Enzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptors

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dc.contributor.authorYonamine, Camila M. [UNIFESP]
dc.contributor.authorKondo, Marcia Y. [UNIFESP]
dc.contributor.authorNering, Marcela B. [UNIFESP]
dc.contributor.authorGouvea, Iuri E. [UNIFESP]
dc.contributor.authorOkamoto, Debora [UNIFESP]
dc.contributor.authorAndrade, Douglas [UNIFESP]
dc.contributor.authorSilva, Jose Alberto A. da
dc.contributor.authorPrieto da Silva, Alvaro R. B.
dc.contributor.authorYamane, Tetsuo
dc.contributor.authorJuliano, Maria A. [UNIFESP]
dc.contributor.authorJuliano, Luiz [UNIFESP]
dc.contributor.authorLapa, Antonio J. [UNIFESP]
dc.contributor.authorHayashi, Mirian A. F. [UNIFESP]
dc.contributor.authorLima-Landman, Maria Teresa R. [UNIFESP]
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.contributor.institutionIPEN
dc.contributor.institutionInst Butantan
dc.contributor.institutionUniv Estado Amazonas
dc.date.accessioned2016-01-24T14:35:23Z
dc.date.available2016-01-24T14:35:23Z
dc.date.issued2014-03-01
dc.description.abstractGyroxin is a serine protease displaying a thrombin-like activity found in the venom of the South American rattlesnake Crotalus durissus terrificus. Typically, intravenous injection of purified gyroxin induces a barrel rotation syndrome in mice. the serine protease thrombin activates platelets aggregation by cleaving and releasing a tethered N-terminus peptide from the G-protein-coupled receptors, known as protease-activated receptors (PARs). Gyroxin also presents pro-coagulant activity suggested to be dependent of PARs activation. in the present work, the effects of these serine proteases, namely gyroxin and thrombin, on PARs were comparatively studied by characterizing the hydrolytic specificity and kinetics using PARs-mimetic FRET peptides. We show for the first time that the short (sh) and long (lg) peptides mimetizing the PAR-1, -2, -3, and -4 activation sites are all hydrolyzed by gyroxin exclusively after the Arg residues. Thrombin also hydrolyzes PAR-1 and -4 after the Arg residue, but hydrolyzes sh and lg PAR-3 after the Lys residue. the k(cat)/K-M values determined for gyroxin using sh and lg PAR-4 mimetic peptides were at least 2150 and 400 times smaller than those determined for thrombin, respectively. for the sh and lg PAR-2 mimetic peptides the k(cat)/K-M values determined for gyroxin were at least 6500 and 2919 times smaller than those determined for trypsin, respectively. the k(cat)/K-M values for gyroxin using the PAR-1 and -3 mimetic peptides could not be determined due to the extreme low hydrolysis velocity. Moreover, the functional studies of the effects of gyroxin on PARs were conducted in living cells using cultured astrocytes, which express all PARs. Despite the ability to cleavage the PAR-I, -2, -3, and -4 peptides, gyroxin was unable to activate the PARs expressed in astrocytes as determined by evaluating the cytosolic calcium mobilization. On the other hand, we also showed that gyroxin is able to interfere with the activation of PAR-1 by thrombin or by synthetic PAR-1 agonist in cultured astrocytes. Taken together, the data presented here allow us showing that gyroxin cleaves PARs-mimetic peptides slowly and it does not induce activation of PARs in astrocytes. Although gyroxin does not mobilize calcium it was shown to interfere with PARs activation by thrombin and PAR-1 agonist. the determination of gyroxin enzymatic specificity and kinetics on PAR-1, -2, -3, and -4 will potentially help to fill the gap in the knowledge in this field, as the PARs are still believed to have a key role for the gyroxin biological effects. (C) 2013 Elsevier B.V. All rights reserved.en
dc.description.affiliationUniversidade Federal de São Paulo, Depto Farmacol, São Paulo, Brazil
dc.description.affiliationUniversidade Federal de São Paulo, Depto Biofis, São Paulo, Brazil
dc.description.affiliationIPEN, Ctr Biotecnol, São Paulo, Brazil
dc.description.affiliationInst Butantan, Lab Genet, São Paulo, Brazil
dc.description.affiliationUniv Estado Amazonas, Escola Super Ciencias Saude, INCT, Manaus, Amazonas, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Depto Farmacol, São Paulo, Brazil
dc.description.affiliationUnifespUniversidade Federal de São Paulo, Depto Biofis, São Paulo, Brazil
dc.description.sourceWeb of Science
dc.description.sponsorshipCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
dc.description.sponsorshipConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
dc.description.sponsorshipFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
dc.format.extent64-71
dc.identifierhttp://dx.doi.org/10.1016/j.toxicon.2013.12.002
dc.identifier.citationToxicon. Oxford: Pergamon-Elsevier B.V., v. 79, p. 64-71, 2014.
dc.identifier.doi10.1016/j.toxicon.2013.12.002
dc.identifier.issn0041-0101
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/37501
dc.identifier.wosWOS:000332444100008
dc.language.isoeng
dc.publisherElsevier B.V.
dc.relation.ispartofToxicon
dc.rightsinfo:eu-repo/semantics/restrictedAccess
dc.rights.licensehttp://www.elsevier.com/about/open-access/open-access-policies/article-posting-policy
dc.subjectAstrocytesen
dc.subjectBarrel rotation syndromeen
dc.subjectGyroxinen
dc.subjectProtease-activated receptorsen
dc.subjectSnake toxinen
dc.subjectSerine proteaseen
dc.titleEnzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptorsen
dc.typeinfo:eu-repo/semantics/article
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