- Email: [email protected]
24. Purification, biochemical and molecular characterization of an L-amino acid oxidase from Cerastes cerastes snake venom
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Abstracts from 21st Meeting of the French Society of Toxinology (SFET) / Toxicon 91 (2014) 164e184
23. A novel toxin variant from the Androctonus australis hector scorpion venom selectively activates Kv7.4 channel Z. Landoulsi a, F. Miceli b, A. Palmese c, A. Amoresano c, G. Marino c, M. El Ayeb a, M. Taglialatela b, d, e, R. Benkhalifa a Laboratoire des Venins et Mol ecules Th erapeutiques, Institut Pasteur de Tunis, Universit e Tunis-El Manar, Tunis-Belv ed ere, Tunisia b Div. Pharmacology, Dept. of Neuroscience, University of Naples Federico II, Naples, Italy c Dept. of Chemical Sciences, University of Naples Federico II, Naples, Italy d Dept. of Medicine and Health Science, University of Molise, Campobasso, Italy (M.T.) e Unidad de Biofísica, Consejo Superior de Investigaciones Cientificas e Universidad del Pais Vasco, Leioa, Spain a
Kv7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells, skeletal and smooth muscle cells. Openers of Kv7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K+ channels; therefore, a combined purification/screening procedure has been optimized to identify specific modulator(s) of Kv7.4 channels from the venom of the North African scorpion Androctonus australis hector. We report, here, the isolation and functional characterization of AaTXKb(2-64), which acted as the first peptide activator of Kv7.4 channels. In particular, in both Xenopus oocytes and mammalian CHO cells, AaTXKb(2-64), hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologoulsyexpressed Kv7.4 channels. AaTXKb(2-64) also activated Kv7.3, Kv7.2/3, and Kv7.5/3 channels, whereas homomeric Kv1.1, Kv7.1 and Kv7.2 channels were unaffected. We anticipate that these results might prove useful to develop novel pharmacological tools allowing subtypeselective targeting of Kv7 channels. http://dx.doi.org/10.1016/j.toxicon.2014.08.031
Abstracts of posters 24. Purification, biochemical and molecular characterization of an L-amino acid oxidase from Cerastes cerastes snake venom Z. Abdelkafi-Koubaa a, J. Jebali a, M. Morjen a, H. Othman a, R. Zouari-Kesentini a, I. Aissa b, A. Bazaa a, H. Majdoub c, N. Srairi-Abid a, Y. Gargouri b, M. El Ayeb a, N. Marrakchi a, d Laboratoire des Venins et Mol ecules Th erapeutiques, Institut Pasteur de Tunis, 13 Place Pasteur, 1002 Tunis Belv ed ere, Tunisia b Laboratoire de Biochimie et de G enie Enzymatique des Lipases, Ecole Nationale d’Ing enieurs de Sfax (ENIS), Route de Soukra, 3038 Sfax, Universit e de Sfax, Tunisia c USCR S equenceur de Prot eines, Facult e des Sciences de Sfax, Route de Soukra 3000 Sfax, Tunisia d Facult e de Medecine de Tunis, Tunis, Tunisia a
L-amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. In the present study, an L-amino acid oxidase from Cerastes cerastes snake venom (designated as CC-LAAO) was purified to a high degree of molecular homogeneity. The isolation of CC-LAAO involved three chromatographic steps: molecular exclusion on Sephadex G-75 column, ion-exchange on Resource Q column and affinity chromatography on a HiTrap Heparin HP column. CC-LAAO is a homodimeric glycoprotein presented an estimated molecular weight of 58 kDa in SDS-PAGE under either reduced and non reduced conditions. The 30 N-terminal amino acid sequence of CCLAAO shares high similarity with other snake venom LAAOs. Unlike known SV-LAAOs, CC-LAAO displayed its maximal activity at 50 C and not at 37 C. This enzyme displayed a Michaelis-Menten behavior with an optimum pH of 8. Kinetics studies showed that the enzyme displays high specificity towards hydrophobic L-amino acids. The best substrates are L-Met, L-Leu and L-Trp. The complete CCLAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1569-bp, which encodes a protein of 518 amino acids comprising a signal peptide of 20 amino acids and 498-residues mature protein. Tertiary model structure of CC-LAAO was generated by homology modeling. http://dx.doi.org/10.1016/j.toxicon.2014.08.032
25. Xestospongin B blocks muscle-type nicotinic acetylcholine receptors with low affinity a oz a, S. Bossi a, E. Benoit a, D. Servent b, J. Molgo R. Ara a
CNRS, Institut de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie et D eveloppement, UPR 3294, 91198 Gif sur Yvette, France CEA, iBiTec-S/SIMOPRO, LabEx LERMIT, 91191 Gif-sur-Yvette, France
b
Xestospongins are a group of natural macrocyclic bis-1oxaquinolizidines alkaloids isolated from marine sponges (Xestospongia species). Xestospongin A, C and D were shown to antagonize the calcium-release action of inositol-1,4,5trisphosphate (IP3) from rabbit cerebellum endoplasmic reticulum vesicles, but with a different potency (IC50 ¼ 358 2500 nM). In contrast to xestospongin C which is an allosteric antagonist of IP3 receptors, xestospongin B was shown to behave as a competitive inhibitor of IP3 receptors in cultured rat myotubes, isolated myonuclei, and neuroblastoma NG108-15 cells (Jaimovich et al., 2005). Its atypical pharmacological profile was also evident when analyzing the effects of xestospongin B on neuromuscular transmission using isolated mouse neuromuscular preparations. Intracellular electrophysiological recordings showed that xestospongin B reduced the amplitude of miniature-endplate potentials resulting from the quantal acetylcholine release and the subsequent activation of nicotinic acetylcholine receptors (nAChR) at the neuromuscular junction. To confirm the latter results we performed voltage-clamp recordings on Xenopus laevis oocytes having incorporated the Torpedo marmorata muscle-type nAChR. Xestospongin B blocked in a dose-
Abstracts from 21st Meeting of the French Society of Toxinology (SFET) / Toxicon 91 (2014) 164e184
23. A novel toxin variant from the Androctonus australis hector scorpion venom selectively activates Kv7.4 channel Z. Landoulsi a, F. Miceli b, A. Palmese c, A. Amoresano c, G. Marino c, M. El Ayeb a, M. Taglialatela b, d, e, R. Benkhalifa a Laboratoire des Venins et Mol ecules Th erapeutiques, Institut Pasteur de Tunis, Universit e Tunis-El Manar, Tunis-Belv ed ere, Tunisia b Div. Pharmacology, Dept. of Neuroscience, University of Naples Federico II, Naples, Italy c Dept. of Chemical Sciences, University of Naples Federico II, Naples, Italy d Dept. of Medicine and Health Science, University of Molise, Campobasso, Italy (M.T.) e Unidad de Biofísica, Consejo Superior de Investigaciones Cientificas e Universidad del Pais Vasco, Leioa, Spain a
Kv7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells, skeletal and smooth muscle cells. Openers of Kv7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K+ channels; therefore, a combined purification/screening procedure has been optimized to identify specific modulator(s) of Kv7.4 channels from the venom of the North African scorpion Androctonus australis hector. We report, here, the isolation and functional characterization of AaTXKb(2-64), which acted as the first peptide activator of Kv7.4 channels. In particular, in both Xenopus oocytes and mammalian CHO cells, AaTXKb(2-64), hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologoulsyexpressed Kv7.4 channels. AaTXKb(2-64) also activated Kv7.3, Kv7.2/3, and Kv7.5/3 channels, whereas homomeric Kv1.1, Kv7.1 and Kv7.2 channels were unaffected. We anticipate that these results might prove useful to develop novel pharmacological tools allowing subtypeselective targeting of Kv7 channels. http://dx.doi.org/10.1016/j.toxicon.2014.08.031
Abstracts of posters 24. Purification, biochemical and molecular characterization of an L-amino acid oxidase from Cerastes cerastes snake venom Z. Abdelkafi-Koubaa a, J. Jebali a, M. Morjen a, H. Othman a, R. Zouari-Kesentini a, I. Aissa b, A. Bazaa a, H. Majdoub c, N. Srairi-Abid a, Y. Gargouri b, M. El Ayeb a, N. Marrakchi a, d Laboratoire des Venins et Mol ecules Th erapeutiques, Institut Pasteur de Tunis, 13 Place Pasteur, 1002 Tunis Belv ed ere, Tunisia b Laboratoire de Biochimie et de G enie Enzymatique des Lipases, Ecole Nationale d’Ing enieurs de Sfax (ENIS), Route de Soukra, 3038 Sfax, Universit e de Sfax, Tunisia c USCR S equenceur de Prot eines, Facult e des Sciences de Sfax, Route de Soukra 3000 Sfax, Tunisia d Facult e de Medecine de Tunis, Tunis, Tunisia a
L-amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding a-ketoacid with hydrogen peroxide and ammonia production. In the present study, an L-amino acid oxidase from Cerastes cerastes snake venom (designated as CC-LAAO) was purified to a high degree of molecular homogeneity. The isolation of CC-LAAO involved three chromatographic steps: molecular exclusion on Sephadex G-75 column, ion-exchange on Resource Q column and affinity chromatography on a HiTrap Heparin HP column. CC-LAAO is a homodimeric glycoprotein presented an estimated molecular weight of 58 kDa in SDS-PAGE under either reduced and non reduced conditions. The 30 N-terminal amino acid sequence of CCLAAO shares high similarity with other snake venom LAAOs. Unlike known SV-LAAOs, CC-LAAO displayed its maximal activity at 50 C and not at 37 C. This enzyme displayed a Michaelis-Menten behavior with an optimum pH of 8. Kinetics studies showed that the enzyme displays high specificity towards hydrophobic L-amino acids. The best substrates are L-Met, L-Leu and L-Trp. The complete CCLAAO cDNA was cloned from the venom gland total RNA preparations. The cDNA sequence contains an open-reading frame (ORF) of 1569-bp, which encodes a protein of 518 amino acids comprising a signal peptide of 20 amino acids and 498-residues mature protein. Tertiary model structure of CC-LAAO was generated by homology modeling. http://dx.doi.org/10.1016/j.toxicon.2014.08.032
25. Xestospongin B blocks muscle-type nicotinic acetylcholine receptors with low affinity a oz a, S. Bossi a, E. Benoit a, D. Servent b, J. Molgo R. Ara a
CNRS, Institut de Neurobiologie Alfred Fessard, Laboratoire de Neurobiologie et D eveloppement, UPR 3294, 91198 Gif sur Yvette, France CEA, iBiTec-S/SIMOPRO, LabEx LERMIT, 91191 Gif-sur-Yvette, France
b
Xestospongins are a group of natural macrocyclic bis-1oxaquinolizidines alkaloids isolated from marine sponges (Xestospongia species). Xestospongin A, C and D were shown to antagonize the calcium-release action of inositol-1,4,5trisphosphate (IP3) from rabbit cerebellum endoplasmic reticulum vesicles, but with a different potency (IC50 ¼ 358 2500 nM). In contrast to xestospongin C which is an allosteric antagonist of IP3 receptors, xestospongin B was shown to behave as a competitive inhibitor of IP3 receptors in cultured rat myotubes, isolated myonuclei, and neuroblastoma NG108-15 cells (Jaimovich et al., 2005). Its atypical pharmacological profile was also evident when analyzing the effects of xestospongin B on neuromuscular transmission using isolated mouse neuromuscular preparations. Intracellular electrophysiological recordings showed that xestospongin B reduced the amplitude of miniature-endplate potentials resulting from the quantal acetylcholine release and the subsequent activation of nicotinic acetylcholine receptors (nAChR) at the neuromuscular junction. To confirm the latter results we performed voltage-clamp recordings on Xenopus laevis oocytes having incorporated the Torpedo marmorata muscle-type nAChR. Xestospongin B blocked in a dose-