Advertisement

Archives of Virology

, Volume 164, Issue 4, pp 1159–1171 | Cite as

Expression, purification and virucidal activity of two recombinant isoforms of phospholipase A2 from Crotalus durissus terrificus venom

  • Raquel Rinaldi Russo
  • Nilton Nascimento dos Santos Júnior
  • Adélia Cristina Oliveira Cintra
  • Luiz Tadeu Moraes Figueiredo
  • Suely Vilela Sampaio
  • Victor Hugo AquinoEmail author
Original Article

Abstract

The global emergence and re-emergence of arthropod-borne viruses (arboviruses) over the past four decades have become a public health crisis of international concern, especially in tropical and subtropical countries. A limited number of vaccines against arboviruses are available for use in humans; therefore, there is an urgent need to develop antiviral compounds. Snake venoms are rich sources of bioactive compounds with potential for antiviral prospection. The major component of Crotalus durissus terrificus venom is a heterodimeric complex called crotoxin, which is constituted by an inactive peptide (crotapotin) and a phospholipase A2 (PLA2-CB). We showed previously the antiviral effect of PLA2-CB against dengue virus, yellow fever virus and other enveloped viruses. The aims of this study were to express two PLA2-CB isoforms in a prokaryotic system and to evaluate their virucidal effects. The sequences encoding the PLA2-CB isoforms were optimized and cloned into a plasmid vector (pG21a) for recombinant protein expression. The recombinant proteins were expressed in the E. coli BL21(DE3) strain as insoluble inclusion bodies; therefore, the purification was performed under denaturing conditions, using urea for protein solubilization. The solubilized proteins were applied to a nickel affinity chromatography matrix for binding. The immobilized recombinant proteins were subjected to an innovative protein refolding step, which consisted of the application of a decreasing linear gradient of urea and dithiothreitol (DTT) concentrations in combination with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS) as a protein stabilizer. The refolded recombinant proteins showed phospholipase activity and virucidal effects against chikungunya virus, dengue virus, yellow fever virus and Zika virus.

Abbreviations

DENV

Dengue virus

ZIKV

Zika virus

YFV

Yellow fever virus

CHIKV

Chikungunya virus

PLA2

Phospholipase A2

sPLA2

Secreted phospholipase A2

svPLA2

Snake venom phospholipase A2

CA

Crotapotin

PLA2-CB

Phospholipase A2 crotoxin B

PLA2-CB1

Phospholipase A2 crotoxin B isoform 1

PLA2-CB2

Phospholipase A2 crotoxin B isoform 2

rPLA2-CB1

Recombinant phospholipase A2 crotoxin B isoform 1

rPLA2-CB2

Phospholipase A2 crotoxin B isoform 2

sn-2

Stereo-specific number 2

6xHis

Polyhistidine tag

LB

Luria-Bertani medium

TBS

Tris-buffered saline

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

DMSO

Dimethyl sulfoxide

CC50

50% cytotoxic concentration

PFU

Plaque-forming units

IPTG

Isopropyl β-D-1-thiogalactopyranoside

BCIP/NBT

5-Bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium

PMSF

Phenylmethylsulfonyl fluoride

DTT

Dithiothreitol

SDS

Sodium dodecyl sulfate

PAGE

Polyacrylamide gel electrophoresis

IgG

Immunoglobulin G

SD

Standard deviation

CHAPS

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate

Notes

Funding

This work was supported by the Sao Paulo Research Foundation (FAPESP), grant no. 2014/02438-6) and the National Council of Technological and Scientific Development (CNPq) (grant no. 479512/2012-4). RR was supported by a FAPESP scholarship (grant no. 2012/12605-1) and VHA holds a CNPq-PQ fellowship (grant no. 306471/2017-5).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal experiments

All animal experiments were performed according to the guidelines of the Brazilian College of Animal Experimentation and were approved by the Ethical Committee on Animal Experimentation of the Campus of Ribeirao Preto, University of Sao Paulo (CEUA/USP-RP, Permit. Nº 12.1.1854.53.8).

Supplementary material

705_2019_4172_MOESM1_ESM.pdf (172 kb)
Supplementary material 1 (PDF 171 kb)
705_2019_4172_MOESM2_ESM.pdf (476 kb)
Supplementary material 2 (PDF 475 kb)

References

  1. 1.
    Alibolandi M, Mirzahoseini H (2011) Chemical assistance in refolding of bacterial inclusion bodies. Biochem Res Int 2011:631607CrossRefPubMedCentralGoogle Scholar
  2. 2.
    Amaral JK, Schoen RT (2018) Chikungunya in Brazil: rheumatologists on the front line. J Rheumatol 45:1491–1492CrossRefGoogle Scholar
  3. 3.
    Bouchier C, Boulain JC, Bon C, Ménez A (1991) Analysis of cDNAs encoding the two subunits of crotoxin, a phospholipase A2 neurotoxin from rattlesnake venom: the acidic non enzymatic subunit derives from a phospholipase A2-like precursor. Biochim Biophys Acta 1088:401–408CrossRefGoogle Scholar
  4. 4.
    Burleson FG, Chambers TM, Wiedbrauk DL (1992) Virology: a laboratory manual. Academic Press, San DiegoGoogle Scholar
  5. 5.
    Castillo JC, Vargas LJ, Segura C, Gutiérrez JM, Pérez JC (2012) In vitro antiplasmodial activity of phospholipases A2 and a phospholipase homologue isolated from the venom of the snake Bothrops asper. Toxins (Basel) 4:1500–1516CrossRefGoogle Scholar
  6. 6.
    Chen YH, Wang YM, Hseu MJ, Tsai IH (2004) Molecular evolution and structure-function relationships of crotoxin-like and asparagine-6-containing phospholipases A2 in pit viper venoms. Biochem J 381:25–34CrossRefPubMedCentralGoogle Scholar
  7. 7.
    Chioato L, Ward RJ (2003) Mapping structural determinants of biological activities in snake venom phospholipases A2 by sequence analysis and site directed mutagenesis. Toxicon 42:869–883CrossRefGoogle Scholar
  8. 8.
    Chiou YL, Cheng YC, Kao PH, Wang JJ, Chang LS (2008) Mutations on the N-terminal region abolish differentially the enzymatic activity, membrane-damaging activity and cytotoxicity of Taiwan cobra phospholipase A2. Toxicon 51:270–279CrossRefGoogle Scholar
  9. 9.
    Collins ND, Barrett AD (2017) Live attenuated yellow fever 17D vaccine: a legacy vaccine still controlling outbreaks in modern day. Curr Infect Dis Rep 19:14CrossRefPubMedCentralGoogle Scholar
  10. 10.
    Dennis EA, Cao J, Hsu YH, Magrioti V, Kokotos G (2011) Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chem Rev 111:6130–6185CrossRefPubMedCentralGoogle Scholar
  11. 11.
    Doley R, Kini RM (2009) Protein complexes in snake venom. Cell Mol Life Sci 66:2851–2871CrossRefGoogle Scholar
  12. 12.
    Faure G, Harvey AL, Thomson E, Saliou B, Radvanyi F, Bon C (1993) Comparison of crotoxin isoforms reveals that stability of the complex plays a major role in its pharmacological action. Eur J Biochem 214:491–496CrossRefGoogle Scholar
  13. 13.
    Faure G, Choumet V, Bouchier C, Camoin L, Guillaume JL, Monegier B, Vuilhorgne M, Bon C (1994) The origin of the diversity of crotoxin isoforms in the venom of Crotalus durissus terrificus. Eur J Biochem 223:161–164CrossRefGoogle Scholar
  14. 14.
    Faure G, Xu H, Saul FA (2011) Crystal structure of crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric β-neurotoxin. J Mol Biol 412:176–191CrossRefGoogle Scholar
  15. 15.
    Ferré H, Ruffet E, Nielsen LL, Nissen MH, Hobley TJ, Thomas OR, Buus S (2005) A novel system for continuous protein refolding and on-line capture by expanded bed adsorption. Protein Sci 14:2141–2153CrossRefPubMedCentralGoogle Scholar
  16. 16.
    Figueiredo LT (2015) The recent arbovirus disease epidemic in Brazil. Rev Soc Bras Med Trop.  https://doi.org/10.1590/0037-8682-0179-2015 Google Scholar
  17. 17.
    Figueiredo LT (2016) How are so many foreign arboviruses introduced in Brazil? Rev Soc Bras Med Trop.  https://doi.org/10.1590/0037-8682-0499-2016 Google Scholar
  18. 18.
    Francischetti IM, Gombarovits ME, Valenzuela JG, Carlini CR, Guimarães JA (2000) Intraspecific variation in the venoms of the South American rattlesnake (Crotalus durissus terrificus). Comp Biochem Physiol C Toxicol Pharmacol 127:23–36Google Scholar
  19. 19.
    Hendon RA, Fraenkel-Conrat H (1971) Biological roles of the two components of crotoxin. Proc Natl Acad Sci USA 68:1560–1563CrossRefGoogle Scholar
  20. 20.
    Hjelmeland LM (1980) A nondenaturing zwitterionic detergent for membrane biochemistry: design and synthesis. Proc Natl Acad Sci USA 77:6368–6370CrossRefGoogle Scholar
  21. 21.
    Jaenicke R (1982) Folding and association of proteins. Biophys Struct Mech 8:231–256CrossRefGoogle Scholar
  22. 22.
    Kang TS, Georgieva D, Genov N, Murakami MT, Sinha M, Kumar RP, Kaur P, Kumar S, Dey S, Sharma S, Vrielink A, Betzel C, Takeda S, Arni RK, Singh TP, Kini RM (2011) Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis. FEBS J 278:4544–4576CrossRefGoogle Scholar
  23. 23.
    Long KC, Ziegler SA, Thangamani S, Hausser NL, Kochel TJ, Higgs S, Tesh RB (2011) Experimental transmission of Mayaro virus by Aedes aegypti. Am J Trop Med Hyg 85:750–757CrossRefPubMedCentralGoogle Scholar
  24. 24.
    Lowe R, Barcellos C, Brasil P, Cruz OG, Honório NA, Kuper H, Carvalho MS (2018) The Zika virus epidemic in Brazil: from discovery to future implications. Int J Environ Res Public Health 15(1):96CrossRefPubMedCentralGoogle Scholar
  25. 25.
    Lôbo de Araújo A, Radvanyi F (1987) Determination of phospholipase A2 activity by a colorimetric assay using a pH indicator. Toxicon 25:1181–1188CrossRefGoogle Scholar
  26. 26.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63CrossRefGoogle Scholar
  27. 27.
    Muller VD, Russo RR, Cintra AC, Sartim MA, ReM Alves-Paiva, Figueiredo LT, Sampaio SV, Aquino VH (2012) Crotoxin and phospholipases A2 from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses. Toxicon 59:507–515CrossRefGoogle Scholar
  28. 28.
    Muller VD, Soares RO, dos Santos NN, Trabuco AC, Cintra AC, Figueiredo LT, Caliri A, Sampaio SV, Aquino VH (2014) Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope. PLoS One 9:e112351CrossRefPubMedCentralGoogle Scholar
  29. 29.
    Murakami M, Sato H, Miki Y, Yamamoto K, Taketomi Y (2015) A new era of secreted phospholipase A2 (sPLA2). J Lipid Res 56:1248–1261CrossRefPubMedCentralGoogle Scholar
  30. 30.
    Nunes DC, Figueira MM, Lopes DS, De Souza DL, Izidoro LF, Ferro EA, Souza MA, Rodrigues RS, Rodrigues VM, Yoneyama KA (2013) BnSP-7 toxin, a basic phospholipase A2 from Bothrops pauloensis snake venom, interferes with proliferation, ultrastructure and infectivity of Leishmania (Leishmania) amazonensis. Parasitology 140:844–854CrossRefGoogle Scholar
  31. 31.
    Pereañez JA, Gómez ID, Patiño AC (2012) Relationship between the structure and the enzymatic activity of crotoxin complex and its phospholipase A2 subunit: an in silico approach. J Mol Graph Model 35:36–42CrossRefGoogle Scholar
  32. 32.
    Quach ND, Arnold RD, Cummings BS (2014) Secretory phospholipase A2 enzymes as pharmacological targets for treatment of disease. Biochem Pharmacol 90:338–348CrossRefPubMedCentralGoogle Scholar
  33. 33.
    Reeks TA, Fry BG, Alewood PF (2015) Privileged frameworks from snake venom. Cell Mol Life Sci 72:1939–1958CrossRefGoogle Scholar
  34. 34.
    Rodrigues RS, Izidoro LF, de Oliveira RJ, Sampaio SV, Soares AM, Rodrigues VM (2009) Snake venom phospholipases A2: a new class of antitumor agents. Protein Pept Lett 16:894–898CrossRefGoogle Scholar
  35. 35.
    Russo RR, Müller VDM, Cintra ACO, Figueiredo LTM, Sampaio SV, Aquino VH (2014) Phospholipase A2 crotoxin B isolated from the venom of Crotalus durissus terrificus exert antiviral effect against dengue virus and yellow fever virus through its catalytic activity. J Virol Antivir Res 3:1Google Scholar
  36. 36.
    Rübsamen K, Breithaupt H, Habermann E (1971) Biochemistry and pharmacology of the crotoxin complex. I. Subfractionation and recombination of the crotoxin complex. Naunyn Schmiedebergs Arch Pharmacol 270:274–288CrossRefGoogle Scholar
  37. 37.
    Sakkas H, Bozidis P, Franks A, Papadopoulou C (2018) Oropouche fever: a review. Viruses 10(4):175Google Scholar
  38. 38.
    Samy RP, Gopalakrishnakone P, Stiles BG, Girish KS, Swamy SN, Hemshekhar M, Tan KS, Rowan EG, Sethi G, Chow VT (2012) Snake venom phospholipases A(2): a novel tool against bacterial diseases. Curr Med Chem 19:6150–6162CrossRefGoogle Scholar
  39. 39.
    Seto M, Ogawa T, Kodama K, Muramoto K, Kanayama Y, Sakai Y, Chijiwa T, Ohno M (2008) A novel recombinant system for functional expression of myonecrotic snake phospholipase A(2) in Escherichia coli using a new fusion affinity tag. Protein Expr Purif 58:194–202CrossRefGoogle Scholar
  40. 40.
    Sharp TM, Tomashek KM, Read JS, Margolis HS, Waterman SH (2017) A new look at an old disease: recent insights into the global epidemiology of dengue. Curr Epidemiol Rep 4:11–21CrossRefPubMedCentralGoogle Scholar
  41. 41.
    Silveira LB, Marchi-Salvador DP, Santos-Filho NA, Silva FP, Marcussi S, Fuly AL, Nomizo A, da Silva SL, Stábeli RG, Arantes EC, Soares AM (2013) Isolation and expression of a hypotensive and anti-platelet acidic phospholipase A2 from Bothrops moojeni snake venom. J Pharm Biomed Anal 73:35–43CrossRefGoogle Scholar
  42. 42.
    Slotta KH, Fraenkel-Conrat H (1938) Schlangengiffe, III: mitteilung reiningung und crystallization des klappershclangengiffes. Berichte der Deutschen Chemischen Gesellschaft 71:1076–1081CrossRefGoogle Scholar
  43. 43.
    Smith HE (2007) The transcriptional response of Escherichia coli to recombinant protein insolubility. J Struct Funct Genom 8:27–35CrossRefGoogle Scholar
  44. 44.
    Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130CrossRefGoogle Scholar
  45. 45.
    Wilder-Smith A, Gubler DJ, Weaver SC, Monath TP, Heymann DL, Scott TW (2017) Epidemic arboviral diseases: priorities for research and public health. Lancet Infect Dis 17:e101–e106CrossRefGoogle Scholar
  46. 46.
    Yunes Quartino PJ, Barra JL, Fidelio GD (2012) Cloning and functional expression of secreted phospholipases A(2) from Bothrops diporus (Yarará Chica). Biochem Biophys Res Commun 427:321–325CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Raquel Rinaldi Russo
    • 1
  • Nilton Nascimento dos Santos Júnior
    • 2
  • Adélia Cristina Oliveira Cintra
    • 3
  • Luiz Tadeu Moraes Figueiredo
    • 4
  • Suely Vilela Sampaio
    • 3
  • Victor Hugo Aquino
    • 1
    Email author
  1. 1.Laboratory of Virology, Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirao PretoUniversity of Sao PauloRibeirao PretoBrazil
  2. 2.Laboratory of Neuro-immuno-endocrinology, Department of Neurosciences and Behavioral Sciences, Ribeirao Preto Medical SchoolUniversity of São PauloRibeirao PretoBrazil
  3. 3.Laboratory of Toxinology, Department of Clinical Analyses, Toxicology and Food Sciences, School of Pharmaceutical Sciences of Ribeirao PretoUniversity of Sao PauloRibeirao PretoBrazil
  4. 4.Virology Research Center, Ribeirao Preto Medical SchoolUniversity of Sao PauloRibeirao PretoBrazil

Personalised recommendations