Antibodies against a single fraction of Micrurus dumerilii venom neutralize the lethal effect of whole venom.

The coralsnake Micrurus dumerilii (Elapidae) is reported to cause envenomings of medical importance. Previous studies characterized the protein composition of its venom, with phospholipase A2 (PLA2) proteins the most abundant. However, it is unknown which venom components are responsible for its lethal toxicity. Fractionation of M. dumerilii venom from Colombia was carried out using RP-HPLC and each fraction was screened for lethal effect in mice at a dose of 20 µg by intraperitoneal route. Results showed that only one fraction, F9, was lethal. This fraction displayed PLA2 activity, induced indirect hemolysis in vitro, as well as edema and myotoxicity in vivo. SDS-PAGE of unreduced F9 evidenced two bands of 8 and 15 kDa, respectively, consistent with the detection of proteins with masses of 13,217.77 Da, 7144.06 Da, and 7665.55 Da. Tryptic digestion of F9 followed by nESI-MS/MS revealed peptide sequences matching proteins of the three-finger toxin (3FTx) and PLA2 families. Immunization of a rabbit with F9 proteins elicited antibody titers up to 1:10,000 by ELISA. After serum fractionation with caprylic acid, the obtained IgG was able to neutralize the lethal effect of the complete venom of M. dumerilii using a challenge of 2 ×LD50 at the IgG/venom ratio of 50:1 (w/w). In conclusion, present results show that the lethal effect of M. dumerilii venom in mice is mainly driven by one fraction which contains 3FTx and PLA2 proteins. The antibodies produced against this fraction cross-recognized other PLA2s and neutralized the lethal effect of whole M. dumerilii venom, pointing out to the potential usefulness of F9 as a relevant antigen for improving current coral snake antivenoms.

Heterologous Expression and Immunogenic Potential of the Most Abundant Phospholipase A2 from Coral Snake Micrurus dumerilii to Develop Antivenoms.

Micrurus dumerilii is a coral snake of clinic interest in Colombia. Its venom is mainly composed of phospholipases A2 being MdumPLA2 the most abundant protein. Nevertheless, Micrurus species produce a low quantity of venom, which makes it difficult to produce anticoral antivenoms. Therefore, in this work, we present the recombinant expression of MdumPLA2 to evaluate its biological activities and its immunogenic potential to produce antivenoms. For this, a genetic construct rMdumPLA2 was cloned into the pET28a vector and expressed heterologously in bacteria. His-rMdumPLA2 was extracted from inclusion bodies, refolded in vitro, and isolated using affinity and RP-HPLC chromatography. His-rMdumPLA2 was shown to have phospholipase A2 activity, a weak anticoagulant effect, and induced myonecrosis and edema. The anti-His-rMdumPLA2 antibodies produced in rabbits recognized native PLA2, the complete venom of M. dumerilii, and a phospholipase from another species of the Micrurus genus. Antibodies neutralized 100% of the in vitro phospholipase activity of the recombinant toxin and a moderate percentage of the myotoxic activity of M. dumerilii venom in mice. These results indicate that His-rMdumPLA2 could be used as an immunogen to improve anticoral antivenoms development. This work is the first report of an M. dumerilii functional recombinant PLA2.

Characterization of a Lab-Scale Process to Produce Whole IgG Antivenom Covering Scorpion Stings by Genus Tityus and Centruroides of Colombia.

Scorpion stings are a public health event in Colombia lacking official epidemiological data, and are considered a medical emergency. Despite the two local producers of antivenoms, neither of them is currently manufacturing scorpion antivenoms. We present the characterization of a lab-scale process to produce the first specific scorpion antivenom for Colombia, formulated to cover scorpion stings produced by Tityus pachyurus, Tityus asthenes, Tityus fuhrmanii, Centruroides spp. To do so, rabbits were immunized by subcutaneous injection with each venom using an immunization program of 3 months. After each rabbit reached the required IgG concentration, rabbits were bled, and plasma was separated by decantation under refrigeration. Immunoglobulins were purified from each hyperimmune plasma using a methodology including precipitation with ammonium sulfate, thermocoagulation, and purification through an ultrafiltration process using a ready-to-use and reusable laboratory crossflow tangential cassette with a polyethersulfone membrane. Each hyperimmune plasma was processed by being separated and freeze-dried at the end of the process. Rabbits were able to produce specific IgG antibodies recognizing the respective immunization venom; even an in vitro interspecies cross-recognition was detected. The separation and purification processes allowed us to obtain IgG products without considerable contaminants (except for albumin). The process was characterized, and critical stages were identified.

Immunorecognition and Neutralization of Crotalus durissus cumanensis Venom by a Commercial Antivenom Produced in Colombia.

In Colombia, on average 2.9% of the nearly 5600 snakebite events that occur annually involve the rattlesnake Crotalus durissus cumanensis. The envenomation by this snake is mainly characterized by neurotoxicity and the main toxin is crotoxin (~64.7% of the total venom). The Instituto Nacional de Salud (INS) produces a polyvalent antivenom aimed at the treatment of bothropic, crotalid, and lachesic envenomations; nonetheless, its immune reactivity profile and neutralizing capacity over biological activities of the C. d. cumanensis venom has been poorly evaluated. In this sense, the study aims: (1) to describe an in-depth exploration of its immunoreactivity through second-generation antivenomics and HPLC fraction-specific ELISA immunoprofiles; and (2) to evaluate the neutralization pattern of the rattlesnake venom in vitro and in vivo biological activities. The results obtained showed a variable recognition of crotoxin subunits, in addition to a molecular mass-dependent immunoreactivity pattern in which the disintegrins were not recognized, and snake venom metalloproteinases and L-amino acid oxidases were the most recognized. Additionally, a high neutralization of proteolytic and coagulant activities was observed, but not over the PLA2 activity. Further, the median effective dose against C. d. cumanensis venom lethality was 962 µL of antivenom per mg of venom. In conclusion, (1) the antivenom recognition over the crotoxin and the disintegrins of the C. d. cumanensis should be improved, thus aiming upcoming efforts for the exploration of new techniques and approaches in antivenom production in Colombia, and (2) the neutralization activity of the antivenom seems to follow the molecular mass-dependent recognition pattern, although other explanations should be explored.

Anti-Neurotoxins from Micrurus mipartitus in the Development of Coral Snake Antivenoms.

In Colombia, the genus Micrurus includes 30 species, of which M. mipartitus and M. dumerilii are the most widely distributed. Micrurus causes less than 3% of the approximately 5000 cases of snakebite per year. The elapid envenomation caused by the snakes from the Micrurus genus, are characterized by the severity of their clinical manifestations, due to the venom neurotoxic components such as three-finger toxins (3FTx) and phospholipases (PLA2). The treatment for snakebites is the administration of specific antivenoms, however, some of them have limitations in their neutralizing ability. A strategy proposed to improve antivenoms is to produce antibodies against the main components of the venom. The aim of this work was to produce an antivenom, using an immunization protocol including the main 3FTx and PLA2 responsible for M. mipartitus lethality. The antibody titers were determined by ELISA in rabbits' serum. The immunized animals elicited a response against toxins and whole venom. The Immunoglobulin G (IgGs) obtained were able to neutralize the lethal effect of their homologous toxins. A combination of antivenom from M. mipartitus with antitoxins improved their neutralizing ability. In the same way, a mixture of anti 3FTx and PLA2 protected the mice from a 1.5 median lethal dose (LD50) of M. mipartitus venom. The results showed that this might be a way to improve antibody titers specificity against the relevant toxins in M. mipartitus venom and indicated that there is a possibility to develop and use recombinant 3FTx and PLA2 toxins as immunogens to produce antivenoms. Additionally, this represents an alternative to reduce the amount of venom used in anti-coral antivenom production.

Novel three-finger toxins from Micrurus dumerilii and Micrurus mipartitus coral snake venoms: Phylogenetic relationships and characterization of Clarkitoxin-I-Mdum.

Micrurus mipartitus and M. dumerilii are the most medically important coral snakes in Colombia. Proteomic characterization of their venoms has previously shown that proteins of the three-finger toxin (3FTx) family are abundant components, especially in M. mipartitus (61%) and to a lesser extent in M. dumerilii (28%). In order to increase knowledge on these toxins, in this work a major 3FTx of M. dumerilii venom (8% of the venom proteins), named Clarkitoxin-I-Mdum, was isolated and characterized. Its amino acid sequence comprises 66 residues, with an isotope-averaged molecular mass of 7537 ±â€¯2 Da and a theoretical pI of 9.36, presenting the conserved pattern of eight cysteines that classifies it as a short-chain (type I) 3FTx. Clarkitoxin-I-Mdum was not lethal to mice by intravenous or intracerebroventricular route and was not cytolytic to myogenic cells in vitro. On the other hand, five coding sequences for 3FTxs were obtained from the venom gland of M. mipartitus. These novel toxin sequences were named Mm3FTx-01 to Mm3FTx-05, all of them also presenting the eight conserved cysteines of short-chain 3FTxs. Phylogenetic analysis revealed high variability of 3FTxs from Micrurus, and ELISA using antibodies raised to the major 3FTxs from M. mipartitus and M. dumerilii confirmed their immunochemical divergence. These results highlight the relevance of performing further studies aiming at a deeper understanding of the functional and antigenic relationships among specific Micrurus toxins, with important implications for the production of antivenoms.

L-amino acid oxidase isolated from Micrurus mipartitus snake venom (MipLAAO) specifically induces apoptosis in acute lymphoblastic leukemia cells mostly via oxidative stress-dependent signaling mechanism.

The effect of Micrurus mipartitus snake venom as a therapeutic alternative for T-acute lymphoblastic leukemia (ALL) is still unknown. This study was aimed to evaluate the cytotoxic effect of M. mipartitus snake venom and a new L-amino acid oxidase (LAAO), named MipLAAO, on human peripheral blood lymphocytes (PBL) and on T-ALL cells (Jurkat), and its mechanism of action. PBL and Jurkat cells were treated with venom and MipLAAO, and morphological changes in the cell nucleus/DNA, mitochondrial membrane potential, levels of intracellular reactive oxygen species and cellular apoptosis markers were determined by fluorescence microscopy, flow cytometry and pharmacological inhibition. Venom and MipLAAO induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose-response manner. Additionally, venom and MipLAAO increased dichlorofluorescein fluorescence intensity, indicative of H2O2 production, increased DJ-1 Cys106-sulfonate, as a marker of intracellular stress and induced the up-regulation of PUMA, p53 and phosphorylation of c-JUN. Additionally, it increased the expression of apoptotic CASPASE-3. In conclusion, M. mipartitus venom and MipLAAO selectively induces apoptosis in Jurkat cells through a H2O2-mediated signaling pathway dependent mostly on CASPASE-3 pathway. Our findings support the potential use of M. mipartitus snake venom compounds as a potential treatment for T-ALL.

Inhibition of a Snake Venom Metalloproteinase by the Flavonoid Myricetin.

Most of the snakebite envenomations in Central and South America are caused by species belonging to Bothrops genus. Their venom is composed mainly by zinc-dependent metalloproteinases, responsible of the hemorrhage characteristic of these envenomations. The aim of this study was to determine the inhibitory ability of ten flavonoids on the in-vitro proteolytic activity of Bothrops atrox venom and on the hemorrhagic, edema-forming and myonecrotic activities of Batx-I, the most abundant metalloproteinase isolated from this venom. Myricetin was the most active compound, exhibiting an IC 50 value of 150 µ M and 1021 µ M for the inhibition of proteolytic and hemorrhagic activity, respectively. Independent injection experiments, with a concentration of 1600 µ M of myricetin administered locally, immediately after toxin injection, demonstrated a reduction of 28 ± 6 % in the hemorrhagic lesion. Additionally, myricetin at concentrations 800, 1200 and 1600 µ M promoted a reduction in plasma creatine kinase activity induced by Batx-I of 21 ± 2 % , 60 ± 5 % and 63 ± 2 % , respectively. Molecular dynamics simulations coupled with the adaptive biasing method suggest that myricetin can bind to the metalloproteinase active site via formation of hydrogen bonds between the hydroxyl groups 3', 4' and 5' of the benzyl moiety and amino acid Glu143 of the metalloproteinase. The hydroxyl substitution pattern of myricetin appears to be essential for its inhibitory activity. Based on this evidence, myricetin constitutes a candidate for the development of inhibitors to reduce local tissue damage in snakebite envenomations.

MipLAAO, a new L-amino acid oxidase from the redtail coral snake Micrurus mipartitus.

L-amino acid oxidases (LAAOs) are ubiquitous enzymes in nature. Bioactivities described for these enzymes include apoptosis induction, edema formation, induction or inhibition of platelet aggregation, as well as antiviral, antiparasite, and antibacterial actions. With over 80 species, Micrurus snakes are the representatives of the Elapidae family in the New World. Although LAAOs in Micrurus venoms have been predicted by venom gland transcriptomic studies and detected in proteomic studies, no enzymes of this kind have been previously purified from their venoms. Earlier proteomic studies revealed that the venom of M. mipartitus from Colombia contains ∼4% of LAAO. This enzyme, here named MipLAAO, was isolated and biochemically and functionally characterized. The enzyme is found in monomeric form, with an isotope-averaged molecular mass of 59,100.6 Da, as determined by MALDI-TOF. Its oxidase activity shows substrate preference for hydrophobic amino acids, being optimal at pH 8.0. By nucleotide sequencing of venom gland cDNA of mRNA transcripts obtained from a single snake, six isoforms of MipLAAO with minor variations among them were retrieved. The deduced sequences present a mature chain of 483 amino acids, with a predicted pI of 8.9, and theoretical masses between 55,010.9 and 55,121.0 Da. The difference with experimentally observed mass is likely due to glycosylation, in agreement with the finding of three putative N-glycosylation sites in its amino acid sequence. A phylogenetic analysis of MmipLAAO placed this new enzyme within the clade of homologous proteins from elapid snakes, characterized by the conserved Serine at position 223, in contrast to LAAOs from viperids. MmipLAAO showed a potent bactericidal effect on S. aureus (MIC: 2 µg/mL), but not on E. coli. The former activity could be of interest to future studies assessing its potential as antimicrobial agent.

Isolation and Functional Characterization of an Acidic Myotoxic Phospholipase A2 from Colombian Bothrops asper Venom.

Myotoxic phospholipases A2 (PLA2) are responsible for many clinical manifestations in envenomation by Bothrops snakes. A new myotoxic acidic Asp49 PLA2 (BaCol PLA2) was isolated from Colombian Bothrops asper venom using reverse-phase high performance liquid chromatography (RP-HPLC). BaCol PLA2 had a molecular mass of 14,180.69 Da (by mass spectrometry) and an isoelectric point of 4.4. The complete amino acid sequence was obtained by cDNA cloning (GenBank accession No. MF319968) and revealed a mature product of 124 amino acids with Asp at position 49. BaCol PLA2 showed structural homology with other acidic PLA2 isolated from Bothrops venoms, including a non-myotoxic PLA2 from Costa Rican B. asper. In vitro studies showed cell membrane damage without exposure of phosphatidylserine, an early apoptosis hallmark. BaCol PLA2 had high indirect hemolytic activity and moderate anticoagulant action. In mice, BaCol PLA2 caused marked edema and myotoxicity, the latter seen as an increase in plasma creatine kinase and histological damage to gastrocnemius muscle fibers that included vacuolization and hyalinization necrosis of the sarcoplasm.

Purification of nasulysin-1: A new toxin from Porthidium nasutum snake venom that specifically induces apoptosis in leukemia cell model through caspase-3 and apoptosis-inducing factor activation.

Nasulysin-1, a new zinc-metalloproteinase from the snake venom of the hognose pit viper Porthidium nasutum, was purified to homogeneity using molecular exclusion chromatography and high performance liquid chromatography on a reverse phase column. The molecular mass of the purified enzyme was 25,900 kDa and pI 4.1, as determined by 1D and 2D polyacrylamide gel electrophoresis. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the N-terminal amino acid sequence (1FSPRYIELVVVADHGMFKKYNSNLNTIR28; 1TASLANLEVWSK12; 1DLLPR6) of the purified nasulysin-1, shows close structural homology with other snake venom metalloproteinases isolated from different snake venoms. The purified nasulysin-1 showed specific apoptosis-inducing activity in Jurkat and K562 cells, a T-cell acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (AML) cell model, respectively, without affecting the viability of human lymphocyte cells. After 48 h treatment, nasulysin-1 (20 µg/mL) induced loss of the mitochondrial membrane potential (ΔΨm), activated the apoptosis-inducing factor (AIF), activated the protease caspase-3, and induced chromatin condensation and DNA fragmentation, all hallmarks of apoptosis. These results strongly suggest that nasulysin-1 selectively induces apoptosis to eliminate leukemia cells. Thus, these data warrant further investigation into the use of the metalloproteinase protein, nasulysin-1 as a potential therapeutic agent for treating leukemia.

Integrative characterization of the venom of the coral snake Micrurus dumerilii (Elapidae) from Colombia: Proteome, toxicity, and cross-neutralization by antivenom.

In Colombia, nearly 2.8% of the 4200 snakebite accidents recorded annually are inflicted by coral snakes (genus Micrurus). Micrurus dumerilii has a broad distribution in this country, especially in densely populated areas. The proteomic profile of its venom was here studied by a bottom-up approach combining RP-HPLC, SDS-PAGE and MALDI-TOF/TOF. Venom proteins were assigned to eleven families, the most abundant being phospholipases A2 (PLA2; 52.0%) and three-finger toxins (3FTx; 28.1%). This compositional profile shows that M. dumerilii venom belongs to the 'PLA2-rich' phenotype, in the recently proposed dichotomy for Micrurus venoms. Enzymatic and toxic venom activities correlated with protein family abundances. Whole venom induced a conspicuous myotoxic, cytotoxic and anticoagulant effect, and was mildly edematogenic and proteolytic, whereas it lacked hemorrhagic activity. Some 3FTxs and PLA2s reproduced the lethal effect of venom. A coral snake antivenom to Micrurus nigrocinctus demonstrated significant cross-recognition of M. dumerilii venom proteins, and accordingly, ability to neutralize its lethal effect. The combined compositional, functional, and immunological data here reported for M. dumerilii venom may contribute to a better understanding of these envenomings, and support the possible use of anti-M. nigrocinctus coral snake antivenom in their treatment. BIOLOGICAL SIGNIFICANCE: Coral snakes represent a highly diversified group of elapids in the New World, with nearly 70 species within the genus Micrurus. Owing to their scarce yields, the biochemical composition and toxic activities of coral snake venoms have been less well characterized than those of viperid species. In this work, an integrative view of the venom of M. dumerilii, a medically relevant coral snake from Colombia, was obtained by a combined proteomic, functional, and immunological approach. The venom contains proteins from at least eleven families, with a predominance of phospholipases A2 (PLA2), followed by three-finger toxins (3FTx). According to its compositional profile, M. dumerilii venom can be grouped with those of several Micrurus species from North and Central America that present a PLA2-predominant phenotype, to date it is the most southerly coral snake species to do so. Other coral snake species that a 'PLA2-rich' venom, M. dumerilii venom contains both components that form MitTx, a pain-inducing heterodimeric complex recently characterized from the venom of Micrurus tener, also present in Micrurus mosquitensis and M. nigrocinctus venoms. In addition to a lethal three-finger toxin, PLA2s participate in the toxicity of M. dumerilii venom, some of them displaying ability to induce cytolysis, muscle necrosis, and lethality to mice. An antivenom to M. nigrocinctus demonstrated significant cross-recognition of M. dumerilii venom proteins, and accordingly, ability to neutralize its lethal effect, being of potential therapeutic usefulness in these envenomings.

Characterization of the most promising fraction of Swietenia macrophylla active against myotoxic phospholipases A2: identification of catechin as one of the active compounds / Caracterización de la fracción más promisoria de Swietenia macrophylla activa contra fosfolipasas A2 miotoxicas: identificación de catequina como uno de los compuestos activos

Background: The pharmacological effects produced by snakebite accidents involve the actions of several enzymes, of which those of the phospholipases A2 (PLA2) exhibit a wide variety of effects such as edema and myotoxicity. Some plant extracts have been antagonists of crude snake venoms and toxins. Based on promising bioactivity, Swietenia macrophylla King was selected for further studies. Objective: The purpose of this study was to identify the PLA2 inhibitors present in the crude extract of S. macrophylla that could be promising leads in neutralizing the local effects of ophidian accidents. Methods: Bioassay-guided fractionation of the ethanolic extract of the leaves of S. macrophylla lead to the detection of (+)-catechin, characterized through gas chromatography coupled with mass spectrometry (GC-MS), and confirmed by HPLC. The PLA2 inhibitory activity was measured with the Dole method and a spectrophotometric assay with 4-Nitro-3 octanoyloxy-benzoic acid (4N3OBA). Cytotoxicity was done on C2C12 murine myoblast. Results: Fraction F5 and (+)-Catechin inhibited the PLA2 activity of B. asper venom, in a dosedependent way. In addition, (+) Catechin showed an inhibition level of 83.1 ± 3.1 % of the enzymati activity of one PLA2 purified from the venom of Crotalus durissus cumanensis using 4N3OBA as substrate. Also the ethanolic extract and fraction F5 showed inhibition of the cytotoxicity induced by the Bothrops atrox venom and their Lys 49 PLA2 (80 and 100% respectively). Molecular docking results suggested that OH from 4´ and 5' carbons of (+)-catechin could form hydrogen bonds with carboxylate moiety of residue Asp49, while OH from 5 could form a hydrogen bond with Asn 6. Additional Van der Waals interactions were also proposed. Conclusion: Swietenia macrophylla exhibited strong inhibitory activity against PLA2s enzymes. Catechin, one of the components in the active fraction F5, is proposed as being partially responsible for the bioactivity.

Antecedentes: Los efectos farmacológicos producidos en el accidente ofídico implican la acción de varias enzimas, como las fosfolipasas A2 (PLA2), que exhiben una amplia variedad de efectos como edema y miotoxicidad. Algunos extractos de plantas han demostrado ser antagonistas de los venenos crudos y sus toxinas. En base a una bioactividad promisoria previa, Swietenia macrophylla King fue seleccionada para estudios posteriores. Objetivo: El propósito de este estudio fue identificar metabolitos inhibidores de PLA2 presentes en el extracto crudo de S. macrophylla que podrían ser prometedores en la neutralización de los efectos locales del accidente ofídico. Métodos: Un fraccionamiento biodirigido del extracto etanólico de hojas de S. macrophylla llevó a la detección de la (+)-catequina mediante cromatografía de gases acoplada a espectrometría de masas (GC-MS), y se confirmó mediante HPLC. La actividad inhibidora de PLA2 se determinó por el método de Dole y un ensayo espectrofotométrico con Ácido 4-Nitro-3-octanoiloxibenzoico (4N3OBA). La citotoxicidad se determinó en mioblastos C2C12 murinos. Resultados: La fracción F5 de S. macrophylla, mostró la mayor inhibición del veneno de Bothrops asper y PLA2s aisladas de B. atrox y Crotalus durissus cumanensis e inhibición completa de la citotoxicidad. La (+)-catequina fue el metabolito más abundante en F5, mostró una inhibición de PLA2 de 89,8%, 81,1% y 74,3%, a diferentes relaciones de veneno: catequina. Mediante estudios de docking molecular se demostró la unión de la (+)-catequina al sitio activo de la PLA2. Conclusión: Swietenia macrophylla exhibió una fuerte actividad inhibitoria sobre las enzimas PLA2s. La (+)-catequina, uno de los metabolitos presentes en la fracción activa F5, se propone como uno de los compuestos responsables de la bioactividad.

Isolation and functional characterization of a basic phospholipase A2 from Colombian Bothrops asper venom / Aislamiento y caracterización funcional de una fosfolipasa A2 básica del veneno de Bothrops asper de Colombia

Antecedentes: las mordeduras de serpientes representan un problema de salud pública relevante en muchas regiones del mundo, particularmente en los países tropicales y subtropicales de África, Asia, América Latina y Oceanía. Los venenos de serpientes son mezclas complejas de enzimas y proteínas tóxicas, donde los componentes más importantes y abundantes que dañan los músculos en los venenos de serpientes son las fosfolipasas A2 (PLA2). Objetivo: Aislar y caracterizar una fosfolipasa A2 del veneno de Venezuela Bothrops para obtener información sobre la composición del veneno de esta especie. Materiales y métodos: La cromatografía de intercambio catiónico seguida de HPLC de fase inversa se usó para purificar la proteína. La espectrometría de masas se utilizó para determinar su masa molecular. La caracterización bioquímica se realizó utilizando un sustrato sintético (ácido 4-nitro-3-octanoiloxi-benzoico). La actividad miotóxica y de inducción de edema de la toxina se probó en ratones, midiendo la actividad de la creatina quinasa plasmática y el diámetro de la almohadilla de la pata, respectivamente. Además, se examinó la actividad citotóxica de mioblastos y miotubos C2C12 del músculo esquelético murino. Resultados: se purificó una PLA2 de Bothrops asper veneno de Colombia (BaspCol-PLA2). Su masa molecular fue de 13974.6 Da. La enzima hidrolizó un sustrato sintético con un KM de 3.11 mM y un VMax de 4.47 nmol / min, mostrando una actividad máxima a 40 ° C y a pH 8.0. La PLA2 requería Ca2 + para la actividad. La adición de Mg2 +, Cd2 +, Mn2 + y Zn2 + (10 mM) en presencia de baja concentración de Ca2 + (1 mM) disminuyó la actividad de la enzima. La sustitución de Ca2 + por los cationes divalentes mencionados también redujo la actividad a niveles similares a los de la ausencia de Ca2 +. Tres fragmentos internos (CCFVHDCCYGK, AAAI / LCFRDNI / LNTYNDKK, DAAI / LCFR) identificada mediante un análisis de espectrometría de masas mostró similitud con las PLA2 de B. asper informadas previamente. En ratones, BaspCol-PLA2 indujo un notable efecto miotóxico local y un edema moderado en la almohadilla del pie. In vitro, esta enzima indujo un efecto citotóxico tanto en los mioblastos como en los miotubos. Además, se clasificó como PLA2 débilmente anticoagulante, mostrando este efecto en concentraciones entre 3 y 10 µg / mL cuando se usa plasma humano. Conclusiones: Una PLA2 se purificó y se llamó BaspCol-PLA2, esta enzima mostró actividad catalítica y una masa molecular de 13974.6 Da. La toxina mostró actividades miotóxicas, formadoras de edema, anticoagulantes y citotóxicas. BaspCol-PLA2 indujo un notable efecto miotóxico local y un edema moderado en la almohadilla del pie. In vitro, esta enzima indujo un efecto citotóxico tanto en los mioblastos como en los miotubos. Además, se clasificó como PLA2 débilmente anticoagulante, mostrando este efecto en concentraciones entre 3 y 10 µg / mL cuando se usa plasma humano. Conclusiones: Una PLA2 se purificó y se llamó BaspCol-PLA2, esta enzima mostró actividad catalítica y una masa molecular de 13974.6 Da. La toxina mostró actividades miotóxicas, formadoras de edema, anticoagulantes y citotóxicas. BaspCol-PLA2 indujo un notable efecto miotóxico local y un edema moderado en la almohadilla del pie. In vitro, esta enzima indujo un efecto citotóxico tanto en los mioblastos como en los miotubos. Además, se clasificó como PLA2 débilmente anticoagulante, mostrando este efecto en concentraciones entre 3 y 10 µg / mL cuando se usa plasma humano. Conclusiones: Una PLA2 se purificó y se llamó BaspCol-PLA2, esta enzima mostró actividad catalítica y una masa molecular de 13974.6 Da. La toxina mostró actividades miotóxicas, formadoras de edema, anticoagulantes y citotóxicas. esta enzima mostró actividad catalítica y masa molecular de 13974.6 Da. La toxina mostró actividades miotóxicas, formadoras de edema, anticoagulantes y citotóxicas. esta enzima mostró actividad catalítica y masa molecular de 13974.6 Da. La toxina mostró actividades miotóxicas, formadoras de edema, anticoagulantes y citotóxicas.

Inhibitory effects of Swietenia macrophylla on myotoxic phospholipases A2

Activity-guided fractionation of an ethanol-soluble extract of the leaves of Swietenia macrophylla King, Meliaceae, led to several fractions. As a result, sample Sm13-16, 23 had the most promising activity against phospholipases A2 (PLA2), Asp49 and Lys49 types. This fraction inhibited PLA2 activity of the Asp49 PLA2, when aggregated substrate was used. On the other hand, this activity was weakly neutralized when monodispersed substrate was used. In addition, Sm13-16, 23 inhibited, in a dose dependent manner, the cytotoxicity, myotoxicity and edema induced by PLA2s, as well as the anticoagulant activity of Asp49 PLA2. Overall, this fraction exhibited a better inhibition of the toxic activities induced by the Lys49 PLA2 than those caused by the Asp49 PLA2. The spectral data of Sm13-16, 23 suggested the presence of aromatic compounds (UV λ max (nm) 655, 266, and 219; IR λ max KBr (cm-1): ~ 3600-3000 (OH), 2923.07 and 1438.90 (C-H), 1656.69 (C = O), 1618.63 and 1607.67 (C-O), 1285.47772.60). We suggest that phenolic compounds could interact and inhibit the toxins by several mechanisms. Further analysis of the compounds present in the active fraction could be a relevant contribution in the treatment of accidents caused by snake envenomation.

Glycolic acid inhibits enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper snake venom: insights from docking and molecular modeling.

Glycolic acid (GA) (2-Hydroxyethanoic acid) is widely used as chemical peeling agent in Dermatology and, more recently, as a therapeutic and cosmetic compound in the field of skin care and disease treatment. In this work we tested the inhibitory ability of glycolic acid on the enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper venom, which induces a variety of toxic actions. Glycolic acid inhibited the proteolytic activity of BaP1 on azocasein, with an IC50 of 1.67 mM. The compound was also effective at inhibiting the hemorrhagic activity of BaP1 in skin and muscle in experiments involving preincubation of enzyme and inhibitor prior to injection. When BaP1 was injected i.m. and then, at the same site, different concentrations of glycolic acid were administered at either 0 or 5 min, 7 mM solutions of the inhibitor partially abrogated hemorrhagic activity when administered at 0 min. Moreover, glycolic acid inhibited, in a concentration-dependent manner, edema-forming activity of BaP1 in the footpad. In order to have insights on the mode of action of glycolic acid, UV-vis and intrinsic fluorescence studies were performed. Results of these assays suggest that glycolic acid interacts directly with BaP1 and chelates the Zn²âº ion at the active site. These findings were supported by molecular docking results, which suggested that glycolic acid forms hydrogen bonds with residues Glu143, Arg110 and Ala111 of the enzyme. Additionally, molecular modeling results suggest that the inhibitor chelates Zn²âº, with a distance of 3.58 Å, and may occupy part of substrate binding cleft of BaP1. Our results suggest that glycolic acid is a candidate for the development of inhibitors to be used in snakebite envenomation.

Cloning and characterization of an antibacterial L-amino acid oxidase from Crotalus durissus cumanensis venom.

An L-amino acid oxidase (LAAO) from Crotalus durissus cumanensis venom (CdcLAAO) was purified to homogeneity using a combination of size-exclusion and ion exchange chromatographies. CdcLAAO is a monomeric protein exhibiting an apparent molecular mass of 55 kDa and a calculated pI of 8. Its complete 498-amino-acid sequence was deduced through cDNA and protein sequencing. The enzyme oxidized L-Leu with K(m) and a V(Max) of 9.23 µM and 0.46 µM/min respectively, and exhibited Kcat and a Kcat/K(m) of 1.8 s(-1) and 195 mM(-1)s(-1). CdcLAAO inhibited in a dose-dependent manner the growth of Staphylococcus aureus and Acinetobacter baumannii. The inhibitory effect was more significant on S. aureus, with a Minimal Inhibitory Concentration (MIC) of 8 µg/mL and Minimal Bactericidal Concentration (MBC) of 16 µg/mL, than against A. baumannii, with a MIC of 16 µg/mL and MBC of 32 µg/mL. However, against Escherichia coli CdcLAAO did not show inhibitory capacity at the concentrations tested (2-128 µg/mL). CdcLAAO did not exhibit cytotoxic activity on the mouse myoblast cell line C(2)C(12) and on peripheral blood mononuclear cell (PBMC).

Snake venomics across genus Lachesis. Ontogenetic changes in the venom composition of Lachesis stenophrys and comparative proteomics of the venoms of adult Lachesis melanocephala and Lachesis acrochorda.

We report the proteomic analysis of ontogenetic changes in venom composition of the Central American bushmaster, Lachesis stenophrys, and the characterization of the venom proteomes of two congeneric pitvipers, Lachesis melanocephala (black-headed bushmaster) and Lachesis acrochorda (Chochoan bushmaster). Along with the previous characterization of the venom proteome of Lachesis muta muta (from Bolivia), our present outcome enables a comparative overview of the composition and distribution of the toxic proteins across genus Lachesis. Comparative venomics revealed the close kinship of Central American L. stenophrys and L. melanocephala and support the elevation of L. acrochorda to species status. Major ontogenetic changes in the toxin composition of L. stenophrys venom involves quantitative changes in the concentration of vasoactive peptides and serine proteinases, which steadily decrease from birth to adulthood, and age-dependent de novo biosynthesis of Gal-lectin and snake venom metalloproteinases (SVMPs). The net result is a shift from a bradykinin-potentiating and C-type natriuretic peptide (BPP/C-NP)-rich and serine proteinase-rich venom in newborns and 2-years-old juveniles to a (PI>PIII) SVMP-rich venom in adults. Notwithstanding minor qualitative and quantitative differences, the venom arsenals of L. melanocephala and L. acrochorda are broadly similar between themselves and also closely mirror those of adult L. stenophrys and L. muta venoms. The high conservation of the overall composition of Central and South American bushmaster venoms provides the ground for rationalizing the "Lachesis syndrome", characterized by vagal syntomatology, sensorial disorders, hematologic, and cardiovascular manifestations, documented in envenomings by different species of this wide-ranging genus. This finding let us predict that monospecific Lachesic antivenoms may exhibit paraspecificity against all congeneric species.

Mipartoxin-I, a novel three-finger toxin, is the major neurotoxic component in the venom of the redtail coral snake Micrurus mipartitus (Elapidae).

The major venom component of Micrurus mipartitus, a coral snake distributed from Nicaragua to northern South America, was characterized biochemically and functionally. This protein, named mipartoxin-I, is a novel member of the three-finger toxin superfamily, presenting the characteristic cysteine signature and amino acid sequence length of the short-chain, type-I, α-neurotoxins. Nevertheless, it varies considerably from related toxins, with a sequence identity not higher than 70% in a multiple alignment of 67 proteins within this family. Its observed molecular mass (7030.0) matches the value predicted by its amino acid sequence, indicating lack of post-translational modifications. Mipartoxin-I showed a potent lethal effect in mice (intraperitoneal median lethal dose: 0.06 µg/g body weight), and caused a clear neuromuscular blockade on both avian and mouse nerve-muscle preparations, presenting a post-synaptic action through the cholinergic nicotinic receptor. Since mipartoxin-I is the most abundant (28%) protein in M. mipartitus venom, it should play a major role in its toxicity, and therefore represents an important target for developing a therapeutic antivenom, which is very scarce or even unavailable in the regions where this snake inhabits. The structural information here provided might help in the preparation of a synthetic or recombinant immunogen to overcome the limited venom availability.

An acidic phospholipase A2 with antibacterial activity from Porthidium nasutum snake venom.

Snake venoms are complex mixtures of proteins among which both basic and acidic phospholipases A(2) (PLA(2)s) can be found. Basic PLA(2)s are usually responsible for major toxic effects induced by snake venoms, while acidic PLA(2)s tend to have a lower toxicity. A novel PLA(2), here named PnPLA(2), was purified from the venom of Porthidium nasutum by means of RP-HPLC on a C18 column. PnPLA(2) is an acidic protein with a pI of 4.6, which migrates as a single band under both non-reducing and reducing conditions in SDS-PAGE. PnPLA(2) had a molecular mass of 15,802.6 Da, determined by ESI-MS. Three tryptic peptides of this protein were characterized by HPLC-nESI-MS/MS, and N-terminal sequencing by direct Edman degradation showing homology to other acidic PLA(2)s from viperid venoms. PnPLA(2) displayed indirect hemolytic activity in agarose erythrocyte-egg yolk gels and bactericidal activity against Staphylococcus aureus in a dose-dependent manner, with a MIC and MBC of 32 µg/mL. In addition, PnPLA(2) showed a potent inhibitory effect on platelet aggregation with doses up to 40 µg/mL. This acidic PLA(2), in contrast to basic enzymes isolated from other viperid snake venoms, was not cytotoxic to murine skeletal muscle myoblasts C(2)C(12). This is the first report on a bactericidal protein of Porthidium nasutum venom.