Three-Finger Viper Toxins – cDNA Cloning and Expression in E. coli Using a Chimeric (Hybrid) Construction with a Partner Protein SUMO

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Abstract

Three-finger toxins (TFTs) form one of the most abundant families of toxins in snake venoms. TFTs are common for most Elapid venoms, but are almost never found in viper venoms. Using the venom glands of the vipers Vipera nikolskii and V. berus, 21 cDNA clones encoding this group of toxins were obtained. The amino acid sequences of 9 TFTs were deduced from the obtained cDNA sequences. The identified sequences have signal peptides containing 19-21 amino acid residues, followed by a mature protein consisting of 67 residues. All viper TFTs belong to the group of non-conventional toxins, and their sequences contain 9 cysteine residues. The TFT encoded by one of the transcripts was obtained by heterologous expression in E. coli cells as a fusion protein with the plant partner protein SUMO, followed by cleavage with the specific plant protease BdSENP1 and chromatographic purification. The structure of the obtained protein was confirmed by mass spectrometry. Analysis of its biological activity showed that this toxin is a weak antagonist of nicotinic acetylcholine receptors of the neuronal α7 and α3β2 subtypes. Using the fusion protein with SUMO, we also attempted to obtain the TFT Aze-2 of the viper Azemiops feae, the amino acid sequence of which was previously established by us as a result of transcriptome analysis of the venom gland of A. feae, and the protein itself was identified in minimal quantities in the venom of this snake. However, a toxin exactly corresponding in mass to Aze-2 could not be obtained using this approach. Thus, as a result of the work, the amino acid sequences of 9 viper TFTs were established, one of which was obtained by gene expression in E. coli cells and showed the ability to interact with nicotinic acetylcholine receptors of the neuronal α7 and α3β2 subtypes.

About the authors

D. A Sukhov

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences; MIREA – Russian Technological University

Moscow, Russia; Moscow, Russia

L. O Ojomoko

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

I. V Shelukhina

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

M. V Vladykina

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

V. Yu Kost

Weizmann Institute of Science

Department of Chemical and Structural Biology Rehovot, Israel

R. Kh Ziganshin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

O. V Geraskina

M.V. Lomonosov Moscow State University, Faculty of Biology

Moscow, Russia

S. V Balandin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

T. V Ovchinnikova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

V. I Tsetlin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

Yu. N Utkin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: yutkin@yandex.ru
Moscow, Russia

References

  1. Utkin Y., Sunagar K., Jackson T.N., Reeks T., Fry B.G. // In Venomous Reptiles and Their Toxins: Evolution, Pathophysiology and Biodiscovery. Ed. Fry B.G. New York: Oxford University Press, 2015. P. 215– 227.
  2. Dubovskii P.V., Utkin Y.N. // Toxins (Basel). 2024. V. 16. P. 262. https://doi.org/10.3390/toxins16060262
  3. Nirthanan S., Gopalakrishnakone P., Gwee M.C., Khoo H.E., Kini R.M. // Toxicon. 2003. V. 41. P. 397– 407. https://doi.org/10.1016/s0041-0101(02)00388-4
  4. Heyborne W.H., Mackessy S.P. // Toxicon. 2021. V. 190. P. 22–30. https://doi.org/10.1016/j.toxicon.2020.12.002
  5. Srodawa K., Cerda P.A., Davis Rabosky A.R., Crowe-Riddell J.M. // Toxins (Basel). 2023. V. 15. P. 523. https://doi.org/10.3390/toxins15090523
  6. Junqueira-de-Azevedo I.L., Ching A.T., Carvalho E., Faria F., Nishiyama M.Y. Jr., Ho P.L., Diniz M.R. // Genetics. 2006. V. 173. P. 877–889. https://doi.org/10.1534/genetics.106.056515
  7. Pahari S., Mackessy S.P., Kini R.M. // BMC Mol. Biol. 2007. V. 8. P. 115. https://doi.org/10.1186/1471-2199-8-115
  8. Nicolau C.A., Carvalho P.C., Junqueira-de-Azevedo I.L., Teixeira-Ferreira A., Junqueira M., Perales J., Neves- Ferreira A.G., Valente R.H. // J. Proteomics. 2017. V. 151. P. 214–231. https://doi.org/10.1016/j.jprot.2016.06.029
  9. Babenko V.V., Ziganshin R.H., Weise C., Dyachenko I., Shaykhutdinova E., Murashev A.N., Zhmak M., Starkov V., Hoang A.N., Tsetlin V., Utkin Y. // Biomedicines. 2020. V. 8. P. 249. https://doi.org/10.3390/biomedicines8080249
  10. Dingwoke E.J., Adamude F.A., Mohamed G., Klein A., Salihu A., Abubakar M.S., Sallau A.B. // Biochem. Biophys. Rep. 2021. V. 28. P. 101164. https://doi.org/10.1016/j.bbrep.2021.101164
  11. Kovalchuk S.I., Ziganshin R.H., Starkov V.G., Tsetlin V.I., Utkin Y.N. // Toxins (Basel). 2016. V. 8. P. 105. https://doi.org/10.3390/toxins8040105
  12. Makarova Y.V., Kryukova E.V., Shelukhina I.V., Lebedev D.S., Andreeva T.V., Ryazantsev D.Y., Balandin S.V., Ovchinnikova T.V., Tsetlin V.I., Utkin Y.N. // Dokl. Biochem. Biophys. 2018. V. 479. P. 127–130. https://doi.org/10.1134/S1607672918020205
  13. Malakhov M.P., Mattern M.R., Malakhova O.A., Drinker M., Weeks S.D., Butt T.R. // J. Struct. Funct. Genomic. 2004. V. 5. P. 75–86. https://doi.org/10.1023/B:JSFG.0000029237.70316.52
  14. Kuo D., Nie M., Courey A.J. // Methods Mol. Biol. 2014. V. 1177. P. 71–80. https://doi.org/10.1007/978-1-4939-1034-2_6
  15. Frey S., Görlich D. // J. Chromatogr. A. 2014. V. 1337. P. 95–105. https://doi.org/10.1016/j.chroma.2014.02.029
  16. Doley R., Tram N.N., Reza M.A., Kini R.M. // BMC Evol. Biol. 2008. V. 8. P. 70. https://doi.org/10.1186/1471-2148-8-70
  17. Chang L.S., Lin S.R., Chang C.C. // Arch. Biochem. Biophys. 1998. V. 354. P. 1–8. https://doi.org/10.1006/abbi.1998.0660
  18. Fry B.G., Scheib H., van der Weerd L., Young B., McNaughtan J., Ramjan S.F., Vidal N., Poelmann R.E., Norman J.A. // Mol. Cell Proteomics. 2008. V. 7. P. 215–246. https://doi.org/10.1074/mcp.M700094-MCP200
  19. Modahl C.M., Mackessy S.P. // PLoS Negl. Trop. Dis. 2016. V. 10. P. e0004587. https://doi.org/10.1371/journal.pntd.0004587
  20. Modahl C.M., Mrinalini, Frietze S., Mackessy S.P. // Proc. Biol. Sci. 2018. V. 285. P. 20181003. https://doi.org/10.1098/rspb.2018.1003
  21. Escherichia coli [gbbct]: 8087 CDS's (2330943 codons) // Codon Usage Database. https://www.kazusa.or.jp/codon/cgi-bin/showcodon. cgi?species=37762
  22. Carpanta V., Clement H., Arenas I., Corzo G. // Biochem. Biophys. Res. Commun. 2024. V. 732. P. 150420. https://doi.org/10.1016/j.bbrc.2024.150420
  23. Drake A.F., Dufton M.J., Hider R.C. // Eur. J. Biochem. 1980. V. 105. P. 623–630. https://doi.org/10.1111/j.1432-1033.1980.tb04540.x
  24. Micsonai A., Wien F., Kernya L., Lee Y.H., Goto Y., Réfrégiers M., Kardos J. // Proc. Natl. Acad. Sci. USA. 2015. V. 112. P. E3095–E3103. https://doi.org/10.1073/pnas.1500851112
  25. Micsonai A., Moussong É., Wien F., Boros E., Vadászi H., Murvai N., Lee Y.H., Molnár T., Réfrégiers M., Goto Y., Tantos Á., Kardos J. // Nucleic Acids Res. 2022. V. 50(W1). P. W90–W98. https://doi.org/10.1093/nar/gkac345
  26. Feofanov A.V., Sharonov G.V., Dubinnyi M.A., Astapova M.V., Kudelina I.A., Dubovskii P.V., Rodionov D.I., Utkin Y.N., Arseniev A.S. // Biochemistry (Moscow). 2004. V. 69. P. 1148–1157. https://doi.org/10.1023/b:biry.0000046890.46901.7e
  27. Severyukhina M.S., Ojomoko L.O., Shelukhina I.V., Kudryavtsev D.S., Kryukova E.V., Epifanova L.A., Denisova D.A., Averin A.S., Ismailova A.M., Shaykhutdinova E.R., Dyachenko I.A., Egorova N.S., Murashev A.N., Tsetlin V.I., Utkin Y.N. // Int. J. Biol. Macromol. 2025. V. 288. P. 138626. https://doi.org/10.1016/j.ijbiomac.2024.138626
  28. Otto P., Kephart D., Bitner R., Huber S., Volkerding K. // Promega Notes. 1998. V. 69. P. 19–23.
  29. Sambrook J., Fritsch E.F., Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989.
  30. Ryabinin V.V., Ziganshin R.H., Starkov V.G., Tsetlin V.I., Utkin Y.N. // Russ. J. Bioorg. Chem. 2019. V. 45. P. 107–121. https://doi.org/10.1134/S1068162019020109
  31. Rappsilber J., Mann M., Ishihama Y. // Nat. Protoc. 2007. V. 2. P. 1896–1906. https://doi.org/10.1038/nprot.2007.261
  32. Geyer P.E., Kulak N.A., Pichler G., Holdt L.M., Teupser D., Mann M. // Cell Syst. 2016. V. 2. P. 185– 195. https://doi.org/10.1016/j.cels.2016.01.002
  33. Ma B., Zhang K., Hendrie C., Liang C., Li M., Doherty-Kirby A., Lajoie G. // Rapid Commun. Mass Spectrom. 2003. V. 17. P. 2337–2342. https://doi.org/10.1002/rcm.1198
  34. Lebedev D.S., Kryukova E.V., Ivanov I.A., Egorova N.S., Timofeev N.D., Spirova E.N., Tufanova E.Y., Siniavin A.E., Kudryavtsev D.S., Kasheverov I.E., Zouridakis M., Katsarava R., Zavradashvili N., Iagorshvili I., Tzartos S.J., Tsetlin V.I. // Mol. Pharmacol. 2019. V. 96. P. 664–673. https://doi.org/10.1124/mol.119.117713

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