Advertisement

Amino Acids

, Volume 50, Issue 7, pp 885–895 | Cite as

Short-chain consensus alpha-neurotoxin: a synthetic 60-mer peptide with generic traits and enhanced immunogenic properties

  • Guillermo de la Rosa
  • Ligia L. Corrales-García
  • Ximena Rodriguez-Ruiz
  • Estuardo López-Vera
  • Gerardo Corzo
Original Article

Abstract

The three-fingered toxin family and more precisely short-chain α-neurotoxins (also known as Type I α-neurotoxins) are crucial in defining the elapid envenomation process, but paradoxically, they are barely neutralized by current elapid snake antivenoms. This work has been focused on the primary structural identity among Type I neurotoxins in order to create a consensus short-chain α-neurotoxin with conserved characteristics. A multiple sequence alignment considering the twelve most toxic short-chain α-neurotoxins reported from the venoms of the elapid genera Acanthophis, Oxyuranus, Walterinnesia, Naja, Dendroaspis and Micrurus led us to propose a short-chain consensus α-neurotoxin, here named ScNtx. The synthetic ScNtx gene was de novo constructed and cloned into the expression vector pQE30 containing a 6His-Tag and an FXa proteolytic cleavage region. Escherichia coli Origami cells transfected with the pQE30/ScNtx vector expressed the recombinant consensus neurotoxin in a soluble form with a yield of 1.5 mg/L of culture medium. The 60-amino acid residue ScNtx contains canonical structural motifs similar to α-neurotoxins from African elapids and its LD50 of 3.8 µg/mice is similar to the most toxic short-chain α-neurotoxins reported from elapid venoms. Furthermore, ScNtx was also able to antagonize muscular, but not neuronal, nicotinic acetylcholine receptors (nAChR). Rabbits immunized with ScNtx were able to immune-recognize short-chain α-neurotoxins within whole elapid venoms. Type I neurotoxins are difficult to isolate and purify from natural sources; therefore, the heterologous expression of molecules such ScNtx, bearing crucial motifs and key amino acids, is a step forward to create common immunogens for developing cost-effective antivenoms with a wider spectrum of efficacy, quality and strong therapeutic value.

Keywords

Antisera Elapid Micrurus α-Neurotoxin Synthetic gene Recombinant Three finger toxins 

Notes

Acknowledgements

This work received funding from the Dirección General de Asuntos del Personal Académico (DGAPA-UNAM) Grant number IN203118, and from SEP-CONACyT grant number 240616 awarded to GC. We acknowledge Dr. Alejandro Alagón and BSc. Felipe Olvera for providing elapid venoms and technical support, respectively. The patent filling advice from MBA Mario Trejo and M.Sc. Martin Patiño is also greatly acknowledged. We greatly appreciate the English grammar edition from Dr. Christopher David Wood. GDLR is a doctoral student from “Programa de Doctorado en Ciencias Bioquímicas” at the Universidad Nacional Autónoma de México (UNAM) and received a fellowship (No. 367094) from CONACYT.

Author contributions

GD designed, expressed and evaluated, biologically and immunologically, the ScNtx, and wrote the manuscript; LCG constructed and cloned the ScNtx; XRR and ELV performed the pharmacological characterization; GC designed CD experiments, reviewed and wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest

Ethical approval

No experiments with humans were performed. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed and procedures performed in the present study involving animals were done so in accordance with the bioethical standards at the “Instituto de Biotecnología”.

Informed consent

The authors confirm that this work is original and new, and it is not under consideration elsewhere.

References

  1. Antil S, Servent D, Ménez A (1999) Variability among the sites by which curaremimetic toxins bind to Torpedo acetylcholine receptor, as revealed by identification of the functional residues of a-cobratoxin. J Biol Chem.  https://doi.org/10.1074/jbc.274.49.34851 PubMedCrossRefGoogle Scholar
  2. Ariaratnam CA, Sheriff MHR, Theakston RDG, Warrell DA (2008) Distinctive epidemiologic and clinical features of common krait (Bungarus caeruleus) bites in Sri Lanka. Am J Trop Med Hyg 79:458–462 (79/3/458 [pii]) PubMedCrossRefGoogle Scholar
  3. Arias HR (1997) Topology of ligand binding sites on the nicotinic acetylcholine receptor. Brian Res Rev.  https://doi.org/10.1016/s0165-0173(97)00020-9 CrossRefGoogle Scholar
  4. Barber CM, Isbister GK, Hodgson WC (2013) Alpha neurotoxins. Toxicon 66:47–58.  https://doi.org/10.1016/j.toxicon.2013.01.019 CrossRefPubMedGoogle Scholar
  5. Bourne Y, Talley T, Hansen S et al (2005a) Crystal structure of a Cbtx-AChBP complex reveals essential interactions between snake alpha-neurotoxins and nicotinic receptors. EMBO J 24:1512–1522CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bourne Y, Talley TT, Hansen SB et al (2005b) Crystal structure of a Cbtx-AChBP complex reveals essential interactions between snake a-neurotoxins and nicotinic receptors. Euro Mol Biol Org.  https://doi.org/10.1038/sj.emboj.7600620 CrossRefGoogle Scholar
  7. Calvete JJ (2013) Snake venomics: from the inventory of toxins to biology. Toxicon 75:44–62.  https://doi.org/10.1016/j.toxicon.2013.03.020 CrossRefPubMedGoogle Scholar
  8. Cartier GE, Yoshikami D, Gray WR et al (1996) A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors. J Biol Chem 271:7522–7528CrossRefPubMedGoogle Scholar
  9. Chan YS, Cheung RCF, Xia L et al (2016) Snake venom toxins: toxicity and medicinal applications. Appl Microbiol Biotechnol.  https://doi.org/10.1007/s00253-016-7610-9 CrossRefPubMedGoogle Scholar
  10. Chang CC (1999) Looking back on the discovery of alpha-bungarotoxin. J Biomed Sci 6:368–375. https://doi.org/25412Google Scholar
  11. Clement H, Flores V, De la Rosa G et al (2016) Heterologous expression, protein folding and antibody recognition of a neurotoxin from the Mexican coral snake Micrurus laticorallis. J Venom Anim Toxins Incl Trop Dis 22:25.  https://doi.org/10.1186/s40409-016-0080-9 CrossRefPubMedPubMedCentralGoogle Scholar
  12. de Weille JR, Schweitz H, Maes P et al (1991) Calciseptine, a peptide isolated from black mamba venom, is a specific blocker of the L-type calcium channel. Proc Natl Acad Sci USA 88:2437–2440.  https://doi.org/10.1073/pnas.88.6.2437 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Diochot S, Alloui A, Rodrigues P et al (2016) Analgesic effects of mambalgin peptide inhibitors of acid-sensing ion channels in inflammatory and neuropathic pain. Pain 157:552–559.  https://doi.org/10.1097/j.pain.0000000000000397 CrossRefPubMedGoogle Scholar
  14. Dufton MJ (1984) Classification of elapid snake neurotoxins and cytotoxins according to chain length: evolutionary implications. J Mol Evol 20:128–134.  https://doi.org/10.1007/bf02257373 CrossRefPubMedGoogle Scholar
  15. Dufton MJ, Hider RC (1988) Structure and pharmacology of elapid cytotoxins. Pharmacol Ther 36:1–40.  https://doi.org/10.1016/0163-7258(88)90111-8 CrossRefPubMedGoogle Scholar
  16. Engmark M, Jespersen MC, Lomonte B et al (2017) High-density peptide microarray exploration of the antibody response in a rabbit immunized with a neurotoxic venom fraction. Toxicon 138:151–158.  https://doi.org/10.1016/j.toxicon.2017.08.028 CrossRefPubMedGoogle Scholar
  17. Fruchart-Gaillard C, Mourier G, Marquer C et al (2006) How three-finger-fold toxins interact with various cholinergic receptors. J Mol Neurosci 30:7–8.  https://doi.org/10.1385/jmn:30:1:7 CrossRefPubMedGoogle Scholar
  18. Fruchart-Gaillard C, Mourier G, Blanchet G et al (2012) Engineering of three-finger fold toxins creates ligands with original pharmacological profiles for muscarinic and adrenergic receptors. PLoS One 7:e39166.  https://doi.org/10.1371/journal.pone.0039166 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hifumi T, Sakai A, Kondo Y et al (2015) Venomous snake bites: clinical diagnosis and treatment. J Intensive Care.  https://doi.org/10.1186/s40560-015-0081-8 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Hung HT, Höjer J, Du NT (2009) Clinical features of 60 consecutive ICU-treated patients envenomed by Bungarus multicinctus. Southeast Asian J Trop Med Public Health 40:518–524PubMedGoogle Scholar
  21. Israeli E (2012) Capsule: black mamba venom peptides target acid-sensing ion channels to abolish pain. Isr Med Assoc J 14:762.  https://doi.org/10.1038/nature11494 CrossRefGoogle Scholar
  22. Kini RM (2002) Invited Paper : Animal Toxins of Asia and Australia. Molecular moulds with multiple missions : functional sites in three-finger toxins, pp 815–822Google Scholar
  23. Kini RM (2011) Evolution of three-finger toxins—a versatile mini protein scaffold. Acta Chim Slov 58:693–701PubMedGoogle Scholar
  24. Kini RM, Doley R (2010) Structure, function and evolution of three-finger toxins: mini proteins with multiple targets. Toxicon 56:855–867.  https://doi.org/10.1016/j.toxicon.2010.07.010 CrossRefPubMedGoogle Scholar
  25. Kolaskar AS, Tongaonkar PC (1990) A semi-empirical method for prediction of antigenic detetermininants on protein antigens. Febbs Lett 276:172–174CrossRefGoogle Scholar
  26. Kozminsky-Atias A, Zilberberg N (2012) Molding the business end of neurotoxins by diversifying evolution. FASEB J 26:576–586.  https://doi.org/10.1096/fj.11-187179 CrossRefPubMedGoogle Scholar
  27. Laemmli UK (1970) 1970 Nature publishing group. Group 227:680–685.  https://doi.org/10.1038/227680a0 CrossRefGoogle Scholar
  28. Lesovoy DM, Bocharov EV, Lyukmanova EN et al (2009) Specific membrane binding of neurotoxin II can facilitate its delivery to acetylcholine receptor. Biophys J 97:2089–2097.  https://doi.org/10.1016/j.bpj.2009.07.037 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Liukmanova EN, Shulepko MA, Shenkarev ZO et al (2010) The in vitro production of three-finger neurotoxins from snake venoms with a high abundance of disulfide bonds. Problems and their solutions. Bioorg Khim 36:149–158PubMedGoogle Scholar
  30. Marchot P, Bourne Y, Prowse CN et al (1998) Inhibition of mouse acetylcholinesterase by fasciculin: crystal structure of the complex and mutagenesis of fasciculin. Toxicon 36:1613–1622.  https://doi.org/10.1016/s0041-0101(98)00154-8 CrossRefPubMedGoogle Scholar
  31. Naimuddin M, Kobayashi S, Tsutsui C et al (2011) Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling. Mol Brain 4:2.  https://doi.org/10.1186/1756-6606-4-2 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Nirthanan S, Gopalakrishnakone P, Gwee MCE et al (2003) Non-conventional toxins from Elapid venoms. Toxicon 41:397–407.  https://doi.org/10.1016/s0041-0101(02)00388-4 CrossRefPubMedGoogle Scholar
  33. Osaka H, Malany S, Molles BE et al (2000) Pairwise electrostatic interactions between $α$-neurotoxins and $α$, $β$, and $γ$ subunits of the nicotinic acetylcholine receptor. J Bio Chem.  https://doi.org/10.1074/jbc.275.8.5478 CrossRefGoogle Scholar
  34. Oyama E, Takahashi H (2015) Purification and characterization of two platelet-aggregation inhibitors, named angustatin and H-toxin TA(2), from the venom of Dendroaspis angusticeps. Toxicon 93:61–67.  https://doi.org/10.1016/j.toxicon.2014.11.002 CrossRefPubMedGoogle Scholar
  35. Petras D, Heiss P, Süssmuth RD, Calvete JJ (2015) Venom proteomics of Indonesian King Cobra, Ophiophagus hannah: integrating top-down and bottom-up approaches. J Proteome Res.  https://doi.org/10.1021/acs.jproteome.5b00305 PubMedCrossRefGoogle Scholar
  36. Pillet L, Tremeau O, Ducancel F, Drevet P et al (1993) Genetic engineering of snake toxins. Role of invariant residues in the structural and functional properties of a curaremimetic toxin, as probed by site-directed mutagenesis. J Biol Chem 268(2):909–916PubMedGoogle Scholar
  37. Rajagopalan N, Pung YF, Zhu YZ et al (2007) Beta-cardiotoxin: a new three-finger toxin from Ophiophagus hannah (king cobra) venom with beta-blocker activity. FASEB J 21:3685–3695.  https://doi.org/10.1096/fj.07-8658com CrossRefPubMedGoogle Scholar
  38. Ranawaka UK, Lalloo DG, de Silva HJ (2013) Neurotoxicity in snakebite-the limits of our knowledge. PLoS Negl Trop Dis.  https://doi.org/10.1371/journal.pntd.0002302 PubMedPubMedCentralCrossRefGoogle Scholar
  39. Redi F (1668) Osservazioni intorno alle vipere. In: All’Insegna della Stella. https://archive.org/details/osservazioniint00redigoog
  40. Reeks TA, Fry BG, Alewood PF (2015) Privileged frameworks from snake venom. Cell Mol Life Sci 72:1939–1958.  https://doi.org/10.1007/s00018-015-1844-z CrossRefPubMedGoogle Scholar
  41. Ricciardi A (2000) Do structural deviations between toxins adopting the same fold reflect functional differences? J Biol Chem 275:18302–18310.  https://doi.org/10.1074/jbc.275.24.18302 CrossRefPubMedGoogle Scholar
  42. Rosenthal JA, Levandoski MM, Chang B et al (1999) The functional role of positively charged amino acid side chains in α- bungarotoxin revealed by site-directed mutagenesis of a His-tagged recombinant α-bungarotoxin. Biochemistry 38:7847–7855.  https://doi.org/10.1021/bi990045g CrossRefPubMedGoogle Scholar
  43. Samson A, Scherf T, Eisenstein M et al (2002) The mechanism for acetylcholine receptor inhibition by alpha-neurotoxins and species-specific resistance to alpha-bungarotoxin revealed by NMR. Neuron.  https://doi.org/10.1016/s0896-6273(02)00773-0 PubMedCrossRefGoogle Scholar
  44. Sewall H (1887) Experiments on the preventive inoculation of rattlesnake venom. J Physiol 8:203–210CrossRefPubMedPubMedCentralGoogle Scholar
  45. Silva A, Hodgson W, Isbister G (2016) Cross-neutralisation of in vitro neurotoxicity of asian and australian snake neurotoxins and venoms by different antivenoms. Toxins (Basel) 8:302.  https://doi.org/10.3390/toxins8100302 CrossRefGoogle Scholar
  46. Sunagar K, Jackson TNW, Undheim EAB et al (2013) Three-fingered RAVERs: rapid accumulation of variations in exposed residues of snake venom toxins. Toxins (Basel) 5:2172–2208.  https://doi.org/10.3390/toxins5112172 CrossRefGoogle Scholar
  47. Teixeira-Clerc F, Ménez A, Kessler P (2002) How do short neurotoxins bind to a muscular-type nicotinic acetylcholine receptor? J Biol Chem 277:25741–25747.  https://doi.org/10.1074/jbc.m200534200 CrossRefPubMedGoogle Scholar
  48. Utkin YN (2013) Three-finger toxins, a deadly weapon of elapid venom–milestones of discovery. Toxicon 62:50–55.  https://doi.org/10.1016/j.toxicon.2012.09.007 CrossRefPubMedGoogle Scholar
  49. Vázquez H, Olvera F, Paniagua-Solís J et al (2010) Pharmacokinetics in rabbits and anti-sphingomyelinase D neutralizing power of Fab, F(ab’)2, IgG and IgG(T) fragments from hyper immune equine plasma. Int Immunopharmacol 10:447–454.  https://doi.org/10.1016/j.intimp.2010.01.005 CrossRefPubMedGoogle Scholar
  50. Waterhouse AM, Procter JB, Martin DMA et al (2009) Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191.  https://doi.org/10.1093/bioinformatics/btp033 CrossRefPubMedPubMedCentralGoogle Scholar
  51. WHO (1981) Progress in the characterization of venoms and standardization of antivenoms. WHO Offset Publ., Geneva, pp 1–44Google Scholar
  52. Yang D, Deuis J, Dashevsky D et al (2016) The snake with the scorpion’s sting: novel three-finger toxin sodium channel activators from the venom of the long-glanded blue coral snake (Calliophis bivirgatus). Toxins (Basel) 8:303.  https://doi.org/10.3390/toxins8100303 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Departamento de Medicina Molecular y Bioprocesos, Instituto de BiotecnologíaUniversidad Nacional Autónoma de México, UNAMCuernavacaMexico
  2. 2.Departamento de Alimentos, Facultad de Ciencias Farmacéuticas y AlimentariasUniversidad de AntioquiaMedellínColombia
  3. 3.Instituto de Ciencias del Mar y Limnología/Posgrado en Ciencias del Mar y LimnologiaUniversidad Nacional Autónoma de México, UNAMMexico CityMexico
  4. 4.Institute of Biotechnology-UNAMCuernavacaMexico

Personalised recommendations