Skip to main content
SpringerLink
Log in

Key amino acid residues involved in mammalian and insecticidal activities of Magi4 and Hv1b, cysteine-rich spider peptides from the δ-atracotoxin family

  • Original Article
  • Published:
Amino Acids Aims and scope Submit manuscript

Abstract

δ-Atracotoxins, also known as δ-hexatoxins, are spider neurotoxic peptides, lethal to both vertebrates and insects. Their mechanism of action involves the binding to of the S3/S4 loop of the domain IV of the voltage-gated sodium channels (Nav). Because of the chemical difficulties of synthesizing folded synthetic δ-atracotoxins correctly, here we explore an expression system that is designed to produce biologically active recombinant δ-atracotoxins, and a number of variants, in order to establish certain amino acids implicated in the pharmacophore of this lethal neurotoxin. In order to elucidate and verify which amino acid residues play a key role that is toxic to vertebrates and insects, amino acid substitutes were produced by aligning the primary structures of several lethal δ-atracotoxins with those of δ-atracotoxins-Hv1b; a member of the δ-atracotoxin family that has low impact on vertebrates and is not toxic to insects. Our findings corroborate that the substitutions of the amino acid residue Y22 from δ-atracotoxin-Mg1a (Magi4) to K22 in δ-atracotoxin-Hv1b reduces its mammalian activity. Moreover, the substitutions of the amino acid residues Y22 and N26 from δ-atracotoxin-Mg1a (Magi4) to K22 and N26 in δ-atracotoxin-Hv1b reduces its insecticidal activity. Also, the basic residues K4 and R5 are important for keeping such insecticidal activity. Structural models suggest that such residues are clustered onto two bioactive surfaces, which share similar areas, previously reported as bioactive surfaces for scorpion α-toxins. Furthermore, these bioactive surfaces were also found to be similar to those found in related spider and anemone toxins, which affect the same Nav receptor, indicating that these motifs are important not only for scorpion but may be also for animal toxins that affect the S3/S4 loop of the domain IV of the Nav.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Alewood D, Birinyi-Strachan LC, Norton RS, Nicholson GM, Pallaghy PK, Alewood PF (2003) Synthesis and characterization of δ-Atracotoxin-Ar1a, the lethal neurotoxin from venom of the sydney funnel-web spider (Atrax robustus). Biochemistry 42:12933–12940

    Article  CAS  Google Scholar 

  • Clairfeuille T, Cloake A, Infield DT, Llongueras JP, Arthur CP, Li ZR, Jian Y, Martin-Eauclaire MF, Bougis PE, Ciferri C, Ahern CA, Bosmans F, Hackos DH, Rohou A, Payandeh J (2019) Structural basis of alpha-scorpion toxin action on Nav channels. Science 363(6433):5873

    Article  Google Scholar 

  • Corzo G, Escoubas P, Villegas E, Karbat I, Gordon D, Gurevitz M, Nakajima T, Gilles N (2005) A spider toxin that induces a typical effect of scorpion alpha-toxins but competes with beta-toxins on binding to insect sodium channels. Biochemistry 44:1542–1549

    Article  CAS  Google Scholar 

  • Corzo G, Sabo JK, Bosmans F, Billen B, Villegas E, Tytgat J, Norton RS (2007) Solution structure and alanine scan of a spider toxin that affects the activation of mammalian voltage-gated sodium channels. J Biol Chem 282(7):4643–4652

    Article  CAS  Google Scholar 

  • Dias-Kadambi BL, Drum CL, Hanck DA, Blumenthal KM (1996) Leucine 18, a hydrophobic residue essential for high affinity binding of anthopleurin B to the voltage-sensitive sodium channel. J Biol Chem 19(271):9422–9428

    Article  Google Scholar 

  • Dixon WJ (1965) The Up-and-Down method for small samples. J Am Stat Assoc 60:967–978

    Article  Google Scholar 

  • Fujiwara K, Toda H, Ikeguchi M (2012) Dependence of alpha-helical and beta-sheet amino acid propensities on the overall protein fold type. BMC Struct Biol 12:18

    Article  CAS  Google Scholar 

  • Gordon D, Karbat I, Ilan N, Cohen L, Kahn R, Gilles N, Dong K, Stuhmer W, Tytgat J, Gurevitz M (2007) The differential preference of scorpion alpha-toxins for insect or mammalian sodium channels: implications for improved insect control. Toxicon 49(4):452–472

    Article  CAS  Google Scholar 

  • Gunning SJ, Chong Y, Khalife AA, Hains PG, Broady KW, Nicholson GM (2003) Isolation of δ-missulenatoxin-Mb1a, the major vertebrate-active spider δ-toxin from the venom of Missulena bradleyi (Actinopodidae). FEBS Lett 554(1–2):211–218

    Article  CAS  Google Scholar 

  • Khera PK, Blumenthal KM (1994) Role of the cationic residues arginine 14 and lysine 48 in the function of the cardiotonic polypeptide anthopleurin B. J Biol Chem 14(269):921–925

    Google Scholar 

  • Klint JK, Senff S, Rupasinghe DB, Er SY, Herzig V, Nicholson GM, King GF (2012) Spider-venom peptides that target voltage-gated sodium channels: pharmacological tools and potential therapeutic leads. Toxicon 60(4):478–491

    Article  CAS  Google Scholar 

  • Little MJ, Wilson H, Zappia C, Cestèle S, Tyler MI, Martin-Eauclaire M-F, Gordond D, Nicholson GM (1998) δ-Atracotoxins from Australian funnel-web spiders compete with scorpion α-toxin binding on both rat brain and insect sodium channels. FEBS Lett 439:246–252

    Article  CAS  Google Scholar 

  • Liu H, Naismith JH (2008) An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol 8:91

    Article  Google Scholar 

  • Moran Y, Gordon D, Gurevitz M (2009) Sea anemone toxins affecting voltage-gated sodium channels–molecular and evolutionary features. Toxicon 54(8):1089–1101

    Article  CAS  Google Scholar 

  • Nicholson GM, Graudins A (2002) Spiders of medical importance in the Asia-Pacific: atracotoxin, latrotoxin and related spider neurotoxins. Clin Exp Pharmacol Physiol 29:785–794

    Article  CAS  Google Scholar 

  • Pineda SS, Undheim EA, Rupasinghe DB, Ikonomopoulou MP, King GF (2014) Spider venomics: implications for drug discovery. Future Med Chem 6(15):1699–1714

    Article  CAS  Google Scholar 

  • Rychlik W (2007) OLIGO 7 primer analysis software, in methods in molecular biology Vol. 402: PCR Primer Design. Humana Press Inc., Totowa, pp 35–39

    Google Scholar 

  • Seibert AL, Liu J, Hanck DA, Blumenthal KM (2004a) Role of Asn-16 and Ser-19 in anthopleurin B binding Implications for the electrostatic nature of Na(V) site. Biochemistry 8:7082–7089

    Article  Google Scholar 

  • Seibert AL, Liu J, Hanck DA, Blumenthal KM (2004b) Role of Asn-16 and Ser-19 in Anthopleurin B Binding. Implications for the Electrostatic Nature of NaV Site 3. Biochemistry 43:7082–7089

    Article  CAS  Google Scholar 

  • Szeto TH, Smith R, Connor M, Christie MJ, King GF, Nicholson GM (2000) Isolation and pharmacological characterisation of δ-atracotoxin-Hv1b, a vertebrate-selective sodium channel toxin. FEBS Lett 470:293–299

    Article  CAS  Google Scholar 

  • Wilson DTR (2016) The venom of Australian spiders. In: Gopalakrishnakone P, Corzo G, Lima ME, Diego‐García E (eds) Spider venoms, toxinology series. Springer, Netherlands, pp 21–43

    Chapter  Google Scholar 

  • Yamaji N, Little MJ, Nishio H, Billen B, Villegas E, Nishiuchi Y, Tytgat J, Nicholson GM, Corzo G (2009) Synthesis, solution structure, and phylum selectivity of a spider delta-toxin that slows inactivation of specific voltage-gated sodium channel subtypes. J Biol Chem 284(36):24568–24582

    Article  CAS  Google Scholar 

  • Zhang Y, Skolnick J (2005) TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res 33(7):2302–2309

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We acknowledge Dr. Fernando Zamudio, Dra. Gloria Saab and M. en C. Pavel A. Montero from Instituto de Biotecnología UNAM for mass spectrometric, circular dichroism and bioinformatic determinations, respectively. We additionally acknowledge Dr. Paul Gaytán, M.C. Eugenio López-Bustos and Q.I. Santiago Becerra from Unidad de Síntesis y Secuenciación de ADN at Instituto de Biotecnología. This work received funding from the Dirección General de Asuntos del Personal Académico (DGAPA-UNAM) grant number IN203118 awarded to GC. JB is a doctoral student from Programa de Doctorado en Ciencias Biomédicas at the Universidad Nacional Autónoma de México (UNAM) and was supported by CONACyT-México (Fellowship No. 415092).

Author information

Authors and Affiliations

  1. Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad, 2001, Apartado Postal 510-3, 62210, Cuernavaca Mor., México

    Jesús Borrego, Herlinda Clement, Ligia-Luz Corrales-García, Iván Arenas & Gerardo Corzo

  2. Departamento de Alimentos, Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia, AA 1226, Medellín, Colombia

    Ligia-Luz Corrales-García

Authors
  1. Jesús Borrego
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Herlinda Clement
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ligia-Luz Corrales-García
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Iván Arenas
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gerardo Corzo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JB designed, cloned, expressed and evaluated, biologically the δ-ACTXs, and wrote the manuscript; HC and LCG constructed and cloned the δ-ACTX; IA performed the protein purification; GC designed CD experiments, reviewed and wrote the manuscript.

Corresponding author

Correspondence to Gerardo Corzo.

Ethics declarations

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 in accordance with the bioethical standards at the “Instituto de Biotecnología—UNAM.”

Informed consent

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

Additional information

Handling Editor: M. S. Palma.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 240 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Borrego, J., Clement, H., Corrales-García, LL. et al. Key amino acid residues involved in mammalian and insecticidal activities of Magi4 and Hv1b, cysteine-rich spider peptides from the δ-atracotoxin family. Amino Acids 52, 465–475 (2020). https://doi.org/10.1007/s00726-020-02825-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00726-020-02825-4

Keywords

Search

Navigation