Journal of Molecular Evolution

, Volume 74, Issue 5–6, pp 297–309 | Cite as

Molecular Phylogeny, Classification and Evolution of Conopeptides

  • N. Puillandre
  • D. Koua
  • P. Favreau
  • B. M. Olivera
  • R. Stöcklin


Conopeptides are toxins expressed in the venom duct of cone snails (Conoidea, Conus). These are mostly well-structured peptides and mini-proteins with high potency and selectivity for a broad range of cellular targets. In view of these properties, they are widely used as pharmacological tools and many are candidates for innovative drugs. The conopeptides are primarily classified into superfamilies according to their peptide signal sequence, a classification that is thought to reflect the evolution of the multigenic system. However, this hypothesis has never been thoroughly tested. Here we present a phylogenetic analysis of 1,364 conopeptide signal sequences extracted from GenBank. The results validate the current conopeptide superfamily classification, but also reveal several important new features. The so-called “cysteine-poor” conopeptides are revealed to be closely related to “cysteine-rich” conopeptides; with some of them sharing very similar signal sequences, suggesting that a distinction based on cysteine content and configuration is not phylogenetically relevant and does not reflect the evolutionary history of conopeptides. A given cysteine pattern or pharmacological activity can be found across different superfamilies. Furthermore, a few conopeptides from GenBank do not cluster in any of the known superfamilies, and could represent yet-undefined superfamilies. A clear phylogenetically based classification should help to disentangle the diversity of conopeptides, and could also serve as a rationale to understand the evolution of the toxins in the numerous other species of conoideans and venomous animals at large.


Cone snails Conus Conoidea Cys-pattern Venom Molecular evolution 



We are grateful to the European Commission for financial support. This study has been performed as a part of the CONCO cone snail genome project for health ( within the 6th Framework Program (LIFESCIHEALTH-6 Integrated Project LSHB-CT-2007, contract number 037592). We are also grateful to Frédérique Lisacek from the Swiss Institute of Bioinformatics for ongoing help. We would like to thank Dr Ron Hogg of OmniScience SA for editorial support.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

239_2012_9507_MOESM1_ESM.xls (200 kb)
Appendix 1: List of analysed sequences with superfamily assignation, GenBank numbers, Cys-pattern, species from which the sequence originated and corresponding feeding type F: Fish-hunting species; M: Mollusc-hunting species; W: Worm-hunting species). (XLS 200 kb)


  1. Aguilar MB, Lopez-Vera E, Ortiz E, Becerril B, Possani LD, Olivera BM, de la Heimer Cotera EP (2005) A novel conotoxin from Conus delessertii with posttranslationally modified lysine residues. Biochemistry 44:11130–11136PubMedCrossRefGoogle Scholar
  2. Aguilar MB, Chan de la Rosa RA, Falcon A, Olivera BM, de la Heimer Cotera EP (2009) Peptide pal9a from the venom of the turrid snail Polystira albida from the Gulf of Mexico: purification, characterization, and comparison with P-conotoxin-like (framework IX) conoidean peptides. Peptides 30:467–476PubMedCrossRefGoogle Scholar
  3. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: signalP 3.0. J Mol Biol. 340(4):783–795PubMedCrossRefGoogle Scholar
  4. Biass D, Dutertre S, Gerbault A, Menou J-L, Offord R, Favreau P, Stöcklin R (2009) Comparative proteomic study of the venom of the piscivorous cone snail Conus consors. J Proteomics 72:210–218PubMedCrossRefGoogle Scholar
  5. Biggs JS, Watkins M, Puillandre N, Ownby JP, Lopez-Vera E, Christensen S, Moreno KJ, Bernaldez J, Licea-Navarro A, Showers Corneli P, Olivera BM (2010) Evolution of Conus peptide toxins: analysis of Conus californicus Reeve, 1844. Mol Phylogenet Evol 56:1–12PubMedCrossRefGoogle Scholar
  6. Blunt JW, Copp BR, Keyzers RA, Munro MH, Prinsep MR (2012) Marine natural products. Nat Prod Rep 29:144–222PubMedCrossRefGoogle Scholar
  7. Bouchet P, Lozouet P, Sysoev AV (2009) An inordinate fondness for turrids. Deep Sea Res II 56:1724–1731CrossRefGoogle Scholar
  8. Cabang AP, Imperial JS, Gajewiak J, Watkins M, Showers Corneli P, Olivera BM, Concepcion GP (2011) Characterization of a venom peptide from a crassispirid gastropod. Toxicon 58:672–680PubMedCrossRefGoogle Scholar
  9. Chang C, Duda TF (2012) Extensive and continuous duplication facilitates rapid evolution and diversification of gene families. Mol Biol Evol. Advance accessGoogle Scholar
  10. Conticello SG, Pilpel Y, Glusman G, Fainzilber M (2000) Position-specific codon conservation in hypervariable gene families. Trends Genet 16:57–59PubMedCrossRefGoogle Scholar
  11. Conticello SG, Gilad Y, Avidan N, Ben-Asher E, Levy Z, Fainzilber M (2001) Mechanisms for evolving hypervariability: the case of conopeptides. Mol Biol Evol 18:120–131PubMedCrossRefGoogle Scholar
  12. Craig AG, Zafaralla G, Cruz LJ, Santos AD, Hillyard DR, Dykert J, Rivier J, Gray WR, Imperial J, DelaCruz RG, Sporning A, Terlau H, West PJ, Yoshikami D, Olivera BM (1998) An O-glycosylated neuroexcitatory Conus peptide. Biochemistry 37:16019–16025PubMedCrossRefGoogle Scholar
  13. Craig AG, Norberg T, Griffin D, Hoeger C, Akhtar M, Schmidt K, Low W, Dykert J, Richelsoni E, Navarro V, Mazella J, Watkins M, Hillyard DR, Imperial J, Cruz LJ, Olivera BM (1999) Contulakin-G, an O-glycosylated invertebrate neurotensin. J Biol Chem 274:13752–13759PubMedCrossRefGoogle Scholar
  14. Daly NL, Craik DJ (2009) Structural studies of conotoxins. IUBMB Life 61:144–150PubMedCrossRefGoogle Scholar
  15. Davis J, Jones A, Lewis RJ (2009) Remarkable inter- and intra-species complexity of conotoxins revealed by LC/MS. Peptides 30:1222–1227PubMedCrossRefGoogle Scholar
  16. Duda TF (2008) Differentiation of venoms of predatory marine gastropods: divergence of orthologous toxin genes of closely related Conus species with different dietary specializations. J Mol Evol 67:315–321PubMedCrossRefGoogle Scholar
  17. Duda TF, Kohn AJ (2005) Species-level phylogeography and evolutionary history of the hyperdiverse marine gastropod genus Conus. Mol Phylogenet Evol 34:257–272PubMedCrossRefGoogle Scholar
  18. Duda JTF, Lee T (2009) Ecological release and venom evolution of a predatory marine Snail at Easter Island. PLoS One 4:e5558PubMedCrossRefGoogle Scholar
  19. Duda TF, Palumbi SR (1999) Molecular genetics of ecological diversification: duplication and rapid evolution of toxin genes of the venomous gastropod Conus. Proc Natl Acad Sci 96:6820–6823PubMedCrossRefGoogle Scholar
  20. Duda TF, Palumbi SR (2000) Evolutionary diversification of multigene families: allelic selection of toxins in predatory cone snails. Mol Biol Evol 17:1286–1293PubMedCrossRefGoogle Scholar
  21. Duda TF, Palumbi SR (2004) Gene expression and feeding ecology: evolution of piscivory in the venomous gastropod genus Conus. Proc Royal Soc B 271:1165–1174CrossRefGoogle Scholar
  22. Duda TF, Remigio A (2008) Variation and evolution of toxin gene expression patterns of six closely related venomous marine snails. Mol Ecol 17:3018–3032PubMedCrossRefGoogle Scholar
  23. Dutertre S, Biass D, Stöcklin R, Favreau P (2010) Dramatic intraspecimen variations within the injected venom of Conus consors: an unsuspected contribution to venom diversity. Toxicon 55:1453–1462PubMedCrossRefGoogle Scholar
  24. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797PubMedCrossRefGoogle Scholar
  25. Espiritu DJD, Watkins M, Dia-Monje V, Cartier GE, Cruz LE, Olivera BM (2001) Venomous cone snails: molecular phylogeny and the generation of toxin diversity. Toxicon 39:1899–1916PubMedCrossRefGoogle Scholar
  26. Favreau P, Stöcklin R (2009) Marine snail venoms: use and trends in receptor and channel neuropharmacology. Curr Opin Pharmacol 9:594–601PubMedCrossRefGoogle Scholar
  27. Favreau P, Benoit E, Hocking E, Carlier L, D’hoedt D, Leipold E, Markgraf D, Schlumberger S, Cordova M, Gaertner H, Paolini-Bertrand M, Hartley O, Tytgat J, Heinemann S, Bertrand D, Boelens R, Stöcklin R, Molgo J (2012) A novel mu-conopeptide, CnIIIC, exerts potent and preferential inhibition of NaV1.2/1.4 channels and blocks neuronal nicotinic acetylcholine receptors. Br J Pharmacol (in press)Google Scholar
  28. Fedosov AE (2007) Anatomy of accessory rhynchodeal organs of Veprecula vepratica and Tritonoturris subrissoides: new types of foregut morphology in Raphitominae (Conoidea). Ruthenica 17:33–41Google Scholar
  29. Fedosov A, Kantor Y (2008) Toxoglossan gastropods of the subfamily Crassispirinae (Turridae) lacking a radula, and a discussion of the status of the subfamily Zemaciinae. J Mollusc Stud 74:27–35CrossRefGoogle Scholar
  30. Gayler K, Sandall D, Greening D, Keays D, Polidano M, Livett B, Down J, Satkunanathan N, Khalil Z (2005) Molecular prospecting for drugs from the sea. IEEE Eng Med Biol Mag 24:79–84PubMedCrossRefGoogle Scholar
  31. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  32. Han TS, Teichert RW, Olivera BM, Bulaj G (2008a) Conus venoms—a rich source of peptide-based therapeutics. Curr Pharm Des 14:2462–2479PubMedCrossRefGoogle Scholar
  33. Han Y, Huang F, Jiang H, Liu L, Wang Q, Wang Y, Shao X, Chi C, Du W, Wang C (2008b) Purification and structural characterization of a d-amino acid-containing conopeptide, conomarphin, from Conus marmoreus. FEBS J 275:1976–1987PubMedCrossRefGoogle Scholar
  34. Heralde FM, Imperial J, Bandyopadhyay P, Olivera BM, Concepcion GP, Santos AD (2008) A rapidly diverging superfamily of peptide toxins in venomous Gemmula species. Toxicon 51:890–897PubMedCrossRefGoogle Scholar
  35. Holford M, Puillandre N, Terryn Y, Cruaud C, Olivera BM, Bouchet P (2009) Evolution of the Toxoglossa venom apparatus as inferred by molecular phylogeny of the Terebridae. Mol Biol Evol 26:15–25PubMedCrossRefGoogle Scholar
  36. Hopkins C, Grilley M, Miller C, Shon K-J, Cruz LJ, Gray WR, Dykert J, Rivier J, Yoshikami D, Olivera BM (1995) A new family of Conus peptides targeted to the nicotinic acetylcholine receptor. J Biol Chem 270:22361–22367PubMedCrossRefGoogle Scholar
  37. Hu H, Bandyopadhyay PK, Olivera BM, Yandell M (2011) Characterization of the Conus bullatus genome and its venom-duct transcriptome. BMC Genomics 12:60PubMedCrossRefGoogle Scholar
  38. Huelsenbeck JP, Ronquist F, Hall B (2001) MrBayes: bayesian inference of phylogeny. Bioinformatics 17:754–755PubMedCrossRefGoogle Scholar
  39. Imperial JS, Watkins M, Chen P, Hillyard DR, Cruz LJ, Olivera BM (2003) The augertoxins: biochemical characterization of venom components from the toxoglossate gastropod Terebra subulata. Toxicon 42:391–398PubMedCrossRefGoogle Scholar
  40. Imperial JS, Kantor Y, Watkins M, Heralde FM, Stevenson B, Chen P, Hansson K, Stenflo J, Ownby J-P, Bouchet P, Olivera BM (2007) Venomous auger snail Hastula (Impages) hectica (Linnaeus 1758): molecular phylogeny, foregut anatomy and comparative toxinology. J Exp Zool 308B:744–756CrossRefGoogle Scholar
  41. Jakubowski JA, Kelley WP, Sweedler JV, Gilly WF, Schulz JR (2005) Intraspecific variation of venom injected by fish-hunting Conus snails. J Exp Biol 208:2873–2883PubMedCrossRefGoogle Scholar
  42. Jimenez EC, Olivera BM, Teichert RW (2007) αC-conotoxin PrXA: a new family of nicotinic acetylcholine receptor antagonists. Biochemistry 46:8717–8724PubMedCrossRefGoogle Scholar
  43. Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. CABIOS 8:275–282PubMedGoogle Scholar
  44. Kaas Q, Westermann JC, Craik DJ (2010) Conopeptide characterization and classifications: an analysis using ConoServer. Toxicon 55:1491–1509PubMedCrossRefGoogle Scholar
  45. Keane TM, Creevey CJ, Pentony MM, Naughton TJ, McInerney JO (2006) Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified. BMC Evol Biol 6:1–17CrossRefGoogle Scholar
  46. Koua D, Brauer A, Laht S, Kaplinski L, Favreau P, Remm M, Lisacek F, Stöcklin R (2012) ConoDictor: a tool for prediction of conopeptide superfamilies. Nucleic Acids Res (in press)Google Scholar
  47. Kraus NJ, Showers Corneli P, Watkins M, Bandyopadhyay PK, Seger J, Olivera BM (2011) Against expectation: a short sequence with high signal elucidates cone snail phylogeny. Mol Phylogenet Evol 58:383–389PubMedCrossRefGoogle Scholar
  48. Laht S, Koua D, Kaplinski L, Lisacek F, Stöcklin R, Remm M (2011) Identification and classification of conopeptides using profile Hidden Markov models. Biochim Biophys Acta 1824:488–492PubMedGoogle Scholar
  49. Leary D, Vierros M, Hamon G, Arico S, Monagle C (2009) Marine genetic resources: a review of scientific and commercial interest. Mar Policy 33:183–194CrossRefGoogle Scholar
  50. Lewis RJ (2012) Discovery and development of the χ-conopeptide class of analgesic peptides. Toxicon 59(4):524–528PubMedCrossRefGoogle Scholar
  51. Lin H, Li Q-Z (2007) Predicting conotoxin superfamily and family by using pseudo amino acid composition and modified Mahalanobis discriminant. Biochem Biophys Res Commun 354:548–551PubMedCrossRefGoogle Scholar
  52. Lopez-Vera E, de la Heimer Cotera EP, Maillo M, Riesgo-Escovar JR, Olivera BM, Aguilar MB (2004) A novel structure class of toxins: the methionine-rich peptides from the venoms of turrid marine snails (Mollusca, Conoidea). Toxicon 43:365–374PubMedCrossRefGoogle Scholar
  53. McGivern JG (2007) Ziconotide: a review of its pharmacology and use in the treatment of pain. Neuropsychiatr Dis Treat 3:69–85PubMedCrossRefGoogle Scholar
  54. Medinskaya AI, Sysoev A (2003) The anatomy of Zemacies excelsa, with a description of a new subfamily of Turridae (Gastropoda, Conoidea). Ruthenica 13:81–87Google Scholar
  55. Mena EE, Gullak MF, Pagnozzi MJ, Richter KE, Rivier J, Cruz LJ, Olivera BM (1990) Conantokin-G: a novel peptide antagonist to the N-methyl-d-aspartic acid (NMDA) receptor. Neurosci Lett 118:241–244PubMedCrossRefGoogle Scholar
  56. Menez A, Stocklin R, Mebs D (2006) Venomics’ or: the venomous systems genome project. Toxicon 47:255–259PubMedCrossRefGoogle Scholar
  57. Miljanich GP (2004) Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. Curr Med Chem 11:3029–3040PubMedGoogle Scholar
  58. Molinski TF, Dalisay DS, Lievens SL, Saludes JP (2009) Drug development from marine natural products. Nat Rev Drug Discov 8:69–85PubMedCrossRefGoogle Scholar
  59. Möller C, Melaun C, Castillo C, Díaz ME, Renzelman CM, Estrada O, Kuch U, Lokey S, Marí F (2010) Functional hypervariability and gene diversity of cardioactive neuropeptides. J Biol Chem 285:40673–40680PubMedCrossRefGoogle Scholar
  60. Mondal S, Bhavna R, Babu RM, Ramakumar S (2006) Pseudo amino acid composition and multi-class support vector machines approach for conotoxin superfamily classification. J Theor Biol 243:252–260PubMedCrossRefGoogle Scholar
  61. Norton RS, Olivera BM (2006) Conotoxins down under. Toxicon 48:780–798PubMedCrossRefGoogle Scholar
  62. Olivera BM (2002) Conus venom peptides: reflections from the biology of clades and species. Annu Rev Ecol Syst 33:25–47CrossRefGoogle Scholar
  63. Olivera BM (2006) Conus peptides: biodiversity-based discovery and exogenomics. J Biol Chem 281:31173–31177PubMedCrossRefGoogle Scholar
  64. Olivera BM, Walker C, Cartier GE, Hooper D, Santos AD, Schoenfeld R, Shetty R, Watkins M, Bandyopadhyay PK, Hillyard DR (1999) Speciation of cone snails and interspecific hyperdivergence of their venom peptides. Potential evolutionary significance of introns. Ann NY Acad Sci 870:223–237PubMedCrossRefGoogle Scholar
  65. Pi C, Liu J, Peng C, Liu Y, Jiang X, Zhao Y, Tang S, Wang L, Dong M, Chen S, Xu A (2006) Diversity and evolution of conotoxins based on gene expression profiling of Conus litteratus. Genomics 88:809–819PubMedCrossRefGoogle Scholar
  66. Puillandre N, Holford M (2010) The Terebridae and teretoxins: combining phylogeny and anatomy for concerted discovery of bioactive compounds. BMC Chem Biol 10:7PubMedCrossRefGoogle Scholar
  67. Puillandre N, Watkins M, Olivera BM (2010) Evolution of Conus peptide genes: duplication and positive selection in the A-superfamily. J Mol Evol 70:190–202CrossRefGoogle Scholar
  68. Puillandre N, Kantor Y, Sysoev A, Couloux A, Meyer C, Rawlings T, Todd JA, Bouchet P (2011) The dragon tamed? A molecular phylogeny of the Conoidea (Mollusca, Gastropoda). J Mollusc Stud 77:259–272CrossRefGoogle Scholar
  69. Quinton L, Gilles N, De Pauw E (2009) TxXIIIA, an atypical homodimeric conotoxin found in the Conus textile venom. J Proteomics 72:219–226PubMedCrossRefGoogle Scholar
  70. Rambaut A, Drummond AJ (2007) Tracer v1.4. Available from
  71. Rojas A, Feregrino A, Ibarra-Alvarado C, Aguilar MB, Falcon A, de la Heimer Cotera EP (2008) Pharmacological characterization of venoms obtained from Mexican toxoglossate gastropods on isolated guinea pig ileum. J Venom Anim Toxins Incl Trop Dis 14:497–513CrossRefGoogle Scholar
  72. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCrossRefGoogle Scholar
  73. Terrat Y, Biass D, Dutertre S, Favreau P, Remm M, Stöcklin R, Piquemal D, Ducancel F (2011) High-resolution picture of a venom gland transcriptome: case study with the marine snail Conus consors. Toxicon 59:34–46PubMedCrossRefGoogle Scholar
  74. Ueberheide BM, Fenyo D, Alewood PF, Chait BT (2009) Rapid sensitive analysis of cysteine rich peptide venom components. Proc Natl Acad Sci 106:6910–6915PubMedCrossRefGoogle Scholar
  75. Violette A, Leonardi A, Piquemal D, Terrat Y, Biass D, Dutertre S, Noguier F, Ducancel F, Stöcklin R, Križaj I, Favreau P (2012) Recruitment of glycosyl hydrolase proteins in a cone snail venomous arsenal: further insights into biomolecular features of Conus venoms. Mar Drugs 10:258–280PubMedCrossRefGoogle Scholar
  76. Walker CS, Jensen S, Ellison M, Matta JA, Lee WY, Imperial JS, Duclos N, Brockie PJ, Madsen DM, Isaac JT, Olivera BM, Maricq AV (2009) A novel Conus snail polypeptide causes excitotoxicity by blocking desensitization of AMPA receptors. Curr Biol 19:900–908PubMedCrossRefGoogle Scholar
  77. Wang Q, Jiang H, Hana Y-H, Yuan DD, Chi C-W (2008) Two different groups of signal sequence in M-superfamily conotoxins. Toxicon 51:813–822PubMedCrossRefGoogle Scholar
  78. Watkins M, Hillyard DR, Olivera BM (2006) Genes expressed in a Turrid venom duct: divergence and similarity to conotoxins. J Mol Evol 62:247–256PubMedCrossRefGoogle Scholar
  79. Zhangsun D, Luo S, Wu Y, Xiaopeng Z, Hu Y, Xie L (2006) Novel O-superfamily conotoxins identified by cDNA cloning from three vermivorous Conus species. Chem Biol Drug Des 68:256–265PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • N. Puillandre
    • 1
    • 2
  • D. Koua
    • 1
    • 3
  • P. Favreau
    • 1
  • B. M. Olivera
    • 2
  • R. Stöcklin
    • 1
  1. 1.Atheris LaboratoriesGenevaSwitzerland
  2. 2.Department of BiologyUniversity of UtahSalt Lake CityUSA
  3. 3.Swiss Institute of BioinformaticsGenevaSwitzerland

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