Purification and enzymatic characterization of a novel metalloprotease from Lachesis muta rhombeata snake venom

Lachesis muta rhombeata (Lmr) is the largest venomous snake in Latin America and its venom contains mainly enzymatic components, such as serine and metalloproteases, L-amino acid oxidase and phospholipases A2. Metalloproteases comprise a large group of zinc-dependent proteases that cleave basement membrane components such as fibronectin, laminin and collagen type IV. These enzymes are responsible for local and systemic changes, including haemorrhage, myonecrosis and inflammation. This study aimed the isolation and enzymatic characterization of the first metalloprotease (Lmr-MP) from Lmr venom (LmrV). Methods and results: Lmr-MP was purified through two chromatographic steps and submitted to enzymatic characterization. It showed proteolytic activity on azocasein with maximum activity at pH 7.0-9.0. It was inhibited by EDTA (a metal chelator that removes zinc, which is essential for enzymatic activity) and no effect was observed with PMSF, iodoacetic acid or pepstatin (inhibitors of serine, cysteine and aspartyl proteases, respectively). Ca2+, Mg2+ and Ba2+ ions increased its activity, while Al3+, Cu2+, Ni2+ and Zn2+ inhibited it. Additionally, ZnCl2 showed a dose dependent inhibition of the enzyme. Lmr-MP activity was also evaluated upon chromogenic substrates for plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) showing the highest activity on S-2302. The activity in different solutions (5 mM or 50 mM ammonium bicarbonate, pH 7.8; 0.1% trifluoroacetic acid + 50% acetonitrile; phosphate buffer saline, pH 7.4; 50 mM sodium acetate, pH 4.0 or ammonium acetate pH 4.5) was also evaluated and the results showed that its activity was abolished at acidic pHs. Its molecular mass (22,858 Da) was determined by MALDI-TOF and about 90% of its primary structure was verified by high-resolution mass spectrometry using HCD and ETD fragmentations and database search against the sequence of closely related species. It is a novel enzyme which shared high identity with other snake venom metalloproteases (svMPs) belonging to the P-I group. Conclusion: The purification procedure achieved a novel pure highly active metalloprotease from LmrV. This new molecule can help to understand the metalloproteases mechanisms of action, the Lachesis envenoming, as well as to open new perspectives for its use as therapeutic tools.(AU)

Experimental Lachesis muta rhombeata envenomation and effects of soursop (Annona muricata) as natural antivenom

Background In the Atlantic forest of the North and Northeast regions of Brazil, local population often uses the fruit juice and the aqueous extract of leaves of soursop (Annona muricata L.) to treat Lachesis muta rhombeata envenomation. Envenomation is a relevant health issue in these areas, especially due to its severity and because the production and distribution of antivenom is limited in these regions. The aim of the present study was to evaluate the relevance of the use of soursop leaf extract and its juice against envenomation by Lachesis muta rhombeata. Methods We evaluated the biochemical, hematological and hemostatic parameters, the blood pressure, the inflammation process and the lethality induced by Lachesis muta rhombeata snake venom. We also assessed the action of the aqueous extract of leaves (AmL) and juice (AmJ) from A. muricata on the animal organism injected with L. m. rhombeata venom (LmrV) in the laboratory environment. Results LmrV induced a decrease of total protein, albumin and glucose; and increase of creatine kinase, aspartate aminotransferase, and urea concentrations. It provoked hemoconcentration followed by reduction of hematocrit, an increase in prothrombin time and partial thromboplastin time and a decrease of the blood pressure. LmrV induced the release of interleukin-6, an increase in neutrophils and changes in the serum protein profile, characteristic of the acute inflammatory process. LD50 values were similar for the groups injected with LmrV and treated or untreated with AmJ and AmL. Both treatments play a role on the maintenance of blood glucose, urea and coagulation parameters and exert a protective action against the myotoxicity. However, they seem to worsen the hypotension caused by LmrV. Conclusion The treatments with AmJ and AmL present some beneficial actions, but they might intensify some effects of the venom. Therefore, additional studies on A. muricata are necessary to enable its use as natural antivenom for bushmaster snakebite.(AU)

Inhibitory effect of a Brazilian marine brown alga Spatoglossum schröederi on biological activities of Lachesis muta snake venom

The ability of crude extracts of the brown seaweed Spatoglossum schröederi to counteract some of the biological activities of Lachesis muta snake venom was evaluated. In vitro assays showed that only the extract of S. schröederi prepared in ethyl acetate was able to inhibit the clotting of fibrinogen induced by L. muta venom. On the other hand, all extracts were able to inhibit partially the hemolysis caused by venom and those prepared in dichloromethane or ethyl acetate fully neutralized the proteolysis and hemorrhage produced by the venom. Moreover, the dichloromethane or ethyl acetate extracts inhibited the hemolysis induced by an isolated phospholipase A2 from L. muta venom, called LM-PLA2-I. In contrast, the hexane extract failed to protect mice from hemorrhage or to inhibit proteolysis and clotting. These results show that the polarity of the solvent used to prepare the extracts of S. schröederi algae influenced the potency of the inhibitory effect of the biological activities induced by L. muta venom. Thus, the seaweed S. schröederi may be a promising source of natural inhibitors of the enzymes involved in biological activities of L. muta venom.

Anti-snake venom effect of secodolastane diterpenes isolated from Brazilian marine brown alga Canistrocarpus cervicornis against Lachesis muta venom

The effect of a Brazilian algae extract and also a mixture of two secodolastane diterpenes (linearol/isolinearol) that were isolated from the marine brown alga Canistrocarpus cervicornis were evaluated against biological activities of Lachesis muta snake venom. In vitro assays showed that the crude extract and the diterpenes were able to inhibit the clotting and proteolytic activity induced by L. muta crude venom, but not the hemolytic activity. However, only the diterpenes inhibited the hemolysis caused by a purified phospholipase A2 previously isolated from L. muta venom, denoted LM-PLA2-I. Interestingly, the crude algal extract and the diterpenes were able to protect mice from hemorrhage induced by L. muta venom. Thus, we may conclude that marine algae are rich and powerful sources of molecules that may be used against L. muta accidents in order to improve treatment of envenomation by this snake.

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.

Antiophidian properties of plant extracts against Lachesis muta venom

Snakebites comprise a serious health problem in several countries due to their global incidence, which exceeds 2.5 million per year, and the elevated number of victim fatalities. To counteract envenomations, antivenoms have been used regularly for more than a century. Apart from side effects including anaphylactic reactions, antivenoms are not able to efficiently neutralize local tissue damage, which contributes to increasing the severity and morbidity observed in patients. This fact, in turn, may be responsible for economic hardship, particularly in rural populations of developing countries. In the present work, we evaluated the antiophidian properties of 12 Brazilian plant extracts against the hemolytic, coagulant, hemorrhagic and proteolytic effects of Lachesis muta venom. Taken together, our data revealed that most of these aqueous products were capable of inhibiting those activities at different levels, except for Sapindus saponaria extract. In contrast, Stryphnodendron barbatiman extract completely neutralized all the analyzed biological activities. Thus, we may conclude that Brazilian flora may also be useful against L. muta accidents.

Purificação e caracterização bioquímica-funcional de duas toxinas (serinas proteinases) do veneno de Lachesis muta rhombeata / Purification and biochemical characterization of two toxins-functional (serine proteinases) of Lachesis muta rhombeata

Duas toxinas foram purificadas do veneno de Lachesis muta rhombeata com uma rendimento de aproximadamente 74 a 76%, empregando uma etapa de focalização isoelétrica preparativa seguida por gel filtração (HPLC)...Quando injetada em camundongos (0,25 ug/g), a LMR 47 induziu episódios de opistótono e giros, evidenciando a atividade giroxina. A análise histopatológica mostrou microhemorragias focais e outras alterações celulares a nível do cerebelo. A injeção intraplantar de 10ug da proteína, LMR 32, em camundongos induziu um incremento do volume (edema) de 45% e a inoculação de 0,1 e 0,25 ug/g da mesma proteína em ratos causou uma queda significativa na pressão arterial.