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Svein Valla - One of the best experts on this subject based on the ideXlab platform.
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structural and functional characterization of the r modules in alginate c 5 epimerases alge4 and alge6 from azotobacter vinelandii
Journal of Biological Chemistry, 2014Co-Authors: Svein Valla, Gudmund Skjakbraek, Anne Tondervik, Olav Andreas Aarstad, Edith Buchinger, Daniel H Knudsen, Manja A Behrens, Jan Skov Pedersen, Reinhard WimmerAbstract:The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7). These epimerases are responsible for the epimerization of β-d-mannuronic Acid (M) to α-L-Guluronic Acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.
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isolation of mutant alginate lyases with cleavage specificity for di guluronic Acid linkages
Journal of Biological Chemistry, 2010Co-Authors: Anne Tondervik, Svein Valla, Gudmund Skjakbraek, Helga Ertesvag, Geir Klinkenberg, Olav Andreas Aarstad, Finn Drablos, Trond E Ellingsen, Havard SlettaAbstract:Abstract Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1–4-linked β-d-mannuronic Acid (M) and α-L-Guluronic Acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ∼7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca2+, one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ∼10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II′ suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure.
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characterization of three new azotobacter vinelandii alginate lyases one of which is involved in cyst germination
Journal of Bacteriology, 2009Co-Authors: Martin Gimmestad, Helga Ertesvag, Britt Iren Glaerum Svanem, Olav Andreas Aarstad, Tonje Marita Bjerkan Heggeset, Svein VallaAbstract:Alginates are polysaccharides composed of 1-4-linked β-d-mannuronic Acid and α-L-Guluronic Acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2+. All three enzymes preferably cleave the bond between guluronic Acid and mannuronic Acid, resulting in a guluronic Acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.
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Characterization of three new Azotobacter vinelandii alginate lyases, one of which is involved in cyst germination
2009Co-Authors: Martin Gimmestad, Helga Ertesvag, Olav Andreas Aarstad, Tonje Marita Bjerkan Heggeset, Britt Iren, Glærum Svanem, Svein VallaAbstract:Alginates are polysaccharides composed of 1-4-linked -D-mannuronic Acid and -L-Guluronic Acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a -elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2. All three enzymes preferably cleave the bond between guluronic Acid and mannuronic Acid, resulting in a guluronic Acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinat
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structural and mutational characterization of the catalytic a module of the mannuronan c 5 epimerase alge4 from azotobacter vinelandii
Journal of Biological Chemistry, 2008Co-Authors: Henriette J Rozeboom, Svein Valla, Tonje M Bjerkan, Kor H Kalk, Synnove Holtan, Bauke W DijkstraAbstract:Abstract Alginate is a family of linear copolymers of (1→4)-linked β-d-mannuronic Acid and its C-5 epimer α-L-Guluronic Acid. The polymer is first produced as polymannuronic Acid and the guluronic Acid residues are then introduced at the polymer level by mannuronan C-5-epimerases. The structure of the catalytic A-module of the Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 has been determined by x-ray crystallography at 2.1-A resolution. AlgE4A folds into a right-handed parallel β-helix structure originally found in pectate lyase C and subsequently in several polysaccharide lyases and hydrolases. The β-helix is composed of four parallel β-sheets, comprising 12 complete turns, and has an amphipathic α-helix near the N terminus. The catalytic site is positioned in a positively charged cleft formed by loops extending from the surface encompassing Asp152, an amino Acid previously shown to be important for the reaction. Site-directed mutagenesis further implicates Tyr149, His154, and Asp178 as being essential for activity. Tyr149 probably acts as the proton acceptor, whereas His154 is the proton donor in the epimerization reaction.
Helga Ertesvag - One of the best experts on this subject based on the ideXlab platform.
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functional characterization of three azotobacter chroococcum alginate modifying enzymes related to the azotobacter vinelandii alge mannuronan c 5 epimerase family
Scientific Reports, 2020Co-Authors: Agnieszka Maria Gawin, Olav Andreas Aarstad, Finn Lillelund Aachmann, Lisa Tietze, Trygve Brautaset, Helga ErtesvagAbstract:Bacterial alginate initially consists of 1-4-linked β-D-mannuronic Acid residues (M) which can be later epimerized to α-L-Guluronic Acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A. chroococcum genes, here designated AcalgE1, AcalgE2 and AcalgE3, were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions - 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.
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isolation of mutant alginate lyases with cleavage specificity for di guluronic Acid linkages
Journal of Biological Chemistry, 2010Co-Authors: Anne Tondervik, Svein Valla, Gudmund Skjakbraek, Helga Ertesvag, Geir Klinkenberg, Olav Andreas Aarstad, Finn Drablos, Trond E Ellingsen, Havard SlettaAbstract:Abstract Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1–4-linked β-d-mannuronic Acid (M) and α-L-Guluronic Acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ∼7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca2+, one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ∼10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II′ suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure.
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characterization of three new azotobacter vinelandii alginate lyases one of which is involved in cyst germination
Journal of Bacteriology, 2009Co-Authors: Martin Gimmestad, Helga Ertesvag, Britt Iren Glaerum Svanem, Olav Andreas Aarstad, Tonje Marita Bjerkan Heggeset, Svein VallaAbstract:Alginates are polysaccharides composed of 1-4-linked β-d-mannuronic Acid and α-L-Guluronic Acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a β-elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2+. All three enzymes preferably cleave the bond between guluronic Acid and mannuronic Acid, resulting in a guluronic Acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2 mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinate poorly compared to wild-type cells.
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Characterization of three new Azotobacter vinelandii alginate lyases, one of which is involved in cyst germination
2009Co-Authors: Martin Gimmestad, Helga Ertesvag, Olav Andreas Aarstad, Tonje Marita Bjerkan Heggeset, Britt Iren, Glærum Svanem, Svein VallaAbstract:Alginates are polysaccharides composed of 1-4-linked -D-mannuronic Acid and -L-Guluronic Acid. The polymer can be degraded by alginate lyases, which cleave the polysaccharide using a -elimination reaction. Two such lyases have previously been identified in the soil bacterium Azotobacter vinelandii, as follows: the periplasmic AlgL and the secreted bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7. In this work, we describe the properties of three new lyases from this bacterium, AlyA1, AlyA2, and AlyA3, all of which belong to the PL7 family of polysaccharide lyases. One of the enzymes, AlyA3, also contains a C-terminal module similar to those of proteins secreted by a type I secretion system, and its activity is stimulated by Ca2. All three enzymes preferably cleave the bond between guluronic Acid and mannuronic Acid, resulting in a guluronic Acid residue at the new reducing end, but AlyA3 also degrades the other three possible bonds in alginate. Strains containing interrupted versions of alyA1, alyA3, and algE7 were constructed, and their phenotypes were analyzed. Genetically pure alyA2mutants were not obtained, suggesting that this gene product may be important for the bacterium during vegetative growth. After centrifugation, cultures from the algE7 mutants form a large pellet containing alginate, indicating that AlgE7 is involved in the release of alginate from the cells. Upon encountering adverse growth conditions, A. vinelandii will form a resting stage called cyst. Alginate is a necessary part of the protective cyst coat, and we show here that strains lacking alyA3 germinat
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construction and analyses of hybrid azotobacter vinelandii mannuronan c 5 epimerases with new epimerization pattern characteristics
Biochemical Journal, 2004Co-Authors: Tonje M Bjerkan, Svein Valla, Gudmund Skjakbraek, Wenche Strand, Bjorn E Lillehov, Helga ErtesvagAbstract:The secreted mannuronan C-5 epimerases from Azotobacter vinelandii form a family of seven homologous modular type enzymes, which appear to have evolved through duplications and point mutations in the individual modules. The catalytic A modules of these enzymes are responsible for generating the characteristic sequence distribution patterns of G residues in the industrially important polymer alginate by epimerizing M (β-D-mannuronic Acid) moieties to G (α-L-Guluronic Acid). Forty-six different hybrid enzymes were constructed by exchanging parts of the sequences encoding the A modules of AlgE2 (generates consecutive stretches of G residues) and AlgE4 (generates alternating structures). These hybrid enzymes introduce a variety of new monomer-sequence patterns into their substrates, and some regions important for the subsite specificity or processivity of the enzymes were identified. By using time-resolved NMR spectroscopy, it became clear that the rates for introducing alternating structures and consecutive stretches of G residues are different for each enzyme, and that it is the ratio between these rates that determines the overall epimerization pattern. These findings open up new possibilities in biotechnology and in studies of the many biological functions of alginates.
Gudmund Skjakbraek - One of the best experts on this subject based on the ideXlab platform.
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structural and functional characterization of the r modules in alginate c 5 epimerases alge4 and alge6 from azotobacter vinelandii
Journal of Biological Chemistry, 2014Co-Authors: Svein Valla, Gudmund Skjakbraek, Anne Tondervik, Olav Andreas Aarstad, Edith Buchinger, Daniel H Knudsen, Manja A Behrens, Jan Skov Pedersen, Reinhard WimmerAbstract:The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7). These epimerases are responsible for the epimerization of β-d-mannuronic Acid (M) to α-L-Guluronic Acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.
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heparin like properties of sulfated alginates with defined sequences and sulfation degrees
Biomacromolecules, 2014Co-Authors: Oystein Arlov, Anders Sundan, Terje Espevik, Gudmund SkjakbraekAbstract:Sulfated glycosaminoglycans have a vast range of protein interactions relevant to the development of new biomaterials and pharmaceuticals, but their characterization and application is complicated mainly due to a high structural variability and the relative difficulty to isolate large quantities of structurally homogeneous samples. Functional and versatile analogues of heparin/heparan sulfate can potentially be created from sulfated alginates, which offer structure customizability through targeted enzymatic epimerization and precise tuning of the sulfation degree. Alginates are linear polysaccharides consisting of β-d-mannuronic Acid (M) and α-L-Guluronic Acid (G), derived from brown algae and certain bacteria. The M/G ratio and distribution of blocks are critical parameters for the physical properties of alginates and can be modified in vitro using mannuronic-C5-epimerases to introduce sequence patterns not found in nature. Alginates with homogeneous sequences (poly-M, poly-MG, and poly-G) and similar mo...
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heparin like properties of sulfated alginates with defined sequences and sulfation degrees
Biomacromolecules, 2014Co-Authors: Oystein Arlov, Anders Sundan, Terje Espevik, Finn Lillelund Aachmann, Gudmund SkjakbraekAbstract:Sulfated glycosaminoglycans have a vast range of protein interactions relevant to the development of new biomaterials and pharmaceuticals, but their characterization and application is complicated mainly due to a high structural variability and the relative difficulty to isolate large quantities of structurally homogeneous samples. Functional and versatile analogues of heparin/heparan sulfate can potentially be created from sulfated alginates, which offer structure customizability through targeted enzymatic epimerization and precise tuning of the sulfation degree. Alginates are linear polysaccharides consisting of β-D-mannuronic Acid (M) and α-L-Guluronic Acid (G), derived from brown algae and certain bacteria. The M/G ratio and distribution of blocks are critical parameters for the physical properties of alginates and can be modified in vitro using mannuronic-C5-epimerases to introduce sequence patterns not found in nature. Alginates with homogeneous sequences (poly-M, poly-MG, and poly-G) and similar molecular weights were chemically sulfated and structurally characterized by the use of NMR and elemental analysis. These sulfated alginates were shown to bind and displace HGF from the surface of myeloma cells in a manner similar to heparin. We observed dependence on the sulfation degree (DS) as well as variation in efficacy based on the alginate monosaccharide sequence, relating to relative flexibility and charge density in the polysaccharide chains. Co-incubation with human plasma showed complement compatibility of the alginates and lowering of soluble terminal complement complex levels by sulfated alginates. The sulfated polyalternating (poly-MG) alginate proved to be the most reproducible in terms of precise sulfation degrees and showed the greatest relative degree of complement inhibition and HGF interaction, maintaining high activity at low DS values.
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alginates induce legumain activity in raw 264 7 cells and accelerate autoactivation of prolegumain
Bioactive Carbohydrates and Dietary Fibre, 2013Co-Authors: Lise Berven, Gudmund Skjakbraek, Oystein Arlov, Finn Lillelund Aachmann, Rigmor Solberg, Hong Hoa Thi Truong, Wolfgang Eggejacobsen, Harald Thidemann Johansen, Anne Berit SamuelsenAbstract:Alginates belong to a family of linear co-polymers of 1→4-linked β-d-mannuronic Acid (M) and α-L-Guluronic Acid (G). Here we show that alginates are able to increase the activity of the lysosomal cysteine protease legumain in RAW 264.7 cells, but with different potency; Alginates with 75% M>60% M≈50% M>100% M≈94% M. Activity is lost upon treatment with alginate lyase and chemical reduction of alginate carboxylic Acid groups showing that molecular size as well at the polymer charges/conformation are essential for activity. Heparin and a sulfated alginate were the most potent activators of legumain activity, but the least potent TNF-α inducing agents tested. Alginate with 94% M had the highest TNF-α inducing activity in RAW 264.7 cells. Legumain participates in several biological and pathological processes including tumor invasion and metastasis. It is synthesized as a zymogen and undergoes pH-dependent autoactivation of the proform in order to reach an enzymatically active form. Alginates that may enter the lysosomes via micropinocytosis interact with prolegumain directly. Here we show that alginates through ionic interactions accelerate the autocatalytic activation of prolegumain at pH 4.0 and 5.0. At pH 5.5 only the alginate with 75% M was able to promote prolegumain activation.
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alginate sequencing an analysis of block distribution in alginates using specific alginate degrading enzymes
Biomacromolecules, 2012Co-Authors: Olav Andreas Aarstad, Anne Tondervik, Havard Sletta, Gudmund SkjakbraekAbstract:Distribution and proportion of β-d-mannuronic and α-L-Guluronic Acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, which is based on NMR, provides a statistical description of alginates. In this work, a method was developed that also gives information of the distribution of block lengths of each of the three block types (M, G, and MG blocks). This was achieved using a combination of alginate lyases with different substrate specificities, including a novel lyase that specifically cleaves diguluronic Acid linkages. Reaction products and isolated fragments of alginates degraded with these lyases were subsequently analyzed with 1H NMR, HPAEC-PAD, and SEC-MALLS. The method was applied on three seaweed alginates with large differences in sequence parameters (FG = 0.32 to 0.67). All samples contained considerable amounts of extremely long G blocks (DP > 100). The finding of long M blocks (DP ≥ 90) suggests that also algal epimerase...
Abbas Mirshafiey - One of the best experts on this subject based on the ideXlab platform.
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evaluation of the oral administration of α l guluronic Acid on cox 1 and cox 2 gene expression profile in ankylosing spondylitis patients
Drug Development Research, 2021Co-Authors: Arezoo Sadoughi, Sepideh Nazeri, Reza Mansouri, Abbas MirshafieyAbstract:Ankylosing spondylitis (AS) is a chronic autoimmune arthritis disease with a genetic background, affecting the skeletal axis, sacroiliac, and peripheral joints. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the first-line treatment for AS to alleviate the inflammation and pain. Despite the beneficial effect, their use is accompanied by a wide variety of possible side effects in the gastrointestinal and kidneys. The α-L-Guluronic Acid (G2013) is a new nonsteroidal anti-inflammatory patented (PCT/EP2017/067920) drug, which has shown its anti-inflammatory properties in the previous investigations. The present study revealed the oral administration effect of G2013 on COX-1 and COX-2 gene expression in AS patients. The blood samples of twelve 18-45 years old patients suffering AS and BASDAI >4, and BASFI >4, before and after 12 weeks of treatment with G2013 and 12 blood samples of healthy volunteers were collected and the effect of G2013 on the gene expression of COX-1 and COX-2 enzymes were assessed by Real-Time PCR. The results indicate that G2013 is able to reduce the gene expression level of COX-1 and COX-2 enzymes in treated AS patients compared to healthy control. Statistically significant differences were not observed between the treatment and the healthy control groups. According to the findings, G2013 might be categorized and introduced as a novel NSAID for the treatment of AS.
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effects of guluronic Acid g2013 on gene expression of tlr2 tlr4 myd88 tnf α and cd52 in multiple sclerosis under in vitro conditions
Immunopharmacology and Immunotoxicology, 2019Co-Authors: Seyedeh Masoomeh Noorbakhsh, Zahra Aghazadeh, Mona Oraei, Alireza Razavi, Nahid Beladi Moghadam, Payam Saadat, Mostafa Hoseini, Maryam Mobini, Abbas MirshafieyAbstract:AbstractContext: Multiple sclerosis (MS) is an autoimmune and chronic inflammatory disease of CNS. The α-L-Guluronic Acid (G2013) as novel NSAID with immunomodulatory effects has shown its positive...
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evaluation of the acute and 28 day sub acute intravenous toxicity of α l guluronic Acid alg g2013 in mice
Drug and Chemical Toxicology, 2019Co-Authors: Ahmad Mahdianshakib, Mohammad Sadegh Hashemzadeh, Ali Anissian, Mona Oraei, Abbas MirshafieyAbstract:α-L-Guluronic Acid (ALG; G2013) has been previously introduced as a new anti-inflammatory agent with promising therapeutic effects. Thus, in the present study, we aimed to evaluate the acute and su...
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the immunomodulatory role of g2013 α l guluronic Acid on the expression of tlr2 and tlr4 in ht29 cell line
Current Drug Discovery Technologies, 2019Co-Authors: Hamid Farhang, Laleh Sharifi, Saied Bokaie, Mona Moshiri, Mohammad Mehdi Soltan Dallal, Zahra Norouzbabaie, Somaye Aletaha, Seyed Jalal Zargar, Abbas MirshafieyAbstract:Background The non-steroidal anti-inflammatory drugs (NSAIDs) play crucial role in the controlling of inflammatory diseases. Due to the vast side effects of NSAIDs, its use is limited. G2013 or α-L-Guluronic Acid is a new NSAID with immunomodulatory features. Objectives Considering the leading role of TLRs in inflammatory responses, in this study, we aimed to evaluate G2013 cytotoxicity and its effect on the expression of TLR2 and TLR4 molecules. Methods HEK293-TLR2 and HEK293-TLR4 cells were cultured and seeded on 96-well cell plate, and MTT assay was performed for detecting the viability of the cells after treatment with different concentrations of G2013. HT29 cells were grown and treated with low and high doses of G2013. After total RNA extraction and cDNA synthesis, quantitative real-time PCR were performed to assess the TLR2 and TLR4 mRNA synthesis. Results We found that concentrations of ≤125 µg/ml of G2013 had no apparent cytotoxicity effect on the HEK293-TLR2 and -TLR4 cells. Our results indicated that after G2013 treatment (5 µg/ml) in HT29 cells, TLR2 and TLR4 mRNA expression decreased significantly compared with the untreated control group (p=0.02 and p=0.001 respectively). Conclusion The results of this study revealed that G2013 can down regulate the TLR2 and TLR4 gene expression and exerts its inhibitory effect. Our findings are parallel to our previous finding which showed G2013 ability to down regulate the signaling pathway of TLRs. However, further studies are needed to identify the molecular mechanism of G2013.
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antagonistic property of g2013 α l guluronic Acid on gene expression of myd88 tollip and nf κb in hek293 tlr2 and hek293 tlr4
Endocrine‚ Metabolic & Immune Disorders-Drug Targets, 2019Co-Authors: Laleh Sharifi, Asghar Aghamohammadi, Saied Bokaie, Gholamreza Azizi, Somaye Aletaha, Razieh Bigdeli, Vahid Asgary, Mohammad Hossein Asgardoon, Abbas MirshafieyAbstract:INTRODUCTION Inhibition of Toll-like receptors (TLRs) signaling plays a crucial role in suppressing the inflammation and available data presenting G2013 as an immunomodulatory agent, therefore, we designed this study to answer whether G2013 can affect the signaling pathway of TLR2 and TLR4. METHODS Cytotoxicity study of G2013 was performed by MTT assay. HEK293 TLR2 and HEK293 TLR4 cell lines were cultured and treated with low dose (5µg/ml) and high dose (25µg/ml) of G2013 for 24 hours. Gene expressions of MyD88, Tollip, and NF-κB were defined by quantitative real-time PCR. RESULTS The cytotoxicity assay showed that the concentrations lesser than 125μg/ml of G3012 had no apparent cytotoxicity, however, the concentrations of 5µg/ml and 25µg/ml could suppress the mRNA expression of MyD88, Tollip and NF-κB in HEK293 TLR2 and HEK293 TLR4 cell lines. CONCLUSION in our study, we verified the linkage between the immunosuppressive property of G2013 and TLR2, TLR4 signaling cascade; but so far, the specific target of G2013 and its molecular mechanism has not been detected yet. We recommend further studies on other Patten Recognition Receptors (PRRs)and other mechanisms of inflammation like oxidative stress to be conducted in the future.
Olav Andreas Aarstad - One of the best experts on this subject based on the ideXlab platform.
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functional characterization of three azotobacter chroococcum alginate modifying enzymes related to the azotobacter vinelandii alge mannuronan c 5 epimerase family
Scientific Reports, 2020Co-Authors: Agnieszka Maria Gawin, Olav Andreas Aarstad, Finn Lillelund Aachmann, Lisa Tietze, Trygve Brautaset, Helga ErtesvagAbstract:Bacterial alginate initially consists of 1-4-linked β-D-mannuronic Acid residues (M) which can be later epimerized to α-L-Guluronic Acid (G). The family of AlgE mannuronan C-5-epimerases from Azotobacter vinelandii has been extensively studied, and three genes putatively encoding AlgE-type epimerases have recently been identified in the genome of Azotobacter chroococcum. The three A. chroococcum genes, here designated AcalgE1, AcalgE2 and AcalgE3, were recombinantly expressed in Escherichia coli and the gene products were partially purified. The catalytic activities of the enzymes were stimulated by the addition of calcium ions in vitro. AcAlgE1 displayed epimerase activity and was able to introduce long G-blocks in the alginate substrate, preferentially by attacking M residues next to pre-existing G residues. AcAlgE2 and AcAlgE3 were found to display lyase activities with a substrate preference toward M-alginate. AcAlgE2 solely accepted M residues in the positions - 1 and + 2 relative to the cleavage site, while AcAlgE3 could accept either M or G residues in these two positions. Both AcAlgE2 and AcAlgE3 were bifunctional and could also catalyze epimerization of M to G. Together, we demonstrate that A. chroococcum encodes three different AlgE-like alginate-modifying enzymes and the biotechnological and biological impact of these findings are discussed.
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structural and functional characterization of the r modules in alginate c 5 epimerases alge4 and alge6 from azotobacter vinelandii
Journal of Biological Chemistry, 2014Co-Authors: Svein Valla, Gudmund Skjakbraek, Anne Tondervik, Olav Andreas Aarstad, Edith Buchinger, Daniel H Knudsen, Manja A Behrens, Jan Skov Pedersen, Reinhard WimmerAbstract:The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1–7). These epimerases are responsible for the epimerization of β-d-mannuronic Acid (M) to α-L-Guluronic Acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6.
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alginate sequencing an analysis of block distribution in alginates using specific alginate degrading enzymes
Biomacromolecules, 2012Co-Authors: Olav Andreas Aarstad, Anne Tondervik, Havard Sletta, Gudmund SkjakbraekAbstract:Distribution and proportion of β-d-mannuronic and α-L-Guluronic Acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, which is based on NMR, provides a statistical description of alginates. In this work, a method was developed that also gives information of the distribution of block lengths of each of the three block types (M, G, and MG blocks). This was achieved using a combination of alginate lyases with different substrate specificities, including a novel lyase that specifically cleaves diguluronic Acid linkages. Reaction products and isolated fragments of alginates degraded with these lyases were subsequently analyzed with 1H NMR, HPAEC-PAD, and SEC-MALLS. The method was applied on three seaweed alginates with large differences in sequence parameters (FG = 0.32 to 0.67). All samples contained considerable amounts of extremely long G blocks (DP > 100). The finding of long M blocks (DP ≥ 90) suggests that also algal epimerase...
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Alginate sequencing: an analysis of block distribution in alginates using specific alginate degrading enzymes.
Biomacromolecules, 2011Co-Authors: Olav Andreas Aarstad, Anne Tondervik, Havard Sletta, Gudmund Skjåk-brækAbstract:Distribution and proportion of β-d-mannuronic and α-L-Guluronic Acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, ...
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isolation of mutant alginate lyases with cleavage specificity for di guluronic Acid linkages
Journal of Biological Chemistry, 2010Co-Authors: Anne Tondervik, Svein Valla, Gudmund Skjakbraek, Helga Ertesvag, Geir Klinkenberg, Olav Andreas Aarstad, Finn Drablos, Trond E Ellingsen, Havard SlettaAbstract:Abstract Alginates are commercially valuable and complex polysaccharides composed of varying amounts and distribution patterns of 1–4-linked β-d-mannuronic Acid (M) and α-L-Guluronic Acid (G). This structural variability strongly affects polymer physicochemical properties and thereby both commercial applications and biological functions. One promising approach to alginate fine structure elucidation involves the use of alginate lyases, which degrade the polysaccharide by cleaving the glycosidic linkages through a β-elimination reaction. For such studies one would ideally like to have different lyases, each of which cleaves only one of the four possible linkages in alginates: G-G, G-M, M-G, and M-M. So far no lyase specific for only G-G linkages has been described, and here we report the construction of such an enzyme by mutating the gene encoding Klebsiella pneumoniae lyase AlyA (a polysaccharide lyase family 7 lyase), which cleaves both G-G and G-M linkages. After error-prone PCR mutagenesis and high throughput screening of ∼7000 lyase mutants, enzyme variants with a strongly improved G-G specificity were identified. Furthermore, in the absence of Ca2+, one of these lyases (AlyA5) was found to display no detectable activity against G-M linkages. G-G linkages were cleaved with ∼10% of the optimal activity under the same conditions. The substitutions conferring altered specificity to the mutant enzymes are located in conserved regions in the polysaccharide lyase family 7 alginate lyases. Structure-function analyses by comparison with the known three-dimensional structure of Sphingomonas sp. A1 lyase A1-II′ suggests that the improved G-G specificity might be caused by increased affinity for nonproductive binding of the alternating G-M structure.
