[en] Phosphodiesterases are exonucleases that sequentially hydrolyse phosphodiester bonds of polynucleotides from the 3'-end and release 5-mononucleotides. After more than one decade without any advance in the study of Bothropic phosphodiesterases, we described here the isolation of the first phosphodiesterase from Bothrops jararacussu, which we named Bj-PDE. A five-step column chromatography procedure (size exclusion, hydrophobic interaction, cation exchange, lentil lectin affinity, and blue sepharose affinity) enabled isolation of Bj-PDE with preserved and stable enzymatic activity (bis(p-nitrophenyl) phosphate substrate), Km = 6.9 mM (± 0.7 mM), kcat/Km = 1.7 × 104 M-1 s-1 (± 0.2 × 104 M-1 s-1), MW = 116 kDa (SDS-PAGE), optimum activity around 45 °C at pH 8.0, and stability for 81 days at different storage temperatures (8, -20, and - 80 °C). Ca2+ and Mg2+ ions positively influenced Bj-PDE activity, while EDTA had the opposite action. Zn2+ restored >50 % of enzyme activity after its inhibition by EDTA. The Bj-PDE partial sequence identified by mass spectrometry was very similar to the sequence of BATXPDE1 from Bothrops atrox, which was evolutionarily close to this new PDE. Therefore, our study represents an important progress on the isolation of this minor toxin and sheds new lights on the properties and bioprospection of bothropic phosphodiesterases.
Precision for document type :
Review article
Disciplines :
Chemistry
Author, co-author :
Gobbi Amorim, Fernanda ; Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Silva, Thiago Abrahão; Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n°, 14040-903 Ribeirão Preto, SP, Brazil. Electronic address: thiago@fcfrp.usp.br
de Oliveira Almeida, Gabriela; Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n°, 14040-903 Ribeirão Preto, SP, Brazil. Electronic address: gabi.almeida@usp.br
Redureau, Damien ; Université de Liège - ULiège > Département de chimie (sciences) > Laboratoire de spectrométrie de masse (L.S.M.)
Cabral, Hamilton; Department of BioMolecular Sciences, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil. Electronic address: hamilton@fcfrp.usp.br
Quinton, Loïc ; Université de Liège - ULiège > Département de chimie (sciences) > Chimie biologique
Sampaio, Suely Vilela; Department of Clinical Analysis, Toxicology and Food Science, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n°, 14040-903 Ribeirão Preto, SP, Brazil. Electronic address: suvilela@usp.br
Language :
English
Title :
Isolation and characterization of the first phosphodiesterase (Bj-PDE) from the venom of Bothrops jararacussu snake.
Publication date :
30 April 2023
Journal title :
International Journal of Biological Macromolecules
CNPq - National Council for Scientific and Technological Development [BR]
Funding text :
We thank Mrs. Nanou Tanteliarisoa Haingo and Mrs. Lisette Trzpiot from the MSLab (ULiège) for their technical support in sample preparation, as well as to Mr. Maximilien Fléron and Mrs. Dominique Baiwir for their technical assistance in mass spectrometry analysis at the GIGA-Proteomics Facility (ULiège, EDRF funding). This study was supported by The National Council for Scientific and Technological Development, Brazil (CNPq – PQ 2018, grant #305282/2018-2). Q-Exactive mass spectrometer was supported by European Regional Development Fund (ERDF) and the Walloon Region grants, and Peaks X+ version Studio 10.5 software was supported by ERDF grant: BIOMED HUB Technology Support (number 2.2.1/996). F.G.A. performed the structural characterization by mass spectrometry and analysed data together with L.Q. and T.A.S. G.O.A. purified the protein and assessed its biological activities. T.A.S. analysed data and plotted the graphs. D.R. performed the phylogeny analysis. F.G.A and T.A.S wrote the paper. L.Q. and S.V.S. gave technical support. S.V.S. and H.C. devised the research project and supervised the experiments. S.V.S. searched for funding and revised the final manuscript. All authors read and approved the final manuscript.This study was supported by The National Council for Scientific and Technological Development , Brazil (CNPq – PQ 2018, grant # 305282/2018-2 ). Q-Exactive mass spectrometer was supported by European Regional Development Fund (ERDF) and the Walloon Region grants, and Peaks X+ version Studio 10.5 software was supported by ERDF grant: BIOMED HUB Technology Support (number 2.2.1/996 ).
Ferraz, C.R., Arrahman, A., Xie, C., Casewell, N.R., Lewis, R.J., Kool, J., Cardoso, F.C., Multifunctional toxins in snake venoms and therapeutic implications: from pain to hemorrhage and necrosis. Front. Ecol. Evol., 7, 2019, 218, 10.3389/fevo.2019.00218.
Ullah, A., Ullah, K., Ali, H., Betzel, C., Ur Rehman, S., The sequence and a three-dimensional structural analysis reveal substrate specificity among snake venom phosphodiesterases. Toxins, 11(11), 2019, 625, 10.3390/toxins11110625.
Boldrini-Franca, J., Cologna, C.T., Pucca, M.B., Bordon, K.D.C.F., Amorim, F.G., Anjolette, F.A.P., Cordeiro, F.A., Wiezel, A., Cerni, F.A., Junior, E.L.P., Shibao, P.Y.T., Ferreira, I.G., Oliveira, I.S.D., Cardoso, I.A., Arantes, E.C., Minor snake venom proteins: structure, function and potential applications. Biochim. Biophys. Acta Gen. Subj. 1861:4 (2017), 824–838, 10.1016/j.bbagen.2016.12.022.
Dhananjaya, B.L., D'souza, C.J.M., An overview on nucleases (DNase, RNase, and phosphodiesterase) in snake venoms. Biochem. Mosc. 75:1 (2010), 1–6, 10.1134/S0006297910010013.
Aird, S.D., Ophidian envenomation strategies and the role of purines. Toxicon 40:4 (2002), 335–393, 10.1016/S0041-0101(01)00232-X.
Weldon, C.L., Mackessy, S.P., Biological and proteomic analysis of venom from the puerto rican racer (Alsophis portoricensis: Dipsadidae). Toxicon 55:2–3 (2010), 558–569, 10.1016/j.toxicon.2009.10.010.
Amorim, F.G., Morandi-Filho, R., Fujimura, P.T., Ueira-Vieira, C., Sampaio, S.V., New findings from the first transcriptome of the Bothrops moojeni snake venom gland. Toxicon 140 (2017), 105–117, 10.1016/j.toxicon.2017.10.025.
Amorim, F.G., Costa, T.R., Baiwir, D., De Pauw, E., Quinton, L., Sampaio, S.V., Proteopeptidomic, functional and immunoreactivity characterization of Bothrops moojeni snake venom: influence of snake gender on venom composition. Toxins, 10(5), 2018, 177, 10.3390/toxins10050177.
Kunalan, S., Othman, I., Syed Hassan, S., Hodgson, W.C., Proteomic characterization of two medically important malaysian snake venoms, Calloselasma rhodostoma (Malayan pit Viper) and Ophiophagus hannah (King Cobra). Toxins, 10(11), 2018, 434, 10.3390/toxins10110434.
Gren, E.C., Kitano, E.S., Andrade-Silva, D., Iwai, L.K., Reis, M.S., Menezes, M.C., Serrano, S.M., Comparative analysis of the high molecular mass subproteomes of eight bothrops snake venoms. Comp. Biochem. Physiol. D: Genomics Proteomics 30 (2019), 113–121, 10.1016/j.cbd.2019.01.012.
de Oliveira, I.S., Cardoso, I.A., Bordon, K.D.C.F., Carone, S.E.I., Boldrini-França, J., Pucca, M.B., Zoccal, K.F., Faccioli, L.H., Sampaio, S.V., Rosa, J.C., Arantes, E.C., Global proteomic and functional analysis of Crotalus durissus collilineatus individual venom variation and its impact on envenoming. J. Proteome 191 (2019), 153–165, 10.1016/j.jprot.2018.02.020.
Valério, A.A., Corradini, A.C., Panunto, P.C., Mello, S.M., Hyslop, S., Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom. J. Protein Chem. 21:8 (2002), 495–503, 10.1023/A:1022414503995.
Al-Saleh, S., Khan, S.U., Ashraf, M., Biochemical characterization and some biological properties of the phosphodiesterase I purified from agistrodon bilineatus venom. Natl. Inst. Sci. Commun. Policy Res. 46:3 (2009), 221–229 http://nopr.niscpr.res.in/handle/123456789/4583.
Al-Saleh, S.S., Khan, S.U., Purification and characterization of phosphodiesterase I from Walterinnesia aegyptia venom. Prep. Biochem. Biotechnol. 41:3 (2011), 262–277, 10.1080/10826068.2011.575319.
Peng, L., Xu, X., Shen, D., Zhang, Y., Song, J., Yan, X., Guo, M., Purification and partial characterization of a novel phosphodiesterase from the venom of Trimeresurus stejnegeri: inhibition of platelet aggregation. Biochimie 93:9 (2011), 1601–1609, 10.1016/j.biochi.2011.05.027.
Mitra, J., Bhattacharyya, D., Phosphodiesterase from Daboia russelli russelli venom: purification, partial characterization and inhibition of platelet aggregation. Toxicon 88 (2014), 1–10, 10.1016/j.toxicon.2014.06.004.
Trummal, K., Aaspõllu, A., Tõnismägi, K., Samel, M., Subbi, J., Siigur, J., Siigur, E., Phosphodiesterase from Vipera lebetina venom–structure and characterization. Biochimie 106 (2014), 48–55, 10.1016/j.biochi.2014.07.020.
Kiheli, H., Chérifi, F., Ameziani, M., Saoud, S., Hariti, G., Laraba-Djebari, F., Isolation and characterization of CD39-like phosphodiesterase (Cc-PDE) from Cerastes cerastes venom: molecular inhibitory mechanism of antiaggregation and anticoagulation. Protein Pept. Lett. 28:4 (2021), 426–441, 10.2174/0929866527666200813200148.
de Oliveira, I.S., Pucca, M.B., Wiezel, G.A., Cardoso, I.A., Bordon, K.D.C.F., Sartim, M.A., Kalogeropoulos, K., Ahmadi, S., Baiwir, D., Nonato, M.C., Sampaio, S.V., Laustsen, A.H., Keller, U.A.D., Quinton, L., Arantes, E.C., Unraveling the structure and function of CdcPDE: a novel phosphodiesterase from Crotalus durissus collilineatus snake venom. Int. J. Biol. Macromol. 178 (2021), 180–192, 10.1016/j.ijbiomac.2021.02.120.
Smith, P.E., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:1 (1985), 76–85, 10.1016/0003-2697(85)90442-7.
Laemmli, U.K., Beguin, F., Gujer-Kellenberger, G., A factor preventing the major head protein of bacteriophage T4 from random aggregation. J. Mol. Biol. 47:1 (1970), 69–85, 10.1016/0022-2836(70)90402-X.
Ma, B., Zhang, K., Hendrie, C., Liang, C., Li, M., Doherty-Kirby, A., Lajoie, G., PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun. Mass Spectrom. 17:20 (2003), 2337–2342, 10.1002/rcm.1196.
Sherkhane, A.S., Somnath, W., Gomase, V.S., Study of conserved domain across divergent phylogenetic lineages of long neurotoxin from genus naja (Elapidae Family). J. Phylogenet. Evol. Biol., 2014, 1–4, 10.4172/2329-9002.1000127.
Ministério da Saúde. Boletim Epidemiológico 09 Vol. 51Mar. 2020 - Acidentes ofídicos no Brasil 2018. https://www.gov.br/saude/pt-br/assuntos/saude-de-a-a-z/a/animais-peconhentos/acidentes-por-abelhas-1/arquivos/boletim-epidemiologico-09-vol-51mar-2020-acidentes-ofidicos-no-brasil-2018.pdf/view, 2020. (Accessed 17 June 2021)
Rodrigues, C.F.B., Zdenek, C.N., Bourke, L.A., Seneci, L., Chowdhury, A., Freitas-de-Sousa, L.A., Menezes, F.D.A., Moura-da-Silva, A.M., Tanaka-Azevedo, A.M., Fry, B.G., Clinical implications of ontogenetic differences in the coagulotoxic activity of Bothrops jararacussu venoms. Toxicol. Lett. 348 (2021), 59–72, 10.1016/j.toxlet.2021.05.005.
Uzair, B., Khan, B.A., Sharif, N., Shabbir, F., Menaa, F., Phosphodiesterases (PDEs) from snake venoms: therapeutic applications. Protein Pept. Lett. 25:7 (2018), 612–618, 10.2174/0929866525666180628160616.
Björk, W., Purification of phosphodiesterase from Bothrops atrox venom, with special consideration of the elimination of monophosphatases. J. Biol. Chem. 238:7 (1963), 2487–2490, 10.1016/S0021-9258(19)67998-6.
Frischauf, A.M., Eckstein, F., Purification of a phosphodiesterase from Bothrops atrox venom by affinity chromatography. Eur. J. Biochem. 32:3 (1973), 479–485, 10.1111/j.1432-1033.1973.tb02631.x.
Philipps, G.R., Purification and characterization of phosphodiesterase I from Bothrops atrox. Biochim. Biophys. Acta, Nucleic Acids Protein Synth. 432:2 (1976), 237–244, 10.1016/0005-2787(76)90165-9.
Sulkowski, E., Björk, W., Laskowski, M. Sr., A specific and nonspecific alkaline monophosphatase in the venom of Bothrops atrox and their occurrence in the purified venom phosphodiesterase. J. Biol. Chem. 238:7 (1963), 2477–2486, 10.1016/S0021-9258(19)67997-4.
Fulkerson, Z., Wu, T., Sunkara, M., Vander Kooi, C., Morris, A.J., Smyth, S.S., Binding of autotaxin to integrins localizes lysophosphatidic acid production to platelets and mammalian cells. J. Biol. Chem. 286:40 (2011), 34654–34663, 10.1074/jbc.M111.276725.
Betts, M.J., Russell, R.B., Amino acid properties and consequences of substitutions. Bioinforma. Geneticists, 317, 2003, 289, 10.1002/0470867302.
