Molecular Cloning and Structural Modelling of Gamma-Phospholipase A2 Inhibitors from Bothrops Atrox and Micrurus Lemniscatus
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International Journal of Biological Macromolecules 103 (2017) 525–532 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ournal homepage: www.elsevier.com/locate/ijbiomac Molecular cloning and structural modelling of gamma-phospholipase A2 inhibitors from Bothrops atrox and Micrurus lemniscatus snakes a b b b Carina G. Picelli , Rafael J. Borges , Carlos A.H. Fernandes , Fabio M. Matioli , a a a a,c,d Carla F.C. Fernandes , Juliana C. Sobrinho , Rudson J. Holanda , Luiz S. Ozaki , a a b a Anderson M. Kayano , Leonardo A. Calderon , Marcos R.M. Fontes , Rodrigo G. Stábeli , a,e,∗ Andreimar M. Soares a Centro de Estudos de Biomoléculas Aplicadas a Saúde, CEBio, Fundac¸ ão Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia and Departamento de Medicina, Universidade Federal de Rondônia, UNIR, 76812-245 Porto Velho, RO, Brazil b Departamento de Física e Biofísica, Instituto de Biociências, Universidade Estadual Paulista, UNESP, Botucatu, SP, Brazil c Center for the Study of Biological Complexity, Life Sciences, Virginia Commonwealth University, 23284, Richmond, USA d PVE-CNPq, Brazil e Centro Universitário São Lucas, UNISL, Porto Velho, RO, Brazil a r t i c l e i n f o a b s t r a c t Article history: Phospholipases A2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential Received 30 January 2017 role in this resistance. These endogenous inhibitors may be classified by their fold in PLI␣, PLI and Received in revised form 3 May 2017 ␥ PLI . Phospholipases A2 (PLA2s) develop myonecrosis in snake envenomation, a consequence that is Accepted 15 May 2017 not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two Available online 18 May 2017 PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of speci- mens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLI␥ Keywords: gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies Snakes showed elevated identity with inhibitor PLI␥ gene sequences from other snake species. Molecular models Bothrops atrox of translated inhibitors’ gene sequences resemble canonical three finger fold from PLI␥ and support the Micrurus lemniscatus Inhibitors of phospholipases A2 hypothesis that the decapeptide (residues 107–116) may be responsible for PLA2 inhibition. Structural Gamma type phospholipase A2 inhibitors studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment. © 2017 Elsevier B.V. All rights reserved. 1. Introduction An important part of the effects described above is due to the action of phospholipases A2 (PLA2s), which are a superfamily of Snake venom cause physiological disorder in their prey or bitten enzymes that specifically catalyze the hydrolysis of the 2-acyl victims. The disturbances are mainly related to the action of toxins ester bond of glycero-phospholipids in cell membranes, result- that possess affinity to receptors or ion channels and of enzymes ing in lysophospholipids and fatty acids such as arachidonic acid. with catalytic activity, which promote homeostatic deviation of the These metabolites act primarily as precursors of inflammatory central and peripheral nervous systems, and cardiovascular, neuro- mediators (eicosanoids), which are all important in intracellular motor, and blood coagulation systems. Furthermore, tissue necrosis signaling pathways such as neuronal transmission, mitogenesis, around the bite, mainly common in Bothrops accidents, may result smooth muscle contraction, and platelet activation [3,4]. Because in permanent sequelae [1,2]. of their importance in inflammatory events, this family of proteins is also well studied in autoimmune and inflammatory diseases. PLA2s and other snake venom toxins can be inhibited by differ- ent molecules that vary in nature, including synthetic or natural chemical agents of animal and plant origin. For example, syn- ∗ Corresponding author at: Centro de Estudos de Biomoléculas Aplicadas a Saúde, thetic low molecular weight heparin, p-bromophenacyl bromide, CEBio, Fundac¸ ão Oswaldo Cruz, FIOCRUZ, Fiocruz Rondônia and Departamento de tetracycline, mono/polyclonal antibodies, and natural flavonoids Medicina, Universidade Federal de Rondônia, UNIR, 76812-245 Porto Velho, RO, Brazil. and alkaloids extracted from plants and molecules stemming from E-mail address: andreimar.soares@fiocruz.br (A.M. Soares). marine organisms, are inhibitors of PLA2 from snake venoms [5]. http://dx.doi.org/10.1016/j.ijbiomac.2017.05.076 0141-8130/© 2017 Elsevier B.V. All rights reserved. 526 C.G. Picelli et al. / International Journal of Biological Macromolecules 103 (2017) 525–532 Table 1 Molecular structures with inhibitory activities of phospholipase Oligonucleotide primers sequences used for the amplification of B. atrox. A2 and metalloproteinase are also known to be found in snakes. These molecules are related to the self-envenoming prevention PLI (ATG) A − SENSE 5 -ATGAAATCYCTACACACCATCTGCC-3 PLI (ATG) B − SENSE 5 ATGAAATCYCTACAGATCATCTGTCTTC-3 mechanism [6]. Besides snakes, other animals, mammals, fish and PLI (STOP) A − ANTISENSE 5 -ATCAGAGGCTTGCCAATCTGATG-3 some invertebrates have been studied for natural resistance against PLI (STOP) B − ANTISENSE 5 -TTATTGTTTTTCAACTTGGATGGC-3 snake venoms [7], fungi and bacteria [8]. PLI (STOP) C − ANTISENSE 5 -GGTGACGGAATTATTCRGAAGGTG-3 PLA2 inhibitors (PLIs) isolated from snake blood may present differences among different snake species PLIs [9]. These molecules are oligomeric, globular, acidic glycoproteins with molecular mass to the manufacturer’s protocols. The purified RNA samples were ◦ − between 75 and 180 kDa. The mechanism of action of secreted PLA2 stored at 80 C until use. inhibitors is through the formation of a soluble complex between the inhibitor and the target enzyme [10]. These inhibitors are clas- 2.3. Cloning of the PLA2 inhibitor genes and sequencing sified into three groups, ␣,  and ␥, based on their structural characteristics and may be present in a single snake, whether poi- Oligonucleotide primers (Table 1) for the polymerase chain sonous or not [11]. Inhibitors from the three groups have been reaction (PCR) were designed based on known sequences of PLA2 ␥ isolated from diverse snake species and families [10,12]. genes, corresponding to type inhibitors and deposited at NCBI The PLI␥s possess the highest inhibitory action among the three (The National Center for Biotechnology Information) GenBank groups [13]. They have inhibitory activities on acidic and basic (AB462512, AF211168, AF211166, AF211165, AF211163, AJ249829, group II as well as on those of group III PLA2s [14] and are acidic gly- AB021425, AB559509, EU155177, EU155169 e AB003472). First coproteins oligomeric, with molecular weights of each monomer stranded cDNA was synthesized from the purified RNAs of the ® between 20 and 31 kDa. The PLI␥s have been classified into two snake specimens liver tissues using the Super Script III First-strand ␥ subgroups, PLI I and II, according to their amino acid sequence, Synthesis System (Thermo Fisher Scientific) and the kit’s oligo(dT) biochemical characteristics and inhibition profile. Members of the primer. The PLI genes were subsequently amplified using the “pool” ␥ ␥ PLI I subgroup have a heteromeric composition and two subunits of PLI designed primers (Table 1) at a final concentration of 0.2 M, (A and B) with less than 33% identity to each other and inhibit in a volume of containing 5 L of the first stranded cDNA reac- TM groups I, II, and III PLA2s. Members of the PLI␥ II subgroup pref- tion mixture, 200 M dNTPs and one unit of HotMaster Taq DNA ◦ erentially inhibits those PLA2s of group II and are constituted of a polymerase (5 Prime). PCR conditions were: 95 C for 3 min and 30 ◦ ◦ ◦ single subunit [7,13,15,16]. However, recently the possibility of a cycles at 95 C for 30 s, 55 C for 30 s and 70 C for one minute. ® second subunit in a PLI␥ II from Crotalus durissus terrificus has been The amplified fragments were cloned into pGEM -T Easy Vector raised [17]. (Promega Co, Madison, WI, USA). The cloned inserts were purified PLIs are interesting candidates for designing drugs or even as a by colony PCR using the vector primers T7 and SP6 and the ampli- tool in envenoming treatment, since they inhibit PLA2 activity and fied fragments sent for sequencing. Sequencing was performed ® consequently many of the important symptoms of snakebite acci- in an ABI Prism 377 automatic sequencer (Thermo Fisher Scien- − dents [13,18]. Furthermore, the study of PLA2 inhibitors is a critical tific)at the company Genomic Engenharia Molecular in São Paulo, area of research because of the pharmacological potential of these SP, Brazil. components in the treatment of inflammation and as a tool to inves- tigate the role of PLA2s in physiological functions of cell death and 2.4. Alignment and phylogenetic analysis injury [19]. In recent years, synthetic derived peptides from PLI␥s ␥ were showed to have therapeutic potential in PLA2-related dis- Different PLI were obtained from NCBI GenBank database using eases and as antivenom-like bioactive molecules for snake venom BLASTP algorithm [22] and BLOSUM62 substitution matrix, with B. neutralization [20,21]. atrox PLI sequence as query. The minimum e-value presented by the 74 In this study, we performed molecular cloning of PLI␥s of the selected sequences was 1e- . Multiple sequence alignment was Amazonian occurring snake species, Bothrops atrox and Micrurus executed using the online program Muscle [23] and phylogenetic lemninscatus, and build in silico models of their structures. tree was build based on the Bayesian method Monte Carlo-Markov chain (MCMC) using the program MrBayes 3.2.6 [24]. Two concur- rent MCMC runs of 500,000 generations were performed using four 2. Material and methods progressively heated chains with a heating parameter of 0.2, tree sampling every 100 generations and a burn-in setting of 250 trees. 2.1. Ethical compliance After, the phylogenetic tree was visualized using Mesquite v.