Full text |
|
Research article - Vol. 25, 2019 |
Proteome of fraction from Tityus serrulatusvenom reveals new enzymes and toxins
1 Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n, Monte Alegre, Ribeirão Preto, SP 14040-903, Brazil.
2 University of Vila Velha, Vila Velha, ES, Brazil.
3 Laboratory of mass spectrometry, MolSys Research Unit, Liège Université, Liège, Belgium.
ABSTRACT
Background:
Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities.
Methods:
Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database.
Results:
Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%).
Conclusions:
The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.
Keywords: Tityus serrulatus; scorpion venom; enzymes; proteases; ACE inhibitors; proteome
Received: July 30, 2018.
Accepted: October 08, 2018.
Correspondence:ecabraga@fcfrp.usp.br
Competing interests The authors declare that there are no competing interests.
Authors' contributions FGA and HLT performed the biochemical characterization and purification of Ts venom. Both authors contributed equally to this work. MD and HTL performed the shotgun proteomic experiments. FGA analyzed the proteomic data and wrote the manuscript. CTC, EDP and LQ supervised the mass spectrometry assays and data analysis. ECA and FGA are designer of the research, ECA searched for funding and supervised the experiments related to the venom fractionation and biochemical characterizations. All authors read, corrected and approved the final manuscript.