Fibrinogen-clotting enzyme, pictobin, from Bothrops pictus snake venom. Structural and functional characterization

Dan E. Vivas-Ruiz, Gustavo A. Sandoval, Edgar Gonzalez-Kozlova, Jacquelyne Zarria-Romero, Fanny Lazo, Edith Rodríguez, Henrique P.B. Magalhães, Carlos Chávez-Olortegui, Luciana S. Oliveira, Valeria G. Alvarenga, Félix A. Urra, Jorge Toledo, Armando Yarlequé, Johannes A. Eble, Eladio F. Sanchez

Research output: Contribution to journalArticlepeer-review

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Abstract

A thrombin-like enzyme, pictobin, was purified from Bothrops pictus snake venom. It is a 41-kDa monomeric glycoprotein as showed by mass spectrometry and contains approx. 45% carbohydrate by mass which could be removed with N-glycosidase. Pictobin coagulates plasma and fibrinogen, releasing fibrinopeptide A and induces the formation of a friable/porous fibrin network as visualized by SEM. The enzyme promoted platelet aggregation in human PRP and defibrination in mouse model and showed catalytic activity on chromogenic substrates S-2266, S-2366, S-2160 and S-2238. Pictobin interacts with the plasma inhibitor α2-macroglobulin, which blocks its interaction with fibrinogen but not with the small substrate BApNA. Heparin does not affect its enzymatic activity. Pictobin cross reacted with polyvalent bothropic antivenom, and its deglycosylated form reduced its catalytic action and antivenom reaction. In breast and lung cancer cells, pictobin inhibits the fibronectin-stimulated migration. Moreover, it produces strong NADH oxidation, mitochondrial depolarization, ATP decrease and fragmentation of mitochondrial network. These results suggest by first time that a snake venom serinprotease produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics. Structural model of pictobin reveals a conserved chymotrypsin fold β/β hydrolase. These data indicate that pictobin has therapeutic potential in the treatment of cardiovascular disorders and metastatic disease.

Original languageEnglish
Pages (from-to)779-795
Number of pages17
JournalInternational Journal of Biological Macromolecules
Volume153
DOIs
StatePublished - 15 Jun 2020

Bibliographical note

Funding Information:
We thank S. Gontijo for her technical assistance. This work was supported by Convenio de Cooperación Bilateral CONCYTEC (Perú) – CNPq (Brazil), Grant 490269/2013-3 , Fundação de Amparo y Pesquisa do Estado de Minas Gerais ( FAPEMIG , Brazil, Grants APQ-01858-15 , AUC-00022-16 ), Programa de Pos Graduação in Toxinology , Instituto Butantan , SP. Programa Nacional de Innovación para la Competitividad y Productividad – Innóvate Perú (Contrato N° 131- FINCyT-2013 ),Vicerrectorado de Investigación y Posgrado – Universidad Nacional Mayor de San Marcos , Perú (Proyectos N° B19101621 , B17101271 ) and partially supported by FONDECYT -Chile postdoctoral fellowship # 3170813 , CONICYT-Chile PCI-Biotechnology # Redbio0027 , FONDECYT -Chile: 1181823 , ICM P09-015-F , EQM140038 and EQM140156 . This report is part of a PhD Thesis of Dan Vivas-Ruiz, Post Graduate School in Biological Sciences, UNMSM. E.F.S, is Research Member of CNPq. J.A.E. received financial support for the German-Brazilian cooperation with E.F.S. from Deutsche Forschungsgemeinschaft (DFG-grant Eb177/13-1 ).

Funding Information:
We thank S. Gontijo for her technical assistance. This work was supported by Convenio de Cooperaci?n Bilateral CONCYTEC (Per?) ? CNPq (Brazil), Grant 490269/2013-3, Funda??o de Amparo y Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil, Grants APQ-01858-15, AUC-00022-16), Programa de Pos Gradua??o in Toxinology, Instituto Butantan, SP. Programa Nacional de Innovaci?n para la Competitividad y Productividad ? Inn?vate Per? (Contrato N? 131- FINCyT-2013),Vicerrectorado de Investigaci?n y Posgrado ? Universidad Nacional Mayor de San Marcos, Per? (Proyectos N? B19101621, B17101271) and partially supported by FONDECYT-Chile postdoctoral fellowship #3170813, CONICYT-Chile PCI-Biotechnology #Redbio0027, FONDECYT-Chile: 1181823, ICM P09-015-F, EQM140038 and EQM140156. This report is part of a PhD Thesis of Dan Vivas-Ruiz, Post Graduate School in Biological Sciences, UNMSM. E.F.S, is Research Member of CNPq. J.A.E. received financial support for the German-Brazilian cooperation with E.F.S. from Deutsche Forschungsgemeinschaft (DFG-grant Eb177/13-1). The authors declared there is no conflict of interest.

Publisher Copyright:
© 2020 Elsevier B.V.

Keywords

  • Bothrops pictus
  • Cancer cells
  • Mitochondrial bioenergetics
  • Oxidative phosphorylation
  • Snake venom
  • Thrombin-like enzyme

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