View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Toxicon 99 (2015) 109e117 Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes T.M. Mendes a, P.T.C. Guimaraes-Okamoto~ b, R.A. Machado-de-Avila a, D. Oliveira a, * M.M. Melo c, Z.I. Lobato c, E. Kalapothakis d,C.Chavez-Ol ortegui a, a Departamento de Bioquímica-Imunologia, Instituto de Ci^encias Biologicas, Universidade Federal de Minas Gerais, CP: 486, CEP: 31270-901 Belo Horizonte, MG, Brazil b Unesp-Faculdade de Medicina Veterinaria e Zootecnia de Botucatu, Distrito de Rubiao~ Júnior s/n, CEP: 18618-970 Botucatu, SP, Brazil c Escola de Veterinaria e Universidade Federal de Minas Gerais, CP: 567, CEP: 30123-970 Belo Horizonte, MG, Brazil d Departamento de Biologia Geral, Instituto de Ci^encias Biologicas, Universidade Federal de Minas Gerais, CP: 486, CEP: 31270-901 Belo Horizonte, MG, Brazil article info abstract Article history: This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus Received 7 August 2014 fasciolatus, which is an endemic species found in the central Brazil (States of Goias and Minas Gerais), Received in revised form being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic 24 March 2015 to mice being the LD is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 Accepted 25 March 2015 50 fractions ranged in size from 6 to 10e80 kDa. Sheep were employed for anti-T. fasciolatus venom serum Available online 27 March 2015 production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Keywords: fi Tityus fasciolatus scorpion venom Using known primers for T. serrulatus toxins, we have identi ed three toxins sequences from T. fasciolatus fi fi Toxins venom. Linear epitopes of these toxins were localized and fty- ve overlapping pentadecapeptides Anti-T. fasciolatus sera covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane Epitope mapping (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction scorpion accidents (Lira-da-Silva et al., 2000). In Northern region, all scorpions causing accidents are identified as T. obscurus (Pardal Tityus fasciolatus is an endemic species found in central Brazil et al., 2003). (Goias) and part of Minas Gerais (Minas Triangle), being responsible The scorpion venom is a complex mixture composed of many for many cases of accidents in this area (Lourenço, 1995, 2003). basic proteins of low molecular mass, amino acids, inorganic salts, Tityus serrulatus (yellow scorpion), Tityus bahiensis (scorpion- lipids, nucleotides and biogenic amines. Generally, it lacks proteo- brown), Tityus stigmurus (yellow scorpion-of-northeast) and Tityus lytic activity, hemolytic, cholinesterase, phospholipase and fibri- obscurus (black scorpion-the-amazon) are the main species of the nolytic activities (Zlotkin, 1978; Possani, 1984). However, toxic genus Tityus of public health importance in Brazil (Ministry of effects are mainly caused by a class of proteins called neurotoxins, Health (2011)). The T. serrulatus and T. bahiensis are considered which act on various ion channels (Possani et al., 1977, 1999; the most common species responsible for the vast majority of Wagner et al., 2003; Vasconcellos et al., 2005; Kahn et al., 2009). scorpion stings in Brazil (Ministry of Health (2011)). However, in The scorpion venoms of the same genus have similar components; Brazilian Northeast, T. stigmurus was responsible for 51.2% of the toxins have high homology in their amino acid sequences and structural features and also are antigenically very similar (Possani et al., 1992, 1999; Batista et al., 2007; Gao et al., 2008; Borges * Corresponding author. Departamento de Bioquímica e Imunologia, Instituto de et al., 2008, 2010; Chen and Khung, 2012a, 2012b). ^ Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. The recommended treatment in a scorpion sting event is the E-mail address: [email protected] (C. Chavez-Ol ortegui). http://dx.doi.org/10.1016/j.toxicon.2015.03.018 0041-0101/© 2015 Elsevier Ltd. All rights reserved. 110 T.M. Mendes et al. / Toxicon 99 (2015) 109e117 specific antiserum (Ministry of Health (2011)). In Brazil currently The managements and procedures involving animal experi- have two sera treatments for accidents with scorpions: anti- ments were approved by Ethics Committee on Animal Experiments scorpion (produced with the whole venom of T. serrulatus) and of UFMG (CETEA) (protocol number 131/2006). anti-arachnid (produced with a mixture of venoms of spiders and scorpion T. serrulatus), which are used in all cases of accidents 2.2. ELISA and western blotting analyses involving scorpions, regardless of the species (Ministry of Health (2011)). The T. serrulatus venom is utilized solely in serum pro- Falcon flexible microtitration plates purchased from Becton duction because of its medical importance and the well-established Dickinson Labware Europe (Becton Dickinson France S. A.) were cross-reactivity between venoms of Tityus genus (Nishikawa et al., coated overnight at 4 C with 100 mlof5mg/mL solution of 1994; D'Suze et al., 2007). However, no cross-neutralization study T. fasciolatus venom or T. serrulatus venom in 20 mM sodium bi- has been done, with the exception of T. bahiensis venom. It was carbonate buffer, pH 9,6. Serum anti-T. serrulatus and anti- demonstrated that this venom is well neutralized by serum anti- T. fasciolatus venom, diluted 1:100, were tested for recognition T. serrulatus (Nishikawa et al., 1994). against whole venom of two scorpions. The assays were performed Evaluation of the neutralizing ability of individual antivenoms is as described previously by Chavez-Olortegui et al. (1991). Absor- a necessary qualification step before they can be release for ther- bance values were determined at 492 nm with a Titertek Multiscan apeutic usage. In Brazil, an ED50 0.5 mg of T. serrulatus venom per spectrophotometer. All measurements were made in duplicate. milliliter of serum is the required minimum potency (Maria et al., The T. fasciolatus venom and T. serrulatus venom were solubi- 2005). lized in reducing sample buffer and run on 15% SDS-PAGE In toxinology field, synthetic peptides that mimic epitopes have (Laemmli, 1970). Two gels were made, after electrophoresis, one been used as antigen for serum therapy and vaccines development was stained with Coomassie Blue G-250 for identification of protein (Calderon-Aranda et al., 1995; Alvarenga et al., 2002; Machado bands and other was transferred to a 0.45 mm nitrocellulose Avila et al., 2004; Felicori et al., 2009; Duarte et al., 2010; Dias- membrane as described by Alvarenga et al. (2002). This was sub- Lopes et al., 2010), and to evaluate antivemons neutralizing po- sequently used to verify the pattern of recognition of venoms tency (Maria et al., 2005; Ramada et al., 2013). The spot synthesis is against anti-T. serrulatus and anti-T. fasciolatus sera. The assays were an easy technique for the positionally addressable, parallel chem- performed as described previously by Guatimosin et al. (1999) and ical synthesis on a membrane support (Frank, 1992; Molina et al., sera were used at a dilution of 1:200. 1996) and subsequently was applied to identification of peptides and amino acids from the antibody paratope that contribute to 2.3. In vitro neutralization assay antigen binding (Laune et al., 2002). Through this technique, several studies have defined synthetic peptides able to neutralize The neutralization assay was adapted from Mendes et al. (2008). venom's toxic effects (Alvarenga et al., 2005; Felicori et al., 2006; We used 56 male CF1 Swiss mice, weighing between 18 and 22 g. A Duarte et al., 2010; Dias-Lopes et al., 2010). vial of serum produced by FUNED 5 mg is capable of neutralizing Therefore, the aim of this work is the molecular and immuno- the venom of the scorpion T. serrulatus (1 mg/mL). The dose of the logical study of the venom T. fasciolatus and identification of venom of T. fasciolatus used, 134.2 mg/mouse (minimum mortal important structural regions to produce neutralizing antibodies. dose that kills 100% e MMD determined in the study of LD50) was For this purpose, we used primers of known toxins from incubated for one hour at 37 C with 200 mlor100ml of scorpion T. serrulatus to verify and obtain homologous sequences present in anti-serum and PBS. The dose of the venom of T. serrulatus used, the venom of T. fasciolatus.Wehavefind amino acid sequences of 52.65 mg/mouse (equivalent to the study of MMD determined three T. fasciolatus toxins and synthesized a set of overlapping LD50) was incubated for one hour at 37 C with 200 mlor100mlof peptides bounded to cellulose membrane (spot-synthesis tech- scorpion anti-serum and PBS. The groups were assembled accord- nique) to identify linear epitopes recognized by antibodies obtained ing to Table 2. Were used four animals per group and inoculations by immunization of sheep with T. fasciolatus crude venom. were done subcutaneously. The count of survivors was realized 48 h after the challenge. 2. Materials and methods 2.4. Molecular identification of toxins from T. fasciolatus venom 2.1. Animals, venoms and antivenoms 2.4.1. Extraction and purification of RNA Scorpion venom was extracted from T. fasciolatus, kindly pro- All procedures were performed at 4 C, using water treated with vided by the Centre of Zoonosis Control (Ituiutaba, MG, Brazil), and diethyl pyrocarbonate (DEPC).