Purification and biochemical characterization of three myotoxins from Bothrops mattogrossensis snake venom with toxicity against Leishmania and tumor cells.

Andréa A De Moura,Sulamita S Setúbal,Carla F C Fernandes,João G Ribeiro,Auro Nomizo,Andreimar M Soares,Neuza B Barros,Saulo L Da Silva,Roberto Nicolete,Laura A Moura,Rodrigo G Stábeli,Andre L Fuly,George A Oliveira,Juliana P Zuliani,Anderson M Kayano,Leonardo A Calderon

doi:10.1155/2014/195356

Abstract

Bothrops mattogrossensis snake is widely distributed throughout eastern South America and is responsible for snakebites in this region. This paper reports the purification and biochemical characterization of three new phospholipases A2 (PLA2s), one of which is presumably an enzymatically active Asp49 and two are very likely enzymatically inactive Lys49 PLA2 homologues. The purification was obtained after two chromatographic steps on ion exchange and reverse phase column. The 2D SDS-PAGE analysis revealed that the proteins have pI values around 10, are each made of a single chain, and have molecular masses near 13 kDa, which was confirmed by MALDI-TOF mass spectrometry. The N-terminal similarity analysis of the sequences showed that the proteins are highly homologous with other Lys49 and Asp49 PLA2s from Bothrops species. The PLA2s isolated were named BmatTX-I (Lys49 PLA2-like), BmatTX-II (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2). The PLA2s induced cytokine release from mouse neutrophils and showed cytotoxicity towards JURKAT (leukemia T) and SK-BR-3 (breast adenocarcinoma) cell lines and promastigote forms of Leishmania amazonensis. The structural and functional elucidation of snake venoms components may contribute to a better understanding of the mechanism of action of these proteins during envenomation and their potential pharmacological and therapeutic applications.

Highlights

Snake venoms contain a complex mixture of components with a wide range of biological and pharmacological activities
The present study describes for the first time the isolation, identification, and functional characterization of three myotoxic phospholipases A2, named: BmatTX-I (Lys49 Phospholipase A2 (PLA2)-like), BmatTXII (Lys49 PLA2-like), and BmatTX-III (Asp49 PLA2) and evaluates their activity against Leishmania and tumor cells
Coagulation activity was confirmed after incubation of different concentrations of B. mattogrossensis crude venom with plasma in which the minimum coagulation dose capable of promoting coagulation in less than 1 min was 0.325 μg of protein

Summary

Introduction

Snake venoms contain a complex mixture of components with a wide range of biological and pharmacological activities. More than 90% of their dry weight is composed of proteins, including a variety of enzymes, such as phospholipases A2, proteases (metallo and serine), L-amino acid oxidases, esterases, as well as many other nonenzymatic proteins and peptides [1,2,3]. These proteins and peptides can be grouped into a small number of superfamilies based on remarkable similarities in their primary, secondary, and tertiary structures, while showing distinct pharmacologic effects [3]. PLA2’s involvement in a variety of inflammatory diseases and accidents caused by venomous animals has raised medical and scientific interest in this enzyme [7, 8]

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Conclusion