Pictolysin-III, a Hemorrhagic Type-III Metalloproteinase Isolated from Bothrops pictus (Serpentes: Viperidae) Venom, Reduces Mitochondrial Respiration and Induces Cytokine Secretion in Epithelial and Stromal Cell Lines.

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.

Functional, immunological characterization, and anticancer activity of BaMtx: A new Lys49- PLA2 homologue isolated from the venom of Peruvian Bothrops atrox snake (Serpentes: Viperidae).

Bothorps atrox is responsible for most of the ophidism cases in Perú. As part of the envenoming, myotoxicity is one of the most recurrent and destructive effects. In this study, a myotoxin, named BaMtx, was purified from B. atrox venom to elucidate its biological, immunological, and molecular characteristics. BaMtx was purified using CM-Sephadex-C-25 ion-exchange resin and SDS-PAGE analysis showed a unique protein band of 13 kDa or 24 kDa under reducing or non-reducing conditions, respectively. cDNA sequence codified a 122-aa mature protein with high homology with other Lys49-PLA2s; modeled structure showed a N-terminal helix, a ß-wing region, and a C-terminal random coil. This protein has a poor phospholipase A2 enzymatic activity. BaMtx has myotoxic (DMM = 12.30 ± 0.95 µg) and edema-forming (DEM = 26.00 ± 1.15 µg) activities. Rabbit immunization with purified enzyme produced anti-BaMtx antibodies that reduced 50.28 ± 10.15% of myotoxic activity and showed significant cross-reactivity against B. brazili and B pictus venoms. On the other hand, BaMtx exhibits mild anti-proliferative and anti-migratory effects on breast cancer cells, affecting the ROS and NADH levels, which may reduce mitochondrial respiration. These results contribute to the understanding of B. atrox Lys49-PLA2 effects and establish the anticancer potential de BaMtx.

Fibrinogen-clotting enzyme, pictobin, from Bothrops pictus snake venom. Structural and functional characterization.

A thrombin-like enzyme, pictobin, was purified from Bothrops pictus snake venom. It is a 41-kDa monomeric glycoprotein as showed by mass spectrometry and contains approx. 45% carbohydrate by mass which could be removed with N-glycosidase. Pictobin coagulates plasma and fibrinogen, releasing fibrinopeptide A and induces the formation of a friable/porous fibrin network as visualized by SEM. The enzyme promoted platelet aggregation in human PRP and defibrination in mouse model and showed catalytic activity on chromogenic substrates S-2266, S-2366, S-2160 and S-2238. Pictobin interacts with the plasma inhibitor α2-macroglobulin, which blocks its interaction with fibrinogen but not with the small substrate BApNA. Heparin does not affect its enzymatic activity. Pictobin cross reacted with polyvalent bothropic antivenom, and its deglycosylated form reduced its catalytic action and antivenom reaction. In breast and lung cancer cells, pictobin inhibits the fibronectin-stimulated migration. Moreover, it produces strong NADH oxidation, mitochondrial depolarization, ATP decrease and fragmentation of mitochondrial network. These results suggest by first time that a snake venom serinprotease produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics. Structural model of pictobin reveals a conserved chymotrypsin fold ß/ß hydrolase. These data indicate that pictobin has therapeutic potential in the treatment of cardiovascular disorders and metastatic disease.

Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergón) Induces Glycoprotein VI Shedding and Impairs Platelet Function.

Atroxlysin-III (Atr-III) was purified from the venom of Bothrops atrox. This 56-kDa protein bears N-linked glycoconjugates and is a P-III hemorrhagic metalloproteinase. Its cDNA-deduced amino acid sequence reveals a multidomain structure including a proprotein, a metalloproteinase, a disintegrin-like and a cysteine-rich domain. Its identity with bothropasin and jararhagin from Bothrops jararaca is 97% and 95%, respectively. Its enzymatic activity is metal ion-dependent. The divalent cations, Mg2+ and Ca2+, enhance its activity, whereas excess Zn2+ inhibits it. Chemical modification of the Zn2+-complexing histidine residues within the active site by using diethylpyrocarbonate (DEPC) inactivates it. Atr-III degrades plasma fibronectin, type I-collagen, and mainly the α-chains of fibrinogen and fibrin. The von Willebrand factor (vWF) A1-domain, which harbors the binding site for GPIb, is not hydrolyzed. Platelets interact with collagen via receptors for collagen, glycoprotein VI (GPVI), and α2ß1 integrin. Neither the α2ß1 integrin nor its collagen-binding A-domain is fragmented by Atr-III. In contrast, Atr-III cleaves glycoprotein VI (GPVI) into a soluble ~55-kDa fragment (sGPVI). Thereby, it inhibits aggregation of platelets which had been stimulated by convulxin, a GPVI agonist. Selectively, Atr-III targets GPVI antagonistically and thus contributes to the antithrombotic effect of envenomation by Bothrops atrox.

L-amino acid oxidase from Bothrops atrox snake venom triggers autophagy, apoptosis and necrosis in normal human keratinocytes.

Snake venom L-amino acid oxidases (LAAOs) are flavoproteins, which perform diverse biological activities in the victim such as edema, myotoxicity and cytotoxicity, contributing to the development of clinical symptoms of envenomation. LAAO cytotoxicity has been described, but the temporal cascade of events leading to cell death has not been explored so far. This study evaluates the involvement of LAAO in dermonecrosis in mice and its cytotoxic effects in normal human keratinocytes, the major cell type in the epidermis, a tissue that undergoes extensive necrosis at the snakebite site. Pharmacological inhibition by the antioxidant NAC (N-acetyl cysteine) prevented B. atrox venom-induced necrosis. Consistent with the potential role of oxidative stress in wounding, treatment with purified LAAO decreased keratinocyte viability with an Effective Concentration (EC50) of 5.1 µg/mL. Cytotoxicity caused by LAAO was mediated by H2O2 and treated cells underwent autophagy, followed by apoptosis and necrosis. LAAO induced morphological alterations that precede cell death. Our results show the chronological events leading to cell death and the temporal resolution from autophagy, apoptosis and necrosis as distinct mechanisms triggered by LAAO. Fluorescently-labelled LAAO was efficiently and rapidly internalized by keratinocytes, suggesting that catalysis of intracellular substrates may contribute to LAAO toxicity. A better understanding of LAAO cytotoxicity and its mechanism of action will help to identify potential therapeutic strategies to ameliorate localized snake envenomation symptoms.

Proteomic profile, biological activities and antigenic analysis of the venom from Bothriopsis bilineata smaragdina ("loro machaco"), a pitviper snake from Peru.

In order to determine Bothriopsis bilineata smaragdina venom (BbsV) composition, proteomic approaches were performed. Venom components were analyzed by RP-HPLC, SDS- PAGE and nano LC on line with LTQ Orbitrap XL. Results showed a total of 189 identified proteins, grouped into 11 different subgroups, which include snake venom metalloproteinases (SVMPs, 54.67%), snake C-type lectins (Snaclecs, 15.78%), snake venom serine proteinases (SVSPs, 14.69%), cystein-rich secretory proteins (CRISP, 2.61%), phospholipases A2 (PLA2, 1.14%), phosphodiesterase (PDE, 1.17%), venom endothelial growth factor (VEGF, 1.06%) 5'nucleotidases (0.33%), L-amino acid oxidases (LAAOs, 0.28%) and other proteins. In vitro enzymatic activities (SVMP, SVSP, LAAO, Hyal and PLA2) of BbsV were also analyzed. BbsV showed high SVSP activity but low PLA2 activity, when compared to other Bothrops venoms. In vivo, BbsV induced hemorrhage and edema in mice and showed intraperitoneal median lethal dose (LD50) of 92.74 (± 0.15) µg/20 g of mice. Furthermore, BbsV reduced cell viability when incubated with VERO cells. Peruvian and Brazilian bothropic antivenoms recognize BbsV proteins, as detected by ELISA and Western Blotting. Both antivenoms were able to neutralize in vivo edema and hemorrhage. SIGNIFICANCE: In Peru, snakebite is a public health problem, especially in the rain forest, as a result of progressive colonization of this geographical area. This country is the second in Latin America, after Brazil, to exhibit the largest variety of venomous snakes. B. atrox and B. b. smaragdina snakes are sympatric species in Peruvian Amazon region and are responsible for approximately 95% of the envenomings reported in this region. B. b. smaragdina may cause a smaller share (3 to 38%) of those accidents, due to its arboreal habits, that make human encounters with these snakes less likely to happen. Despite B. b. smaragdina recognized medical importance, its venom composition and biological activities have been poorly studied. Furthermore, BbsV is not a component of the antigenic pool used to produce the corresponding Peruvian bothropic antivenom (P-BAV). Our results not only provide new insights on BbsV composition and biological activity, but also demonstrate that both P-BAV and B-BAV polyvalent antivenoms have a considerable recognition of proteins from BbsV and, more importantly, neutralized hemorrhage and edema, the main local effects of bothropic envenomation.

A novel fibrinolytic metalloproteinase, barnettlysin-I from Bothrops barnetti (Barnett´s pitviper) snake venom with anti-platelet properties.

BACKGROUND: Viperid snake venoms contain active components that interfere with hemostasis. We report a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), isolated from the venom of Bothrops barnetti and evaluated its fibrinolytic and antithrombotic potential. METHODS: Bar-I was purified using a combination of molecular exclusion and cation-exchange chromatographies. We describe some biochemical features of Bar-I associated with its effects on hemostasis and platelet function. RESULTS: Bar-I is a 23.386 kDa single-chain polypeptide with pI of 6.7. Its sequence (202 residues) shows high homology to other members of the SVMPs. The enzymatic activity on dimethylcasein (DMC) is inhibited by metalloproteinase inhibitors e.g. EDTA, and by α2-macroglobulin. Bar-I degrades fibrin and fibrinogen dose- and time-dependently by cleaving their α-chains. Furthermore, it hydrolyses plasma fibronectin but not laminin nor collagen type I. In vitro Bar-I dissolves fibrin clots made either from purified fibrinogen or from whole blood. In contrast to many other P-I SVMPs, Bar-I is devoid of hemorrhagic activity. Also, Bar-I dose- and time-dependently inhibits aggregation of washed human platelets induced by vWF plus ristocetin and collagen (IC50=1.3 and 3.2 µM, respectively), presumably Bar-I cleaves both vWF and GPIb. Thus, it effectively inhibits vWF-induced platelet aggregation. Moreover, this proteinase cleaves the collagen-binding α2-A domain (160 kDa) of α2ß1-integrin. This explains why it additionally inhibits collagen-induced platelet activation. CONCLUSION: A non-hemorrhagic but fibrinolytic metalloproteinase dissolves fibrin clots in vitro and impairs platelet function. GENERAL SIGNIFICANCE: This study provides new opportunities for drug development of a fibrinolytic agent with antithrombotic effect.

Caracterización de la enzima similar a trombina del veneno de Bothrops pictus "jergón de costa" / Characterization of thrombin like enzyme from Bothrops pictus venom

Realizar una caracterización bioquímica y molecular del principio coagulante del veneno de Bothrops pictus. Materiales y métodos. Se realizó la amplificación del gen a partir de cDNA, se analizó la homología de la secuencia nucleotídica y de la proteína deducida. Se procedió a purificar la enzima para los análisis de secuenciación directa N terminal de los primeros 20 aminoácidos y los ensayos de coagulación sobre plasma humano y fibrinógeno humano, por otro lado, se evaluó el patrón de corte del fibrinógeno por medio de PAGE SDS y la actividad defibrinogenante en roedores albinos (18-22 g). Se determinó el contenido de carbohidratos asociados, el efecto de inhibidores clásicos de proteasas y el efecto de iones bajo la forma de cloruros. Resultados. La enzima mostró homología en la estructura primaria con otras TLEs reportadas para la familia Viperidae, la dosis coagulante mínima (DCM) sobre plasma y fibrinógeno humano fue de 18 y 6 ug respectivamente y su potencia coagulante fue de 131,1 NHI unidades de trombina. La enzima se mostró estable a condiciones fisiológicas y prescinde de iones para su actividad. Los carbohidratos asociados detectados fueron hexosas (25,76%), hexosaminas (13,1%) y ácido siálico (0,76%). Los agentes fluoruro de fenil metil sulfonil floruro (PMSF) ditiotreitol (DTT) fueron los principales inhibidores de la actividad enzimática en tanto que la heparina no tuvo efecto inhibidor. Conclusiones. El principio coagulante del veneno de Bothrops pictus es una enzima similar a trombina...

To perform a biochemical and molecular characterization of the coagulant principle from Bothrops pictus venom. Materials and methods. We amplified the genetic sequence of this enzyme from cDNA and analyzed the homology of its nucleotide sequence and its deduced protein. This enzyme was also purified for N-terminal sequencing of first 20 amino acids and for coagulation assays using human plasma and human fibrinogen. Furthermore, cleavage pattern on fibrinogen was evaluated using SDS-PAGE and defibrinogenant activity on white mice (18-22 g). Finally, associated carbohydrate content, effect of protease inhibitors and chloride ions on its enzymatic activity were analyzed. Results. The Thrombin-like Enzyme from Bothrops pictus showed homology at primary level of structure with other previously reported TLEs from Viperidae family. Minimum Coagulant Dosis (MCD) on plasma and human fibrinogen were 18 and 6 ug, respectively, and its coagulant potency was 131.1 NHI Thrombin units. This TLE was stable under physiological conditions and chloride ions are not necessary for its activity. Detected associated carbohydrates were hexoses (25.76%), hexosamines (13.12%) and sialic acid (0.76%). Phenyl methyl sulphonyl fluoride (PMSF) and dithiothreitol (DTT) were the main inhibitors of its enzymatic activity, but heparin had no inhibitor effect. Conclusions. The coagulant principle of Bothrops pictus venom is a Thrombin-like enzyme...

[Potential use of snake venom components in cancer treatment]. / Uso potencial de componentes del veneno de serpiente en el tratamiento del cáncer.

Cancer can develop to the extent tumor cells grow, divide and grow into other body tissues. Integrins are a family of cell-surface heterodimeric receptors that play an important role in the development of tumor angiogenesis, growth and metastasis, thus being recognized as an attractive therapeutic target. Snake venom contains low-molecular weight peptides known as "disintegrins" that bind to integrins with high affinity, and prevent their action in cancer. In the next article, we go over the results of investigations, both in vitro and in vivo, which have shown promising results, thus revealing that the use of disintegrins could be a promising alternative for the treatment of different neoplasias.

Uso potencial de componentes del veneno de serpiente en el tratamiento del cáncer / Potential use of snake venom components in cancer treatment

El desarrollo del cáncer es posible en la medida que las células tumorales proliferen, se dispersen e invadan otros tejidos del cuerpo. Las integrinas son una familia de receptores heterodiméricos de superficie celular que cumplen un papel crucial en el desarrollo de la angiogénesis, crecimiento y metástasis de un tumor señalándolas como un atractivo blanco terapéutico. Los venenos de serpientes contienen péptidos de bajo peso molecular conocidos como desintegrinas, las que se unen con una alta afinidad a las integrinas e inhiben su accionar en un proceso cancerígeno. En el siguiente articulo revisamos los resultados de investigaciones, tanto in vitro como in vivo, que han mostrado resultados promisorios, por lo cual el uso de las desintegrinas podrían constituir una alternativa promisoria para el tratamiento de diversas neoplasias.

Cancer can develop to the extent tumor cells grow, divide and grow into other body tissues. Integrins are a family of cell-surface heterodimeric receptors that play an important role in the development of tumor angiogenesis, growth and metastasis, thus being recognized as an attractive therapeutic target. Snake venom contains low-molecular weight peptides known as “disintegrins” that bind to integrins with high affinity, and prevent their action in cancer. In the next article, we go over the results of investigations, both in vitro and in vivo, which have shown promising results, thus revealing that the use of disintegrins could be a promising alternative for the treatment of different neoplasias.

Variaciones en las actividades enzimáticas del veneno de la serpiente Bothrops atrox "jergón", de tres zonas geográficas del Perú / Variation of the enzymatic activity of Bothrops atrox "jergon" snake venom from three geographic regions, Peru

Objetivos. Estudiar la variabilidad en la composición y actividades enzimáticas entre venenos de ejemplares adultos de Bothrops atrox. Materiales y métodos. Se emplearon venenos de serpientes adultas procedentes de Amazonas, Junín y Ucayali. A cada una de las muestras se les realizó el análisis del contenido proteico y del número de bandas por PAGESDS, así como las actividades de fosfolipasa A2, hemolítica indirecta, amidolítica, coagulante, hemorrágica y proteolítica sobre caseína y mediante zimograma; además, se hicieron ensayos de inmunodifusión y neutralización in vitro con el suero antibotrópico polivalente del Instituto Nacional de Salud de Perú. Resultados. Las actividades amidolítica, coagulante, hemorrágica, proteolítica mediante zimograma, fosfolipasa A2 y hemolítica indirecta fueron variables, evidenciándose en las tres últimas una mayor actividad en los venenos de Amazonas, mientras que en la cantidad de proteína, bandas electroforéticas y actividad proteolítica sobre caseína no se observaron diferencias. Con respecto a las pruebas de neutralización, 0,5 dosis del antiveneno fueron suficientes para neutralizar con eficacia (más del 50%) la actividad coagulante y fosfolipasa A2 de todas las muestras analizadas. Conclusiones. Algunas propiedades biológicas del veneno de ejemplares adultos de Bothrops atrox de Perú son variables, sin que ello afecte la neutralización in vitro por parte del suero antibotrópico polivalente sobre las actividades coagulante y fosfolipasa A2 del veneno.

Objectives. To study the variability in the composition and enzymatic activity of venom from adult Bothrops atrox specimens. Materials and methods. We used venoms from adult snakes from Amazonas, Junín and Ucayali. Each of the venom samples underwent analysis for protein and number of bands by pagesds. Phospholipase A2, hemolytic, amidolytic, coagulant, hemorrhagic activity were analyzed, also and proteolytic activity on casein and by zymogram. Additionally, immunodiffusion and neutralization assays in vitro were done with a polyvalent botropic serum from the national institute of health of Peru. Results. The amidolytic, coagulant, hemorrhagic, proteolytic by zymogram, phospholipase A2, and indirect hemolytic activity were variable, demonstrating increased activity in the venoms from Amazonas, regarding proteolytic by zymogram, phospholipase A2, and indirect hemolytic activity. While the amount of protein electrophoretic bands and proteolytic activity on casein did not demonstrated differences. Regarding neutralization tests, a 0.5 dose of antivenom was sufficient to effectively neutralize (>50%) the coagulant activity and phospholipase A2 of all samples analyzed. Conclusions. Some biological properties of the venom from adult Bothrops atrox of Peru are variable, without interference with the in vitro neutralization by the polyvalent botropic serum on coagulant and phospholipase A2 properties of the venom.

Eficacia experimental de anticuerpos IgY producidos en huevos, contra el veneno de la serpiente peruana Bothrops atrox / Experimental efficacy of IgY antibodies produced in eggs against the venom of the Peruvian snake Bothrops atrox

Objetivos. Desarrollar un protocolo de inmunización para producir inmunoglobulinas IgY de origen aviar contra el veneno de la serpiente peruana Bothrops atrox y evaluar la capacidad neutralizante. Materiales y métodos. Se inmunizaron seis gallinas de postura de la raza hy line brown con 500 μg/dosis de veneno de B. atrox en un periodo de dos meses. Cada semana, los huevos fueron colectados para el aislamiento de inmunoglobulinas IgY a partir de la yema, usando dos pasos consecutivos con αcido caprνlico y sulfato de amonio. La detecciσn de anticuerpos se realizσ por inmunodifusiσn doble mientras que el tνtulo y reactividad cruzada se determinaron por las técnicas de ELISA y Western blot. El cálculo de DL50 y de la DE50 del antiveneno IgY producido se realizó utilizando el método de Probits. Resultados. La masa de anticuerpos aislados fue de 8,5 ± 1,35 mg de IgY/mL de yema. Asimismo, la DE50 del antiveneno aviar fue calculada en 575 μL de antiveneno/mg de veneno. Adicionalmente, los ensayos de reactividad cruzada mostraron que el veneno de B. atrox comparte mas epνtopes comunes con el veneno de B. brazili (47%) que con otros veneno del mismo género, en tanto que los venenos de Lachesis muta (19%) y Crotalus durissus (12%) mostraron una baja reactividad cruzada. Conclusiones. Se ha obtenido IgY purificada contra el veneno de B. atrox con capacidad neutralizante y se ha demostrado su utilidad como herramienta inmunoanalítica para evaluar la reactividad cruzada con venenos de otras especies.

Objectives. To develop an immunization protocol in order to produce avian IgY immunoglobulins against Bothrops atrox Peruvian snake venom and to evaluate its neutralizing capacity. Materials and methods. Six Hy Line Brown hens were immunized each two weeks using 500μg/doses of B. atrox venom in a period of two months. Each week, eggs were collected for IgY isolation from yolk using two consecutive steps with caprilic acid and ammonium sulfate. Detection of IgY anti-B. atrox were performed by double immunodiffusion, whereas title and cross-reactivity were analyzed using ELISA and Western Blot technics, respectively. Furthermore, letal dose (DL50) and Medium Effective Dose (DE50) were obtained by Probit analysis. Results. As a result of this protocol, chicken IgY’s were obtained in a concentration of 8,5 ± 1,35 mg/yolk mL. DE50 from avian antivenom was 575 μL/venom mg. Cross-reactivity studies showed Bothrops atrox venom share more commom epitopes with Bothrops brazili (47%) than others Bothrops venoms showing Lachesis muta (19%) and Crotalus durissus (12%) venoms a low crossing reactivity, instead. Conclusions. Using this procedure, we could purify chicken IgY with a neutralizant capacity of B. atrox venom which is comparable to the antivenom of equine origin and demonstrate its capacity as a immunoanalitical tool to evaluate the cross reactivity with others peruvian snakes.

Exploring the proteomes of the venoms of the Peruvian pit vipers Bothrops atrox, B. barnetti and B. pictus.

We report the comparative proteomic characterization of the venoms of Bothrops atrox, B. barnetti and B. pictus. The venoms were subjected to RP-HPLC and the resulting fractions analyzed by SDS-PAGE. The proteins were cut from the gels, digested with trypsin and identified via peptide mass fingerprint and manual sequencing of selected peptides by MALDI-TOF/TOF mass spectrometry. Around 20-25 proteins were identified belonging to only 6-7 protein families. Metalloproteinases of the classes P-I and P-III were the most abundant proteins in all venoms (58-74% based on peak area A214 nm), followed by phospholipases-A(2) (6.4-14%), disintegrins (3.2-9%) and serine proteinases (7-11%), and some of these proteins occurred in several isoforms. In contrast cysteine-rich secretory proteins and L-amino acid oxidases appeared only as single isoforms and were found only in B. atrox and B. barnetti. C-type lectins were also detected in all venoms but at low levels (~ 5%). Furthermore, the venoms contain variable numbers of peptides (<3 kDa) and non-protein compounds which were not considered in this work. The protein composition of the investigated Bothrops species is in agreement with their pharmacological and pathological effects.

Clonaje y caracterización molecular in silico de un transcrito de fosfolipasa A2 aislado del veneno de la serpiente peruana Lachesis muta / Molecular cloning and characterization in silico of phospholipase A2 transcripto isolated from Lachesis muta peruvian snake venom

Objetivo. Aislar y caracterizar in silico un transcrito del gen de fosfolipasa A2 (PLA2) aislado del veneno de Lachesis muta de la Amazonía peruana. Materiales y métodos. Se amplificó el transcrito del gen sPLA2 mediante la técnica de RT-PCR a partir de RNA total utilizando cebadores específicos, el producto de DNA amplificado se insertó en el vector pGEM para su posterior secuenciación. Mediante análisis bioinformático de la secuencia nucleotídica se determinó un marco de lectura abierta de 414 nucleótidos que codifica 138 aminoácidos, incluyendo16 aminoácidos del péptido señal, el peso molecular y el pI fueron de 13 976 kDa y 5,66 respectivamente. Resultados. La secuencia aminoacídica denominada Lm-PLA2- Perú, contiene Asp49, así como Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 importantes para la actividad enzimática. La comparación de Lm-PLA2-Perú con las secuencias aminoacídicas de los bancos de datos mostró 93 por ciento de similitud con las sPLA2 de Lachesis stenophrys y más del 80 por ciento con otras sPLA2 de venenos de la familia Viperidae. El análisis filogenético de la secuencia nucleotídica del transcrito del gen sPLA2 indica que Lm-PLA2-Perú se agrupa con otras sPLA2 [Asp49] ácidas previamente aisladas del veneno de Bothriechis schlegelii con un 89 por ciento de identidad. El modelaje tridimensional de Lm-PLA2-Perú, presenta una estructura característica de sPLA2 del Grupo II formada por tres hélices-α, una lámina-β, una hélice corta y un lazo de unión con calcio. Conclusión. La secuencia nucleotídica corresponde al primer transcripto del gen de PLA2 clonado a partir del veneno de la serpiente Lachesis muta, que habita en la selva del Perú.

Objective. Isolate and characterize in silico gene phospholipase A2 (PLA2) isolated from Lachesis muta venom of the Peruvian Amazon. Material and methods. Technique RT-PCR from total RNA was using specific primers, the amplified DNA product was inserted into the pGEM vector for subsequent sequencing. By bioinformatic analysis identified an open reading frame of 414 nucleotides that encoded 138 amino acids including a signal peptide of 16 aminoacids, molecular weight and pI were 13 976 kDa and 5.66 respectively. Results. The aminoacid sequence was called Lm-PLA2-Peru, contains an aspartate at position 49, this aminoacid in conjunction with other conserved residues such as Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 are important for enzymatic activity. The comparison with the amino acid sequence data banks showed of similarity between PLA2 from Lachesis stenophrys (93 percent) and other PLA2 snake venoms and over 80 percent of other sPLA2 family Viperidae venoms. A phylogenetic analysis showed that Lm-PLA2-Peru grouped with other acidic [Asp49] sPLA2 previously isolated from Bothriechis schlegelii venom showing 89 percent nucleotide sequence identity. Finally, the computer modeling indicated that enzyme had the characteristic structure of sPLA2 group II that consisted of three α-helices, a β-wing, a short helix and a calcium-binding loop. Conclusion. The nucleotide sequence corresponding to the first transcript of gene from PLA2 cloned of Lachesis muta venom, snake from the Peruvian rainforest.

Características bioquímicas y evaluación preclínica de un antiveneno botrópico liofilizado contra el veneno de la serpiente Bothrops atrox / Biochemicals characteristics and pre-clinical testing of lyophilized bothropic antivenom against bothrops atrox snake venom

Se han estudiado las características bioquímicas y la capacidad neutralizante del antiveneno botrópico liofilizado producido por el Instituto Nacional de Salud (Lima, Perú), se encontró que posee 51,4 mg/mL de proteínas, las preparaciones liofilizadas se reconstituyen en un periodo de 10 min alcanzando valores de Abs600nm y pH de 0,091 y 7,0, respectivamente. Para el caso de las actividades tóxicas delveneno en estudio se obtuvieron valores de toxicidad DL50: 3,33 μg/g ratón, dosis hemorrágica mínima: 4,10 mas o menos 0,64 μg, dosis miotóxica mínima 30,2 mas o menos 2,5 μg, dosis coagulante mínima: 4,50 mas o menos 0,6 μg y dosis defibrinante mínima: 8 μg, y valores de dosis efectiva del antiveneno evaluado de 140,48 (120,09-164,33), 230,67 mas o menos11,78, 316,56 mas o menos 40,31, 105,5 mas o menos 4,2 y 500 μL antiveneno/mg veneno, respectivamente, lo cual indica que posee capacidad para neutralizar tales parámetros. Por estas razones se concluye que el producto biológico investigado cumple con los requerimientos de la Organización Mundial de la Salud (OMS) para ser considerado un antiveneno neutralizante de las principales actividades biológicas antes señaladas.

Biochemical features and neutralizing capacity of lyophilized bothropic antivenom elaborated by the Peruvian National Health Institute (Lima, Peru). It was found that the antivenom protein contents is 51.4 mg/mL. Lyophilized preparations can be reconstituted in 10 minutes, reaching Abs600nm and pH values reported as 0.091 and 7.0, respectively. Regarding toxicity of the venom for mice, LD50 was 3.33 μg,minimal hemorrhagic dose was 4.10 more or less 0.64 μg, minimal myotoxic dose was 30.2 more or less 2.5 μg, minimal coagulant dose was 4.50 more or less 0.6 μg, and the minimal defibrinating dose was 8 μg; and the effective dose values of the antivenom for the aforementioned parameters were140.48 (120.09-164.33), 230.67 more or less 11.78, 316.56 more or less 40.31, 105.5 more or less 4.2, and 500 μL antivenom/mg venom, respectively, indicating that this preparation has the ability to neutralize each of the parameters tested. For these reasons we conclude that the investigated product complies with the World Health Organization (WHO) requirements to be considered an effective antivenom capable of neutralizing themain biological activities previously mentioned.

Acción del antiveneno botrópico polivalente sobre las actividades proteolíticas presentes en los venenos de serpientes peruanas / Action of polivalent bothropic antivenom on proteolytic activities from peruvian snake venoms

Los venenos de las serpientes peruanas causantes de la mayoría de accidentes ofídicos, contienen enzimas proteolíticas que pueden degradar proteínas tisulares y plasmáticas, así como causar hipotensión y coagulación sanguínea. Objetivos. Evaluar la capacidadinhibitoria del antiveneno botrópico polivalente al estado líquido producido por el Instituto Nacional de Salud del Perú (INS) sobre las actividades caseinolítica, coagulante y amidolítica de los venenos de Bothrops atrox, Bothrops brazili, Bothrops pictus y Bothrops barnetti. Materiales y métodos. Se usaron en cada caso sustratos como caseína, fibrinógeno bovino y el cromógeno benzoil-arginil-p-nitroanilida(BApNA) respectivamente, y se midieron los cambios en los valores de la actividad enzimática a ½, 1 y 2 dosis del antiveneno tanto al estado natural como calentado a 37 °C durante cinco días. Resultados. La actividad caseinolítica es la más resistente a la inhibición especialmente por el suero no calentado en tanto que, la actividad amidolítica fue severamente inhibida principalmente en los venenosde B. pictus y B. atrox. Así mismo la actividad coagulante fue totalmente inhibida en el veneno de B. pictus, mostrándose a su vez unaelevada inhibición sobre los venenos de B. brazili y B. atrox. Para las actividades coagulante y amidolítica, los sueros calentados fueron menos efectivos que aquellos al estado natural. Conclusiones. El suero antibotrópico polivalente producido por el INS es efectivo parainhibir las actividades proteolíticas de los venenos de las serpientes peruanas ensayadas.

Peruvian snake venoms responsible for most of ophidism accidents, contain proteolytic enzymes that can degrade tissue and plasmatic proteins, as well as cause hypotension and blood coagulation. Objectives. The inhibiting capacity of liquid polyvalent bothropic antivenom produced by Instituto Nacional de Salud (INS), has been evaluated on caseinolytic, coagulant and amidolytic activities on Bothrops atrox,Bothrops brazili, Bothrops pictus and Bothrops barnetti venoms. Material and methods. Using in each case casein, bovine fibrinogen and the chromogenic substrate BApNA respectively, measuring changes in values of enzymatic activity at ½, 1 and 2 doses of either natural and heating (incubated at 37 °C during five days) antivenom. Results. Caseinolytic activity is more resistant to inhibition especially by thenatural antivenom, amidolytic activity was severely inhibited mainly in B. pictus and B. atrox venoms. Also coagulant activity was totallyinhibited in B. pictus venom, being high on B. brazili and B. atrox venoms. For coagulant and amidolytic activities, heated antivenom was less effective than natural one. Conclusions. The bothropic antivenom produced by INS is effective to inhibit the proteolytic activity from Peruvian snake venoms tested.

Efecto del antiveneno botrópico sobre las actividades de fosfolipasa A2, l-aminoácido oxidasa y hialuronidasa de los venenos de serpientes peruanas / Effect of polivalent bothropic antivenom on phospholipase a2, L- amino acid oxidase and hyaluronidase from peruvian snake venom

Las serpientes Bothrops sp. causan el mayor número de casos de ofidismo en el Perú, su veneno contiene enzimas que participan en la difusión de la ponzoña, así como en sus efectos miotóxicos, edemáticos y de alteración en la agregación plaquetaria. Objetivos. Evaluar el efecto del antiveneno botrópico polivalente al estado líquido producido por el Instituto Nacional de Salud (INS) sobre la fosfolipasaA2 (PLA2), L-aminoácido oxidasa (LAO) y hialuronidasa (HA) de los venenos de B. atrox, B. barnetti, B. brazili y B. pictus. Materiales y métodos. La PLA2 fue determinada por el retardo en el tiempo de coagulación de una emulsión lipoproteica al 45 por ciento, LAO usando Lleucina como substrato en presencia de O-dianisidina y HA empleando ácido hialurónico y el reactivo turbidimétrico BCTA, se usó para cada enzima ½, 1 y 2 dosis del antiveneno al estado natural o calentado a 37 °C durante cinco días ensayados por triplicado. Resultados. HA fue la enzima más neutralizada por el antiveneno, todos los venenos con excepción de B. brazili fueron totalmente inhibidos a cualquierdosis. Para LAO se tuvieron valores de inhibición de 68 a 100% usando dos dosis del antiveneno, mientras que PLA2 fue la menos inhibida (70 a 80 por ciento) a dos dosis. Con el antiveneno calentado se registró una disminución del efecto inhibitorio encontrado inicialmente. Conclusiones. La medición de la HA podría servir como indicador in vitro de la potencia del antiveneno, el antiveneno producido por el INS guarda las condiciones in vitro de inhibición de tres de las principales actividades de los venenos de serpientes peruanas.

Bothrops sp. snakes causing the largest number of cases of ophidism in Peru, their venom contain several enzymes related to poison spreading, miotoxic and platelet aggregation disturbances. Objectives. The inhibiting capacity of liquid polivalent bothropic antivenomfrom Instituto Nacional de Salud (INS) has been evaluated on phospholipase A2 (PLA2), L amino acid oxidase (LAO) and hyaluronidase activities using B. atrox, B. barnetti, B. brazili and B. pictus venoms. Material and methods. In each case on 45 per cent egg yolk lipoprotein, Lleucina and O-dianisidine, as well as hyaluronic acid as substrates respectively, using for each enzyme ½, 1 and 2 doses of either naturaland heated (37 °C during five days) antivenom, assayed in triplicate. Results. HA was more neutralized enzyme for antivenin, all venomswith the exception of B. brazili were totally inhibited at any dose. For LAO had values of inhibition of 68 to 100 per cent using two doses of the antivenin, PLA2 was the least inhibited (70 to 80 per cent) to two doses. With the heated antivenin was a decline of the inhibitory effect initially found. Conclusions. The measurement of the HA might serve as an indicator of the in vitro potency of antivenins, the bothropic antiveninproduced by INS keeping in vitro conditions for inhibition of three major activities of the Peruvian snake venoms.

Aislamiento y algunas propiedades de la atroxina, una proteinasa del veneno de la serpiente peruana Bothrops atrox "Jergon" / Isolation and some properties of the proteinasa atroxin from the venom of the sanke Bothrops atrox

A proteolytic enzyme from the venom of Bothrops atrox snake was isolated. It was designed as Atroxin, and three chromatography steps were used to purification: ion exchange chromatography on DEAE-Sephadex A-50 equilibrated with 0.05 M Tris HCl buffer, 1 mM CaCl2 pH 7.4, followed by gel filtration on Sephadex G-50 andSephadex G-100, respectively, using the same buffer. The enzyme was recovered with a 7.4 folds and 11 percent of yield. It had a high activity on casein being 7.4 optimus pH. A molecular weight was 19.9 Kd calculated by polyacrilamide gel electrophoresis, and head treatment showed that the enzyme preserves its activity in the range of 37-45 degrees C, while it was decrease when the temperature values were higher. On the other hand, 0.133 mumoles of Ca2+ and Mg2+, and Zn2+ ions (0.266 mumoles) were activators, while EDTA (0.20 mumoles) and sodium azide (0.053 mumoles) were inhibitors. The enzymatic activity was not affected by glicerol(1.33 mumoles) and phenyl methyl sulphonyl fluoride(PSMF) (0.16 mumoles). In addition, iodoacetic acid (0.08 mumoles) was slight inhibitor, but 0.16 mumoles of p-tosyl-1-lysine chloromethyl ketone (TLCK) was activator. Biological assays on mice showed that atroxin produced hemorrhagic and necrosis after 24 h of injection, which was increased by 5 mM calcium chloride

Aislamiemto y algunas propiedades de la proteinasa I del veneno de la serpiente peruana Lachesis muta / Isolation and various properties of proteinase I from the venom of the Peruvian snake Lachesis muta

Se ha purificado una enzima proteolítica con elevada actividad sobre caseína a partir del veneno de la serpiente Lachesis muta. Esta proteína denominada "Proteinasa I" fue obtenida mediante una cromatografía de filtración sobre sephadex G-100 a pH 6.5 con buffer acetato de amonio 0.1 seguida de una cromatografía de intercambio iónico con DEAE-celulosa a pH 7.5 y una recromatografía en DEAE-celulosa a pH 9.0 y 7.8. La proteína aislada mostró un banda homogenea en electroforeis en gel de poliacrilamida. El peso molecular calculado por filtración en gel fue 26,100 y su pH óptimo 8.4. La ctividad enzimática no fue alterada depués de un calentamiento a 45-C por 10 minutos, mientras que a 70- sólo se registró un 4%de actividad. La enzima no ataca ésteres sintético como TAME y BAEE, no es afectada por lo iones de calcio y carecen de actividad hemorrágica