Positions under positive selection--key for selectivity and potency of scorpion alpha-toxins.

Alpha-neurotoxins target voltage-gated sodium channels (Na(v)s) and constitute an important component in the venom of Buthidae scorpions. These toxins are short polypeptides highly conserved in sequence and three-dimensional structure, and yet they differ greatly in activity and preference for insect and various mammalian Na(v)s. Despite extensive studies of the structure-function relationship of these toxins, only little is known about their evolution and phylogeny. Using a broad data set based on published sequences and rigorous cloning, we reconstructed a reliable phylogenetic tree of scorpion alpha-toxins and estimated the evolutionary forces involved in the diversification of their genes using maximum likelihood-based methods. Although the toxins are largely conserved, four positions were found to evolve under positive selection, of which two (10 and 18; numbered according to LqhalphaIT and Lqh2 from the Israeli yellow scorpion Leiurus quinquestriatus hebraeus) have been previously shown to affect toxin activity. The putative role of the other two positions (39 and 41) was analyzed by mutagenesis of Lqh2 and LqhalphaIT. Whereas substitution P41K in Lqh2 did not alter its activity, substitution K41P in LqhalphaIT significantly decreased the activity at insect and mammalian Na(v)s. Surprisingly, not only that substitution A39L in both toxins increased their activity by 10-fold but also LqhalphaIT(A39L) was active at the mammalian brain channel rNa(v)1.2a, which otherwise is hardly affected by LqhalphaIT, and Lqh2(A39L) was active at the insect channel, DmNa(v)1, which is almost insensitive to Lqh2. Thus, position 39 is involved not only in activity but also in toxin selectivity. Overall, this study describes evolutionary forces involved in the diversification of scorpion alpha-toxins, highlights the key role of positions under positive selection for selectivity and potency, and raises new questions as to the toxin-channel face of interaction.

[1]  A. Hughes,et al.  Looking for Darwin in all the wrong places: the misguided quest for positive selection at the nucleotide sequence level , 2007, Heredity.

[2]  Louis W. Chang,et al.  Handbook of Neurotoxicology , 1995, Humana Press.

[3]  O. Cheneval,et al.  Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: case applications with single arthropod specimen. , 2006, Toxicon : official journal of the International Society on Toxinology.

[4]  M. Elazar,et al.  BjalphaIT: a novel scorpion alpha-toxin selective for insects--unique pharmacological tool. , 2005, Insect biochemistry and molecular biology.

[5]  L. Ouafik,et al.  Characterisation of the gene encoding the alpha-toxin Amm V from the scorpion Androctonus mauretanicus mauretanicus. , 2001, Toxicon : official journal of the International Society on Toxinology.

[6]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[7]  S. Heinemann,et al.  The unique pharmacology of the scorpion α‐like toxin Lqh3 is associated with its flexible C‐tail , 2007 .

[8]  S. Palumbi,et al.  Molecular genetics of ecological diversification: duplication and rapid evolution of toxin genes of the venomous gastropod Conus. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  C. Chi,et al.  Exploration of the functional site of a scorpion alpha-like toxin by site-directed mutagenesis. , 2003, Biochemistry.

[10]  Shunyi Zhu,et al.  Adaptive Evolution of Scorpion Sodium Channel Toxins , 2004, Journal of Molecular Evolution.

[11]  H. Drobecq,et al.  New toxins acting on sodium channels from the scorpion Leiurus quinquestriatus hebraeus suggest a clue to mammalian vs insect selectivity. , 1998, Toxicon : official journal of the International Society on Toxinology.

[12]  Michael Emerman,et al.  Positive selection of primate TRIM5alpha identifies a critical species-specific retroviral restriction domain. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Gordon,et al.  In vitro folding and functional analysis of an anti-insect selective scorpion depressant neurotoxin produced in Escherichia coli. , 1997, Protein expression and purification.

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

[15]  H. Karoui,et al.  Molecular cloning and functional expression of the alpha-scorpion toxin BotIII: pivotal role of the C-terminal region for its interaction with voltage-dependent sodium channels , 2004, Peptides.

[16]  Oren Froy,et al.  Molecular Basis of the High Insecticidal Potency of Scorpion α-Toxins* , 2004, Journal of Biological Chemistry.

[17]  H. Vacher,et al.  Characterization of Amm VIII from Androctonus mauretanicus mauretanicus: a new scorpion toxin that discriminates between neuronal and skeletal sodium channels. , 2003, The Biochemical journal.

[18]  E. Redaelli,et al.  Isolation and characterization of a novel toxin from the venom of the spider Grammostola rosea that blocks sodium channels. , 2007, Toxicon.

[19]  D. Gordon,et al.  The selectivity of scorpion alpha-toxins for sodium channel subtypes is determined by subtle variations at the interacting surface. , 2003, Toxicon : official journal of the International Society on Toxinology.

[20]  W. Stühmer,et al.  The insecticidal potential of scorpion beta-toxins. , 2007, Toxicon : official journal of the International Society on Toxinology.

[21]  M. Gurevitz,et al.  Advances in molecular genetics of scorpion neurotoxins , 1994 .

[22]  L. Reed,et al.  A SIMPLE METHOD OF ESTIMATING FIFTY PER CENT ENDPOINTS , 1938 .

[23]  M. Elazar,et al.  BjαIT: a novel scorpion α-toxin selective for insects—unique pharmacological tool , 2005 .

[24]  M. Hattori,et al.  Accelerated evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. Benveniste,et al.  Common Features in the Functional Surface of Scorpion β-Toxins and Elements That Confer Specificity for Insect and Mammalian Voltage-gated Sodium Channels* , 2005, Journal of Biological Chemistry.

[26]  Genomic characterisation of the toxin Amm VIII from the scorpion Androctonus mauretanicus mauretanicus. , 2006, Toxicon : official journal of the International Society on Toxinology.

[27]  D. Gordon,et al.  The FASEB Journal • FJ Express Full-Length Article , 2022 .

[28]  W. Stühmer,et al.  The insecticidal potential of scorpion β-toxins , 2007 .

[29]  Yong-hua Ji,et al.  The modulation effects of BmK I, an α-like scorpion neurotoxin, on voltage-gated Na+ currents in rat dorsal root ganglion neurons , 2005, Neuroscience Letters.

[30]  M. Nei,et al.  Prospects for inferring very large phylogenies by using the neighbor-joining method. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[31]  O. Froy,et al.  Dynamic Diversification from a Putative Common Ancestor of Scorpion Toxins Affecting Sodium, Potassium, and Chloride Channels , 1999, Journal of Molecular Evolution.

[32]  P. Bougis,et al.  New analysis of the toxic compounds from the Androctonus mauretanicus mauretanicus scorpion venom. , 2008, Toxicon : official journal of the International Society on Toxinology.

[33]  S. Liang,et al.  Molecular diversity and evolution of cystine knot toxins of the tarantula Chilobrachys jingzhao , 2008, Cellular and Molecular Life Sciences.

[34]  Ziheng Yang.  PAML 4: phylogenetic analysis by maximum likelihood. , 2007, Molecular biology and evolution.

[35]  D. Gordon,et al.  Domain 2 of Drosophila Para Voltage-Gated Sodium Channel Confers Insect Properties to a Rat Brain Channel , 2002, The Journal of Neuroscience.

[36]  D. Mishmar,et al.  Assembling an arsenal, the scorpion way , 2008, BMC Evolutionary Biology.

[37]  Yehu Moran,et al.  Molecular analysis of the sea anemone toxin Av3 reveals selectivity to insects and demonstrates the heterogeneity of receptor site-3 on voltage-gated Na+ channels. , 2007, The Biochemical journal.

[38]  S. Lukyanov,et al.  An improved PCR method for walking in uncloned genomic DNA. , 1995, Nucleic acids research.

[39]  B. Gantenbein,et al.  The phylogeographic importance of the Strait of Gibraltar as a gene flow barrier in terrestrial arthropods: a case study with the scorpion Buthus occitanus as model organism. , 2003, Molecular phylogenetics and evolution.

[40]  W. Stühmer,et al.  The differential preference of scorpion alpha-toxins for insect or mammalian sodium channels: implications for improved insect control. , 2007, Toxicon : official journal of the International Society on Toxinology.

[41]  J. Finnerty,et al.  Concerted evolution of sea anemone neurotoxin genes is revealed through analysis of the Nematostella vectensis genome. , 2008, Molecular biology and evolution.

[42]  O. Froy,et al.  Molecular basis of the high insecticidal potency of scorpion alpha-toxins. , 2004, The Journal of biological chemistry.

[43]  W. Catterall,et al.  Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion α-Toxins* , 2009, The Journal of Biological Chemistry.

[44]  S. Heinemann,et al.  The unique pharmacology of the scorpion alpha-like toxin Lqh3 is associated with its flexible C-tail. , 2007, The FEBS journal.

[45]  Yehu Moran,et al.  Expression and mutagenesis of the sea anemone toxin Av2 reveals key amino acid residues important for activity on voltage-gated sodium channels. , 2006, Biochemistry.