Please use this identifier to cite or link to this item: http://repositorio.unifesp.br/handle/11600/37501
Title: Enzyme specificity and effects of gyroxin, a serine protease from the venom of the South American rattlesnake Crotalus durissus terrificus, on protease-activated receptors
Authors: Yonamine, Camila M. [UNIFESP]
Kondo, Marcia Y. [UNIFESP]
Nering, Marcela B. [UNIFESP]
Gouvea, Iuri E. [UNIFESP]
Okamoto, Debora [UNIFESP]
Andrade, Douglas [UNIFESP]
Silva, Jose Alberto A. da
Prieto da Silva, Alvaro R. B.
Yamane, Tetsuo
Juliano, Maria A. [UNIFESP]
Juliano, Luiz [UNIFESP]
Lapa, Antonio J. [UNIFESP]
Hayashi, Mirian A. F. [UNIFESP]
Lima-Landman, Maria Teresa R. [UNIFESP]
Universidade Federal de São Paulo (UNIFESP)
IPEN
Inst Butantan
Univ Estado Amazonas
Keywords: Astrocytes
Barrel rotation syndrome
Gyroxin
Protease-activated receptors
Snake toxin
Serine protease
Issue Date: 1-Mar-2014
Publisher: Elsevier B.V.
Citation: Toxicon. Oxford: Pergamon-Elsevier B.V., v. 79, p. 64-71, 2014.
Abstract: Gyroxin 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.
URI: http://repositorio.unifesp.br/handle/11600/37501
ISSN: 0041-0101
Other Identifiers: http://dx.doi.org/10.1016/j.toxicon.2013.12.002
Appears in Collections:Em verificação - Geral

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