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Yuji Nagashima - One of the best experts on this subject based on the ideXlab platform.
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antibacterial action of l amino acid Oxidase from the skin mucus of rockfish sebastes schlegelii
Comparative Biochemistry and Physiology B, 2008Co-Authors: Yoichiro Kitani, Nobuyo Kikuchi, Guohua Zhang, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo Shiomi, Yuji NagashimaAbstract:Abstract L -Amino acid Oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H 2 O 2 generated in the oxidative process of L -amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H 2 O 2 . SSAP inhibited potently the growth of Aeromonas salmonicida , Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 μg/mL, respectively. H 2 O 2 inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P . damselae subsp. piscicida and Escherichia coli , SSAP was demonstrated to bind to P . damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H 2 O 2 induced cell surface damage to A. salmonicida , remarkable elongation of P . damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.
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identification of an antibacterial protein as l amino acid Oxidase in the skin mucus of rockfish sebastes schlegeli
FEBS Journal, 2007Co-Authors: Yoichiro Kitani, Guohua Zhang, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo Shiomi, Chihiro Tsukamoto, Hiroshi Nagai, Masami Ishida, Yuji NagashimaAbstract:Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3′ RACE, 5′ RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid Oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H2O2-generation assay and substrate specificity for only l-Lys with a Km of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H2O2 is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.
Ene Siigur - One of the best experts on this subject based on the ideXlab platform.
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L-Amino acid Oxidase from Naja naja oxiana venom.
Comparative Biochemistry and Physiology B, 2007Co-Authors: Mari Samel, Heiki Vija, Kulli Tonismagi, Gunilla Ronnholm, Juri Siigur, Nisse Kalkkinen, Ene SiigurAbstract:Abstract A new l -amino acid Oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l -amino acid Oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (− 20 °C, − 70 °C) and loses its activity by heating at 70 °C. Specific substrates for the isolated protein are l -phenylalanine, l -tryptophan, l -methionine and l -leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive ( Bacillus subtilis ) and Gram-negative ( Escherichia coli ) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC 50 of 0.094 μM and 0.036 μM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.
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l amino acid Oxidase from vipera lebetina venom isolation characterization effects on platelets and bacteria
Toxicon, 2006Co-Authors: Kulli Tonismagi, Mari Samel, Katrin Trummal, Gunilla Ronnholm, Juri Siigur, Nisse Kalkkinen, Ene SiigurAbstract:Abstract The l -amino acid Oxidase from Vipera lebetina venom was purified to homogeneity using combination of size exclusion, ion exchange and hydrophobic chromatography. The monomeric molecular mass of the homodimeric enzyme is 60.9 kDa. The N-terminal and the tryptic peptides share high homology with other snake venom l -amino acid Oxidases. The enzyme displays high specificity towards hydrophobic l -amino acids, the best substrates are l -Met, l -Trp, l -Leu followed by l -His, l -Phe, l -Arg and l -Ile. Six substrates—Gly, l -Ser, l -Thr, l -Pro, l -Cys, l -Asp—were not oxidized. The enzyme has antimicrobial activity inhibiting the growth of both Gram-negative and Gram-positive bacteria. V. lebetina LAAO dose-dependently inhibited platelet aggregation induced by ADP or collagen. In case of ADP-induced aggregation the inhibitory effect was more pronounced on the second wave of aggregation.
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isolation and characterization of an apoptotic and platelet aggregation inhibiting l amino acid Oxidase from vipera berus berus common viper venom
Biochimica et Biophysica Acta, 2006Co-Authors: Mari Samel, Heiki Vija, Gunilla Ronnholm, Juri Siigur, Nisse Kalkkinen, Ene SiigurAbstract:Abstract An l -amino acid Oxidase was isolated from the venom of the common viper Vipera berus berus by a three-step procedure combining gel filtration, ion exchange and hydrophobic chromatography. The enzyme is a non-covalently bound homodimer with a monomeric molecular mass of 57.7 kDa. The N-terminal amino acid sequence and the internal peptide sequences show close structural homology with other snake venom l -amino acid Oxidases. The purified protein catalyzed oxidative desamination of l -amino acids, the most specific substrate is l -Phe. The best substrates among the studied 20 amino acids were: l -Met, l -Leu, l -Phe, l -Ile, l -Arg and l -His. Five amino acids, l -Ser, l -Pro, Gly, l -Thr and l -Cys, were not oxidized. The enzyme inhibited ADP-induced platelet aggregation dose-dependently with an IC50 of 0.07 μM. The effect was neutralized by catalase. V. berus berus LAAO induced apoptosis in cultured HeLa and K562 cells as shown by DNA fragmentation gel pattern. The induction of apoptosis was inhibited by catalase.
Maxey C M Chung - One of the best experts on this subject based on the ideXlab platform.
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purification and properties of the l amino acid Oxidase from malayan pit viper calloselasma rhodostoma venom
Archives of Biochemistry and Biophysics, 1994Co-Authors: Gnanajothy Ponnudurai, Maxey C M ChungAbstract:Abstract The L-amino acid Oxidase of Malayan pit viper ( Calloselasma rhodostoma ) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was 132,000 as determined by Sephadex G-200 gel filtration chromatography and 66,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a glycoprotein, has an isoelectric point of 4.4, and contains 2 mol of flavin mononucleotide per mole of enzyme. The N-terminal amino acid sequence of the enzyme was A-D-D-R-N-P-L-A-E-E-F-Q-E-N-N-Y-E-E-F-L. Kinetic studies suggest the presence of a alkyl side-chain binding site in the enzyme and that the binding site comprises at least four hydrophobic subsites. The characteristics of the binding site differ slightly from those of cobra venom L-amino acid Oxidases.
Sandro Ghisla - One of the best experts on this subject based on the ideXlab platform.
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l-Amino acid Oxidase isolated from Calloselasma rhodostoma snake venom induces cytotoxicity and apoptosis in JAK2V617F-positive cell lines
Revista Brasileira De Hematologia E Hemoterapia, 2016Co-Authors: Cristiane Fernandes De Freitas Tavares, Thaís Fontanezi Maciel, Sandra Mara Burin, Luciana Ambrósio, Sandro Ghisla, Suely V. Sampaio, Fabíola Attié De CastroAbstract:BACKGROUND: Myeloproliferative neoplasms are Philadelphia chromosome-negative diseases characterized by hyperproliferation of mature myeloid cells, associated or not with the Janus kinase 2 tyrosine kinase mutation, JAK2V617F. As there is no curative therapy, researchers have been investigating new drugs to treat myeloproliferative neoplasms, including l-amino acid Oxidase from Calloselasma rhodostoma snake venom (CR-LAAO), which is a toxin capable of eliciting apoptosis in several tumor cell lines. OBJECTIVE: To evaluate the effects of l-amino acid Oxidase from C. rhodostoma snake venom in the apoptotic machinery of JAK2-mutated cell lines. METHODS: The HEL 92.1.7 and SET-2 cell lines were cultured with l-amino acid Oxidase and catalase for 12 h at 37 °C in 5% carbon dioxide. The cell viability was assessed by the multi-table tournament method, the level of apoptosis was measured by flow cytometry, and the expression of cysteine-dependent aspartate-specific proteases and cleaved Poly(ADP-ribose) polymerase were analyzed by Western blotting. RESULTS: l-Amino acid Oxidase from C. rhodostoma snake venom was cytotoxic to HEL 92.1.7 and SET-2 cells (50% inhibitory concentration = 0.15 µg/mL and 1.5 µg/mL, respectively) and induced apoptosis in a concentration-dependent manner. Cell treatment with catalase mitigated the l-amino acid Oxidase toxicity, indicating that hydrogen peroxide is a key component of its cytotoxic effect.The activated caspases 3 and 8 expression and cleaved PARP in HEL 92.1.7 and SET-2 cells confirmed the apoptosis activation by CR-LAAO. CONCLUSIONS: l-Amino acid Oxidase from C. rhodostoma snake venom is a potential antineoplastic agent against HEL 92.1.7 and SET-2 JAK2V617F-positive cells as it activates the extrinsic apoptosis pathway.
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induction of apoptosis in yeast by l amino acid Oxidase from the malayan pit viper calloselasma rhodostoma
Yeast, 2008Co-Authors: Sandro Ghisla, Sudharsana Rao Ande, Heike Fussi, Heide Knauer, Michael Murkovic, Kaiuwe Frohlich, Peter MacherouxAbstract:Here we report for the first time that L-amino acid Oxidase (LAAO), a major component of snake venom, induces apoptosis in yeast. The causative agent for induction of apoptosis has been shown to be hydrogen peroxide, produced by the enzymatic activity of LAAO. However, the addition of catalase, a specific hydrogen peroxide scavenger, does not prevent cell demise completely. Intriguingly, depletion of leucine from the medium by LAAO and the interaction of LAAO with yeast cells are shown to be the major factors responsible for cell demise in the presence of catalase. Copyright © 2008 John Wiley & Sons, Ltd.
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structure and characterization of the glycan moiety of l amino acid Oxidase from the malayan pit viper calloselasma rhodostoma
FEBS Journal, 2001Co-Authors: Armin Geyer, Sandro Ghisla, Teresa B Fitzpatrick, Peter D Pawelek, Karina Kitzing, Alice Vrielink, Peter MacherouxAbstract:Ophidian L-Amino-Acid Oxidase (L-Amino-Acid oxygen:oxidoreductase, deaminating, EC 1.4.3.2) is found in the venom of many poisonous snakes (crotalids, elapids and viperids). This FAD-dependent glycoprotein has been studied from several snake species (e.g. Crotalus adamanteus, Crotalus atrox and Calloselasma rhodostoma) in detail with regard to the biochemical and enzymatic properties. The nature of glycosylation, however, as well as the chemical structure(s) of the attached oligosaccharide(s) are unknown. In view of the putative involvement of the glycan moiety in the biological effects of ophidian L-Amino-Acid Oxidase, notably the apoptotic activity of the enzyme, structural knowledge is needed to evaluate its exact function. In this study we report on the glycosylation of L-Amino-Acid Oxidase from the venom of the Malayan pit viper (Calloselasma rhodostoma). Its glycosylation is remarkably homogeneous with the major oligosaccharide accounting for approximately 90% of the total sugar content. Based on detailed analysis of the isolated oligosaccharide by 2D NMR spectroscopies and MALDI-TOF mass spectrometry the glycan is identified as a bis-sialylated, biantennary, core-fucosylated dodecasaccharide. The biological significance of this finding is discussed in light of the biological activities of the enzyme.
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l amino acid Oxidase from the malayan pit viper calloselasma rhodostoma comparative sequence analysis and characterization of active and inactive forms of the enzyme
FEBS Journal, 2001Co-Authors: Peter Macheroux, Oliver Seth, Claus Bollschweiler, Margarete Schwarz, Manfred Kurfurst, Lochun Au, Sandro GhislaAbstract:Here we report the cDNA-deduced amino-acid sequence of L-Amino-Acid Oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FAD-dependent ophidian LAAOs to FAD-dependent Oxidases such as monoamine Oxidases, d-amino-acid Oxidases and tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine Oxidases, tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl−, prevent pH-induced inactivation. LAAO exhibits typical flavoprotein Oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.
Yoichiro Kitani - One of the best experts on this subject based on the ideXlab platform.
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antibacterial action of l amino acid Oxidase from the skin mucus of rockfish sebastes schlegelii
Comparative Biochemistry and Physiology B, 2008Co-Authors: Yoichiro Kitani, Nobuyo Kikuchi, Guohua Zhang, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo Shiomi, Yuji NagashimaAbstract:Abstract L -Amino acid Oxidase (LAO) shows broadly antibacterial activity against Gram-positive and Gram-negative bacteria by H 2 O 2 generated in the oxidative process of L -amino acids. However, LAO (termed SSAP) isolated from the rockfish Sebastes schlegelii skin mucus acted selectively on Gram-negative bacteria. Therefore, this study was undertaken to clarify the antibacterial action of SSAP as compared with H 2 O 2 . SSAP inhibited potently the growth of Aeromonas salmonicida , Photobacterium damselae subsp. piscicida and Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) of 0.078, 0.16 and 0.63 μg/mL, respectively. H 2 O 2 inhibited the growth of both Gram-positive and Gram-negative bacteria with an MIC ranging from 0.31 to 2.5 mM. When SSAP was incubated with P . damselae subsp. piscicida and Escherichia coli , SSAP was demonstrated to bind to P . damselae subsp. piscicida but not to E. coli by Western blotting and LAO activity measurement. These results show that the bacteria binding activity may be involved in the bacterial cell selectivity of SSAP. Electron microscopic observation of A. salmonicida, P. damselae subsp. piscicida and V. parahaemolyticus revealed that the treatments with SSAP and H 2 O 2 induced cell surface damage to A. salmonicida , remarkable elongation of P . damselae subsp. piscicida bodies and pores into V. parahaemolyticus cells.
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identification of an antibacterial protein as l amino acid Oxidase in the skin mucus of rockfish sebastes schlegeli
FEBS Journal, 2007Co-Authors: Yoichiro Kitani, Guohua Zhang, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo Shiomi, Chihiro Tsukamoto, Hiroshi Nagai, Masami Ishida, Yuji NagashimaAbstract:Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3′ RACE, 5′ RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid Oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H2O2-generation assay and substrate specificity for only l-Lys with a Km of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H2O2 is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.
