Structural determinants of the hyperalgesic activity of myotoxic Lys49-phospholipase A₂

Vanessa Olzon Zambelli¹, Lucimara Chioato², Vanessa Pacciari Gutierrez¹, Richard John Ward² , Yara Cury¹

 

1 Butantan Institute, Special Laboratory for Pain and Signaling, Av. Vital Brazil, 1500, São Paulo, SP CEP 05503-900, Brazil.

2 Department of Chemistry, School of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP, Brazil.

 

ABSTRACT

Background

Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A₂ (Lys49-PLA₂) 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-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 PLA₂ 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 PLA₂ catalytic 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.

 

Key words: Lys49-Phospholipase A2; Hyperalgesia; Site-directed mutagenesis; Myotoxic effect; Edema; Membrane damage.

 

Funding

This work was supported by a grant from the State of São Paulo Research Foundation (FAPESP, grant no. 2002/12906-0) and by Butantan Foundation, São Paulo, Brazil.

 

Received: September 1, 2016.

Revised: February 1, 2017.

Accepted: February 10, 2017.

 

Correspondence: yara.cury@butantan.gov.br; yarac57@gmail.com

 

Authors’ contributions

YC, RJW and VOZ conceived the project. VOZ, LC, VPG and YC designed and performed the experiments and carried out data analysis. VOZ wrote the manuscript with input from RJW. All authors read and approved the final manuscript.

 

Competing interests

The authors declare that they have no competing interests.

 

Consent for publication

Not applicable.

 

Ethics approval

All animal tests were conducted in accordance with the guidelines of the International Association for the Study of Pain [20]. The present study was approved by the Institutional Animal Care Committee of the Butantan Institute (CEUAIB, protocol number 118/2002).