower in liver (1.1) and heart (1.7). The UDP-N-acetylglucosamine/UDP-glucuronic acid ratio was also constant, between 9.7 and 7.7, except in liver with the ratio as low as 1.8. The distinct UDP-glucose/galactose ratio, and the abundance of UDP-glucuronic acid may reflect the specific role of liver in glycogen synthesis, and metabolism of hormones and xenobiotics, respectively, using these UDP-sugars as substrates. Copyright © 2013 Elsevier B.V. All rights reserved.

Polymorphisms of Glutathione S-transferases Omega-1 among ethnic populations in China

PubMed Central

Fu, Songbo; Wu, Jie; Chen, Feng; Sun, Dianjun; Fu, Songbin

2008-01-01

Background Glutathione S-transferases (GSTs) is a genetic factor for many diseases and exhibits great diversities among various populations. We assessed association of the genotypes of Glutathione S-transferases Omega-1 (GSTO1) A140D with ethnicity in China. Results Peripheral blood samples were obtained from 1314 individuals from 14 ethnic groups. Polymorphisms of GSTO1 A140D were measured using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Logistic regression was employed to adjustment for regional factor. The frequency of GSTO1 140A allele was 15.49% in the total 14 ethnic populations. Compared to Han ethnic group, two ethnic populations were more likely to have AA or CA genotype [odds ratio (OR): 1.77, 95% confidence interval (95% CI): 1.05–2.98 for Uygur and OR: 1.78, 95% CI: 1.18–2.69 for Hui]. However, there were no statistically significant differences across 14 ethnic groups when region factor was adjusted. In Han ethnicity, region was significantly associated with AA or CA genotype. Han individuals who resided in North-west of China were more likely to have these genotypes than those in South of China (OR: 1.63, 95% CI: 1.21–2.20). Conclusion The prevalence of the GSTO1 140A varied significantly among different regional populations in China, which showed that geography played a more important role in the population differentiation for this allele than the ethnicity/race. PMID:18400112

Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase.

PubMed

Becerra-Arteaga, Alejandro; Shuler, Michael L

2007-08-15

We report for the first time that culture conditions, specifically culture medium supplementation with nucleotide-sugar precursors, can alter significantly the N-linked glycosylation of a recombinant protein in plant cell culture. Human secreted alkaline phosphatase produced in tobacco NT1 cell suspension cultures was used as a model system. Plant cell cultures were supplemented with ammonia (30 mM), galactose (1 mM) and glucosamine (10 mM) to improve the extent of N-linked glycosylation. The highest levels of cell density and active extracellular SEAP in supplemented cultures were on average 260 g/L and 0.21 U/mL, respectively, compared to 340 g/L and 0.4 U/mL in unsupplemented cultures. The glycosylation profile of SEAP produced in supplemented cultures was determined via electrospray ionization mass spectrometry with precursor ion scanning and compared to that of SEAP produced in unsupplemented cultures. In supplemented and unsupplemented cultures, two biantennary complex-type structures terminated with one or two N-acetylglucosamines and one paucimannosidic glycan structure comprised about 85% of the SEAP glycan pool. These three structures contained plant-specific xylose and fucose residues and their relative abundances were affected by each supplement. High mannose structures (6-9 mannose residues) accounted for the remaining 15% glycans in all cases. The highest proportion (approximately 66%) of a single complex-type biantennary glycan structure terminated in both antennae by N- acetylglucosamine was obtained with glucosamine supplementation versus only 6% in unsupplemented medium. This structure is amenable for in vitro modification to yield a more human-like glycan and could serve as a route to plant cell culture produced therapeutic glycoproteins. (c) 2007 Wiley Periodicals, Inc.

Sequential high-content profiling of the IgG-autoantibody repertoire reveals novel antigens in rheumatoid arthritis.

PubMed

Vordenbäumen, Stefan; Lueking, Angelika; Budde, Petra; Zucht, Hans-Dieter; Goehler, Heike; Brinks, Ralph; Fischer-Betz, Rebecca; Richter, Jutta; Bleck, Ellen; Detert, Jacqueline; Langer, Hans-Eckhard; Sörgel, Anne; Burmester, Gerd-Rüdiger; Schulz-Knappe, Peter; Schneider, Matthias

2016-10-12

The aim was to identify novel diagnostic autoantibody candidates for rheumatoid arthritis (RA) by comprehensive screening for autoreactivity. We incubated 5892 recombinant proteins coupled to fluorescent beads, with patients' sera for the detection of IgG-autoantibodies in three independent patient cohorts: A (n = 72 patients with established RA); B/B- (n = 116 patients with early RA (B) and n = 51 CCP-negative patients with early RA from B (B-)); and C (n = 184 patients with early seronegative RA), in comparison to matched healthy controls. Intersects of significantly increased autoantibodies as determined by the Mann-Whitney test were sought. Screening of 5892 antigens in RA cohorts A and B, or the seronegative cohorts B- and C revealed intersects of 23 and 13 significantly increased autoantibodies, respectively. Reactivity to three antigens was increased in all cohorts tested: N-acetylglucosamine-1-phosphate transferase, gamma subunit (GNPTG), heterogeneous nuclear ribonucleoprotein A1-like 2 (HNRNPA1), and insulin-like growth factor binding protein 2 (IGFBP2). Comprehensive sequential screening for autoantibodies reveals novel candidates for diagnostic markers in both seropositive and seronegative RA and suggests new fields of research into the pathogenesis of RA.

Demystifying O-GlcNAcylation: hints from peptide substrates.

PubMed

Shi, Jie; Ruijtenbeek, Rob; Pieters, Roland J

2018-03-22

O-GlcNAcylation, analogous to phosphorylation, is an essential post-translational modification of proteins at Ser/Thr residues with a single β-N-acetylglucosamine moiety. This dynamic protein modification regulates many fundamental cellular processes and its deregulation has been linked to chronic diseases such as cancer, diabetes and neurodegenerative disorders. Reversible attachment and removal of O-GlcNAc is governed only by O-GlcNAc transferase and O-GlcNAcase, respectively. Peptide substrates, derived from natural O-GlcNAcylation targets, function in the catalytic cores of these two enzymes by maintaining interactions between enzyme and substrate, which makes them ideal models for the study of O-GlcNAcylation and deglycosylation. These peptides provide valuable tools for a deeper understanding of O-GlcNAc processing enzymes. By taking advantage of peptide chemistry, recent progress in the study of activity and regulatory mechanisms of these two enzymes has advanced our understanding of their fundamental specificities as well as their potential as therapeutic targets. Hence, this review summarizes the recent achievements on this modification studied at the peptide level, focusing on enzyme activity, enzyme specificity, direct function, site-specific antibodies and peptide substrate-inspired inhibitors.

Neuroantibodies (NAB) in African-American Children: Associations with Gender, Glutathione-S-Transferase (GST)Pi Polymorphisms (SNP) and Heavy Metals

EPA Science Inventory

CONTACT (NAME ONLY): Hassan El-Fawal Abstract Details PRESENTATION TYPE: Platform or Poster CURRENT CATEGORY: Neurodegenerative Disease | Biomarkers | Neurotoxicity, Metals KEYWORDS: Autoantibodies, Glutathione-S-Transferase, DATE/TIME LAST MODIFIED: DATE/TIME SUBMITTED: Abs...

Glutathione Transferase from Trichoderma virens Enhances Cadmium Tolerance without Enhancing Its Accumulation in Transgenic Nicotiana tabacum

PubMed Central

Dixit, Prachy; Mukherjee, Prasun K.; Ramachandran, V.; Eapen, Susan

2011-01-01

Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and

Vanillin as a modulator agent in SMART test: inhibition in the steps that precede N-methyl-N-nitrosourea-, N-ethyl-N-nitrosourea-, ethylmethanesulphonate- and bleomycin-genotoxicity.

PubMed

Sinigaglia, Marialva; Lehmann, Maurício; Baumgardt, Paula; do Amaral, Viviane Souza; Dihl, Rafael Rodrigues; Reguly, Maria Luíza; de Andrade, Heloísa Helena Rodrigues

2006-09-05

Vanillin (VA), the world's major flavoring compound used in food industry and confectionery products - that has antimutagenic and anticarcinogenic activity against a variety of mutagenic/carcinogenic agents - was tested for the interval between the formation of premutational lesion and it is finalization as a DNA lesion. The overall findings using co-treatment protocols in SMART test suggest that VA can lead to a significant protection against the general genotoxicity of ethylmethanesulphonate (EMS), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU) and bleomycin sulphate (BLEO). Considering MNU, ENU and EMS the desmutagenic activity observed could result from VA-stimulation of detoxification, via induction of glutathione S-transferase. However, the protector effect related to BLEO could be attributed to its powerful scavenger ability, which has the potential to prevent oxidative damage induced by BLEO.

Clonorchis sinensis omega-class glutathione transferases are reliable biomarkers for serodiagnosis of clonorchiasis and opisthorchiasis.

PubMed

Kim, J-G; Ahn, C-S; Sripa, B; Eom, K S; Kang, I; Sohn, W-M; Nawa, Y; Kong, Y

2018-04-09

To determine the potential for immunodiagnostic application of two recombinant forms of Clonorchis sinensis omega-class glutathione transferases (rCsGSTo1 and rCsGSTo2) against human small liver-fluke C. sinensis and Opisthorchis viverrini infections. Specific antibody levels against rCsGSTo1 and rCsGSTo2 in patients' sera of egg-positive opisthorchiasis (n = 87) and clonorchiasis (n = 120), as well as those in sera from patients with other helminthic infections (n = 252) and healthy controls (n = 40) were retrospectively analysed by ELISA. We observed highly positive correlation coefficients between specific antibody levels against rCsGSTo1 and rCsGSTo2 and egg counts per gramme of faeces (EPG) of patients with opisthorchiasis (n = 87; r = 0.88 for rCsGSTo1 and r = 0.90 for rCsGSTo2). Sera from opisthorchiasis patients whose EPG counts >100 (n = 43) revealed high antibody titres against both antigens. Patients' sera with low EPG counts (<100, n = 44) also exhibited reliable sensitivities of 93.2% and 97.7% for rCsGSTo1 and rCsGSTo2, respectively. Sera from clonorchiasis patients showed sensitivities of 90% (108/120 samples) and 89.2% (107/120 sera) for rCsGSTo1 and rCsGSTo2. Overall diagnostic sensitivities for liver-fluke infections were 92.3% for rCsGSTo1 (191/207 samples) and 93.2% for rCsGSTo2 (193/207 samples). Specificities were 89.7% (rCsGSTo1) and 97.6% (rCsGSTo2). Detection of specific antibody levels against rCsGSTo1 or rCsGSTo2 might be promising for the serodiagnosis of patients infected with these two phylogenetically close carcinogenic liver-flukes. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

O-linked N-acetyl-glucosamine deposition in placental proteins varies according to maternal glycemic levels.

PubMed

Dela Justina, Vanessa; Dos Passos Junior, Rinaldo R; Bressan, Alecsander F; Tostes, Rita C; Carneiro, Fernando S; Soares, Thaigra S; Volpato, Gustavo T; Lima, Victor Vitorino; Martin, Sebastian San; Giachini, Fernanda R

2018-05-07

Hyperglycemia increases glycosylation with O-linked N‑acetyl‑glucosamine (O-GlcNAc) contributing to placental dysfunction and fetal growth impairment. Our aim was to determine how O-GlcNAc levels are affected by hyperglycemia and the O-GlcNAc distribution in different placental regions. Female Wistar rats were divided into the following groups: severe hyperglycemia (>300 mg/dL; n = 5); mild hyperglycemia (>140 mg/dL, at least than two time points during oral glucose tolerance test; n = 7) or normoglycemia (<120 mg/dL; n = 6). At 21 days of pregnancy, placental tissue was collected and processed for morphometry and immunohistochemistry analyses, or properly stored at -80 °C for protein quantification by western blot. Placental index was increased only in severe hyperglycemic rats. Morphometric analysis showed increased junctional zone and decreased labyrinth region in placentas exclusively from the severe hyperglycemic group. Proteins targeted by O-GlcNAc were detected in all regions, with increased O-GlcNAc levels in the hyperglycemic group compared to control and mild hyperglycemic rats. Proteins in endothelial and trophoblast cells were the main target for O-GlcNAc. Whereas no changes in O-GlcNAc transferase (OGT) expression were detected, O-GlcNAcase (OGA) expression was reduced in placentas from the severe hyperglycemic group and augmented in placentas from the mild hyperglycemic group, compared with their respective control groups. Placental O-GlcNAc overexpression may contribute to placental dysfunction, as indicated by the placental index. Additionally, morphometric alterations, occurring simultaneously with increased O-GlcNAc accumulation in the placental tissue may contribute to placental dysfunction during hyperglycemia. Copyright © 2017. Published by Elsevier Inc.

Development of a cell transducible RhoA inhibitor TAT-C3 transferase and its encapsulation in biocompatible microspheres to promote survival and enhance regeneration of severed neurons.

PubMed

Tan, Elaine Y M; Law, Janice W S; Wang, Chi-Hwa; Lee, Alan Y W

2007-12-01

Neurons in post-traumatized mammalian central nervous system show only limited degree of regeneration, which can be attributed to the presence of neurite outgrowth inhibitors in damaged myelin and glial scar, and to the apoptosis of severed central neurons and glial cells during secondary Wallerian degeneration. RhoA GTPase has been implicated as the common denominator in these counter-regeneration events, which shows significant and persistent up-regulation for weeks in injured spinal cord and cerebral infarct after stroke. While the exoenzyme C3 transferase is a potent RhoA inhibitor, its extremely low efficiency of cell entry and degradation in vivo has restricted the therapeutic value. This study aims to circumvent these problems by developing a membrane-permeating form of C3 transferase and a biopolymer-based microsphere depot system for sustainable controlled release of the protein. A membrane-permeating form of C3 transferase was developed by fusing a Tat (trans-activating transcription factor) transduction domain of human immunodeficiency virus to its amino terminal using standard molecular cloning techniques. After confirming efficient cell entry into epithelial and neuroblastoma cells, the resulting recombinant protein TAT-C3 was encapsulated in biocompatible polymer poly(D,L -lactide-co-glycolide) in the form of microspheres by a water-in-oil-in-water (W/O/W) emulsion method. By blending capped and uncapped form of the polymer at different ratios, TAT-C3 protein release profile was modified to suit the expression pattern of endogenous RhoA during CNS injuries. Bioactivity of TAT-C3 released from microspheres was assessed by RhoA ribosylation assay. In contrast to wild-type C3 transferase, the modified TAT-C3 protein was found to efficiently enter NIH3T3 and N1E-115 neuroblastoma cells as early as 6 hours of incubation. The fusion of TAT sequence to C3 transferase imposed no appreciable effects on its biological activity in promoting neurite outgrowth

Combined glutathione S transferase M1/T1 null genotypes is associated with type 2 diabetes mellitus

PubMed Central

POROJAN, MIHAI D.; BALA, CORNELIA; ILIES, ROXANA; CATANA, ANDREEA; POPP, RADU A.; DUMITRASCU, DAN L.

2015-01-01

Background Due to new genetic insights, a considerably large number of genes and polymorphic gene variants are screened and linked with the complex pathogenesis of type 2 diabetes (DM). Our study aimed to investigate the association between the two isoforms of the glutathione S-transferase genes (Glutathione S transferase isoemzyme type M1- GSTM1 and Glutathione S transferase isoemzyme type T1-GSTT1) and the prevalence of DM in the Northern Romanian population. Methods We conducted a cross-sectional, randomized, case-control study evaluating the frequency of GSTM1 and GSTT1 null alleles in patients diagnosed with DM. A total of 106 patients diagnosed with DM and 124 healthy controls were included in the study. GSTM1 and GSTT1 null alleles genotyping was carried out using Multiplex PCR amplification of relevant gene fragments, followed by gel electrophoresis analysis of the resulting amplicons. Results Molecular analysis did not reveal an increased frequency of the null GSTM1 and GSTT1 alleles (mutant genotypes) respectively in the DM group compared to controls (p=0.171, OR=1.444 CI=0.852–2.447; p=0.647, OR=0.854, CI=0.436–1.673). Nevertheless, the combined GSTM1/GSTT1 null genotypes were statistically significantly higher in DM patients compared to control subjects (p=0.0021, OR=0.313, CI=0.149–0.655) Conclusions The main finding of our study is that the combined, double GSTM1/GSTT1 null genotypes are to be considered among the polymorphic genetic risk factors for type 2 DM. PMID:26528065

Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro.

PubMed

Trombley, Michael P; Post, Deborah M B; Rinker, Sherri D; Reinders, Lorri M; Fortney, Kate R; Zwickl, Beth W; Janowicz, Diane M; Baye, Fitsum M; Katz, Barry P; Spinola, Stanley M; Bauer, Margaret E

2015-01-01

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.

Computer-Aided Structural Engineering (CASE) Project: State of the Art on Expert Systems Applications in Design, Construction and Maintenance of Structures

DTIC Science & Technology

1989-09-01

OGT, F1EPQRTJTL4, W" - 3^ n"r-- n *ON EXPERT SYSTEMS IN DESIGN, CONSTRUCTION AND’, IWAJNTENANCE-OF STRUCTURES Arockiasamy, Sunghoon Lee Clepartrhent...based expert system applications in the areas of structural design, design standards, and construction planning. This study will aid in the development...of a comprehensive expert system for tvical hydraulic structures. Funding for this report was provided by the US Army Engineer Waterways Experiment

Dexamethasone-Induced Skeletal Muscle Atrophy Increases O-GlcNAcylation in C2C12 Cells.

PubMed

Massaccesi, Luca; Goi, Giancarlo; Tringali, Cristina; Barassi, Alessandra; Venerando, Bruno; Papini, Nadia

2016-08-01

Skeletal muscle atrophy is a well-known adverse effect of chronic treatment with glucocorticoids and it also occurs when stress conditions such as sepsis and cachexia increase the release of endogenous glucocorticoids. Although the mechanisms of action of these hormones have been elucidated, the possible molecular mechanisms causing atrophy are not yet fully understood. The involvement of the O-GlcNAcylation process has recently been reported in disuse atrophy. O-GlcNAcylation, a regulatory post-translational modification of nuclear and cytoplasmic proteins consists in the attachment of O-GlcNAc residues on cell proteins and is regulated by two enzymes: O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA). O-GlcNAcylation plays a crucial role in many cellular processes and it seems to be related to skeletal muscle physiological function. The aim of this study is to investigate the involvement of O-GlcNAcylation in glucocorticoid-induced atrophy by using an "in vitro" model, achieved by treatment of C2C12 with 10 μM dexamethasone for 48 h. In atrophic condition, we observed that O-GlcNAc levels in cell proteins increased and concomitantly protein phosphorylation on serine and threonine residues decreased. Analysis of OGA expression at mRNA and protein levels showed a reduction in this enzyme in atrophic myotubes, whereas no significant changes of OGT expression were found. Furthermore, inhibition of OGA activity by Thiamet G induced atrophy marker expression. Our current findings suggest that O-GlcNAcylation is involved in dexamethasone-induced atrophy. In particular, we propose that the decrease in OGA content causes an excessive and mostly durable level of O-GlcNAc residues on sarcomeric proteins that might modify their function and stability. J. Cell. Biochem. 117: 1833-1842, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Characterization of a grape class IV chitinase.

PubMed

Vincenzi, Simone; Bierma, Jan; Wickramasekara, Samanthi I; Curioni, Andrea; Gazzola, Diana; Bakalinsky, Alan T

2014-06-18

A chitinase was purified from Vitis vinifera Manzoni Bianco grape juice and characterized. On the basis of proteomic analysis of tryptic peptides, a significant match identified the enzyme as a type IV grape chitinase previously found in juices of other V. vinifera varieties. The optimal pH and temperature for activity toward colloidal chitin were found to be 6 and 30 °C, respectively. The enzyme was found to hydrolyze chitin and oligomers of N-acetylglucosamine, generating N,N'-diacetylchitobiose and N-acetylglucosamine as products, but was inactive toward N,N'-diacetylchitobiose. The enzyme exhibited both endo- and exochitinase activities. Because yeast contains a small amount of chitin in the cell wall, the possibility of growth inhibition was tested. At a concentration and pH expected in ripe grapes, no inhibition of wine yeast growth by the chitinase was observed.

Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains.

PubMed

Li, Shan; Zhang, Li; Yao, Qing; Li, Lin; Dong, Na; Rong, Jie; Gao, Wenqing; Ding, Xiaojun; Sun, Liming; Chen, Xing; Chen, She; Shao, Feng

2013-09-12

The tumour necrosis factor (TNF) family is crucial for immune homeostasis, cell death and inflammation. These cytokines are recognized by members of the TNF receptor (TNFR) family of death receptors, including TNFR1 and TNFR2, and FAS and TNF-related apoptosis-inducing ligand (TRAIL) receptors. Death receptor signalling requires death-domain-mediated homotypic/heterotypic interactions between the receptor and its downstream adaptors, including TNFR1-associated death domain protein (TRADD) and FAS-associated death domain protein (FADD). Here we discover that death domains in several proteins, including TRADD, FADD, RIPK1 and TNFR1, were directly inactivated by NleB, an enteropathogenic Escherichia coli (EPEC) type III secretion system effector known to inhibit host nuclear factor-κB (NF-κB) signalling. NleB contained an unprecedented N-acetylglucosamine (GlcNAc) transferase activity that specifically modified a conserved arginine in these death domains (Arg 235 in the TRADD death domain). NleB GlcNAcylation (the addition of GlcNAc onto a protein side chain) of death domains blocked homotypic/heterotypic death domain interactions and assembly of the oligomeric TNFR1 complex, thereby disrupting TNF signalling in EPEC-infected cells, including NF-κB signalling, apoptosis and necroptosis. Type-III-delivered NleB also blocked FAS ligand and TRAIL-induced cell death by preventing formation of a FADD-mediated death-inducing signalling complex (DISC). The arginine GlcNAc transferase activity of NleB was required for bacterial colonization in the mouse model of EPEC infection. The mechanism of action of NleB represents a new model by which bacteria counteract host defences, and also a previously unappreciated post-translational modification.

Cloning and overexpression of beta-N-acetylglucosaminidase encoding gene nagA from Aspergillus oryzae and enzyme-catalyzed synthesis of human milk oligosaccharide.

PubMed

Matsuo, Ichiro; Kim, Sunhwa; Yamamoto, Yuichi; Ajisaka, Katsumi; Maruyama, Jun-ich; Nakajima, Harushi; Kitamoto, Katsuhiko

2003-03-01

We isolated a beta-N-acetylglucosaminidase encoding gene from the filamentous fungus Aspergillus oryzae, and designated it nagA. The nagA gene encoded a polypeptide of 600 amino acids with significant similarity to glucosaminidases and hexosaminidases of various eukaryotes. A. oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture. The beta-N-acetylglucosaminidase was purified from crude extracts of the solid culture by column chromatographies on Q-Sepharose and Sephacryl S-200. This enzyme was used for synthesis of lacto-N-triose II, which is contained in human milk. By reverse hydrolysis reaction, lacto-N-triose II and its positional isomer were synthesized from lactose and D-N-acetylglucosamine in 0.21% and 0.15% yield, respectively.

The Drosophila protein palmitoylome: Characterizing palmitoyl-thioesterases and DHHC palmitoyl-transferases

PubMed Central

Bannan, Barbra A.; Van Etten, Jamie; Kohler, John A.; Tsoi, Yui; Hansen, Nicole M.; Sigmon, Stacey; Fowler, Elizabeth; Buff, Haley; Williams, Tiffany S.; Ault, Jeffrey G.; Glaser, Robert L.; Korey, Christopher A.

2010-01-01

Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization, RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627, and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male-specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2), and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation. PMID:18719403

Glutathione S-transferases act as isomerases in isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

PubMed

Chen, H; Juchau, M R

1997-11-01

A discovery that rapid enzymic isomerization of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) can be catalysed by purified hepatic glutathione S-transferases (GSTs; EC 2.5.1.18) from rat is now reported. Rates of cis-trans isomerization were determined quantitatively by HPLC. GST-catalysed reactions reached equilibrium rapidly, in marked contrast with uncatalysed or GSH-catalysed isomerizations. The GST-catalysed reaction exhibited substrate saturation kinetics with a Km of approx. 8 microM. The maximal velocity of the reaction and the catalytic efficiency of GSTs were determined. The initial rate of the reaction increased linearly as a function of enzyme concentration. Catalysis by GSTs was independent of the presence of GSH, indicating that GSTs act as GSH-independent isomerases as well as transferases. Incubation with guanidine (7-8 M) or heat-inactivation of GSTs (100 degrees C for 3 min) decreased isomerase activities by approx. 50% and 75% respectively. The same heat treatment did not significantly inhibit isomerization catalysed by GSH and apoferritin, indicating that the observed decrease in isomerase activity by heat inactivation was not primarily due to oxidation of protein thiol groups in the GSTs. The specific activity of GSTs was approx. 23- and 340-fold those of GSH and apoferritin respectively when comparisons were made on the basis of free thiol concentrations, indicating that free thiol in GSTs cannot account for the majority of observed isomerase activities and suggesting that specific conformations of GSTs are important for such activities. Complete inhibition of the reaction by low concentrations of N-ethylmaleimide (10 microM) demonstrated that intact protein thiols are required for the isomerase activities of GSTs.

Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in Haloarcula hispanica.

PubMed

Borjian, Farshad; Johnsen, Ulrike; Schönheit, Peter; Berg, Ivan A

2017-01-01

Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria ) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ∼40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to β-methylmalyl-CoA. The cleavage of β-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C 4 -dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica , succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii , and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as

Bacterial synthesis of N-hydroxycinnamoyl phenethylamines and tyramines.

PubMed

Sim, Geun Young; Yang, So-Mi; Kim, Bong Gyu; Ahn, Joong-Hoon

2015-10-13

Hydroxycinnamic acids (HCAs) including cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid, are C6-C3 phenolic compounds that are synthesized via the phenylpropanoid pathway. HCAs serve as precursors for the synthesis of lignins, flavonoids, anthocyanins, stilbenes and other phenolic compounds. HCAs can also be conjugated with diverse compounds including quinic acid, hydroxyl acids, and amines. Hydroxycinnamoyl (HC) amine conjugates such as N-HC tyramines and N-HC phenethylamines have been considered as potential starting materials to develop antiviral and anticancer drugs. We synthesized N-HC tyramines and N-HC phenethylamines using three different approaches in Escherichia coli. Five N-HC phenethylamines and eight N-HC tyramines were synthesized by feeding HCAs and phenethylamine or tyramine to E. coli harboring 4CL (encoding 4-coumarate CoA:ligase) and either SHT (encoding phenethylamine N-HC transferase) or THT (encoding tyramine N-HC transferase). Also, N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine were synthesized from p-coumaric acid using E. coli harboring an additional gene, PDC (encoding phenylalanine decarboxylase) or TDC (encoding tyrosine decarboxylase). Finally, we synthesized N-(p-coumaroyl) phenethylamine and N-(p-coumaroyl) tyramine from glucose by reconstructing the metabolic pathways for their synthesis in E. coli. Productivity was maximized by optimizing the cell concentration and incubation temperature. We reconstructed the metabolic pathways for synthesis of N-HC tyramines and N-HC phenethylamines by expressing several genes including 4CL, TST or SHT, PDC or TDC, and TAL (encoding tyrosine ammonia lyase) and engineering the shikimate metabolic pathway to increase endogenous tyrosine concentration in E. coli. Approximately 101.9 mg/L N-(p-coumaroyl) phenethylamine and 495.4 mg/L N-(p-coumaroyl) tyramine were synthesized from p-coumaric acid. Furthermore, 152.5 mg/L N-(p-coumaroyl) phenethylamine and 94.7 mg/L N

The synthesis of ethacrynic acid thiazole derivatives as glutathione S-transferase pi inhibitors.

PubMed

Li, Ting; Liu, Guyue; Li, Hongcai; Yang, Xinmei; Jing, Yongkui; Zhao, Guisen

2012-04-01

Glutathione S-transferase pi (GSTpi) is a phase II enzyme which protects cells from death and detoxifies chemotherapeutic agents in cancer cells. Ethacrynic acid (EA) is a weak GSTpi inhibitor. Structure modifications were done to improve the ability of EA to inhibit GSTpi activity. Eighteen EA thiazole derivatives were designed and synthesized. Compounds 9a, 9b and 9c with a replacement of carboxyl group of EA by a heterocyclic thiazole exhibited improvement over EA to inhibit GSTpi activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

Effects of high-intensity intermittent training on carnitine palmitoyl transferase activity in the gastrocnemius muscle of rats

PubMed Central

Carnevali, L.C.; Eder, R.; Lira, F.S.; Lima, W.P.; Gonçalves, D.C.; Zanchi, N.E.; Nicastro, H.; Lavoie, J.M.; Seelaender, M.C.L.

2012-01-01

We examined the capacity of high-intensity intermittent training (HI-IT) to facilitate the delivery of lipids to enzymes responsible for oxidation, a task performed by the carnitine palmitoyl transferase (CPT) system in the rat gastrocnemius muscle. Male adult Wistar rats (160-250 g) were randomly distributed into 3 groups: sedentary (Sed, N = 5), HI-IT (N = 10), and moderate-intensity continuous training (MI-CT, N = 10). The trained groups were exercised for 8 weeks with a 10% (HI-IT) and a 5% (MI-CT) overload. The HI-IT group presented 11.8% decreased weight gain compared to the Sed group. The maximal activities of CPT-I, CPT-II, and citrate synthase were all increased in the HI-IT group compared to the Sed group (P < 0.01), as also was gene expression, measured by RT-PCR, of fatty acid binding protein (FABP; P < 0.01) and lipoprotein lipase (LPL; P < 0.05). Lactate dehydrogenase also presented a higher maximal activity (nmol·min−1·mg protein−1) in HI-IT (around 83%). We suggest that 8 weeks of HI-IT enhance mitochondrial lipid transport capacity thus facilitating the oxidation process in the gastrocnemius muscle. This adaptation may also be associated with the decrease in weight gain observed in the animals and was concomitant to a higher gene expression of both FABP and LPL in HI-IT, suggesting that intermittent exercise is a “time-efficient” strategy inducing metabolic adaptation. PMID:22735180

One-to-One Computing and Student Achievement in Ohio High Schools

ERIC Educational Resources Information Center

Williams, Nancy L.; Larwin, Karen H.

2016-01-01

This study explores the impact of one-to-one computing on student achievement in Ohio high schools as measured by performance on the Ohio Graduation Test (OGT). The sample included 24 treatment schools that were individually paired with a similar control school. An interrupted time series methodology was deployed to examine OGT data over a period…

ROLE OF STEROID HORMONES AND DECIDUAL INDUCTION IN THE REGULATION OF ADENOSINE DIPHOSPHORIBOSYL TRANSFERASE ACTIVITY IN RAT ENDOMETRIUM

EPA Science Inventory

To assess the effect of ovarian steroid hormones on enzyme activity, adenosine diphosphoribosyl transferase (ADPRT) was measured in endometrial nuclei isolated on estrus and on d 4 from rats ovariectomized on estrus (d 0) and treated d 0-3 with (a) vehicle, (b) 1 ug estrone/d (E)...

BIOTRANSFORMATION AND GENOTOXICITY OF THE DRINKING WATER DISINFECTION BYPRODUCT BROMODICHLOROMETHANE: DNA BINDING MEDIATED BY GLUTATHIONE TRANSFERASE THETA 1-1

EPA Science Inventory

The drinking water disinfection byproduct bromodichloromethane (CHBrCl2) was
previously shown to be mutagenic in Salmonella typhimurium that overexpress rat glutathione
transferase theta 1-1 (GSTT1-1). Several experimental approaches were undertaken in this study
to inve...

Dimethyl adenosine transferase (KsgA) deficiency in Salmonella Enteritidis confers susceptibility to high osmolarity and virulence attenuation in chickens

USDA-ARS?s Scientific Manuscript database

: Dimethyladenosine transferase (KsgA) performs diverse roles in bacteria including ribosomal maturation, DNA mismatch repair, and synthesis of KsgA is responsive to antibiotics and cold temperature. We previously showed that ksgA mutation in Salmonella Enteritidis results in impaired invasiveness i...

A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

PubMed

Singh, Sudhir; Singh, Chhaya; Tripathi, Anil Kumar

2014-05-01

The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

The in vivo Pig-a gene mutation assay, a potential tool for regulatory safety assessment.

PubMed

Dobrovolsky, Vasily N; Miura, Daishiro; Heflich, Robert H; Dertinger, Stephen D

2010-01-01

The Pig-a (phosphatidylinositol glycan, Class A) gene codes for a catalytic subunit of the N-acetylglucosamine transferase complex involved in an early step of glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Pig-a is the only gene involved in GPI anchor synthesis that is on the X-chromosome, and research into the origins of an acquired genetic disease involving GPI anchor deficiency (paroxysmal nocturnal hemoglobinuria) indicates that cells lacking GPI anchors, or GPI-anchored cell surface proteins, almost always have mutations in the Pig-a gene. These properties of the Pig-a gene and the GPI anchor system have been exploited in a series of assays for measuring in vivo gene mutation in blood cells from humans, rats, mice, and monkeys. In rats, flow cytometric measurement of Pig-a mutation in red blood cells requires microliter volumes of blood and data can be generated in hours. Spontaneous mutant frequencies are relatively low (<5 × 10(-6)) and rats treated with multiple doses of the potent mutagen, N-ethyl-N-nitrosourea, display Pig-a mutant frequencies that are close to the sum of the frequencies produced by the individual exposures. A general observation is that induced mutant frequencies are manifested earlier in reticulocytes (about 2 weeks after treatment) than in total red blood cells (about 2 months after exposure). Based on data from a limited number of test agents, the assay shows promise for regulatory applications, including integration of gene mutation measurement into repeat-dose toxicology studies.

MOF Acetyl Transferase Regulates Transcription and Respiration in Mitochondria.

PubMed

Chatterjee, Aindrila; Seyfferth, Janine; Lucci, Jacopo; Gilsbach, Ralf; Preissl, Sebastian; Böttinger, Lena; Mårtensson, Christoph U; Panhale, Amol; Stehle, Thomas; Kretz, Oliver; Sahyoun, Abdullah H; Avilov, Sergiy; Eimer, Stefan; Hein, Lutz; Pfanner, Nikolaus; Becker, Thomas; Akhtar, Asifa

2016-10-20

A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

Transgenic expression of a maize geranyl geranyl transferase gene sequence in maize callus increases resistance to ear rot pathogens

USDA-ARS?s Scientific Manuscript database

Determining the genes responsible for pest resistance in maize can allow breeders to develop varieties with lower losses and less contamination with undesirable toxins. A gene sequence coding for a geranyl geranyl transferase-like protein located in a fungal ear rot resistance quantitative trait loc...

The N-end rule pathway and regulation by proteolysis

PubMed Central

Varshavsky, Alexander

2011-01-01

The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. The main determinant of an N-degron is a destabilizing N-terminal residue of a protein. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system and consists of two branches, the Ac/N-end rule and the Arg/N-end rule pathways. The Ac/N-end rule pathway targets proteins containing Nα-terminally acetylated (Nt-acetylated) residues. The Arg/N-end rule pathway recognizes unacetylated N-terminal residues and involves N-terminal arginylation. Together, these branches target for degradation a majority of cellular proteins. For example, more than 80% of human proteins are cotranslationally Nt-acetylated. Thus, most proteins harbor a specific degradation signal, termed AcN-degron, from the moment of their birth. Specific N-end rule pathways are also present in prokaryotes and in mitochondria. Enzymes that produce N-degrons include methionine-aminopeptidases, caspases, calpains, Nt-acetylases, Nt-amidases, arginyl-transferases, and leucyl-transferases. Regulated degradation of specific proteins by the N-end rule pathway mediates a legion of physiological functions, including the sensing of heme, oxygen, and nitric oxide; selective elimination of misfolded proteins; the regulation of DNA repair, segregation, and condensation; the signaling by G proteins; the regulation of peptide import, fat metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis, neurogenesis, and cardiovascular development; and the functioning of adult organs, including the pancreas and the brain. Discovered 25 years ago, this pathway continues to be a fount of biological insights. PMID:21633985

Downregulation of glutathione S-transferase pi in asthma contributes to enhanced oxidative stress.

PubMed

Schroer, Kathy T; Gibson, Aaron M; Sivaprasad, Umasundari; Bass, Stacey A; Ericksen, Mark B; Wills-Karp, Marsha; Lecras, Tim; Fitzpatrick, Anne M; Brown, Lou Ann S; Stringer, Keith F; Hershey, Gurjit K Khurana

2011-09-01

Glutathione S-transferase pi (GSTPi) is the predominant redox regulator in the lung. Although evidence implicates an important role for GSTPi in asthma, the mechanism for this has remained elusive. We sought to determine how GSTPi is regulated in asthma and to elucidate its role in maintaining redox homeostasis. We elucidated the regulation of GSTPi in children with asthma and used murine models of asthma to determine the role of GSTPi in redox homeostasis. Our findings demonstrate that GSTPi transcript levels are markedly downregulated in allergen- and IL-13-treated murine models of asthma through signal transducer and activator of transcription 6-dependent and independent pathways. Nuclear factor erythroid 2-related factor 2 was also downregulated in these models. The decrease in GSTPi expression was associated with decreased total glutathione S-transferase activity in the lungs of mice. Examination of cystine intermediates uncovered a functional role for GSTPi in regulating cysteine oxidation, whereby GSTPi-deficient mice exhibited increased oxidative stress (increase in percentage cystine) compared with wild-type mice after allergen challenge. GSTPi expression was similarly downregulated in children with asthma. These data collectively suggest that downregulation of GSTPi after allergen challenge might contribute to the asthma phenotype because of disruption of redox homeostasis and increased oxidative stress. Furthermore, GSTPi might be an important therapeutic target for asthma, and evaluation of GSTPi expression might prove beneficial in identifying patients who would benefit from therapy targeting this pathway. Copyright © 2011 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

The effects of endosulfan on cytochrome P450 enzymes and glutathione S-transferases in zebrafish (Danio rerio) livers.

PubMed

Dong, Miao; Zhu, Lusheng; Shao, Bo; Zhu, Shaoyuan; Wang, Jun; Xie, Hui; Wang, Jinhua; Wang, Fenghua

2013-06-01

Endosulfan, an organochlorine pesticide, has been used worldwide in the past decades. The present study was performed to investigate the effect of endosulfan on liver microsomal cytochrome P450 (CYP) enzymes and glutathione S-transferases (GST) in zebrafish. Male and female zebrafish were separated and exposed to a control and four concentrations of endosulfan (0.01, 0.1, 1, and 10μgL(-1)) and were sampled on days 7, 14, 21, and 28. After exposure to endosulfan, the content of CYP increased and later gradually fell back to control level in most sampling time intervals. A similar tendency was also found in the activities of NADPH-P450 reductase (NCR), aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND). GST activities were generally higher in treatment groups than control groups. Regarding sex-based differences, the induction degree of the activity of NCR was generally higher in males than females. Similar differences were also found on the 28th day in the activities of APND and ERND, as well as GST activity on the 7th day. Overall, the present results demonstrate the toxicity at low doses of endosulfan and indicated marked induction of CYP and GST enzymes in zebrafish liver. Copyright © 2012 Elsevier Inc. All rights reserved.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

PubMed

Matsumoto, M; Imagawa, M; Aoki, Y

2000-07-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression.

Epidermal growth factor regulation of glutathione S-transferase gene expression in the rat is mediated by class Pi glutathione S-transferase enhancer I.

PubMed Central

Matsumoto, M; Imagawa, M; Aoki, Y

2000-01-01

Using chloramphenicol acetyltransferase assays we showed that epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), and 3,3',4,4',5-pentachlorobiphenyl (PenCB) induce class Pi glutathione S-transferase (GSTP1) in primary cultured rat liver parenchymal cells. GSTP1 enhancer I (GPEI), which is required for the stimulation of GSTP1 expression by PenCB, also mediates EGF and TGF alpha stimulation of GSTP1 gene expression. However, hepatocyte growth factor and insulin did not stimulate GPEI-mediated gene expression. On the other hand, the antioxidant reagents butylhydroxyanisole and t-butylhydroquinone, stimulated GPEI-mediated gene expression, but the level of GSTP1 mRNA was not elevated. Our observations suggest that EGF and TGF alpha induce GSTP1 by the same signal transduction pathway as PenCB. Since the sequence of GPEI is similar to that of the antioxidant responsive element (ARE), some factors which bind to ARE might play a role in GPEI-mediated gene expression. PMID:10861232

Glutathione S-transferases as antioxidant defence agents confer pyrethroid resistance in Nilaparvata lugens.

PubMed Central

Vontas, J G; Small, G J; Hemingway, J

2001-01-01

Selection of a laboratory colony of the brown planthopper Nilaparvata lugens with the pyrethroids permethrin and lambda-cyhalothrin increased its resistance to both insecticides. Biochemical analysis and synergistic studies with metabolic inhibitors indicated that elevated glutathione S-transferases (GSTs) with a predominant peroxidase activity conferred resistance to both pyrethroids, whereas esterases conferred part of the resistance to permethrin. Purified esterases hydrolysed permethrin at a slow rate, but incubation of either pyrethroid or their primary metabolites with partially purified GSTs had no effect on the metabolic profile. Although GSTs were sensitive to inhibition by both pyrethroids, they did not serve as binding proteins, as previously hypothesized [Grant and Matsumura (1988) Insect Biochem. 18, 615-622]. We demonstrate that pyrethroids, in addition to their neurotoxic effect, induce oxidative stress and lipid peroxidation in insects. Pyrethroid exposure induced lipid peroxides, protein oxidation and depleted reduced glutathione. Elevated GSTs in the resistant strains attenuated the pyrethroid-induced lipid peroxidation and reduced mortality, whereas their in vivo inhibition eliminated their protective role. We therefore hypothesize that the main role of elevated GSTs in conferring resistance in N. lugens is through protecting tissues from oxidative damage. Our study extends the GSTs' range of efficacy to pyrethroid insecticides and possibly explains the role of elevated GSTs in other pyrethroid-resistant insects. PMID:11415437

Sphingobium sp. SYK-6 LigG involved in lignin degradation is structurally and biochemically related to the glutathione transferase ω class.

PubMed

Meux, Edgar; Prosper, Pascalita; Masai, Eiji; Mulliert, Guillermo; Dumarçay, Stéphane; Morel, Mélanie; Didierjean, Claude; Gelhaye, Eric; Favier, Frédérique

2012-11-16

SpLigG is one of the three glutathione transferases (GSTs) involved in the process of lignin breakdown in the soil bacterium Sphingobium sp. SYK-6. Sequence comparisons showed that SpLigG and several proteobacteria homologues form an independent cluster within cysteine-containing GSTs. The relationship between SpLigG and other GSTs was investigated. The X-ray structure and biochemical properties of SpLigG indicate that this enzyme belongs to the omega class of glutathione transferases. However, the hydrophilic substrate binding site of SpLigG, together with its known ability to stereoselectively deglutathionylate the physiological substrate α-glutathionyl-β-hydroxypropiovanillone, argues for broadening the definition of the omega class. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Isolation and structures of glycoprotein-derived free oligosaccharides from the unfertilized eggs of Scyliorhinus caniculus. Characterization of the sequences galactose(alpha 1-4)galactose(beta 1-3)-N-acetylglucosamine and N-acetylneuraminic acid(alpha 2-6)galactose(beta 1-3)-N-acetylglucosamine.

PubMed

Plancke, Y; Delplace, F; Wieruszeski, J M; Maes, E; Strecker, G

1996-01-15

As previously reported [Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H. & Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K. & Inoue, Y. (1989) J. Biol. Chem. 264, 18520-185261, the unfertilized eggs of two different species of fresh-water fish, Plecoglossus altivelis and Tribodolon hakonensis, contain relatively large amounts of free sialooligosaccharides. These oligosaccharides were found to derive from glycophosphoproteins, owing to the activity of a peptide - N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase [Iwasaki, M., Seko, A., Kitajima, K., Inoue, Y. & Inoue, S. (1992) J. Biol. Chem. 267, 24287-24296; Seko, A., Kitajima, K., Inoue, Y. & Inoue, S. (1991) J. Biol. Chem. 266, 22110-22114]. Here we describe a new type of free oligosaccharides, isolated from unfertilized eggs of Scyliorhinus caniculus. From the structural analysis, based upon 1H-NMR spectroscopy, the following glycan units are proposed.[Formula: see text

Function and phylogeny of bacterial butyryl-CoA:acetate transferases and their diversity in the proximal colon of swine

USDA-ARS?s Scientific Manuscript database

Studying the host-associated butyrate-producing bacterial community is important because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl-coA:acetate transferase (2.3.8.3) as a the main gene for butyrate production in intestinal ecosystems; h...

Glutathione S-transferases act as isomerases in isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

PubMed Central

Chen, H; Juchau, M R

1997-01-01

A discovery that rapid enzymic isomerization of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) can be catalysed by purified hepatic glutathione S-transferases (GSTs; EC 2.5.1.18) from rat is now reported. Rates of cis-trans isomerization were determined quantitatively by HPLC. GST-catalysed reactions reached equilibrium rapidly, in marked contrast with uncatalysed or GSH-catalysed isomerizations. The GST-catalysed reaction exhibited substrate saturation kinetics with a Km of approx. 8 microM. The maximal velocity of the reaction and the catalytic efficiency of GSTs were determined. The initial rate of the reaction increased linearly as a function of enzyme concentration. Catalysis by GSTs was independent of the presence of GSH, indicating that GSTs act as GSH-independent isomerases as well as transferases. Incubation with guanidine (7-8 M) or heat-inactivation of GSTs (100 degrees C for 3 min) decreased isomerase activities by approx. 50% and 75% respectively. The same heat treatment did not significantly inhibit isomerization catalysed by GSH and apoferritin, indicating that the observed decrease in isomerase activity by heat inactivation was not primarily due to oxidation of protein thiol groups in the GSTs. The specific activity of GSTs was approx. 23- and 340-fold those of GSH and apoferritin respectively when comparisons were made on the basis of free thiol concentrations, indicating that free thiol in GSTs cannot account for the majority of observed isomerase activities and suggesting that specific conformations of GSTs are important for such activities. Complete inhibition of the reaction by low concentrations of N-ethylmaleimide (10 microM) demonstrated that intact protein thiols are required for the isomerase activities of GSTs. PMID:9581548

THE PRESENCE OF A GROUP A VARIANT-LIKE ANTIGEN IN STREPTOCOCCI OF OTHER GROUPS WITH SPECIAL REFERENCE TO GROUP N

PubMed Central

Elliott, S. D.; Hayward, John; Liu, T. Y.

1971-01-01

A Group A variant-like antigen has been detected in streptococci belonging to Groups D, E, G, M, and N. In Groups D and N the variant-like antigen was located in the streptococcal cell walls. In two strains of Group N streptococci (C559 and B209) the cell walls were chemically different and serologically distinct. In strain C559 N-acetylgalactosamine, and in strain B209, N-acetylglucosamine were the major determinants of serological specificity. The cell walls of strain C559 contained at least three serologically reactive components: a rhamnose-containing fraction that precipitated with an antiserum to Group A-variant carbohydrate; a strain-specific polysaccharide composed of galactosamine and glucosamine, both in the N-acetylated form and probably polymerized with an unidentified phosphorylated substance; and a component of unknown composition serologically related to a Group D streptococcus strain C3 (S. durans). An analogy is drawn between the cell wall structure in streptococcus and Salmonella. PMID:5111438

Characterization of a Grape Class IV Chitinase

PubMed Central

2015-01-01

A chitinase was purified from Vitis vinifera Manzoni Bianco grape juice and characterized. On the basis of proteomic analysis of tryptic peptides, a significant match identified the enzyme as a type IV grape chitinase previously found in juices of other V. vinifera varieties. The optimal pH and temperature for activity toward colloidal chitin were found to be 6 and 30 °C, respectively. The enzyme was found to hydrolyze chitin and oligomers of N-acetylglucosamine, generating N,N′-diacetylchitobiose and N-acetylglucosamine as products, but was inactive toward N,N′-diacetylchitobiose. The enzyme exhibited both endo- and exochitinase activities. Because yeast contains a small amount of chitin in the cell wall, the possibility of growth inhibition was tested. At a concentration and pH expected in ripe grapes, no inhibition of wine yeast growth by the chitinase was observed. PMID:24845689

Thr105Ile (rs11558538) polymorphism in the histamine-1-methyl-transferase (HNMT) gene and risk for restless legs syndrome.

PubMed

Jiménez-Jiménez, Félix Javier; García-Martín, Elena; Alonso-Navarro, Hortensia; Martínez, Carmen; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Rojo-Sebastián, Ana; Rubio, Lluisa; Ortega-Cubero, Sara; Pastor, Pau; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Albea, Esteban; Agúndez, José A G

2017-03-01

A recent meta-analysis suggests an association between the rs11558538 single nucleotide polymorphism in the histamine-N-methyl-transferase (HNMT) gene and the risk for Parkinson's disease. Based on the possible relationship between PD and restless legs syndrome (RLS), we tried to establish whether rs11558538 SNP is associated with the risk for RLS. We studied the genotype and allelic variant frequencies of HNMT rs11558538 SNP 205 RLS patients and 410 healthy controls using a TaqMan assay. The frequencies of the HNMT rs11558538 genotypes allelic variants were similar between RLS patients and controls, and were not influenced by gender, family history of RLS, or RLS severity. RLS patients carrying the genotype rs11558538TT had an earlier age at onset, but this finding was based on three subjects only. These results suggest a lack of major association between HNMT rs11558538 SNP and the risk for RLS.

N-Glycosylation Profiling of Porcine Reproductive and Respiratory Syndrome Virus Envelope Glycoprotein 5

PubMed Central

Li, Juan; Tao, Shujuan; Orlando, Ron; Murtaugh, Michael P.

2015-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense ssRNA virus whose envelope contains four glycoproteins and three nonglycosylated proteins. Glycans of major envelope glycoprotein 5 (GP5) are proposed as important for virus assembly and entry into permissive cells. Structural characterization of GP5 glycans would facilitate the mechanistic understanding of these processes. Thus, we purified the PRRSV type 2 prototype strain, VR2332, and analyzed the virion-associated glycans by both biochemical and mass spectrometric methods. Endoglycosidase digestion showed that GP5 was the primary protein substrate, and that the carbohydrate moieties were primarily complex-type N-glycans. Mass spectrometric analysis (HPLC-ESI-MS/MS) of GP5 N-glycans revealed an abundance of N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) oligomers in addition to sialic acids. GlcNAc and LacNAc accessibility to ligands was confirmed by lectin co-precipitation. Our findings help to explain PRRSV infection of cells lacking sialoadhesin and provide a glycan database to facilitate molecular structural studies of PRRSV. PMID:25726973

Host Glycan Sugar-Specific Pathways in Streptococcus pneumonia: Galactose as a Key Sugar in Colonisation and Infection

PubMed Central

Paixão, Laura; Oliveira, Joana; Veríssimo, André; Vinga, Susana; Lourenço, Eva C.; Ventura, M. Rita; Kjos, Morten; Veening, Jan-Willem; Fernandes, Vitor E.; Andrew, Peter W.; Yesilkaya, Hasan; Neves, Ana Rute

2015-01-01

The human pathogen Streptococcus pneumoniae is a strictly fermentative organism that relies on glycolytic metabolism to obtain energy. In the human nasopharynx S. pneumoniae encounters glycoconjugates composed of a variety of monosaccharides, which can potentially be used as nutrients once depolymerized by glycosidases. Therefore, it is reasonable to hypothesise that the pneumococcus would rely on these glycan-derived sugars to grow. Here, we identified the sugar-specific catabolic pathways used by S. pneumoniae during growth on mucin. Transcriptome analysis of cells grown on mucin showed specific upregulation of genes likely to be involved in deglycosylation, transport and catabolism of galactose, mannose and N acetylglucosamine. In contrast to growth on mannose and N-acetylglucosamine, S. pneumoniae grown on galactose re-route their metabolic pathway from homolactic fermentation to a truly mixed acid fermentation regime. By measuring intracellular metabolites, enzymatic activities and mutant analysis, we provide an accurate map of the biochemical pathways for galactose, mannose and N-acetylglucosamine catabolism in S. pneumoniae. Intranasal mouse infection models of pneumococcal colonisation and disease showed that only mutants in galactose catabolic genes were attenuated. Our data pinpoint galactose as a key nutrient for growth in the respiratory tract and highlights the importance of central carbon metabolism for pneumococcal pathogenesis. PMID:25826206

Sialoglycoproteins and N-glycans from secreted exosomes of ovarian carcinoma cells.

PubMed

Escrevente, Cristina; Grammel, Nicolas; Kandzia, Sebastian; Zeiser, Johannes; Tranfield, Erin M; Conradt, Harald S; Costa, Júlia

2013-01-01

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.

Catalysis by the second class of tRNA(m1G37) methyl transferase requires a conserved proline.

PubMed

Christian, Thomas; Evilia, Caryn; Hou, Ya-Ming

2006-06-20

The enzyme tRNA(m1G37) methyl transferase catalyzes the transfer of a methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37, which is 3' to the anticodon sequence and whose modification is important for maintaining the reading frame fidelity. While the enzyme in bacteria is highly conserved and is encoded by the trmD gene, recent studies show that the counterpart of this enzyme in archaea and eukarya, encoded by the trm5 gene, is unrelated to trmD both in sequence and in structure. To further test this prediction, we seek to identify residues in the second class of tRNA(m1G37) methyl transferase that are required for catalysis. Such residues should provide mechanistic insights into the distinct structural origins of the two classes. Using the Trm5 enzyme of the archaeon Methanocaldococcus jannaschii (previously MJ0883) as an example, we have created mutants to test many conserved residues for their catalytic potential and substrate-binding capabilities with respect to both AdoMet and tRNA. We identified that the proline at position 267 (P267) is a critical residue for catalysis, because substitution of this residue severely decreases the kcat of the methylation reaction in steady-state kinetic analysis, and the k(chem) in single turnover kinetic analysis. However, substitution of P267 has milder effect on the Km and little effect on the Kd of either substrate. Because P267 has no functional side chain that can directly participate in the chemistry of methyl transfer, we suggest that its role in catalysis is to stabilize conformations of enzyme and substrates for proper alignment of reactive groups at the enzyme active site. Sequence analysis shows that P267 is embedded in a peptide motif that is conserved among the Trm5 family, but absent from the TrmD family, supporting the notion that the two families are descendants of unrelated protein structures.

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase π

PubMed Central

Ralat, Luis A.; Colman, Roberta F.

2003-01-01

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase π (GST π): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST π with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST π at pH 7.0 and 25°C as assayed using mBBr as substrate, with a lesser effect on the enzyme’s use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST π with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites. PMID:14573868

Monobromobimane occupies a distinct xenobiotic substrate site in glutathione S-transferase pi.

PubMed

Ralat, Luis A; Colman, Roberta F

2003-11-01

Monobromobimane (mBBr), functions as a substrate of porcine glutathione S-transferase pi (GST pi): The enzyme catalyzes the reaction of mBBr with glutathione. S-(Hydroxyethyl)bimane, a nonreactive analog of monobromobimane, acts as a competitive inhibitor with respect to mBBr as substrate but does not affect the reaction of GST pi with another substrate, 1-chloro-2,4-dinitrobenzene (CDNB). In the absence of glutathione, monobromobimane inactivates GST pi at pH 7.0 and 25 degrees C as assayed using mBBr as substrate, with a lesser effect on the enzyme's use of CDNB as substrate. These results indicate that the sites occupied by CDNB and mBBr are not identical. Inactivation is proportional to the incorporation of 2 moles of bimane/mole of subunit. Modification of GST pi with mBBr does not interfere with its binding of 8-anilino-1-naphthalene sulfonate, indicating that this hydrophobic site is not the target of monobromobimane. S-Methylglutathione and S-(hydroxyethyl)bimane each yield partial protection against inactivation and decrease reagent incorporation, while glutathionyl-bimane protects completely against inactivation. Peptide analysis after trypsin digestion indicates that mBBr modifies Cys45 and Cys99 equally. Modification of Cys45 is reduced in the presence of S-methylglutathione, indicating that this residue is at or near the glutathione binding region. In contrast, modification of Cys99 is reduced in the presence of S-(hydroxyethyl)bimane, suggesting that this residue is at or near the mBBr xenobiotic substrate binding site. Modification of Cys99 can best be understood by reaction with monobromobimane while it is bound to its xenobiotic substrate site in an alternate orientation. These results support the concept that glutathione S-transferase accomplishes its ability to react with a diversity of substrates in part by harboring distinct xenobiotic substrate sites.

S-Nitrosation destabilizes glutathione transferase P1-1.

PubMed

Balchin, David; Stoychev, Stoyan H; Dirr, Heini W

2013-12-23

Protein S-nitrosation is a post-translational modification that regulates the function of more than 500 human proteins. Despite its apparent physiological significance, S-nitrosation is poorly understood at a molecular level. Here, we investigated the effect of S-nitrosation on the activity, structure, stability, and dynamics of human glutathione transferase P1-1 (GSTP1-1), an important detoxification enzyme ubiquitous in aerobes. S-Nitrosation at Cys47 and Cys101 reduces the activity of the enzyme by 94%. Circular dichroism spectroscopy, acrylamide quenching, and amide hydrogen-deuterium exchange mass spectrometry experiments indicate that the loss of activity is caused by the introduction of local disorder at the active site of GSTP1-1. Furthermore, the modification destabilizes domain 1 of GSTP1-1 against denaturation, smoothing the unfolding energy landscape of the protein and introducing a refolding defect. In contrast, S-nitrosation at Cys101 alone introduces a refolding defect in domain 1 but compensates by stabilizing the domain kinetically. These data elucidate the physical basis for the regulation of GSTP1-1 by S-nitrosation and provide general insight into the consequences of S-nitrosation on protein stability and dynamics.

Madumycin II inhibits peptide bond formation by forcing the peptidyl transferase center into an inactive state

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osterman, Ilya A.; Khabibullina, Nelli F.; Komarova, Ekaterina S.

The emergence of multi-drug resistant bacteria is limiting the effectiveness of commonly used antibiotics, which spurs a renewed interest in revisiting older and poorly studied drugs. Streptogramins A is a class of protein synthesis inhibitors that target the peptidyl transferase center (PTC) on the large subunit of the ribosome. In this work, we have revealed the mode of action of the PTC inhibitor madumycin II, an alanine-containing streptogramin A antibiotic, in the context of a functional 70S ribosome containing tRNA substrates. Madumycin II inhibits the ribosome prior to the first cycle of peptide bond formation. It allows binding of themore » tRNAs to the ribosomal A and P sites, but prevents correct positioning of their CCA-ends into the PTC thus making peptide bond formation impossible. We also revealed a previously unseen drug-induced rearrangement of nucleotides U2506 and U2585 of the 23S rRNA resulting in the formation of the U2506•G2583 wobble pair that was attributed to a catalytically inactive state of the PTC. The structural and biochemical data reported here expand our knowledge on the fundamental mechanisms by which peptidyl transferase inhibitors modulate the catalytic activity of the ribosome.« less

Introducing N-glycans into natural products through a chemoenzymatic approach.

PubMed

Huang, Wei; Ochiai, Hirofumi; Zhang, Xinyu; Wang, Lai-Xi

2008-11-24

The present study describes an efficient chemoenzymatic method for introducing a core N-glycan of glycoprotein origin into various lipophilic natural products. It was found that the endo-beta-N-acetylglucosaminidase from Arthrobactor protophormiae (Endo-A) had broad substrate specificity and can accommodate a wide range of glucose (Glc)- or N-acetylglucosamine (GlcNAc)-containing natural products as acceptors for transglycosylation, when an N-glycan oxazoline was used as a donor substrate. Using lithocholic acid as a model compound, we have shown that introduction of an N-glycan could be achieved by a two-step approach: chemical glycosylation to introduce a monosaccharide (Glc or GlcNAc) as a handle, and then Endo-A catalyzed transglycosylation to accomplish the site-specific N-glycan attachment. For those natural products that already carry terminal Glc or GlcNAc residues, direct enzymatic transglycosylation using sugar oxazoline as the donor substrate was achievable to introduce an N-glycan. It was also demonstrated that simultaneous double glycosylation could be fulfilled when the natural product contains two Glc residues. This chemoenzymatic method is concise, site-specific, and highly convergent. Because N-glycans of glycoprotein origin can serve as ligands for diverse lectins and cell-surface receptors, introduction of a defined N-glycan into biologically significant natural products may bestow novel properties onto these natural products for drug discovery and development.

Novosphingobium aromaticivorans uses a Nu-class glutathione S-transferase as a glutathione lyase in breaking the β-aryl ether bond of lignin

PubMed Central

Kontur, Wayne S.; Bingman, Craig A.; Olmsted, Charles N.; Wassarman, Douglas R.; Ulbrich, Arne; Gall, Daniel L.; Smith, Robert W.; Yusko, Larissa M.; Fox, Brian G.; Noguera, Daniel R.; Coon, Joshua J.; Donohue, Timothy J.

2018-01-01

As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the β-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the β-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-β-guaiacyl ether (GGE) and found that Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate β-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S-transferase NaGSTNu is the only enzyme needed to remove glutathione from both (R)- and (S)-GS-HPV in N. aromaticivorans. We solved the crystal structure of NaGSTNu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr51, Tyr166, and Tyr224) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6 (which can also break the β-aryl ether bond) and Escherichia coli (which cannot break the β-aryl ether bond) can also cleave (R)- and (S)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S-transferases. PMID:29449375

O-GlcNAcase Fragment Discovery with Fluorescence Polarimetry.

PubMed

Borodkin, Vladimir S; Rafie, Karim; Selvan, Nithya; Aristotelous, Tonia; Navratilova, Iva; Ferenbach, Andrew T; van Aalten, Daan M F

2018-05-18

The attachment of the sugar N-acetyl-D-glucosamine (GlcNAc) to specific serine and threonine residues on proteins is referred to as protein O-GlcNAcylation. O-GlcNAc transferase (OGT) is the enzyme responsible for carrying out the modification, while O-GlcNAcase (OGA) reverses it. Protein O-GlcNAcylation has been implicated in a wide range of cellular processes including transcription, proteostasis, and stress response. Dysregulation of O-GlcNAc has been linked to diabetes, cancer, and neurodegenerative and cardiovascular disease. OGA has been proposed to be a drug target for the treatment of Alzheimer's and cardiovascular disease given that increased O-GlcNAc levels appear to exert a protective effect. The search for specific, potent, and drug-like OGA inhibitors with bioavailability in the brain is therefore a field of active research, requiring orthogonal high-throughput assay platforms. Here, we describe the synthesis of a novel probe for use in a fluorescence polarization based assay for the discovery of inhibitors of OGA. We show that the probe is suitable for use with both human OGA, as well as the orthologous bacterial counterpart from Clostridium perfringens, CpOGA, and the lysosomal hexosaminidases HexA/B. We structurally characterize CpOGA in complex with a ligand identified from a fragment library screen using this assay. The versatile synthesis procedure could be adapted for making fluorescent probes for the assay of other glycoside hydrolases.

N-Acetylglucosamine Inhibits LuxR, LasR and CviR Based Quorum Sensing Regulated Gene Expression Levels

PubMed Central

Kimyon, Önder; Ulutürk, Zehra İ.; Nizalapur, Shashidhar; Lee, Matthew; Kutty, Samuel K.; Beckmann, Sabrina; Kumar, Naresh; Manefield, Mike

2016-01-01

N-acetyl glucosamine, the monomer of chitin, is an abundant source of carbon and nitrogen in nature as it is the main component and breakdown product of many structural polymers. Some bacteria use N-acyl-L-homoserine lactone (AHL) mediated quorum sensing (QS) to regulate chitinase production in order to catalyze the cleavage of chitin polymers into water soluble N-acetyl-D-glucosamine (NAG) monomers. In this study, the impact of NAG on QS activities of LuxR, LasR, and CviR regulated gene expression was investigated by examining the effect of NAG on QS regulated green fluorescent protein (GFP), violacein and extracellular chitinase expression. It was discovered that NAG inhibits AHL dependent gene transcription in AHL reporter strains within the range of 50–80% reduction at low millimolar concentrations (0.25–5 mM). Evidence is presented supporting a role for both competitive inhibition at the AHL binding site of LuxR type transcriptional regulators and catabolite repression. Further, this study shows that NAG down-regulates CviR induced violacein production while simultaneously up-regulating CviR dependent extracellular enzymes, suggesting that an unknown NAG dependent regulatory component influences phenotype expression. The quorum sensing inhibiting activity of NAG also adds to the list of compounds with known quorum sensing inhibiting activities. PMID:27602027

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singhal, Sharad S., E-mail: ssinghal@coh.org; Singh, Sharda P.; Singhal, Preeti

2015-12-15

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxidesmore » and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes — higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article. - Highlights: • GSTs are the major

Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis.

PubMed

Chaouch, Melek; Fathallah-Mili, Akila; Driss, Mehdi; Lahmadi, Ramzi; Ayari, Chiraz; Guizani, Ikram; Ben Said, Moncef; Benabderrazak, Souha

2013-03-01

Discrimination of the Old World Leishmania parasites is important for diagnosis and epidemiological studies of leishmaniasis. We have developed PCR assays that allow the discrimination between Leishmania major, Leishmania tropica and Leishmania infantum Tunisian species. The identification was performed by a simple PCR targeting cysteine protease B (cpb) gene copies. These PCR can be a routine molecular biology tools for discrimination of Leishmania spp. from different geographical origins and different clinical forms. Our assays can be an informative source for cpb gene studying concerning drug, diagnostics and vaccine research. The PCR products of the cpb gene and the N-acetylglucosamine-1-phosphate transferase (nagt) Leishmania gene were sequenced and aligned. Phylogenetic trees of Leishmania based cpb and nagt sequences are close in topology and present the classic distribution of Leishmania in the Old World. The phylogenetic analysis has enabled the characterization and identification of different strains, using both multicopy (cpb) and single copy (nagt) genes. Indeed, the cpb phylogenetic analysis allowed us to identify the Tunisian Leishmania killicki species, and a group which gathers the least evolved isolates of the Leishmania donovani complex, that was originated from East Africa. This clustering confirms the African origin for the visceralizing species of the L. donovani complex. Copyright © 2012 Elsevier B.V. All rights reserved.

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Low, Wai Yee; Feil, Susanne C.; Ng, Hooi Ling

2010-06-14

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 {angstrom} resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model ofmore » the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional {sup 1}H,{sup 15}N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT binding support the predicted binding site. We also show that the ancestral GSTD1 is likely to have possessed DDT dehydrochlorinase activity because both GSTD1 from D. melanogaster and its sibling species, Drosophila simulans, have this activity.« less

Testing the Effects of SIAH Ubiquitin E3 Ligases on Lysine Acetyl Transferases.

PubMed

Hagenbucher, Jan; Stekman, Hilda; Rodriguez-Gil, Alfonso; Kracht, Michael; Schmitz, M Lienhard

2017-01-01

The family of seven-in-absentia (SIAH) ubiquitin E3 ligases functions in the control of numerous key signaling pathways. These enzymes belong to the RING (really interesting new gene) group of E3 ligases and mediate the attachment of ubiquitin chains to substrates, which then leads to their proteasomal degradation. Here, we describe a protocol that allows measuring SIAH-mediated ubiquitination and degradation of its client proteins as exemplified by acetyl transferases using simple overexpression experiments. The impact of SIAH expression on the relative amounts of target proteins and their mRNAs can be quantified by Western blotting and quantitative PCR (qPCR) as described here.

Differential substrate behaviours of ethylene oxide and propylene oxide towards human glutathione transferase theta hGSTT1-1.

PubMed

Thier, R; Wiebel, F A; Bolt, H M

1999-11-01

The transformation of ethylene oxide (EO), propylene oxide (PO) and 1-butylene oxide (1-BuO) by human glutathione transferase theta (hGSTT1-1) was studied comparatively using 'conjugator' (GSTT1 + individuals) erythrocyte lysates. The relative sequence of velocity of enzymic transformation was PO > EO > 1-BuO. The faster transformation of PO compared to EO was corroborated in studies with human and rat GSTT1-1 (hGSTT1-1 and rGSTT1-1, respectively) expressed by Salmonella typhimurium TA1535. This sequence of reactivities of homologous epoxides towards GSTT1-1 contrasts to the sequence observed in homologous alkyl halides (methyl bromide, MBr; ethyl bromide, EtBr; n-propyl bromide, PrBr) where the relative sequence MeBr > EtBr > PrBr is observed. The higher reactivity towards GSTT1-1 of propylene oxide compared to ethylene oxide is consistent with a higher chemical reactivity. This is corroborated by experimental data of acid-catalysed hydrolysis of a number of aliphatic epoxides, including ethylene oxide and propylene oxide and consistent with semi-empirical molecular orbital modelings.

The effect of menadione on glutathione S-transferase A1 (GSTA1): c-Jun N-terminal kinase (JNK) complex dissociation in human colonic adenocarcinoma Caco-2 cells.

PubMed

Adnan, Humaira; Antenos, Monica; Kirby, Gordon M

2012-10-02

Glutathione S-transferases (GSTs) act as modulators of mitogen-activated protein kinase signal transduction pathways via a mechanism involving protein-protein interactions. We have demonstrated that GSTA1 forms complexes with JNK and modifies JNK activation during cellular stress, but the factors that influence complex association and dissociation are unknown. We hypothesized that menadione causes dissociation of GSTA1-JNK complexes, activates JNK, and the consequences of menadione exposure depend on GSTA1 expression. We demonstrate that menadione causes GSTA1-JNK dissociation and JNK activation in preconfluent Caco-2 cells, whereas postconfluent cells are resistant to this effect. Moreover, preconfluent cells are more sensitive than postconfluent cells to menadione-induced cytotoxicity. Activation of JNK is transient since removal of menadione causes GSTA1 to re-associate with JNK reducing cytotoxicity. Over-expression and knockdown of GSTA1 did not alter JNK activation by menadione or sensitivity to menadione-induced cytotoxicity. These results indicate that GSTA1-JNK complex integrity does not affect the ability of menadione to activate JNK. N-acetyl cysteine prevents GSH depletion and blocks menadione-induced complex dissociation, JNK activation and inhibits menadione-induced cytotoxicity. JNK activation and inhibits menadione-induced cytotoxicity. The data suggest that the mechanism of menadione-induced JNK activation involves the production of reactive oxygen species, likely superoxide anion, and intracellular GSH levels play an important role in preventing GSTA1-JNK complex dissociation, subsequent JNK activation and induction of cytotoxicity. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Aedes aegypti juvenile hormone acid methyl transferase, the ultimate enzyme in the biosynthetic pathway of juvenile hormone III, exhibits substrate control

USDA-ARS?s Scientific Manuscript database

We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzym...

Cysteine S-glycosylation, a new post-translational modification found in glycopeptide bacteriocins.

PubMed

Stepper, Judith; Shastri, Shilpa; Loo, Trevor S; Preston, Joanne C; Novak, Petr; Man, Petr; Moore, Christopher H; Havlíček, Vladimír; Patchett, Mark L; Norris, Gillian E

2011-02-18

O-Glycosylation is a ubiquitous eukaryotic post-translational modification, whereas early reports of S-linked glycopeptides have never been verified. Prokaryotes also glycosylate proteins, but there are no confirmed examples of sidechain glycosylation in ribosomal antimicrobial polypeptides collectively known as bacteriocins. Here we show that glycocin F, a bacteriocin secreted by Lactobacillus plantarum KW30, is modified by an N-acetylglucosamine β-O-linked to Ser18, and an N-acetylhexosamine S-linked to C-terminal Cys43. The O-linked N-acetylglucosamine is essential for bacteriostatic activity, and the C-terminus is required for full potency (IC(50) 2 nM). Genomic context analysis identified diverse putative glycopeptide bacteriocins in Firmicutes. One of these, the reputed lantibiotic sublancin, was shown to contain a hexose S-linked to Cys22. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Acetate Utilization and Butyryl Coenzyme A (CoA):Acetate-CoA Transferase in Butyrate-Producing Bacteria from the Human Large Intestine

PubMed Central

Duncan, Sylvia H.; Barcenilla, Adela; Stewart, Colin S.; Pryde, Susan E.; Flint, Harry J.

2002-01-01

Seven strains of Roseburia sp., Faecalibacterium prausnitzii, and Coprococcus sp. from the human gut that produce high levels of butyric acid in vitro were studied with respect to key butyrate pathway enzymes and fermentation patterns. Strains of Roseburia sp. and F. prausnitzii possessed butyryl coenzyme A (CoA):acetate-CoA transferase and acetate kinase activities, but butyrate kinase activity was not detectable either in growing or in stationary-phase cultures. Although unable to use acetate as a sole source of energy, these strains showed net utilization of acetate during growth on glucose. In contrast, Coprococcus sp. strain L2-50 is a net producer of acetate and possessed detectable butyrate kinase, acetate kinase, and butyryl-CoA:acetate-CoA transferase activities. These results demonstrate that different functionally distinct groups of butyrate-producing bacteria are present in the human large intestine. PMID:12324374

Characterization of a Glucosamine/Glucosaminide N-Acetyltransferase of Clostridium acetobutylicum▿†

PubMed Central

Reith, Jan; Mayer, Christoph

2011-01-01

Many bacteria, in particular Gram-positive bacteria, contain high proportions of non-N-acetylated amino sugars, i.e., glucosamine (GlcN) and/or muramic acid, in the peptidoglycan of their cell wall, thereby acquiring resistance to lysozyme. However, muramidases with specificity for non-N-acetylated peptidoglycan have been characterized as part of autolytic systems such as of Clostridium acetobutylicum. We aim to elucidate the recovery pathway for non-N-acetylated peptidoglycan fragments and present here the identification and characterization of an acetyltransferase of novel specificity from C. acetobutylicum, named GlmA (for glucosamine/glucosaminide N-acetyltransferase). The enzyme catalyzes the specific transfer of an acetyl group from acetyl coenzyme A to the primary amino group of GlcN, thereby generating N-acetylglucosamine. GlmA is also able to N-acetylate GlcN residues at the nonreducing end of glycosides such as (partially) non-N-acetylated peptidoglycan fragments and β-1,4-glycosidically linked chitosan oligomers. Km values of 114, 64, and 39 μM were determined for GlcN, (GlcN)2, and (GlcN)3, respectively, and a 3- to 4-fold higher catalytic efficiency was determined for the di- and trisaccharides. GlmA is the first cloned and biochemically characterized glucosamine/glucosaminide N-acetyltransferase and a member of the large GCN5-related N-acetyltransferases (GNAT) superfamily of acetyltransferases. We suggest that GlmA is required for the recovery of non-N-acetylated muropeptides during cell wall rescue in C. acetobutylicum. PMID:21784938

Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins.

PubMed

Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

2007-08-01

INTRODUCTIONGlutathione-S-transferase (GST) fusion proteins have had a wide range of applications since their introduction as tools for synthesis of recombinant proteins in bacteria. GST was originally selected as a fusion moiety because of several desirable properties. First and foremost, when expressed in bacteria alone, or as a fusion, GST is not sequestered in inclusion bodies (in contrast to previous fusion protein systems). Second, GST can be affinity-purified without denaturation because it binds to immobilized glutathione, which provides the basis for simple purification. Consequently, GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis. This article describes the use of GST fusion proteins as probes for the identification of protein-protein interactions.

A Xanthomonas uridine 5'-monophosphate transferase inhibits plant immune kinases.

PubMed

Feng, Feng; Yang, Fan; Rong, Wei; Wu, Xiaogang; Zhang, Jie; Chen, She; He, Chaozu; Zhou, Jian-Min

2012-04-15

Plant innate immunity is activated on the detection of pathogen-associated molecular patterns (PAMPs) at the cell surface, or of pathogen effector proteins inside the plant cell. Together, PAMP-triggered immunity and effector-triggered immunity constitute powerful defences against various phytopathogens. Pathogenic bacteria inject a variety of effector proteins into the host cell to assist infection or propagation. A number of effector proteins have been shown to inhibit plant immunity, but the biochemical basis remains unknown for the vast majority of these effectors. Here we show that the Xanthomonas campestris pathovar campestris type III effector AvrAC enhances virulence and inhibits plant immunity by specifically targeting Arabidopsis BIK1 and RIPK, two receptor-like cytoplasmic kinases known to mediate immune signalling. AvrAC is a uridylyl transferase that adds uridine 5'-monophosphate to and conceals conserved phosphorylation sites in the activation loop of BIK1 and RIPK, reducing their kinase activity and consequently inhibiting downstream signalling.

Glutathione S-transferase mediates an ageing response to mitochondrial dysfunction

PubMed Central

Dancy, Beverley M.; Brockway, Nicole; Ramadasan-Nair, Renjini; Yang, Yoing; Sedensky, Margaret M.; Morgan, Philip G.

2016-01-01

To understand primary mitochondrial disease, we utilized a complex I-deficient Caenorhabditis elegans mutant, gas-1. These animals strongly upregulate the expression of gst-14 (encoding a glutathione S-transferase). Knockdown of gst-14 dramatically extends the lifespan of gas-1 and increases hydroxynonenal (HNE) modified mitochondrial proteins without improving complex I function. We observed no change in reactive oxygen species levels as measured by Mitosox staining, consistent with a potential role of GST-14 in HNE clearance. The upregulation of gst-14 in gas-1 animals is specific to the pharynx. These data suggest that an HNE-mediated response in the pharynx could be beneficial for lifespan extension in the context of complex I dysfunction in C. elegans. Thus, whereas HNE is typically considered damaging, our work is consistent with recent reports of its role in signaling, and that in this case, the signal is pro-longevity in a model of mitochondrial dysfunction. PMID:26704446

Differences in response of glucuronide and glutathione conjugating enzymes to aflatoxin B/sub 1/ and N-acetylaminofluorene in underfed rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rajpurohit, R.; Krishnaswamy, K.

Changes in the hepatic drug/xenobiotic-metabolizing enzymes in underfed rats exposed to aflatoxin B/sub 1/ and N-acetylaminofluorene were investigated. Neither carcinogen, fed at the level of 10 ..mu..g and 0.667 mg per 100 g body weight, respectively, over a period of 3 wk, had any significant influence on cytochrome P-450 and aryl hydrocarbon hydroxylase in the undernourished rats. Significantly low activities of UDP-glucuronyltransferase and glutathione S-transferase were observed in food-restricted animals fed on aflatoxin B/sub 1/. N-acetylaminofluorene, on the other hand stimulated both the enzyme activities in the underfed group, to as much observed in the respective well-fed treated group. UDP-Glucuronyltransferasemore » and glutathione S-transferase in undernutrition seem to respond differently to aflatoxin B/sub 1/ and N-acetylaminofluorene. Further studies are needed to assess the possible consequences of such alterations.« less

1H NMR studies of molecular interaction of D-glucosamine and N-acetyl-D-glucosamine with capsaicin in aqueous and non-aqueous media.

PubMed

Higuera-Ciapara, Inocencio; Virués, Claudia; Jiménez-Chávez, Marcela; Martínez-Benavidez, Evelin; Hernández, Javier; Domínguez, Zaira; López-Rendón, Roberto; Velázquez, Enrique F; Inoue, Motomichi

2017-11-27

Complex formation of D-glucosamine (Gl) and N-acetyl-D-glucosamine (AGl) with capsaicin (Cp) were studied by 1 H NMR titrations in H 2 O-d 2 and DMSO-d 6 ; capsaicin is the major bioactive component of chili peppers. Every titration curve has been interpreted by formulating a suitable model for the reaction equilibrium, to elucidate intermolecular interactions. In DMSO, glucosamine cations associate with each other to yield linear aggregates, and undergo pseudo-1:1-complexation with capsaicin, the formation constant being ca. 30 M -1 . N-Acetylglucosamine, without self-association, forms a 2:1-complex AGl 2 Cp with the stability of ca. 70 M -2 . These complexations are achieved by intermolecular hydrogen bonds. In D 2 O, glucosamine undergoes reversible protonation equilibrium between Gl 0 and GlH + with the logarithmic protonation constants log K D  = 8.63 for α-glucosamine and 8.20 for β-isomer. Both anomeric isomers of deprotonated glucosamine form Gl 0 Cp-type complexes of capsaicin, in a competitive manner, with a formation constant of 1040 M -1 for the α-glucosamine complex and 830 M -1 for the β-complex; the anomeric carbons result in the difference in thermodynamic stability. The reactant molecules are closed up by the solvent-exclusion effect and/or the van der Waals interaction; the resulting pair is stabilized by intermolecular hydrogen bonding within a local water-free space between the component molecules. By contrast, neither protonated glucosamine (GlH + ) nor N-acetylglucosamine yields a capsaicin complex with the definite stoichiometry. The monosaccharides recognize capsaicin under only a controlled condition; the same phenomena are predicted for biological systems and nanocarriers based on polysaccharides such as chitosan. Copyright © 2017 Elsevier Ltd. All rights reserved.

Endothelial cell palmitoylproteomics identifies novel lipid modified targets and potential substrates for protein acyl transferases

PubMed Central

Marin, Ethan P.; Derakhshan, Behrad; Lam, TuKiet T.; Davalos, Alberto; Sessa, William C.

2012-01-01

Rationale Protein S-palmitoylation is the post-translational attachment of a saturated 16-carbon palmitic acid to a cysteine side chain via a thioester bond. Palmitoylation can affect protein localization, trafficking, stability, and function. The extent and roles of palmitoylation in endothelial cell (EC) biology is not well understood, in part due to technological limits on palmitoylprotein detection. Objective To develop a method using acyl-biotinyl exchange (ABE) technology coupled with mass spectrometry to globally isolate and identify palmitoylproteins in EC. Methods and Results More than 150 putative palmitoyl proteins were identified in EC using ABE and mass spectrometry. Among the novel palmitoylproteins identified is superoxide dismutase 1 (SOD1), an intensively studied enzyme that protects all cells from oxidative damage. Mutation of cysteine 6 prevents palmitoylation, leads to reduction in SOD1 activity in vivo and in vitro, and inhibits nuclear localization, thereby supporting a functional role for SOD1 palmitoylation. Moreover, we used ABE to search for substrates of particular protein acyl transferases in EC. We found that palmitoylation of the cell adhesion protein PECAM1 is dependent on the protein acyl transferase ZDHHC21. We show that knockdown of ZDHHC21 leads to reduced levels of PECAM1 at the cell surface. Conclusions Our data demonstrate the utility of EC palmitoylproteomics to reveal new insights into the role of this important post-translational lipid modification in EC biology. PMID:22496122

Aniline exposure associated with up-regulated transcriptional responses of three glutathione S-transferase Delta genes in Drosophila melanogaster.

PubMed

Chan, Wen-Chiao; Chien, Yi-Chih; Chien, Cheng-I

2015-03-01

Complex transcriptional profile of glutathione S-transferase Delta cluster genes occurred in the developmental process of the fruit fly Drosophila melanogaster. The purpose of this project was to quantify the expression levels of Gst Delta class genes altered by aniline exposure and to understand the relationship between aniline dosages and the variation of Gst Delta genes expressed in D. melanogaster. Using RT-PCR expression assays, the expression patterns of the transcript mRNAs of the glutathione S-transferase Delta genes were revealed and their expression levels were measured at eggs, larvae, pupae and adults. The adult stage was selected for further dose-response assays. After analysis, the results indicated that three Gst Delta genes (Gst D2, Gst D5 and Gst D6) were found to show a peak of up-regulated transcriptional response at 6-8h of exposure of aniline. Furthermore, the dose-response relationship of their induction levels within the dose regiments (from 1.2 to 2.0 μl/tube) had been measured. The expression patterns and annotations of these genes were discussed in the context. Copyright © 2015 Elsevier B.V. All rights reserved.

Protein O-linked ß-N-acetylglucosamine: A novel effector of cardiomyocyte metabolism and function

PubMed Central

Darley-Usmar, Victor M.; Ball, Lauren E.; Chatham, John C.

2014-01-01

The post-translational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAc) is emerging as an important mechanism for the regulation of numerous biological processes critical for normal cell function. Active synthesis of O-GlcNAc is essential for cell viability and acute activation of pathways resulting in increased protein O-GlcNAc levels improves the tolerance of cells to a wide range of stress stimuli. Conversely sustained increases in O-GlcNAc levels have been implicated in numerous chronic disease states, especially as a pathogenic contributor to diabetic complications. There has been increasing interest in the role of O-GlcNAc in the heart and vascular system and acute activation of O-GlcNAc levels have been shown to reduce ischemia/reperfusion injury attenuate vascular injury responses as well mediate some of the detrimental effects of diabetes and hypertension on cardiac and vascular function. Here we provide an overview of our current understanding of pathways regulating protein O-GlcNAcylation, summarize the different methodologies for identifying and characterizing O-GlcNAcylated proteins and subsequently focus on two emerging areas: 1) the role of O-GlcNAc as a potential regulator of cardiac metabolism and 2) the cross talk between O-GlcNAc and reactive oxygen species. PMID:21878340

Expression Patterns of Glutathione Transferase Gene (GstI) in Maize Seedlings Under Juglone-Induced Oxidative Stress

PubMed Central

Sytykiewicz, Hubert

2011-01-01

Juglone (5-hydroxy-1,4-naphthoquinone) has been identified in organs of many plant species within Juglandaceae family. This secondary metabolite is considered as a highly bioactive substance that functions as direct oxidant stimulating the production of reactive oxygen species (ROS) in acceptor plants. Glutathione transferases (GSTs, E.C.2.5.1.18) represent an important group of cytoprotective enzymes participating in detoxification of xenobiotics and limiting oxidative damages of cellular macromolecules. The purpose of this study was to investigate the impact of tested allelochemical on growth and development of maize (Zea mays L.) seedlings. Furthermore, the effect of juglone-induced oxidative stress on glutathione transferase (GstI) gene expression patterns in maize seedlings was recorded. It was revealed that 4-day juglone treatment significantly stimulated the transcriptional activity of GstI in maize seedlings compared to control plants. By contrast, at the 6th and 8th day of experiments the expression gene responses were slightly lower as compared with non-stressed seedlings. Additionally, the specific gene expression profiles, as well as the inhibition of primary roots and coleoptile elongation were proportional to juglone concentrations. In conclusion, the results provide strong molecular evidence that allelopathic influence of juglone on growth and development of maize seedlings may be relevant with an induction of oxidative stress in acceptor plants. PMID:22174645

Fucosylation of xyloglucan: localization of the transferase in dictyosomes of pea stem cells. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Camirand, A.; Brummell, D.; MacLachlan, G.

1987-07-01

Microsomal membranes from elongating regions of etiolated Pisum sativum stems were separated by rate-zonal centrifugation on Renografin gradients. The transfer of labeled fucose and xylose from GDP-(/sup 14/C) fucose and UDP-(/sup 14/C)xylose to xyloglucan occurred mainly in dictyosome-enriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with (/sup 3/H)fucose suggest that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.

A Second β-Hexosaminidase Encoded in the Streptococcus pneumoniae Genome Provides an Expanded Biochemical Ability to Degrade Host Glycans*

PubMed Central

Robb, Melissa; Robb, Craig S.; Higgins, Melanie A.; Hobbs, Joanne K.; Paton, James C.; Boraston, Alisdair B.

2015-01-01

An important facet of the interaction between the pathogen Streptococcus pneumoniae (pneumococcus) and its human host is the ability of this bacterium to process host glycans. To achieve cleavage of the glycosidic bonds in host glycans, S. pneumoniae deploys a wide array of glycoside hydrolases. Here, we identify and characterize a new family 20 glycoside hydrolase, GH20C, from S. pneumoniae. Recombinant GH20C possessed the ability to hydrolyze the β-linkages joining either N-acetylglucosamine or N-acetylgalactosamine to a wide variety of aglycon residues, thus revealing this enzyme to be a generalist N-acetylhexosaminidase in vitro. X-ray crystal structures were determined for GH20C in a ligand-free form, in complex with the N-acetylglucosamine and N-acetylgalactosamine products of catalysis and in complex with both gluco- and galacto-configured inhibitors O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino N-phenyl carbamate (PUGNAc), O-(2-acetamido-2-deoxy-d-galactopyranosylidene)amino N-phenyl carbamate (GalPUGNAc), N-acetyl-d-glucosamine-thiazoline (NGT), and N-acetyl-d-galactosamine-thiazoline (GalNGT) at resolutions from 1.84 to 2.7 Å. These structures showed N-acetylglucosamine and N-acetylgalactosamine to be recognized via identical sets of molecular interactions. Although the same sets of interaction were maintained with the gluco- and galacto-configured inhibitors, the inhibition constants suggested preferred recognition of the axial O4 when an aglycon moiety was present (Ki for PUGNAc > GalPUGNAc) but preferred recognition of an equatorial O4 when the aglycon was absent (Ki for GalNGT > NGT). Overall, this study reveals GH20C to be another tool that is unique in the arsenal of S. pneumoniae and that it may implement the effort of the bacterium to utilize and/or destroy the wide array of host glycans that it may encounter. PMID:26491009

Golgi Traffic and Integrity Depend on N-Myristoyl Transferase-1 in Arabidopsis[W

PubMed Central

Renna, Luciana; Stefano, Giovanni; Majeran, Wojciech; Micalella, Chiara; Meinnel, Thierry; Giglione, Carmela; Brandizzi, Federica

2013-01-01

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified. Using a confocal microscopy forward genetics screen for the identification of Arabidopsis thaliana secretory mutants, we isolated STINGY, a recessive mutant with defective Golgi traffic and integrity. We mapped STINGY to a substitution at position 160 of Arabidopsis NMT1 (NMT1A160T). In vitro kinetic studies with purified NMT1A160T enzyme revealed a significant reduction in its activity due to a remarkable decrease in affinity for both myristoyl-CoA and peptide substrates. We show here that this recessive mutation is responsible for the alteration of Golgi traffic and integrity by predominantly affecting the Golgi membrane/cytosol partitioning of ADP-ribosylation factor proteins. Our results provide important functional insight into N-myristoylation in plants by ascribing postembryonic functions of Arabidopsis NMT1 that involve regulation of the functional and morphological integrity of the plant endomembranes. PMID:23673980

The Disruptive Effect of Lysozyme on the Bacterial Cell Wall Explored by an "In-Silico" Structural Outlook

ERIC Educational Resources Information Center

Primo, Emiliano D.; Otero, Lisandro H.; Ruiz, Francisco; Klinke, Sebastián; Giordano, Walter

2018-01-01

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall…

Glutathione Transferase as a Potential Marker for Gut Epithelial Injury versus the Protective Role of Breast Milk sIgA in Infants with Rota Virus Gastroenteritis

PubMed Central

Sherif, Lobna S.; Raouf, Randaa K. Abdel; Sayede, Rokaya M. El; Wakkadd, Amany S. El; Shoaib, Ashraf R.; Ali, Hanan M.; Refay, Amira S. El

2015-01-01

BACKGROUND: Secretory immunoglobulin A (SIgA) plays an important protective role in the recognition and clearance of enteric pathogens. AIM: This study was designed to assess if mucosal integrity “measured by secretory IgA (SIgA)” is a protective factor from more epithelial alteration “measured by glutathione transferase” in infants with Rota gastroenteritis and its relation to infants’ feeding pattern. PATIENTS AND METHODS: This study was conducted on 79 infants aged 6 months and less from those diagnosed as having gastroenteritis and admitted to Gastroenteritis Department in Abo El Rish Pediatric Hospital, Cairo University. Plasma glutathione s-transferases and Stool SIgA were measured using ELISA technique. Rota virus detection was done by Reverse transcriptase PCR. RESULTS: SIgA was found to be significantly positive in exclusive breast fed infants, Glutathione transferase was significantly more frequently positive in Rota positive cases than Rota negative cases by Reverse transcriptase PCR. A significant negative correlation between Glutathione transferase and Secretory IgA was found, (p < 0.05). CONCLUSION: Breast feeding should be encouraged and highly recommended in the first two years of life as it provides Secretory IgA to breast fed infants who in turn protect them against epithelial damage caused by Rota viral gastroenteritis. PMID:27275307

Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.

PubMed

Vontas, John G; Small, Graham J; Nikou, Dimitra C; Ranson, Hilary; Hemingway, Janet

2002-03-01

A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens.

A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

PubMed Central

Rajatileka, Shavanti; Steven, Andrew; Hemingway, Janet; Ranson, Hilary; Paine, Mark; Vontas, John

2010-01-01

Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control. PMID:20824165

O-GlcNAc cycling in the developing, adult and geriatric brain.

PubMed

Lagerlöf, Olof

2018-06-01

Hundreds of proteins in the nervous system are modified by the monosaccharide O-GlcNAc. A single protein is often O-GlcNAcylated on several amino acids and the modification of a single site can play a crucial role for the function of the protein. Despite its complexity, only two enzymes add and remove O-GlcNAc from proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Global and local regulation of these enzymes make it possible for O-GlcNAc to coordinate multiple cellular functions at the same time as regulating specific pathways independently from each other. If O-GlcNAcylation is disrupted, metabolic disorder or intellectual disability may ensue, depending on what neurons are affected. O-GlcNAc's promise as a clinical target for developing drugs against neurodegenerative diseases has been recognized for many years. Recent literature puts O-GlcNAc in the forefront among mechanisms that can help us better understand how neuronal circuits integrate diverse incoming stimuli such as fluctuations in nutrient supply, metabolic hormones, neuronal activity and cellular stress. Here the functions of O-GlcNAc in the nervous system are reviewed.

GRASP55 Senses Glucose Deprivation through O-GlcNAcylation to Promote Autophagosome-Lysosome Fusion.

PubMed

Zhang, Xiaoyan; Wang, Leibin; Lak, Behnam; Li, Jie; Jokitalo, Eija; Wang, Yanzhuang

2018-04-23

The Golgi apparatus is the central hub for protein trafficking and glycosylation in the secretory pathway. However, how the Golgi responds to glucose deprivation is so far unknown. Here, we report that GRASP55, the Golgi stacking protein located in medial- and trans-Golgi cisternae, is O-GlcNAcylated by the O-GlcNAc transferase OGT under growth conditions. Glucose deprivation reduces GRASP55 O-GlcNAcylation. De-O-GlcNAcylated GRASP55 forms puncta outside of the Golgi area, which co-localize with autophagosomes and late endosomes/lysosomes. GRASP55 depletion reduces autophagic flux and results in autophagosome accumulation, while expression of an O-GlcNAcylation-deficient mutant of GRASP55 accelerates autophagic flux. Biochemically, GRASP55 interacts with LC3-II on the autophagosomes and LAMP2 on late endosomes/lysosomes and functions as a bridge between LC3-II and LAMP2 for autophagosome and lysosome fusion; this function is negatively regulated by GRASP55 O-GlcNAcylation. Therefore, GRASP55 senses glucose levels through O-GlcNAcylation and acts as a tether to facilitate autophagosome maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification and characterization of the zebrafish glutathione S-transferase Pi-1.

PubMed

Abunnaja, Maryam S; Kurogi, Katsuhisa; Mohammed, Yasir I; Sakakibara, Yoichi; Suiko, Masahito; Hassoun, Ezdihar A; Liu, Ming-Cheh

2017-10-01

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined. © 2017 Wiley Periodicals, Inc.

Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B.

PubMed

Borowiak, Maria; Fischer, Jennie; Hammerl, Jens A; Hendriksen, Rene S; Szabo, Istvan; Malorny, Burkhard

2017-12-01

Plasmid-mediated mobilized colistin resistance is currently known to be caused by phosphoethanolamine transferases termed MCR-1, MCR-2, MCR-3 and MCR-4. However, this study focuses on the dissection of a novel resistance mechanism in mcr-1-, mcr-2- and mcr-3-negative d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B (Salmonella Paratyphi B dTa+) isolates with colistin MIC values >2 mg/L. A selected isolate from the strain collection of the German National Reference Laboratory for Salmonella was investigated by WGS and bioinformatical analysis to identify novel phosphoethanolamine transferase genes involved in colistin resistance. Subsequently PCR screening, S1-PFGE and DNA-DNA hybridization were performed to analyse the prevalence and location of the identified mcr-5 gene. Cloning and transformation experiments in Escherichia coli DH5α and Salmonella Paratyphi B dTa+ control strains were carried out and the activity of MCR-5 was determined in vitro by MIC testing. In this study, we identified a novel phosphoethanolamine transferase in 14 mcr-1-, mcr-2- and mcr-3-negative Salmonella Paratyphi B dTa+ isolates with colistin MIC values >2 mg/L that were received during 2011-13. The respective gene, further termed as mcr-5 (1644 bp), is part of a 7337 bp transposon of the Tn3 family and usually located on related multi-copy ColE-type plasmids. Interestingly, in one isolate an additional subclone with a chromosomal location of the mcr-5 transposon was observed. Our findings suggest that the transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety, prevalence and impact on public health. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Orotate phosphoribosyl transferase mRNA expression and the response of cholangiocarcinoma to 5-fluorouracil

PubMed Central

Hahnvajanawong, Chariya; Chaiyagool, Jariya; Seubwai, Wunchana; Bhudhisawasdi, Vajarabhongsa; Namwat, Nisana; Khuntikeo, Narong; Sripa, Banchob; Pugkhem, Ake; Tassaneeyakul, Wichittra

2012-01-01

AIM: To determine whether expression of certain enzymes related to 5-fluorouracil (5-FU) metabolism predicts 5-FU chemosensitivity in cholangiocarcinoma (CCA). METHODS: The histoculture drug response assay (HDRA) was performed using surgically resected CCA tissues. Tumor cell viability was determined morphologically with hematoxylin and eosin- and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling-stained tissues. The mRNA expression of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) was determined with real-time reverse transcriptase-polymerase chain reaction. The levels of gene expression and the sensitivity to 5-FU were evaluated. RESULTS: Twenty-three CCA tissues were obtained from patients who had been diagnosed with intrahepatic CCA and who underwent surgical resection at Srinagarind Hospital, Khon Kaen University from 2007 to 2009. HDRA was used to determine the response of these CCA tissues to 5-FU. Based on the dose-response curve, 200 μg/mL 5-FU was selected as the test concentration. The percentage of inhibition index at the median point was selected as the cut-off point to differentiate the responding and non-responding tumors to 5-FU. When the relationship between TP, OPRT, TS and DPD mRNA expression levels and the sensitivity of CCA tissues to 5-FU was examined, only OPRT mRNA expression was significantly correlated with the response to 5-FU. The mean expression level of OPRT was significantly higher in the responder group compared to the non-responder group (0.41 ± 0.25 vs 0.22 ± 0.12, P < 0.05). CONCLUSION: OPRT mRNA expression may be a useful predictor of 5-FU chemosensitivity of CCA. Whether OPRT mRNA could be used to predict the success of 5-FU chemotherapy in CCA patients requires confirmation in patients. PMID:22912546

Hypermethylation of the Human Glutathione S-Transferase-π Gene (GSTP1) CpG Island Is Present in a Subset of Proliferative Inflammatory Atrophy Lesions but Not in Normal or Hyperplastic Epithelium of the Prostate

PubMed Central

Nakayama, Masashi; Bennett, Christina J.; Hicks, Jessica L.; Epstein, Jonathan I.; Platz, Elizabeth A.; Nelson, William G.; De Marzo, Angelo M.

2003-01-01

Somatic inactivation of the glutathione S-transferase-π gene (GSTP1) via CpG island hypermethylation occurs early during prostate carcinogenesis, present in ∼70% of high-grade prostatic intraepithelial neoplasia (high-grade PIN) lesions and more than 90% of adenocarcinomas. Recently, there has been a resurgence of the concept that foci of prostatic atrophy (referred to as proliferative inflammatory atrophy or PIA) may be precursor lesions for the development of prostate cancer and/or high-grade PIN. Many of the cells within PIA lesions contain elevated levels of GSTP1, glutathione S-transferase-α (GSTA1), and cyclooxygenase-II proteins, suggesting a stress response. Because not all PIA cells are positive for GSTP1 protein, we hypothesized that some of the cells within these regions acquire GSTP1 CpG island hypermethylation, increasing the chance of progression to high-grade PIN and/or adenocarcinoma. Separate regions (n =199) from 27 formalin-fixed paraffin-embedded prostates were microdissected by laser-capture microdissection (Arcturus PixCell II). These regions included normal epithelium (n = 48), hyperplasticepithelium from benign prostatic hyperplasia nodules (n = 22), PIA (n = 64), high-grade PIN (n = 32), and adenocarcinoma (n = 33). Genomic DNA was isolated and assessed for GSTP1 CpG island hypermethylation by methylation-specific polymerase chain reaction. GSTP1 CpG island hypermethylation was not detected in normal epithelium (0 of 48) or in hyperplastic epithelium (0 of 22), but was found in 4 of 64 (6.3%) PIA lesions. The difference in the frequency of GSTP1 CpG island hypermethylation between normal or hyperplastic epithelium and PIA was statistically significant (P = 0.049). Similar to studies using nonmicrodissected cases, hypermethylation was found in 22 of 32 (68.8%) high-grade PIN lesions and in 30 of 33 (90.9%) adenocarcinoma lesions. Unlike normal or hyperplastic epithelium, GSTP1 CpG island hypermethylation can be detected in some PIA

Reviewing Hit Discovery Literature for Difficult Targets: Glutathione Transferase Omega-1 as an Example.

PubMed

Xie, Yiyue; Dahlin, Jayme L; Oakley, Aaron J; Casarotto, Marco G; Board, Philip G; Baell, Jonathan B

2018-05-10

Early stage drug discovery reporting on relatively new or difficult targets is often associated with insufficient hit triage. Literature reviews of such targets seldom delve into the detail required to critically analyze the associated screening hits reported. Here we take the enzyme glutathione transferase omega-1 (GSTO1-1) as an example of a relatively difficult target and review the associated literature involving small-molecule inhibitors. As part of this process we deliberately pay closer-than-usual attention to assay interference and hit quality aspects. We believe this Perspective will be a useful guide for future development of GSTO1-1 inhibitors, as well serving as a template for future review formats of new or difficult targets.

Recent advances in methods for the analysis of protein o-glycosylation at proteome level.

PubMed

You, Xin; Qin, Hongqiang; Ye, Mingliang

2018-01-01

O-Glycosylation, which refers to the glycosylation of the hydroxyl group of side chains of Serine/Threonine/Tyrosine residues, is one of the most common post-translational modifications. Compared with N-linked glycosylation, O-glycosylation is less explored because of its complex structure and relatively low abundance. Recently, O-glycosylation has drawn more and more attention for its various functions in many sophisticated biological processes. To obtain a deep understanding of O-glycosylation, many efforts have been devoted to develop effective strategies to analyze the two most abundant types of O-glycosylation, i.e. O-N-acetylgalactosamine and O-N-acetylglucosamine glycosylation. In this review, we summarize the proteomics workflows to analyze these two types of O-glycosylation. For the large-scale analysis of mucin-type glycosylation, the glycan simplification strategies including the ''SimpleCell'' technology were introduced. A variety of enrichment methods including lectin affinity chromatography, hydrophilic interaction chromatography, hydrazide chemistry, and chemoenzymatic method were introduced for the proteomics analysis of O-N-acetylgalactosamine and O-N-acetylglucosamine glycosylation. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An OGA-Resistant Probe Allows Specific Visualization and Accurate Identification of O-GlcNAc-Modified Proteins in Cells.

PubMed

Li, Jing; Wang, Jiajia; Wen, Liuqing; Zhu, He; Li, Shanshan; Huang, Kenneth; Jiang, Kuan; Li, Xu; Ma, Cheng; Qu, Jingyao; Parameswaran, Aishwarya; Song, Jing; Zhao, Wei; Wang, Peng George

2016-11-18

O-linked β-N-acetyl-glucosamine (O-GlcNAc) is an essential and ubiquitous post-translational modification present in nucleic and cytoplasmic proteins of multicellular eukaryotes. The metabolic chemical probes such as GlcNAc or GalNAc analogues bearing ketone or azide handles, in conjunction with bioorthogonal reactions, provide a powerful approach for detecting and identifying this modification. However, these chemical probes either enter multiple glycosylation pathways or have low labeling efficiency. Therefore, selective and potent probes are needed to assess this modification. We report here the development of a novel probe, 1,3,6-tri-O-acetyl-2-azidoacetamido-2,4-dideoxy-d-glucopyranose (Ac 3 4dGlcNAz), that can be processed by the GalNAc salvage pathway and transferred by O-GlcNAc transferase (OGT) to O-GlcNAc proteins. Due to the absence of a hydroxyl group at C4, this probe is less incorporated into α/β 4-GlcNAc or GalNAc containing glycoconjugates. Furthermore, the O-4dGlcNAz modification was resistant to the hydrolysis of O-GlcNAcase (OGA), which greatly enhanced the efficiency of incorporation for O-GlcNAcylation. Combined with a click reaction, Ac 3 4dGlcNAz allowed the selective visualization of O-GlcNAc in cells and accurate identification of O-GlcNAc-modified proteins with LC-MS/MS. This probe represents a more potent and selective tool in tracking, capturing, and identifying O-GlcNAc-modified proteins in cells and cell lysates.

Gene Polymorphisms of Glutathione S-Transferase T1/M1 in Egyptian Children and Adolescents with Type 1 Diabetes Mellitus.

PubMed

Barseem, Naglaa; Elsamalehy, Mona

2017-06-01

Oxidative stress plays an important role in the pathogenesis of type 1 diabetes mellitus (T1DM). To evaluate the association of glutathione S-transferase mu 1 (GST M1) and glutathione S-transferase theta 1 (GST T1) polymorphisms with development of T1DM and disease-related risk factors. Measurement of fasting glucose, serum creatinine, lipid profile, and glycosylated hemoglobin (HbA1c), as well as evaluation of GST T1 and M1 genetic polymorphisms using polymerase chain reaction were done in 64 diabetic children and 41 controls. The diabetic group had significantly higher fasting glucose, HbA1c, and cholesterol levels. GST T1 null genotype was more frequent in the diabetic than the control group with 4.2-fold increased risk of T1DM (odds ratio=4.2; 95% confidence interval=1.6-11.5; p=0.03). Significant positive associations were found with lipid profile, HbA1c, and duration of illness but not with age, age at onset, and body mass index. Gene polymorphisms of the enzyme GST are associated with development of T1DM and disease-related risk factors.

N-glycosylation by N-acetylglucosaminyltransferase V enhances the interaction of CD147/basigin with integrin β1 and promotes HCC metastasis.

PubMed

Cui, Jian; Huang, Wan; Wu, Bo; Jin, Jin; Jing, Lin; Shi, Wen-Pu; Liu, Zhen-Yu; Yuan, Lin; Luo, Dan; Li, Ling; Chen, Zhi-Nan; Jiang, Jian-Li

2018-05-01

While the importance of protein N-glycosylation in cancer cell migration is well appreciated, the precise mechanisms by which N-acetylglucosaminyltransferase V (GnT-V) regulates cancer processes remain largely unknown. In the current study, we report that GnT-V-mediated N-glycosylation of CD147/basigin, a tumor-associated glycoprotein that carries β1,6-N-acetylglucosamine (β1,6-GlcNAc) glycans, is upregulated during TGF-β1-induced epithelial-to-mesenchymal transition (EMT), which correlates with tumor metastasis in patients with hepatocellular carcinoma (HCC). Interruption of β1,6-GlcNAc glycan modification of CD147/basigin decreased matrix metalloproteinase (MMP) expression in HCC cell lines and affected the interaction of CD147/basigin with integrin β1. These results reveal that β1,6-branched glycans modulate the biological function of CD147/basigin in HCC metastasis. Moreover, we showed that the PI3K/Akt pathway regulates GnT-V expression and that inhibition of GnT-V-mediated N-glycosylation suppressed PI3K signaling. In summary, β1,6-branched N-glycosylation affects the biological function of CD147/basigin and these findings provide a novel approach for the development of therapeutic strategies targeting metastasis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Increased resistance to acetaminophen hepatotoxicity in mice lacking glutathione S-transferase Pi

PubMed Central

Henderson, Colin J.; Wolf, C. Roland; Kitteringham, Neil; Powell, Helen; Otto, Diana; Park, B. Kevin

2000-01-01

Overdose of acetaminophen, a widely used analgesic drug, can result in severe hepatotoxicity and is often fatal. This toxic reaction is associated with metabolic activation by the P450 system to form a quinoneimine metabolite, N-acetyl-p-benzoquinoneimine (NAPQI), which covalently binds to proteins and other macromolecules to cause cellular damage. At low doses, NAPQI is efficiently detoxified, principally by conjugation with glutathione, a reaction catalyzed in part by the glutathione S-transferases (GST), such as GST Pi. To assess the role of GST in acetaminophen hepatotoxicity, we examined acetaminophen metabolism and liver damage in mice nulled for GstP (GstP1/P2(−/−)). Contrary to our expectations, instead of being more sensitive, GstP null mice were highly resistant to the hepatotoxic effects of this compound. No significant differences between wild-type (GstP1/P2(+/+)) mice and GstP1/P2(−/−) nulls in either the rate or route of metabolism, particularly to glutathione conjugates, or in the levels of covalent binding of acetaminophen-reactive metabolites to cellular protein were observed. However, although a similar rapid depletion of hepatic reduced glutathione (GSH) was found in both GstP1/P2(+/+) and GstP1/P2(−/−) mice, GSH levels only recovered in the GstP1/P2(−/−) mice. These data demonstrate that GstP does not contribute in vivo to the formation of glutathione conjugates of acetaminophen but plays a novel and unexpected role in the toxicity of this compound. This study identifies new ways in which GST can modulate cellular sensitivity to toxic effects and suggests that the level of GST Pi may be an important and contributing factor in the sensitivity of patients with acetaminophen-induced hepatotoxicity. PMID:11058152

Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum.

PubMed

Liebau, Eva; De Maria, Francesca; Burmeister, Cora; Perbandt, Markus; Turella, Paola; Antonini, Giovanni; Federici, Giorgio; Giansanti, Francesco; Stella, Lorenzo; Lo Bello, Mario; Caccuri, Anna Maria; Ricci, Giorgio

2005-07-15

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Analysis of agalacto-IgG in rheumatoid arthritis using surface plasmon resonance.

PubMed

Liljeblad, M; Lundblad, A; Påhlsson, P

2000-05-01

It is well established that IgG from rheumatoid arthritis (RA) patients are less galactosylated than IgG from normal individuals. Determination of agalacto-IgG may therefore aid in diagnosis and treatment of RA. The decrease in galactosylation of IgG leads to an increase in terminal N-acetylglucosamine residues, which can be detected using a specific lectin from Psathyrella velutina. In the present study IgG from RA and control serum was purified using affinity chromatography. The samples were then, after reduction, analyzed on a BIOCORE 2000 system with immobilized Psathyrella velutina lectin. Using this technique it was possible to discriminate between IgG from RA patients and IgG from control individuals with respect to its content of IgG with terminal N-acetylglucosamine. The affinity biosensor technique makes it possible to detect binding without labeling or using secondary antibodies.

A closer look at the nitrogen next door: 1H-15N NMR methods for glycosaminoglycan structural characterization

NASA Astrophysics Data System (ADS)

Langeslay, Derek J.; Beni, Szabolcs; Larive, Cynthia K.

2012-03-01

Recently, experimental conditions were presented for the detection of the N-sulfoglucosamine (GlcNS) NHSO3- or sulfamate 1H and 15N NMR resonances of the pharmaceutically and biologically important glycosaminoglycan (GAG) heparin in aqueous solution. In the present work, we explore further the applicability of nitrogen-bound proton detection to provide structural information for GAGs. Compared to the detection of 15N chemical shifts of aminosugars through long-range couplings using the IMPACT-HNMBC pulse sequence, the more sensitive two-dimensional 1H-15N HSQC-TOCSY experiments provided additional structural data. The IMPACT-HNMBC experiment remains a powerful tool as demonstrated by the spectrum measured for the unsubstituted amine of 3-O-sulfoglucosamine (GlcN(3S)), which cannot be observed with the 1H-15N HSQC-TOCSY experiment due to the fast exchange of the amino group protons with solvent. The 1H-15N HSQC-TOCSY NMR spectrum reported for the mixture of model compounds GlcNS and N-acetylglucosamine (GlcNAc) demonstrate the broad utility of this approach. Measurements for the synthetic pentasaccharide drug Arixtra® (Fondaparinux sodium) in aqueous solution illustrate the power of this NMR pulse sequence for structural characterization of highly similar N-sulfoglucosamine residues in GAG-derived oligosaccharides.

Serum glutathione S-transferase Pi as predictor of the outcome and acute kidney injury in premature newborns.

PubMed

Stojanović, Vesna D; Barišić, Nenad A; Radovanović, Tanja D; Kovač, Nataša B; Djuran, Jelena D; Antić, Amira Peco E; Doronjski, Aleksandra D

2018-07-01

The incidence of acute kidney injury (AKI) among the neonates treated at the Neonatal Intensive Care Unit is high with high mortality rates. Glutathione S-transferase (GST) class Pi plays an important role in the protection of cells from cytotoxic and oncogenic agents. The aim of the study was to examine whether the levels of serum glutathione S-transferase Pi (GST Pi) determined after birth have any predictive value for the outcome and development of AKI in premature neonates. The prospective study included 36 premature neonates. The data about morbidity was gathered for all the neonates included in the study. The blood samples were taken in the first 6 h of life and GST Pi levels were measured. The mean values and standard deviations of GST Pi among the neonates who died and who survived were 1.904 ± 0.4535 vs 1.434 ± 0.444 ng/ml (p = 0.0128). Logistic regression revealed a statistically significant, positive correlation between GST Pi levels and death (p = 0.0180, OR7.5954; CI 1.4148-40.7748).The mean value of GST Pi levels in the neonates with AKI was higher than in neonates without AKI (p = 0.011). The conclusion of our study is that high levels of serum GST Pi in the first 6 h after birth are associated with an increased mortality and development of AKI in prematurely born neonates.

p-Coumaroyl-CoA:monolignol transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon

PubMed Central

Petrik, Deborah L; Karlen, Steven D; Cass, Cynthia L; Padmakshan, Dharshana; Lu, Fachuang; Liu, Sarah; Le Bris, Philippe; Antelme, Sébastien; Santoro, Nicholas; Wilkerson, Curtis G; Sibout, Richard; Lapierre, Catherine; Ralph, John; Sedbrook, John C

2014-01-01

Grass lignins contain substantial amounts of p-coumarate (pCA) that acylate the side-chains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages; however p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was involved specifically in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no (<0.5%) residual pCA on lignin, and BdPMT RNAi plants had levels as low as 10% of wild-type, whereas the amounts of pCA acylating arabinosyl units on arabinoxylans in these PMT mutant plants remained unchanged. pCA acylation of lignin from BdPMT-overexpressing plants was found to be more than three-fold higher than that of wild-type, but again the level on arabinosyl units remained unchanged. Taken together, these data are consistent with a defined role for grass PMT genes in encoding BAHD (BEAT, AHCT, HCBT, and DAT) acyltransferases that specifically acylate monolignols with pCA and produce monolignol p-coumarate conjugates that are used for lignification in planta. PMID:24372757

Identification of a novel gene cluster in the upstream region of the S-layer gene sbpA involved in cell wall metabolism of Lysinibacillus sphaericus CCM 2177 and characterization of the recombinantly produced autolysin and pyruvyl transferase.

PubMed

Pleschberger, Magdalena; Hildner, Florian; Rünzler, Dominik; Gelbmann, Nicola; Mayer, Harald F; Sleytr, Uwe B; Egelseer, Eva M

2013-05-01

The S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 assembles into a square (p4) lattice structure and recognizes a pyruvylated secondary cell wall polymer (SCWP) as the proper anchoring structure to the rigid cell wall layer. Sequencing of 8,004 bp in the 5'-upstream region of the S-layer gene sbpA led to five ORFs-encoding proteins involved in cell wall metabolism. After cloning and heterologous expression of ORF1 and ORF5 in Escherichia coli, the recombinant autolysin rAbpA and the recombinant pyruvyl transferase rCsaB were isolated, purified, and correct folding was confirmed by circular dichroism. Although rAbpA encoded by ORF1 showed amidase activity, it could attack whole cells of Ly. sphaericus CCM 2177 only after complete extraction of the S-layer lattice. Despite the presence of three S-layer-homology motifs on the N-terminal part, rAbpA did not show detectable affinity to peptidoglycan-containing sacculi, nor to isolated SCWP. As the molecular mass of the autolysin lies above the molecular exclusion limit of the S-layer, AbpA is obviously trapped within the rigid cell wall layer by the isoporous protein lattice. Immunogold-labeling of ultrathin-sectioned whole cells of Ly. sphaericus CCM 2177 with a polyclonal rabbit antiserum raised against rCsaB encoded by ORF5, and cell fractionation experiments demonstrated that the pyruvyl transferase was located in the cytoplasm, but not associated with cell envelope components including the plasma membrane. In enzymatic assays, rCsaB clearly showed pyruvyl transferase activity. By using RT-PCR, specific transcripts for each ORF could be detected. Cotranscription could be confirmed for ORF2 and ORF3.

Recombinant fungal lectin as a new tool to investigate O-GlcNAcylation processes.

PubMed

Machon, Oriane; Baldini, Steffi F; Ribeiro, João P; Steenackers, Agata; Varrot, Annabelle; Lefebvre, Tony; Imberty, Anne

2017-01-01

Glycosylation is a group of post-translational modifications that displays a large variety of structures and are implicated in a plethora of biological processes. Therefore, studying glycosylation requires different technical approaches and reliable tools, lectins being part of them. Here, we describe the use of the recombinant mushroom lectin PVL to discriminate O-GlcNAcylation, a modification consisting in the attachment of a single N-acetylglucosamine residue to proteins confined within the cytosolic, nuclear and mitochondrial compartments. Recombinant PVL (Psathyrella velutina lectin) (rPVL) displays significantly stronger affinity for GlcNAc over Neu5Ac residues as verified by thermal shift assays and surface plasmon resonance experiments, being therefore an excellent alternative to WGA (wheat germ agglutinin). Labeling of rPVL with biotin or HRP (horseradish peroxidase) allows its useful and efficient utilization by western blot. The staining of whole cell lysates with  labeled-rPVL was dramatically decreased in response to O-GlcNAc transferase knockdown and seen to increase after pharmacological blockade of O-GlcNAcase. Also, HRP-rPVL seemed to be more sensitive than the anti-O-GlcNAc antibody RL2. Thus, rPVL is a potent new tool to selectively detect O-GlcNAcylated proteins. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The 30 kDa protein co-purified with chick liver glutathione S-transferases is a carbonyl reductase.

PubMed

Tsai, S P; Wang, L Y; Yeh, H I; Tam, M F

1996-02-08

An unidentified 30 kDa protein was co-purified with chick liver glutathione S-transferases from S-hexylglutathione affinity column. The protein was isolated to apparent homogeneity with chromatofocusing. The molecular mass of the protein was determined to be 30 277 +/- 3 dalton by mass spectrometry. The protein was digested with Achromobacter proteinase I. Amino-acid sequence analyses of the resulting peptides show a high degree of identity with those of human carbonyl reductase. The protein is active with menadione as substrate. Thus, it is identified as chick liver carbonyl reductase.

The Extracellular Wall-Bound β-N-Acetylglucosaminidase from Lactobacillus casei Is Involved in the Metabolism of the Human Milk Oligosaccharide Lacto-N-Triose

PubMed Central

Bidart, Gonzalo N.; Rodríguez-Díaz, Jesús

2015-01-01

Human milk oligosaccharides (HMOs) are considered to play a key role in establishing and maintaining the infant gut microbiota. Lacto-N-triose forms part of both type 1 and type 2 HMOs and also of the glycan moieties of glycoproteins. Upstream of the previously characterized gene cluster involved in lacto-N-biose and galacto-N-biose metabolism from Lactobacillus casei BL23, there are two genes, bnaG and manA, encoding a β-N-acetylglucosaminidase precursor and a mannose-6-phosphate isomerase, respectively. In this work, we show that L. casei is able to grow in the presence of lacto-N-triose as a carbon source. Inactivation of bnaG abolished the growth of L. casei on this oligosaccharide, demonstrating that BnaG is involved in its metabolism. Interestingly, whole cells of a bnaG mutant were totally devoid of β-N-acetylglucosaminidase activity, suggesting that BnaG is an extracellular wall-attached enzyme. In addition to hydrolyzing lacto-N-triose into N-acetylglucosamine and lactose, the purified BnaG enzyme also catalyzed the hydrolysis of 3′-N-acetylglucosaminyl-mannose and 3′-N-acetylgalactosaminyl-galactose. L. casei can be cultured in the presence of 3′-N-acetylglucosaminyl-mannose as a carbon source, but, curiously, the bnaG mutant strain was not impaired in its utilization. These results indicate that the assimilation of 3′-N-acetylglucosaminyl-mannose is independent of BnaG. Enzyme activity and growth analysis with a manA-knockout mutant showed that ManA is involved in the utilization of the mannose moiety of 3′-N-acetylglucosaminyl-mannose. Here we describe the physiological role of a β-N-acetylglucosaminidase in lactobacilli, and it supports the metabolic adaptation of L. casei to the N-acetylglucosaminide-rich gut niche. PMID:26546429

Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs.

PubMed Central

Varner, Amanda S; Ducker, Charles E; Xia, Zuping; Zhuang, Yan; De Vos, Mackenzie L; Smith, Charles D

2003-01-01

The covalent attachment of palmitate to proteins commonly occurs on cysteine residues near either N-myristoylated glycine residues or C-terminal farnesylated cysteine residues. It therefore seems likely that multiple palmitoyl-acyl transferase (PAT) activities exist to recognize and modify these distinct palmitoylation motifs. To evaluate this possibility, two synthetic peptides representing these palmitoylation motifs, termed MyrGCK(NBD) and FarnCNRas(NBD), were used to characterize PAT activity under a variety of conditions. The human tumour cell lines MCF-7 and Hep-G2 each demonstrated high levels of PAT activity towards both peptides. In contrast, normal mouse fibroblasts (NIH/3T3 cells) demonstrated PAT activity towards the myristoylated substrate peptide but not the farnesylated peptide, while ras -transformed NIH/3T3 cells were able to palmitoylate the FarnCNRas(NBD) peptide. The kinetic parameters for PAT activity were determined using membranes from MCF-7 cells, and indicated that the K (m) values for palmitoyl-CoA were identical for PAT activity towards the two substrate peptides; however, the K (m) for MyrGCK(NBD) was 5-fold lower than the K (m) for FarnCNRas(NBD). PAT activity towards the two substrate peptides was dose-dependently inhibited by 2-bromopalmitate and 3-(1-oxo-hexadecyl)oxiranecarboxamide (16C; IC(50) values of approx. 4 and 1.3 microM, respectively); however, 2-bromopalmitate was found to be uncompetitive with respect to palmitoyl-CoA, whereas 16C was competitive. To seek additional evidence for multiple PATs, the effects of altering the assay conditions on the palmitoylation of MyrGCK(NBD) and FarnCNRas(NBD) were compared. PAT activity towards the two peptide substrates was modulated similarly by changing the ionic strength or incubation temperature, or by the addition of dithiothreitol. In contrast, the enzymic palmitoylation of the two peptides was differentially affected by N -ethylmaleimide and thermal denaturation. Overall, these

Purification, molecular cloning and heterologous expression of a glutathione S-transferase involved in insecticide resistance from the rice brown planthopper, Nilaparvata lugens.

PubMed Central

Vontas, John G; Small, Graham J; Nikou, Dimitra C; Ranson, Hilary; Hemingway, Janet

2002-01-01

A novel glutathione S-transferase (GST)-based pyrethroid resistance mechanism was recently identified in Nilaparvata lugens [Vontas, Small and Hemingway (2001) Biochem. J. 357, 65-72]. To determine the nature of GSTs involved in conferring this resistance, the GSTs from resistant and susceptible strains of N. lugens were partially purified by anion exchange and affinity chromatography. The majority of peroxidase activity, previously correlated with resistance, was confined to the fraction that bound to the affinity column, which was considerably elevated in the resistant insects. A cDNA clone encoding a GST (nlgst1-1) - the first reported GST sequence from Hemiptera with up to 54% deduced amino-acid identity with other insect class I GSTs - was isolated from a pyrethroid-resistant strain. Northern analysis showed that nlgst1-1 was overexpressed in resistant insects. nlgst1-1 was expressed in Escherichia coli, purified and characterized. The ability of the recombinant protein to bind to the S-hexylglutathione affinity matrix, its substrate specificities and its immunological properties confirmed that this GST was one from the elevated subset of N. lugens GSTs. Peroxidase activity of the recombinant nlgst1-1 indicated that it had a role in resistance, through detoxification of lipid peroxidation products induced by pyrethroids. Southern analysis of genomic DNA from the resistant and susceptible strains indicated that GST-based insecticide resistance may be associated with gene amplification in N. lugens. PMID:11853540

Echinococcus granulosus: Evidence of a heterodimeric glutathione transferase built up by phylogenetically distant subunits.

PubMed

Arbildi, Paula; La-Rocca, Silvana; Lopez, Veronica; Da-Costa, Natalia; Fernandez, Veronica

2017-01-01

In the cestode parasite Echinococcus granulosus, three phylogenetically distant cytosolic glutathione transferases (GSTs) (EgGST1, 2 and 3) were identified. Interestingly, the C-terminal domains of EgGST3 and EgGST2 but not EgGST1, exhibit all amino acids involved in Sigma-class GST dimerization. Here, we provide evidence indicating that EgGST2 and EgGST3 naturally form a heterodimeric structure (EgGST2-3), and also we report the enzymatic activity of the recombinant heterodimer. EgGST2-3 might display novel properties able to influence the infection establishment. This is the first report of a stable heterodimeric GST built up by phylogenetically distant subunits. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of a dual domain phosphoglycosyl transferase reveals a ping-pong mechanism with a covalent enzyme intermediate

PubMed Central

Das, Debasis; Kuzmic, Petr

2017-01-01

Phosphoglycosyl transferases (PGTs) are integral membrane proteins with diverse architectures that catalyze the formation of polyprenol diphosphate-linked glycans via phosphosugar transfer from a nucleotide diphosphate-sugar to a polyprenol phosphate. There are two PGT superfamilies that differ significantly in overall structure and topology. The polytopic PGT superfamily, represented by MraY and WecA, has been the subject of many studies because of its roles in peptidoglycan and O-antigen biosynthesis. In contrast, less is known about a second, extensive superfamily of PGTs that reveals a core structure with dual domain architecture featuring a C-terminal soluble globular domain and a predicted N-terminal membrane-associated domain. Representative members of this superfamily are the Campylobacter PglCs, which initiate N-linked glycoprotein biosynthesis and are implicated in virulence and pathogenicity. Despite the prevalence of dual domain PGTs, their mechanism of action is unknown. Here, we present the mechanistic analysis of PglC, a prototypic dual domain PGT from Campylobacter concisus. Using a luminescence-based assay, together with substrate labeling and kinetics-based approaches, complementary experiments were carried out that support a ping-pong mechanism involving a covalent phosphosugar intermediate for PglC. Significantly, mass spectrometry-based approaches identified Asp93, which is part of a highly conserved AspGlu dyad found in all dual domain PGTs, as the active-site nucleophile of the enzyme involved in the formation of the covalent adduct. The existence of a covalent phosphosugar intermediate provides strong support for a ping-pong mechanism of PglC, differing fundamentally from the ternary complex mechanisms of representative polytopic PGTs. PMID:28630348

Regional differences in lectin binding patterns of vestibular hair cells

NASA Technical Reports Server (NTRS)

Baird, R. A.; Schuff, N. R.; Bancroft, J.

1993-01-01

Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylglucosamine (WGA), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not strain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type I hair cells while labeling, as in the bullfrog, Type II hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

Novel type of murein transglycosylase in Escherichia coli.

PubMed Central

Höltje, J V; Mirelman, D; Sharon, N; Schwarz, U

1975-01-01

The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed. PMID:357

Glutathione -S-Transferase μ 1 Regulates Vascular Smooth Muscle Cell Proliferation, Migration, and Oxidative Stress

PubMed Central

Yang, Yanqiang; Parsons, Kelly K.; Chi, Liqun; Malakauskas, Sandra M.; Le, Thu H.

2009-01-01

Glutathione S-transferase μ-1, GSTM1, belongs to a superfamily of glutathione-S-transferases that metabolize a broad range of reactive oxygen species (ROS) and xenobiotics. Across species, genetic variants that result in decreased expression of the Gstm1 gene are associated with increased susceptibility for vascular diseases, including atherosclerosis in humans. We previously identified Gstm1 as a positional candidate in our gene mapping study for susceptibility to renal vascular injury characterized by medial hypertrophy and hyperplasia of the renal vessels. To determine the role of Gstm1 in vascular smooth muscle cells (VSMCs), we isolated VSMCs from mouse aortas. We demonstrate that VSMCs from the susceptible C57BL/6 mice have reduced expression of Gstm1 mRNA and its protein product compared to that of the resistant 129 mice. After serum stimulation, C57BL/6 VSMCs proliferate and migrate at a much faster rate than 129 VSMCs. Furthermore, C57BL/6 VSMCs have higher levels of ROS, and exhibit exaggerated p38 MAPK phosphorylation after exposure to H2O2. To establish causality, we show that knockdown of Gstm1 by siRNA results in increased proliferation of VSMCs in a dose dependent manner, as well as in increased ROS levels and VSM cell migration. Moreover, Gstm1 siRNA causes increased p38 MAPK phosphorylation, and attenuates the anti-proliferative effect of TEMPOL. Our data suggest that Gstm1 is a novel regulator of VSMC proliferation and migration through its role in handling ROS. Genetic variants that cause a decremental change in expression of Gstm1 may permit an environment of exaggerated oxidative stress, leading to susceptibility to vascular remodeling and atherosclerosis. PMID:19822795

Identification and clarification of the role of key active site residues in bacterial glutathione S-transferase zeta/maleylpyruvate isomerase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Ti; Li, De-Feng; Zhou, Ning-Yi, E-mail: n.zhou@pentium.whiov.ac.cn

2011-07-08

Highlights: {yields} Application of site-directed mutagenesis to probe the active site residues of glutathione-dependent maleylpyruvate isomerase. {yields} Two conserved residues, Arg8 and Arg176, in zeta class glutathione S-transferases are critical for maleylpyruvate orientation and enolization. {yields} Arg109, found exclusively in NagL, participates in k{sub cat} regulation. {yields} The T11A mutant exhibited a significantly decreased K{sub m} value for glutathione with little impact on maleylpyruvate kinetics. {yields} The Thr11 residue appears to have significance in the evolution of glutathione S-transferase classes. -- Abstract: The maleylpyruvate isomerase NagL from Ralstonia sp. strain U2, which has been structurally characterized previously, catalyzes the isomerizationmore » of maleylpyruvate to fumarylpyruvate. It belongs to the class zeta glutathione S-transferases (GSTZs), part of the cytosolic GST family (cGSTs). In this study, site-directed mutagenesis was conducted to probe the functions of 13 putative active site residues. Steady-state kinetic information for mutants in the reduced glutathione (GSH) binding site, suggested that (a) Gln64 and Asp102 interact directly with the glutamyl moiety of glutathione, (b) Gln49 and Gln64 are involved in a potential electron-sharing network that influences the ionization of the GSH thiol. The information also suggests that (c) His38, Asn108 and Arg109 interact with the GSH glycine moiety, (d) His104 has a role in the ionization of the GSH sulfur and the stabilization of the maleyl terminal carboxyl group in the reaction intermediate and (e) Arg110 influences the electron distribution in the active site and therefore the ionization of the GSH thiolate. Kinetic data for mutants altered in the substrate-binding site imply that (a) Arg8 and Arg176 are critical for maleylpyruvate orientation and enolization, and (b) Arg109 (exclusive to NagL) participates in k{sub cat} regulation. Surprisingly, the T11A mutant

Pharmacogenetics of azathioprine in inflammatory bowel disease: A role for glutathione-S-transferase?

PubMed Central

Stocco, Gabriele; Pelin, Marco; Franca, Raffaella; De Iudicibus, Sara; Cuzzoni, Eva; Favretto, Diego; Martelossi, Stefano; Ventura, Alessandro; Decorti, Giuliana

2014-01-01

Azathioprine is a purine antimetabolite drug commonly used to treat inflammatory bowel disease (IBD). In vivo it is active after reaction with reduced glutathione (GSH) and conversion to mercaptopurine. Although this reaction may occur spontaneously, the presence of isoforms M and A of the enzyme glutathione-S-transferase (GST) may increase its speed. Indeed, in pediatric patients with IBD, deletion of GST-M1, which determines reduced enzymatic activity, was recently associated with reduced sensitivity to azathioprine and reduced production of azathioprine active metabolites. In addition to increase the activation of azathioprine to mercaptopurine, GSTs may contribute to azathioprine effects even by modulating GSH consumption, oxidative stress and apoptosis. Therefore, genetic polymorphisms in genes for GSTs may be useful to predict response to azathioprine even if more in vitro and clinical validation studies are needed. PMID:24707136

Superoxide dismutase, catalase, glutathione peroxidase and gluthatione S-transferases M1 and T1 gene polymorphisms in three Brazilian population groups.

PubMed

de Oliveira Hiragi, Cássia; Miranda-Vilela, Ana Luisa; Rocha, Dulce Maria Sucena; de Oliveira, Silviene Fabiana; Hatagima, Ana; de Nazaré Klautau-Guimarães, Maria

2011-01-01

Antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX1) reduce the oxidation rates in the organism. Gluthatione S-transferases (GSTs) play a vital role in phase 2 of biotransformation of many substances. Variation in the expression of these enzymes suggests individual differences for the degree of antioxidant protection and geographical differences in the distribution of these variants. We described the distribution frequency of CAT (21A/T), SOD2 (Ala9Val), GPX1 (Pro198Leu), GSTM1 and GSTT1 polymorphisms in three Brazilian population groups: Kayabi Amerindians (n = 60), Kalunga Afro-descendants (n = 72), and an urban mixed population from Federal District (n = 162). Frequencies of the variants observed in Kalunga (18% to 58%) and Federal District (33% to 63%) were similar to those observed in Euro and Afro-descendants, while in Kayabi (3% to 68%), depending on the marker, frequencies were similar to the ones found in different ethnic groups. Except for SOD2 in all population groups studied here, and for GPX1 in Kalunga, the genotypic distributions were in accordance with Hardy-Weinberg Equilibrium. These data can clarify the contribution of different ethnicities in the formation of mixed populations, such as that of Brazil. Moreover, outcomes will be valuable resources for future functional studies and for genetic studies in specific populations. If these studies are designed to comprehensively explore the role of these genetic polymorphisms in the etiology of human diseases they may help to prevent inconsistent genotype-phenotype associations in pharmacogenetic studies.

Analysis of Protein Adduction Kinetics by Quantitative Mass Spectrometry. Competing Adduction Reactions of Glutathione-S-Transferase P1-1 with Electrophiles

PubMed Central

Orton, Christopher R.; Liebler, Daniel C.

2007-01-01

Defining the mechanisms and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. Here we describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein. Adducts are formed by electrophiles at Cys14 and Cys47 on the metabolic enzyme glutathione-S-transferase P1-1 and modification is accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples in liquid chromatography-tandem mass spectrometry analyses using a multiple reaction monitoring method. This approach was used to measure rate constants for adduction at both positions with two different model electrophiles, N-iodoacetyl-N-biotinylhexylenediamine and 1-biotinamido-4-(4′-[maleimidoethyl-cyclohexane]-carboxamido)butane. The results indicate that Cys47 was approximately 2–3-fold more reactive toward both electrophiles than was Cys14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system and with previously reported trends in reactivity of these sites. Kinetic analyses of protein modification reactions provide a means of evaluating the selectivity of reactive mediators of chemical toxicity. PMID:17433278

N-Glycan Modification in Aspergillus Species▿

PubMed Central

Kainz, Elke; Gallmetzer, Andreas; Hatzl, Christian; Nett, Juergen H.; Li, Huijuan; Schinko, Thorsten; Pachlinger, Robert; Berger, Harald; Reyes-Dominguez, Yazmid; Bernreiter, Andreas; Gerngross, Tillmann; Wildt, Stefan; Strauss, Joseph

2008-01-01

The production by filamentous fungi of therapeutic glycoproteins intended for use in mammals is held back by the inherent difference in protein N-glycosylation and by the inability of the fungal cell to modify proteins with mammalian glycosylation structures. Here, we report protein N-glycan engineering in two Aspergillus species. We functionally expressed in the fungal hosts heterologous chimeric fusion proteins containing different localization peptides and catalytic domains. This strategy allowed the isolation of a strain with a functional α-1,2-mannosidase producing increased amounts of N-glycans of the Man5GlcNAc2 type. This strain was further engineered by the introduction of a functional GlcNAc transferase I construct yielding GlcNAcMan5GlcNac2 N-glycans. Additionally, we deleted algC genes coding for an enzyme involved in an early step of the fungal glycosylation pathway yielding Man3GlcNAc2 N-glycans. This modification of fungal glycosylation is a step toward the ability to produce humanized complex N-glycans on therapeutic proteins in filamentous fungi. PMID:18083888

INDUCTION OF DNA-PROTEIN CROSSLINKS BY THE METABOLISM OF DICHLOROMETHANE IN V79 CELL LINES TRANSFECTED WITH THE MURINE GLUTATHIONE-S-TRANSFERASE THETA 1 GENE

EPA Science Inventory

Dichloromethane (DCM) is considered a probable human carcinogen. Laboratory studies have shown an increased incidence of lung and liver cancer in mice but not in rats or hamsters. Despite the correlation between metabolism of DCM by the glutathione-S-transferase (GST) pathway and...

[Investigation of biofilm formation properties of staphylococcus isolates].

PubMed

Öcal, Duygu Nilüfer; Dolapçı, İştar; Karahan, Zeynep Ceren; Tekeli, Alper

2017-01-01

Biofilm production is an important virulence factor which allows staphylococci to adhere to medical devices. The principal component of biofilm is a "polysaccharide intercellular adhesin (PIA)" which is composed of a beta-1,6-N-acetylglucosamine polymer synthesized by an enzyme (N-acetylglucosamine transferase) encoded by the ica operon found on the bacterial chromosome. This operon is composed of four genes (A, B, C, and D), and a transposable element IS256. In this study, we aimed to determine the biofilm production characteristics of invasive/non-invasive staphylococcus isolates and different staphylococcus species. Biofilm production of 166 staphylococci was phenotypically investigated on Congo Red Agar (CRA); the presence of icaA, icaD and IS256 genes were investigated by polymerase chain reaction (PCR). 74 of the isolates (44.6%) were identified as methicillin resistant Staphylococcus aureus (MRSA), 25 (15.1%) as methicillin sensitive S.aureus (MSSA), 25 (37.3%) as Staphylococcus hominis, 20 (12%) as S.epidermidis, ten (15%) as Staphylococcus haemolyticus, nine (13.4%) as Staphylococcus capitis, two (3%) Staphylococcus saprophyticus and one (1.5%) as Staphylococcus warnerii. Of the MRSA strains, 52 were isolated from blood and 22 from nose; all MSSA strains were isolated from nose cultures. Coagulase-negative staphylococci (CoNS) strains were composed of invasive and non-invasive strains isolated from nose, catheter tip and blood cultures from patients with catheter. Production with CRA method was found to be statistically significant in invasive isolates (p< 0.001). It is concluded that; as the biofilm formation capacity of invasive isolates can cause refractory infections and the importance of carriage and hospital infections of these bacteria, it is important to prevent the spread of these isolates. A combination of phenotypic and genotypic tests is recommended for the investigation of biofilm formation in staphylococci. 40.3% of the CoNS isolates, and 85

Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae

PubMed Central

May, Meghan; Brown, Daniel R.

2008-01-01

We explored the genetic basis for intraspecific variation in mycoplasmal sialidase activity that correlates with virulence, and its potentially advantageous linkage to nutrient catabolism. Polymorphism in N-acetylneuraminate scavenging and degradation genes (sialidase, N-acetylneuraminate lyase, N-acetylmannosamine kinase, N-acetylmannosamine-6-phosphate epimerase, N-acetylglucosamine-6-phosphate deacetylase, and glucosamine-6-phosphate deaminase) was evident among eight strains of the avian pathogen Mycoplasma synoviae. Most differences were single nucleotide polymorphisms, ranging from 0.34 ± 0.04 substitutions per 100 bp for N-acetylmannosamine kinase to 0.65 ± 0.03 for the single-copy sialidase gene nanI. Missense mutations were twice as common as silent mutations in nanI; 26% resulted in amino acids dissimilar to consensus; and there was a 12-base deletion near the nanI promoter in strain WVU1853T, supporting a complex genetic basis for differences in sialidase activity. Two strains had identical frameshifts in the N-acetylneuraminate lyase gene nanA, resulting in nonsense mutations, and both had downstream deletions in nanA. Such genetic lesions uncouple extracellular liberation of sialic acid from generation of fructose-6-phosphate and pyruvate via intracellular N-acetylneuraminate degradation. Retention of nanI by such strains, but not others in the M. synoviae phylogenetic cluster, is evidence that sialidase has an important non-nutritional role in the ecology of M. synoviae and certain other mycoplasmas. PMID:18490131

Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

PubMed Central

Marita, Jane M; Hatfield, Ronald D; Rancour, David M; Frost, Kenneth E

2014-01-01

Grasses, such as Zea mays L. (maize), contain relatively high levels of p-coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls. PMID:24654730

Role of N-acetylglucosaminidase and N-acetylmuramidase activities in Enterococcus faecalis peptidoglycan metabolism.

PubMed

Mesnage, Stéphane; Chau, Françoise; Dubost, Lionel; Arthur, Michel

2008-07-11

Identification of the full complement of peptidoglycan hydrolases detected by zymogram in Enterococcus faecalis extracts led to the characterization of two novel hydrolases that we named AtlB and AtlC. Both enzymes have a similar modular organization comprising a central catalytic domain fused to two LysM peptidoglycan-binding modules. AtlB and AtlC displayed N-acetylmuramidase activity, as demonstrated by tandem mass spectrometry analyses of peptidoglycan fragments generated by the purified enzymes. The genes encoding AtlB and AtlC were deleted either alone or in combination with the gene encoding AtlA, a previously described N-acetylglucosaminidase. No autolytic activity was detected in the triple mutant indicating that AtlA, AtlB, and AtlC account for the major hydrolytic activities in E. faecalis. Analysis of cell size distribution by flow cytometry showed that deletion of atlA resulted in the formation of long chains. Thus, AtlA digests the septum and is required for cell separation after cell division. We found that AtlB could act as a surrogate for AtlA, although the enzyme was less efficient at septum digestion. Deletion of atlC had no impact on cell morphology. Labeling of the peptidoglycan with N-[14C]acetylglucosamine revealed an unusually slow turnover as compared with model organisms, almost completely dependent upon the combined activities of AtlA and AtlB. In contrast to atlA, the atlB and atlC genes are located in putative prophages. Because AtlB and AtlC were produced in the absence of cell lysis or production of phage progeny, these enzymes may have been hijacked by E. faecalis to contribute to peptidoglycan metabolism.

Phosphoneurofilament heavy chain and N-glycomics from the cerebrospinal fluid in amyotrophic lateral sclerosis.

PubMed

Gonçalves, Margarida; Tillack, Linda; de Carvalho, Mamede; Pinto, Susana; Conradt, Harald S; Costa, Júlia

2015-01-01

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron for which no clinically validated biomarkers have been identified. We have quantified by ELISA the biomarker phosphoneurofilament heavy chain (pNFH) in the cerebrospinal fluid (CSF) of ALS patients (n=29) and age-matched control patients with other diseases (n=19) by ELISA. Furthermore, we compared protein N-glycosylation of the CSF in ALS patients and controls, by applying a glycomics approach based on liquid chromatography and mass spectrometry. pNFH levels were significantly higher in ALS patients in comparison with controls (P<0.0001) in particular in fast progressors. The N-glycans found in the CSF were predominantly complex diantennary with sialic acid in α2,3- and α2,6-linkage, and bisecting N-acetylglucosamine-containing structures as well as peripherally fucosylated structures were found. As compared with controls the ALS group had a significant increase of a peak composed of the monosialylated diantennary glycans A2G2S(6)1 and FA2G2S(3)1 (P=0.0348). Our results underscore the value of pNFH as a biomarker in ALS. In addition, we identified a variation of the N-glycosylation pattern in ALS, suggesting that this change should be explored in future studies as potential biomarker. Copyright © 2014 Elsevier B.V. All rights reserved.

Association of manganese superoxide dismutase and glutathione S-transferases genotypes with myocardial infarction in patients with type 2 diabetes mellitus.

PubMed

Kariž, Stojan; Nikolajević Starčević, Jovana; Petrovič, Daniel

2012-10-01

In the present study we investigated the association between genetic polymorphisms with functional effects on redox regulation: Val16Ala of manganese superoxide dismutase (MnSOD), polymorphic deletions of glutathione S-transferases M1 (GSTM1) and T1 (GSTT1) and Ile105Val of glutathione S-transferase P1 (GSTP1) and myocardial infarction (MI) in a group of patients with type 2 diabetes mellitus. The study population consisted of 463 Caucasian subjects with type 2 diabetes mellitus of more than 10 years' duration: 206 patients with MI and 257 patients with no history of coronary artery disease (CAD). Genotypes were determined by polymerase chain reaction (PCR) with restriction fragment length polymorphism (RFLP) and with multiplex PCR. The genotype distributions of tested single nucleotide polymorphisms did not show significant difference between cases and controls. After adjustment for age, gender, smoking, BMI, duration of diabetes and lipid parameters carriers of GSTM1/GSTT1-null haplotype showed an increased risk for MI (OR=3.22, 95% CI 1.37-5.04, p=0.03). The GSTM1/GSTT1 haplotype might be a genetic risk factor for MI in patients with type 2 diabetes mellitus. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Maize w3 disrupts homogentisate solanesyl transferase (ZmHst) and reveals a plastoquinone-9 independent path for phytoene desaturation and tocopherol accumulation in kernels

USDA-ARS?s Scientific Manuscript database

Maize white seedling 3 (w3) has been used to study carotenoid deficiency for almost 100 years, although its genetic basis remained unknown. We show here that w3 phenotype is caused by disruption of homogentisate solanesyl transferase (HST), which catalyzes the first committed step in plastoquinone-9...

Study of trans-trans farnesol effect on hyphae formation by Yarrowia lipolytica.

PubMed

Nunes, Patrícia Martins Botelho; da Rocha, Silvia Maria; Amaral, Priscilla Filomena Fonseca; da Rocha-Leão, Maria Helena Miguez

2013-12-01

Dimorphism is an ability of certain fungi related to its adaptation to the environment and provides a selective advantage under stress conditions and is associated to the development of human diseases. Hyphae inducing- and inhibitory-effect of farnesol on hyphae formation by the dimorphic yeast Yarrowia lipolytica was evaluated through digital image analysis. The agitation speed of the culture was the most effective hyphae inducer in comparison to bovine calf serum and N-acetylglucosamine. In low agitation system, bovine calf serum was more effective for hyphae formation inducing 57 % of hyphae transition. Farnesol inhibited hyphae formation even in low concentration (300 μM) and this effect increased with increasing concentrations. In the presence of N-acetylglucosamine, this effect was more evident in comparison to the presence of bovine calf serum, which might have protected the cells from farnesol. Digital image analysis was an important tool to evaluate this phenomenon.

A phase I trial of the novel farnesyl protein transferase inhibitor, BMS-214662, in combination with paclitaxel and carboplatin in patients with advanced cancer.

PubMed

Dy, Grace K; Bruzek, Laura M; Croghan, Gary A; Mandrekar, Sumithra; Erlichman, Charles; Peethambaram, Prema; Pitot, Henry C; Hanson, Lorelei J; Reid, Joel M; Furth, Alfred; Cheng, Shinta; Martell, Robert E; Kaufmann, Scott H; Adjei, Alex A

2005-03-01

This phase I study was conducted to determine the toxicities, pharmacokinetics, and pharmacodynamics of BMS-214662, a farnesyl transferase inhibitor, in combination with paclitaxel and carboplatin, in patients with advanced solid tumors. Patients with solid tumors received one of six escalating dose levels of BMS-214662 infused over 1 hour given following paclitaxel and carboplatin on the first day of a 21-day cycle. Toxicities were graded by the National Cancer Institute common toxicity criteria and recorded as maximum grade per patient for each treatment cycle. Inhibition of farnesyl transferase activity in peripheral blood mononuclear cells (PBMCs) was evaluated. Accumulation of unfarnesylated HDJ-2 in PBMCs of patients was evaluated as a marker of farnesyl transferase inhibition by BMS-214662. Thirty patients received 141 cycles of treatment through six dose levels. Dose-limiting toxicities were neutropenia, thrombocytopenia, nausea, and vomiting. There was no pharmacokinetic interaction between BMS-214662 and paclitaxel. The maximum tolerated dose was established as BMS-214662 (160 mg/m(2)), paclitaxel (225 mg/m(2)) and carboplatin (area under the curve = 6 on day 1), every 21 days. Inhibition of HDJ-2 farnesylation in PBMCs of patients was shown. One measurable partial response was observed in a patient with taxane-resistant esophageal cancer. There was partial regression of evaluable disease in two other patients (endometrial and ovarian cancer). Stable disease (> 4 cycles) occurred in eight other patients. The combination of BMS-214662 with paclitaxel and carboplatin was well tolerated, with broad activity in solid tumors. There was no correlation between dose level and accumulation of unfarnesylated HDJ-2 in PBMCs nor tumor response.

Glutathione S-transferase P protects against cyclophosphamide-induced cardiotoxicity in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conklin, Daniel J., E-mail: dj.conklin@louisville.edu; Institute of Molecular Cardiology, University of Louisville, Louisville, KY 40292; Haberzettl, Petra

2015-06-01

High-dose chemotherapy regimens using cyclophosphamide (CY) are frequently associated with cardiotoxicity that could lead to myocyte damage and congestive heart failure. However, the mechanisms regulating the cardiotoxic effects of CY remain unclear. Because CY is converted to an unsaturated aldehyde acrolein, a toxic, reactive CY metabolite that induces extensive protein modification and myocardial injury, we examined the role of glutathione S-transferase P (GSTP), an acrolein-metabolizing enzyme, in CY cardiotoxicity in wild-type (WT) and GSTP-null mice. Treatment with CY (100–300 mg/kg) increased plasma levels of creatine kinase-MB isoform (CK·MB) and heart-to-body weight ratio to a significantly greater extent in GSTP-null thanmore » WT mice. In addition to modest yet significant echocardiographic changes following acute CY-treatment, GSTP insufficiency was associated with greater phosphorylation of c-Jun and p38 as well as greater accumulation of albumin and protein–acrolein adducts in the heart. Mass spectrometric analysis revealed likely prominent modification of albumin, kallikrein-1-related peptidase, myoglobin and transgelin-2 by acrolein in the hearts of CY-treated mice. Treatment with acrolein (low dose, 1–5 mg/kg) also led to increased heart-to-body weight ratio and myocardial contractility changes. Acrolein induced similar hypotension in GSTP-null and WT mice. GSTP-null mice also were more susceptible than WT mice to mortality associated with high-dose acrolein (10–20 mg/kg). Collectively, these results suggest that CY cardiotoxicity is regulated, in part, by GSTP, which prevents CY toxicity by detoxifying acrolein. Thus, humans with low cardiac GSTP levels or polymorphic forms of GSTP with low acrolein-metabolizing capacity may be more sensitive to CY toxicity. - Graphical abstract: Cyclophosphamide (CY) treatment results in P450-mediated metabolic formation of phosphoramide mustard and acrolein (3-propenal). Acrolein is either metabolized

Effect of a glutathione S-transferase inhibitor on oxidative stress and ischemia-reperfusion-induced apoptotic signalling of cultured cardiomyocytes

PubMed Central

Röth, E; Marczin, N; Balatonyi, B; Ghosh, S; Kovács, V; Alotti, N; Borsiczky, B; Gasz, B

2011-01-01

Oxidative stress and ischemia-reperfusion (I/R) injury are crucial in the pathogenesis of cardiovascular diseases. The antioxidant glutathione S-transferase (GST) is responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. The main objective of the present study was to examine the effect of GST inhibition (with the administration of ethacrynic acid [EA]) on the viability and apoptosis of cardiomyocytes when these cells are exposed to various stress components of I/R and mitogen-activated protein kinase (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase [ERK]) inhibitors. The primary culture of neonatal rat cardiomyocytes was divided into six experimental groups: control group of cells (group 1), cells exposed to H2O2 (group 2), I/R (group 3), I/R and EA (group 4), H2O2 coupled with EA (group 5), and EA alone (group 6). The viability of cardiomyocytes was determined using a colorimetric MTT assay. The apoptosis ratio was evaluated via fluorescein isothiocyanate-labelled annexin V and propidium iodide staining. c-Jun N-terminal kinase, p38, Akt/protein kinase B and ERK/p42-p44 transcription factors were monitored with flow cytometry. c-Jun N-terminal kinase activation increased due to GST inhibition during I/R. EA administration led to a significant increase in p38 activation following both H2O2 treatment and I/R. ERK phosphorylation increased when GST was exposed to I/R. A pronounced decrease in Akt phosphorylation was observed when cells were cotreated with EA and H2O2. GST plays an important role as a regulator of mitogen-activated protein kinase pathways in I/R injury. PMID:22065940

Single-cell immunofluorescence assay for terminal transferase: human leukaemic and non-leukaemic cells.

PubMed

Okamura, S; Crane, F; Jamal, N; Messner, H A; Mak, T W

1980-02-01

The characteristics of a single-cell immunofluorescence assay for terminal deoxynucleotidyl transferase (terminal transferase, TdT) is described. The data indicate that the single-cell immunofluorescence assay is highly efficient and specific for the detection of cells containing TdT. Using this assay, we have examined 124 marrow or peripheral-blood samples from 104 patients with or without haematological malignancies. Results indicate that TdT(+) cells from 6% to 100% were found in the following patients: 34/40 samples from patients with ALL at the time of diagnosis or during relapse; 2/3 patients with acute undifferentiated leukaemia; 2/3 patients with acute myelomonocytic leukaemia; 1/24 patients with acute myeloblastic leukaemia; 1/5 patients with chronic myelocytic leukaemia (CML) in blastic crisis; and 2/2 patients with diffuse lymphoblastic lymphoma. In contrast less than 1% of TdT(+) cells were found in 20 marrow or peripheral-blood samples from ALL patients in complete remission; 8 patients with CML in chronic phase; 2 patients with myeloma; 1 sample from a patient with Hodgkin's disease, peripheral-blood samples from 7 normal donors and marrow samples from 6 patients without haematological malignancies. TdT(+) cells were also found in association with cells with lymphoblast morphology. The TdT(+) cells in marrow were shown to be directly correlated with the percentage of morphological lymphoblasts, with a Spearman rank coefficient of 0·81, significant at a 0·001 level. In 2 longitudinal studies of 2 ALL patients with TdT(+) cells at diagnosis, the percentage TdT(+) cells also changed in parallel with the proportion of lymphoblasts. However, studies of 2 other patients with morphologically diagnosed ALL with < 1% TdT(+) cells at diagnosis also showed < 1% TdT(+) cells throughout the period studied, indicating a stable phenotype of blast cells in these patients. The single-cell immunofluorescence assay for TdT, which requires < 0·1% of the cells used in

Inhibition of the recombinant cattle tick Rhipicephalus (Boophilus) annulatus glutathione S-transferase.

PubMed

Guneidy, Rasha A; Shahein, Yasser E; Abouelella, Amira M K; Zaki, Eman R; Hamed, Ragaa R

2014-09-01

Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes severe production losses in the cattle industry. This study aims to evaluate the in vitro effects of tannic acid, hematin (GST inhibitors) and different plant extracts (rich in tannic acid) on the activity of the recombinant glutathione S-transferase enzyme of the Egyptian cattle tick R. annulatus (rRaGST), in order to confirm their ability to inhibit the parasitic essential detoxification enzyme glutathione S-transferase. Extraction with 70% ethanol of Hibiscus cannabinus (kenaf flowers), Punica granatum (red and white pomegranate peel), Musa acuminata (banana peel) (Musaceae), Medicago sativa (alfalfa seeds), Tamarindus indicus (seed) and Cuminum cyminum (cumin seed) were used to assess: (i) inhibitory capacities of rRaGST and (ii) their phenolic and flavonoid contents. Ethanol extraction of red pomegranate peel contained the highest content of phenolic compounds (29.95mg gallic acid/g dry tissue) compared to the other studied plant extracts. The highest inhibition activities of rRaGST were obtained with kenaf and red pomegranate peel (P. granatum) extracts with IC50 values of 0.123 and 0.136mg dry tissue/ml, respectively. Tannic acid was the more effective inhibitor of rRaGST with an IC50 value equal to 4.57μM compared to delphinidine-HCl (IC50=14.9±3.1μM). Gossypol had a weak inhibitory effect (IC50=43.7μM), and caffeic acid had almost no effect on tick GST activity. The IC50 values qualify ethacrynic acid as a potent inhibitor of rRaGST activity (IC50=0.034μM). Cibacron blue and hematin showed a considerable inhibition effect on rRaGST activity, and their IC50 values were 0.13μM and 7.5μM, respectively. The activity of rRaGST was highest for CDNB (30.2μmol/min/mg protein). The enzyme had also a peroxidatic activity (the specific activity equals 26.5μmol/min/mg protein). Both tannic acid and hematin inhibited rRaGST activity non-competitively with respect to GSH and

Glutathione-S-transferase P protects against endothelial dysfunction induced by exposure to tobacco smoke.

PubMed

Conklin, Daniel J; Haberzettl, Petra; Prough, Russell A; Bhatnagar, Aruni

2009-05-01

Exposure to tobacco smoke impairs endothelium-dependent arterial dilation. Reactive constituents of cigarette smoke are metabolized and detoxified by glutathione-S-transferases (GSTs). Although polymorphisms in GST genes are associated with the risk of cancer in smokers, the role of these enzymes in regulating the cardiovascular effects of smoking has not been studied. The P isoform of GST (GSTP), which catalyzes the conjugation of electrophilic molecules in cigarette smoke such as acrolein, was expressed in high abundance in the mouse lung and aorta. Exposure to tobacco smoke for 3 days (5 h/day) decreased total plasma protein. These changes were exaggerated in GSTP(-/-) mice. Aortic rings isolated from tobacco smoke-exposed GSTP(-/-) mice showed greater attenuation of ACh-evoked relaxation than those from GSTP(+/+) mice. The lung, plasma, and aorta of mice exposed to tobacco smoke or acrolein (for 5 h) accumulated more acrolein-adducted proteins than those tissues of mice exposed to air, indicating that exposure to tobacco smoke results in the systemic delivery of acrolein. Relative to GSTP(+/+) mice, modification of some proteins by acrolein was increased in the aorta of GSTP(-/-) mice. Aortic rings prepared from GSTP(-/-) mice that inhaled acrolein (1 ppm, 5 h/day for 3 days) or those exposed to acrolein in an organ bath showed diminished ACh-induced arterial relaxation more strongly than GSTP(+/+) mice. Acrolein-induced endothelial dysfunction was prevented by pretreatment of the aorta with N-acetylcysteine. These results indicate that GSTP protects against the endothelial dysfunction induced by tobacco smoke exposure and that this protection may be related to the detoxification of acrolein or other related cigarette smoke constituents.

Differential transcription of cytochrome P450s and glutathione S transferases in DDT-susceptible and resistant Drosophila melanogaster strains in response to DDT and oxidative stress

USDA-ARS?s Scientific Manuscript database

Metabolic DDT resistance in Drosophila melanogaster has previously been associated with constitutive over-transcription of cytochrome P450s. Increased P450 activity has also been associated with increased oxidative stress. In contrast, over-transcription of glutathione S transferases (GSTs) has been...

Cold sensitivity in rice (Oryza sativa L.) is strongly correlated with a naturally occurring I99V mutation in the multifunctional glutathione transferase isoenzyme GSTZ2

USDA-ARS?s Scientific Manuscript database

GSTZs (zeta class glutathione transferases) belong to a highly conserved subfamily of soluble GSTs found in species ranging from fungi and plants to animals. GSTZ is identical to MAAI (maleylacetoacetate isomerase), which functions in tyrosine catabolism by catalyzing the isomerization of MAA (maley...

Protein Glycosylation in Archaea: A Post-Translational Modification to Enhance Extremophilic Protein Stability

DTIC Science & Technology

2010-01-15

Analysis of the chemical composition of the Asn-linked polysaccharides decorating many archaeal proteins has revealed the use of a wider variety of sugar...reminiscent of the eukaryal glycan-charged lipid, linked to a variety of monosaccharides , including glucose, mannose, and N-acetylglucosamine (GlcNAc

Role of glutathione S-transferase Pi in cisplatin-induced nephrotoxicity.

PubMed

Townsend, Danyelle M; Tew, Kenneth D; He, Lin; King, Jarrod B; Hanigan, Marie H

2009-02-01

One of the dose-limiting toxicities of cisplatin is nephrotoxicity. Renal toxicity is localized to quiescent proximal tubule cells, where the formation of DNA-adducts cannot account for the dose-limiting toxicity. Our earlier results have shown that a glutathione conjugate of cisplatin is metabolized to a nephrotoxicant via gamma-glutamyl transpeptidase (GGT) and a cysteine S-conjugate beta-lyase. The present study was designed to evaluate the potential role of glutathione S-transferase Pi (GSTP) in the initial steps of the bioactivation of cisplatin. Wild-type mice and mice deficient in both murine GSTP genes (GstP1/P2) were treated with cisplatin. Toxicity in both male and female mice was evaluated 5 days after treatment and renal damage was most severe in wild-type male mice. Wild-type males have approximately 10-fold higher levels of GSTP expression in the liver than females, suggesting that hepatic GSTP in the wild-type males contributed to the formation of the nephrotoxic platinum-glutathione conjugate. In GstP1/P2 null mice the gender difference in toxicity was eliminated. Our data show that GSTP expression is a determinant in cisplatin-induced nephrotoxicity and its levels contribute to sex-dependent differences.

Role of Glutathione S-Transferase Pi in Cisplatin Induced Nephrotoxicity

PubMed Central

Townsend, Danyelle M.; Tew, Kenneth D.; He, Lin; King, Jarrod B.; Hanigan, Marie H.

2009-01-01

SUMMARY One of the dose-limiting toxicities of cisplatin is nephrotoxicity. Renal toxicity is localized to quiescent proximal tubule cells, where the formation of DNA-adducts cannot account for the dose-limiting toxicity. Our earlier results have shown that a glutathione-conjugate of cisplatin is metabolized to a nephrotoxicant via gamma-glutamyltranspeptidase (GGT) and a cysteine S-conjugate beta-lyase. The present study was designed to evaluate the potential role of glutathione-S-transferase Pi (GSTP) in the initial steps of the bioactivation of cisplatin. Wild-type mice and mice deficient in both murine GSTP genes (GstP1/P2) were treated with cisplatin. Toxicity in both male and female mice was evaluated 5 days after treatment and renal damage was most severe in wild-type male mice. Wild-type males have ~10-fold higher levels of GSTP expression in the liver than females, suggesting that hepatic GSTP in the wild-type males contributed to the formation of the nephrotoxic platinum-glutathione conjugate. In GstP1/P2 null mice the gender difference in toxicity was eliminated. Our data show that GSTP expression is a determinant in cisplatin-induced nephrotoxicity and its levels contribute to sex-dependent differences. PMID:18819770

Glutathione S-transferase activity in follicular fluid from women undergoing ovarian stimulation: role in maturation.

PubMed

Meijide, Susana; Hernández, M Luisa; Navarro, Rosaura; Larreategui, Zaloa; Ferrando, Marcos; Ruiz-Sanz, José Ignacio; Ruiz-Larrea, M Begoña

2014-10-01

Female infertility involves an emotional impact for the woman, often leading to a state of anxiety and low self-esteem. The assisted reproduction techniques (ART) are used to overcome the problem of infertility. In a first step of the in vitro fertilization therapy women are subjected to an ovarian stimulation protocol to obtain mature oocytes, which will result in competent oocytes necessary for fertilization to occur. Ovarian stimulation, however, subjects the women to a high physical and psychological stress, thus being essential to improve ART and to find biomarkers of dysfunction and fertility. GSH is an important antioxidant, and is also used in detoxification reactions, catalysed by glutathione S-transferases (GST). In the present work, we have investigated the involvement of GST in follicular maturation. Patients with fertility problems and oocyte donors were recruited for the study. From each woman follicles at two stages of maturation were extracted at the preovulatory stage. Follicular fluid was separated from the oocyte by centrifugation and used as the enzyme source. GST activity was determined based on its conjugation with 3,4-dichloronitrobenzene and the assay was adapted to a 96-well microplate reader. The absorbance was represented against the incubation time and the curves were adjusted to linearity (R(2)>0.990). Results showed that in both donors and patients GST activity was significantly lower in mature oocytes compared to small ones. These results suggest that GST may play a role in the follicle maturation by detoxifying xenobiotics, thus contributing to the normal development of the oocyte. Supported by FIS/FEDER (PI11/02559), Gobierno Vasco (Dep. Educación, Universiades e Investigación, IT687-13), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01). Copyright © 2014. Published by

Genetic polymorphisms in glutathione-S-transferases are associated with anxiety and mood disorders in nicotine dependence

PubMed Central

Pizzo de Castro, Márcia Regina; Ehara Watanabe, Maria Angelica; Losi Guembarovski, Roberta; Odebrecht Vargas, Heber; Vissoci Reiche, Edna Maria; Kaminami Morimoto, Helena; Dodd, Seetal; Berk, Michael

2014-01-01

Background Nicotine dependence is associated with an increased risk of mood and anxiety disorders and suicide. The primary hypothesis of this study was to identify whether the polymorphisms of two glutathione-S-transferase enzymes (GSTM1 and GSTT1 genes) predict an increased risk of mood and anxiety disorders in smokers with nicotine dependence. Materials and methods Smokers were recruited at the Centre of Treatment for Smokers. The instruments were a sociodemographic questionnaire, Fagerström Test for Nicotine Dependence, diagnoses of mood disorder and nicotine dependence according to DSM-IV (SCID-IV), and the Alcohol, Smoking and Substance Involvement Screening Test. Anxiety disorder was assessed based on the treatment report. Laboratory assessment included glutathione-S-transferases M1 (GSTM1) and T1 (GSTT1), which were detected by a multiplex-PCR protocol. Results Compared with individuals who had both GSTM1 and GSTT1 genes, a higher frequency of at least one deletion of the GSTM1 and GSTT1 genes was identified in anxious smokers [odds ratio (OR)=2.21, 95% confidence interval (CI)=1.05–4.65, P=0.034], but there was no association with bipolar and unipolar depression (P=0.943). Compared with nonanxious smokers, anxious smokers had a greater risk for mood disorders (OR=4.67; 95% CI=2.24–9.92, P<0.001), lung disease (OR=6.78, 95% CI=1.95–23.58, P<0.003), and suicide attempts (OR=17.01, 95% CI=2.23–129.91, P<0.006). Conclusion This study suggests that at least one deletion of the GSTM1 and GSTT1 genes represents a risk factor for anxious smokers. These two genes may modify the capacity for the detoxification potential against oxidative stress. PMID:24637631

Identification and suppression of the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase in Zea mays L.

PubMed

Marita, Jane M; Hatfield, Ronald D; Rancour, David M; Frost, Kenneth E

2014-06-01

Grasses, such as Zea mays L. (maize), contain relatively high levels of p-coumarates (pCA) within their cell walls. Incorporation of pCA into cell walls is believed to be due to a hydroxycinnamyl transferase that couples pCA to monolignols. To understand the role of pCA in maize development, the p-coumaroyl CoA:hydroxycinnamyl alcohol transferase (pCAT) was isolated and purified from maize stems. Purified pCAT was subjected to partial trypsin digestion, and peptides were sequenced by tandem mass spectrometry. TBLASTN analysis of the acquired peptide sequences identified a single full-length maize cDNA clone encoding all the peptide sequences obtained from the purified enzyme. The cDNA clone was obtained and used to generate an RNAi construct for suppressing pCAT expression in maize. Here we describe the effects of suppression of pCAT in maize. Primary screening of transgenic maize seedling leaves using a new rapid analytical platform was used to identify plants with decreased amounts of pCA. Using this screening method, mature leaves from fully developed plants were analyzed, confirming reduced pCA levels throughout plant development. Complete analysis of isolated cell walls from mature transgenic stems and leaves revealed that lignin levels did not change, but pCA levels decreased and the lignin composition was altered. Transgenic plants with the lowest levels of pCA had decreased levels of syringyl units in the lignin. Thus, altering the levels of pCAT expression in maize leads to altered lignin composition, but does not appear to alter the total amount of lignin present in the cell walls. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Oxidative stress markers and genetic polymorphisms of glutathione S-transferase T1, M1, and P1 in a subset of children with autism spectrum disorder in Lagos, Nigeria.

PubMed

Oshodi, Y; Ojewunmi, O; Oshodi, T A; Ijarogbe, G T; Ogun, O C; Aina, O F; Lesi, Fea

2017-09-01

The role of oxidative stress has been identified in the development of autism spectrum disorder (ASD), and polymorphisms of glutathione S-transferase have been associated with some diseases linked to oxidative stress. Hence, we evaluated the serum levels of oxidative stress markers and investigated genetic polymorphisms of glutathione S-transferase associated with autism. Forty-two children clinically diagnosed with ASD using the Diagnostic and Statistical Manual for Mental Disorders (DSM-5) criteria and a clinical interview were included in the study. Twenty-three age-matched controls without any known genetic/developmental disorder were also recruited. Oxidative stress markers along with the genetic polymorphisms of glutathione S-transferase were determined. Reduced glutathione in ASD patients was significantly lower than the control (P = 0.008), whereas other oxidative stress markers measured were not significantly different in both the control and case populations. The frequencies of GSTT1 and GSTM1 null genotypes were lower among the controls compared with the cases, however, no association risk was observed. The observed risk of carrying Val/Val genotype among the cases was approximately six times that of the controls. Individuals with ASD showed a significant diminished level of reduced glutathione, however, the distribution of GSTT1, GSTM1, and GSTP1 polymorphisms was not found to be associated with autism in this study population.

Amitriptyline may have a supportive role in cancer treatment by inhibiting glutathione S-transferase pi (GST-π) and alpha (GST-α).

PubMed

Kulaksiz-Erkmen, Gulnihal; Dalmizrak, Ozlem; Dincsoy-Tuna, Gamze; Dogan, Arın; Ogus, I Hamdi; Ozer, Nazmi

2013-02-01

A tricyclic anti-depressant, amitriptyline, is a highly prescribed drug for cancer patients for mood elevation but there are limited studies about the interaction of amitriptyline with glutathione S-transferases pi (GST-π) and glutathione S-transferases alpha (GST-α). GST isozymes have been implicated in chemotherapeutic drug resistance. We demonstrated that the concentration dependent inhibition of GST-π and GST-α by amitriptyline followed inverse hyperbolic inhibition curves with IC(50) values of 5.54 and 8.32 mM, respectively. When the varied substrate was GSH, amitriptyline inhibited both isozymes competitively and similar K(i) values were found for GST-π (K(i) = 1.61 ± 0.17 mM) and GST-α (K(i) = 1.45 ± 0.20 mM). On the other hand, when the varied substrate was CDNB, the inhibition types were non-competitive for GST-π (K(i) = 1.98 ± 0.31 mM) and competitive for GST-α (K(i) = 1.57 ± 0.16 mM). Amitriptyline, in addition to its antidepressant effect, might also have a minor supportive role on the effectiveness of the anticancer drugs by decreasing their elimination through inhibiting GST-π and GST-α.

Terminal deoxynucleotidyl transferase in the diagnosis of leukemia and malignant lymphoma.

PubMed

Kung, P C; Long, J C; McCaffrey, R P; Ratliff, R L; Harrison, T A; Baltimore, D

1978-05-01

Neoplastic cells from 253 patients with leukemia and 46 patients with malignant lymphoma were studied for the presence of terminal deoxynucleotidyl transferase (TdT) by biochemical and fluorescent antibody technics. TdT was detected in circulating blast cells from 73 of 77 patients with acute lymphoblastic leukemia, 24 of 72 patients with chronic myelogenous leukemia examined during the blastic phase of the disorder and in cell suspensions of lymph nodes from nine of nine patients with diffuse lymphoblastic lymphoma. Blast cells from six of 10 patients with acute undifferentiated leukemia were TdT positive, but the enzyme was found in only two of 55 patients with acute myeloblastic leukemia. TdT was not detected in other lymphocytic or granulocytic leukemias or in other types of malignant lymphomas. The fluorescent antibody assay for TdT permits rapid and specific identification of the enzyme in single cells. The TdT assay is clinically useful in confirming the diagnosis of acute lymphoblastic leukemia, evaluating patients with blastic chronic myelogenous leukemia, and distinguishing patients with lymphoblastic lymphoma, whose natural history includes rapid extranodal dissemination, from patients with other poorly differentiated malignant lymphomas.

Glutathione-s-transferase-pi expression in early breast cancer: association with outcome and response to chemotherapy.

PubMed

Arun, Banu K; Granville, Laura A; Yin, Guosheng; Middleton, Lavinia P; Dawood, Shaheenah; Kau, Shu-Wan; Kamal, Arif; Hsu, Limin; Hortobagyi, Gabriel N; Sahin, Aysegul A

2010-06-01

Glutathione-S-transferase-pi (GST-pi) is a detoxification enzyme expressed in breast cancer; however its involvement in chemotherapy sensitivity and prognosis is not well understood. We evaluated the expression of GSTpi and its predictive role of chemotherapy response. Breast tumor samples from 166 patients at stage I/II of the disease were immunostained for GST-pi, and the expression was 96 %. There was a trend toward improved disease-free survival with high GST-pi expression (p =.09). There was a statistically non-significant association between high GST-pi expression and improved outcome with adjuvant chemotherapy (p =.055). Further studies should evaluate the role of GST-pi expression in relation to response to different chemotherapies.

S-Glutathionylation of Keap1: a new role for glutathione S-transferase pi in neuronal protection.

PubMed

Carvalho, Andreia Neves; Marques, Carla; Guedes, Rita C; Castro-Caldas, Margarida; Rodrigues, Elsa; van Horssen, Jack; Gama, Maria João

2016-05-01

Oxidative stress is a key pathological feature of Parkinson's disease (PD). Glutathione S-transferase pi (GSTP) is a neuroprotective antioxidant enzyme regulated at the transcriptional level by the antioxidant master regulator nuclear factor-erythroid 2-related factor 2 (Nrf2). Here, we show for the first time that upon MPTP-induced oxidative stress, GSTP potentiates S-glutathionylation of Kelch-like ECH-associated protein 1 (Keap1), an endogenous repressor of Nrf2, in vivo. S-glutathionylation of Keap1 leads to Nrf2 activation and subsequently increases expression of GSTP. This positive feedback regulatory loop represents a novel mechanism by which GSTP elicits antioxidant protection in the brain. © 2016 Federation of European Biochemical Societies.

Isolation and purification of glutathione S-transferases from Brachionus plicatilis and B. calyciflorus (Rotifera).

PubMed

Bowman, B P; Snell, T W; Cochrane, B J

1990-01-01

1. The enzyme glutathione S-transferase (GST), a critical element in xenobiotic metabolism, was isolated from the marine rotifer Brachionus plicatilis and its freshwater congener B. calyciflorus. 2. In B. plicatilis, GST comprised 4.2% of cytosolic protein and was present as three separate isozymes with mol. wts 30,000, 31,400 and 33,700. Specific activity of crude homogenates was 56 nmol min-1 mg-1 protein, while that of affinity chromatography purified GST was 1850. 3. In B. calyciflorus, GST was present as two isozymes with mol. wts of 26,300 and 28,500, representing 1.0% of cytosolic protein. Crude GST specific activity was 1750 nmol min-1 mg-1 protein and purified was 72,400. 4. Rotifer GSTs are unusual because they are monomers whereas all other animals thus far investigated posses dimeric GSTs.

Chitin synthases are required for survival, fecundity and egg-hatch in the red flour beetle, Tribolium castaneum

USDA-ARS?s Scientific Manuscript database

The synthesis of chitin, the Beta-1,4-linked polymer of N-acetylglucosamine, is catalyzed by chitin synthase (CHS). Chitin is essential for the structural integrity of the exoskeletal cuticle and midgut peritrophic membrane (PM) of insects. To study the functions of the two chitin synthase genes, ...

Fragmentation of negative ions from N-linked carbohydrates, part 4. Fragmentation of complex glycans lacking substitution on the 6-antenna.

PubMed

Harvey, David J; Jaeken, Jaak; Butler, Mike; Armitage, Alison J; Rudd, Pauline M; Dwek, Raymond A

2010-05-01

Negative ion CID spectra of N-linked glycans released from glycoproteins contain many ions that are diagnostic for specific structural features such as the detailed arrangement of antennae and the location of fucose residues. Identification of such ions requires reference glycans that are often difficult to acquire in a pure state. The recent acquisition of a sample of N-glycans from a patient lacking the enzyme N-acetylglucosaminyltransferase-2 provided an opportunity to investigate fragmentation of glycans lacking a 6-antenna. These glycans contained one or two galactose-N-acetylglucosamine-chains attached to the 3-linked mannose residue of the trimannosyl-chitobiose core with and without fucose substitution. The spectra from the patient sample clearly defined the antenna distribution and showed striking differences from the spectra of isomeric compounds obtained from normal subjects. Furthermore, they provided additional information on previously identified antenna-specific fragment ions and indicated the presence of additional ions that were diagnostic of fucose substitution. Glycans obtained from such enzyme-deficient patients can, thus, be a valuable way of obtaining spectra of specific isomers in a relatively pure state for interpretation of mass spectra. 2010 John Wiley & Sons, Ltd.

WGA-based lectin affinity gel electrophoresis: A novel method for the detection of O-GlcNAc-modified proteins.

PubMed

Kubota, Yuji; Fujioka, Ko; Takekawa, Mutsuhiro

2017-01-01

Post-translational modification with O-linked β-N-acetylglucosamine (O-GlcNAc) occurs selectively on serine and/or threonine residues of cytoplasmic and nuclear proteins, and dynamically regulates their molecular functions. Since conventional strategies to evaluate the O-GlcNAcylation level of a specific protein require time-consuming steps, the development of a rapid and easy method for the detection and quantification of an O-GlcNAcylated protein has been a challenging issue. Here, we describe a novel method in which O-GlcNAcylated and non-O-GlcNAcylated forms of proteins are separated by lectin affinity gel electrophoresis using wheat germ agglutinin (WGA), which primarily binds to N-acetylglucosamine residues. Electrophoresis of cell lysates through a gel containing copolymerized WGA selectively induced retardation of the mobility of O-GlcNAcylated proteins, thereby allowing the simultaneous visualization of both the O-GlcNAcylated and the unmodified forms of proteins. This method is therefore useful for the quantitative detection of O-GlcNAcylated proteins.

Structural basis for the interaction of antibiotics with peptidyl transferase center in eubacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schlunzen, Frank; Zarivach, Raz; Harms, Jörg

2009-10-07

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome-antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavitymore » and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg{sup +2} ions for the binding of some drugs. This structural analysis should facilitate rational drug design.« less

Glutathione S-transferase-encoding gene as a potential probe for environmental bacterial isolates capable of degrading polycyclic aromatic hydrocarbons.

PubMed Central

Lloyd-Jones, G; Lau, P C

1997-01-01

Homologs of the glutathione S-transferase (GST)-encoding gene were identified in a collection of aromatic hydrocarbon-degrading Sphingomonas spp. isolated from New Zealand, Antarctica, and the United States by using PCR primers designed from the GST-encoding gene of Sphingomonas paucimobilis EPA505. Sequence analysis of PCR fragments generated from these isolates and of the GST gene amplified from DNA extracted from polycyclic aromatic hydrocarbon (PAH)-contaminated soil revealed a high degree of conservation, which may make the GST-encoding gene a potentially useful marker for PAH-degrading bacteria. PMID:9251217

Dual Catalytic Activity of Hydroxycinnamoyl-Coenzyme A Quinate Transferase from Tomato Allows It to Moonlight in the Synthesis of Both Mono- and Dicaffeoylquinic Acids1[W][OPEN

PubMed Central

Moglia, Andrea; Lanteri, Sergio; Comino, Cinzia; Hill, Lionel; Knevitt, Daniel; Cagliero, Cecilia; Rubiolo, Patrizia; Bornemann, Stephen

2014-01-01

Tomato (Solanum lycopersicum), like other Solanaceous species, accumulates high levels of antioxidant caffeoylquinic acids, which are strong bioactive molecules and protect plants against biotic and abiotic stresses. Among these compounds, the monocaffeoylquinic acids (e.g. chlorogenic acid [CGA]) and the dicaffeoylquinic acids (diCQAs) have been found to possess marked antioxidative properties. Thus, they are of therapeutic interest both as phytonutrients in foods and as pharmaceuticals. Strategies to increase diCQA content in plants have been hampered by the modest understanding of their biosynthesis and whether the same pathway exists in different plant species. Incubation of CGA with crude extracts of tomato fruits led to the formation of two new products, which were identified by liquid chromatography-mass spectrometry as diCQAs. This chlorogenate:chlorogenate transferase activity was partially purified from ripe fruit. The final protein fraction resulted in 388-fold enrichment of activity and was subjected to trypsin digestion and mass spectrometric sequencing: a hydroxycinnamoyl-Coenzyme A:quinate hydroxycinnamoyl transferase (HQT) was selected as a candidate protein. Assay of recombinant HQT protein expressed in Escherichia coli confirmed its ability to synthesize diCQAs in vitro. This second activity (chlorogenate:chlorogenate transferase) of HQT had a low pH optimum and a high Km for its substrate, CGA. High concentrations of CGA and relatively low pH occur in the vacuoles of plant cells. Transient assays demonstrated that tomato HQT localizes to the vacuole as well as to the cytoplasm of plant cells, supporting the idea that in this species, the enzyme catalyzes different reactions in two subcellular compartments. PMID:25301886

Regulation of chlorogenic acid biosynthesis by hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase in Lonicera japonica.

PubMed

Zhang, Jingru; Wu, Minlin; Li, Weidong; Bai, Genben

2017-12-01

For many centuries, Lonicera japonica has been used as an effective herb for the treatment of inflammation and swelling because of the presence of bioactive components such as chlorogenic acid (CGA). To clarify the relationship between L. japonica hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase (HQT) gene expression and CGA content, an HQT eukaryotic expression system was constructed using Gateway cloning. L. japonica callus transformed with HQT was obtained using Agrobacterium tumefaciens-mediated transformation. We found a positive correlation between CGA content, determined by High-Performance Liquid Chromatography (HPLC), and the expression of HQT, analyzed by semi-quantitative RT-PCR. This study demonstrates that the HQT gene positively regulates CGA synthesis and lays the foundation for further study into enhancing efficacious components of medicinal plants. Copyright © 2017. Published by Elsevier Masson SAS.

Beta-propeller crystal structure of Psathyrella velutina lectin: an integrin-like fungal protein interacting with monosaccharides and calcium.

PubMed

Cioci, Gianluca; Mitchell, Edward P; Chazalet, Valerie; Debray, Henri; Oscarson, Stefan; Lahmann, Martina; Gautier, Catherine; Breton, Christelle; Perez, Serge; Imberty, Anne

2006-04-14

The lectin from the mushroom Psathyrella velutina recognises specifically N-acetylglucosamine and N-acetylneuraminic acid containing glycans. The crystal structure of the 401 amino acid residue lectin shows that it adopts a very regular seven-bladed beta-propeller fold with the N-terminal region tucked into the central cavity around the pseudo 7-fold axis. In the complex with N-acetylglucosamine, six monosaccharides are bound in pockets located between two consecutive propeller blades. Due to the repeats shown by the sequence the binding sites are very similar. Five hydrogen bonds between the protein and the sugar hydroxyl and N-acetyl groups stabilize the complex, together with the hydrophobic interactions with a conserved tyrosine and histidine. The complex with N-acetylneuraminic acid shows molecular mimicry with the same hydrogen bond network, but with different orientations of the carbohydrate ring in the binding site. The beta-hairpin loops connecting the two inner beta-strands of each blade are metal binding sites and two to three calcium ions were located in the structure. The multispecificity and high multivalency of this mushroom lectin, combined with its similarity to the extracellular domain of an important class of cell adhesion molecules, integrins, are another example of the outstanding success of beta-propeller structures as molecular binding machines in nature.

YrdC exhibits properties expected of a subunit for a tRNA threonylcarbamoyl transferase.

PubMed

Harris, Kimberly A; Jones, Victoria; Bilbille, Yann; Swairjo, Manal A; Agris, Paul F

2011-09-01

The post-transcriptional nucleoside modifications of tRNA's anticodon domain form the loop structure and dynamics required for effective and accurate recognition of synonymous codons. The N(6)-threonylcarbamoyladenosine modification at position 37 (t(6)A(37)), 3'-adjacent to the anticodon, of many tRNA species in all organisms ensures the accurate recognition of ANN codons by increasing codon affinity, enhancing ribosome binding, and maintaining the reading frame. However, biosynthesis of this complex modification is only partially understood. The synthesis requires ATP, free threonine, a single carbon source for the carbamoyl, and an enzyme yet to be identified. Recently, the universal protein family Sua5/YciO/YrdC was associated with t(6)A(37) biosynthesis. To further investigate the role of YrdC in t(6)A(37) biosynthesis, the interaction of the Escherichia coli YrdC with a heptadecamer anticodon stem and loop of lysine tRNA (ASL(Lys)(UUU)) was examined. YrdC bound the unmodified ASL(Lys)(UUU) with high affinity compared with the t(6)A(37)-modified ASL(Lys)(UUU) (K(d) = 0.27 ± 0.20 μM and 1.36 ± 0.39 μM, respectively). YrdC also demonstrated specificity toward the unmodified versus modified anticodon pentamer UUUUA and toward threonine and ATP. The protein did not significantly alter the ASL architecture, nor was it able to base flip A(37), as determined by NMR, circular dichroism, and fluorescence of 2-aminopuine at position 37. Thus, current data support the hypothesis that YrdC, with many of the properties of a putative threonylcarbamoyl transferase, most likely functions as a component of a heteromultimeric protein complex for t(6)A(37) biosynthesis.

Ferrocene labelings as inhibitors and dual electrochemical sensors of human glutathione S-transferase P1-1.

PubMed

Martos-Maldonado, Manuel C; Quesada-Soriano, Indalecio; García-Maroto, Federico; Vargas-Berenguel, Antonio; García-Fuentes, Luís

2012-12-01

The inhibitory and sensor properties of two ferrocene conjugates, in which the ferrocene and glutathione are linked through a spacer arm of different length and chemical structure, on human Pi glutathione S-transferase, were examined by activity assays, ITC, fluorescence spectroscopy and voltammetry. Such ferrocene conjugates are strong competitive inhibitors of this enzyme with an enhanced binding affinity, the one bearing the longest spacer arm being the most potent inhibitor. Voltammetric measurements showed a strong decrease of the peak current intensity and an increase of the oxidation potential upon binding of ferrocene-glutathione conjugates to GST P1-1 showing that both conjugates can be used as dual electrochemical sensors for GST P1-1. Copyright © 2012 Elsevier Ltd. All rights reserved.

Preferential glutathione conjugation of a reverse diol epoxide compared to a bay region diol epoxide of phenanthrene in human hepatocytes: relevance to molecular epidemiology studies of glutathione-s-transferase polymorphisms and cancer.

PubMed

Hecht, Stephen S; Berg, Jeannette Zinggeler; Hochalter, J Bradley

2009-03-16

Bay region diol epoxides are recognized ultimate carcinogens of polycyclic aromatic hydrocarbons (PAH), and in vitro studies have demonstrated that they can be detoxified by conjugation with glutathione, leading to the widely investigated hypothesis that individuals with low activity forms of glutathione-S-transferases are at higher risk of PAH induced cancer, a hypothesis that has found at most weak support in molecular epidemiology studies. A weakness in this hypothesis was that the mercapturic acids resulting from the conjugation of PAH bay region diol epoxides had never been identified in human urine. We recently analyzed smokers' urine for mercapturic acids derived from phenanthrene, the simplest PAH with a bay region. The only phenanthrene diol epoxide-derived mercapturic acid in smokers' urine was produced from the reverse diol epoxide, anti-phenanthrene-3,4-diol-1,2-epoxide (11), not the bay region diol epoxide, anti-phenanthrene-1,2-diol-3,4-epoxide (10), which does not support the hypothesis noted above. In this study, we extended these results by examining the conjugation of phenanthrene metabolites with glutathione in human hepatocytes. We identified the mercapturic acid N-acetyl-S-(r-4,t-2,3-trihydroxy-1,2,3,4-tetrahydro-c-1-phenanthryl)-L-cysteine (14a), (0.33-35.9 pmol/mL at 10 microM 8, 24 h incubation, N = 10) in all incubations with phenanthrene-3,4-diol (8) and the corresponding diol epoxide 11, but no mercapturic acids were detected in incubations with phenanthrene-1,2-diol (7), and only trace amounts were observed in incubations with the corresponding bay region diol epoxide 10. Taken together with our previous results, these studies clearly demonstrate that glutathione conjugation of a reverse diol epoxide of phenanthrene is favored over conjugation of a bay region diol epoxide. Since reverse diol epoxides of PAH are generally weakly or nonmutagenic/carcinogenic, these results, if generalizable to other PAH, do not support the widely held

Methods for improving enzymatic trans-glycosylation for synthesis of human milk oligosaccharide biomimetics.

PubMed

Zeuner, Birgitte; Jers, Carsten; Mikkelsen, Jørn Dalgaard; Meyer, Anne S

2014-10-08

Recently, significant progress has been made within enzymatic synthesis of biomimetic, functional glycans, including, for example, human milk oligosaccharides. These compounds are mainly composed of N-acetylglucosamine, fucose, sialic acid, galactose, and glucose, and their controlled enzymatic synthesis is a novel field of research in advanced food ingredient chemistry, involving the use of rare enzymes, which have until now mainly been studied for their biochemical significance, not for targeted biosynthesis applications. For the enzymatic synthesis of biofunctional glycans reaction parameter optimization to promote "reverse" catalysis with glycosidases is currently preferred over the use of glycosyl transferases. Numerous methods exist for minimizing the undesirable glycosidase-catalyzed hydrolysis and for improving the trans-glycosylation yields. This review provides an overview of the approaches and data available concerning optimization of enzymatic trans-glycosylation for novel synthesis of complex bioactive carbohydrates using sialidases, α-l-fucosidases, and β-galactosidases as examples. The use of an adequately high acceptor/donor ratio, reaction time control, continuous product removal, enzyme recycling, and/or the use of cosolvents may significantly improve trans-glycosylation and biocatalytic productivity of the enzymatic reactions. Protein engineering is also a promising technique for obtaining high trans-glycosylation yields, and proof-of-concept for reversing sialidase activity to trans-sialidase action has been established. However, the protein engineering route currently requires significant research efforts in each case because the structure-function relationship of the enzymes is presently poorly understood.

Electron transfer dissociation (ETD): The mass spectrometric breakthrough essential for O-GlcNAc protein site assignments – A study of the O-GlcNAcylated protein Host Cell Factor C1

PubMed Central

Myers, Samuel A.; Daou, Salima; Affar, El Bachir; Burlingame, AL

2014-01-01

The development of electron-based, unimolecular dissociation mass spectrometric methods, i.e. electron capture and electron transfer dissociation (ECD and ETD, respectively), has greatly increased the speed and reliability of labile post-translational modification (PTM) site assignment. The field of intracellular O-GlcNAc (O-linked N-acetylglucosamine) signaling has especially advanced with the advent of ETD mass spectrometry. Only within the last five years have proteomic-scale experiments utilizing ETD allowed the assignment of hundreds of O-GlcNAc sites within cells and subcellular structures. Our ability to identify and unambiguously assign the site of O-GlcNAc modifications using ETD is rapidly increasing our understanding of this regulatory glycosylation and its potential interaction with other PTMs. Here, we discuss the advantages of using ETD, complimented with collisional-activation mass spectrometry (CID/CAD), in a study of O-GlcNAc modified peptides of the extensively O-GlcNAcylated protein Host Cell Factor C1 (HCF-1). HCF-1 is a transcriptional co-regulator, forms a stable complex with O-GlcNAc transferase and is involved in control of cell cycle progression. ETD, along with higher energy collisional dissociation (HCD) mass spectrometry, was employed to assign the PTMs of the HCF-1 protein isolated from HEK293T cells. These include nineteen sites of O-GlcNAcylation, two sites of phosphorylation and two sites bearing dimethylarginine, and showcase the residue-specific, PTM complexity of this regulator of cell proliferation. PMID:23335398

Structural analysis of the asparagine-linked oligosaccharides of cholinesterases. N-linked carbohydrates of cholinesterases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saxena, A.; Doctor, B.P.

1995-12-31

Cholinesterases are serine esterases that hydrolyse choline esters faster than other substrates. They are highly glycosylated proteins with up to 24% of their molecular weight constituted of carbohydrates. Here we report the results of our studies on the glycosylation of fetal bovine serum acetylcholinesterase (FBS AChE) and horse serum butyrylcholinesterase (Eq BChE). Analysis of the monosaccharide content of the two enzymes indicated that Eq BChE contained 520 nmoles of monosaccharide/mg protein, as compared to 1290 nmoles/mg protein for Eq BChE. Both enzymes contained mannose, galactose, N-acetylglucosamine and sialic acid. Fucose was present in Eq BChE only. The structures of themore » two major oligosaccharides from FBS AChE and one major oligosaccharide from Eq BChE were determined and found to be very similar except that one of the oligosaccharides from FBS AChE contained a galactose alphal-3 galactose betal-4-determinant which has been identified as a potentially immunogenic determinant.« less

Glutathione S-transferase M1 (GSTM1) polymorphisms and lung cancer: a literature-based systematic HuGE review and meta-analysis.

PubMed

Carlsten, C; Sagoo, G S; Frodsham, A J; Burke, W; Higgins, J P T

2008-04-01

Multiple genes have been studied for potential associations with lung cancer. The gene most frequently associated with increased risk has been glutathione S-transferase M1 (GSTM1). The glutathione S-transferase enzyme family is known to catalyze detoxification of electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins, and products of oxidative stress. In this review, the authors summarize the available evidence associating lung cancer with the GSTM1 gene. They describe results from an updated meta-analysis of 98 published genetic association studies investigating the relation between the GSTM1 null variant and lung cancer risk including 19,638 lung cancer cases and 25,266 controls (counting cases and controls in each study only once). All studies considered, the GSTM1 null variant was associated with an increased risk of lung cancer (odds ratio (OR) = 1.22, 95% confidence interval (CI): 1.14, 1.30), but no increase in risk was seen (OR = 1.01, 95% CI: 0.91, 1.12) when only the five largest studies (>500 cases each) were considered. Furthermore, while GSTM1 null status conferred a significantly increased risk of lung cancer to East Asians (OR = 1.38, 95% CI: 1.24, 1.55), such a genotype did not confer increased risk to Caucasians. More data regarding the predictive value of GSTM1 genetic testing are needed before population-based testing may be reasonably considered.

Efficacy of a new oral lubricant solution in the management of psychotropic drug-induced xerostomia: a randomized controlled trial.

PubMed

Mouly, Stéphane J; Orler, Jean-Baptiste; Tillet, Yves; Coudert, Anne-Claude; Oberli, Frantz; Preshaw, Phillip; Bergmann, Jean-François

2007-10-01

Xerostomia is a subjective sensation of mouth dryness often occurring as an unwanted effect of psychotropic drugs. The clinical efficacy and acceptability of a new oxygenated glycerol triester (OGT) oral spray (1 or 2 sprays up to 4 times daily) in the treatment of xerostomia was compared with those of a commercially available artificial saliva substitute (ASS [Saliveze]) in a 2-week, open-labeled, randomized, parallel-group study. Clinical assessment of xerostomia included evaluation of mouth dryness by means of a 10-cm-long visual analog scale, objective blinded assessment of the oral tissue condition by a dental hygienist by means of a 4-point ordinal scale, and subjective patient-based assessment of dry mouth symptoms by means of dichotomous responses to a questionnaire. [Day 14 - baseline] patient-based mouth dryness score was the primary end point. Seventy-four patients (41 women and 33 men, 44 +/- 15 years) undergoing long-term psychotropic drug treatment were consecutively enrolled. At day 14, OGT resulted in better efficacy than ASS in mouth dryness score (mean difference, 1.2 +/- 0.4; P = 0.006), speech difficulties (mean difference, 1.2 +/- 0.4; P = 0.005), taste (mean difference, 1.1 +/- 0.4; P = 0.02), and overall mouth condition (mean difference, 1.4 +/- 0.9; P = 0.005). Taste of OGT was better than that of ASS (mean difference, 1.4 +/- 0.6; P = 0.04), as was OGT acceptability (mean difference, 1.4 +/- 0.9; P = 0.005). Oxygenated glycerol triester lubricant oral spray was superior to a commercially available ASS in improving xerostomia and overall condition of the oral tissue.

The N-glycan processing enzymes α-mannosidase and β-D-N-acetylhexosaminidase are involved in ripening-associated softening in the non-climacteric fruits of capsicum

PubMed Central

Ghosh, Sumit; Meli, Vijaykumar S.; Kumar, Anil; Thakur, Archana; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

2011-01-01

Excessive softening of fruits during the ripening process leads to deterioration. This is of significant global importance as softening-mediated deterioration leads to huge postharvest losses. N-glycan processing enzymes are reported to play an important role during climacteric fruit softening: however, to date these enzymes have not been characterized in non-climacteric fruit. Two ripening-specific N-glycan processing enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex), have been identified and targeted to enhance the shelf life in non-climacteric fruits such as capsicum (Capsicum annuum). The purification, cloning, and functional characterization of α-Man and β-Hex from capsicum, which belong to glycosyl hydrolase (GH) families 38 and 20, respectively, are described here. α-Man and β-Hex are cell wall glycoproteins that are able to cleave terminal α-mannose and β-D-N-acetylglucosamine residues of N-glycans, respectively. α-Man and β-Hex transcripts as well as enzyme activity increase with the ripening and/or softening of capsicum. The function of α-Man and β-Hex in capsicum softening is investigated through RNA interference (RNAi) in fruits. α-Man and β-Hex RNAi fruits were approximately two times firmer compared with the control and fruit deterioration was delayed by approximately 7 d. It is shown that silencing of α-Man and β-Hex enhances fruit shelf life due to the reduced degradation of N-glycoproteins which resulted in delayed softening. Altogether, the results provide evidence for the involvement of N-glycan processing in non-climacteric fruit softening. In conclusion, genetic engineering of N-glycan processing can be a common strategy in both climacteric and non-climacteric species to reduce the post-harvest crop losses. PMID:21030387

Assessing Diversity of DNA Structure-Related Sequence Features in Prokaryotic Genomes

PubMed Central

Huang, Yongjie; Mrázek, Jan

2014-01-01

Prokaryotic genomes are diverse in terms of their nucleotide and oligonucleotide composition as well as presence of various sequence features that can affect physical properties of the DNA molecule. We present a survey of local sequence patterns which have a potential to promote non-canonical DNA conformations (i.e. different from standard B-DNA double helix) and interpret the results in terms of relationships with organisms' habitats, phylogenetic classifications, and other characteristics. Our present work differs from earlier similar surveys not only by investigating a wider range of sequence patterns in a large number of genomes but also by using a more realistic null model to assess significant deviations. Our results show that simple sequence repeats and Z-DNA-promoting patterns are generally suppressed in prokaryotic genomes, whereas palindromes and inverted repeats are over-represented. Representation of patterns that promote Z-DNA and intrinsic DNA curvature increases with increasing optimal growth temperature (OGT), and decreases with increasing oxygen requirement. Additionally, representations of close direct repeats, palindromes and inverted repeats exhibit clear negative trends with increasing OGT. The observed relationships with environmental characteristics, particularly OGT, suggest possible evolutionary scenarios of structural adaptation of DNA to particular environmental niches. PMID:24408877

Phylogenetic characterization of Clonorchis sinensis proteins homologous to the sigma-class glutathione transferase and their differential expression profiles.

PubMed

Bae, Young-An; Kim, Jeong-Geun; Kong, Yoon

2016-01-01

Glutathione transferase (GST) is one of the major antioxidant proteins with diverse supplemental activities including peroxidase, isomerase, and thiol transferase. GSTs are classified into multiple classes on the basis of their primary structures and substrate/inhibitor specificity. However, the evolutionary routes and physiological environments specific to each of the closely related bioactive enzymes remain elusive. The sigma-like GSTs exhibit amino acid conservation patterns similar to the prostaglandin D synthases (PGDSs). In this study, we analyzed the phylogenetic position of the GSTs of the biocarcinogenic liver fluke, Clonorchis sinensis. We also observed induction profile of the GSTs in association with the parasite's maturation and in response to exogenous oxidative stresses, with special attention to sigma-class GSTs and PGDSs. The C. sinensis genome encoded 12 GST protein species, which were separately assigned to cytosolic (two omega-, one zeta-, two mu-, and five sigma-class), mitochondrial (one kappa-class), and microsomal (one membrane-associated proteins in eicosanoid and glutathione metabolism-like protein) GST families. Multiple sigma GST (or PGDS) orthologs were also detected in Opisthorchis viverrini. Other trematode species possessed only a single sigma-like GST gene. A phylogenetic analysis demonstrated that one of the sigma GST lineages duplicated in the common ancestor of trematodes were specifically expanded in the opisthorchiids, but deleted in other trematodes. The induction profiles of these sigma GST genes along with the development and aging of C. sinensis, and against various exogenous chemical stimuli strongly suggest that the paralogous sigma GST genes might be undergone specialized evolution to cope with the diverse hostile biochemical environments within the mammalian hepatobiliary ductal system. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of human β-N-acetylhexosaminidase B in yeast eases the search for selective inhibitors.

PubMed

Krejzová, Jana; Kulik, Natallia; Slámová, Kristýna; Křen, Vladimír

2016-07-01

Human lysosomal β-N-acetylhexosaminidases from the family 20 of glycoside hydrolases are dimeric enzymes catalysing the cleavage of terminal β-N-acetylglucosamine and β-N-acetylgalactosamine residues from a broad spectrum of glycoconjugates. Here, we present a facile, robust, and cost-effective extracellular expression of human β-N-acetylhexosaminidase B in Pichia pastoris KM71H strain. The prepared Hex B was purified in a single step with 33% yield obtaining 10mg of the pure enzyme per 1L of the culture media. The enzyme was used in the inhibition assays with the known mechanism-based inhibitor NAG-thiazoline and a wide variety of its derivatives in the search for specific inhibitors of the human GH20 β-N-acetylhexosaminidases over the human GH84 β-N-acetylglucosaminidase, which was expressed, purified and used in the inhibition experiments as well. Moreover, enzyme-inhibitor complexes were analysed employing computational tools in order to reveal the structural basis of the results of the inhibition assays, showing the importance of water-mediated interactions between the enzyme and respective ligands. The presented method for the heterologous expression of human Hex B is robust, it significantly reduces the costs and equipment demands in comparison to the expression in mammalian cell lines. This will enhance accessibility of this human enzyme to the broad scientific community and may speed up the research of specific inhibitors of this physiologically important glycosidase family. Copyright © 2016 Elsevier Inc. All rights reserved.

Glutathione-S-transferases pi, alpha, mu and mdr1 mRNA expression in normal lymphocytes and chronic lymphocytic leukemia.

PubMed

Marie, J P; Simonin, G; Legrand, O; Delmer, A; Faussat, A M; Lewis, A D; Sikic, B I; Zittoun, R

1995-10-01

Chronic B cell lymphoproliferative disorders are frequently sensitive to alkylating agents. To assess the glutathione-S-transferases (GSTs) gene expression in B tumoral lymphocytes, possibly responsible for this sensitivity, we developed a sensitive RT-PCR assay for the three isoenzymes GST pi, GST mu and GST alpha mRNA. Normal B and T lymphocytes from 11 blood donors were separated by magnetic beads and tested with this assay. The GST pi was the most abundant transferase, and was detected in all B and T cell samples. GST mu was undetectable ('null' phenotype) in 6/11 normal donors, either in B or T cells. GST alpha was very stable from donor to donor, and was highly correlated between B and T cells of the same individual (P < 0.0001). There is no correlation between the three isoenzymes, and between each isoenzyme and mdr1 gene expression. Twenty-three B lymphoproliferative disorders (20 B-CLL, 3 CD5- chronic lymphoproliferative syndromes) were tested with the same technique. An average decrease of 57% of the GST pi expression was noted in the mononuclear cells of these patients (P < 0.02), with no differences between the untreated and treated cases. The GST alpha and mdr1 mRNA levels did not differ from normal B lymphocytes, but the proportion of patients with no detectable expression of GST mu is lower than in the control (13%). Interestingly, the low content of GST pi in B-CLL could explain the frequent sensitivity of this disease to alkylating agents.

New high-performance liquid chromatography assay for glycosyltransferases based on derivatization with anthranilic acid and fluorescence detection.

PubMed

Anumula, Kalyan Rao

2012-07-01

Assays were developed using the unique labeling chemistry of 2-aminobenzoic acid (2AA; anthranilic acid, AA) for measuring activities of both β1-4 galactosyltransferase (GalT-1) and α2-6 sialyltransferase (ST-6) by high-performance liquid chromatography (HPLC) with fluorescence detection (Anumula KR. 2006. Advances in fluorescence derivatization methods for high-performance liquid chromatographic analysis of glycoprotein carbohydrates. Anal Biochem. 350:1-23). N-Acetylglucosamine (GlcNAc) and N-acetyllactosamine were used as acceptors and uridine diphosphate (UDP)-galactose and cytidine monophosphate (CMP)-N-acetylneuraminic acid (NANA) as donors for GalT-1 and ST-6, respectively. Enzymatic products were labeled in situ with AA and were separated from the substrates on TSKgel Amide 80 column using normal-phase conditions. Enzyme units were determined from the peak areas by comparison with the concomitantly derivatized standards Gal-β1-4GlcNAc and NANA-α2-6 Gal-β1-4GlcNAc. Linearity (time and enzyme concentration), precision (intra- and interassay) and reproducibility for the assays were established. The assays were found to be useful in monitoring the enzyme activities during isolation and purification. The assays were highly sensitive and performed equal to or better than the traditional radioactive sugar-based measurements. The assay format can also be used for measuring the activity of other transferases, provided that the carbohydrate acceptors contain a reducing end for labeling. An assay for glycoprotein acceptors was developed using IgG. A short HPLC profiling method was developed for the separation of IgG glycans (biantennary G0, G1, G2, mono- and disialylated), which facilitated the determination of GalT-1 and ST-6 activities in a rapid manner. Furthermore, this profiling method should prove useful for monitoring the changes in IgG glycans in clinical settings.

Biosynthesis of glycoproteins in the human pathogenic fungus Sporothrix schenckii: synthesis of dolichol phosphate mannose and mannoproteins by membrane-bound and solubilized mannosyl transferases.

PubMed

Ruiz-Baca, Estela; Villagómez-Castro, Julio C; Leal-Morales, Carlos A; Sabanero-López, Myrna; Flores-Carreón, Arturo; López-Romero, Everardo

2005-01-01

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg2+ and Mn2+. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A-Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26-112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.

Trivalent chromium inhibits TSP-1 expression, proliferation, and O-GlcNAc signaling in vascular smooth muscle cells in response to high glucose in vitro.

PubMed

Ganguly, Rituparna; Sahu, Soumyadip; Chavez, Ronaldo J; Raman, Priya

2015-01-15

Trivalent chromium (Cr(3+)) is a mineral nutrient reported to have beneficial effects in glycemic and cardiovascular health. In vitro and in vivo studies suggest that Cr(3+) supplementation reduces the atherogenic potential and lowers the risk of vascular inflammation in diabetes. However, effects of Cr(3+) in vascular cells under conditions of hyperglycemia, characteristic of diabetes, remain unknown. In the present study we show that a therapeutically relevant concentration of Cr(3+) (100 nM) significantly downregulates a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in human aortic smooth muscle cells (HASMC) stimulated with high glucose in vitro. Promoter-reporter assays reveal that this downregulation of TSP-1 expression by Cr(3+) occurs at the level of transcription. The inhibitory effects of Cr(3+) on TSP-1 were accompanied by significant reductions in O-glycosylation of cytoplasmic and nuclear proteins. Using Western blotting and immunofluorescence studies, we demonstrate that reduced protein O-glycosylation by Cr(3+) is mediated via inhibition of glutamine: fructose 6-phosphate amidotransferase, a rate-limiting enzyme of the hexosamine pathway, and O-linked N-acetylglucosamine (O-GlcNAc) transferase, a distal enzyme in the pathway that controls intracellular protein O-glycosylation. Additionally, we found that Cr(3+) attenuates reactive oxygen species formation in glucose-stimulated HASMC, suggesting an antioxidant effect. Finally, we report an antiproliferative effect of Cr(3+) that is specific for high glucose and conditions triggering elevated protein O-glycosylation. Taken together, these findings provide the first cellular evidence for a novel role of Cr(3+) to modulate aberrant vascular smooth muscle cell function associated with hyperglycemia-induced vascular complications. Copyright © 2015 the American Physiological Society.

Phi Class of Glutathione S-transferase Gene Superfamily Widely Exists in Nonplant Taxonomic Groups.

PubMed

Munyampundu, Jean-Pierre; Xu, You-Ping; Cai, Xin-Zhong

2016-01-01

Glutathione S-transferases (GSTs) constitute a superfamily of enzymes involved in detoxification of noxious compounds and protection against oxidative damage. GST class Phi (GSTF), one of the important classes of plant GSTs, has long been considered as plant specific but was recently found in basidiomycete fungi. However, the range of nonplant taxonomic groups containing GSTFs remains unknown. In this study, the distribution and phylogenetic relationships of nonplant GSTFs were investigated. We identified GSTFs in ascomycete fungi, myxobacteria, and protists Naegleria gruberi and Aureococcus anophagefferens. GSTF occurrence in these bacteria and protists correlated with their genome sizes and habitats. While this link was missing across ascomycetes, the distribution and abundance of GSTFs among ascomycete genomes could be associated with their lifestyles to some extent. Sequence comparison, gene structure, and phylogenetic analyses indicated divergence among nonplant GSTFs, suggesting polyphyletic origins during evolution. Furthermore, in silico prediction of functional partners suggested functional diversification among nonplant GSTFs.

Phi Class of Glutathione S-transferase Gene Superfamily Widely Exists in Nonplant Taxonomic Groups

PubMed Central

Munyampundu, Jean-Pierre; Xu, You-Ping; Cai, Xin-Zhong

2016-01-01

Glutathione S-transferases (GSTs) constitute a superfamily of enzymes involved in detoxification of noxious compounds and protection against oxidative damage. GST class Phi (GSTF), one of the important classes of plant GSTs, has long been considered as plant specific but was recently found in basidiomycete fungi. However, the range of nonplant taxonomic groups containing GSTFs remains unknown. In this study, the distribution and phylogenetic relationships of nonplant GSTFs were investigated. We identified GSTFs in ascomycete fungi, myxobacteria, and protists Naegleria gruberi and Aureococcus anophagefferens. GSTF occurrence in these bacteria and protists correlated with their genome sizes and habitats. While this link was missing across ascomycetes, the distribution and abundance of GSTFs among ascomycete genomes could be associated with their lifestyles to some extent. Sequence comparison, gene structure, and phylogenetic analyses indicated divergence among nonplant GSTFs, suggesting polyphyletic origins during evolution. Furthermore, in silico prediction of functional partners suggested functional diversification among nonplant GSTFs. PMID:26884677

Electrochemical DNA biosensor based on grafting-to mode of terminal deoxynucleoside transferase-mediated extension.

PubMed

Chen, Jinyuan; Liu, Zhoujie; Peng, Huaping; Zheng, Yanjie; Lin, Zhen; Liu, Ailin; Chen, Wei; Lin, Xinhua

2017-12-15

Previously reported electrochemical DNA biosensors based on in-situ polymerization approach reveal that terminal deoxynucleoside transferase (TdTase) has good amplifying performance and promising application in the design of electrochemical DNA biosensor. However, this method, in which the background is significantly affected by the amount of TdTase, suffers from being easy to produce false positive result and poor stability. Herein, we firstly present a novel electrochemical DNA biosensor based on grafting-to mode of TdTase-mediated extension, in which DNA targets are polymerized in homogeneous solution and then hybridized with DNA probes on BSA-based DNA carrier platform. It is surprising to find that the background in the grafting-to mode of TdTase-based electrochemical DNA biosensor have little interference from the employed TdTase. Most importantly, the proposed electrochemical DNA biosensor shows greatly improved detection performance over the in-situ polymerization approach-based electrochemical DNA biosensor. Copyright © 2017 Elsevier B.V. All rights reserved.

Nuclear glutathione S-transferase pi prevents apoptosis by reducing the oxidative stress-induced formation of exocyclic DNA products.

PubMed

Kamada, Kensaku; Goto, Shinji; Okunaga, Tomohiro; Ihara, Yoshito; Tsuji, Kentaro; Kawai, Yoshichika; Uchida, Koji; Osawa, Toshihiko; Matsuo, Takayuki; Nagata, Izumi; Kondo, Takahito

2004-12-01

We previously found that nuclear glutathione S-transferase pi (GSTpi) accumulates in cancer cells resistant to anticancer drugs, suggesting that it has a role in the acquisition of resistance to anticancer drugs. In the present study, the effect of oxidative stress on the nuclear translocation of GSTpi and its role in the protection of DNA from damage were investigated. In human colonic cancer HCT8 cells, the hydrogen peroxide (H(2)O(2))-induced increase in nuclear condensation, the population of sub-G(1) peak, and the number of TUNEL-positive cells were observed in cells pretreated with edible mushroom lectin, an inhibitor of the nuclear transport of GSTpi. The DNA damage and the formation of lipid peroxide were dependent on the dose of H(2)O(2) and the incubation time. Immunological analysis showed that H(2)O(2) induced the nuclear accumulation of GSTpi but not of glutathione peroxidase. Formation of the 7-(2-oxo-hepyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine adduct by the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with 2'-deoxyguanosine was inhibited by GSTpi in the presence of glutathione. The conjugation product of 4-oxo-2-nonenal, a lipid aldehyde of 13-HPODE, with GSH in the presence of GSTpi, was identified by LS/MS. These results suggested that nuclear GSTpi prevents H(2)O(2)-induced DNA damage by scavenging the formation of lipid-peroxide-modified DNA.

Prognostic significance of the null genotype of glutathione S-transferase-T1 in patients with acute myeloid leukemia: increased early death after chemotherapy.

PubMed

Naoe, T; Tagawa, Y; Kiyoi, H; Kodera, Y; Miyawaki, S; Asou, N; Kuriyama, K; Kusumoto, S; Shimazaki, C; Saito, K; Akiyama, H; Motoji, T; Nishimura, M; Shinagawa, K; Ueda, R; Saito, H; Ohno, R

2002-02-01

We investigated the prognostic significance of genetic polymorphism in glutathione-S transferase mu 1 (GSTM1), glutathione-S transferase theta 1 (GSTT1), NAD(P)H:quinone oxidoreductase (NQO1) and myeloperoxidase (MPO), the products of which are associated with drug metabolism as well as with detoxication, in 193 patients with de novo acute myeloid leukemia (AML) other than M3. Of the patients, 64.2% were either homozygous or heterozygous for GSTT1 (GSTT1(+)), while 35.8% showed homozygous deletions of GSTT1 (GSTT1(-)). The GSTT1(-) group had a worse prognosis than the GSTT1(+) group (P = 0.04), whereas other genotypes did not affect the outcome. Multivariate analysis revealed that GSTT1(-) was an independent prognostic factor for overall survival (relative risk: 1.53; P = 0.026) but not for disease-free survival of 140 patients who achieved complete remission (CR). The rate of early death after the initiation of chemotherapy was higher in the GSTT1(-) group than the GSTT1(+) group (within 45 days after initial chemotherapy, P = 0.073; within 120 days, P = 0.028), whereas CR rates and relapse frequencies were similar. The null genotype of GSTT1 might be associated with increased toxicity after chemotherapy.

Placental biomarkers of PAH exposure and glutathione-S-transferase biotransformation enzymes in an obstetric population from Tijuana, Baja California, Mexico.

PubMed

Dodd-Butera, Teresa; Quintana, Penelope J E; Ramirez-Zetina, Martha; Batista-Castro, Ana C; Sierra, Maria M; Shaputnic, Carolyn; Garcia-Castillo, Maura; Ingmanson, Sonja; Hull, Stacy

2017-01-01

Environmental exposures along the US-Mexico border have the potential to adversely affect the maternal-fetal environment. The purpose of this study was to assess placental biomarkers of environmental exposures in an obstetric population at the California-Baja California border in relation to detoxifying enzymes in the placenta and nutritional status. This study was conducted on consenting, full-term, obstetric patients (n=54), delivering in a hospital in Tijuana, Baja California (BC), Mexico. Placental polyaromatic hydrocarbon (PAH)-DNA adducts were measured in addition to placental glutathione-S-transferase (GST) activity and genotype, maternal serum folate, and maternal and umbilical cord blood lead and cadmium levels. A questionnaire was administered to the mothers to determine maternal occupation in a maquiladora, other exposures, and obstetric indicators. In univariate analysis, maternal serum folate levels were inversely correlated with total PAH-DNA adducts (rho=-0.375, p=0.007); adduct #1 (rho=-0.388, p=0.005); and adduct #3 (rho =-0.430, p=0.002). Maternal lead levels were significantly positively correlated with cord blood lead levels (rho=0.512, p<0.001). Cadmium levels were generally very low but significantly higher in mothers exposed to environmental tobacco smoke (ETS) (either at work or at home, n=10). In multivariate analysis, only maternal serum folate levels remained as a significant negative predictor of total DNA-PAH adducts levels in placenta. These findings affirm that placental tissue is a valuable and readily available source of human tissue for biomonitoring; and indicate that further study of the role of nutrition in detoxification and mitigation of environmental exposures in pregnant women is warranted. Copyright © 2016 Elsevier Inc. All rights reserved.

Human acetyl-CoA:glucosamine-6-phosphate N-acetyltransferase 1 has a relaxed donor specificity and transfers acyl groups up to four carbons in length.

PubMed

Brockhausen, Inka; Nair, Dileep G; Chen, Min; Yang, Xiaojing; Allingham, John S; Szarek, Walter A; Anastassiades, Tassos

2016-04-01

Glucosamine-6-phosphate N-acetyltransferase1 (GNA1) catalyses the transfer of an acetyl group from acetyl coenzyme A (AcCoA) to glucosamine-6-phosphate (GlcN6P) to form N-acetylglucosamine-6-phosphate (GlcNAc6P), which is an essential intermediate in UDP-GlcNAc biosynthesis. An analog of GlcNAc, N-butyrylglucosamine (GlcNBu) has shown healing properties for bone and articular cartilage in animal models of arthritis. The goal of this work was to examine whether GNA1 has the ability to transfer a butyryl group from butyryl-CoA to GlcN6P to form GlcNBu6P, which can then be converted to GlcNBu. We developed fluorescent and radioactive assays and examined the donor specificity of human GNA1. Acetyl, propionyl, n-butyryl, and isobutyryl groups were all transferred to GlcN6P, but isovaleryl-CoA and decanoyl-CoA did not serve as donor substrates. Site-specific mutants were produced to examine the role of amino acids potentially affecting the size and properties of the AcCoA binding pocket. All of the wild type and mutant enzymes showed activities of both acetyl and butyryl transfer and can therefore be used for the enzymatic synthesis of GlcNBu for biomedical applications.

Organometallic ruthenium anticancer complexes inhibit human glutathione-S-transferase π.

PubMed

Lin, Yu; Huang, Yongdong; Zheng, Wei; Wang, Fuyi; Habtemariam, Abraha; Luo, Qun; Li, Xianchan; Wu, Kui; Sadler, Peter J; Xiong, Shaoxiang

2013-11-01

The organometallic ruthenium(II) anticancer complexes [(η(6)-arene)Ru(en)Cl](+) (arene = p-cymene (1), biphenyl (2) or 9,10-dihydrophenanthrene (3); en = ethylenediamine), exhibit in vitro and in vivo anticancer activities. In the present work, we show that they inhibit human glutathione-S-transferase π (GSTπ) with IC50 values of 59.4 ± 1.3, 63.2 ± 0.4 and 37.2 ± 1.1 μM, respectively. Mass spectrometry revealed that complex 1 binds to the S-donors of Cys15, Cys48 within the G-site and Cys102 at the interface of the GSTπ dimer, while complex 2 binds to Cys48 and Met92 at the dimer interface and complex 3 to Cys15, Cys48 and Met92. Moreover, the binding of complex 1 to Cys15 and Cys102, complex 2 to Cys48 and complex 3 to Cys15 induces the irreversible oxidation of the coordinated thiolates to sulfenates. Molecular modeling studies indicate that the coordination of the {(arene)Ru(en)}(2+) fragment to Cys48 blocks the hydrophilic G-site sterically, perhaps preventing substrate from proper positioning and accounting for the reduction in enzymatic activity of ruthenated GSTπ. The binding of the ruthenium arene complexes to Cys102 or Met92 disrupts the dimer interface which is an essential structural feature for the proper functioning of GSTπ, perhaps also contributing to the inhibition of GSTπ. © 2013.

Development of pyrethroid-like fluorescent substrates for glutathione S-transferase

PubMed Central

Huang, Huazhang; Yao, Hongwei; Liu, Jun-Yan; Samra, Aman I.; Kamita, Shizuo G.; Cornel, Anthony J.; Hammock, Bruce D.

2012-01-01

The availability of highly sensitive substrates is critical for the development of precise and rapid assays for detecting changes in glutathione S-transferase (GST) activity that are associated with GST-mediated metabolism of insecticides. In this study, six pyrethroid-like compounds were synthesized and characterized as substrates for insect and mammalian GSTs. All of the substrates were esters composed of the same alcohol moiety, 7-hydroxy-4-methylcoumarin, and acid moieties that structurally mimic some commonly used pyrethroid insecticides including cypermethrin and cyhalothrin. CpGSTD1, a recombinant Delta class GST from the mosquito Culex pipiens, metabolized our pyrethroid-like substrates with both chemical and geometric (i.e., the cis-isomers were metabolized at 2- to 5-fold higher rates than the corresponding trans-isomers) preference. A GST preparation from mouse liver also metabolized most of our pyrethroid-like substrates with both chemical and geometric preference but at 10- to 170-fold lower rates. CpGSTD1 and mouse GSTs metabolized CDNB, a general GST substrate, at more than 200-fold higher rates than our novel pyrethroid-like substrates. There was a 10-fold difference in the specificity constant (kcat/KM ratio) of CpGSTD1 for CDNB and those of CpGSTD1 for cis-DCVC and cis-TFMCVC suggesting that cis-DCVC and cis-TFMCVC may be useful for the detection of GST-based metabolism of pyrethroids in mosquitoes. PMID:23000005

Alternate gram staining technique using a fluorescent lectin.

PubMed Central

Sizemore, R K; Caldwell, J J; Kendrick, A S

1990-01-01

Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin. By exploiting this phenomenon, an alternative Gram staining technique has been developed. Images PMID:1697149

Roles for stress-inducible lambda glutathione transferases in flavonoid metabolism in plants as identified by ligand fishing.

PubMed

Dixon, David P; Edwards, Robert

2010-11-19

The glutathione transferases (GSTs) of plants are a superfamily of abundant enzymes whose roles in endogenous metabolism are largely unknown. For example, the lambda class of GSTs (GSTLs) have members that are selectively induced by chemical stress treatments and based on their enzyme chemistry are predicted to have roles in redox homeostasis. However, using conventional approaches these functions have yet to be determined. To address this, recombinant GSTLs from wheat and Arabidopsis were tagged with a Strep tag and after affinity-immobilization, incubated with extracts from Arabidopsis, tobacco, and wheat. Bound ligands were then recovered by solvent extraction and identified by mass spectrometry (MS). With the wheat enzyme TaGSTL1, the ligand profiles obtained with in vitro extracts from tobacco closely matched those observed after the protein had been expressed in planta, demonstrating that these associations were physiologically representative. The stress-inducible TaGSTL1 was found to selectively recognize flavonols (e.g. taxifolin; K(d) = 25 nM), with this binding being dependent upon S-glutathionylation of an active site cysteine. In the case of the wheat extracts, this selectivity in ligand recognitions lead to the detection of flavonols that had not been previously described in this cereal. Subsequent in vitro assays showed that the co-binding of flavonols, such as quercetin, to the thiolated TaGSTL1 represented an intermediate step in the reduction of the respective S-glutathionylated quinone derivatives to yield free flavonols. These results suggest a novel role for GSTLs in maintaining the flavonoid pool under stress conditions.