From the venom of the Bothrops pictus snake, an endemic species from Peru, we recently have described toxins that inhibited platelet aggregation and cancer cell migration. In this work, we characterize a novel P-III class snake venom metalloproteinase, called pictolysin-III (Pic-III). It is a 62 kDa proteinase that hydrolyzes dimethyl casein, azocasein, gelatin, fibrinogen, and fibrin. The cations Mg²⁺ and Ca²⁺ enhanced its enzymatic activity, whereas Zn²⁺ inhibited it. In addition, EDTA and marimastat were also effective inhibitors. The amino acid sequence deduced from cDNA shows a multidomain structure that includes a proprotein, metalloproteinase, disintegrin-like, and cysteine-rich domains. Additionally, Pic-III reduces the convulxin- and thrombin-stimulated platelet aggregation and in vivo, it has hemorrhagic activity (DHM = 0.3 µg). In epithelial cell lines (MDA-MB-231 and Caco-2) and RMF-621 fibroblast, it triggers morphological changes that are accompanied by a decrease in mitochondrial respiration, glycolysis, and ATP levels, and an increase in NAD(P)H, mitochondrial ROS, and cytokine secretion. Moreover, Pic-III sensitizes to the cytotoxic BH3 mimetic drug ABT-199 (Venetoclax) in MDA-MB-231 cells. To our knowledge, Pic-III is the first SVMP reported with action on mitochondrial bioenergetics and may offer novel opportunities for promising lead compounds that inhibit platelet aggregation or ECM–cancer-cell interactions.

RNA purification and cDNA synthesis represents the starting point for molecular analyses of snake venom proteins-enzymes. Usually, the sacrifice of snakes is necessary for venom gland extraction to identify protein-coding transcripts; however, the venom can be used as a source of transcripts. Although there are methods for obtaining RNA from venom, no comparative analysis has been conducted in the Bothrops genus. In the present study, we compared four commercial methods for RNA purification and cDNA synthesis from venom (liquid, lyophilized, or long-term storage) of four clinically relevant species of Peruvian Bothrops. Our results show that the TRIzol method presents the highest yield of RNA purified from venom (59 ± 11 ng/100 µL or 10 mg). The SuperScript First-Strand Synthesis System kit produced high amounts of cDNA (3.2 ± 1.2 ng cDNA/ng RNA), and the highest value was from combination with the Dynabeads mRNA DIRECT kit (4.8 ± 2.0 ng cDNA/ng RNA). The utility of cDNA was demonstrated with the amplification of six relevant toxins: thrombin-like enzymes, P-I and P-III metalloproteinases, acid and basic phospholipases A2, and disintegrins. To our knowledge, this is the first comparative study of RNA purification and cDNA synthesis methodologies from Bothrops genus venom.

Viperid snake venoms are notably abundant in metalloproteinases (proteins) (SVMPs), which are primarily responsible for inducing hemorrhage and disrupting the hemostatic process and tissue integrity in envenomed victims. In this study, barnettlysin-III (Bar-III), a hemorrhagic P-III SVMP, was purified from the venom of the Peruvian snake Bothrops barnetti. Bar-III has a molecular mass of approximately 50 kDa and is a glycosylation-dependent functional metalloproteinase. Some biochemical properties of Bar-III, including the full amino acid sequence deduced from its cDNA, are reported. Its enzymatic activity is increased by Ca²⁺ ions and inhibited by an excess of Zn²⁺. Synthetic metalloproteinase inhibitors and EDTA also inhibit its proteolytic action. Bar-III degrades several plasma and ECM proteins, including fibrin(ogen), fibronectin, laminin, and nidogen. Platelets play a key role in hemostasis and thrombosis and in other biological process, such as inflammation and immunity, and platelet activation is driven by the platelet signaling receptors, glycoprotein (GP)Ib-IX-V, which binds vWF, and GPVI, which binds collagen. Moreover, Bar-III inhibits vWF- and convulxin-induced platelet aggregation in human washed platelets by cleaving the recombinant A1 domain of vWF and GPVI into a soluble ectodomain fraction of ~55 kDa (sGPVI). Bar-III does not reduce the viability of cultured endothelial cells; however, it interferes with the adhesion of these cells to fibronectin, vitronectin, and RGD peptides, as well as their migration profile. Bar-III binds specifically to the surface of these cells, and part of this interaction involves α5β1 integrin receptors. These results contribute to a better comprehension of the pathophysiology of snakebite accidents/incidents and could be used as a tool to explore novel and safer anti-venom therapeutics.

Introducción: La expresión recombinante de toxinas de origen ofídico ha permitido potenciar su estudio tanto estructural y funcional, especialmente en sus aplicaciones biomédicas. Pictobina es una enzima similar a trombina del veneno de B. pictus, especie endémica del Perú, que presenta un alto potencial. Objetivo: producir la versión recombinante de Pictobina (rPictobina) en el modelo eucariótico Piccha pastoris, Métodos: Se inició con las síntesis comerciales de la secuencia nucleotídica de Pictobina (750 pb), que incorpora los sitios de restricción EcoR I y Not I. Esta secuencia fue amplificada por PCR, insertada en el vector pPICZα-C y clonada en E. coli OneShot TOP10. Posteriormente, se amplificó el plásmido pPICZα-C–rPictobina para su transformación y expresión en el sistema Pichia pastoris GS115. Resultados: La expresión se logró tras la inducción con metanol al 0,5% durante 7 días. El rendimiento de expresión fue de 0,4 mg/L en P. pastoris. Adicionalmente, el análisis por SDS-PAGE reveló que rPictobina tiene una masa molecular de 49 kDa, y mostró actividad amidolítica pero no acción coagulante sobre el fibrinógeno. Los análisis por ELISA y Western Blot mostraron un bajo reconocimiento de la versión recombinante por los anticuerpos anti-veneno tal de B. pictus. Conclusión: Se logró la producción de la versión recombinante de Pictobina, la cual posee actividad sobre sustrato sintético BApNA específico para serina proteasas, esto permite dar los primeros pasos para la producción heteróloga de esta prometedora biomolécula.