Caracterização proteômica e biológica da peçonha de escorpiões do gênero Tityus

Abstract

Scorpions of the genus Tityus belong to the Buthidae family and are responsible for most of the reported envenomation accidents in Brazil. Envenomation is often characterized by local symptoms as pain, paresthesia, edema and erythema. Scorpion venoms are complex mixtures containing many toxic peptides and proteins. The study of toxic composition of scorpion venom is especially important for the development of effective treatments for cases of scorpionism. Additionally, toxins are molecules that may to have potential biotechnological applications. In this study, we present the proteomic and functional characterization of the Brazilian scorpions venoms Tityus stigmurus, Tityus neglectus and Tityus pusillus, species widely distributed in the northeastern of the Brazil. T. stigmurus is recognized as the main responsible for medical poisoning in the Northeast region. Although studies have been reported for this species, a large number of components of the venom remain unknown. Tityus neglectus and Tityus pusillus do not present study of chemical or biological characterization of the venom. The proteomic characterization of venoms were performed by bottom-up proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS) performed in a LTQ (Linear Trap Quadrupole) Orbitrap Velos. Peptide spectrum matching (PSM) search was performed using the PatternLab platform, and de novo sequencing using PEAKS Studio 8.0. The databases containing all the protein entries for order Scorpiones from UniProtKB (UniPro Knowledgebase) and NCBI (National Center for Biotechnology Information), and protein amounts was estimated using the normalized spectral abundance factor (NSAF). For the functional characterization, enzymatic assays as phospholipase A2, hyaluronidase and fibrinogenolytic, PT and aPTT tests, antimicrobial activity, cell viability assay, nitric oxide (NO) production, lethality determination and evaluation of effects of envenomation were performed. The proteome analyzes for T. stigmurus revealed the presence of 2 new families of toxins, serine proteases and phospholipases A2, described for the first time in this venom. The ions channels modulators peptides account for approximately 70% of the peptides identified, outlining the importance of neurotoxins. In the venoms of T. neglectus and T. pusillus, families of proteins such as NaTx, KTx, CRISPs, AMPs, hypotensins, serine protease inhibitors, serine proteases, metalloproteases, phospholipases A2 and hyaluronidases were identified. The three venoms presented hyaluronidase and fibrinogenolytic activity with the prominent participation of metalloprotease enzymes. The antimicrobial activity was not verified, and the action of phospholipases was verified only in T. neglectus venom. Cytotoxic activity was verified in human embryonic kidney (Hek) cells for T. stigmurus and T. neglectus venoms. T. neglectus also demonstrated cytotoxicity for macrophages (RAW 264.7) and fibroblasts (3T3), as well as modulating effect of NO production. The T. stigmurus venom showed high toxicity to BALB/c mice (LD50 = 0.575 mg/kg) and the effects of poisoning were described. The biological tests associated with the technology of proteomic analysis contributed to a better elucidation of composition and characterization of T. stigmurus venom. The approach presented in this study comprised the most complete description of the molecular repertoire for T. stigmurus scorpion, which together with T. neglectus and T. pusillus constitute sources of molecules with potential pharmacological and/or biotechnological application.