Molecular Characterization of a Three-disulfide Bridges Beta-like Neurotoxin from Androctonus crassicauda Scorpion Venom

Document Type: Original Articles

Author

Department of Basic Sciences, Biochemistry and Molecular Biology Section, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran

Abstract

Scorpion venom is the richest source of peptide toxins with high levels of specific interactions with different ion-channel membrane proteins. The present study involved the amplification and sequencing of a 310-bp cDNA fragment encoding a beta-like neurotoxin active on sodium ion-channel from the venom glands of scorpion Androctonus crassicauda belonging to the Buthidae family using reverse transcription polymerase chain reaction (RT-PCR) technique. The amplified complementary DNA (cDNA) fragment had a coding sequence of 240 bp. The deduced precursor open-reading frame was composed of 80 amino acid residues contain a signal peptide of 22 amino acid residues, followed by a mature toxin of 58 amino acids. It had a molecular mass of 6.84 kDa and isoelectric point of 4.58. The sequence similarity search revealed several matches with the scorpion toxin-like domain of toxin-3 superfamily with a homology range of 35- 75%. Multiple alignments and secondary structure prediction demonstrated that the toxin peptide deduced from the amplified cDNA was related to the long-chain neurotoxins in size but stabilized by three disulfide bridges instead of four. The level of difference implies that the corresponding genes have originated from a common ancestor. This level of difference may also confirm an evolutionary link between the ‘short-chain’ and ‘long-chain’ toxins. The analysis showed one major segment within this neurotoxin with maximal hydrophilicity which was predicted to be antigenic by inducing an antibody response.

Keywords

Main Subjects


Article Title [French]

Caractérisation Moléculaire d'un Tri-disulfure Pontant la Neurotoxine de Type Bêta du Venin de Scorpion Androctonus crassicauda

Abstract [French]

Le venin de scorpion est la source la plus riche en toxines peptidiques avec des niveaux élevés d’interactions spécifiques avec différentes protéines membranaires des canaux ioniques. La présente étude porte sur l’amplification et le séquençage par la technique de transcription en chaîne à la polymérase (RT-PCR) d’un fragment d’ADNc de 310 pb codant une neurotoxine de type bêta active exprimée au niveau du canal ionique sodique de la glande à venin du scorpion Androctonus crassicauda appartenant à la famille des Buthidae eL'ADN complémentaire amplifié (ADNc) avait une séquence codante de 240 pb. Le cadre de lecture ouverte du précurseur déduit contenait 80 résidus d’acides aminés composés d’un peptide signal de 22 résidus d’acides aminés et d’une toxine mature de 58 acides aminés. Cette toxine avait une masse moléculaire de 6,84 kDa et un point isoélectrique de 4,58. La recherche de similarité de séquence a révélé plusieurs correspondances avec un domaine d ‘une toxine de scorpion appartenant à la superfamille de la toxine-3 avec une plage d'homologie de 35 à 75%. Les alignements multiples et la prédiction de la structure secondaire ont démontré que le peptide de la toxine déduit de l'ADNc amplifié était lié aux neurotoxines à longue chaîne, mais stabilisé par trois ponts disulfures au lieu de quatre. Le niveau de différence implique que les gènes correspondants proviennent d'un ancêtre commun. Ce niveau de différence peut également confirmer un lien évolutif entre les toxines «à chaîne courte» et «à chaîne longue». Nos analyses ont montré qu'un segment majeur de cette neurotoxine présentant une hydrophilie maximale était antigénique et induisait une réponse anticorps.

Keywords [French]

  • Androctonus crassicauda
  • Bêta-neurotoxine
  • Ponts disulfure
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J MolBiol 215, 403-410.

Bougis, P.E., Rochat, H., Smith, L.A., 1989. Precursors of Androctonus australis scorpion neurotoxins. Structures of precursors, processing outcomes, and expression of a functional recombinant toxin II. J Biol Chem 264, 19259-19265.

Carbone, E., Wanke, E., Prestipino, G., Possani, L.D., Maelicke, A., 1982. Selective blockage of voltage-dependent K+ channels by a novel scorpion toxin. Nature 296, 90-91.

DeBin, J.A., Maggio, J.E., Strichartz, G.R., 1993. Purification and characterization of chlorotoxin, a chloride channel ligand from the venom of the scorpion. Am J Physiol 264, C361-369.

Farajzadeh-Sheikh, A., Jolodar, A., Ghaemmaghami, S., 2013. Sequence characterization of cDNA sequence of encoding of an antimicrobial Peptide with no disulfide bridge from the Iranian mesobuthus eupeus venomous glands. Iran Red Crescent Med J 15, 36-41.

Finn, R.D., Tate, J., Mistry, J., Coggill, P.C., Sammut, S.J., Hotz, H.R., et al., 2008. The Pfam protein families database. Nucleic Acids Res 36, D281-288.

Jouirou, B., Mouhat, S., Andreotti, N., De Waard, M., Sabatier, J.M., 2004. Toxin determinants required for interaction with voltage-gated K+ channels. Toxicon 43, 909-914.

Jover, E., Couraud, F., Rochat, H., 1980. Two types of scorpion neurotoxins characterized by their binding to two separate receptor sites on rat brain synaptosomes. Biochem BiophResCom 95, 1607-1614.

Kelley, L.A., Sternberg, M.J., 2009. Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4, 363-371.

Kobayashi, Y., Takashima, H., Tamaoki, H., Kyogoku, Y., Lambert, P., Kuroda, H., et al., 1991. The cystine-stabilized alpha-helix: a common structural motif of ion-channel blocking neurotoxic peptides. Biopolymers 31, 1213-1220.

Kolaskar, A.S., Tongaonkar, P.C., 1990. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276, 172-174.

Kopeyan, C., Martinez, G., Rochat, H., 1979. Amino acid sequence of neurotoxin III of the scorpion Androctonus austrialis Hector. Eur J Biochem 94, 609-615.

Lebrun, B., Romi-Lebrun, R., Martin-Eauclaire, M.F., Yasuda, A., Ishiguro, M., Oyama, Y., et al., 1997. A four-disulphide-bridged toxin, with high affinity towards voltage-gated K+ channels, isolated from Heterometrus spinnifer (Scorpionidae) venom. Biochem J 328 ( Pt 1), 321-327.

Loret, E.P., Mansuelle, P., Rochat, H., Granier, C., 1990. Neurotoxins active on insects: amino acid sequences, chemical modifications, and secondary structure estimation by circular dichroism of toxins from the scorpion Androctonus australis Hector. Biochemistry 29, 1492-1501.

Martin-Eauclaire, M.F., Ceard, B., Bosmans, F., Rosso, J.P., Tytgat, J., Bougis, P.E., 2005. New "Birtoxin analogs" from Androctonus australis venom. Biochem Biophys Res Commun 333, 524-530.

Nakagawa, Y., Lee, Y.M., Lehmberg, E., Herrmann, R., Herrmann, R., Moskowitz, H., et al., 1997. Anti-insect toxin 5 (AaIT5) from Androctonus australis. Eur J Biochem 246, 496-501.

Rochat, C., Sampieri, F., Rochat, H., Miranda, F., Lissitzky, S., 1972. Iodination of neurotoxins I and II of the scorpion Androctonus australis Hector. Biochimie 54, 445-449.

Srinivasan, K.N., Gopalakrishnakone, P., Tan, P.T., Chew, K.C., Cheng, B., Kini, R.M., et al., 2002. SCORPION, a molecular database of scorpion toxins. Toxicon 40, 23-31.

Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.

Valdivia, H.H., Possani, L.D., 1998. Peptide Toxins as Probes of Ryanodine Receptor Structure and Function. Trends CardiovasMed 8, 111-118.

von Heijne, G., 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14, 4683-4690.

Zeng, X.C., Corzo, G., Hahin, R., 2005. Scorpion venom peptides without disulfide bridges. IUBMB Life 57, 13-21.

Zeng, X.C., Luo, F., Li, W.X., 2006. Molecular dissection of venom from Chinese scorpion Mesobuthus martensii: identification and characterization of four novel disulfide-bridged venom peptides. Peptides 27, 1745-1754.

Zeng, X.C., Wang, S.X., Zhu, Y., Zhu, S.Y., Li, W.X., 2004. Identification and functional characterization of novel scorpion venom peptides with no disulfide bridge from Buthus martensii Karsch. Peptides 25, 143-150.

Zhu, S., Gao, B., 2006. Molecular characterization of a new scorpion venom lipolysis activating peptide: Evidence for disulfide bridge-mediated functional switch of peptides. FEBS Lett 580, 6825-6836.

Zlotkin, E., Martinez, G., Rochat, H., Miranda, F., 1975. A protein toxic to crustacea from the venom of the scorpion Androctonus australis. Insect Biochemistry 5, 243-250.

Zlotkin, E., Rochat, H., Kopeyan, Miranda, F., Lissitzky, S., 1971. Purification and properties of the insect toxin from the venom of the scorpion Androctonus australis Hector. Biochimie 53, 1073-1078.