Natterins, a new class of proteins with kininogenase activity characterized from Thalassophryne nattereri fish venom
Introduction
In venomous animals, evolution has done an extraordinary job of making toxins with a wide variety of sequence motifs, which are essential for targeting important physiological components with affinity high enough to kill or immobilize preys and predators. Therefore, venoms are extensively studied revealing tools to understand physiological pathways and eventually discover new drugs. Moreover, the knowledge of toxin function helps in the understanding and treating the human victims inflicted by venomous animals. Amongst venomous animals, several species of fishes can produce severe human envenoming and interesting toxins have already been described from their venoms [1], [2], [3]. In the North and Northeast of Brazil, Thalassophryne nattereri fishes, belonging to Batrachoididae family, are responsible for several accidents amongst fishermen [4]. Human accidents occur by contact with the fish spines located on their dorsum and both sides of the head, which are connected to the venom glands. Envenoming symptoms include severe edema and pain followed by a fast settling necrosis, both in human victims and experimental animals [5]. It is estimated that hundreds of accidents occur every year and the incidence is underestimated because patients seldom look for medical care due to its lack of efficacy.
T. nattereri venom is composed of proteins endowed with proteolytic and myotoxic properties, but devoid of phospholipase A2 activity [6]. Analysis of its local effects showed myotoxicity with difficult muscle regeneration [7]. The blood flow at microvessels was also impaired with stasis and presence of thrombi in venules, focal transient constrictions in arterioles and increased vascular permeability. Venom lacked a direct pro-coagulant activity, but exerted a strong cytolytic action on platelets and endothelial cells in vitro [8]. Recently, we also have demonstrated a kininogenase activity in the whole venom, which was correlated, by the use of specific antagonists, with the major symptoms of envenoming including local edema and nociception [9].
With the purpose of understanding the nature of T. nattereri venom toxicity, we carried out a structural characterization of the major venom toxins through proteomic and transcriptomic techniques. Under these approaches we isolated the major venom toxin with a kininogenase activity as well as its and other related cDNA sequences from the venom gland cDNA library. These are novel sequences in databanks, with minor resemblance to other proteinases, characterizing a new family of proteins named natterins, presenting a kininogenase activity.
Section snippets
Characterization of venom toxins
T. nattereri venom was collected from 40 specimens collected at Mundaú lagoon, State of Alagoas, Brazil. Venom was extracted from the openings at the tip of the spines by applying pressure at their bases. Venom was pooled and conserved at –80 °C before use. For toxin purification, 3 mg venom was diluted in 500 ml 50 mM Tris–HCl pH 8.0 and subjected to fractionation in a Mono-S FPLC-column equilibrated with the same buffer, and eluted with a linear gradient from 0 to 2 M NaCl in the same buffer
Analysis of the main venom toxins
T. nattereri venom was fractionated by current chromatographic methods and the main fractions characterized for primary structure and screened for the most important venom activities: induction of edema, nociception and kininogenase activity. Following a cation-exchange chromatography (FPLC, Mono-S column, pH 8.0), five fractions were isolated: two acidic/neutral (MS1, MS2) collected before the initiation of the NaCl gradient and three basic (MS3, MS4 and MS5) fractions collected with NaCl
Discussion
Biochemical characterization of major toxins from venom mixtures may lead to unknown components with important biological applications. Additionally, it is essential for understanding the mechanisms of action, structure/function relationships, cloning and production of recombinant proteins. In this paper, we used a combined approach of pharmacological, proteomic and transcriptomic techniques, which resulted in the description of natterins, a new class of kininogenases with completely distinct
Acknowledgements
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Brazilian Research Council (CNPq).
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