Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom

Menaldo, Danilo L.; Jacob-Ferreira, Anna L.; Bernardes, Carolina P.; Cintra, Adélia Cristina Oliveira; Sampaio, Suely Vilela

doi:10.1186/s40409-015-0027-6

Cited by 31 publications

(18 citation statements)

References 69 publications

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“…In this paper we describe unique activities of a PI-class SVMP isolated from Bothrops atrox venom, Atroxlysin-Ia, that induces strong hemorrhage and dermonecrosis in the skin of mice. Other PI-class SVMPs have already been isolated from B. atrox venom as HI-5 [ 31 ], Batx-I [ 32 ] and Batroxase [ 23 , 33 ]. Batroxase showed 89% identity with Atroxlysin-Ia, while HI-5 and Batx-I have not a complete amino acid sequence and thus alignment was not possible.…”

Section: Discussionmentioning

confidence: 99%

Insights into the Mechanisms Involved in Strong Hemorrhage and Dermonecrosis Induced by Atroxlysin-Ia, a PI-Class Snake Venom Metalloproteinase

Freitas-de-Sousa

Colombini

Lopes-Ferreira

et al. 2017

Toxins

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Hemorrhage is the most prominent effect of snake venom metalloproteinases (SVMPs) in human envenomation. The capillary injury is a multifactorial effect caused by hydrolysis of the components of the basement membrane (BM). The PI and PIII classes of SVMPs are abundant in viperid venoms and hydrolyze BM components. However, hemorrhage is associated mostly with PIII-class SVMPs that contain non-catalytic domains responsible for the binding of SVMPs to BM proteins, facilitating enzyme accumulation in the tissue and enhancing its catalytic efficiency. Here we report on Atroxlysin-Ia, a PI-class SVMP that induces hemorrhagic lesions in levels comparable to those induced by Batroxrhagin (PIII-class), and a unique SVMP effect characterized by the rapid onset of dermonecrotic lesions. Atroxlysin-Ia was purified from B. atrox venom, and sequence analyses indicated that it is devoid of non-catalytic domains and unable to bind to BM proteins as collagen IV and laminin in vitro or in vivo. The presence of Atroxlysin-Ia was diffuse in mice skin, and localized mainly in the epidermis with no co-localization with BM components. Nevertheless, the skin lesions induced by Atroxlysin-Ia were comparable to those induced by Batroxrhagin, with induction of leukocyte infiltrates and hemorrhagic areas soon after toxin injection. Detachment of the epidermis was more intense in skin injected with Atroxlysin-Ia. Comparing the catalytic activity of both toxins, Batroxrhagin was more active in the hydrolysis of a peptide substrate while Atroxlysin-Ia hydrolyzed more efficiently fibrin, laminin, collagen IV and nidogen. Thus, the results suggest that Atroxlysin-Ia bypasses the binding step to BM proteins, essential for hemorrhagic lesions induced by PII-and P-III class SVMPs, causing a significantly fast onset of hemorrhage and dermonecrosis, due to its higher proteolytic capacity on BM components.

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Section: Discussionmentioning

confidence: 99%

Insights into the Mechanisms Involved in Strong Hemorrhage and Dermonecrosis Induced by Atroxlysin-Ia, a PI-Class Snake Venom Metalloproteinase

Freitas-de-Sousa

Colombini

Lopes-Ferreira

et al. 2017

Toxins

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“…BJ-PLA 2 -I showed high catalytic activity as evaluated by the phospholipase assays, which is consistent with the presence of the Asp49 residue and its classification as an acidic PLA 2 . Several other acidic PLA 2 s from Bothrops venoms have been described as exerting high catalytic activity, including Bl-PLA 2 ( B. leucurus ) [ 30 ], Bp-PLA 2 ( B. pauloensis ) [ 31 ], BpirPLA 2 -I ( B. pirajai ) [ 32 ], BmooPLA 2 ( B. moojeni ) [ 33 ], BE-I-PLA 2 ( B. erithromelas ) [ 34 ], MTX-I ( B. brazili ) [ 35 ] and BatroxPLA 2 ( B. atrox ) [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

“…The phospholipase activity of the chromatographic fractions and BJ-PLA 2 -I was evaluated on egg yolk-agar plates, following the methodology described by Gutiérrez et al [ 21 ], with modifications by Menaldo et al [ 22 ]. Assessed samples included a negative control of phosphate buffered saline (PBS), a positive control of B. jararaca venom (15 μg) and different quantities of BJ-PLA 2 -I (0.08–2.5 μg), all diluted in PBS.…”

Section: Methodsmentioning

confidence: 99%

Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom

Cedro

Menaldo

Costa

et al. 2018

J Venom Anim Toxins Incl Trop Dis

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BackgroundSnake venom phospholipases A2 (PLA2s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects. This work aimed at the isolation and functional characterization of a PLA2 isolated from Bothrops jararaca venom, named BJ-PLA2-I.Methods and ResultsFor its purification, three consecutive chromatographic steps were used (Sephacryl S-200, Source 15Q and Mono Q 5/50 GL). BJ-PLA2-I showed acidic characteristics, with pI~ 4.4 and molecular mass of 14.2 kDa. Sequencing resulted in 60 amino acid residues that showed high similarity to other Bothrops PLA2s, including 100% identity with BJ-PLA2, an Asp49 PLA2 previously isolated from B. jararaca venom. Being an Asp49 PLA2, BJ-PLA2-I showed high catalytic activity, and also inhibitory effects on the ADP-induced platelet aggregation. Its inflammatory characterization showed that BJ-PLA2-I was able to promote leukocyte migration in mice at different concentrations (5, 10 and 20 μg/mL) and also at different response periods (2, 4 and 24 h), mainly by stimulating neutrophil infiltration. Furthermore, increased levels of total proteins, IL-6, IL-1β and PGE2 were observed in the inflammatory exudate induced by BJ-PLA2-I, while nitric oxide, TNF-α, IL-10 and LTB4 levels were not significantly altered. This toxin was also evaluated for its cytotoxic potential on normal (PBMC) and tumor cell lines (HL-60 and HepG2). Overall, BJ-PLA2-I (2.5–160 μg/mL) promoted low cytotoxicity, with cell viabilities mostly varying between 70 and 80% and significant values obtained for HL-60 and PBMC only at the highest concentrations of the toxin evaluated.ConclusionsBJ-PLA2-I was characterized as an acidic Asp49 PLA2 that induces acute local inflammation and low cytotoxicity. These results should contribute to elucidate the action mechanisms of snake venom PLA2s.

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“…The envenomation caused by Lachesis genus represents 4.5 % of all registered snakebites in Brazil and is characterized by the so-called “ Lachesis Syndrome” [ 4 ]. Within the first few minutes after the bite, the victim is affected by agonizing burning throbbing local pain and edema, followed by intense inflammation, bleeding disorders, clotting disorders, kidney malfunction, myotoxicity and autonomic syndrome evidenced by sweating, nausea, vomiting, abdominal cramps, diarrhea, hypotension and bradycardia [ 5 – 9 ].…”

Section: Introductionmentioning

confidence: 99%

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

Cremonez

Leite

Bordon

et al. 2016

J Venom Anim Toxins Incl Trop Dis

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BackgroundIn 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.MethodsWe 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.ResultsLmrV 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.ConclusionThe 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.

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Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom

Cited by 31 publications

References 69 publications

Insights into the Mechanisms Involved in Strong Hemorrhage and Dermonecrosis Induced by Atroxlysin-Ia, a PI-Class Snake Venom Metalloproteinase

Insights into the Mechanisms Involved in Strong Hemorrhage and Dermonecrosis Induced by Atroxlysin-Ia, a PI-Class Snake Venom Metalloproteinase

Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom

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

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