Glycoside hydrolase gene transcription by Alicyclobacillus acidocaldarius during growth on wheat arabinoxylan and monosaccharides: a proposed xylan hydrolysis mechanism

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Lee, Brady D.; Apel, William A.; Sheridan, Peter P.; DeVeaux, Linda C.

2018-04-16

Background Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Results Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside hydrolases were down-regulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. Conclusions Glycoside hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside hydrolases was not tested as part of the research discussed, many of the glycoside hydrolases found in the A. acidocaldarius Type Strain appear to have a broader

Random transposon mutagenesis of the Saccharopolyspora erythraea genome reveals additional genes influencing erythromycin biosynthesis

Science.gov (United States)

Fedashchin, Andrij; Cernota, William H.; Gonzalez, Melissa C.; Leach, Benjamin I.; Kwan, Noelle; Wesley, Roy K.; Weber, J. Mark

2015-01-01

A single cycle of strain improvement was performed in Saccharopolyspora erythraea mutB and 15 genotypes influencing erythromycin production were found. Genotypes generated by transposon mutagenesis appeared in the screen at a frequency of ∼3%. Mutations affecting central metabolism and regulatory genes were found, as well as hydrolases, peptidases, glycosyl transferases and unknown genes. Only one mutant retained high erythromycin production when scaled-up from micro-agar plug fermentations to shake flasks. This mutant had a knockout of the cwh1 gene (SACE_1598), encoding a cell-wall-associated hydrolase. The cwh1 knockout produced visible growth and morphological defects on solid medium. This study demonstrated that random transposon mutagenesis uncovers strain improvement-related genes potentially useful for strain engineering. PMID:26468041

Experimental mixture design as a tool to enhance glycosyl hydrolases production by a new Trichoderma harzianum P49P11 strain cultivated under controlled bioreactor submerged fermentation.

Science.gov (United States)

Delabona, Priscila da Silva; Farinas, Cristiane Sanchez; Lima, Deise Juliana da Silva; Pradella, José Geraldo da Cruz

2013-03-01

This work investigates the glycosyl hydrolase (GH) profile of a new Trichoderma harzianum strain cultivated under controlled bioreactor submerged fermentation. The influence of different medium components (delignified steam-exploded sugarcane bagasse, sucrose, and soybean flour) on GH biosynthesis was assessed using experimental mixture design (EMD). Additionally, the effect of increased component concentrations in culture media selected from the EMD was studied. It was found that that a mixed culture medium could significantly maximize GH biosynthesis rate, especially for xylanase enzymes which achieved a 2-fold increment. Overall, it was demonstrated that T. harzianumP49P11 enzymes have a great potential to be used in the deconstruction of biomass. Copyright © 2012 Elsevier Ltd. All rights reserved.

Control of mucin-type O-glycosylation

DEFF Research Database (Denmark)

Bennett, Eric P; Mandel, Ulla; Clausen, Henrik

2012-01-01

residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue...... in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The Gal...

Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing

NARCIS (Netherlands)

Timal, Sharita; Hoischen, Alexander; Lehle, Ludwig; Adamowicz, Maciej; Huijben, Karin; Sykut-Cegielska, Jolanta; Paprocka, Justyna; Jamroz, Ewa; van Spronsen, Francjan J.; Koerner, Christian; Gilissen, Christian; Rodenburg, Richard J.; Eidhof, Ilse; Van den Heuvel, Lambert; Thiel, Christian; Wevers, Ron A.; Morava, Eva; Veltman, Joris; Lefeber, Dirk J.

2012-01-01

Congenital disorders of glycosylation type I (CDG-I) form a growing group of recessive neurometabolic diseases. Identification of disease genes is compromised by the enormous heterogeneity in clinical symptoms and the large number of potential genes involved. Until now, gene identification included

Phylogenetic diversity and environment-specific distributions of glycosyl hydrolase family 10 xylanases in geographically distant soils.

Directory of Open Access Journals (Sweden)

Guozeng Wang

Full Text Available BACKGROUND: Xylan is one of the most abundant biopolymers on Earth. Its degradation is mediated primarily by microbial xylanase in nature. To explore the diversity and distribution patterns of xylanase genes in soils, samples of five soil types with different physicochemical characters were analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Partial xylanase genes of glycoside hydrolase (GH family 10 were recovered following direct DNA extraction from soil, PCR amplification and cloning. Combined with our previous study, a total of 1084 gene fragments were obtained, representing 366 OTUs. More than half of the OTUs were novel (identities of <65% with known xylanases and had no close relatives based on phylogenetic analyses. Xylanase genes from all the soil environments were mainly distributed in Bacteroidetes, Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Dictyoglomi and some fungi. Although identical sequences were found in several sites, habitat-specific patterns appeared to be important, and geochemical factors such as pH and oxygen content significantly influenced the compositions of xylan-degrading microbial communities. CONCLUSION/SIGNIFICANCE: These results provide insight into the GH 10 xylanases in various soil environments and reveal that xylan-degrading microbial communities are environment specific with diverse and abundant populations.

Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis

NARCIS (Netherlands)

Visser, H.; Vreugdenhil, S.; Bont, de J.A.M.; Verdoes, J.C.

2000-01-01

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of

Steady state kinetic analysis of substrate specificity of glycoside hydrolases from families 13 and 38

DEFF Research Database (Denmark)

Nielsen, Jonas Willum

Glycosidases are widespread in nature, where they perform a diverse range of functions. The glycoside hydrolase (GH) family 38, α-mannosidase II enzymes play a crucial role in mammalian cells, in the maturation of N-glycosylated proteins in the Golgi apparatus and in catabolism in cytosol...

Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

Science.gov (United States)

Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

2003-03-01

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodri;guez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128-8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase.

Variation in bleomycin hydrolase gene is associated with reduced survival after chemotherapy for testicular germ cell cancer

NARCIS (Netherlands)

de Haas, Esther C.; Zwart, Nynke; Meijer, Coby; Nuver, Janine; Boezen, H. Marike; Suurmeijer, Albert J. H.; Hoekstra, Harald J.; van der Steege, Gerrit; Sleijfer, Dirk Th.; Gietema, Jourik A.

2008-01-01

Purpose Response to chemotherapy may be determined by gene polymorphisms involved in metabolism of cytotoxic drugs. A plausible candidate is the gene for bleomycin hydrolase (BLMH), an enzyme that inactivates bleomycin, an essential component of chemotherapy regimens for disseminated testicular

Regulatory regions in the rat lactase-phlorizin hydrolase gene that control cell-specific expression

NARCIS (Netherlands)

Verhave, Menno; Krasinski, Stephen D.; Christian, Sara I.; van Schaik, Sandrijn; van den Brink, Gijs R.; Doting, Edwina M. H.; Maas, Saskia M.; Wolthers, Katja C.; Grand, Richard J.; Montgomery, Robert K.

2004-01-01

OBJECTIVES: Lactase-phlorizin hydrolase (LPH) is an enterocyte-specific gene whose expression has been well-characterized, not only developmentally but also along the crypt-villus axis and along the length of the small bowel. Previous studies from the authors' laboratory have demonstrated that 2 kb

Peptidoglycan Hydrolases of Escherichia coli

Science.gov (United States)

van Heijenoort, Jean

2011-01-01

Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

Genes Involved in the Endoplasmic Reticulum N-Glycosylation Pathway of the Red Microalga Porphyridium sp.: A Bioinformatic Study

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Oshrat Levy-Ontman

2014-02-01

Full Text Available N-glycosylation is one of the most important post-translational modifications that influence protein polymorphism, including protein structures and their functions. Although this important biological process has been extensively studied in mammals, only limited knowledge exists regarding glycosylation in algae. The current research is focused on the red microalga Porphyridium sp., which is a potentially valuable source for various applications, such as skin therapy, food, and pharmaceuticals. The enzymes involved in the biosynthesis and processing of N-glycans remain undefined in this species, and the mechanism(s of their genetic regulation is completely unknown. In this study, we describe our pioneering attempt to understand the endoplasmic reticulum N-Glycosylation pathway in Porphyridium sp., using a bioinformatic approach. Homology searches, based on sequence similarities with genes encoding proteins involved in the ER N-glycosylation pathway (including their conserved parts were conducted using the TBLASTN function on the algae DNA scaffold contigs database. This approach led to the identification of 24 encoded-genes implicated with the ER N-glycosylation pathway in Porphyridium sp. Homologs were found for almost all known N-glycosylation protein sequences in the ER pathway of Porphyridium sp.; thus, suggesting that the ER-pathway is conserved; as it is in other organisms (animals, plants, yeasts, etc..

Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

Science.gov (United States)

Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

2015-01-01

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

Expression of Nudix hydrolase genes in barley under UV irradiation

Science.gov (United States)

Tanaka, Sayuri; Sugimoto, Manabu; Kihara, Makoto

Seed storage and cultivation should be necessary to self-supply foods when astronauts would stay and investigate during long-term space travel and habitation in the bases on the Moon and Mars. Thought the sunlight is the most importance to plants, both as the ultimate energy source and as an environmental signal regulating growth and development, UV presenting the sunlight can damage many aspects of plant processes at the physiological and DNA level. Especially UV-C, which is eliminated by the stratospheric ozone layer, is suspected to be extremely harmful and give a deadly injury to plants in space. However, the defense mechanism against UV-C irradiation damage in plant cells has not been clear. In this study, we investigated the expression of Nudix hydrolases, which defense plants from biotic / abiotic stress, in barley under UV irradiation. The genes encoding the amino acid sequences, which show homology to those of 28 kinds of Nudix hydrolases in Arabidopsis thaliana, were identified in the barley full-length cDNA library. BLAST analysis showed 14 kinds of barley genes (HvNUDX1-14), which encode the Nudix motif sequence. A phylogenetic tree showed that HvNUDX1, HvNUDX7, HvNUDX9 and HvNUDX11 belonged to the ADP-ribose pyrophosphohydrolase, ADP-sugar pyrophosphohydrolase, NAD(P)H pyrophosphohydrolase and FAD pyrophosphohydrolase subfamilies, respectively, HvNUDX3, HvNUDX6, and HvNUDX8 belonged to the Ap _{n}A pyrophosphohydrolase subfamilies, HvNUDX5 and HvNUDX14 belonged to the coenzyme A pyrophosphohydrolase subfamilies, HvNUDX12 and HvNUDX13 belonged to the Ap _{4}A pyrophosphohydrolase subfamilies. Induction of HvNUDX genes by UV-A (340nm), UV-B (312nm), and UV-C (260nm) were analyzed by quantitative RT-PCR. The results showed that HvNUDX4 was induced by UV-A and UV-B, HvNUDX6 was induced by UV-B and UV-C, and HvNUDX7 and HvNUDX14 were induced by UV-C, significantly. Our results suggest that the response of HvNUDXs to UV irradiation is different by UV

Molecular cloning of cellulase genes from indigenous bacterial isolates

International Nuclear Information System (INIS)

Jong Bor Chyan; Pauline Liew Woan Ying; Mat Rasol Awang

2006-01-01

Indigenous cellulolytic bacterial isolates having high activities in degrading carboxymethyl cellulose (CMC) were isolated from local environments. Identification of these isolates were performed by molecular techniques. By using polymerase chain reaction (PCR) techniques, PCR products encoding cellulase gene were amplified from the total genomic DNAs. Purified PCR product was successfully cloned and expressed in Escherichia coli host system. The complete nucleotide sequences of the cellulase genes determined. The analysis of amino acid sequences deduced from the genes indicated that the cloned DNA fragments show high homology to those of endoglucanase genes of family GH5. All cloned genes consist of an N-terminal signal peptide, a catalytic domain of family 5 glycosyl hydrolase and a cellulose-binding domain of family III. (Author)

Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

Science.gov (United States)

Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

2015-05-01

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Site-specific protein O-glycosylation modulates proprotein processing - Deciphering specific functions of the large polypeptide GalNAc-transferase gene family

DEFF Research Database (Denmark)

Schjoldager, Katrine Ter-Borch Gram; Clausen, Henrik

2012-01-01

Posttranslational modifications (PTMs) greatly expand the function and regulation of proteins, and glycosylation is the most abundant and diverse PTM. Of the many different types of protein glycosylation, one is quite unique; GalNAc-type (or mucin-type) O-glycosylation, where biosynthesis...... and considerable redundancy. Recently we have begun to uncover human diseases associated with deficiencies in GalNAc-T genes (GALNTs). Thus deficiencies in individual GALNTs produce cell and protein specific effects and subtle distinct phenotypes such as hyperphosphatemia with hyperostosis (GALNT3...

Genomic analysis of Bacillus subtilis lytic bacteriophage ϕNIT1 capable of obstructing natto fermentation carrying genes for the capsule-lytic soluble enzymes poly-γ-glutamate hydrolase and levanase.

Science.gov (United States)

Ozaki, Tatsuro; Abe, Naoki; Kimura, Keitarou; Suzuki, Atsuto; Kaneko, Jun

2017-01-01

Bacillus subtilis strains including the fermented soybean (natto) starter produce capsular polymers consisting of poly-γ-glutamate and levan. Capsular polymers may protect the cells from phage infection. However, bacteriophage ϕNIT1 carries a γ-PGA hydrolase gene (pghP) that help it to counteract the host cell's protection strategy. ϕNIT had a linear double stranded DNA genome of 155,631-bp with a terminal redundancy of 5,103-bp, containing a gene encoding an active levan hydrolase. These capsule-lytic enzyme genes were located in the possible foreign gene cluster regions between central core and terminal redundant regions, and were expressed at the late phase of the phage lytic cycle. All tested natto origin Spounavirinae phages carried both genes for capsule degrading enzymes similar to ϕNIT1. A comparative genomic analysis revealed the diversity among ϕNIT1 and Bacillus phages carrying pghP-like and levan-hydrolase genes, and provides novel understanding on the acquisition mechanism of these enzymatic genes.

Deciphering a pathway of Halobacterium salinarum N-glycosylation

Science.gov (United States)

Kandiba, Lina; Eichler, Jerry

2015-01-01

Genomic analysis points to N-glycosylation as being a common posttranslational modification in Archaea. To date, however, pathways of archaeal N-glycosylation have only been described for few species. With this in mind, the similarities of N-linked glycans decorating glycoproteins in the haloarchaea Haloferax volcanii and Halobacterium salinarum directed a series of bioinformatics, genetic, and biochemical experiments designed to describe that Hbt. salinarum pathway responsible for biogenesis of one of the two N-linked oligosaccharides described in this species. As in Hfx. volcanii, where agl (archaeal glycosylation) genes that encode proteins responsible for the assembly and attachment of a pentasaccharide to target protein Asn residues are clustered in the genome, Hbt. salinarum also contains a group of clustered homologous genes (VNG1048G-VNG1068G). Introduction of these Hbt. salinarum genes into Hfx. volcanii mutant strains deleted of the homologous sequence restored the lost activity. Moreover, transcription of the Hbt. salinarum genes in the native host, as well as in vitro biochemical confirmation of the predicted functions of several of the products of these genes provided further support for assignments made following bioinformatics and genetic experiments. Based on the results obtained in this study, the first description of an N-glycosylation pathway in Hbt. salinarum is offered. PMID:25461760

Mice lacking lipid droplet-associated hydrolase, a gene linked to human prostate cancer, have normal cholesterol ester metabolism

DEFF Research Database (Denmark)

Kory, Nora; Grond, Susanne; Kamat, Siddhesh S

2017-01-01

Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids...

Genetic variation in the bleomycin hydrolase gene and bleomycin-induced pulmonary toxicity in germ cell cancer patients

NARCIS (Netherlands)

Nuver, J; Lutke-Holzik, MF; van Zweeden, M; Hoekstra, HJ; Meijer, C; Suurmeijer, AJH; Hofstra, RM; Sluiter, WJ; Sleijfer, D; Gietema, JA; Groen, Hendricus; Groen, Herman

Objective Use of bleomycin as a cytotoxic agent is limited by its pulmonary toxicity. Bleomycin is mainly excreted by the kidneys, but can also be inactivated by bleomycin hydrolase (BMH). An 1450A > G polymorphic site in the BMH gene results in an amino acid substitution in the C-terminal domain of

Cocaine Hydrolase Gene Transfer Demonstrates Cardiac Safety and Efficacy against Cocaine-Induced QT Prolongation in Mice

OpenAIRE

Murthy, Vishakantha; Reyes, Santiago; Geng, Liyi; Gao, Yang; Brimijoin, Stephen

2016-01-01

Cocaine addiction is associated with devastating medical consequences, including cardiotoxicity and risk-conferring prolongation of the QT interval. Viral gene transfer of cocaine hydrolase engineered from butyrylcholinesterase offers therapeutic promise for treatment-seeking drug users. Although previous preclinical studies have demonstrated benefits of this strategy without signs of toxicity, the specific cardiac safety and efficacy of engineered butyrylcholinesterase viral delivery remains...

N-linked glycosylation of the immunoglobulin variable region

NARCIS (Netherlands)

van de Bovenkamp, Fleur S.; Derksen, Ninotska I. L.; Ooijevaar-de Heer, Pleuni; van Schie, Karin A.; Kruithof, Simone; Berkowska, Magdalena A.; van der Schoot, C. Ellen; Ijspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E. M.; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S. Marieke; Vidarsson, Gestur; Rispens, Theo

2018-01-01

N-glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we

Glycoside hydrolases having multiple hydrolase activities

Energy Technology Data Exchange (ETDEWEB)

Chen, Zhiwei; Friedland, Gregory D.; Chhabra, Swapnil R.; Chivian, Dylan C.; Simmons, Blake A

2017-08-08

Glycoside hydrolases having at least two different hydrolytic activities are provided. In one embodiment, an isolated recombinant hydrolase having at least two activities selected from a group including asparagine derivatives, glutamine derivatives, and histidine derivatives is provided. Further, a method of generating free sugars from a mixture comprising asparagine derivatives, glutamine derivatives, and histidine derivatives is provided.

The role of glycosylation in breast cancer metastasis and cancer control

Directory of Open Access Journals (Sweden)

Alexandra eKölbl

2015-10-01

Full Text Available AbstractGlycosylation and its correlation to the formation of remote metastasis in breast cancer had been an important scientific topic in the last 25 years. With the development of new analytical techniques new insights were gained on the mechanisms underlying metastasis formation and the role of aberrant glycosylation within. Mucin-1 and Galectin were recognized as key players in glycosylation. Interestingly, aberrant carbohydrate structures seem to support the development of brain metastasis in breast cancer patients, as changes in glycosylation structures facilitate an overcoming of blood-brain barrier. Changes in the gene expression of glycosyltransferases are the leading cause for a modification of carbohydrate chains, so that also altered gene expression plays a role for glycosylation. In consequence, glycosylation and changes within can be useful for cancer diagnosis, determination of tumour stage and prognosis, but can as well be targets for therapeutic strategies. Thus, further research on this topic would worth wile for cancer combating.

Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules.

Science.gov (United States)

Kersten, Roland D; Ziemert, Nadine; Gonzalez, David J; Duggan, Brendan M; Nizet, Victor; Dorrestein, Pieter C; Moore, Bradley S

2013-11-19

Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experiment-guided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways from genome-sequenced microbes by targeting glycosyl groups in microbial metabolomes. Microbial GNPs consist of aglycone and glycosyl structure groups in which the sugar unit(s) are often critical for the GNP's bioactivity, e.g., by promoting binding to a target biomolecule. GNPs are a structurally diverse class of molecules with important pharmaceutical and agrochemical applications. Herein, O- and N-glycosyl groups are characterized in their sugar monomers by tandem mass spectrometry (MS) and matched to corresponding glycosylation genes in secondary metabolic pathways by a MS-glycogenetic code. The associated aglycone biosynthetic genes of the GNP genotype then classify the natural product to further guide structure elucidation. We highlight the glycogenomic strategy by the characterization of several bioactive glycosylated molecules and their gene clusters, including the anticancer agent cinerubin B from Streptomyces sp. SPB74 and an antibiotic, arenimycin B, from Salinispora arenicola CNB-527.

Distribution and function of carbamate hydrolase genes cehA and mcd in soils: the distinct role of soil pH.

Science.gov (United States)

Rousidou, Constantina; Karaiskos, Dionysis; Myti, Despoina; Karanasios, Evangelos; Karas, Panagiotis A; Tourna, Maria; Tzortzakakis, Emmanuel A; Karpouzas, Dimitrios G

2017-01-01

Synthetic carbamates constitute a significant pesticide group with oxamyl being a leading compound in the nematicide market. Oxamyl degradation in soil is mainly microbially mediated. However, the distribution and function of carbamate hydrolase genes (cehA, mcd, cahA) associated with the soil biodegradation of carbamates is not yet clear. We studied oxamyl degradation in 16 soils from a potato monoculture area in Greece where oxamyl is regularly used. Oxamyl showed low persistence (DT50 2.4-26.7 days). q-PCR detected the cehA and mcd genes in 10 and three soils, respectively. The abundance of the cehA gene was positively correlated with pH, while both cehA abundance and pH were negatively correlated with oxamyl DT50. Amongst the carbamates used in the study region, oxamyl stimulated the abundance and expression only of the cehA gene, while carbofuran stimulated the abundance and expression of both genes. The cehA gene was also detected in pristine soils upon repeated treatments with oxamyl and carbofuran and only in soils with pH ≥7.2, where the most rapid degradation of oxamyl was observed. These results have major implications regarding the maintenance of carbamate hydrolase genes in soils, have practical implications regarding the agricultural use of carbamates, and provide insights into the evolution of cehA. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

How to find soluble proteins: a comprehensive analysis of alpha/beta hydrolases for recombinant expression in E. coli

Directory of Open Access Journals (Sweden)

Barth Sandra

2005-04-01

Full Text Available Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5:443–448. We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used

Coexpression of bile salt hydrolase gene and catalase gene remarkably improves oxidative stress and bile salt resistance in Lactobacillus casei.

Science.gov (United States)

Wang, Guohong; Yin, Sheng; An, Haoran; Chen, Shangwu; Hao, Yanling

2011-08-01

Lactic acid bacteria (LAB) encounter various types of stress during industrial processes and gastrointestinal transit. Catalase (CAT) and bile salt hydrolase (BSH) can protect bacteria from oxidative stress or damage caused by bile salts by decomposing hydrogen peroxide (H(2)O(2)) or deconjugating the bile salts, respectively. Lactobacillus casei is a valuable probiotic strain and is often deficient in both CAT and BSH. In order to improve the resistance of L. casei to both oxidative and bile salts stress, the catalase gene katA from L. sakei and the bile salt hydrolase gene bsh1 from L. plantarum were coexpressed in L. casei HX01. The enzyme activities of CAT and BSH were 2.41 μmol H(2)O(2)/min/10(8) colony-forming units (CFU) and 2.11 μmol glycine/min/ml in the recombinant L. casei CB, respectively. After incubation with 8 mM H(2)O(2), survival ratio of L. casei CB was 40-fold higher than that of L. casei CK. Treatment of L. casei CB with various concentrations of sodium glycodeoxycholate (GDCA) showed that ~10(5) CFU/ml cells survived after incubation with 0.5% GDCA, whereas almost all the L. casei CK cells were killed when treaded with 0.4% GDCA. These results indicate that the coexpression of CAT and BSH confers high-level resistance to both oxidative and bile salts stress conditions in L. casei HX01.

Bovine glycomacropeptide promotes the growth of Bifidobacterium longum ssp. infantis and modulates its gene expression.

Science.gov (United States)

O'Riordan, N; O'Callaghan, J; Buttò, L F; Kilcoyne, M; Joshi, L; Hickey, R M

2018-05-23

Bovine milk glycomacropeptide (GMP) is derived from κ-casein, with exclusively o-linked glycosylation. Glycomacropeptide promoted the growth of Bifidobacterium longum ssp. infantis in a concentration-dependent manner, and this activity was lost following periodate treatment of the GMP (GMP-P), which disables biological recognition of the conjugated oligosaccharides. Transcriptional analysis of B. longum ssp. infantis following exposure to GMP revealed a substantial response to GMP relative to bacteria treated with GMP-P, with a greater number of differentially expressed transcripts and larger fold changes versus the control. Therefore, stimulation of B. longum ssp. infantis growth by GMP is intrinsically linked to the peptide's O-linked glycosylation. The pool of differentially expressed transcripts included 2 glycoside hydrolase (family 25) genes, which were substantially upregulated following exposure to GMP, but not GMP-P. These GH25 genes were present in duplicated genomic islands that also contained genes encoding fibronectin type III binding domain proteins and numerous phage-related proteins, all of which were also upregulated. Homologs of this genomic arrangement were present in other Bifidobacterium species, which suggest it may be a conserved domain for the utilization of glycosylated peptides. This study provides insights into the molecular basis for the prebiotic effect of bovine milk GMP on B. longum ssp. infantis. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose.

OpenAIRE

Yagi, Toshihiro; Baroja-Fernández, Edurne; Yamamoto, Ryuji; Muñoz, Francisco José; Akazawa, Takashi; Hong, Kyoung Su; Pozueta-Romero, Javier

2003-01-01

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney ( Sus scrofa ). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna,...

N-glycosylation in sugarcane

Directory of Open Access Journals (Sweden)

Maia Ivan G.

2001-01-01

Full Text Available The N-linked glycosylation of secretory and membrane proteins is the most complex posttranslational modification known to occur in eukaryotic cells. It has been shown to play critical roles in modulating protein function. Although this important biological process has been extensively studied in mammals, much less is known about this biosynthetic pathway in plants. The enzymes involved in plant N-glycan biosynthesis and processing are still not well defined and the mechanism of their genetic regulation is almost completely unknown. In this paper we describe our first attempt to understand the N-linked glycosylation mechanism in a plant species by using the data generated by the Sugarcane Expressed Sequence Tag (SUCEST project. The SUCEST database was mined for sugarcane gene products potentially involved in the N-glycosylation pathway. This approach has led to the identification and functional assignment of 90 expressed sequence tag (EST clusters sharing significant sequence similarity with the enzymes involved in N-glycan biosynthesis and processing. The ESTs identified were also analyzed to establish their relative abundance.

Cell Surface Glycosylation Is Required for Efficient Mating of Haloferax volcanii

Directory of Open Access Journals (Sweden)

Yarden Shalev

2017-07-01

Full Text Available Halophilic archaea use a fusion-based mating system for lateral gene transfer across cells, yet the molecular mechanisms involved remain unknown. Previous work implied that cell fusion involves cell–cell recognition since fusion occurs more efficiently between cells from the same species. Long believed to be restricted only to Eukarya, it is now known that cells of all three domains of life perform N-glycosylation, the covalent attachment of glycans to select target asparagine residues in proteins, and that this post-translational modification is common for archaeal cell surface proteins. Here, we show that differences in glycosylation of the Haloferax volcanii surface-layer glycoprotein, brought about either by changing medium salinity or by knocking out key glycosylation genes, reduced mating success. Thus, different glycosylation patterns are likely to underlie mating preference in halophilic archaea, contributing to speciation processes.

Molecular Cloning and Nucleotide Sequence of the Gene Encoding the Major Peptidoglycan Hydrolase of Lactococcus lactis, a Muramidase Needed for Cell Separation

NARCIS (Netherlands)

Buist, Girbe; Kok, Jan; Leenhouts, Kees J.; Dabrowska, Magdalena; Venema, Gerhardus; Haandrikman, Alfred J.

A gene of Lactococcus lactis subsp, cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells,

Friend or foe? Evolutionary history of glycoside hydrolase family 32 genes encoding for sucrolytic activity in fungi and its implications for plant-fungal symbioses

Directory of Open Access Journals (Sweden)

James Timothy Y

2009-06-01

Full Text Available Abstract Background Many fungi are obligate biotrophs of plants, growing in live plant tissues, gaining direct access to recently photosynthesized carbon. Photosynthate within plants is transported from source to sink tissues as sucrose, which is hydrolyzed by plant glycosyl hydrolase family 32 enzymes (GH32 into its constituent monosaccharides to meet plant cellular demands. A number of plant pathogenic fungi also use GH32 enzymes to access plant-derived sucrose, but less is known about the sucrose utilization ability of mutualistic and commensal plant biotrophic fungi, such as mycorrhizal and endophytic fungi. The aim of this study was to explore the distribution and abundance of GH32 genes in fungi to understand how sucrose utilization is structured within and among major ecological guilds and evolutionary lineages. Using bioinformatic and PCR-based analyses, we tested for GH32 gene presence in all available fungal genomes and an additional 149 species representing a broad phylogenetic and ecological range of biotrophic fungi. Results We detected 9 lineages of GH32 genes in fungi, 4 of which we describe for the first time. GH32 gene number in fungal genomes ranged from 0–12. Ancestral state reconstruction of GH32 gene abundance showed a strong correlation with nutritional mode, and gene family expansion was observed in several clades of pathogenic filamentous Ascomycota species. GH32 gene number was negatively correlated with animal pathogenicity and positively correlated with plant biotrophy, with the notable exception of mycorrhizal taxa. Few mycorrhizal species were found to have GH32 genes as compared to other guilds of plant-associated fungi, such as pathogens, endophytes and lichen-forming fungi. GH32 genes were also more prevalent in the Ascomycota than in the Basidiomycota. Conclusion We found a strong signature of both ecological strategy and phylogeny on GH32 gene number in fungi. These data suggest that plant biotrophic fungi

ClbS Is a Cyclopropane Hydrolase That Confers Colibactin Resistance.

Science.gov (United States)

Tripathi, Prabhanshu; Shine, Emilee E; Healy, Alan R; Kim, Chung Sub; Herzon, Seth B; Bruner, Steven D; Crawford, Jason M

2017-12-13

Certain commensal Escherichia coli contain the clb biosynthetic gene cluster that codes for small molecule prodrugs known as precolibactins. Precolibactins are converted to colibactins by N-deacylation; the latter are postulated to be genotoxic and to contribute to colorectal cancer formation. Though advances toward elucidating (pre)colibactin biosynthesis have been made, the functions and mechanisms of several clb gene products remain poorly understood. Here we report the 2.1 Å X-ray structure and molecular function of ClbS, a gene product that confers resistance to colibactin toxicity in host bacteria and which has been shown to be important for bacterial viability. The structure harbors a potential colibactin binding site and shares similarity to known hydrolases. In vitro studies using a synthetic colibactin analog and ClbS or an active site residue mutant reveal cyclopropane hydrolase activity that converts the electrophilic cyclopropane of the colibactins into an innocuous hydrolysis product. As the cyclopropane has been shown to be essential for genotoxic effects in vitro, this ClbS-catalyzed ring-opening provides a means for the bacteria to circumvent self-induced genotoxicity. Our study provides a molecular-level view of the first reported cyclopropane hydrolase and support for a specific mechanistic role of this enzyme in colibactin resistance.

N-Glycosylation of Carnosinase Influences Protein Secretion and Enzyme Activity Implications for Hyperglycemia

NARCIS (Netherlands)

Riedl, Eva; Koeppel, Hannes; Pfister, Frederick; Peters, Verena; Sauerhoefer, Sibylle; Sternik, Paula; Brinkkoetter, Paul; Zentgraf, Hanswalter; Navis, Gerjan; Henning, Robert H.; Van Den Born, Jacob; Bakker, Stephan J. L.; Janssen, Bart; van der Woude, Fokko J.; Yard, Benito A.

OBJECTIVE-The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1

Glycosylation Engineering

DEFF Research Database (Denmark)

Clausen, Henrik; Wandall, Hans H.; Steentoft, Catharina

2017-01-01

Knowledge of the cellular pathways of glycosylation across phylogeny provides opportunities for designing glycans via genetic engineering in a wide variety of cell types including bacteria, fungi, plant cells, and mammalian cells. The commercial demand for glycosylation engineering is broad......, including production of biological therapeutics with defined glycosylation (Chapter 57). This chapter describes how knowledge of glycan structures and their metabolism (Parts I–III of this book) has led to the current state of glycosylation engineering in different cell types. Perspectives for rapid...

Cloning, expression and mutation of a triazophos hydrolase gene from Burkholderia sp. SZL-1.

Science.gov (United States)

Zhang, Hao; Li, Qiang; Guo, Su-Hui; Cheng, Ming-Gen; Zhao, Meng-Jun; Hong, Qing; Huang, Xing

2016-06-01

Triazophos is a broad-spectrum and highly effective insecticide, and the residues of triazophos have been frequently detected in the environment. A triazophos-degrading bacterium, Burkholderia sp. SZL-1, was isolated from a long-term triazophos-polluted soil. Strain SZL-1 could hydrolyze triazophos to 1-phenyl-3-hydroxy-1,2,4-triazole, which was further utilized as the carbon sources for growth. The triazophos hydrolase gene trhA, cloned from strain SZL-1, was expressed and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. TrhA is 55 kDa and displays maximum activity at 25°C, pH 8.0. This enzyme still has nearly 60% activity at the range of 15°C-50°C for 30 min. TrhA was mutated by sequential error prone PCR and screened for improved activity for triazophos degradation. One purified variant protein (Val89-Gly89) named TrhA-M1 showed up to 3-fold improvement in specific activity against triazophos, and the specificity constants of Kcat and Kcat/Km for TrhA-M1 were improved up to 2.3- and 8.28-fold, respectively, compared to the wild-type enzyme. The results in this paper provided potential material for the contaminated soil remediation and hydrolase genetic structure research. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

International Nuclear Information System (INIS)

Kilcoyne, Michelle; Sharma, Shashank; McDevitt, Niamh; O’Leary, Claire; Joshi, Lokesh; McMahon, Siobhán S.

2012-01-01

Highlights: ► Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. ► Neuronal glycosylation in injury and after ChABC treatment is unknown. ► In silico mining verified that glyco-related genes were differentially regulated after SCI. ► In vitro model system revealed abnormal sialylation in an injured environment. ► The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually α-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

Energy Technology Data Exchange (ETDEWEB)

Kilcoyne, Michelle; Sharma, Shashank [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McDevitt, Niamh; O' Leary, Claire [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland); Joshi, Lokesh [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McMahon, Siobhan S., E-mail: siobhan.mcmahon@nuigalway.ie [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland)

2012-04-13

Highlights: Black-Right-Pointing-Pointer Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. Black-Right-Pointing-Pointer Neuronal glycosylation in injury and after ChABC treatment is unknown. Black-Right-Pointing-Pointer In silico mining verified that glyco-related genes were differentially regulated after SCI. Black-Right-Pointing-Pointer In vitro model system revealed abnormal sialylation in an injured environment. Black-Right-Pointing-Pointer The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually {alpha}-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment

Diagnostic serum glycosylation profile in patients with intellectual disability as a result of MAN1B1 deficiency

DEFF Research Database (Denmark)

Van Scherpenzeel, Monique; Timal, Sharita; Rymen, Daisy

2014-01-01

Congenital disorders of glycosylation comprise a group of genetic defects with a high frequency of intellectual disability, caused by deficient glycosylation of proteins and lipids. The molecular basis of the majority of the congenital disorders of glycosylation type I subtypes, localized...... in the cytosol and endoplasmic reticulum, has been solved. However, elucidation of causative genes for defective Golgi glycosylation (congenital disorders of glycosylation type II) remains challenging because of a lack of sufficiently specific diagnostic serum methods. In a single patient with intellectual...... disability, whole-exome sequencing revealed MAN1B1 as congenital disorder of glycosylation type II candidate gene. A novel mass spectrometry method was applied for high-resolution glycoprofiling of intact plasma transferrin. A highly characteristic glycosylation signature was observed with hybrid type N...

High-throughput screening for gene libraries expressing carbohydrate hydrolase activity

NARCIS (Netherlands)

Leemhuis, Hans; Euverink, Gert-Jan W.; Dijkhuizen, Lubbert

2003-01-01

A simple and fast method is described allowing screening of large number of Escherichia coli clones (4000 per day) for the presence of functional or improved carbohydrate hydrolase enzymes. The procedure is relatively cheap and has the advantage that carbohydrate degrading activity can be directly

Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes

Science.gov (United States)

Zhang, Jun; Zhang, Lei; Geng, Alei; Liu, Fanghua; Zhao, Guoping; Wang, Shengyue; Zhou, Zhihua; Yan, Xing

2015-01-01

Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to Clostridium straminisolvens and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in E. coli demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future. PMID:26070087

Gene-centric metagenomics of the fiber-adherent bovine rumen microbiome reveals forage specific glycoside hydrolases.

Science.gov (United States)

Brulc, Jennifer M; Antonopoulos, Dionysios A; Miller, Margret E Berg; Wilson, Melissa K; Yannarell, Anthony C; Dinsdale, Elizabeth A; Edwards, Robert E; Frank, Edward D; Emerson, Joanne B; Wacklin, Pirjo; Coutinho, Pedro M; Henrissat, Bernard; Nelson, Karen E; White, Bryan A

2009-02-10

The complex microbiome of the rumen functions as an effective system for the conversion of plant cell wall biomass to microbial protein, short chain fatty acids, and gases. As such, it provides a unique genetic resource for plant cell wall degrading microbial enzymes that could be used in the production of biofuels. The rumen and gastrointestinal tract harbor a dense and complex microbiome. To gain a greater understanding of the ecology and metabolic potential of this microbiome, we used comparative metagenomics (phylotype analysis and SEED subsystems-based annotations) to examine randomly sampled pyrosequence data from 3 fiber-adherent microbiomes and 1 pooled liquid sample (a mixture of the liquid microbiome fractions from the same bovine rumens). Even though the 3 animals were fed the same diet, the community structure, predicted phylotype, and metabolic potentials in the rumen were markedly different with respect to nutrient utilization. A comparison of the glycoside hydrolase and cellulosome functional genes revealed that in the rumen microbiome, initial colonization of fiber appears to be by organisms possessing enzymes that attack the easily available side chains of complex plant polysaccharides and not the more recalcitrant main chains, especially cellulose. Furthermore, when compared with the termite hindgut microbiome, there are fundamental differences in the glycoside hydrolase content that appear to be diet driven for either the bovine rumen (forages and legumes) or the termite hindgut (wood).

The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

Science.gov (United States)

Sedbrook, John C.; Carroll, Kathleen L.; Hung, Kai F.; Masson, Patrick H.; Somerville, Chris R.

2002-01-01

To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation.

Science.gov (United States)

Könitzer, Jennifer D; Müller, Markus M; Leparc, Germán; Pauers, Martin; Bechmann, Jan; Schulz, Patrick; Schaub, Jochen; Enenkel, Barbara; Hildebrandt, Tobias; Hampel, Martin; Tolstrup, Anne B

2015-09-01

Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

OpenAIRE

Oguro, Ami; Imaoka, Susumu

2012-01-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hyd...

Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Lack, Nathan; Lowe, Edward D.; Liu, Jie; Eltis, Lindsay D.; Noble, Martin E. M.; Sim, Edith; Westwood, Isaac M.

2007-01-01

The structure of HsaD, a carbon–carbon bond serine hydrolase involved in steroid catabolism that is critical for the survival of M. tuberculosis inside human macrophages, has been solved by X-ray crystallography. Data were collected at the Diamond Light Source in Oxfordshire, England: this paper describes one of the first structures determined at the new synchrotron. Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta-cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon–carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35 Å resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors’ knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors

Altered protein glycosylation predicts Alzheimer's disease and modulates its pathology in disease model Drosophila.

Science.gov (United States)

Frenkel-Pinter, Moran; Stempler, Shiri; Tal-Mazaki, Sharon; Losev, Yelena; Singh-Anand, Avnika; Escobar-Álvarez, Daniela; Lezmy, Jonathan; Gazit, Ehud; Ruppin, Eytan; Segal, Daniel

2017-08-01

The pathological hallmarks of Alzheimer's disease (AD) are pathogenic oligomers and fibrils of misfolded amyloidogenic proteins (e.g., β-amyloid and hyper-phosphorylated tau in AD), which cause progressive loss of neurons in the brain and nervous system. Although deviations from normal protein glycosylation have been documented in AD, their role in disease pathology has been barely explored. Here our analysis of available expression data sets indicates that many glycosylation-related genes are differentially expressed in brains of AD patients compared with healthy controls. The robust differences found enabled us to predict the occurrence of AD with remarkable accuracy in a test cohort and identify a set of key genes whose expression determines this classification. We then studied in vivo the effect of reducing expression of homologs of 6 of these genes in transgenic Drosophila overexpressing human tau, a well-established invertebrate AD model. These experiments have led to the identification of glycosylation genes that may augment or ameliorate tauopathy phenotypes. Our results indicate that OstDelta, l(2)not and beta4GalT7 are tauopathy suppressors, whereas pgnat5 and CG33303 are enhancers, of tauopathy. These results suggest that specific alterations in protein glycosylation may play a causal role in AD etiology. Copyright © 2017 Elsevier Inc. All rights reserved.

HEK293T cell lines defective for O-linked glycosylation.

Directory of Open Access Journals (Sweden)

James M Termini

Full Text Available Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE, galactokinase 1 (GALK1, and galactokinase 2 (GALK2 genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2, O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

[Blue-light induced expression of S-adenosy-L-homocysteine hydrolase-like gene in Mucor amphibiorum RCS1].

Science.gov (United States)

Gao, Ya; Wang, Shu; Fu, Mingjia; Zhong, Guolin

2013-09-04

To determine blue-light induced expression of S-adenosyl-L-homocysteine hydrolase-like (sahhl) gene in fungus Mucor amphibiorum RCS1. In the random process of PCR, a sequence of 555 bp was obtained from M. amphibiorum RCS1. The 555 bp sequence was labeled with digoxin to prepare the probe for northern hybridization. By northern hybridization, the transcription of sahhl gene was analyzed in M. amphibiorum RCS1 mycelia culture process from darkness to blue light to darkness. Simultaneously real-time PCR method was used to the sahhl gene expression analysis. Compared with the sequence of sahh gene from Homo sapiens, Mus musculus and some fungi species, a high homology of the 555 bp sequence was confirmed. Therefore, the preliminary confirmation has supported that the 555 bp sequence should be sahhl gene from M. amphibiorum RCS1. Under the dark pre-culture in 24 h, a large amounts of transcript of sahhl gene in the mycelia can be detected by northern hybridization and real-time PCR in the condition of 24 h blue light. But a large amounts of transcript of sahhl gene were not found in other detection for the dark pre-culture of 48 h, even though M. amphibiorum RCS1 mycelia were induced by blue light. Blue light can induce the expression of sahhl gene in the vigorous growth of M. amphibiorum RCS1 mycelia.

Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa

KAUST Repository

Shih, P. B.; Yang, J.; Morisseau, C.; German, J. B.; Scott-Van Zeeland, A. A.; Armando, A. M.; Quehenberger, O.; Bergen, A. W.; Magistretti, Pierre J.; Berrettini, W.; Halmi, K. A.; Schork, N.; Hammock, B. D.; Kaye, W.

2015-01-01

Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product

In silico analysis of β-mannanases and β-mannosidase from Aspergillus flavus and Trichoderma virens UKM1

Science.gov (United States)

Yee, Chai Sin; Murad, Abdul Munir Abdul; Bakar, Farah Diba Abu

2013-11-01

A gene encoding an endo-β-1,4-mannanase from Trichoderma virens UKM1 (manTV) and Aspergillus flavus UKM1 (manAF) was analysed with bioinformatic tools. In addition, A. flavus NRRL 3357 genome database was screened for a β-mannosidase gene and analysed (mndA-AF). These three genes were analysed to understand their gene properties. manTV and manAF both consists of 1,332-bp and 1,386-bp nucleotides encoding 443 and 461 amino acid residues, respectively. Both the endo-β-1,4-mannanases belong to the glycosyl hydrolase family 5 and contain a carbohydrate-binding module family 1 (CBM1). On the other hand, mndA-AF which is a 2,745-bp gene encodes a protein sequence of 914 amino acid residues. This β-mannosidase belongs to the glycosyl hydrolase family 2. Predicted molecular weight of manTV, manAF and mndA-AF are 47.74 kDa, 49.71 kDa and 103 kDa, respectively. All three predicted protein sequences possessed signal peptide sequence and are highly conserved among other fungal β-mannanases and β-mannosidases.

GtfA and GtfB Are Both Required for Protein O-Glycosylation in Lactobacillus plantarum

Science.gov (United States)

Lee, I-Chiao; van Swam, Iris I.; Tomita, Satoru; Morsomme, Pierre; Rolain, Thomas; Hols, Pascal; Bron, Peter A.

2014-01-01

Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively. PMID:24532775

Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

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Babst Benjamin A

2009-12-01

Full Text Available Abstract Background Phenylpropanoid-derived phenolic glycosides (PGs and condensed tannins (CTs comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM, and a negative effect on cell growth (at 10 mM. The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we

Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes : Prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

NARCIS (Netherlands)

Jiménez Avella, Diego; Dini Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered

Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

International Nuclear Information System (INIS)

Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Sund, Christian J.; Hurley, Margaret M.

2015-01-01

The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

Structural analysis of Clostridium acetobutylicum ATCC 824 glycoside hydrolase from CAZy family GH105

Energy Technology Data Exchange (ETDEWEB)

Germane, Katherine L., E-mail: katherine.germane.civ@mail.mil [Oak Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017 (United States); Servinsky, Matthew D. [US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20783 (United States); Gerlach, Elliot S. [Federal Staffing Resources, 2200 Somerville Road, Annapolis, MD 21401 (United States); Sund, Christian J. [US Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20783 (United States); Hurley, Margaret M., E-mail: katherine.germane.civ@mail.mil [US Army Research Laboratory, 4600 Deer Creek Loop, Aberdeen Proving Ground, MD 21005 (United States); Oak Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017 (United States)

2015-07-29

The crystal structure of the protein product of the C. acetobutylicum ATCC 824 gene CA-C0359 is structurally similar to YteR, an unsaturated rhamnogalacturonyl hydrolase from B. subtilis strain 168. Substrate modeling and electrostatic studies of the active site of the structure of CA-C0359 suggests that the protein can now be considered to be part of CAZy glycoside hydrolase family 105. Clostridium acetobutylicum ATCC 824 gene CA-C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR of Bacillus subtilis strain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA-C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry (http://scripts.iucr.org/cgi-bin/cr.cgi?rm)) from Bacillus subtilis strain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA-C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase from Bacillus subtilis strain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate

Characterization of an ancient lepidopteran lateral gene transfer.

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David Wheeler

Full Text Available Bacteria to eukaryote lateral gene transfers (LGT are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31 from an Enterococcus bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the Bombyx mori genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65-145 million years ago. The most basal species in which the LGT was found is Plutella xylostella (superfamily: Yponomeutoidea. Array data from Bombyx mori shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material.

Isolation of oxamyl-degrading bacteria and identification of cehA as a novel oxamyl hydrolase gene

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Konstantina eRousidou

2016-04-01

Full Text Available Microbial degradation is the main process controlling the environmental dissipation of the nematicide oxamyl. Despite that, little is known regarding the microorganisms involved in its biotransformation. We report the isolation of four oxamyl-degrading bacterial strains from an agricultural soil exhibiting enhanced biodegradation of oxamyl. Multilocus sequence analysis (MLSA assigned the isolated bacteria to different subgroups of the genus Pseudomonas. The isolated bacteria hydrolyzed oxamyl to oxamyl oxime, which was not further transformed, and utilized methylamine as a C and N source. This was further supported by the detection of methylamine dehydrogenase in three of the four isolates. All oxamyl-degrading strains carried a gene highly homologous to a carbamate-hydrolase gene cehA previously identified in carbaryl- and carbofuran-degrading strains. Transcription analysis verified its direct involvement in the hydrolysis of oxamyl. Selected isolates exhibited relaxed degrading specificity and transformed all carbamates tested including the oximino carbamates aldicarb and methomyl (structurally related to oxamyl and the aryl-methyl carbamates carbofuran and carbaryl which share with oxamyl only the carbamate moiety

Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies

Science.gov (United States)

Ogo, Yuko; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Takaiwa, Fumio

2016-01-01

Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20–40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances. PMID:26438413

Flagellar glycosylation in Clostridium botulinum.

Science.gov (United States)

Twine, Susan M; Paul, Catherine J; Vinogradov, Evgeny; McNally, David J; Brisson, Jean-Robert; Mullen, James A; McMullin, David R; Jarrell, Harold C; Austin, John W; Kelly, John F; Logan, Susan M

2008-09-01

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure.

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Chiaki Nagai-Okatani

Full Text Available Targeted proteomics focusing on post-translational modifications, including glycosylation, is a useful strategy for discovering novel biomarkers. To apply this strategy effectively to cardiac hypertrophy and resultant heart failure, we aimed to characterize glycosylation profiles in the left ventricle and plasma of rats with cardiac hypertrophy. Dahl salt-sensitive hypertensive rats, a model of hypertension-induced cardiac hypertrophy, were fed a high-salt (8% NaCl diet starting at 6 weeks. As a result, they exhibited cardiac hypertrophy at 12 weeks and partially impaired cardiac function at 16 weeks compared with control rats fed a low-salt (0.3% NaCl diet. Gene expression analysis revealed significant changes in the expression of genes encoding glycosyltransferases and glycosidases. Glycoproteome profiling using lectin microarrays indicated upregulation of mucin-type O-glycosylation, especially disialyl-T, and downregulation of core fucosylation on N-glycans, detected by specific interactions with Amaranthus caudatus and Aspergillus oryzae lectins, respectively. Upregulation of plasma α-l-fucosidase activity was identified as a biomarker candidate for cardiac hypertrophy, which is expected to support the existing marker, atrial natriuretic peptide and its related peptides. Proteomic analysis identified cysteine and glycine-rich protein 3, a master regulator of cardiac muscle function, as an O-glycosylated protein with altered glycosylation in the rats with cardiac hypertrophy, suggesting that alternations in O-glycosylation affect its oligomerization and function. In conclusion, our data provide evidence of significant changes in glycosylation pattern, specifically mucin-type O-glycosylation and core defucosylation, in the pathogenesis of cardiac hypertrophy and heart failure, suggesting that they are potential biomarkers for these diseases.

A screening method for β-glucan hydrolase employing Trypan Blue-coupled β-glucan agar plate and β-glucan zymography.

Science.gov (United States)

Park, Chang-Su; Yang, Hee-Jong; Kim, Dong-Ho; Kang, Dae-Ook; Kim, Min-Soo; Choi, Nack-Shick

2012-06-01

A new screening method for β-(1,3-1,6) glucan hydrolase was developed using a pure β-glucan from Aureobaisidum pullulans by zymography and an LB-agar plate. Paenibacillus sp. was screened as a producer a β-glucan hydrolase on the Trypan Blue-coupled β-glucan LB-agar plate and the activity of the enzyme was analyzed by SDS-β-glucan zymography. The β-glucan was not hydrolyzed by Bacillus spp. strains, which exhibit cellulolytic activity on CMC zymography. The gene, obtaining by shotgun cloning and encoding the β-glucan hydrolase of Paenibacillus sp. was sequenced.

The heterotaxy gene GALNT11 glycosylates Notch to orchestrate cilia type and laterality

DEFF Research Database (Denmark)

Boskovski, Marko T; Yuan, Shiaulou; Pedersen, Nis Borbye

2013-01-01

to such determination. We previously identified GALNT11 as a candidate disease gene in a patient with heterotaxy, and now demonstrate, in Xenopus tropicalis, that galnt11 activates Notch signalling. GALNT11 O-glycosylates human NOTCH1 peptides in vitro, thereby supporting a mechanism of Notch activation either...... by increasing ADAM17-mediated ectodomain shedding of the Notch receptor or by modification of specific EGF repeats. We further developed a quantitative live imaging technique for Xenopus left-right organizer cilia and show that Galnt11-mediated Notch1 signalling modulates the spatial distribution and ratio...... of motile and immotile cilia at the left-right organizer. galnt11 or notch1 depletion increases the ratio of motile cilia at the expense of immotile cilia and produces a laterality defect reminiscent of loss of the ciliary sensor Pkd2. By contrast, Notch overexpression decreases this ratio, mimicking...

Functional importance of PAI-1 glycosylation

DEFF Research Database (Denmark)

Christensen, Anni; Naessens, Dominik; Skottrup, Peter

2001-01-01

Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-glycosylated PAI-1 could be conferred upon PAI-1 expressed in HEK293 cells by mutational inactivation of one or the other glycosylation site. These findings reveal a novel functional role for glycosylation of a serpin. The glycosylation sites are localised between a-helix H and b-strand 2C and b-strand 3C and a...

The role of epoxide hydrolase Y113H gene variant in pancreatic diseases.

NARCIS (Netherlands)

Ockenga, J.; Strunck, S.; Post, C.; Schulz, H.U.; Halangk, J.; Pfutzer, R.H.; Lohr, M.; Oettle, H.; Kage, A.; Rosendahl, J.; Keim, V.; Drenth, J.P.H.; Jansen, J.B.M.J.; Lochs, H.; Witt, H.

2009-01-01

OBJECTIVES: Chronic pancreatitis (CP) and pancreatic adenocarcinoma (pCA) are associated with risk factors such as alcohol intake and tobacco smoking. Microsomal epoxide hydrolase (EPHX1) is a phase II detoxifying enzyme capable of tobacco-borne toxicant inactivation. We studied the role of the

Adaptive antibody diversification through N-linked glycosylation of the immunoglobulin variable region.

Science.gov (United States)

van de Bovenkamp, Fleur S; Derksen, Ninotska I L; Ooijevaar-de Heer, Pleuni; van Schie, Karin A; Kruithof, Simone; Berkowska, Magdalena A; van der Schoot, C Ellen; IJspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E M; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S Marieke; Vidarsson, Gestur; Rispens, Theo

2018-02-20

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N -linked glycans, a process conditional on the introduction of consensus amino acid motifs ( N -glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N -glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

Response of a diuron-degrading community to diuron exposure assessed by real-time quantitative PCR monitoring of phenylurea hydrolase A and B encoding genes

OpenAIRE

Pesce , S.; Beguet , J.; Rouard , N.; Devers Lamrani , M.; Martin Laurent , F.

2013-01-01

A real-time quantitative PCR method was developed to detect and quantify phenlylurea hydrolase genes' (puhA and puhB) sequences from environmental DNA samples to assess diuron-degrading genetic potential in some soil and sediment microbial communities. In the soil communities, mineralization rates (determined with [ring-14C]-labeled diuron) were linked to diuron-degrading genetic potentials estimated from puhB number copies, which increased following repeated diuron treatments. In the sedimen...

Cloning, expression and structural stability of a cold-adapted ß-Galactosidase from Rahnella sp.R3

Science.gov (United States)

A novel gene was isolated for the first time from a psychrophilic gram-negative bacterium Rahnella sp.R3. It encoded a cold-adapted ß-galactosidase (R-ß-Gal). Recombinant R-ß-Gal was expressed in Escherichia coli BL21 (DE3), purified, and characterized. R-ß-Gal belongs to the glycosyl hydrolase fami...

Variants of glycoside hydrolases

Science.gov (United States)

Teter, Sarah [Davis, CA; Ward, Connie [Hamilton, MT; Cherry, Joel [Davis, CA; Jones, Aubrey [Davis, CA; Harris, Paul [Carnation, WA; Yi, Jung [Sacramento, CA

2011-04-26

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

Molecular phylogeny and predicted 3D structure of plant beta-D-N-acetylhexosaminidase.

Science.gov (United States)

Hossain, Md Anowar; Roslan, Hairul Azman

2014-01-01

beta-D-N-Acetylhexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of β-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. We constructed phylogenetic tree of β-hexosaminidases to analyze the evolutionary history and predicted functions of plant hexosaminidases. Phylogenetic analysis reveals the complex history of evolution of plant β-hexosaminidase that can be described by gene duplication events. The 3D structure of tomato β-hexosaminidase (β-Hex-Sl) was predicted by homology modeling using 1now as a template. Structural conformity studies of the best fit model showed that more than 98% of the residues lie inside the favoured and allowed regions where only 0.9% lie in the unfavourable region. Predicted 3D structure contains 531 amino acids residues with glycosyl hydrolase20b domain-I and glycosyl hydrolase20 superfamily domain-II including the (β/α)8 barrel in the central part. The α and β contents of the modeled structure were found to be 33.3% and 12.2%, respectively. Eleven amino acids were found to be involved in ligand-binding site; Asp(330) and Glu(331) could play important roles in enzyme-catalyzed reactions. The predicted model provides a structural framework that can act as a guide to develop a hypothesis for β-Hex-Sl mutagenesis experiments for exploring the functions of this class of enzymes in plant kingdom.

Efficient synthesis of glycosylated phenazine natural products and analogs with DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors

DEFF Research Database (Denmark)

Laursen, Jane B.; Petersen, Lars; Jensen, K.J.

2003-01-01

Inspired by the occurrence and function of phenazines in natural products, new glycosylated analogs were designed and synthesized. DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors were used in an efficient and easily-handled glycosylation protocol compatible with combinatorial chemistry....... Benzoylated D-glucose, D-galactose and L-quinovose DISAL glycosyl donors were synthesized in high yields and used under mild conditions to glycosylate methyl saphenate and 2-hydroxyphenazine. The glycosides were screened for biological activity and one compound showed inhibitory activity towards topoisomerase...

6-Oxocyclohex-1-ene-1-carbonyl-coenzyme A hydrolases from obligately anaerobic bacteria: characterization and identification of its gene as a functional marker for aromatic compounds degrading anaerobes.

Science.gov (United States)

Kuntze, Kevin; Shinoda, Yoshifumi; Moutakki, Housna; McInerney, Michael J; Vogt, Carsten; Richnow, Hans-Hermann; Boll, Matthias

2008-06-01

In anaerobic bacteria, most aromatic growth substrates are channelled into the benzoyl-coenzyme A (CoA) degradation pathway where the aromatic ring is dearomatized and cleaved into an aliphatic thiol ester. The initial step of this pathway is catalysed by dearomatizing benzoyl-CoA reductases yielding the two electron-reduction product, cyclohexa-1,5-diene-1-carbonyl-CoA, to which water is subsequently added by a hydratase. The next two steps have so far only been studied in facultative anaerobes and comprise the oxidation of the 6-hydroxyl-group to 6-oxocyclohex-1-ene-1-carbonyl-CoA (6-OCH-CoA), the addition of water and hydrolytic ring cleavage yielding 3-hydroxypimelyl-CoA. In this work, two benzoate-induced genes from the obligately anaerobic bacteria, Geobacter metallireducens (bamA(Geo)) and Syntrophus aciditrophicus (bamA(Syn)), were heterologously expressed in Escherichia coli, purified and characterized as 6-OCH-CoA hydrolases. Both enzymes consisted of a single 43 kDa subunit. Some properties of the enzymes are presented and compared with homologues from facultative anaerobes. An alignment of the nucleotide sequences of bamA(Geo) and bamA(Syn) with the corresponding genes from facultative anaerobes identified highly conserved DNA regions, which enabled the discrimination of genes coding for 6-OCH-CoA hydrolases from those coding for related enzymes. A degenerate oligonucleotide primer pair was deduced from conserved regions and applied in polymerase chain reaction reactions. Using these primers, the expected DNA fragment of the 6-OCH-CoA hydrolase genes was specifically amplified from the DNA of nearly all known facultative and obligate anaerobes that use aromatic growth substrates. The only exception was the aromatic compound-degrading Rhodopseudomonas palustris, which uniquely uses a modified benzoyl-CoA degradation pathway. Using the oligonucleotide primers, the expected DNA fragment was also amplified in a toluene-degrading and a m

Congenital disorders of glycosylation: The Saudi experience.

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Alsubhi, Sarah; Alhashem, Amal; Faqeih, Eissa; Alfadhel, Majid; Alfaifi, Abdullah; Altuwaijri, Waleed; Alsahli, Saud; Aldhalaan, Hesham; Alkuraya, Fowzan S; Hundallah, Khalid; Mahmoud, Adel; Alasmari, Ali; Mutairi, Fuad Al; Abduraouf, Hanem; AlRasheed, Layan; Alshahwan, Saad; Tabarki, Brahim

2017-10-01

We retrospectively reviewed Saudi patients who had a congenital disorder of glycosylation (CDG). Twenty-seven Saudi patients (14 males, 13 females) from 13 unrelated families were identified. Based on molecular studies, the 27 CDG patients were classified into different subtypes: ALG9-CDG (8 patients, 29.5%), ALG3-CDG (7 patients, 26%), COG6-CDG (7 patients, 26%), MGAT2-CDG (3 patients, 11%), SLC35A2-CDG (1 patient), and PMM2-CDG (1 patient). All the patients had homozygous gene mutations. The combined carrier frequency of CDG for the encountered founder mutations in the Saudi population is 11.5 per 10,000, which translates to a minimum disease burden of 14 patients per 1,000,000. Our study provides comprehensive epidemiologic information and prevalence figures for each of these CDG in a large cohort of congenital disorder of glycosylation patients. © 2017 Wiley Periodicals, Inc.

N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation.

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Stavenhagen, Kathrin; Kayili, H Mehmet; Holst, Stephanie; Koeleman, Carolien A M; Engel, Ruchira; Wouters, Diana; Zeerleder, Sacha; Salih, Bekir; Wuhrer, Manfred

2018-06-01

Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N - and O -glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O -glycosylated N-terminal region. Five novel and five known O -glycosylation sites were identified, carrying mainly core1-type O -glycans. In addition, we detected a heavily O -glycosylated portion spanning from Thr 82 -Ser 121 with up to 16 O -glycans attached. Likewise, all known six N -glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N -glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of expression and glycosylation of avian metapneumovirus attachment glycoprotein from recombinant baculoviruses.

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Luo, Lizhong; Nishi, Krista; MacLeod, Erin; Sabara, Marta I; Li, Yan

2010-11-01

Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Relative gene expression of bile salt hydrolase and surface proteins in two putative indigenous Lactobacillus plantarum strains under in vitro gut conditions.

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Duary, Raj Kumar; Batish, Virender Kumar; Grover, Sunita

2012-03-01

Probiotic bacteria must overcome the toxicity of bile salts secreted in the gut and adhere to the epithelial cells to enable their better colonization with extended transit time. Expression of bile salt hydrolase and other proteins on the surface of probiotic bacteria can help in better survivability and optimal functionality in the gut. Two putative Lactobacillus plantarum isolates i.e., Lp9 and Lp91 along with standard strain CSCC5276 were used. A battery of six housekeeping genes viz. gapB, dnaG, gyrA, ldhD, rpoD and 16S rRNA were evaluated by using geNorm 3.4 excel based application for normalizing the expression of bile salt hydrolase (bsh), mucus-binding protein (mub), mucus adhesion promoting protein (mapA), and elongation factor thermo unstable (EF-Tu) in Lp9 and Lp91. The maximal level of relative bsh gene expression was recorded in Lp91 with 2.89 ± 0.14, 4.57 ± 0.37 and 6.38 ± 0.19 fold increase at 2% bile salt concentration after 1, 2 and 3 h, respectively. Similarly, mub and mapA genes were maximally expressed in Lp9 at the level of 20.07 ± 1.28 and 30.92 ± 1.51 fold, when MRS was supplemented with 0.05% mucin and 1% each of bile and pancreatin (pH 6.5). However, in case of EF-Tu, the maximal expression of 42.84 ± 5.64 fold was recorded in Lp91 in the presence of mucin alone (0.05%). Hence, the expression of bsh, mub, mapA and EF-Tu could be considered as prospective biomarkers for screening of novel probiotic lactobacillus strains for optimal functionality in the gut.

Isolation of the opdE gene that encodes for a new hydrolase of Enterobacter sp. capable of degrading organophosphorus pesticides.

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Chino-Flores, Concepción; Dantán-González, Edgar; Vázquez-Ramos, Alejandra; Tinoco-Valencia, Raunel; Díaz-Méndez, Rafael; Sánchez-Salinas, Enrique; Castrejón-Godínez, Maria Luisa; Ramos-Quintana, Fernando; Ortiz-Hernández, Maria Laura

2012-06-01

Microbial enzymes that can hydrolyze organophosphorus compounds have been isolated, identified and characterized from different microbial species in order to use them in biodegradation of organophosphorus compounds. We isolated a bacterial strain Cons002 from an agricultural soil bacterial consortium, which can hydrolyze methyl-parathion (MP) and other organophosphate pesticides. HPLC analysis showed that strain Cons002 is capable of degrading pesticides MP, parathion and phorate. Pulsed-field gel electrophoresis and 16S rRNA amplification were performed for strain characterization and identification, respectively, showing that the strain Cons002 is related to the genus Enterobacter sp. which has a single chromosome of 4.6 Mb and has no plasmids. Genomic library was constructed from DNA of Enterobacter sp. Cons002. A gene called opdE (Organophosphate Degradation from Enterobacter) consists of 753 bp and encodes a protein of 25 kDa, which was isolated using activity methods. This gene opdE had no similarity to any genes reported to degrade organophosphates. When kanamycin-resistance cassette was placed in the gene opdE, hydrolase activity was suppressed and Enterobacter sp. Cons002 had no growth with MP as a nutrients source.

Prediction of glycosylation sites using random forests

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Hirst Jonathan D

2008-11-01

Full Text Available Abstract Background Post translational modifications (PTMs occur in the vast majority of proteins and are essential for function. Prediction of the sequence location of PTMs enhances the functional characterisation of proteins. Glycosylation is one type of PTM, and is implicated in protein folding, transport and function. Results We use the random forest algorithm and pairwise patterns to predict glycosylation sites. We identify pairwise patterns surrounding glycosylation sites and use an odds ratio to weight their propensity of association with modified residues. Our prediction program, GPP (glycosylation prediction program, predicts glycosylation sites with an accuracy of 90.8% for Ser sites, 92.0% for Thr sites and 92.8% for Asn sites. This is significantly better than current glycosylation predictors. We use the trepan algorithm to extract a set of comprehensible rules from GPP, which provide biological insight into all three major glycosylation types. Conclusion We have created an accurate predictor of glycosylation sites and used this to extract comprehensible rules about the glycosylation process. GPP is available online at http://comp.chem.nottingham.ac.uk/glyco/.

Endoplasmic reticulum stress and N-glycosylation modulate expression of WFS1 protein

International Nuclear Information System (INIS)

Yamaguchi, Suguru; Ishihara, Hisamitsu; Tamura, Akira; Yamada, Takahiro; Takahashi, Rui; Takei, Daisuke; Katagiri, Hideki; Oka, Yoshitomo

2004-01-01

Mutations of the WFS1 gene are responsible for two hereditary diseases, Wolfram syndrome and low frequency sensorineural hearing loss. The WFS1 protein is a glycoprotein located in the endoplasmic reticulum (ER) membrane but its function is poorly understood. Herein we show WFS1 mRNA and protein levels in pancreatic islets to be increased with ER-stress inducers, thapsigargin and dithiothreitol. Another ER-stress inducer, the N-glycosylation inhibitor tunicamycin, also raised WFS1 mRNA but not protein levels. Site-directed mutagenesis showed both Asn-663 and Asn-748 to be N-glycosylated in mouse WFS1 protein. The glycosylation-defective WFS1 protein, in which Asn-663 and Asn-748 had been substituted with aspartate, exhibited an increased protein turnover rate. Consistent with this, the WFS1 protein was more rapidly degraded in the presence of tunicamycin. These data indicate that ER-stress and N-glycosylation play important roles in WFS1 expression and stability, and also suggest regulatory roles for this protein in ER-stress induced cell death

Hallmarks of glycosylation in cancer.

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Munkley, Jennifer; Elliott, David J

2016-06-07

Aberrant glycosylation plays a fundamental role in key pathological steps of tumour development and progression. Glycans have roles in cancer cell signalling, tumour cell dissociation and invasion, cell-matrix interactions, angiogenesis, metastasis and immune modulation. Aberrant glycosylation is often cited as a 'hallmark of cancer' but is notably absent from both the original hallmarks of cancer and from the next generation of emerging hallmarks. This review discusses how glycosylation is clearly an enabling characteristic that is causally associated with the acquisition of all the hallmark capabilities. Rather than aberrant glycosylation being itself a hallmark of cancer, another perspective is that glycans play a role in every recognised cancer hallmark.

Metagenomic-based study of the phylogenetic and functional gene diversity in Galápagos land and marine iguanas.

Science.gov (United States)

Hong, Pei-Ying; Mao, Yuejian; Ortiz-Kofoed, Shannon; Shah, Rushabh; Cann, Isaac; Mackie, Roderick I

2015-02-01

In this study, a metagenome-based analysis of the fecal samples from the macrophytic algae-consuming marine iguana (MI; Amblyrhynchus cristatus) and terrestrial biomass-consuming land iguanas (LI; Conolophus spp.) was conducted. Phylogenetic affiliations of the fecal microbiome were more similar between both iguanas than to other mammalian herbivorous hosts. However, functional gene diversities in both MI and LI iguana hosts differed in relation to the diet, where the MI fecal microbiota had a functional diversity that clustered apart from the other terrestrial-biomass consuming reptilian and mammalian hosts. A further examination of the carbohydrate-degrading genes revealed that several of the prevalent glycosyl hydrolases (GH), glycosyl transferases (GT), carbohydrate binding modules (CBM), and carbohydrate esterases (CE) gene classes were conserved among all examined herbivorous hosts, reiterating the important roles these genes play in the breakdown and metabolism of herbivorous diets. Genes encoding some classes of carbohydrate-degrading families, including GH2, GH13, GT2, GT4, CBM50, CBM48, CE4, and CE11, as well as genes associated with sulfur metabolism and dehalogenation, were highly enriched or unique to the MI. In contrast, gene sequences that relate to archaeal methanogenesis were detected only in LI fecal microbiome, and genes coding for GH13, GH66, GT2, GT4, CBM50, CBM13, CE4, and CE8 carbohydrate active enzymes were highly abundant in the LI. Bacterial populations were enriched on various carbohydrates substrates (e.g., glucose, arabinose, xylose). The majority of the enriched bacterial populations belong to genera Clostridium spp. and Enterococcus spp. that likely accounted for the high prevalence of GH13 and GH2, as well as the GT families (e.g., GT2, GT4, GT28, GT35, and GT51) that were ubiquitously present in the fecal microbiota of all herbivorous hosts.

Metagenomic-Based Study of the Phylogenetic and Functional Gene Diversity in Galápagos Land and Marine Iguanas

KAUST Repository

Hong, Pei-Ying

2014-12-19

In this study, a metagenome-based analysis of the fecal samples from the macrophytic algae-consuming marine iguana (MI; Amblyrhynchus cristatus) and terrestrial biomass-consuming land iguanas (LI; Conolophus spp.) was conducted. Phylogenetic affiliations of the fecal microbiome were more similar between both iguanas than to other mammalian herbivorous hosts. However, functional gene diversities in both MI and LI iguana hosts differed in relation to the diet, where the MI fecal microbiota had a functional diversity that clustered apart from the other terrestrial-biomass consuming reptilian and mammalian hosts. A further examination of the carbohydrate-degrading genes revealed that several of the prevalent glycosyl hydrolases (GH), glycosyl transferases (GT), carbohydrate binding modules (CBM), and carbohydrate esterases (CE) gene classes were conserved among all examined herbivorous hosts, reiterating the important roles these genes play in the breakdown and metabolism of herbivorous diets. Genes encoding some classes of carbohydrate-degrading families, including GH2, GH13, GT2, GT4, CBM50, CBM48, CE4, and CE11, as well as genes associated with sulfur metabolism and dehalogenation, were highly enriched or unique to the MI. In contrast, gene sequences that relate to archaeal methanogenesis were detected only in LI fecal microbiome, and genes coding for GH13, GH66, GT2, GT4, CBM50, CBM13, CE4, and CE8 carbohydrate active enzymes were highly abundant in the LI. Bacterial populations were enriched on various carbohydrates substrates (e.g., glucose, arabinose, xylose). The majority of the enriched bacterial populations belong to genera Clostridium spp. and Enterococcus spp. that likely accounted for the high prevalence of GH13 and GH2, as well as the GT families (e.g., GT2, GT4, GT28, GT35, and GT51) that were ubiquitously present in the fecal microbiota of all herbivorous hosts.

Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community

Energy Technology Data Exchange (ETDEWEB)

Allgaier, M.; Reddy, A.; Park, J. I.; Ivanova, N.; D' haeseleer, P.; Lowry, S.; Sapra, R.; Hazen, T.C.; Simmons, B.A.; VanderGheynst, J. S.; Hugenholtz, P.

2009-11-15

Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, {approx}10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

Targeted Discovery of Glycoside Hydrolases from a Switchgrass-Adapted Compost Community

Energy Technology Data Exchange (ETDEWEB)

Reddy, Amitha; Allgaier, Martin; Park, Joshua I.; Ivanoval, Natalia; Dhaeseleer, Patrik; Lowry, Steve; Sapra, Rajat; Hazen, Terry C.; Simmons, Blake A.; VanderGheynst, Jean S.; Hugenholtz, Philip

2011-05-11

Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum) and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Smallsubunit (SSU) rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC) with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, ,10percent were putative cellulasesmostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50uC and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

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Caroline da Silva Moraes

2014-08-01

Full Text Available The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females or blood feeders (females only, and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18 and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes.

Expression pattern of glycoside hydrolase genes in Lutzomyia longipalpis reveals key enzymes involved in larval digestion

Science.gov (United States)

Moraes, Caroline da Silva; Diaz-Albiter, Hector M.; Faria, Maiara do Valle; Sant'Anna, Maurício R. V.; Dillon, Rod J.; Genta, Fernando A.

2014-01-01

The sand fly Lutzomyia longipalpis is the most important vector of American Visceral Leishmaniasis. Adults are phytophagous (males and females) or blood feeders (females only), and larvae feed on solid detritus. Digestion in sand fly larvae has scarcely been studied, but some glycosidase activities putatively involved in microorganism digestion were already described. Nevertheless, the molecular nature of these enzymes, as the corresponding genes and transcripts, were not explored yet. Catabolism of microbial carbohydrates in insects generally involves β-1,3-glucanases, chitinases, and digestive lysozymes. In this work, the transcripts of digestive β-1,3-glucanase and chitinases were identified in the L. longipalpis larvae throughout analysis of sequences and expression patterns of glycoside hydrolases families 16, 18, and 22. The activity of one i-type lysozyme was also registered. Interestingly, this lysozyme seems to play a role in immunity, rather than digestion. This is the first attempt to identify the molecular nature of sand fly larval digestive enzymes. PMID:25140153

Impact of the Staphylococcus epidermidis LytSR two-component regulatory system on murein hydrolase activity, pyruvate utilization and global transcriptional profile

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Yu Fangyou

2010-11-01

Full Text Available Abstract Background Staphylococcus epidermidis has emerged as one of the most important nosocomial pathogens, mainly because of its ability to colonize implanted biomaterials by forming a biofilm. Extensive studies are focused on the molecular mechanisms involved in biofilm formation. The LytSR two-component regulatory system regulates autolysis and biofilm formation in Staphylococcus aureus. However, the role of LytSR played in S. epidermidis remained unknown. Results In the present study, we demonstrated that lytSR knock-out in S. epidermidis did not alter susceptibility to Triton X-100 induced autolysis. Quantitative murein hydrolase assay indicated that disruption of lytSR in S. epidermidis resulted in decreased activities of extracellular murein hydrolases, although zymogram showed no apparent differences in murein hydrolase patterns between S. epidermidis strain 1457 and its lytSR mutant. Compared to the wild-type counterpart, 1457ΔlytSR produced slightly more biofilm, with significantly decreased dead cells inside. Microarray analysis showed that lytSR mutation affected the transcription of 164 genes (123 genes were upregulated and 41 genes were downregulated. Specifically, genes encoding proteins responsible for protein synthesis, energy metabolism were downregulated, while genes involved in amino acid and nucleotide biosynthesis, amino acid transporters were upregulated. Impaired ability to utilize pyruvate and reduced activity of arginine deiminase was observed in 1457ΔlytSR, which is consistent with the microarray data. Conclusions The preliminary results suggest that in S. epidermidis LytSR two-component system regulates extracellular murein hydrolase activity, bacterial cell death and pyruvate utilization. Based on the microarray data, it appears that lytSR inactivation induces a stringent response. In addition, LytSR may indirectly enhance biofilm formation by altering the metabolic status of the bacteria.

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

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Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

Cloning and identification of novel hydrolase genes from a dairy cow rumen metagenomic library and characterization of a cellulase gene

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Gong Xia

2012-10-01

Full Text Available Abstract Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

Generation and characterization of epoxide hydrolase 3 (EPHX3-deficient mice.

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Samantha L Hoopes

Full Text Available Cytochrome P450 (CYP epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs, which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH and soluble epoxide hydrolase (EPHX2, sEH were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT. Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic

Molecular Phylogeny and Predicted 3D Structure of Plant beta-D-N-Acetylhexosaminidase

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Md. Anowar Hossain

2014-01-01

Full Text Available beta-D-N-Acetylhexosaminidase, a family 20 glycosyl hydrolase, catalyzes the removal of β-1,4-linked N-acetylhexosamine residues from oligosaccharides and their conjugates. We constructed phylogenetic tree of β-hexosaminidases to analyze the evolutionary history and predicted functions of plant hexosaminidases. Phylogenetic analysis reveals the complex history of evolution of plant β-hexosaminidase that can be described by gene duplication events. The 3D structure of tomato β-hexosaminidase (β-Hex-Sl was predicted by homology modeling using 1now as a template. Structural conformity studies of the best fit model showed that more than 98% of the residues lie inside the favoured and allowed regions where only 0.9% lie in the unfavourable region. Predicted 3D structure contains 531 amino acids residues with glycosyl hydrolase20b domain-I and glycosyl hydrolase20 superfamily domain-II including the (β/α8 barrel in the central part. The α and β contents of the modeled structure were found to be 33.3% and 12.2%, respectively. Eleven amino acids were found to be involved in ligand-binding site; Asp(330 and Glu(331 could play important roles in enzyme-catalyzed reactions. The predicted model provides a structural framework that can act as a guide to develop a hypothesis for β-Hex-Sl mutagenesis experiments for exploring the functions of this class of enzymes in plant kingdom.

Response of a diuron-degrading community to diuron exposure assessed by real-time quantitative PCR monitoring of phenylurea hydrolase A and B encoding genes.

Science.gov (United States)

Pesce, Stéphane; Beguet, Jérémie; Rouard, Nadine; Devers-Lamrani, Marion; Martin-Laurent, Fabrice

2013-02-01

A real-time quantitative PCR method was developed to detect and quantify phenlylurea hydrolase genes' (puhA and puhB) sequences from environmental DNA samples to assess diuron-degrading genetic potential in some soil and sediment microbial communities. In the soil communities, mineralization rates (determined with [ring-¹⁴C]-labeled diuron) were linked to diuron-degrading genetic potentials estimated from puhB number copies, which increased following repeated diuron treatments. In the sediment communities, mineralization potential did not depend solely on the quantity of puhB copies, underlining the need to assess gene expression. In the sediment samples, both puhB copy numbers and mineralization capacities were highly conditioned by whether or not diuron-treated soil was added. This points to transfers of degradative potential from soils to sediments. No puhA gene was detected in soil and sediment DNA extracts. Moreover, some sediments exhibited high diuron mineralization potential even though puhB genes were not detected, suggesting the existence of alternative diuron degradation pathways.

Further characterization of intestinal lactase/phlorizin hydrolase

DEFF Research Database (Denmark)

Skovbjerg, H; Norén, O; Sjöström, H

1982-01-01

Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the pres......Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis...... in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig...... membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase....

Gene cloning, overexpression, and characterization of a xylanase from Penicillium sp. CGMCC 1669.

Science.gov (United States)

Liu, Wanli; Shi, Pengjun; Chen, Qiang; Yang, Peilong; Wang, Guozeng; Wang, Yaru; Luo, Huiying; Yao, Bin

2010-09-01

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml(-1). After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40 degrees C, was stable at acidic buffers of pH 4.5-9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and alpha-chymotrypsin). The specific activity, K (m), and V (max) for oat spelt xylan substrate was 7,988 U mg(-1), 22.2 mg ml(-1), and 15,105.7 micromol min(-1) mg(-1), respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.

A Trapped Covalent Intermediate of a Glycoside Hydrolase on the Pathway to Transglycosylation. Insights from Experiments and Quantum Mechanics/Molecular Mechanics Simulations.

Science.gov (United States)

Raich, Lluís; Borodkin, Vladimir; Fang, Wenxia; Castro-López, Jorge; van Aalten, Daan M F; Hurtado-Guerrero, Ramón; Rovira, Carme

2016-03-16

The conversion of glycoside hydrolases (GHs) into transglycosylases (TGs), i.e., from enzymes that hydrolyze carbohydrates to enzymes that synthesize them, represents a promising solution for the large-scale synthesis of complex carbohydrates for biotechnological purposes. However, the lack of knowledge about the molecular details of transglycosylation hampers the rational design of TGs. Here we present the first crystallographic structure of a natural glycosyl-enzyme intermediate (GEI) of Saccharomyces cerevisiae Gas2 in complex with an acceptor substrate and demonstrate, by means of quantum mechanics/molecular mechanics metadynamics simulations, that it is tuned for transglycosylation (ΔG(⧧) = 12 kcal/mol). The 2-OH···nucleophile interaction is found to be essential for catalysis: its removal raises the free energy barrier significantly (11 and 16 kcal/mol for glycosylation and transglycosylation, respectively) and alters the conformational itinerary of the substrate (from (4)C1 → [(4)E](⧧) → (1,4)B/(4)E to (4)C1 → [(4)H3](⧧) → (4)C1). Our results suggest that changes in the interactions involving the 2-position could have an impact on the transglycosylation activity of several GHs.

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

International Nuclear Information System (INIS)

Kalra, Rajkumar S.; Wadhwa, Renu

2015-01-01

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

Energy Technology Data Exchange (ETDEWEB)

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp [Cell Proliferation Research Group and DBT-AIST International Laboratory for Advanced Biomedicine, National Institute of Advanced Industrial Science and Technology (AIST Central 4), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

Functional importance of PAI-1 glycosylation

DEFF Research Database (Denmark)

Christensen, Anni; Naessens, Dominik; Skottrup, Peter

susceptible PAI-1 variant was not necessarily the one used when raising the antibody. This and other observations indicated that the carbohydrate moieties or the glycosylation sites are unlikely to be part of the epitopes for these antibodies. The antibody susceptibility characteristic for non......Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-linked glycosylation. Biochemical analysis of PAI-1 variants with substitutions of the Asn residues in each of these sites and expression in human embryonic kidney 293 (HEK293) cells showed that only Asn211 and Asn 267, but not Asn331 are glycosylated, and revealed a differential composition of the carbohydrate...

Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community.

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Martin Allgaier

Full Text Available Development of cellulosic biofuels from non-food crops is currently an area of intense research interest. Tailoring depolymerizing enzymes to particular feedstocks and pretreatment conditions is one promising avenue of research in this area. Here we added a green-waste compost inoculum to switchgrass (Panicum virgatum and simulated thermophilic composting in a bioreactor to select for a switchgrass-adapted community and to facilitate targeted discovery of glycoside hydrolases. Small-subunit (SSU rRNA-based community profiles revealed that the microbial community changed dramatically between the initial and switchgrass-adapted compost (SAC with some bacterial populations being enriched over 20-fold. We obtained 225 Mbp of 454-titanium pyrosequence data from the SAC community and conservatively identified 800 genes encoding glycoside hydrolase domains that were biased toward depolymerizing grass cell wall components. Of these, approximately 10% were putative cellulases mostly belonging to families GH5 and GH9. We synthesized two SAC GH9 genes with codon optimization for heterologous expression in Escherichia coli and observed activity for one on carboxymethyl cellulose. The active GH9 enzyme has a temperature optimum of 50 degrees C and pH range of 5.5 to 8 consistent with the composting conditions applied. We demonstrate that microbial communities adapt to switchgrass decomposition using simulated composting condition and that full-length genes can be identified from complex metagenomic sequence data, synthesized and expressed resulting in active enzyme.

Mapping N-linked Glycosylation Sites in the Secretome and Whole Cells of Aspergillus niger Using Hydrazide Chemistry and Mass Spectrometry

Energy Technology Data Exchange (ETDEWEB)

Wang, Lu; Aryal, Uma K.; Dai, Ziyu; Mason, Alisa C.; Monroe, Matthew E.; Tian, Zhixin; Zhou, Jianying; Su, Dian; Weitz, Karl K.; Liu, Tao; Camp, David G.; Smith, Richard D.; Baker, Scott E.; Qian, Weijun

2012-01-01

Protein glycosylation is known to play an essential role in both cellular functions and the secretory pathways; however, little information is available on the dynamics of glycosylated N-linked glycosites of fungi. Herein we present the first extensive mapping of glycosylated N-linked glycosites in industrial strain Aspergillus niger by applying an optimized solid phase enrichment of glycopeptide protocol using hydrazide modified magnetic beads. The enrichment protocol was initially optimized using mouse plasma and A. niger secretome samples, which was then applied to profile N-linked glycosites from both the secretome and whole cell lysates of A. niger. A total of 847 unique N-linked glycosites and 330 N-linked glycoproteins were confidently identified by LC-MS/MS. Based on gene ontology analysis, the identified N-linked glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, golgi apparatus, lysosome, and storage vacuoles. The identified N-linked glycoproteins are involved in a wide range of biological processes including gene regulation and signal transduction, protein folding and assembly, protein modification and carbohydrate metabolism. The extensive coverage of glycosylated N-linked glycosites along with identification of partial N-linked glycosylation in those enzymes involving in different biochemical pathways provide useful information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.

Allelic variation of bile salt hydrolase genes in Lactobacillus salivarius does not determine bile resistance levels.

LENUS (Irish Health Repository)

Fang, Fang

2009-09-01

Commensal lactobacilli frequently produce bile salt hydrolase (Bsh) enzymes whose roles in intestinal survival are unclear. Twenty-six Lactobacillus salivarius strains from different sources all harbored a bsh1 allele on their respective megaplasmids. This allele was related to the plasmid-borne bsh1 gene of the probiotic strain UCC118. A second locus (bsh2) was found in the chromosomes of two strains that had higher bile resistance levels. Four Bsh1-encoding allele groups were identified, defined by truncations or deletions involving a conserved residue. In vitro analyses showed that this allelic variation was correlated with widely varying bile deconjugation phenotypes. Despite very low activity of the UCC118 Bsh1 enzyme, a mutant lacking this protein had significantly lower bile resistance, both in vitro and during intestinal transit in mice. However, the overall bile resistance phenotype of this and other strains was independent of the bsh1 allele type. Analysis of the L. salivarius transcriptome upon exposure to bile and cholate identified a multiplicity of stress response proteins and putative efflux proteins that appear to broadly compensate for, or mask, the effects of allelic variation of bsh genes. Bsh enzymes with different bile-degrading kinetics, though apparently not the primary determinants of bile resistance in L. salivarius, may have additional biological importance because of varying effects upon bile as a signaling molecule in the host.

Glycoside Hydrolase MoGls2 Controls Asexual/Sexual Development, Cell Wall Integrity and Infectious Growth in the Rice Blast Fungus.

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Mengying Li

Full Text Available N-linked glycosylation is a way of glycosylation for newly synthesized protein, which plays a key role in the maturation and transport of proteins. Glycoside hydrolases (GHs are essential in this process, and are involved in processing of N-linked glycoproteins or degradation of carbohydrate structures. Here, we identified and characterized MoGls2 in Magnaporthe oryzae, which is a yeast glucosidase II homolog Gls2 and is required for trimming the final glucose in N-linked glycans and normal cell wall synthesis. Target deletion of MoGLS2 in M. oryzae resulted in a reduced mycelial growth, an increased conidial production, delayed conidial germination and loss the ability of sexual reproduction. Pathogenicity assays revealed that the ΔMogls2 mutant showed significantly decreased in virulence and infectious growth. Further studies showed that the mutant was less sensitive to salt and osmotic stress, and increased sensitivity to cell wall stresses. Additionally, the ΔMogls2 mutant showed a defect in cell wall integrity. Our results indicate that MoGls2 is a key protein for the growth and development of M. oryzae, involving in the regulation of asexual/sexual development, stress response, cell wall integrity and infectious growth.

The phosducin-like protein PhLP1 impacts regulation of glycoside hydrolases and light response in Trichoderma reesei

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Tisch Doris

2011-12-01

Full Text Available Abstract Background In the biotechnological workhorse Trichoderma reesei (Hypocrea jecorina transcription of cellulase genes as well as efficiency of the secreted cellulase mixture are modulated by light. Components of the heterotrimeric G-protein pathway interact with light-dependent signals, rendering this pathway a key regulator of cellulase gene expression. Results As regulators of heterotrimeric G-protein signaling, class I phosducin-like proteins, are assumed to act as co-chaperones for G-protein beta-gamma folding and exert their function in response to light in higher eukaryotes. Our results revealed light responsive transcription of the T. reesei class I phosducin-like protein gene phlp1 and indicate a light dependent function of PhLP1 also in fungi. We showed the functions of PhLP1, GNB1 and GNG1 in the same pathway, with one major output being the regulation of transcription of glycoside hydrolase genes including cellulase genes in T. reesei. We found no direct correlation between the growth rate and global regulation of glycoside hydrolases, which suggests that regulation of growth does not occur only at the level of substrate degradation efficiency. Additionally, PhLP1, GNB1 and GNG1 are all important for proper regulation of light responsiveness during long term exposure. In their absence, the amount of light regulated genes increased from 2.7% in wild type to 14% in Δphlp1. Besides from the regulation of degradative enzymes, PhLP1 was also found to impact on the transcription of genes involved in sexual development, which was in accordance with decreased efficiency of fruiting body formation in Δphlp1. The lack of GNB1 drastically diminished ascospore discharge in T. reesei. Conclusions The heterotrimeric G-protein pathway is crucial for the interconnection of nutrient signaling and light response of T. reesei, with the class I phosducin-like protein PhLP1, GNB1 and GNG1 acting as important nodes, which influence light

Chondrocyte secreted CRTAC1: a glycosylated extracellular matrix molecule of human articular cartilage.

Science.gov (United States)

Steck, Eric; Bräun, Jessica; Pelttari, Karoliina; Kadel, Stephanie; Kalbacher, Hubert; Richter, Wiltrud

2007-01-01

Cartilage acidic protein 1 (CRTAC1), a novel human marker which allowed discrimination of human chondrocytes from osteoblasts and mesenchymal stem cells in culture was so far studied only on the RNA-level. We here describe its genomic organisation and detect a new brain expressed (CRTAC1-B) isoform resulting from alternate last exon usage which is highly conserved in vertebrates. In humans, we identify an exon sharing process with the neighbouring tail-to-tail orientated gene leading to CRTAC1-A. This isoform is produced by cultured human chondrocytes, localized in the extracellular matrix of articular cartilage and its secretion can be stimulated by BMP4. Of five putative O-glycosylation motifs in the last exon of CRTAC1-A, the most C-terminal one is modified according to exposure of serial C-terminal deletion mutants to the O-glycosylation inhibitor Benzyl-alpha-GalNAc. Both isoforms contain four FG-GAP repeat domains and an RGD integrin binding motif, suggesting cell-cell or cell-matrix interaction potential. In summary, CRTAC1 acquired an alternate last exon from the tail-to-tail oriented neighbouring gene in humans resulting in the glycosylated isoform CRTAC1-A which represents a new extracellular matrix molecule of articular cartilage.

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase[S

Science.gov (United States)

Oguro, Ami; Imaoka, Susumu

2012-01-01

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3–7 μM; Vmax, 150–193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism. PMID:22217705

Engineering Mammalian Mucin-type O-Glycosylation in Plants

DEFF Research Database (Denmark)

Yang, Zhang; Drew, Damian P; Jørgensen, Bodil

2012-01-01

-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity...... was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon a2b. In plants, prolines in certain...... classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host...

Proteomics and pathway analysis of N-glycosylated mammary gland proteins in response to Escherichia coli mastitis in cattle.

Science.gov (United States)

Yang, Yongxin; Shen, Weijun; Zhao, Xiaowei; Zhao, Huiling; Huang, Dongwei; Cheng, Guanglong

2014-06-01

The aim of this study was to investigate the N-linked glycosylated protein profile of mammary tissue from healthy cows and cows with mastitis due to Escherichia coli, in order to understand the molecular mechanisms of the host response to mastitis. N-glycopeptides were enriched with a lectin mixture and identified through high-accuracy mass spectrometry. A total of 551 N-glycosylation sites, corresponding to 294 proteins, were identified in the mammary tissues of healthy cows; these glycoproteins were categorised into three functional groups and clustered into 11 specific pathways. A total of 511 N-glycosylation sites, corresponding to 283 glycosylated proteins, were detected in the mammary tissues of cows with E. coli mastitis. There were differences in N-glycosylation sites in 98 proteins in the mammary tissues of healthy cows and cows with mastitis due to E. coli. Most proteins with altered glycosylation were those involved in responses to stress, cell adhesion and the immune response, and were assigned to five specific pathways based on their gene ontology annotation. The results from this study show that the glycosylated protein profile in the mammary tissues of healthy and mastitic cows are different, and altered glycoproteins are associated with several pathways, including the lysosome and O-glycan biosynthesis pathways. Copyright © 2014. Published by Elsevier Ltd.

Trans-species Engineering of Glycosylated Therapeutic Proteins

DEFF Research Database (Denmark)

Yang, Zhang

important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...... understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower......Recombinant expression of therapeutic proteins is one of the major tasks in modern biomedicine. One of the most important factors with respect to therapeutic use in human is posttranslational modifications (PTMs) of the recombinant proteins, of which protein glycosylation is by far the most...

O-Glycosylation Modulates Proprotein Convertase Activation of Angiopoietin-like Protein 3: POSSIBLE ROLE OF POLYPEPTIDE GalNAc-TRANSFERASE-2 IN REGULATION OF CONCENTRATIONS OF PLASMA LIPIDS

DEFF Research Database (Denmark)

Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B; Bennett, Eric Paul

2010-01-01

immediately C-terminal (TT(226)). We developed an in vivo model system in CHO ldlD cells that was used to show that O-glycosylation in the processing site blocked processing of ANGPTL3. Genome-wide SNP association studies have identified the polypeptide GalNAc-transferase gene, GALNT2, as a candidate gene...... for low HDL and high triglyceride blood levels. We hypothesized that the GalNAc-T2 transferase performed critical O-glycosylation of proteins involved in lipid metabolism. Screening of a panel of proteins known to affect lipid metabolism for potential sites glycosylated by GalNAc-T2 led to identification...

Poly(aspartic acid) (PAA) hydrolases and PAA biodegradation: current knowledge and impact on applications.

Science.gov (United States)

Hiraishi, Tomohiro

2016-02-01

Thermally synthesized poly(aspartic acid) (tPAA) is a bio-based, biocompatible, biodegradable, and water-soluble polymer that has a high proportion of β-Asp units and equivalent moles of D- and L-Asp units. Poly(aspartic acid) (PAA) hydrolase-1 and hydrolase-2 are tPAA biodegradation enzymes purified from Gram-negative bacteria. PAA hydrolase-1 selectively cleaves amide bonds between β-Asp units via an endo-type process, whereas PAA hydrolase-2 catalyzes the exo-type hydrolysis of the products of tPAA hydrolysis by PAA hydrolase-1. The novel reactivity of PAA hydrolase-1 makes it a good candidate for a biocatalyst in β-peptide synthesis. This mini-review gives an overview of PAA hydrolases with emphasis on their biochemical and functional properties, in particular, PAA hydrolase-1. Functionally related enzymes, such as poly(R-3-hydroxybutyrate) depolymerases and β-aminopeptidases, are compared to PAA hydrolases. This mini-review also provides findings that offer an insight into the catalytic mechanisms of PAA hydrolase-1 from Pedobacter sp. KP-2.

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide.

Science.gov (United States)

Daubenspeck, James M; Jordan, David S; Simmons, Warren; Renfrow, Matthew B; Dybvig, Kevin

2015-01-01

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.

Identification of the chain-dispersing peptidoglycan hydrolase LytB of Streptococcus gordonii.

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Riccardo Arrigucci

Full Text Available Bacterial cell division ends with the separation of the daughter cells, a process that requires peptidoglycan hydrolases (PGHs. Bacteria lacking cell separating PGHs are impaired in cell separation with the formation of long chains or clusters. We identified a gene in Streptococcus gordonii encoding for a putative glucosaminidase (lytB. The lytB isogenic mutant grew in long bacterial chains and resulted in impaired biofilm formation. Purified recombinant LytB showed a murolytic activity on Micrococcus lysodeikticus cell suspension and was able to disperse the long chains of the mutant, restoring the wild type diplococci/short chain phenotype. LytB protein was localized only in culture supernatant cell fraction of S. gordonii, and co-cultures of wild type and lytB mutant showed a significant reduction of bacterial chain length, indicating that LytB is a secreted enzyme. Our results demonstrate that LytB is a secreted peptidoglycan hydrolase required for S. gordonii cell separation.

Saccharomyces cerevisiae KTR4, KTR5 and KTR7 encode mannosyltransferases differentially involved in the N- and O-linked glycosylation pathways.

Science.gov (United States)

Hernández, Nahúm V; López-Ramírez, Luz A; Díaz-Jiménez, Diana F; Mellado-Mojica, Erika; Martínez-Duncker, Iván; López, Mercedes G; Mora-Montes, Héctor M

2017-10-01

Saccharomyces cerevisiae is a model to understand basic aspects of protein glycosylation pathways. Although these metabolic routes have been thoroughly studied, there are still knowledge gaps; among them, the role of the MNT1/KRE2 gene family. This family is composed of nine members, with only six functionally characterized. The enzymes Ktr1, Ktr3, and Mnt1/Kre2 have overlapping activities in both O-linked and N-linked glycan synthesis; while Ktr2 and Yur1 participate exclusively in the elongation of the N-linked glycan outer chain. KTR6 encodes for a phosphomannosyltransferase that synthesizes the cell wall phosphomannan. Here, we aimed to establish the functional role of KTR4, KTR5 and KTR7 in the protein glycosylation pathways, by using heterologous complementation in Candida albicans null mutants lacking members of the MNT1/KRE2 gene family. The three S. cerevisiae genes restored defects in the C. albicans N-linked glycosylation pathway. KTR5 and KTR7 partially complemented a C. albicans null mutant with defects in the synthesis of O-linked glycans, and only KTR4 fully elongated the O-linked glycans like wild-type cells. Therefore, our results suggest that the three genes have a redundant activity in the S. cerevisiae N-linked glycosylation pathway, but KTR4 plays a major role in O-linked glycan synthesis. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Allelic Variation of Bile Salt Hydrolase Genes in Lactobacillus salivarius Does Not Determine Bile Resistance Levels▿ †

Science.gov (United States)

Fang, Fang; Li, Yin; Bumann, Mario; Raftis, Emma J.; Casey, Pat G.; Cooney, Jakki C.; Walsh, Martin A.; O'Toole, Paul W.

2009-01-01

Commensal lactobacilli frequently produce bile salt hydrolase (Bsh) enzymes whose roles in intestinal survival are unclear. Twenty-six Lactobacillus salivarius strains from different sources all harbored a bsh1 allele on their respective megaplasmids. This allele was related to the plasmid-borne bsh1 gene of the probiotic strain UCC118. A second locus (bsh2) was found in the chromosomes of two strains that had higher bile resistance levels. Four Bsh1-encoding allele groups were identified, defined by truncations or deletions involving a conserved residue. In vitro analyses showed that this allelic variation was correlated with widely varying bile deconjugation phenotypes. Despite very low activity of the UCC118 Bsh1 enzyme, a mutant lacking this protein had significantly lower bile resistance, both in vitro and during intestinal transit in mice. However, the overall bile resistance phenotype of this and other strains was independent of the bsh1 allele type. Analysis of the L. salivarius transcriptome upon exposure to bile and cholate identified a multiplicity of stress response proteins and putative efflux proteins that appear to broadly compensate for, or mask, the effects of allelic variation of bsh genes. Bsh enzymes with different bile-degrading kinetics, though apparently not the primary determinants of bile resistance in L. salivarius, may have additional biological importance because of varying effects upon bile as a signaling molecule in the host. PMID:19592587

Use of full recovery hydrolasing equipment for facility decommissioning - 16325

International Nuclear Information System (INIS)

Martin, Scott A.; Adams, Scott R.

2009-01-01

The removal of surface contamination is a major challenge for nearly all nuclear facilities undergoing, or awaiting, decommissioning. Conventional means of surface decontamination can expose workers to unnecessary hazards, and are often not fit-for-purpose due to size constraints or weight restrictions. Additionally, conventional methods are not always easily deployed remotely due to their complexity or required services. The use of ultra high pressure water for surface decontamination, known as hydrolasing, is recognized as a technology which can be used in various applications requiring surface removal. Hydrolasing is an advantageous technology for many reasons including its versatility, overall simplicity and relative ease of remote deployment. For the nuclear industry, one of the largest challenges with regards to the use of hydrolasing is the requirement for the full recovery of the injected water and removed solids. For nonnuclear applications, there is often no requirement for recovery of the liquid and solid waste, which has led to few system designs which will recover the waste in full. S.A. Robotics' experience with the deployment of ultra high pressure water systems for nuclear applications has shown that full recovery of injected water and removed solids is achievable in both underwater and in-air applications. Innovative equipment and system design have allowed S.A. Robotics' hydrolasing systems to achieve near 100% solid and liquid recovery during concrete hydrolasing. This technology has been deployed for Fluor Hanford at Hanford's K-Basins, as well as for UKAEA as part of the Windscale Piles decommissioning project. The purpose of this paper is to provide a short description of the hydrolasing process and the associated waste issues, describe the unique design features of S.A. Robotics' hydrolasing systems which combat these issues, and provide an overview of two of the hydrolasing projects that S.A. Robotics has completed. (authors)

Carboxylesterase 1 genes

DEFF Research Database (Denmark)

Rasmussen, Henrik Berg; Madsen, Majbritt Busk

2018-01-01

The carboxylesterase 1 gene (CES1) encodes a hydrolase that metabolizes commonly used drugs. The CES1-related pseudogene, carboxylesterase 1 pseudogene 1 (CES1P1), has been implicated in gene exchange with CES1 and in the formation of hybrid genes including the carboxylesterase 1A2 gene (CES1A2...

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.

Science.gov (United States)

Yamakoshi, Yasuo; Nagano, Takatoshi; Hu, Jan Cc; Yamakoshi, Fumiko; Simmer, James P

2011-02-03

Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr200, Thr216 and Thr

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

Directory of Open Access Journals (Sweden)

Yamakoshi Fumiko

2011-02-01

Full Text Available Abstract Background Dentin sialophosphoprotein (Dspp is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp, the N-terminal domain of dentin sialophosphoprotein (Dspp, is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. Results To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were

Glycosylated yellow laccases of the basidiomycete Stropharia aeruginosa.

Science.gov (United States)

Daroch, Maurycy; Houghton, Catharine A; Moore, Jonathan K; Wilkinson, Mark C; Carnell, Andrew J; Bates, Andrew D; Iwanejko, Lesley A

2014-05-10

Here we describe the identification, purification and characterisation of glycosylated yellow laccase proteins from the basidiomycete fungus Stropharia aeruginosa. Biochemical characterisation of two yellow laccases, Yel1p and Yel3p, show that they are both secreted, monomeric, N-glycosylated proteins of molecular weight around 55kDa with substrate specificities typical of laccases, but lacking the absorption band at 612nm typical of the blue laccase proteins. Low coverage, high throughput 454 transcriptome sequencing in combination with inverse-PCR was used to identify cDNA sequences. One of the cDNA sequences has been assigned to the Yel1p protein on the basis of identity between the translated protein sequence and the peptide data from the purified protein, and the full length gene sequence has been obtained. Biochemical properties, substrate specificities and protein sequence data have been used to discuss the unusual spectroscopic properties of S. aeruginosa proteins in the context of recent theories about the differences between yellow and blue laccases. Copyright © 2014 Elsevier Inc. All rights reserved.

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla......The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous...... with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended...

Glycosylation: a hallmark of cancer?

Science.gov (United States)

Vajaria, Bhairavi N; Patel, Prabhudas S

2017-04-01

The hallmarks of cancer are characterized by functional capabilities that allow cancer cells to survive, proliferate and disseminate during the multistep tumorigenesis. Cancer being a cellular disease, changes in cellular glycoproteins play an important role in malignant transformation and cancer progression. The present review summarizes various studies that depicted correlation of glycosylation with tumor initiation, progression and metastasis, which are helpful in early diagnosis, disease monitoring and prognosis. The results are further strengthened by our reports, which depicted alterations in sialylation and fucosylation in different cancers. Alterations in glycosyltransferases are also involved in formation of various tumor antigens (e.g. Sialyl Lewis x) which serves as ligand for the cell adhesion molecule, selectin which is involved in adhesion of cancer cells to vascular endothelium and thus contributes to hematogenous metastasis. Increased glycosylation accompanied by alterations in glycosyltranferases, glycosidases, glycans and mucins (MUC)s are also involved in loss of E-cadherin, a key molecule implicated in metastatic dissemination of cells. The present review also summarizes the correlation of glycosylation with all the hallmarks of cancer. The enormous progress in the design of novel inhibitors of pathway intermediates of sialylation and fucosylation can prove wonders in combating the dreadful disease. The results provide the evidence that altered glycosylation is linked to tumor initiation, progression and metastasis. Hence, it can be considered as a new hallmark of cancer development and strategies to develop novel glycosylation targeted molecules should be strengthened.

Thermophilic and thermoacidophilic glycosylation genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

Science.gov (United States)

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

2016-01-12

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for glycosylating and/or post-translationally modifying proteins using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird?s-Nest Fern

OpenAIRE

Wu, Guan-Long; Kuo, Tzu-Hao; Tsay, Tung-Tsuan; Tsai, Isheng J.; Chen, Peichen J.

2016-01-01

Five Aphelenchoides besseyi isolates collected from bird's-nest ferns or rice possess different parasitic capacities in bird's-nest fern. Two different glycoside hydrolase (GH) 45 genes were identified in the fern isolates, and only one was found in the rice isolates. A Abe GH5-1 gene containing an SCP-like family domain was found only in the fern isolates. Abe GH5-1 gene has five introns suggesting a eukaryotic origin. A maximum likelihood phylogeny revealed that Abe GH5-1 is part of the nem...

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

International Nuclear Information System (INIS)

Jones, Meredith B.; Tomiya, Noboru; Betenbaugh, Michael J.; Krag, Sharon S.

2010-01-01

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man 5 GlcNAc 2 -P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man 9 GlcNAc 2 -P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc 3 Man 9 GlcNAc 2 -P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man 5 GlcNAc 2 -PP-Dol through Glc 1 Man 9 GlcNAc 2 -PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc 3 Man 9 GlcNAc 2 -P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

Energy Technology Data Exchange (ETDEWEB)

Jones, Meredith B., E-mail: mbauman7@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Tomiya, Noboru, E-mail: ntomiya1@jhu.edu [Department of Biology, Johns Hopkins University, 3400 North Charles Street, Mudd Hall 104A, Baltimore, MD 21218 (United States); Betenbaugh, Michael J., E-mail: beten@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Krag, Sharon S., E-mail: skrag@jhsph.edu [Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205 (United States)

2010-04-23

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man{sub 5}GlcNAc{sub 2}-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man{sub 9}GlcNAc{sub 2}-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man{sub 5}GlcNAc{sub 2}-PP-Dol through Glc{sub 1}Man{sub 9}GlcNAc{sub 2}-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

Competition between folding and glycosylation in the endoplasmic reticulum

DEFF Research Database (Denmark)

Holst, B; Bruun, A W; Kielland-Brandt, Morten

1996-01-01

Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new...... acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation...

Comparative Glycoproteome Analysis: Dynamics of Protein Glycosylation during Metamorphic Transition from Pelagic to Benthic Life Stages in Three Invertebrates

KAUST Repository

Chandramouli, Kondethimmanahalli

2012-02-03

The life cycle of most benthic marine invertebrates has two distinct stages: the pelagic larval stage and the sessile juvenile stage. The transition between the larval stage and the juvenile stage is often abrupt and may be triggered by post-translational modification of proteins. Glycosylation, a very important post-translational modification, influences the biological activity of proteins. We used two-dimensional gel electrophoresis (2-DE) followed by glycoprotein-specific fluorescence staining and mass spectrometry with the goal of identifying glycosylation pattern changes during larval settlement and metamorphosis in barnacles, bryozoans, and polychaetes. Our results revealed substantial changes in the protein glycosylation patterns from larval to juvenile stages. Before metamorphosis, the degree of protein glycosylation was high in the barnacle Balanus (=Amphibalanus) amphitrite and the spionid polychaete Pseudopolydora vexillosa, whereas it increased after metamorphosis in the bryozoan Bugula neritina. We identified 19 abundant and differentially glycosylated proteins in these three species. Among the proteins, cellular stress- and metabolism-related proteins exhibited distinct glycosylation in B. amphitrite and B. neritina, whereas fatty acid metabolism-related proteins were abundantly glycosylated in P. vexillosa. Furthermore, the protein and gene expression analysis of some selected glycoproteins revealed that the degree of protein glycosylation did not always complement with transcriptional and translational changes associated with the larval-juvenile transition. The current study provides preliminary information on protein glycosylation in marine invertebrates that will serve as a solid basis for future comprehensive analysis of glycobiology during larval settlement and metamorphosis. © 2011 American Chemical Society.

Isolation and characterization of 9-lipoxygenase and epoxide hydrolase 2 genes: Insight into lactone biosynthesis in mango fruit (Mangifera indica L.).

Science.gov (United States)

Deshpande, Ashish B; Chidley, Hemangi G; Oak, Pranjali S; Pujari, Keshav H; Giri, Ashok P; Gupta, Vidya S

2017-06-01

Uniqueness and diversity of mango flavour across various cultivars are well known. Among various flavour metabolites lactones form an important class of aroma volatiles in certain mango varieties due to their ripening specific appearance and lower odour detection threshold. In spite of their biological and biochemical importance, lactone biosynthetic pathway in plants remains elusive. Present study encompasses quantitative real-time analysis of 9-lipoxygenase (Mi9LOX), epoxide hydrolase 2 (MiEH2), peroxygenase, hydroperoxide lyase and acyl-CoA-oxidase genes during various developmental and ripening stages in fruit of Alphonso, Pairi and Kent cultivars with high, low and no lactone content and explains their variable lactone content. Study also covers isolation, recombinant protein characterization and transient over-expression of Mi9LOX and MiEH2 genes in mango fruits. Recombinant Mi9LOX utilized linoleic and linolenic acids, while MiEH2 utilized aromatic and fatty acid epoxides as their respective substrates depicting their role in fatty acid metabolism. Significant increase in concentration of δ-valerolactone and δ-decalactone upon Mi9LOX over-expression and that of δ-valerolactone, γ-hexalactone and δ-hexalactone upon MiEH2 over-expression further suggested probable involvement of these genes in lactone biosynthesis in mango. Copyright © 2017 Elsevier Ltd. All rights reserved.

Structure of a Trypanosoma brucei α/β-hydrolase fold protein with unknown function

International Nuclear Information System (INIS)

Merritt, Ethan A.; Holmes, Margaret; Buckner, Frederick S.; Van Voorhis, Wesley C.; Quartly, Erin; Phizicky, Eric M.; Lauricella, Angela; Luft, Joseph; DeTitta, George; Neely, Helen; Zucker, Frank; Hol, Wim G. J.

2008-01-01

T. brucei gene Tb10.6k15.0140 codes for an α/β-hydrolase fold protein of unknown function. The 2.2 Å crystal structure shows that members of this sequence family retain a conserved Ser residue at the expected site of a catalytic nucleophile, but that trypanosomatid sequences lack structural homologs for the other expected residues of the catalytic triad. The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 Å using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the α/β-hydrolase fold family. Structural superposition onto representative α/β-hydrolase fold proteins of known function indicates that a possible catalytic nucleophile, Ser116 in the T. brucei protein, lies at the expected location. However, the present structure and by extension the other trypanosomatid members of this sequence family have neither sequence nor structural similarity at the location of other active-site residues typical for proteins with this fold. Together with the presence of an additional domain between strands β6 and β7 that is conserved in trypanosomatid genomes, this suggests that the function of these homologs has diverged from other members of the fold family

Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature

DEFF Research Database (Denmark)

Busk, Peter Kamp; Lange, Mette; Pilgaard, Bo

2014-01-01

The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence....... In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important...

Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity

DEFF Research Database (Denmark)

Norén, K; Hansen, Gert Helge; Clausen, H

1997-01-01

In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks...... the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms...

Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

Science.gov (United States)

Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

2016-05-20

C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

IS30-related transposon mediated insertional inactivation of bile salt hydrolase (bsh1) gene of Lactobacillus plantarum strain Lp20.

Science.gov (United States)

Kumar, Rajesh; Grover, Sunita; Kaushik, Jai K; Batish, Virender Kumar

2014-01-01

Lactobacillus plantarum is a flexible and versatile microorganism that inhabits a variety of niches, and its genome may express up to four bsh genes to maximize its survival in the mammalian gut. However, the ecological significance of multiple bsh genes in L. plantarum is still not clearly understood. Hence, this study demonstrated the disruption of bile salt hydrolase (bsh1) gene due to the insertion of a transposable element in L. plantarum Lp20 - a wild strain of human fecal origin. Surprisingly, L. plantarum strain Lp20 produced a ∼2.0 kb bsh1 amplicon against the normal size (∼1.0 kb) bsh1 amplicon of Bsh(+)L. plantarum Lp21. Strain Lp20 exhibited minimal Bsh activity in spite of having intact bsh2, bsh3 and bsh4 genes in its genome and hence had a Bsh(-) phenotype. Cloning and sequence characterization of Lp20 bsh1 gene predicted four individual open reading frames (ORFs) within this region. BLAST analysis of ORF1 and ORF2 revealed significant sequence similarity to the L. plantarum bsh1 gene while ORF3 and ORF4 showed high sequence homology to IS30-family transposases. Since, IS30-related transposon element was inserted within Lp20 bsh1 gene in reverse orientation (3'-5'), it introduced several stop codons and disrupted the protein reading frames of both Bsh1 and transposase. Inverted terminal repeats (GGCAGATTG) of transposon, mediated its insertion at 255-263 nt and 1301-1309 nt positions of Lp20 bsh1 gene. In conclusion, insertion of IS30 related-transposon within the bsh1 gene sequence of L. plantarum strain Lp20 demolished the integrity and functionality of Bsh1 enzyme. Additionally, this transposon DNA sequence remains active among various Lactobacillus spp. and hence harbors the potential to be explored in the development of efficient insertion mutagenesis system. Copyright © 2013 Elsevier GmbH. All rights reserved.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

DEFF Research Database (Denmark)

Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki

2018-01-01

in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation is not required for transport and cell surface expression and stability...... of these receptors, but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by approximately 5-fold. The molecular basis for this observation is currently unknown, but these findings open up new...

Cloning, characterization, and expression of xyloglucan endotransglucosylase/hydrolase and expansin genes associated with petal growth and development during carnation flower opening

Science.gov (United States)

Harada, Taro; Torii, Yuka; Morita, Shigeto; Onodera, Reiko; Hara, Yoshinao; Yokoyama, Ryusuke; Nishitani, Kazuhiko; Satoh, Shigeru

2011-01-01

Growth of petal cells is a basis for expansion and morphogenesis (outward bending) of petals during opening of carnation flowers (Dianthus caryophyllus L.). Petal growth progressed through elongation in the early stage, expansion with outward bending in the middle stage, and expansion of the whole area in the late stage of flower opening. In the present study, four cDNAs encoding xyloglucan endotransglucosylase/hydrolase (XTH) (DcXTH1–DcXTH4) and three cDNAs encoding expansin (DcEXPA1–DcEXPA3) were cloned from petals of opening carnation flowers and characterized. Real-time reverse transcription-PCR analyses showed that transcript levels of XTH and expansin genes accumulated differently in floral and vegetative tissues of carnation plants with opening flowers, indicating regulated expression of these genes. DcXTH2 and DcXTH3 transcripts were detected in large quantities in petals as compared with other tissues. DcEXPA1 and DcEXPA2 transcripts were markedly accumulated in petals of opening flowers. The action of XTH in growing petal tissues was confirmed by in situ staining of xyloglucan endotransglucosylase (XET) activity using a rhodamine-labelled xyloglucan nonasaccharide as a substrate. Based on the present findings, it is suggested that two XTH genes (DcXTH2 and DcXTH3) and two expansin genes (DcEXPA1 and DcEXPA2) are associated with petal growth and development during carnation flower opening. PMID:20959626

Nutritional Therapies in Congenital Disorders of Glycosylation (CDG

Directory of Open Access Journals (Sweden)

Peter Witters

2017-11-01

Full Text Available Congenital disorders of glycosylation (CDG are a group of more than 130 inborn errors of metabolism affecting N-linked, O-linked protein and lipid-linked glycosylation. The phenotype in CDG patients includes frequent liver involvement, especially the disorders belonging to the N-linked protein glycosylation group. There are only a few treatable CDG. Mannose-Phosphate Isomerase (MPI-CDG was the first treatable CDG by high dose mannose supplements. Recently, with the successful use of d-galactose in Phosphoglucomutase 1 (PGM1-CDG, other CDG types have been trialed on galactose and with an increasing number of potential nutritional therapies. Current mini review focuses on therapies in glycosylation disorders affecting liver function and dietary intervention in general in N-linked glycosylation disorders. We also emphasize now the importance of early screening for CDG in patients with mild hepatopathy but also in cholestasis.

Three-way stabilization of the covalent intermediate in amylomaltase, an alpha-like transglycosylase

NARCIS (Netherlands)

Barends, T.R.M.; Bultema, J.B.; Kaper, T.; Maarel, M.J.E.C. van der; Dijkhuizen, L.; Dijkstra, B.W.

2007-01-01

Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip book movie of the amylomaltase catalytic cycle in atomic

Gut metagenomes of type 2 diabetic patients have characteristic single-nucleotide polymorphism distribution in Bacteroides coprocola.

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Chen, Yaowen; Li, Zongcheng; Hu, Shuofeng; Zhang, Jian; Wu, Jiaqi; Shao, Ningsheng; Bo, Xiaochen; Ni, Ming; Ying, Xiaomin

2017-02-01

Gut microbes play a critical role in human health and disease, and researchers have begun to characterize their genomes, the so-called gut metagenome. Thus far, metagenomics studies have focused on genus- or species-level composition and microbial gene sets, while strain-level composition and single-nucleotide polymorphism (SNP) have been overlooked. The gut metagenomes of type 2 diabetes (T2D) patients have been found to be enriched with butyrate-producing bacteria and sulfate reduction functions. However, it is not known whether the gut metagenomes of T2D patients have characteristic strain patterns or SNP distributions. We downloaded public gut metagenome datasets from 170 T2D patients and 174 healthy controls and performed a systematic comparative analysis of their metagenome SNPs. We found that Bacteroides coprocola, whose relative abundance did not differ between the groups, had a characteristic distribution of SNPs in the T2D patient group. We identified 65 genes, all in B. coprocola, that had remarkably different enrichment of SNPs. The first and sixth ranked genes encode glycosyl hydrolases (GenBank accession EDU99824.1 and EDV02301.1). Interestingly, alpha-glucosidase, which is also a glycosyl hydrolase located in the intestine, is an important drug target of T2D. These results suggest that different strains of B. coprocola may have different roles in human gut and a specific set of B. coprocola strains are correlated with T2D.

Differential Involvement of β-Glucosidases from Hypocrea jecorina in Rapid Induction of Cellulase Genes by Cellulose and Cellobiose

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Zhou, Qingxin; Xu, Jintao; Kou, Yanbo; Lv, Xinxing; Zhang, Xi; Zhao, Guolei; Zhang, Weixin; Chen, Guanjun

2012-01-01

Appropriate perception of cellulose outside the cell by transforming it into an intracellular signal ensures the rapid production of cellulases by cellulolytic Hypocrea jecorina. The major extracellular β-glucosidase BglI (CEL3a) has been shown to contribute to the efficient induction of cellulase genes. Multiple β-glucosidases belonging to glycosyl hydrolase (GH) family 3 and 1, however, exist in H. jecorina. Here we demonstrated that CEL1b, like CEL1a, was an intracellular β-glucosidase displaying in vitro transglycosylation activity. We then found evidence that these two major intracellular β-glucosidases were involved in the rapid induction of cellulase genes by insoluble cellulose. Deletion of cel1a and cel1b significantly compromised the efficient gene expression of the major cellulase gene, cbh1. Simultaneous absence of BglI, CEL1a, and CEL1b caused the induction of the cellulase gene by cellulose to further deteriorate. The induction defect, however, was not observed with cellobiose. The absence of the three β-glucosidases, rather, facilitated the induced synthesis of cellulase on cellobiose. Furthermore, addition of cellobiose restored the productive induction on cellulose in the deletion strains. The results indicate that the three β-glucosidases may not participate in transforming cellobiose beyond hydrolysis to provoke cellulase formation in H. jecorina. They may otherwise contribute to the accumulation of cellobiose from cellulose as inducing signals. PMID:23002106

Halide-mediated regioselective 6-O-glycosylation of unprotected hexopyranosides with perbenzylated glycosyl bromide donors

DEFF Research Database (Denmark)

Niedbal, Dominika Alina; Madsen, Robert

2016-01-01

The regio- and stereoselective glycosylation at the 6-position in 2,3,4,6-unprotected hexopyranosides has been investigated with dibutyltin oxide as the directing agent. Perbenzylated hexopyranosyl bromides were employed as the donors and the glycosylations were promoted by tetrabutylammonium...... bromide. The couplings were completely selective for both glucose and galactose donors and acceptors as long as the stannylene acetal of the acceptor was soluble in dichloromethane. This gave rise to a number of 1,2-cis-linked disaccharides in reasonable yields. Mannose donors and acceptors, on the other...

Glycosylation of the self-recognizing Escherichia coli Ag43 autotransporter protein

DEFF Research Database (Denmark)

Sherlock, O.; Dobrindt, U.; Jensen, J.B.

2006-01-01

a novel member to this exclusive group, namely, antigen 43 (Ag43), a self-recognizing autotransporter protein. By mass spectrometry Ag43 was demonstrated to be glycosylated by addition of heptose residues at several positions in the passenger domain. Glycosylation of Ag43 by the action of the Aah and Tib......C glycosyltransferases was observed in laboratory strains. Importantly, Ag43 was also found to be glycosylated in a wild-type strain, suggesting that Ag43-glycosylation may be a widespread phenomenon. Glycosylation of Ag43 does not seem to interfere with its self-associating properties. However, the glycosylated form...

Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

Science.gov (United States)

de Jongh, Harmen H. J.; Robles, Carlos López; Nordlee, Julie A.; Lee, Poi-Wah; Baumert, Joseph L.; Hamilton, Robert G.; Taylor, Steve L.; Koppelman, Stef J.

2013-01-01

Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein's digestibility is not affected by such processing. PMID:23878817

Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

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Harmen H. J. de Jongh

2013-01-01

Full Text Available Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa, the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein’s digestibility is not affected by such processing.

Functional Analysis of Glycosylation of Zika Virus Envelope Protein

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Camila R. Fontes-Garfias

2017-10-01

Full Text Available Summary: Zika virus (ZIKV infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. : Zika virus (ZIKV causes devastating congenital abnormities and Guillain-Barré syndrome. Fontes-Garfias et al. showed that the glycosylation of ZIKV envelope protein plays an important role in infecting mosquito vectors and pathogenesis in mouse. Keywords: Zika virus, glycosylation, flavivirus entry, mosquito transmission, vaccine

Toward stable genetic engineering of human o-glycosylation in plants

DEFF Research Database (Denmark)

Yang, Zhang; Bennett, Eric Paul; Jørgensen, Bodil

2012-01-01

Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types...... an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating Gal......NAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O...

Meta-analysis of microsomal epoxide hydrolase gene polymorphism and risk of hepatocellular carcinoma.

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Jian-Hong Zhong

Full Text Available BACKGROUND: Hepatocarcinogenesis is a complex process that may be influenced by many factors, including polymorphism in microsomal epoxide hydrolase (mEH. Previous work suggests an association between the Tyr113His and His139Arg mEH polymorphisms and susceptibility to hepatocellular carcinoma (HCC, but the results have been inconsistent. METHODS: PubMed, EMBASE, Google Scholar and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between Tyr113His and His139Arg mEH polymorphism and susceptibility to HCC. Odds ratios (ORs and 95% confidence intervals (95% CIs were calculated. RESULTS: Eleven studies were included in the meta-analysis, involving 1,696 HCC cases and 3,600 controls. The 113His- mEH allele was significantly associated with increased risk of HCC based on allelic contrast (OR = 1.35, 95% CI = 1.04-1.75, p = 0.02, homozygote comparison (OR = 1.65, 95% CI = 1.07-2.54, p = 0.02 and a recessive genetic model (OR = 1.54, 95% CI = 1.21-1.96, p<0.001, while individuals carrying the Arg139Arg mEH genotype had no association with increased or decreased risk of HCC. CONCLUSION: The 113His- allele polymorphism in mEH may be a risk factor for hepatocarcinogenesis, while the mEH 139Arg- allele may not be a risk or protective factor. There is substantial evidence that mEH polymorphisms interact synergistically with other genes and the environment to modulate risk of HCC. Further large and well-designed studies are needed to confirm these conclusions.

Trans-species Engineering of Glycosylated Therapeutic Proteins

DEFF Research Database (Denmark)

Yang, Zhang

eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N...

25-Hydroxycholesterol Inhibition of Lassa Virus Infection through Aberrant GP1 Glycosylation

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Punya Shrivastava-Ranjan

2016-12-01

Full Text Available Lassa virus (LASV infection is a major public health concern due to high fatality rates and limited effective treatment. The interferon-stimulated gene cholesterol 25-hydroxylase (CH25H encodes an enzyme that catalyzes the production of 25-hydroxycholesterol (25HC. 25HC is involved in regulating cholesterol biosynthesis and has recently been identified as a potent antiviral targeting enveloped virus entry. Here, we show a previously unrecognized role of CH25H in inhibiting LASV glycoprotein glycosylation and the production of infectious virus. Overexpression of CH25H or treatment with 25HC decreased LASV G1 glycoprotein N-glycan maturation and reduced the production of infectious LASV. Depletion of endogenous CH25H using small interfering RNA (siRNA enhanced the levels of fully glycosylated G1 and increased infectious LASV production. Finally, LASV particles produced from 25HC-treated cells were found to be less infectious, to incorporate aberrantly glycosylated GP1 species, and to be defective in binding alpha-dystroglycan, an attachment and entry receptor. Our findings identify a novel role for CH25H in controlling LASV propagation and indicate that manipulation of the expression of CH25H or the administration of 25HC may be a useful anti-LASV therapy.

Similarities and Differences in the Glycosylation Mechanisms in Prokaryotes and Eukaryotes

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Anne Dell

2010-01-01

Full Text Available Recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i oligosaccharyltransferase (OST-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain; (iii OST-mediated O-glycosylation which appears to be characteristic of bacteria; and (iv stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER/periplasmic/cytoplasmic membranes, and transferring “en bloc” to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.

Glycosylation of the N-terminal potential N-glycosylation sites in the human α1,3-fucosyltransferase V and -VI (hFucTV and -VI)

DEFF Research Database (Denmark)

Christensen, Lise Lotte; Bross, Peter Gerd; Ørntoft, Torben Falck

2000-01-01

Human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and h......FucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins...... in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced...

Diversity and functions of protein glycosylation in insects.

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Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

2017-04-01

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Sequence analysis and gene expression of putative exo- and endo-glucanases from oil palm (Elaeis guineensis) during fungal infection.

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Yeoh, Keat-Ai; Othman, Abrizah; Meon, Sariah; Abdullah, Faridah; Ho, Chai-Ling

2012-10-15

Glucanases are enzymes that hydrolyze a variety β-d-glucosidic linkages. Plant β-1,3-glucanases are able to degrade fungal cell walls; and promote the release of cell-wall derived fungal elicitors. In this study, three full-length cDNA sequences encoding oil palm (Elaeis guineensis) glucanases were analyzed. Sequence analyses of the cDNA sequences suggested that EgGlc1-1 is a putative β-d-glucan exohydolase belonging to glycosyl hydrolase (GH) family 3 while EgGlc5-1 and EgGlc5-2 are putative glucan endo-1,3-β-glucosidases belonging to GH family 17. The transcript abundance of these genes in the roots and leaves of oil palm seedlings treated with Ganoderma boninense and Trichoderma harzianum was profiled to investigate the involvement of these glucanases in oil palm during fungal infection. The gene expression of EgGlc1-1 in the root of oil palm seedlings was increased by T. harzianum but suppressed by G. boninense; while the gene expression of both EgGlc5-1 and EgGlc5-2 in the roots of oil palm seedlings was suppressed by G. boninense or/and T. harzianum. Copyright © 2012 Elsevier GmbH. All rights reserved.

Glycosylation in HIV-1 envelope glycoprotein and its biological implications

KAUST Repository

Ho, Yung Shwen

2013-08-01

Glycosylation of HIV-1 envelope proteins (Env gp120/gp41) plays a vital role in viral evasion from the host immune response, which occurs through the masking of key neutralization epitopes and the presentation of the Env glycosylation as \\'self\\' to the host immune system. Env glycosylation is generally conserved, yet its continual evolution plays an important role in modulating viral infectivity and Env immunogenicity. Thus, it is believed that Env glycosylation, which is a vital part of the HIV-1 architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may assist in effectively designing Env-based immunogens and in clearly understanding HIV vaccines. This review provides an in-depth perspective of various aspects of Env glycosylation in the context of HIV-1 pathogenesis. © 2013 Future Medicine Ltd.

The role of the active site Zn in the catalytic mechanism of the GH38 Golgi alpha-mannosidase II: Implications from noeuromycin inhibition

DEFF Research Database (Denmark)

Bols, Mikael; Kuntz, Douglas A.; Rose, David R.

2006-01-01

Golgi alpha-mannosidase II (GMII) is a Family 38 glycosyl hydrolase involved in the eukaryotic N-glycosylation pathway in protein synthesis. Understanding of its catalytic mechanism has been of interest for the development of specific inhibitors that could lead to novel anti-metastatic or anti-in...

A xylanase gene directly cloned from the genomic DNA of alkaline wastewater sludge showing application potential in the paper industry.

Science.gov (United States)

Zhao, Yanyu; Luo, Huiying; Meng, Kun; Shi, Pengjun; Wang, Guozeng; Yang, Peilong; Yuan, Tiezheng; Yao, Bin

2011-09-01

A xylanase gene, aws-2x, was directly cloned from the genomic DNA of the alkaline wastewater sludge using degenerated PCR and modified TAIL-PCR. The deduced amino acid sequence of AWS-2x shared the highest identity (60%) with the xylanase from Chryseobacterium gleum belonging to the glycosyl hydrolase GH family 10. Recombinant AWS-2x was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 7.5 and 55 °C, maintained more than 50% of maximal activity when assayed at pH 9.0, and was stable over a wide pH range from 4.0 to 11.0. The specific activity of AWS-2x towards hardwood xylan (beechwood and birchwood xylan) was significantly higher than that to cereal xylan (oat spelt xylan and wheat arabinoxylan). These properties make AWS-2x a potential candidate for application in the pulp and paper industry.

[Non-enzymatic glycosylation of dietary protein in vitro].

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Bednykh, B S; Evdokimov, I A; Sokolov, A I

2015-01-01

Non-enzymatic glycosylation of proteins, based on discovered by Mayarn reaction of carbohydrate aldehyde group with a free amino group of a protein molecule, is well known to experts in biochemistry of food industry. Generated brown solid in some cases give the product marketable qualities--crackling bread--in others conversely, worsen the product. The biological effects of far-advanced products of non-enzymatic protein glycosylation reaction have not been studied enough, although it was reported previously that they are not split by digestive enzymes and couldn't be absorbed by animals. The objective of this work was to compare the depth of glycosylation of different food proteins of animal and vegetable origin. The objects of the study were proteins of animal (casein, lactoglobulin, albumin) and vegetable (soy isolate, proteins of rice flour, buckwheat, oatmeal) origin, glucose and fructose were selected as glycosylation agents, exposure 15 days at 37 degrees C. Lactoglobulin was glycosylated to a lesser extent among the proteins of animal origin while protein of oatmeal was glycosylated in the least degree among vegetable proteins. Conversely, such proteins as casein and soya isolate protein bound rather large amounts of carbohydrates. Fructose binding with protein was generally higher than the binding of glucose. The only exception was a protein of oatmeal. When of glucose and fructose simultaneously presented in the incubation medium, glucose binding usually increased while binding of fructose, in contrast, reduced. According to the total amount of carbohydrate (mcg), which is able to attach a protein (mg) the studied food proteins located in the following order: albumin (38) > soy protein isolate (23) > casein (15,) > whey protein rice flour protein (6) > protein from buckwheat flour (3) > globulin (2) > protein of oatmeal (0.3). The results obtained are to be used to select the optimal combination of proteins and carbohydrates, in which the glycosylation

Functional Analysis of Glycosylation of Zika Virus Envelope Protein.

Science.gov (United States)

Fontes-Garfias, Camila R; Shan, Chao; Luo, Huanle; Muruato, Antonio E; Medeiros, Daniele B A; Mays, Elizabeth; Xie, Xuping; Zou, Jing; Roundy, Christopher M; Wakamiya, Maki; Rossi, Shannan L; Wang, Tian; Weaver, Scott C; Shi, Pei-Yong

2017-10-31

Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Autolysis of dairy leuconostocs and detection of peptidoglycan hydrolases by renaturing SDS-PAGE.

Science.gov (United States)

Cibik, R; Chapot-Chartier, M P

2000-11-01

The autolysis of lactic acid bacteria plays a major role during cheese ripening. The aim of this study was to evaluate the autolytic properties and peptidoglycan hydrolase content of dairy leuconostocs. Autolysis of 59 strains of dairy Leuconostoc was examined under starvation conditions in potassium phosphate buffer. The ability of dairy leuconostocs to lyse is strain dependant and not related to the species. The peptidoglycan hydrolase profile of Leuc. mesenteroides subsp. mesenteroides 10L was analysed by renaturing gel electrophoresis. Two major activity bands migrating at 41 and 52 kDa were observed. According to the specificity analysis, strain 10L seems to contain a glycosidase and an N-acetyl-muramyl-L-alanine amidase, or an endopeptidase. The peptidoglycan hydrolase profiles of various Leuconostoc species were also compared. Several peptidoglycan hydrolase activities could be detected in the different Leuconostoc species. Further characterization of the peptidoglycan hydrolases will help to control autolysis of leuconostocs in cheese.

Importance of N-Glycosylation on CD147 for Its Biological Functions

Science.gov (United States)

Bai, Yang; Huang, Wan; Ma, Li-Tian; Jiang, Jian-Li; Chen, Zhi-Nan

2014-01-01

Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation. PMID:24739808

Importance of N-Glycosylation on CD147 for Its Biological Functions

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Yang Bai

2014-04-01

Full Text Available Glycosylation of glycoproteins is one of many molecular changes that accompany malignant transformation. Post-translational modifications of proteins are closely associated with the adhesion, invasion, and metastasis of tumor cells. CD147, a tumor-associated antigen that is highly expressed on the cell surface of various tumors, is a potential target for cancer diagnosis and therapy. A significant biochemical property of CD147 is its high level of glycosylation. Studies on the structure and function of CD147 glycosylation provide valuable clues to the development of targeted therapies for cancer. Here, we review current understanding of the glycosylation characteristics of CD147 and the glycosyltransferases involved in the biosynthesis of CD147 N-glycans. Finally, we discuss proteins regulating CD147 glycosylation and the biological functions of CD147 glycosylation.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions.

Science.gov (United States)

Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki; Ye, Zilu; Tian, Weihua; Goth, Christoffer K; Lira-Navarrete, Erandi; Pedersen, Nis B; Benito-Vicente, Asier; Martin, Cesar; Uribe, Kepa B; Hurtado-Guerrero, Ramon; Christoffersen, Christina; Seidah, Nabil G; Nielsen, Rikke; Christensen, Erik I; Hansen, Lars; Bennett, Eric P; Vakhrushev, Sergey Y; Schjoldager, Katrine T; Clausen, Henrik

2018-05-11

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O -glycan sites. Moreover, we found that O -glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O -glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O -glycosylation of LDLR-related proteins and identified conserved O -glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O -glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O -glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Links between CD147 Function, Glycosylation, and Caveolin-1

OpenAIRE

Tang, Wei; Chang, Sharon B.; Hemler, Martin E.

2004-01-01

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-cataly...

Cloning and Genomic Organization of a Rhamnogalacturonase Gene from Locally Isolated Strain of Aspergillus niger.

Science.gov (United States)

Damak, Naourez; Abdeljalil, Salma; Taeib, Noomen Hadj; Gargouri, Ali

2015-08-01

The rhg gene encoding a rhamnogalacturonase was isolated from the novel strain A1 of Aspergillus niger. It consists of an ORF of 1.505 kb encoding a putative protein of 446 amino acids with a predicted molecular mass of 47 kDa, belonging to the family 28 of glycosyl hydrolases. The nature and position of amino acids comprising the active site as well as the three-dimensional structure were well conserved between the A. niger CTM10548 and fungal rhamnogalacturonases. The coding region of the rhg gene is interrupted by three short introns of 56 (introns 1 and 3) and 52 (intron 2) bp in length. The comparison of the peptide sequence with A. niger rhg sequences revealed that the A1 rhg should be an endo-rhamnogalacturonases, more homologous to rhg A than rhg B A. niger known enzymes. The comparison of rhg nucleotide sequence from A. niger A1 with rhg A from A. niger shows several base changes. Most of these changes (59 %) are located at the third base of codons suggesting maintaining the same enzyme function. We used the rhamnogalacturonase A from Aspergillus aculeatus as a template to build a structural model of rhg A1 that adopted a right-handed parallel β-helix.

[The role of protein glycosylation in immune system].

Science.gov (United States)

Ząbczyńska, Marta; Pocheć, Ewa

2015-01-01

Glycosylation is one of the most frequent post-translational modifications of proteins. The majority of cell surface and secreted proteins involved in immune response is glycosylated. The structural diversity of glycans depends on monosaccharide composition, type of glycosidic linkage and branching. These structural modifications determine a great variability of glycoproteins. The oligosaccharide components of proteins are regulated mostly by expression of glycosyltransferases and glycosidases and many environmental factors. Glycosylation influences the function of all immune cells. Glycans play a crucial role in intercellular contacts and leukocytes migration. These interactions are important in activation and proliferation of leukocytes and during immune response. The key immune proteins, such as TCR, MHC, TLR and antibodies are glycosylated. Sugars on the surface of pathogens and self-surface glycoproteins are recognized by special carbohydrate binding proteins called lectins. Changes of glycan structure are common in many pathological processes occurring in immune system, therefore they are used as molecular markers of different diseases.

Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts.

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Eklund, Erik A; Merbouh, Nabyl; Ichikawa, Mie; Nishikawa, Atsushi; Clima, Jessica M; Dorman, James A; Norberg, Thomas; Freeze, Hudson H

2005-11-01

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.

O-linked glycosylation of retroviral envelope gene products

Energy Technology Data Exchange (ETDEWEB)

Pinter, A.; Honnen, W.J. (Public Health Research Institute of the City of New York Inc., NY (USA))

1988-03-01

Treatment of ({sup 3}H)glucosamine-labeled Friend mink cell focus-forming virus (FrMCF) gp70 with excess peptide:N-glycanase F (PNGase F) resulted in removal of the expected seven N-linked oligosaccharide chains; however, approximately 10% of the glucosamine label was retained in the resulting 49,000-M{sub r} (49K) product. For ({sup 3}H)mannose-labeled gp70, similar treatment led to removal of all the carbohydrate label from the protein. Prior digestion of the PNGase F-treated gp70 with neuraminidase resulted in an addition size shift, and treatment with O-glycanase led to the removal of almost all of the PNGase F-resistant sugars. These results indicate that gp70 possesses sialic acid-containing O-linked oligosaccharides. Analysis of intracellular env precursors demonstrated that O-linked sugars were present in gPr90{sup env}, the polyprotein intermediate which contains complex sugars, but not in the primary translation product, gPr80{sup env}, and proteolytic digestion studies allowed localization of the O-linked carbohydrates to a 10K region near the center of the gp70 molecule. similar substituents were detected on the gp70s of ecotropic and xenotropic murine leukemia viruses and two subgroups of feline leukemia virus, indicting that O-linked glycosylation is a conserved feature of retroviral env proteins.

O-linked glycosylation of retroviral envelope gene products

International Nuclear Information System (INIS)

Pinter, A.; Honnen, W.J.

1988-01-01

Treatment of [ 3 H]glucosamine-labeled Friend mink cell focus-forming virus (FrMCF) gp70 with excess peptide:N-glycanase F (PNGase F) resulted in removal of the expected seven N-linked oligosaccharide chains; however, approximately 10% of the glucosamine label was retained in the resulting 49,000-M r (49K) product. For [ 3 H]mannose-labeled gp70, similar treatment led to removal of all the carbohydrate label from the protein. Prior digestion of the PNGase F-treated gp70 with neuraminidase resulted in an addition size shift, and treatment with O-glycanase led to the removal of almost all of the PNGase F-resistant sugars. These results indicate that gp70 possesses sialic acid-containing O-linked oligosaccharides. Analysis of intracellular env precursors demonstrated that O-linked sugars were present in gPr90 env , the polyprotein intermediate which contains complex sugars, but not in the primary translation product, gPr80 env , and proteolytic digestion studies allowed localization of the O-linked carbohydrates to a 10K region near the center of the gp70 molecule. similar substituents were detected on the gp70s of ecotropic and xenotropic murine leukemia viruses and two subgroups of feline leukemia virus, indicting that O-linked glycosylation is a conserved feature of retroviral env proteins

Prion propagation in cells expressing PrP glycosylation mutants.

Science.gov (United States)

Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-04-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Les lipases sont des hydrolases atypiques : principales caractéristiques et applications

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Fickers P.

2008-01-01

Full Text Available ipases are atypical hydrolases: principal characteristics and applications. Due to their kinetic and substrate specificities, triacylglycerol acyl-hydrolases or lipases are atypical enzymes. In function of their microenvironment, lipases are able to act as hydrolases in aqueous solution or as biocatalysts in organic synthesis. As hydrolases, they are responsible of the triglycerids catabolism into fatty acids and glycerol. In many organisms, this reaction plays a major role in the fat and lipid metabolism. In addition, lipases are also able to hydrolyse phospholipids and cholesterol esters. In organic solvent, lipases could catalyse reactions such as esterifications, acidolysis or alcoolysis with enantio-, regio- and chimioselectivity. Lipases form a mixed class of enzyme due to their animal, vegetal or microbial origins. All those properties led to the development of many applications in the food and chemical industries but also in the medical and therapeutic field.

Unusual glycosylation of proteins: Beyond the universal sequon and other amino acids.

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Dutta, Devawati; Mandal, Chhabinath; Mandal, Chitra

2017-12-01

Glycosylation of proteins is the most common, multifaceted co- and post-translational modification responsible for many biological processes and cellular functions. Significant alterations and aberrations of these processes are related to various pathological conditions, and often turn out to be disease biomarkers. Conventional N-glycosylation occurs through the recognition of the consensus sequon, asparagine (Asn)-X-serine (Ser)/threonine (Thr), where X is any amino acid except for proline, with N-acetylglucosamine (GlcNAc) as the first glycosidic linkage. Usually, O-glycosylation adds a glycan to the hydroxyl group of Ser or Thr beginning with N-acetylgalactosamine (GalNAc). Protein glycosylation is further governed by additional diversifications in sequon and structure, which are yet to be fully explored. This review mainly focuses on the occurrence of N-glycosylation in non-consensus motifs, where Ser/Thr at the +2 position is substituted by other amino acids. Additionally, N-glycosylation is also observed in other amide/amine group-containing amino acids. Similarly, O-glycosylation occurs at hydroxyl group-containing amino acids other than serine/threonine. The neighbouring amino acids and local structural features around the potential glycosylation site also play a significant role in determining the extent of glycosylation. All of these phenomena that yield glycosylation at the atypical sites are reported in a variety of biological systems, including different pathological conditions. Therefore, the discovery of more novel sequence patterns for N- and O-glycosylation may help in understanding the functions of complex biological processes and cellular functions. Taken together, all these information provided in this review would be helpful for the biological readers. Copyright © 2017 Elsevier B.V. All rights reserved.

Glycosylation analysis and protein structure determination of murine fetal antigen 1 (mFA1)--the circulating gene product of the delta-like protein (dlk), preadipocyte factor 1 (Pref-1) and stromal-cell-derived protein 1 (SCP-1) cDNAs

DEFF Research Database (Denmark)

Krogh, T N; Bachmann, E; Teisner, B

1997-01-01

By means of sequence analysis, murine fetal antigen 1 (mFA1) isolated from Mus musculus amniotic fluid was shown to be the circulating protein of the delta-like protein, stromal-cell-derived protein 1 (SCP-1) and preadipocyte factor 1 (Pref-1) gene products. The protein contains 36 cysteine...... residues arranged in six epidermal-growth-factor-like domains. The purification of several C-terminal peptides of varying lengths showed mFA1 to be C-terminal heterogeneous. O-linked glycosylations of the NeuNAc alpha2-3Gal beta1-3(NeuNAc alpha2-6)GalNAc type were present on all C-terminal peptides...... at residues Thr235, Thr244 and Thr248, although glycosylation on Thr244 was only partial. Three N-linked glycosylations were localized in mFA1 (Asn77, Asn142 and Asn151), two of which (Asn142 and Asn151) were in the unusual Asn-Xaa-Cys motif. Fucosylated biantennary complex-type and small amounts (less than 5...

Microsomal epoxide hydrolase gene polymorphisms and risk of chronic obstructive pulmonary disease: A comprehensive meta-analysis

OpenAIRE

LI, HUI; FU, WEI-PING; HONG, ZE-HUI

2012-01-01

Microsomal epoxide hydrolase (EPHX1) is an enzyme involved in the detoxification the products of smoking and is proposed to be a genetic factor for the development of chronic obstructive pulmonary disease (COPD). Two functional polymorphisms of EPHX1, T113C and A139G, have been analyzed in numerous studies to assess the COPD risk attributed to these variants. However, the conclusions were controversial. We performed a comprehensive meta-analysis to clarify these findings. A total of 24 studie...

Co-silencing of tomato S-adenosylhomocysteine hydrolase genes confers increased immunity against Pseudomonas syringae pv. tomato DC3000 and enhanced tolerance to drought stress

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Li Xiao Hui

2015-09-01

Full Text Available S-adenosylhomocysteine hydrolase (SAHH, catalyzing the reversible hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine, is a key enzyme that maintain the cellular methylation potential in all organisms. We report here the biological functions of tomato SlSAHHs in stress response. The tomato genome contains three SlSAHH genes that encode SlSAHH proteins with high level of sequence identity. qRT-PCR analysis revealed that SlSAHHs responded with distinct expression induction patterns to Pseudomonas syringae pv. tomato (Pst DC3000 and Botrytis cinerea as well as to defense signaling hormones such as salicylic acid, jasmonic acid and a precursor of ethylene. Virus-induced gene silencing-based knockdown of individual SlSAHH gene did not affect the growth performance and the response to Pst DC3000. However, co-silencing of three SlSAHH genes using a conserved sequence led to significant inhibition of vegetable growth. The SlSAHH-co-silenced plants displayed increased resistance to Pst DC3000 but did not alter the resistance to B. cinerea. Co-silencing of SlSAHHs resulted in constitutively activated defense responses including elevated SA level, upregulated expression of defense-related and PAMP-triggered immunity marker genes and increased callose deposition and H2O2 accumulation. Furthermore, the SlSAHH-co-silenced plants also exhibited enhanced drought stress tolerance although they had relatively small roots. These data demonstrate that, in addition to the functions in growth and development, SAHHs also play important roles in regulating biotic and abiotic stress responses in plants.

Microbial biodegradation of biuret: defining biuret hydrolases within the isochorismatase superfamily.

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Robinson, Serina L; Badalamenti, Jonathan P; Dodge, Anthony G; Tassoulas, Lambros J; Wackett, Lawrence P

2018-03-12

Biuret is a minor component of urea fertilizer and an intermediate in s-triazine herbicide biodegradation. The microbial metabolism of biuret has never been comprehensively studied. Here, we enriched and isolated bacteria from a potato field that grew on biuret as a sole nitrogen source. We sequenced the genome of the fastest-growing isolate, Herbaspirillum sp. BH-1 and identified genes encoding putative biuret hydrolases (BHs). We purified and characterized a functional BH enzyme from Herbaspirillum sp. BH-1 and two other bacteria from divergent phyla. The BH enzymes reacted exclusively with biuret in the range of 2-11 µmol min -1 mg -1 protein. We then constructed a global protein superfamily network to map structure-function relationships in the BH subfamily and used this to mine > 7000 genomes. High-confidence BH sequences were detected in Actinobacteria, Alpha- and Beta-proteobacteria, and some fungi, archaea and green algae, but not animals or land plants. Unexpectedly, no cyanuric acid hydrolase homologs were detected in > 90% of genomes with BH homologs, suggesting BHs may have arisen independently of s-triazine ring metabolism. This work links genotype to phenotype by enabling accurate genome-mining to predict microbial utilization of biuret. Importantly, it advances understanding of the microbial capacity for biuret biodegradation in agricultural systems. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

Optimized expression of prolyl aminopeptidase in Pichia pastoris and its characteristics after glycosylation.

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Yang, Hongyu; Zhu, Qiang; Zhou, Nandi; Tian, Yaping

2016-11-01

Prolyl aminopeptidases are specific exopeptidases that catalyze the hydrolysis of the N-terminus proline residue of peptides and proteins. In the present study, the prolyl aminopeptidase gene (pap) from Aspergillus oryzae JN-412 was optimized through the codon usage of Pichia pastoris. Both the native and optimized pap genes were inserted into the expression vector pPIC9 K and were successfully expressed in P. pastoris. Additionally, the activity of the intracellular enzyme expressed by the recombinant optimized pap gene reached 61.26 U mL(-1), an activity that is 2.1-fold higher than that of the native gene. The recombinant enzyme was purified by one-step elution through Ni-affinity chromatography. The optimal temperature and pH of the purified PAP were 60 °C and 7.5, respectively. Additionally, the recombinant PAP was recovered at a yield greater than 65 % at an extremely broad range of pH values from 6 to 10 after treatment at 50 °C for 6 h. The molecular weight of the recombinant PAP decreased from 50 kDa to 48 kDa after treatment with a deglycosylation enzyme, indicating that the recombinant PAP was completely glycosylated. The glycosylated PAP exhibited high thermo-stability. Half of the activity remained after incubation at 50 °C for 50 h, whereas the remaining activity of PAP expressed in E. coli was only 10 % after incubation at 50 °C for 1 h. PAP could be activated by the appropriate salt concentration and exhibited salt tolerance against NaCl at a concentration up to 5 mol L(-1).

Glycosylation Helps Cellulase Enzymes Bind to Plant Cell Walls (Fact Sheet)

Energy Technology Data Exchange (ETDEWEB)

2012-06-01

Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms of life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.

Determination of frequencies of alleles, associated with the pseudodeficiency of lysosomal hydrolases, in population of Ukraine

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N. V. Olkhovych

2016-10-01

Full Text Available The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi­ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W and c.745C>T (R249W in the HEXA gene, c.1726G>A (G576S and c.2065G>A (E689K in the GAA gene, c.937G>T (D313Y in the GLA1 gene and c.898G>A (A300T in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease. The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes was 10.3%. The frequency of c.739C>T (R247W allele, associated with hexo­saminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/c.2065A haplotypes was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y in the GLA1 gene and c.898G>A (A300T in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of

Determination of frequencies of alleles, associated with the pseudodeficiency of lysosomal hydrolases, in population of Ukraine.

Science.gov (United States)

Olkhovych, N V; Gorovenko, N G

2016-01-01

The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi­ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W) and c.745C>T (R249W) in the HEXA gene, c.1726G>A (G576S) and c.2065G>A (E689K) in the GAA gene, c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease). The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes) was 10.3%. The frequency of c.739C>T (R247W) allele, associated with hexo­saminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/c.2065A haplotypes) was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of pseudodeficiency may

N (6-substituted AMPs inhibit mammalian deoxynucleotide N-hydrolase DNPH1.

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Claire Amiable

Full Text Available The gene dnph1 (or rcl encodes a hydrolase that cleaves the 2'-deoxyribonucleoside 5'-monophosphate (dNMP N-glycosidic bond to yield a free nucleobase and 2-deoxyribose 5-phosphate. Recently, the crystal structure of rat DNPH1, a potential target for anti-cancer therapies, suggested that various analogs of AMP may inhibit this enzyme. From this result, we asked whether N (6-substituted AMPs, and among them, cytotoxic cytokinin riboside 5'-monophosphates, may inhibit DNPH1. Here, we characterized the structural and thermodynamic aspects of the interactions of these various analogs with DNPH1. Our results indicate that DNPH1 is inhibited by cytotoxic cytokinins at concentrations that inhibit cell growth.

Surface glycosylation profiles of urine extracellular vesicles.

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Jared Q Gerlach

Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

The impact of nonpolar lipids on the regulation of the steryl ester hydrolases Tgl1p and Yeh1p in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Klein, Isabella; Korber, Martina; Athenstaedt, Karin; Daum, Günther

2017-12-01

In the yeast Saccharomyces cerevisiae degradation of steryl esters is catalyzed by the steryl ester hydrolases Tgl1p, Yeh1p and Yeh2p. The two steryl ester hydrolases Tgl1p and Yeh1p localize to lipid droplets, a cell compartment storing steryl esters and triacylglycerols. In the present study we investigated regulatory aspects of these two hydrolytic enzymes, namely the gene expression level, protein amount, stability and enzyme activity of Tgl1p and Yeh1p in strains lacking both or only one of the two major nonpolar lipids, steryl esters and triacylglycerols. In a strain lacking both nonpolar lipids and consequently lipid droplets, Tgl1p as well as Yeh1p were present at low amount, became highly unstable compared to wild-type cells, and lost their enzymatic activity. Under these conditions both steryl ester hydrolases were retained in the endoplasmic reticulum. The lack of steryl esters alone was not sufficient to cause an altered intracellular localization of Tgl1p and Yeh1p. Surprisingly, the stability of Tgl1p and Yeh1p was markedly reduced in a strain lacking triacylglycerols, but their capacity to mobilize steryl esters remained unaffected. We also tested a possible cross-regulation of Tgl1p and Yeh1p by analyzing the behavior of each hydrolase in the absence of its counterpart steryl ester hydrolases. In summary, this study demonstrates a strong regulation of the two lipid droplet associated steryl ester hydrolases Tgl1p and Yeh1p due to the presence/absence of their host organelle. Copyright © 2017 Elsevier B.V. All rights reserved.

The C-terminal domain of the nuclear factor I-B2 isoform is glycosylated and transactivates the WAP gene in the JEG-3 cells

International Nuclear Information System (INIS)

Mukhopadhyay, Sudit S.; Rosen, Jeffrey M.

2007-01-01

The transcription factor nuclear factor I (NFI) has been shown previously both in vivo and in vitro to be involved in the cooperative regulation of whey acidic protein (WAP) gene transcription along with the glucocorticoid receptor and STAT5. In addition, one of the specific NFI isoforms, NFI-B2, was demonstrated in transient co-transfection experiments in JEG cells, which lack endogenous NFI, to be preferentially involved in the cooperative regulation of WAP gene expression. A comparison of the DNA-binding specificities of the different NFI isoforms only partially explained their differential ability to activate the WAP gene transcription. Here, we analyzed the transactivation regions of two NFI isoforms by making chimeric proteins between the NFI-A and B isoforms. Though, their DNA-binding specificities were not altered as compared to the corresponding wild-type transcription factors, the C-terminal region of the NFI-B isoform was shown to preferentially activate WAP gene transcription in cooperation with GR and STAT5 in transient co-transfection assays in JEG-3 cells. Furthermore, determination of serine and threonine-specific glycosylation (O-linked N-acetylglucosamine) of the C-terminus of the NFI-B isoform suggested that the secondary modification by O-GlcNAc might play a role in the cooperative regulation of WAP gene transcription by NFI-B2 and STAT5

Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair.

Science.gov (United States)

Whitney, John C; Chou, Seemay; Russell, Alistair B; Biboy, Jacob; Gardiner, Taylor E; Ferrin, Michael A; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D

2013-09-13

Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity.

Identification, Structure, and Function of a Novel Type VI Secretion Peptidoglycan Glycoside Hydrolase Effector-Immunity Pair*

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Whitney, John C.; Chou, Seemay; Russell, Alistair B.; Biboy, Jacob; Gardiner, Taylor E.; Ferrin, Michael A.; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D.

2013-01-01

Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity. PMID:23878199

In Silico Investigation of Flavonoids as Potential Trypanosomal Nucleoside Hydrolase Inhibitors

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Christina Hung Hung Ha

2015-01-01

Full Text Available Human African Trypanosomiasis is endemic to 37 countries of sub-Saharan Africa. It is caused by two related species of Trypanosoma brucei. Current therapies suffer from resistance and public accessibility of expensive medicines. Finding safer and effective therapies of natural origin is being extensively explored worldwide. Pentamidine is the only available therapy for inhibiting the P2 adenosine transporter involved in the purine salvage pathway of the trypanosomatids. The objective of the present study is to use computational studies for the investigation of the probable trypanocidal mechanism of flavonoids. Docking experiments were carried out on eight flavonoids of varying level of hydroxylation, namely, flavone, 5-hydroxyflavone, 7-hydroxyflavone, chrysin, apigenin, kaempferol, fisetin, and quercetin. Using AutoDock 4.2, these compounds were tested for their affinity towards inosine-adenosine-guanosine nucleoside hydrolase and the inosine-guanosine nucleoside hydrolase, the major enzymes of the purine salvage pathway. Our results showed that all of the eight tested flavonoids showed high affinities for both hydrolases (lowest free binding energy ranging from −10.23 to −7.14 kcal/mol. These compounds, especially the hydroxylated derivatives, could be further studied as potential inhibitors of the nucleoside hydrolases.

Quantifying risk of penile prosthesis infection with elevated glycosylated hemoglobin.

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Wilson, S K; Carson, C C; Cleves, M A; Delk, J R

1998-05-01

Elevation of glycosylated hemoglobin above levels of 11.5 mg.% has been considered a contraindication to penile prosthesis implantation in diabetic patients. We determine the predictive value of glycosylated hemoglobin A1C in penile prosthesis infections in diabetic and nondiabetic patients to confirm or deny this prevalent opinion. We conducted a 2-year prospective study of 389 patients, including 114 diabetics, who underwent 3-piece penile prosthesis implantation. All patients had similar preoperative preparation without regard to diabetic status, control or glycosylated hemoglobin A1C level. Risk of infection was statistically analyzed for diabetics versus nondiabetics, glycosylated hemoglobin A1C values above and below 11.5 mg.%, insulin dependent versus oral medication diabetics, and fasting blood sugars above and below 180 mg.%. Prosthesis infections developed in 10 diabetics (8.7%) and 11 nondiabetics (4.0%). No increased infection rate was observed in diabetics with high fasting sugars or diabetics on insulin. There was no statistically significant increased infection risk with increased levels of glycosylated hemoglobin A1C among all patients or among only the diabetics. In fact, there was no meaningful difference in the median or mean level of glycosylated hemoglobin A1C in the infected and noninfected patients regardless of diabetes. Use of glycosylated hemoglobin A1C values to identify and exclude surgical candidates with increased risk of infections is not proved by this study. Elevation of fasting sugar or insulin dependence also does not increase risk of infection in diabetics undergoing prosthesis implantation.

Method Development in the Regioselective Glycosylation of Unprotected Carbohydrates

DEFF Research Database (Denmark)

Niedbal, Dominika Alina

and the glycosylations were promoted by tetrabutylammonium bromide. The couplings were completely selective and gave rise to a number of 1,6-linked disaccharides with 1,2- cis-linked orientation. Project 2: Boron-mediated glycosylation of unprotected carbohydrates Boron-mediated regioselective Koenigs...

Peptidoglycan Hydrolases of Local Lactic Acid Bacteria from Kazakh Traditional Food

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Serik Shaikhin

2014-01-01

Full Text Available Introduction: Peptidoglycan (PG is a major component of the cell wall of Gram-positive bacteria and is essential for maintaining the integrity of the bacterial cell and its shape. The bacteria synthesize PG hydrolases, which are capable of cleaving the covalent bonds of PG. They also play an important role in modeling PG, which is required for bacterial growth and division. In an era of increasing antibiotic-resistant pathogens, PG hydrolases that destroy these important structures of the cell wall act as a potential source of new antimicrobials. The aim of this study is to identify the main PG hydrolases of local lactic acid bacteria isolated from traditional foods that enhance probiotic activity of a biological preparation. Methods. Lactococcus lactis 17А and Lactococcus garvieae 19А were isolated from the traditional sausage-like meat product called kazy. They were isolated according to standards methods of microbiology. Genetic identification of the isolates were tested by determining the nucleotide sequences of 16S rDNA. The Republican collection of microorganisms took strains of Lactobacillus casei subsp. Rhamnosus 13-P, L. delbrueckii subsp. lactis CG-1 B-RKM 0044 from cheese, Lactobacillus casei subsp. casei B-RKM 0202 from homemade butter. They used the standard technique of renaturating polyacrylamide gel electrophoresis to detect PG hydrolases activity. Results. According to the profiles of PG hydrolase activity on zymograms, the enzymes of Lactococci 17A and 19A in kazy are similar in electrophoretic mobility to major autolysin AcmA, while the lactobacilli of industrial and home-made dairy products have enzymes similar to extracellular proteins p40 and p75, which have probiotic activity. Conclusions. Use of peptidoglycan hydrolases seems to be an interesting approach in the fight against multi-drug resistant strains of bacteria and could be a valuable tool for the treatment of diseases caused by these microorganisms in Kazakhstan.

Distribution of N-glycosylation sequons in proteins: how apart are they?

DEFF Research Database (Denmark)

Rao, Shyama Prasad; Buus, Ole Thomsen; Wollenweber, Bernd

2011-01-01

of experimentally confirmed eukaryotic N-glycoproteins we analyzed the relative position and distribution of sequons. N-Glycosylation probability was found to be lower in the termini of protein sequences compared to the mid region. N-glycosylated sequons were found much farther from C terminus compared to the N......N-glycosylation is a common protein modification process, which affects a number of properties of proteins. Little is known about the distribution of N-glycosylation sequons, for example, the distance between glycosylated sites and their position in the protein primary sequence. Using a large set......-terminus of the protein sequence and this effect was more pronounced for NXS sequons. The distribution of sequons, modeled based on balls-in-boxes classical occupancy, showed a near-maximum probability. Considerable proportion of sequons was found within a distance of ten amino acids, indicating that the steric hindrance...

Members of the amylovora group of Erwinia are cellulolytic and possess genes homologous to the type II secretion pathway.

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Riekki, R; Palomäki, T; Virtaharju, O; Kokko, H; Romantschuk, M; Saarilahti, H T

2000-07-01

A cellulase-producing clone was isolated from a genomic library of the Erwinia rhapontici (Millard) Burkholder strain NCPPB2989. The corresponding gene, named celA, encodes an endoglucanase (EC 3.2.1.4) with the extremely low pH optimum of 3.4 and a temperature optimum between 40 and 50 degrees C. A single ORF of 999 nt was found to be responsible for the Cel activity. The corresponding protein, named CelA, showed 67% identity to the endoglucanase Y of E. chrysanthemi and 51.5% identity to the endoglucanase of Cellulomonas uda, and thus belongs to the glycosyl hydrolase family 8. The celA gene, or its homologue, was found to be present in all E. rhapontici isolates analysed, in E. chrysanthemi, and in E. amylovora. The presence of plant cell wall-degrading enzymes in the amylovora group of Erwinia spp. had not previously been established. Furthermore, the DNA of both E. rhapontici and E. amylovora was found to exhibit homology to genes encoding the type II (GSP) secretion pathway, which is known to be responsible for extracellular targeting of cellulases and pectinases in Erwinia spp. that cause soft rotting, such as E. carotovora and E. chrysanthemi. Secretion of the CelA protein by E. rhapontici could not be verified. However, the CelA protein itself was found to include the information necessary for heterologous secretion by E. chrysanthemi.

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

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Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-01-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

Effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1

International Nuclear Information System (INIS)

Watanabe, Ayahisa; Nishijima, Ken-ichi; Zhao, Songji; Tamaki, Nagara; Kuge, Yuji; Tanaka, Yoshikazu; Itoh, Takeshi; Takemoto, Hiroshi

2012-01-01

Glycosylation is generally applicable as a strategy for increasing the activity of bioactive proteins. In this study, we examined the effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1 (GLP-1) as a bioactive peptide for type 2 diabetes. Noninvasive imaging studies were performed using a gamma camera after the intravenous administration of 123 I-GLP-1 or 123 I-α2, 6-sialyl N-acetyllactosamine (glycosylated) GLP-1 in rats. In ex vivo biodistribution studies using 125 I-GLP-1 or 125 I-glycosylated GLP-1, organ samples were measured for radioactivity. Plasma samples were added to 15% trichloroacetic acid (TCA) to obtain TCA-insoluble and TCA-soluble fractions. The radioactivity in the TCA-insoluble and TCA-soluble fractions was measured. In the noninvasive imaging studies, a relatively high accumulation level of 123 I-GLP-1 was found in the liver, which is the major organ to eliminate exogenous GLP-1. The area under the time-activity curve (AUC) of 123 I-glycosylated GLP-1 in the liver was significantly lower (89%) than that of 123 I-GLP-1. These results were consistent with those of ex vivo biodistribution studies using 125 I-labeled peptides. The AUC of 125 I-glycosylated GLP-1 in the TCA-insoluble fraction was significantly higher (1.7-fold) than that of GLP-1. This study demonstrated that glycosylation significantly decreased the distribution of radiolabeled GLP-1 into the liver and increased the concentration of radiolabeled GLP-1 in plasma. These results suggested that glycosylation is a useful strategy for decreasing the distribution into the liver of bioactive peptides as desirable pharmaceuticals. (author)

Overexpression of fatty acid amide hydrolase induces early flowering in Arabidopsis thaliana

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Neal D. Teaster

2012-02-01

Full Text Available N-Acylethanolamines (NAEs are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE. In animal systems this reaction is part of the endocannabinoid signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH, which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440 lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA, enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD and inductive long day (LD conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3, were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways.

GH97 is a new family of glycoside hydrolases, which is related to the α-galactosidase superfamily

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Naumoff Daniil G

2005-08-01

Full Text Available Abstract Background As a rule, about 1% of genes in a given genome encode glycoside hydrolases and their homologues. On the basis of sequence similarity they have been grouped into more than ninety GH families during the last 15 years. The GH97 family has been established very recently and initially included only 18 bacterial proteins. However, the evolutionary relationship of the genes encoding proteins of this family remains unclear, as well as their distribution among main groups of the living organisms. Results The extensive search of the current databases allowed us to double the number of GH97 family proteins. Five subfamilies were distinguished on the basis of pairwise sequence comparison and phylogenetic analysis. Iterative sequence analysis revealed the relationship of the GH97 family with the GH27, GH31, and GH36 families of glycosidases, which belong to the α-galactosidase superfamily, as well as a more distant relationship with some other glycosidase families (GH13 and GH20. Conclusion The results of this study show an unexpected sequence similarity of GH97 family proteins with glycoside hydrolases from several other families, that have (β/α8-barrel fold of the catalytic domain and a retaining mechanism of the glycoside bond hydrolysis. These data suggest a common evolutionary origin of glycosidases representing different families and clans.

Regulation of calcium release from the endoplasmic reticulum by the serine hydrolase ABHD2.

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Yun, Bogeon; Lee, HeeJung; Powell, Roger; Reisdorph, Nichole; Ewing, Heather; Gelb, Michael H; Hsu, Ku-Lung; Cravatt, Benjamin F; Leslie, Christina C

2017-09-02

The serine hydrolase inhibitors pyrrophenone and KT195 inhibit cell death induced by A23187 and H 2 O 2 by blocking the release of calcium from the endoplasmic reticulum and mitochondrial calcium uptake. The effect of pyrrophenone and KT195 on these processes is not due to inhibition of their known targets, cytosolic phospholipase A 2 and α/β-hydrolase domain-containing (ABHD) 6, respectively, but represent off-target effects. To identify targets of KT195, fibroblasts were treated with KT195-alkyne to covalently label protein targets followed by click chemistry with biotin azide, enrichment on streptavidin beads and tryptic peptide analysis by mass spectrometry. Although several serine hydrolases were identified, α/β-hydrolase domain-containing 2 (ABHD2) was the only target in which both KT195 and pyrrophenone competed for binding to KT195-alkyne. ABHD2 is a serine hydrolase with a predicted transmembrane domain consistent with its pull-down from the membrane proteome. Subcellular fractionation showed localization of ABHD2 to the endoplasmic reticulum but not to mitochondria or mitochondrial-associated membranes. Knockdown of ABHD2 with shRNA attenuated calcium release from the endoplasmic reticulum, mitochondrial calcium uptake and cell death in fibroblasts stimulated with A23187. The results describe a novel mechanism for regulating calcium transfer from the endoplasmic reticulum to mitochondria that involves the serine hydrolase ABHD2. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystallization of mouse S-adenosyl-l-homocysteine hydrolase

International Nuclear Information System (INIS)

Ishihara, Masaaki; Kusakabe, Yoshio; Ohsumichi, Tsuyoshi; Tanaka, Nobutada; Nakanishi, Masayuki; Kitade, Yukio; Nakamura, Kazuo T.

2010-01-01

Mouse S-adenosyl-l-homocysteine hydrolase has been crystallized in the presence of the reaction product adenosine. Diffraction data to 1.55 Å resolution were collected using synchrotron radiation. S-Adenosyl-l-homocysteine hydrolase (SAHH; EC 3.3.1.1) catalyzes the reversible hydrolysis of S-adenosyl-l-homocysteine to adenosine and l-homocysteine. For crystallographic investigations, mouse SAHH (MmSAHH) was overexpressed in bacterial cells and crystallized using the hanging-drop vapour-diffusion method in the presence of the reaction product adenosine. X-ray diffraction data to 1.55 Å resolution were collected from an orthorhombic crystal form belonging to space group I222 with unit-cell parameters a = 100.64, b = 104.44, c = 177.31 Å. Structural analysis by molecular replacement is in progress

Diagnostic accuracy of urinary prostate protein glycosylation profiling in prostatitis diagnosis.

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Vermassen, Tijl; Van Praet, Charles; Poelaert, Filip; Lumen, Nicolaas; Decaestecker, Karel; Hoebeke, Piet; Van Belle, Simon; Rottey, Sylvie; Delanghe, Joris

2015-01-01

Although prostatitis is a common male urinary tract infection, clinical diagnosis of prostatitis is difficult. The developmental mechanism of prostatitis is not yet unraveled which led to the elaboration of various biomarkers. As changes in asparagine-linked-(N-)-glycosylation were observed between healthy volunteers (HV), patients with benign prostate hyperplasia and prostate cancer patients, a difference could exist in biochemical parameters and urinary N-glycosylation between HV and prostatitis patients. We therefore investigated if prostatic protein glycosylation could improve the diagnosis of prostatitis. Differences in serum and urine biochemical markers and in total urine N-glycosylation profile of prostatic proteins were determined between HV (N=66) and prostatitis patients (N=36). Additionally, diagnostic accuracy of significant biochemical markers and changes in N-glycosylation was assessed. Urinary white blood cell (WBC) count enabled discrimination of HV from prostatitis patients (Pprostatitis patients from HV (Pprostatitis patients compared to HV (Pprostatitis. Further research is required to unravel the developmental course of prostatic inflammation.

The Emerging Importance of IgG Fab Glycosylation in Immunity.

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van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

The apo structure of sucrose hydrolase from Xanthomonas campestris pv. campestris shows an open active-site groove

DEFF Research Database (Denmark)

Champion, Elise; Remaud-Simeon, Magali; Skov, Lars Kobberøe

2009-01-01

Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris...... of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite -1 glucosyl-binding determinants...

Overelaborated synaptic architecture and reduced synaptomatrix glycosylation in a Drosophila classic galactosemia disease model

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Patricia Jumbo-Lucioni

2014-12-01

Full Text Available Classic galactosemia (CG is an autosomal recessive disorder resulting from loss of galactose-1-phosphate uridyltransferase (GALT, which catalyzes conversion of galactose-1-phosphate and uridine diphosphate (UDP-glucose to glucose-1-phosphate and UDP-galactose, immediately upstream of UDP–N-acetylgalactosamine and UDP–N-acetylglucosamine synthesis. These four UDP-sugars are essential donors for driving the synthesis of glycoproteins and glycolipids, which heavily decorate cell surfaces and extracellular spaces. In addition to acute, potentially lethal neonatal symptoms, maturing individuals with CG develop striking neurodevelopmental, motor and cognitive impairments. Previous studies suggest that neurological symptoms are associated with glycosylation defects, with CG recently being described as a congenital disorder of glycosylation (CDG, showing defects in both N- and O-linked glycans. Here, we characterize behavioral traits, synaptic development and glycosylated synaptomatrix formation in a GALT-deficient Drosophila disease model. Loss of Drosophila GALT (dGALT greatly impairs coordinated movement and results in structural overelaboration and architectural abnormalities at the neuromuscular junction (NMJ. Dietary galactose and mutation of galactokinase (dGALK or UDP-glucose dehydrogenase (sugarless genes are identified, respectively, as critical environmental and genetic modifiers of behavioral and cellular defects. Assaying the NMJ extracellular synaptomatrix with a broad panel of lectin probes reveals profound alterations in dGALT mutants, including depletion of galactosyl, N-acetylgalactosamine and fucosylated horseradish peroxidase (HRP moieties, which are differentially corrected by dGALK co-removal and sugarless overexpression. Synaptogenesis relies on trans-synaptic signals modulated by this synaptomatrix carbohydrate environment, and dGALT-null NMJs display striking changes in heparan sulfate proteoglycan (HSPG co-receptor and Wnt

Hydrolase activity in Jerusalem artichoke and chicory

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Klaushofer, H.; Abraham, B.; Leichtfried, G.

1988-03-01

Post-harvest storage of chicory and Jerusalem artichoke and overwintering of Jerusalem artichoke in the soil cause a more or less pronounced shortening of the fructan chain, depending on the variety. The proportion of fructose in the total fructan thus shifts towards glucose. This reduction on the fructose/glucose ratio is undesirable if the intention is to obtain a sweetener of high fructose content. In this work an attempt was made, via the quantity of fructose formed after a 4(3)-hour reaction of a tuber (root) extract with inulin, to assign a characteristic value to the depolymerization tendency of the material in question. However, since the plant extract not only contains enzymes (hydrolase A and B) that shorten the fructan chains but the activity of fructosyltransferase (SST, FFT) and enzymes of microbial origin (inulinase II, invertase) must also be considered, the concept of 'hydrolase activity' used by the authors is essentially an expression of 'total activity'. The activity unit (EU) is defined as the ability to split of 1 ..mu..mol of fructose from (chicory) inulin per minute under experimental conditions. Values of 0.25 to 0.77 EU/g dry solids were found in Jerusalem artichoke. Considerable differences may occur between varieties from the same cultivated area and the same harvest period. With one and the same variety, the activity appears to be subject to marked yearly fluctuations, so that at present, because of hydrolase activity, nothing certain can be said about the depolymerization tendency of a variety.

Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH.

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Vallbracht, Melina; Rehwaldt, Sascha; Klupp, Barbara G; Mettenleiter, Thomas C; Fuchs, Walter

2018-05-01

Many viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reduced in vitro fusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in the Varicellovirus genus, resulted in enhanced in vitro fusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. IMPORTANCE Herpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide

DISAL glycosyl donors for the synthesis of a linear hexasaccharide under mild conditions

DEFF Research Database (Denmark)

Petersen, Lars; Laursen, Jane B.; Larsen, K.

2003-01-01

The new class of glycosyl donors with a methyl 3,5-dinitrosalicylate (DISAL) anomeric leaving group has proved efficient for glycosylation under strictly neutral, mildly basic, or mildly acidic conditions. Here, we report the synthesis of novel DISAL disaccharide glycosyl donors prepared by easy...... nucleophilic aromatic substitution. These DISAL donors proved efficient in the synthesis of a starch-related hexasaccharide under very mild conditions. Glycosylations proceeded with alpha-selectivity and were compatible with Trt protecting groups....

Cloning of a novel xylanase gene from a newly isolated Fusarium sp. Q7-31 and its expression in Escherichia coli

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Zhan-Ling Xie

2012-03-01

Full Text Available A strain of Q7-31 was isolated from Qinghai-Tibet Plateau and was identified as Fusarium sp. based on its morphological characteristics and ITS rDNA gene sequence analysis. It has the highest capacity of degrading cell wall activity compared with other 11 strains. To do research on its xylanase activity of Fusarium sp. Q7-31 while the degrading the rice cell walls, the complete gene xyn8 that encodes endo-1, 4-β-xylanase secreted by Fusarium sp. Q7-31 was cloned and sequenced. The coding region of the gene is separated by two introns of 56bp and 55bp. It encodes 230 amino acid residues of a protein with a calculated molecular weight of 25.7 kDa. The animo acids sequence of xyn8 gene has higher similarity with those of family 11 of glycosyl hydrolases reported from other microorganisms. The nature peptide encodeing cDNA was subcloned into pGEX5x-1 expression vector. The recombinant plasmid was expressed in Escherichia coli BL21-CodonPlus (DE3-RIL, and xylanase activity was measured. The expression fusion protein was identified by SDS-PAGE and Western blotting, a new specific band of about 52kDa was identified when induced by IPTG. Enzyme activity assay verified the recombinants proteins as a xylanase. A maxium activity of 2.34U/ mg, the xylanase had optimal activity at pH 6.0 and temperature 40ºC .

A remote but significant sequence homology between glycoside hydrolase clan GH-H and glycoside hydrolase family GH 31

DEFF Research Database (Denmark)

Janecek, S.; Svensson, Birte; MacGregor, E.A.

2007-01-01

Although both the α-amylase super-family, i.e. the glycoside hydrolase (GH) clan GH-H (the GH families 13, 70 and 77), and family GH31 share some characteristics, their different catalytic machinery prevents classification of GH31 in clan GH-H. A significant but remote evolutionary relatedness is...

HYDROLASING OF CONTAMINATED UNDERWATER BASIN SURFACES AT THE HANFORD K AREA

International Nuclear Information System (INIS)

CHRONISTER, G.B.

2005-01-01

This paper discusses selecting and implementing hydrolasing technology to reduce radioactive contamination in preparing to dispose of the K Basins; two highly contaminated concrete basins at the Hanford Site. A large collection of spent nuclear fuel stored for many years underwater at the K Basins has been removed to stable, dry, safe storage. Remediation activities have begun for the remaining highly contaminated water. sludge, and concrete basin structures. Hydrolasing will be used to decontaminate and prepare the basin structures for disposal

Characterization of a novel xylanase gene from rumen content of Hu sheep.

Science.gov (United States)

Wang, Qian; Luo, Yang; He, Bo; Jiang, Lin-Shu; Liu, Jian-Xin; Wang, Jia-Kun

2015-12-01

A novel xylanase gene, xyn-lxy, was cloned from a metagenomic fosmid library, which was previously constructed from the rumen contents of Hu sheep and was functionally characterized in Escherichia coli. The open reading frame was composed of 1923 bp and encoded for 640 amino acids, including a catalytic domain of glycosyl hydrolase family 10 and carbohydrate-binding module 9. The gene showed 97 % identity with uncultured bacterium Contig1552 but low similarity with xylanases from known cellulolytic-degrading microorganisms in the rumen. The recombinant XYN-LXY showed a specific activity of 664.7 U mg(-1). The optimal temperature and pH of the enzyme were 50 °C and 6.0, respectively. Specifically, XYN-LXY was exclusively activated by Mn(2+) among all of the cations and reducing agents tested in this study. An enzymatic hydrolysis assay revealed that XYN-LXY degraded birchwood xylan into xylooligosaccharide with a low degree of polymerization. After incubation for 4 h, the concentration of the dominant product, xylobiose, was 2.297 ± 0.175 mg ml(-1) (74.07 % of total product) followed by xylose with a concentration of 0.656 ± 0.010 mg ml(-1) (21.14 % of total product). The XYN-LXY exhibited deep degradation effects on the xylan substrate, which were rarely observed with endo-xylanase, making it a promising candidate for industrial application, especially in biofuel production.

α-Amylase: an enzyme specificity found in various families of glycoside hydrolases

DEFF Research Database (Denmark)

Janeček, Štefan; Svensson, Birte; MacGregor, E. Ann

2014-01-01

of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α...... investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases....

Nonenzymatic glycosylation of bovine myelin basic protein

International Nuclear Information System (INIS)

Hitz, J.B.

1987-01-01

In the CNS myelin sheath the nonenzymatic glycosylation reaction (at the early stage of the Amadori product) occurs only with the myelin basic protein and not with the other myelin proteins. This was observed in isolated bovine myelin by in vitro incubation with [ 14 C]-galactose and [ 14 C]-glucose. The respective in-vitro incorporation rates for purified bovine myelin basic protein with D-galactose, D-glucose and D-mannose were 7.2, 2.4 and 2.4 mmoles/mole myelin basic protein per day at 37 0 C. A more rapid, HPLC method was devised and characterized to specifically analyze for the Amadori product. The HPLC method was correlated to the [ 14 C]-sugar incorporation method for myelin basic protein under a set of standard reaction conditions using [ 14 C]-glucose and [ 14 C]-mannose with HPLC values at 1/6 and 1/5 of the [ 14 C]-sugar incorporation method. A novel myelin basic protein purification step has been developed that yields a relativity proteolytic free preparation that is easy to work with, being totally soluble at a neutral pH. Nine new spots appear for a trypsinized glycosylated MBP in the paper peptide map of which eight correspond to positions of the [ 3 H]-labeled Amadori product in affinity isolated peptides. These studies provide a general characterization of and a structural basis for investigations on nonenzymatically glycosylated MBP as well as identifying MBP as the only nonenzymatically glycosylated protein in the CNS myelin sheath which may accumulate during aging, diabetes, and demyelinating diseases in general

The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

Science.gov (United States)

URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

2015-01-01

The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820

Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

Science.gov (United States)

Su, Xiaoyun; Zhang, Jing; Mackie, Roderick I; Cann, Isaac K O

2012-01-01

The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

Directory of Open Access Journals (Sweden)

Xiaoyun Su

Full Text Available The glycoside hydrolases (GH of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

Preparation, crystallization and preliminary X-ray crystallographic studies of diadenosine tetraphosphate hydrolase from Shigella flexneri 2a

Energy Technology Data Exchange (ETDEWEB)

Hu, Wenxin; Wang, Qihai; Bi, Ruchang, E-mail: rcbi@sun5.ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101 (China)

2005-12-01

The 31.3 kDa Ap{sub 4}A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap{sub 4}A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Diadenosine tetraphosphate (Ap{sub 4}A) hydrolase (EC 3.6.1.41) hydrolyzes Ap{sub 4}A symmetrically in prokaryotes. It plays a potential role in organisms by regulating the concentration of Ap{sub 4}A in vivo. To date, no three-dimensional structures of proteins with significant sequence homology to this protein have been determined. The 31.3 kDa Ap{sub 4}A hydrolase from Shigella flexneri 2a has been cloned, expressed and purified using an Escherichia coli expression system. Crystals of Ap{sub 4}A hydrolase have been obtained by the hanging-drop technique at 291 K using PEG 550 MME as precipitant. Ap{sub 4}A hydrolase crystals diffract X-rays to 3.26 Å and belong to space group P2{sub 1}, with unit-cell parameters a = 118.9, b = 54.6, c = 128.5 Å, β = 95.7°.

Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa

KAUST Repository

Shih, P. B.

2015-03-31

Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment.

Cancer associated aberrant protein o-glycosylation can modify antigen processing and immune response

DEFF Research Database (Denmark)

Madsen, Caroline B; Petersen, Cecilie; Lavrsen, Kirstine

2012-01-01

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing......, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/- glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo...

Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent

Science.gov (United States)

Wadzinski, Tyler J.; Steinauer, Angela; Hie, Liana; Pelletier, Guillaume; Schepartz, Alanna; Miller, Scott J.

2018-06-01

Glycosylated natural products and synthetic glycopeptides represent a significant and growing source of biochemical probes and therapeutic agents. However, methods that enable the aqueous glycosylation of endogenous amino acid functionality in peptides without the use of protecting groups are scarce. Here, we report a transformation that facilitates the efficient aqueous O-glycosylation of phenolic functionality in a wide range of small molecules, unprotected tyrosine, and tyrosine residues embedded within a range of complex, fully unprotected peptides. The transformation, which uses glycosyl fluoride donors and is promoted by Ca(OH)2, proceeds rapidly at room temperature in water, with good yields and selective formation of unique anomeric products depending on the stereochemistry of the glycosyl donor. High functional group tolerance is observed, and the phenol glycosylation occurs selectively in the presence of virtually all side chains of the proteinogenic amino acids with the singular exception of Cys. This method offers a highly selective, efficient, and operationally simple approach for the protecting-group-free synthesis of O-aryl glycosides and Tyr-O-glycosylated peptides in water.

[Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins].

Science.gov (United States)

Avanov, A Ia; Lipkind, G M

1990-03-01

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.

A computational framework for the automated construction of glycosylation reaction networks.

Science.gov (United States)

Liu, Gang; Neelamegham, Sriram

2014-01-01

Glycosylation is among the most common and complex post-translational modifications identified to date. It proceeds through the catalytic action of multiple enzyme families that include the glycosyltransferases that add monosaccharides to growing glycans, and glycosidases which remove sugar residues to trim glycans. The expression level and specificity of these enzymes, in part, regulate the glycan distribution or glycome of specific cell/tissue systems. Currently, there is no systematic method to describe the enzymes and cellular reaction networks that catalyze glycosylation. To address this limitation, we present a streamlined machine-readable definition for the glycosylating enzymes and additional methodologies to construct and analyze glycosylation reaction networks. In this computational framework, the enzyme class is systematically designed to store detailed specificity data such as enzymatic functional group, linkage and substrate specificity. The new classes and their associated functions enable both single-reaction inference and automated full network reconstruction, when given a list of reactants and/or products along with the enzymes present in the system. In addition, graph theory is used to support functions that map the connectivity between two or more species in a network, and that generate subset models to identify rate-limiting steps regulating glycan biosynthesis. Finally, this framework allows the synthesis of biochemical reaction networks using mass spectrometry (MS) data. The features described above are illustrated using three case studies that examine: i) O-linked glycan biosynthesis during the construction of functional selectin-ligands; ii) automated N-linked glycosylation pathway construction; and iii) the handling and analysis of glycomics based MS data. Overall, the new computational framework enables automated glycosylation network model construction and analysis by integrating knowledge of glycan structure and enzyme biochemistry. All

A computational framework for the automated construction of glycosylation reaction networks.

Directory of Open Access Journals (Sweden)

Gang Liu

Full Text Available Glycosylation is among the most common and complex post-translational modifications identified to date. It proceeds through the catalytic action of multiple enzyme families that include the glycosyltransferases that add monosaccharides to growing glycans, and glycosidases which remove sugar residues to trim glycans. The expression level and specificity of these enzymes, in part, regulate the glycan distribution or glycome of specific cell/tissue systems. Currently, there is no systematic method to describe the enzymes and cellular reaction networks that catalyze glycosylation. To address this limitation, we present a streamlined machine-readable definition for the glycosylating enzymes and additional methodologies to construct and analyze glycosylation reaction networks. In this computational framework, the enzyme class is systematically designed to store detailed specificity data such as enzymatic functional group, linkage and substrate specificity. The new classes and their associated functions enable both single-reaction inference and automated full network reconstruction, when given a list of reactants and/or products along with the enzymes present in the system. In addition, graph theory is used to support functions that map the connectivity between two or more species in a network, and that generate subset models to identify rate-limiting steps regulating glycan biosynthesis. Finally, this framework allows the synthesis of biochemical reaction networks using mass spectrometry (MS data. The features described above are illustrated using three case studies that examine: i O-linked glycan biosynthesis during the construction of functional selectin-ligands; ii automated N-linked glycosylation pathway construction; and iii the handling and analysis of glycomics based MS data. Overall, the new computational framework enables automated glycosylation network model construction and analysis by integrating knowledge of glycan structure and enzyme

Characterization of three plant biomass-degrading microbial consortia by metagenomics- and metasecretomics-based approaches

DEFF Research Database (Denmark)

Jiménez, Diego Javier; Brossi, Maria Julia de Lima; Schückel, Julia

2016-01-01

). The highest degradation rates of lignin (~59 %) were observed with SG-M, whereas CS-M showed a high consumption of cellulose and hemicellulose. Analyses of the carbohydrate-active enzymes in the three microbial consortia showed the dominance of glycosyl hydrolases (e.g. of families GH3, GH43, GH13, GH10, GH29......), switchgrass (SG-M) and corn stover (CS-M) under aerobic and mesophilic conditions. Molecular fingerprintings, bacterial 16S ribosomal RNA (rRNA) gene amplicon sequencing and metagenomic analyses showed that the three microbial consortia were taxonomically distinct. Based on the taxonomic affiliation...

Effects of anti-cocaine vaccine and viral gene transfer of cocaine hydrolase in mice on cocaine toxicity including motor strength and liver damage.

Science.gov (United States)

Gao, Yang; Geng, Liyi; Orson, Frank; Kinsey, Berma; Kosten, Thomas R; Shen, Xiaoyun; Brimijoin, Stephen

2013-03-25

In developing an vivo drug-interception therapy to treat cocaine abuse and hinder relapse into drug seeking provoked by re-encounter with cocaine, two promising agents are: (1) a cocaine hydrolase enzyme (CocH) derived from human butyrylcholinesterase and delivered by gene transfer; (2) an anti-cocaine antibody elicited by vaccination. Recent behavioral experiments showed that antibody and enzyme work in a complementary fashion to reduce cocaine-stimulated locomotor activity in rats and mice. Our present goal was to test protection against liver damage and muscle weakness in mice challenged with massive doses of cocaine at or near the LD50 level (100-120 mg/kg, i.p.). We found that, when the interceptor proteins were combined at doses that were only modestly protective in isolation (enzyme, 1mg/kg; antibody, 8 mg/kg), they provided complete protection of liver tissue and motor function. When the enzyme levels were ~400-fold higher, after in vivo transduction by adeno-associated viral vector, similar protection was observed from CocH alone. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

A Novel Strategy for Characterization of Glycosylated Proteins Separated by Gel Electrophoresis

DEFF Research Database (Denmark)

Larsen, Martin; Skottrup, Peter; Enghild, Jan Johannes

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins...... graphite powder micro-columns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and would be ideal for proteomics studies and verification of correct glycosylation of recombinant glycoproteins....

Purification and characterization of a GH43 β-xylosidase from Enterobacter sp. identified and cloned from forest soil bacteria.

Science.gov (United States)

Campos, Eleonora; Negro Alvarez, María José; Sabarís di Lorenzo, Gonzalo; Gonzalez, Sergio; Rorig, Marcela; Talia, Paola; Grasso, Daniel H; Sáez, Felicia; Manzanares Secades, Paloma; Ballesteros Perdices, Mercedes; Cataldi, Angel A

2014-01-01

The use of lignocellulosic biomass for second generation biofuels requires optimization of enzymatic breakdown of plant cell walls. In this work, cellulolytic bacteria were isolated from a native and two cultivated forest soil samples. Amplification of glycosyl hydrolases was attempted by using a low stringency-degenerate primer PCR strategy, using total soil DNA and bulk DNA pooled from positive colonies as template. A set of primers was designed based on Acidothermus cellulolyticus genome, by search of conserved domains of glycosyl hydrolases (GH) families of interest. Using this approach, a fragment containing an open reading frame (ORF) with 98% identity to a putative GH43 beta-xylosidase coding gene from Enterobacter cloacae was amplified and cloned. The full protein was expressed in Escherichia coli as N-terminal or C-terminal His-tagged fusions and purified under native conditions. Only N-terminal fusion protein, His-Xyl43, presented beta-xylosidase activity. On pNPX, optimal activity was achieved at pH 6 and 40 °C and Km and Kcat values were 2.92 mM and 1.32 seg(-1), respectively. Activity was also demonstrated on xylobiose (X2), with Km 17.8 mM and Kcat 380 s(-1). These results demonstrated that Xyl43 is a functional beta-xylosidase and it is the first evidence of this activity for Enterobacter sp. Copyright © 2013 Elsevier GmbH. All rights reserved.

Glycosylation patterns of kidney proteins differ in rat diabetic nephropathy.

Science.gov (United States)

Ravidà, Alessandra; Musante, Luca; Kreivi, Marjut; Miinalainen, Ilkka; Byrne, Barry; Saraswat, Mayank; Henry, Michael; Meleady, Paula; Clynes, Martin; Holthofer, Harry

2015-05-01

Diabetic nephropathy often progresses to end-stage kidney disease and, ultimately, to renal replacement therapy. Hyperglycemia per se is expected to have a direct impact on the biosynthesis of N- and O-linked glycoproteins. This study aims to establish the link between protein glycosylation and progression of experimental diabetic kidney disease using orthogonal methods. Kidneys of streptozotocin-diabetic and control rats were harvested at three different time points post streptozotocin injection. A panel of 12 plant lectins was used in the screening of lectin blots. The lectins UEAI, PHA-E, GSI, PNA, and RCA identified remarkable disease-associated differences in glycoprotein expression. Lectin affinity chromatography followed by mass spectrometric analyses led to the identification of several glycoproteins involved in salt-handling, angiogenesis, and extracellular matrix degradation. Our data confirm a substantial link between glycosylation signature and diabetes progression. Furthermore, as suggested by our findings on dipeptidyl peptidase-IV, altered protein glycosylation may reflect changes in biochemical properties such as enzymatic activity. Thus, our study demonstrates the unexplored potential of protein glycosylation analysis in the discovery of molecules linked to diabetic kidney disease.

Molecular cloning, expression, and characterization of four novel thermo-alkaliphilic enzymes retrieved from a metagenomic library.

Science.gov (United States)

Maruthamuthu, Mukil; van Elsas, Jan Dirk

2017-01-01

Enzyme discovery is a promising approach to aid in the deconstruction of recalcitrant plant biomass in an industrial process. Novel enzymes can be readily discovered by applying metagenomics on whole microbiomes. Our goal was to select, examine, and characterize eight novel glycoside hydrolases that were previously detected in metagenomic libraries, to serve biotechnological applications with high performance. Here, eight glycosyl hydrolase family candidate genes were selected from metagenomes of wheat straw-degrading microbial consortia using molecular cloning and subsequent gene expression studies in Escherichia coli. Four of the eight enzymes had significant activities on either p NP-β-d-galactopyranoside, p NP-β-d-xylopyranoside, p NP-α-l-arabinopyranoside or p NP-α-d-glucopyranoside. These proteins, denoted as proteins 1, 2, 5 and 6, were his-tag purified and their nature and activities further characterized using molecular and activity screens with the p NP-labeled substrates. Proteins 1 and 2 showed high homologies with (1) a β-galactosidase (74%) and (2) a β-xylosidase (84%), whereas the remaining two (5 and 6) were homologous with proteins reported as a diguanylate cyclase and an aquaporin, respectively. The β-galactosidase- and β-xylosidase-like proteins 1 and 2 were confirmed as being responsible for previously found thermo-alkaliphilic glycosidase activities of extracts of E. coli carrying the respective source fosmids. Remarkably, the β-xylosidase-like protein 2 showed activities with both p NP-Xyl and p NP-Ara in the temperature range 40-50 °C and pH range 8.0-10.0. Moreover, proteins 5 and 6 showed thermotolerant α-glucosidase activity at pH 10.0. In silico structure prediction of protein 5 revealed the presence of a potential "GGDEF" catalytic site, encoding α-glucosidase activity, whereas that of protein 6 showed a "GDSL" site, encoding a 'new family' α-glucosidase activity. Using a rational screening approach, we identified and

Model-based analysis of N-glycosylation in Chinese hamster ovary cells

DEFF Research Database (Denmark)

Krambeck, Frederick J.; Bennun, Sandra V; Andersen, Mikael Rørdam

2017-01-01

The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower...

Oxytocin analogues with O-glycosylated serine and threonine in position 4

Czech Academy of Sciences Publication Activity Database

Marcinkowska, A.; Borovičková, Lenka; Slaninová, Jiřina; Grzonka, Z.

2007-01-01

Roč. 81, č. 7 (2007), s. 1335-1344 ISSN 0137- 5083 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z90210515 Keywords : oxytocin * glycosylated serin * glycosylated threonin * position 4 Subject RIV: CE - Biochemistry Impact factor: 0.483, year: 2007

Micropinocytic ingestion of glycosylated albumin by isolated microvessels: possible role in pathogenesis of diabetic microangiopathy.

OpenAIRE

Williams, S K; Devenny, J J; Bitensky, M W

1981-01-01

Microvessels isolated from rat epididymal fat exhibit differential vesicular ingestion rates for unmodified and non-enzymatically glycosylated rat albumin. While unmodified rat albumin is excluded from ingestion by endothelial micropinocytic vesicles, glycosylated albumin is avidly taken up by endocytosis. Interaction of albumin and glycosylated albumin with endothelium was studied with a double-label fluorescence assay of micropinocytosis. When glycosylated albumin was present at a concentra...

Molecular cloning and biochemical characterization of an α-amylase family from Aspergillus niger

Directory of Open Access Journals (Sweden)

Junying Wang

2018-03-01

Full Text Available Background: α-Amylase is widely used in the starch processing, food and paper industries, hydrolyzing starch, glycogen and other polysaccharides into glucose, maltose and oligosaccharides. An α-amylase gene family from Aspergillus niger CBS513.88 encode eight putative α-amylases. The differences and similarities, biochemical properties and functional diversity among these eight α-amylases remain unknown. Results: The eight genes were cloned and expressed in Pichia pastoris GS115 by shaking-flask fermentation under the induction of methanol. The sequence alignment, biochemical characterizations and product analysis of starch hydrolysis by these α-amylases were investigated. It is found that the eight α-amylases belonged to three different groups with the typical structure of fungal α-amylase. They exhibited maximal activities at 30–40°C except AmyG and were all stable at acidic pH. Ca2+ and EDTA had no effects on the activities of α-amylases except AmyF and AmyH, indicating that the six amylases were Ca2+ independent. Two novel α-amylases of AmyE and AmyF were found. AmyE hydrolyzed starch into maltose, maltotriose and a small amount of glucose, while AmyF hydrolyzed starch into mainly glucose. The excellent physical and chemical properties including high acidic stability, Ca2+-independent and high maltotriose-forming capacity make AmyE suitable in food and sugar syrup industries. Conclusions: This study illustrates that a gene family can encode multiple enzymes members having remarkable differences in biochemical properties. It provides not only new insights into evolution and functional divergence among different members of an α-amylase family, but the development of new enzymes for industrial application. Keywords: Biochemical properties, Food industry, Fungal α-amylase, Glycosyl hydrolase family, Glycosyl hydrolase family, Industrial application, Paper industry, Recombinant Pichia pastoris, Starch processing, α-amylase cloning

In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins.

Science.gov (United States)

Fang, Pan; Wang, Xin-Jian; Xue, Yu; Liu, Ming-Qi; Zeng, Wen-Feng; Zhang, Yang; Zhang, Lei; Gao, Xing; Yan, Guo-Quan; Yao, Jun; Shen, Hua-Li; Yang, Peng-Yuan

2016-06-21

N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation strategies. Altogether, 13492 N-glycopeptides containing 8386 N-glycosylation sites on 3982 proteins were identified. After evaluating the performance of the above methods, we proposed a simple and efficient workflow for large-scale N-glycosylation site mapping. The optimized workflow yielded 80% of the initially identified N-glycosylation sites with considerably less effort. Analysis of the identified N-glycoproteins revealed that many of the mouse brain proteins are N-glycosylated, including those proteins in critical pathways for nervous system development and neurological disease. Additionally, several important biomarkers of various diseases were found to be N-glycosylated. These data confirm that N-glycosylation is important in both physiological and pathological processes in the brain, and provide useful details about numerous N-glycosylation sites in brain proteins.

Novel microbial epoxide hydrolases for biohydrolysis of glycidyl derivatives

Czech Academy of Sciences Publication Activity Database

Kotík, Michael; Břicháč, Jiří; Kyslík, Pavel

2005-01-01

Roč. 120, - (2005), s. 364-375 ISSN 0168-1656 Institutional research plan: CEZ:AV0Z5020903 Keywords : screening * epoxide hydrolase * biotransformation Subject RIV: EE - Microbiology, Virology Impact factor: 2.687, year: 2005

Structure/functional aspects of the human riboflavin transporter-3 (SLC52A3): role of the predicted glycosylation and substrate-interacting sites.

Science.gov (United States)

Subramanian, Veedamali S; Sabui, Subrata; Teafatiller, Trevor; Bohl, Jennifer A; Said, Hamid M

2017-08-01

The human riboflavin (RF) transporter-3 (hRFVT-3; product of the SLC52A3 gene) plays an essential role in the intestinal RF absorption process and is expressed exclusively at the apical membrane domain of polarized enterocytes. Previous studies have characterized different physiological/biological aspects of this transporter, but nothing is known about the glycosylation status of the hRFVT-3 protein and role of this modification in its physiology/biology. Additionally, little is known about the residues in the hRFVT-3 protein that interact with the ligand, RF. We addressed these issues using appropriate biochemical/molecular approaches, a protein-docking model, and established intestinal/renal epithelial cells. Our results showed that the hRFVT-3 protein is glycosylated and that glycosylation is important for its function. Mutating the predicted N -glycosylation sites at Asn 94 and Asn 168 led to a significant decrease in RF uptake; it also led to a marked intracellular (in the endoplasmic reticulum, ER) retention of the mutated proteins as shown by live-cell confocal imaging studies. The protein-docking model used in this study has identified a number of putative substrate-interacting sites: Ser 16 , Ile 20 , Trp 24 , Phe 142 , Thr 314 , and Asn 315 Mutating these potential interacting sites was indeed found to lead to a significant inhibition in RF uptake and to intracellular (ER) retention of the mutated proteins (except for the Phe 142 mutant). These results demonstrate that the hRFVT-3 protein is glycosylated and this glycosylation is important for its function and cell surface expression. This study also identified a number of residues in the hRFVT-3 polypeptide that are important for its function/cell surface expression.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy.

Science.gov (United States)

Bondt, Albert; Wuhrer, Manfred; Kuijper, T Martijn; Hazes, Johanna M W; Dolhain, Radboud J E M

2016-11-25

Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. IgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from intact IgG were analysed. The data was used to calculate glycosylation traits, and to estimate the level of Fab glycosylation. The overall level of Fab glycosylation was increased in RA patients compared to controls, while no differences in Fab glycosylation patterns were found. For the Fc and intact IgG (Total) previously observed differences in galactosylation and bisection were confirmed. Furthermore, increased galactosylation of Fc and Total were associated with lower disease activity and autoantibody positivity. In addition, the change in Fc galactosylation associated with the change in disease activity during pregnancy and after delivery, while this was not the case for Fab. In contrast to changes in Fc glycosylation, changes in Fab glycosylation are not associated with improvement of RA during pregnancy and arthritis flare after delivery.

Effects of preventing O-glycosylation on the secretion of human chorionic gonadotropin in Chinese hamster ovary cells

International Nuclear Information System (INIS)

Matzuk, M.M.; Krieger, M.; Corless, C.L.; Boime, I.

1987-01-01

Human chorionic gonadotropin (hCG) is a member of a family of heterodimeric glycoprotein hormones that have a common α subunit but differ in their hormone-specific β-subunits. The β subunit of hCG (hCGβ) is unique among the β subunits in that it contains four mucin-like O-linked oligosaccharides attached to a carboxyl-terminal extension. To study the effects of O-glycosylation on the secretion and assembly of hCG, expression vectors containing either hCGβ gene alone or together with the hCGα gene were transfected into a mutant Chinese hamster ovary cell line, 1d1D, which exhibits a reversible defect in O-glycosylation. The results reveal that hCGβ can be secreted normally in the absence of its O-linked oligosaccharides. hCGβ devoid of O-linked carbohydrate can also combine efficiently with hCGα and be secreted as an intact dimer. The authors conclude that in Chinese hamster ovary cells, the hCGβ O-linked chains play no role in the assembly and secretion of hCG. The normal and O-linked oligosaccharide-deficient forms of hCG secreted by these cells should prove useful in examining the role of O-linked chains on the biological function of hCG

Tumor-derived microvesicles mediate human breast cancer invasion through differentially glycosylated EMMPRIN.

Science.gov (United States)

Menck, Kerstin; Scharf, Christian; Bleckmann, Annalen; Dyck, Lydia; Rost, Ulrike; Wenzel, Dirk; Dhople, Vishnu M; Siam, Laila; Pukrop, Tobias; Binder, Claudia; Klemm, Florian

2015-04-01

Tumor cells secrete not only a variety of soluble factors, but also extracellular vesicles that are known to support the establishment of a favorable tumor niche by influencing the surrounding stroma cells. Here we show that tumor-derived microvesicles (T-MV) also directly influence the tumor cells by enhancing their invasion in a both autologous and heterologous manner. Neither the respective vesicle-free supernatant nor MV from benign mammary cells mediate invasion. Uptake of T-MV is essential for the proinvasive effect. We further identify the highly glycosylated form of the extracellular matrix metalloproteinase inducer (EMMPRIN) as a marker for proinvasive MV. EMMPRIN is also present at high levels on MV from metastatic breast cancer patients in vivo. Anti-EMMPRIN strategies, such as MV deglycosylation, gene knockdown, and specific blocking peptides, inhibit MV-induced invasion. Interestingly, the effect of EMMPRIN-bearing MV is not mediated by matrix metalloproteinases but by activation of the p38/MAPK signaling pathway in the tumor cells. In conclusion, T-MV stimulate cancer cell invasion via a direct feedback mechanism dependent on highly glycosylated EMMPRIN. © The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.

Enzymatic hydrolysis of lignocelluloses: Identification of novel cellulase genes from filamentous fungi

DEFF Research Database (Denmark)

Kolasa, Marta; Ahring, Birgitte Kiær; Lübeck, Peter Stephensen

2010-01-01

source. By means of degenerate PCR, specific genes, homologous to the genes of previously classified glycoside hydrolases from CAZY database, are searched for in selected strains of Aspergillus sp., Trichoderma sp. and Penicillium sp. Both methods are anticipated to facilitate identification of target...

N-Glycosylation optimization of recombinant antibodies in CHO cell through process and metabolic engineering

DEFF Research Database (Denmark)

Fan, Yuzhou

, analysis, control and optimization of N-glycosylation were thoroughly reviewed. In particular, how to control and optimize N-glycosylation in CHO cells was exclusively studied. The main focus of this PhD project is to find effective approaches of modulating N-glycosylation of CHO-derived recombinant...... galactose as feed additives, changing process parameters such as seeding density and cultivation duration are all demonstrated to be effective. The causal explanation of their impact on glycosylation can be various, including product, metabolism, proteome and physiology-associated mechanism. In the middle...... part of the thesis, both literature reviews and experimental applications were provided to demonstrate how to use omics data and implement systems biology to understand biological activities, especially N-glycosylation in CHO cells. In the last part of the thesis, the second strategy that apply genetic...

Enzymatic glycosylation of multivalent scaffolds

Czech Academy of Sciences Publication Activity Database

Bojarová, Pavla; Rosencrantz, R. R.; Elling, L.; Křen, Vladimír

2013-01-01

Roč. 42, č. 11 (2013), s. 4774-4797 ISSN 0306-0012 R&D Projects: GA MŠk(CZ) LD13042; GA ČR GAP207/10/0321 Institutional support: RVO:61388971 Keywords : N-ACETYLGLUCOSAMINYLTRANSFERASE-III * MUCIN TANDEM REPEAT * NEIGHBORING RESIDUE GLYCOSYLATION Subject RIV: CC - Organic Chemistry Impact factor: 30.425, year: 2013

COMPARISON OF FRUCTOSAMINE AND GLYCOSYLATED HEMOGLOBIN IN A NON-INSULIN DEPENDENT DIABETIC POPULATION

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M. Amini

1999-08-01

Full Text Available In an attempt to determine the clinical value of frnctosamine assay for monitoring type II diabetic patients, correlation of frnctosamine with glycosylated hemoglobin was studied. 100 patients with type II diabetes mcllitus were compared with 100 normal subjects. Fasting blood glucose, glycosylated hemoglobin, albumin and frnctosamine were measured in alt subjects. In the diabetic patients, a significant correlation was observed between fasting blood glucose and glycosylated hemoglobin (r = 0.64, p < 0.01 and scrum frnctosamine (r = 0.7, P < 0.01. Tlicrc was also a significant correlation between glycosylated hemoglobin and scrum frtictosmine (r = .94, I'<0.01. Frnctosamine, assay can be used as an index of diabetes control.

Importance of glycosylation on function of a potassium channel in neuroblastoma cells.

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M K Hall

Full Text Available The Kv3.1 glycoprotein, a voltage-gated potassium channel, is expressed throughout the central nervous system. The role of N-glycans attached to the Kv3.1 glycoprotein on conducting and non-conducting functions of the Kv3.1 channel are quite limiting. Glycosylated (wild type, partially glycosylated (N220Q and N229Q, and unglycosylated (N220Q/N229Q Kv3.1 proteins were expressed and characterized in a cultured neuronal-derived cell model, B35 neuroblastoma cells. Western blots, whole cell current recordings, and wound healing assays were employed to provide evidence that the conducting and non-conducting properties of the Kv3.1 channel were modified by N-glycans of the Kv3.1 glycoprotein. Electrophoretic migration of the various Kv3.1 proteins treated with PNGase F and neuraminidase verified that the glycosylation sites were occupied and that the N-glycans could be sialylated, respectively. The unglycosylated channel favored a different whole cell current pattern than the glycoform. Further the outward ionic currents of the unglycosylated channel had slower activation and deactivation rates than those of the glycosylated Kv3.1 channel. These kinetic parameters of the partially glycosylated Kv3.1 channels were also slowed. B35 cells expressing glycosylated Kv3.1 protein migrated faster than those expressing partially glycosylated and much faster than those expressing the unglycosylated Kv3.1 protein. These results have demonstrated that N-glycans of the Kv3.1 glycoprotein enhance outward ionic current kinetics, and neuronal migration. It is speculated that physiological changes which lead to a reduction in N-glycan attachment to proteins will alter the functions of the Kv3.1 channel.

Lipase genes in Mucor circinelloides: identification, sub-cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation.

Science.gov (United States)

Zan, Xinyi; Tang, Xin; Chu, Linfang; Zhao, Lina; Chen, Haiqin; Chen, Yong Q; Chen, Wei; Song, Yuanda

2016-10-01

Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the microbial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglycerol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the majority of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/β-hydrolase_3 family were the conserved lipase family in M. circinelloides. Additionally, some lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospholipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

Science.gov (United States)

Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

2018-05-09

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature.

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Peter K Busk

Full Text Available The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.

Modulation of redox homeostasis under suboptimal conditions by Arabidopsis nudix hydrolase 7

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Jambunathan Niranjani

2010-08-01

Full Text Available Abstract Background Nudix hydrolases play a key role in maintaining cellular homeostasis by hydrolyzing various nuceloside diphosphate derivatives and capped mRNAs. Several independent studies have demonstrated that Arabidopsis nudix hydrolase 7 (AtNUDT7 hydrolyzes NADH and ADP-ribose. Loss of function Atnudt7-1 mutant plants (SALK_046441 exhibit stunted growth, higher levels of reactive oxygen species, enhanced resistance to pathogens. However, using the same T-DNA line, two other groups reported that mutant plants do not exhibit any visible phenotypes. In this study we analyze plausible factors that account for differences in the observed phenotypes in Atnudt7. Secondly, we evaluate the biochemical and molecular consequences of increased NADH levels due to loss of function of AtNUDT7 in Arabidopsis. Results We identified a novel conditional phenotype of Atnudt7-1 knockout plants that was contingent upon nutrient composition of potting mix. In nutrient-rich Metro-Mix, there were no phenotypic differences between mutant and wild-type (WT plants. In the nutrient-poor mix (12 parts vermiculite: 3 parts Redi-earth and 1 part sand, mutant plants showed the characteristic stunted phenotype. Compared with WT plants, levels of glutathione, NAD+, NADH, and in turn NADH:NAD+ ratio were higher in Atnudt7-1 plants growing in 12:3:1 potting mix. Infiltrating NADH and ADP-ribose into WT leaves was sufficient to induce AtNUDT7 protein. Constitutive over-expression of AtNudt7 did not alter NADH levels or resistance to pathogens. Transcriptome analysis identified nearly 700 genes differentially expressed in the Atnudt7-1 mutant compared to WT plants grown in 12:3:1 potting mix. In the Atnudt7-1 mutant, genes associated with defense response, proteolytic activities, and systemic acquired resistance were upregulated, while gene ontologies for transcription and phytohormone signaling were downregulated. Conclusions Based on these observations, we conclude that the

Patterns of expression of cell wall related genes in sugarcane

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Lima D.U.

2001-01-01

Full Text Available Our search for genes related to cell wall metabolism in the sugarcane expressed sequence tag (SUCEST database (http://sucest.lbi.dcc.unicamp.br resulted in 3,283 reads (1% of the total reads which were grouped into 459 clusters (potential genes with an average of 7.1 reads per cluster. To more clearly display our correlation coefficients, we constructed surface maps which we used to investigate the relationship between cell wall genes and the sugarcane tissues libraries from which they came. The only significant correlations that we found between cell wall genes and/or their expression within particular libraries were neutral or synergetic. Genes related to cellulose biosynthesis were from the CesA family, and were found to be the most abundant cell wall related genes in the SUCEST database. We found that the highest number of CesA reads came from the root and stem libraries. The genes with the greatest number of reads were those involved in cell wall hydrolases (e.g. beta-1,3-glucanases, xyloglucan endo-beta-transglycosylase, beta-glucosidase and endo-beta-mannanase. Correlation analyses by surface mapping revealed that the expression of genes related to biosynthesis seems to be associated with the hydrolysis of hemicelluloses, pectin hydrolases being mainly associated with xyloglucan hydrolases. The patterns of cell wall related gene expression in sugarcane based on the number of reads per cluster reflected quite well the expected physiological characteristics of the tissues. This is the first work to provide a general view on plant cell wall metabolism through the expression of related genes in almost all the tissues of a plant at the same time. For example, developing flowers behaved similarly to both meristematic tissues and leaf-root transition zone tissues. Besides providing a basis for future research on the mechanisms of plant development which involve the cell wall, our findings will provide valuable tools for plant engineering in the

A zebrafish model of congenital disorders of glycosylation with phosphomannose isomerase deficiency reveals an early opportunity for corrective mannose supplementation

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Jaime Chu

2013-01-01

Individuals with congenital disorders of glycosylation (CDG have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO, which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf, which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy

Macrocyclic bis-thioureas catalyze stereospecific glycosylation reactions.

Science.gov (United States)

Park, Yongho; Harper, Kaid C; Kuhl, Nadine; Kwan, Eugene E; Liu, Richard Y; Jacobsen, Eric N

2017-01-13

Carbohydrates are involved in nearly all aspects of biochemistry, but their complex chemical structures present long-standing practical challenges to their synthesis. In particular, stereochemical outcomes in glycosylation reactions are highly dependent on the steric and electronic properties of coupling partners; thus, carbohydrate synthesis is not easily predictable. Here we report the discovery of a macrocyclic bis-thiourea derivative that catalyzes stereospecific invertive substitution pathways of glycosyl chlorides. The utility of the catalyst is demonstrated in the synthesis of trans-1,2-, cis-1,2-, and 2-deoxy-β-glycosides. Mechanistic studies are consistent with a cooperative mechanism in which an electrophile and a nucleophile are simultaneously activated to effect a stereospecific substitution reaction. Copyright © 2017, American Association for the Advancement of Science.

O-GLYCOBASE version 4.0: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Gupta, Ramneek; Birch, Hanne; Rapacki, Krzysztof

1999-01-01

O-GLYCBASE is a database of glycoproteins with O-linked glycosylation sites. Entries with at least one experimentally verified O-glycosylation site have been complied from protein sequence databases and literature. Each entry contains information about the glycan involved, the species, sequence, ...

Identification of a dithiol-dependent nucleoside triphosphate hydrolase in Sarcocystis neurona.

Science.gov (United States)

Zhang, Deqing; Gaji, Rajshekhar Y; Howe, Daniel K

2006-09-01

A putative nucleoside triphosphate hydrolase (NTPase) gene was identified in a database of expressed sequence tags (ESTs) from the apicomplexan parasite Sarcocystis neurona. Analysis of culture-derived S. neurona merozoites demonstrated a dithiol-dependent NTPase activity, consistent with the presence of a homologue to the TgNTPases of Toxoplasma gondii. A complete cDNA was obtained for the S. neurona gene and the predicted amino acid sequence shared 38% identity with the two TgNTPase isoforms from T. gondii. Based on the obvious homology, the S. neurona protein was designated SnNTP1. The SnNTP1 cDNA encodes a polypeptide of 714 amino acids with a predicted 22-residue signal peptide and an estimated mature molecular mass of 70kDa. Southern blot analysis of the SnNTP1 locus revealed that the gene exists as a single copy in the S. neurona genome, unlike the multiple gene copies that have been observed in T. gondii and Neospora caninum. Analyses of the SnNTP1 protein demonstrated that it is soluble and secreted into the culture medium by extracellular merozoites. Surprisingly, indirect immunofluorescence analysis of intracellular S. neurona revealed apical localisation of SnNTP1 and temporal expression characteristics that are comparable with the microneme protein SnMIC10. The absence of SnNTP1 during much of endopolygeny implies that this protein does not serve a function during intracellular growth and development of S. neurona schizonts. Instead, SnNTP1 may play a role in events that occur during or proximal to merozoite egress from and/or invasion into cells.

SEM visualization of glycosylated surface molecules using lectin-coated microspheres

Science.gov (United States)

Duke, J.; Janer, L.; Campbell, M.

1985-01-01

There are several techniques currently used to localize glycosylated surface molecules by scanning electron microscopy (Grinnell, 1980; Molday, 1976; Linthicum and Sell, 1975; Nicolson, 1974; Lo Buglio, et al, 1972). A simple and rapid method, using a modification of Grinnell's technique is reported here. Essentially, microspheres coated with Concavalin A are used to bind to glycosylated regions of the palatal shelf epithelium and are visualized in the scanning electron microscope (SEM).

Glycosylation of KSHV Encoded vGPCR Functions in Its Signaling and Tumorigenicity

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Hui Wu

2015-03-01

Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV is a tumor virus and the etiologic agent of Kaposi’s Sarcoma (KS. KSHV G protein-coupled receptor (vGPCR is an oncogene that is implicated in malignancies associated with KHSV infection. In this study, we show that vGPCR undergoes extensive N-linked glycosylation within the extracellular domains, specifically asparagines 18, 22, 31 and 202. An immunofluorescence assay demonstrates that N-linked glycosylation are necessary for vGPCR trafficking to the cellular membrane. Employing vGPCR mutants whose glycosylation sites were ablated, we show that these vGPCR mutants failed to activate downstream signaling in cultured cells and were severely impaired to induce tumor formation in the xenograph nude mouse model. These findings support the conclusion that glycosylation is critical for vGPCR tumorigenesis and imply that chemokine regulation at the plasma membrane is crucial for vGPCR mediated signaling.

O-GLYCBASE: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Hansen, Jan; Lund, Ole; Nielsen, Jens O.

1996-01-01

O-GLYCBASE is a comprehensive database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the SWISS-PROT and PIR databases as well as directly from recently published reports. Nineteen percent of the entries extracted from the databases n...... of mucin type O-glycosylation sites in mammalian glycoproteins exclusively from the primary sequence is made available by E-mail or WWW. The O-GLYCBASE database is also available electronically through our WWW server or by anonymous FTP....

Structural and Functional Consequences of Increased Tubulin Glycosylation in Diabetes Mellitus

Science.gov (United States)

Williams, Stuart K.; Howarth, Nancy L.; Devenny, James J.; Bitensky, Mark W.

1982-11-01

The extent of in vitro nonenzymatic glycosylation of purified rat brain tubulin was dependent on time and glucose concentration. Tubulin glycosylation profoundly inhibited GTP-dependent tubulin polymerization. Electron microscopy and NaDodSO4/polyacrylamide gel electrophoresis showed that glycosylated tubulin forms high molecular weight amorphous aggregates that are not disrupted by detergents or reducing agents. The amount of covalently bound NaB3H4-reducible sugars in tubulin recovered from brain of streptozotocin-induced diabetic rats was dramatically increased as compared with tubulin recovered from normal rat brain. Moreover, tubulin recovered from diabetic rat brain exhibited less GTP-induced polymerization than tubulin from nondiabetic controls. The possible implications of these data for diabetic neuropathy are discussed.

Cloning and expression of gene encoding P23 protein from Cryptosporidium parvum

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Dinh Thi Bich Lan

2014-12-01

Full Text Available We cloned the cp23 gene coding P23 (glycoprotein from Cryptosporidium parvum isolated from Thua Thien Hue province, Vietnam. The coding region of cp23 gene from C. parvum is 99% similar with cp23 gene deposited in NCBI (accession number: U34390. SDS-PAGE and Western blot analysis showed that the cp23 gene in E. coli BL21 StarTM (DE3 produced polypeptides with molecular weights of approximately 37, 40 and 49 kDa. These molecules may be non-glycosylated or glycosylated P23 fusion polypeptides. Recombinant P23 protein purified by GST (glutathione S-transferase affinity chromatography can be used as an antigen for C. parvum antibody production as well as to develop diagnostic kit for C. parvum.

O-GLYCBASE version 3.0: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Hansen, Jan; Lund, Ole; Nilsson, Jette

1998-01-01

O-GLYCBASE is a revised database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the literature, and from the sequence databases. Entries include informations about species, sequence, glycosylation sites and glycan type and is fully cr...

Glycosyltransferase Gene Expression Profiles Classify Cancer Types and Propose Prognostic Subtypes

Science.gov (United States)

Ashkani, Jahanshah; Naidoo, Kevin J.

2016-05-01

Aberrant glycosylation in tumours stem from altered glycosyltransferase (GT) gene expression but can the expression profiles of these signature genes be used to classify cancer types and lead to cancer subtype discovery? The differential structural changes to cellular glycan structures are predominantly regulated by the expression patterns of GT genes and are a hallmark of neoplastic cell metamorphoses. We found that the expression of 210 GT genes taken from 1893 cancer patient samples in The Cancer Genome Atlas (TCGA) microarray data are able to classify six cancers; breast, ovarian, glioblastoma, kidney, colon and lung. The GT gene expression profiles are used to develop cancer classifiers and propose subtypes. The subclassification of breast cancer solid tumour samples illustrates the discovery of subgroups from GT genes that match well against basal-like and HER2-enriched subtypes and correlates to clinical, mutation and survival data. This cancer type glycosyltransferase gene signature finding provides foundational evidence for the centrality of glycosylation in cancer.

Patterns of glycemic control using glycosylated hemoglobin in diabetics.

Science.gov (United States)

Kahlon, Arunpreet Singh; Pathak, Rambha

2011-07-01

Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS) is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3%) had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came out to be .311. This correlation was found to be statistically

ORF Alignment: NC_006274 [GENIUS II[Archive

Lifescience Database Archive (English)

Full Text Available NC_006274 gi|52142820 >1kwfA 3 362 53 435 5e-85 ... ref|YP_084010.1| chitosanase; gly...cosyl hydrolases family 8; endoglucanase [Bacillus ... cereus ZK] gb|AAU17839.1| chitosanase; glycosy

Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

Science.gov (United States)

Zhu, Zhikai; Desaire, Heather

2015-07-01

Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains.

Science.gov (United States)

Chigira, Yuko; Oka, Takuji; Okajima, Tetsuya; Jigami, Yoshifumi

2008-04-01

Development of a heterologous system for the production of homogeneous sugar structures has the potential to elucidate structure-function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose. This system allowed identification of an endogenous ability of S. cerevisiae to transport GDP-fucose. Moreover, expression of EGF domain mutants in this system revealed the different contribution of three disulfide bonds to in vivo O-fucosylation. In addition, lectin blotting revealed differences in the ability of fucose-specific lectin to bind the O-fucosylated structure of EGF domains from human factors VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated the addition of N-acetylglucosamine to the EGF domain from factor IX but not from factor VII. The results suggest that engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains.

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

Energy Technology Data Exchange (ETDEWEB)

Palmieri, D. [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy); Valli, M.; Viglio, S. [Department of Biochemistry, University of Pavia (Italy); Ferrari, N. [Istituto Nazionale per la ricerca sul Cancro, Genova (Italy); Ledda, B.; Volta, C. [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy); Manduca, P., E-mail: man-via@unige.it [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy)

2010-03-10

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

International Nuclear Information System (INIS)

Palmieri, D.; Valli, M.; Viglio, S.; Ferrari, N.; Ledda, B.; Volta, C.; Manduca, P.

2010-01-01

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

Identification and characterization of some Aspergillus pectinolytic glycoside hydrolases

NARCIS (Netherlands)

Zandleven, J.S.

2006-01-01

Keywords: Aspergillusniger , Arabidopsis thaliana , homogalacturonan, rhamnogalacturonan, xylogalacturonan, xylogalacturonan hydrolase, exo-polygalacturonasePectinases are used for many food

Enantioselectivity of a recombinant epoxide hydrolase from Agrobacterium radiobacter

NARCIS (Netherlands)

Lutje Spelberg, Jeffrey H.; Rink, Rick; Kellogg, Richard M.; Janssen, Dick B.

1998-01-01

The recombinant epoxide hydrolase from Agrobacterium radiobacter AD1 was used to obtain enantiomerically pure epoxides by means of a kinetic resolution. Epoxides such as styrene oxide and various derivatives thereof and phenyl glycidyl ether were obtained in high enantiomeric excess and in

Construction of a rice glycoside hydrolase phylogenomic database and identification of targets for biofuel research

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Rita eSharma

2013-08-01

Full Text Available Glycoside hydrolases (GH catalyze the hydrolysis of glycosidic bonds in cell wall polymers and can have major effects on cell wall architecture. Taking advantage of the massive datasets available in public databases, we have constructed a rice phylogenomic database of GHs (http://ricephylogenomics.ucdavis.edu/cellwalls/gh/. This database integrates multiple data types including the structural features, orthologous relationships, mutant availability and gene expression patterns for each GH family in a phylogenomic context. The rice genome encodes 437 GH genes classified into 34 families. Based on pairwise comparison with eight dicot and four monocot genomes, we identified 138 GH genes that are highly diverged between monocots and dicots, 57 of which have diverged further in rice as compared with four monocot genomes scanned in this study. Chromosomal localization and expression analysis suggest a role for both whole-genome and localized gene duplications in expansion and diversification of GH families in rice. We examined the meta-profiles of expression patterns of GH genes in twenty different anatomical tissues of rice. Transcripts of 51 genes exhibit tissue or developmental stage-preferential expression, whereas, seventeen other genes preferentially accumulate in actively growing tissues. When queried in RiceNet, a probabilistic functional gene network that facilitates functional gene predictions, nine out of seventeen genes form a regulatory network with the well-characterized genes involved in biosynthesis of cell wall polymers including cellulose synthase and cellulose synthase-like genes of rice. Two-thirds of the GH genes in rice are up regulated in response to biotic and abiotic stress treatments indicating a role in stress adaptation. Our analyses identify potential GH targets for cell wall modification.

Distribution of events of positive selection and population differentiation in a metabolic pathway: the case of asparagine N-glycosylation

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Dall’Olio Giovanni

2012-06-01

Full Text Available Abstract Background Asparagine N-Glycosylation is one of the most important forms of protein post-translational modification in eukaryotes. This metabolic pathway can be subdivided into two parts: an upstream sub-pathway required for achieving proper folding for most of the proteins synthesized in the secretory pathway, and a downstream sub-pathway required to give variability to trans-membrane proteins, and involved in adaptation to the environment and innate immunity. Here we analyze the nucleotide variability of the genes of this pathway in human populations, identifying which genes show greater population differentiation and which genes show signatures of recent positive selection. We also compare how these signals are distributed between the upstream and the downstream parts of the pathway, with the aim of exploring how forces of population differentiation and positive selection vary among genes involved in the same metabolic pathway but subject to different functional constraints. Results Our results show that genes in the downstream part of the pathway are more likely to show a signature of population differentiation, while events of positive selection are equally distributed among the two parts of the pathway. Moreover, events of positive selection are frequent on genes that are known to be at bifurcation points, and that are identified as being in key position by a network-level analysis such as MGAT3 and GCS1. Conclusions These findings indicate that the upstream part of the Asparagine N-Glycosylation pathway has lower diversity among populations, while the downstream part is freer to tolerate diversity among populations. Moreover, the distribution of signatures of population differentiation and positive selection can change between parts of a pathway, especially between parts that are exposed to different functional constraints. Our results support the hypothesis that genes involved in constitutive processes can be expected to show

Fasting serum glucose and glycosylated hemoglobin level in obesity.

Science.gov (United States)

Das, R K; Nessa, A; Hossain, M A; Siddiqui, N I; Hussain, M A

2014-04-01

Obesity is a condition in which the body fat stores are increased to an extent which impairs health and leads to serious health consequences. The amount of body fat is difficult to measure directly, and is usually determined from an indirect measure - the body mass index (BMI). Increased BMI in obese persons is directly associated with an increase in metabolic disease, such as type 2 diabetes mellitus. This Analytical cross sectional study was undertaken to assess the relation between obesity and glycemic control of body by measuring fasting serum glucose and glycosylated hemoglobin. This study was carried out in the Department of Physiology, Mymensingh Medical College, Mymensingh from 1st July 2011 to 30th June 2012 on 120 equally divided male and female persons within the age range of 25 to 55 years. Age more than 55 years and less than 25 years and diagnosed case of Hypothyroidism, Cushing's syndrome, polycystic ovary, Antipsychotic drug user and regular steroid users were excluded. Non probability purposive type of sampling technique was used for selecting the study subjects. Measurement of body mass index was done as per procedure. Fasting serum glucose was estimated by glucose oxidase method and Glycosylated hemoglobin by Boronate Affinity method. Statistical analysis was done by SPSS (version 17.0). Data were expressed as Mean±SE and statistical significance of difference among the groups were calculated by unpaired student's 't' test and Pearson's correlation coefficient tests were done as applicable. The Mean±SE of fasting serum glucose was significant at 1% level (P value obese group of BMI. There was no significant difference of glycosylated hemoglobin level between control and study groups. But there was positive correlation within each group. Fasting serum glucose also showed a bit stronger positive correlation with BMI. Both obese male and female persons showed higher levels of fasting serum glucose and glycosylated hemoglobin. The observed positive

Involvement of Aberrant Glycosylation in Thyroid Cancer

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Eiji Miyoshi

2010-01-01

Full Text Available Glycosylation is one of the most common posttranslational modification reactions and nearly half of all known proteins in eukaryotes are glycosylated. In fact, changes in oligosaccharides structures are associated with many physiological and pathological events, including cell growth, migration and differentiation, and tumor invasion. Therefore, functional glycomics, which is a comprehensive study of the structures and functions of glycans, is attracting the increasing attention of scientists in various fields of life science. In cases of thyroid cancer, the biological characters and prognosis are completely different in each type of histopathology, and their oligosaccharide structures as well as the expression of glycosyltransferases are also different. In this review, we summarized our previous papers on oligosaccharides and thyroid cancers and discussed a possible function of oligosaccharides in the carcinogenesis in thyroid cancer.

Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

DEFF Research Database (Denmark)

Kari, Jeppe; Olsen, Johan Pelck; Borch, Kim

2014-01-01

Cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme mixtures used for deconstruction of lignocellulosic biomass. Iden...

An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.

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Julius W Kim

Full Text Available Vectors based on human adenovirus serotype 5 (HAdV-5 continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting.As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4. This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells.These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

Properties of epoxide hydrolase from the yeast Rhodotorula glutinis

NARCIS (Netherlands)

Ariës-Kronenburg, N.A.E.

2002-01-01

Epoxide hydrolases are ubiquitous enzymes that can be found in nearly all living organisms. Some of the enzymes play an important role in detoxifying xenobiotic and metabolic compounds. Others are important in the growth of organisms like