title: Structural determinants of the hyperalgesic activity of myotoxic Lys49-phospholipase A2.
abstract: BACKGROUND
  Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2(Lys49-PLA2) from the venom ofBothrops jararacussu,which despite of the lack of catalytic activity induces myotoxicity, inflammation and pain. The C-terminal region of the Lys49-PLA2s is important for these effects; however, the amino acid residues that determine hyperalgesia and edema are unknown. The aim of this study was to characterize the structural determinants for the Lys49-PLA2-induced nociception and inflammation.
  
  
  METHODS
  Scanning alanine mutagenesis in the active-site and C-terminal regions of BthTx-I has been used to study the structural determinants of toxin activities. The R118A mutant was employed as this substitution decreases PLA2myotoxicity. In addition, K115A and K116A mutants - which contribute to decrease cytotoxicity - and the K122A mutant - which decreases both myotoxicity and cytotoxicity - were also used. The H48Q mutant - which does not interfere with membrane damage or myotoxic activity - was used to evaluate if the PLA2catalytic site is relevant for the non-catalytic PLA2-induced pain and inflammation. Wistar male rats received intraplantar injections with mutant PLA2. Subsequently, hyperalgesia and edema were evaluated by the paw pressure test and by a plethysmometer. Native and recombinant BthTx-I were used as controls.
  
  
  RESULTS
  Native and recombinant BthTx-I induced hyperalgesia and edema, which peaked at 2 h. The R118A mutant did not induce nociception or edema. The mutations K115A and K116A abolished hyperalgesia without interfering with edema. Finally, the K122A mutant did not induce hyperalgesia and presented a decreased inflammatory response.
  
  
  CONCLUSIONS
  The results obtained with the BthTx-I mutants suggest, for the first time, that there are distinct residues responsible for the hyperalgesia and edema induced by BthTx-I. In addition, we also showed that cytolytic activity is essential for the hyperalgesic effect but not for edematogenic activity, corroborating previous data showing that edema and hyperalgesia can occur in a non-dependent manner. Understanding the structure-activity relationship in BthTx-I has opened new possibilities to discover the target for PLA2-induced pain.
authors.affiliation: Butantan Institute, Special Laboratory for Pain and Signaling, Av. Vital Brazil, 1500, São Paulo, SP CEP 05503-900 Brazil.
authors.forename: Vanessa Olzon
authors.initials: VO
authors.lastname: Zambelli
authors.affiliation: Department of Chemistry, School of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP Brazil.
authors.forename: Lucimara
authors.initials: L
authors.lastname: Chioato
authors.affiliation: Butantan Institute, Special Laboratory for Pain and Signaling, Av. Vital Brazil, 1500, São Paulo, SP CEP 05503-900 Brazil.
authors.forename: Vanessa Pacciari
authors.initials: VP
authors.lastname: Gutierrez
authors.affiliation: Department of Chemistry, School of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP Brazil.
authors.forename: Richard John
authors.initials: RJ
authors.lastname: Ward
authors.affiliation: Butantan Institute, Special Laboratory for Pain and Signaling, Av. Vital Brazil, 1500, São Paulo, SP CEP 05503-900 Brazil.
authors.forename: Yara
authors.initials: Y
authors.lastname: Cury
keywords: Edema; Hyperalgesia; Lys49-Phospholipase A2; Membrane damage; Myotoxic effect; Site-directed mutagenesis