Purification, characterization, and primary structure of four depressant insect-selective neurotoxin analogs from scorpion (Buthus sindicus) venom.

Four depressant insect-selective neurotoxin analogs (termed Bs-dprIT1 to 4) from the venom of the scorpion Buthus sindicus were purified to homogeneity in a single step using reverse-phase HPLC. The molecular masses of the purified toxins were 6820.9, 6892.4, 6714.7, and 6657.1 Da, respectively, as determined by mass spectrometry. These long-chain neurotoxins were potent against insects with half lethal dose values of 67, 81, 103, and 78 ng/100 mg larva and 138, 160, 163, and 142 ng/100 mg cockroach, respectively, but were not lethal to mice even at the highest applied dose of 10 microg/20 g mouse. When injected into blowfly larvae (Sarcophaga falculata), Bs-dprIT1 to 4 induced classical manifestations of depressant toxins, i.e., a slow depressant flaccid paralysis. The primary structures of Bs-dprIT 1 to 4 revealed high sequence homology (60-75%) with other depressant insect toxins isolated from scorpion venoms. Despite the high sequence conservation, Bs-dprIT1 to 4 showed some remarkable features such as (i) the presence of methionine (Met(6) in Bs-dprIT1 and Met(24) in Bs-dprIT2 to 4) and histidine (His(53) and His(57) in Bs-dprIT1) residues, i.e., amino acid residues that are uncommon to this type of toxin; (ii) the substitution of two highly conserved tryptophan residues (Trp43 --> Ala and Trp53 --> His) in the sequence of Bs-dprIT1; and (iii) the occurrence of more positively charged amino acid residues at the C-terminal end than in other depressant insect toxins. Multiple sequence alignment, sequence analysis, sequence-based structure prediction, and 3D homology modeling studies revealed a protein fold and secondary structural elements similar to those of other scorpion toxins affecting sodium channel activation. The electrostatic potential calculated on the surface of the predicted 3D model of Bs-dprIT1 revealed a significant positive patch in the region of the toxin that is supposed to bind to the sodium channel.

[1]  C. Chi,et al.  Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch. , 1999, Toxicon : official journal of the International Society on Toxinology.

[2]  E. Grishin,et al.  Purification and cDNA cloning of an insecticidal protein from the venom of the scorpion Orthochirus scrobiculosus. , 2000, Toxicon : official journal of the International Society on Toxinology.

[3]  D. Babin,et al.  Amino acid sequences of neurotoxic protein variants from the venom of Centruroides sculpturatus Ewing. , 1974, Archives of biochemistry and biophysics.

[4]  H. Rochat,et al.  Two types of scorpion neurotoxins characterized by their binding to two separate receptor sites on rat brain synaptosomes. , 1980, Biochemical and biophysical research communications.

[5]  D. Arad,et al.  Identification of Structural Elements of a Scorpion α-Neurotoxin Important for Receptor Site Recognition* , 1997, The Journal of Biological Chemistry.

[6]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[7]  V. Bindokas,et al.  Functional duality and structural uniqueness of depressant insect-selective neurotoxins. , 1991, Biochemistry.

[8]  V. Tugarinov,et al.  Solution structures of a highly insecticidal recombinant scorpion alpha-toxin and a mutant with increased activity. , 1997, Biochemistry.

[9]  W. Catterall,et al.  Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. , 2000, Biochimie.

[10]  M. Delepierre,et al.  Scorpion toxins specific for Na+-channels. , 1999, European journal of biochemistry.

[11]  A. van Dorsselaer,et al.  Aah VI, a novel, N‐glycosylated anti‐insect toxin from Androctonus australis hector scorpion venom: isolation, characterisation, and glycan structure determination , 1999, FEBS letters.

[12]  M. Corona,et al.  Peptides and genes coding for scorpion toxins that affect ion-channels. , 2000, Biochimie.

[13]  C. Granier,et al.  Biochemical and pharmacological characterization of a depressant insect toxin from the venom of the scorpion Buthacus arenicola. , 1997, European journal of biochemistry.

[14]  Oren Froy,et al.  The Putative Bioactive Surface of Insect-selective Scorpion Excitatory Neurotoxins* , 1999, The Journal of Biological Chemistry.

[15]  B. W. Erickson,et al.  Scorpion toxins from Centruroides noxius and Tityus serrulatus. Primary structures and sequence comparison by metric analysis. , 1985, The Biochemical journal.

[16]  W. Braun,et al.  Two-dimensional 1H nuclear magnetic resonance study of AaH IT, an anti-insect toxin from the scorpion Androctonus australis Hector. Sequential resonance assignments and folding of the polypeptide chain. , 1991, Biochemistry.

[17]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[18]  W. Catterall,et al.  Localization of receptor sites for insect-selective toxins on sodium channels by site-directed antibodies. , 1992, Biochemistry.

[19]  C. Granier,et al.  Primary structure of scorpion anti‐insect toxins isolated from the venom of Leiurus quinquestriatus quinquestriatus , 1990, FEBS letters.

[20]  D. Kadouri,et al.  An excitatory and a depressant insect toxin from scorpion venom both affect sodium conductance and possess a common binding site. , 1985, Archives of biochemistry and biophysics.

[21]  M. Sippl.  Recognition of errors in three‐dimensional structures of proteins , 1993, Proteins.

[22]  S. Marangoni,et al.  Crystal structure of neurotoxin Ts1 from Tityus serrulatus provides insights into the specificity and toxicity of scorpion toxins. , 1999, Journal of molecular biology.

[23]  M. Delepierre,et al.  Primary and NMR three-dimensional structure determination of a novel crustacean toxin from the venom of the scorpion Centruroides limpidus limpidus Karsch. , 1994, Biochemistry.

[24]  B. Neumeister,et al.  Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom. , 2000, Archives of biochemistry and biophysics.

[25]  C. Granier,et al.  An anti-insect toxin purified from the scorpion Androctonus australis Hector also acts on the alpha- and beta-sites of the mammalian sodium channel: sequence and circular dichroism study. , 1991, Biochemistry.

[26]  Xiaoping Zhou,et al.  Purification and N-terminal partial sequence of anti-epilepsy peptide from venom of the scorpion Buthus martensii Karsch. , 1989, The Biochemical journal.

[27]  D. Gordon,et al.  Binding of an α scorpion toxin to insect sodium channels is not dependent on membrane potential , 1993 .

[28]  B. Hammock,et al.  Anti-insect toxin 5 (AaIT5) from Androctonus australis. , 1997, European journal of biochemistry.

[29]  H. Rochat,et al.  Two types of scorpion receptor sites, one related to the activation, the other to the inactivation of the action potential sodium channel. , 1982, Toxicon : official journal of the International Society on Toxinology.

[30]  H. Rochat,et al.  Characterization of toxin III of the scorpion Leiurus quinquestriatus quinquestriatus: a new type of alpha-toxin highly toxic both to mammals and insects. , 1993, Natural toxins.

[31]  O. Froy,et al.  An excitatory scorpion toxin with a distinctive feature: an additional alpha helix at the C terminus and its implications for interaction with insect sodium channels. , 1998, Structure.

[32]  E. Zlotkin,et al.  A scorpion venom neurotoxin paralytic to insects that affects sodium current inactivation: purification, primary structure, and mode of action. , 1990, Biochemistry.

[33]  M. Gurevitz,et al.  The cDNA sequence of a depressant insect selective neurotoxin from the scorpion Buthotus judaicus. , 1991, Toxicon : official journal of the International Society on Toxinology.

[34]  H. Rochat,et al.  Scorpion α‐like toxins, toxic to both mammals and insects, differentially interact with receptor site 3 on voltage‐gated sodium channels in mammals and insects , 1999, The European journal of neuroscience.

[35]  B. Hammock,et al.  A depressant insect-selective toxin analog from the venom of the scorpion Leiurus quinquestriatus hebraeus--purification and structure/function characterization. , 1998, European journal of biochemistry.

[36]  H. Karoui,et al.  A new scorpion venom toxin paralytic to insects that affects Na+ channel activation. Purification, structure, antigenicity and mode of action. , 1996, European journal of biochemistry.

[37]  H. Karoui,et al.  Purification, structure and activity of three insect toxins from Buthus occitanus tunetanus venom. , 1997, Toxicon : official journal of the International Society on Toxinology.

[38]  B. Hammock,et al.  Positive cooperativity among insecticidal scorpion neurotoxins. , 1995, Toxicon : official journal of the International Society on Toxinology.

[39]  H. Rochat,et al.  Covalent structure of the insect toxin of the North African scorpion Androctonus australis Hector. , 2009, International journal of peptide and protein research.

[40]  E. Carlier,et al.  Scorpion Toxins Affecting Sodium Current Inactivation Bind to Distinct Homologous Receptor Sites on Rat Brain and Insect Sodium Channels (*) , 1996, Journal of Biological Chemistry.

[41]  S E Ealick,et al.  Structure of scorpion toxin variant-3 at 1.2 A resolution. , 1992, Journal of molecular biology.

[42]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[43]  H. Rochat,et al.  Primary structure of toxin IV of Leiurus quinquestriatus quinquestriatus , 1985 .

[44]  M. Delepierre,et al.  An insect-specific toxin from Centruroides noxius Hoffmann. cDNA, primary structure, three-dimensional model and electrostatic surface potentials in comparison with other toxin variants. , 1996, European journal of biochemistry.

[45]  H. Rochat,et al.  Tityus serrulatus toxin VII bears pharmacological properties of both beta-toxin and insect toxin from scorpion venoms. , 1986, Biochemical and biophysical research communications.