Crotalus durissus terrificus crotapotin naturally displays preferred positions for amino acid substitutions

 

Laudicéia Alves de Oliveira¹, Rui Seabra Ferreira Jr^1 2, Benedito Barraviera^1 2, Francilene Capel Tavares de Carvalho¹, Luciana Curtolo de Barros², Lucilene Delazari dos Santos^1 2, Daniel Carvalho Pimenta^1 3

 

1 Postgraduate Program in Tropical Diseases, Botucatu Medical School, Sao Paulo State University (UNESP), Botucatu, SP, Brazil.

2 Center for the Studies of Venoms and Venomous Animals (CEVAP), São Paulo State University (UNESP), Botucatu, SP, Brazil.

3 Laboratory of Biochemistry and Biophysics, Butantan Institute, Av. Vital Brazil, 1500, São Paulo, SP CEP 05503-900, Brazil.

 

ABSTRACT

Background

Classically, Crotalus durissus terrificus (Cdt) venom can be described, according to chromatographic criteria, as a simple venom, composed of four major toxins, namely: gyroxin, crotamine, crotoxin and convulxin. Crotoxin is a non-covalent heterodimeric neurotoxin constituted of two subunits: an active phospholipase A₂ and a chaperone protein, termed crotapotin. This molecule is composed of three peptide chains connected by seven disulfide bridges. Naturally occurring variants/isoforms of either crotoxin or crotapotin itself have already been reported.

Methods

The crude Cdt venom was separated by using RP-HPLC and the toxins were identified by mass spectrometry (MS). Crotapotin was purified, reduced and alkylated in order to separate the peptide chains that were further analyzed by mass spectrometry and de novo peptide sequencing.

Results

The RP-HPLC profile of the isolated crotapotin chains already indicated that the α chain would present isoforms, which was corroborated by the MS and tandem mass spectrometry analyses.

Conclusion

It was possible to observe that the Cdt crotapotin displays a preferred amino acid substitution pattern present in the α chain, at positions 31 and 40. Moreover, substitutions could also be observed in β and γ chains (one for each). The combinations of these four different peptides, with the already described chains, would produce ten different crotapotins, which is compatible to our previous observations for the Cdt venom.

 

Key words: Crotalus durissus terrificus; Venom; Crotoxin; Crotapotin; Isoforms

 

Funding

This study was partly supported by grants from the São Paulo Research Foundation (FAPESP - proc. n^o2012/08101-8) to RSFJ and from the Coordination for the Improvement of Higher Education Personnel (CAPES - Edital Toxinologia nº 063/2010, proc. nº 23038.006285/2011-21, AUXPE Toxinologia 1219) to BB. RSFJr is a CNPq DTI fellow researcher (310,395/2014-3). DCP is a fellow CNPq researcher (303,792/2016-7).

 

Received: June 30, 2017.

Revised: November 16, 2017.

Accepted: November 28, 2017.

 

Correspondence: dcpimenta@butantan.gov.br

 

Authors’ contributions

LAO, DCP and LDS designed the study, collected and analyzed data, and wrote the manuscript. FCTC and LCB collected and analyzed data. RSFJ, BB and DCP reviewed the manuscript. All authors read and approved the final version of the manuscript.

 

Ethics approval

All procedures involving snake specimens were in accordance with the ethical standards of the institutional and/or national research committee. The present study was approved by the responsible Ethics Committee on Animal Use of Botucatu Medical School (protocol n^o 1145/2015 - CEUA).

 

Consent for publication

Not applicable.

 

Competing interests

The authors declare that they have no competing interests. One of the authors of this article, Dr. Benedito Barraviera, is the Editor-in-Chief of Journal of Venomous Animals and Toxins including Tropical Diseases. He did not get involved in the peer review process of this manuscript.

 

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