Structural determinants of the hyperalgesic activity of myotoxic Lys49-phospholipase A(2)

Author(s)

Zambelli, Vanessa Olzon ; Chioato, Lucimara ; Gutierrez, Vanessa Pacciari ; Ward, Richard John ; Cury, Yara

• Document type: Journal article
• Source: Journal of Venomous Animals and Toxins including Tropical Diseases; v. 23, n. 1 FEB 10 2017.
• Web of Science Citations: 5

Abstract

Background: Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A(2) (Lys49-PLA(2)) from the venom of Bothrops jararacussu, which despite of the lack of catalytic activity induces myotoxicity, inflammation and pain. The C-terminal region of the Lys49-PLA(2)s 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-PLA(2)-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 PLA(2) myotoxicity. 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 PLA(2) catalytic site is relevant for the non-catalytic PLA(2)-induced pain and inflammation. Wistar male rats received intraplantar injections with mutant PLA(2). 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 PLA(2)-induced pain. (AU)