A New Phospholipase A&lt;sub&gt;2&lt;/sub&gt; from Lachesis &lt;i&gt;muta rhombeata&lt;/i&gt;: Purification, Biochemical and Comparative Characterization with Crotoxin B

Cordeiro, Francielle Almeida; Perini, Tibério Gracco Kazuhiro; Bregge-Silva, Cristiane; Cremonez, Caroline Marroni; Rodrigues, Renata Santos; Boldrini-França, Johara; Bordon, Karla de Castro Figueiredo; Souza, Dayane Lorena Naves de; Achê, David Collares; Rodrigues, Veridiana de Melo; Santos, Wagner Ferreira dos; Rosa, José César; Arantes, Eliane Candiani

doi:10.2174/0929866522666150706112431

Cited by 11 publications

(8 citation statements)

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“…is characterized by intense local pain, oedema and necrosis (Damico et al, 2006;Ferreira et al, 2009;Damico et al, 2012), systemic myotoxicity (Fuly et al, 2000;Fuly et al, 2003;Damico et al, 2006), renal failure (Damico et al, 2007), haemorrhage and coagulopathy (Sánchez et al, 1987;Sánchez et al, 1991;Sánchez et al, 1995;Fuly et al, 1997;Rucavado et al, 1999;Estêvão-Costa et al, 2000;Torres-Huaco et al, 2013), including severe cardiovascular disorders (Diniz and Oliveira, 1992;Giovanni-De-Simone et al, 1997;Dias et al, 2016a;Dias et al, 2016b). Such effects have been associated predominantly with the presence of phospholipases A 2 (PLA 2 ) (Cordeiro et al, 2015;Diniz-Sousa et al, 2018), metalloproteases (Cordeiro et al, 2018) and serine proteases (Wiezel et al, 2019), including biologically active peptides (Graham et al, 2005;Soares et al, 2005;Sanz et al, 2008;Pla et al, 2013;Pinheiro-Júnior et al, 2018), in these venoms.…”

Section: Introductionmentioning

confidence: 99%

Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom

et al. 2022

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Varespladib (VPL) was primarily developed to treat inflammatory disturbances associated with high levels of serum phospholipase A2 (PLA2). VPL has also demonstrated to be a potential antivenom support agent to prevent PLA2-dependent effects produced by snake venoms. In this study, we examined the action of VPL on the coagulant, haemorrhagic and enzymatic activities of Lachesis muta rhombeata (South-American bushmaster) venom. Conventional colorimetric enzymatic assays were performed for PLA2, caseinolytic and esterasic activities; in vitro coagulant activities for prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in rat citrated plasma through a quick timer coagulometer, whereas the dimensions of haemorrhagic haloes obtained after i.d. injections of venom in Wistar rats were determined using ImageJ software. Venom (1 mg/ml) exhibited accentuated enzymatic activities for proteases and PLA2in vitro, with VPL abolishing the PLA2 activity from 0.01 mM; VPL did not affect caseinolytic and esterasic activities at any tested concentrations (0.001–1 mM). In rat citrated plasma in vitro, VPL (1 mM) alone efficiently prevented the venom (1 mg/ml)-induced procoagulant disorder associated to extrinsic (PT) pathway, whereas its association with a commercial antivenom successfully prevented changes in both intrinsic (aPTT) and extrinsic (PT) pathways; commercial antivenom by itself failed to avoid the procoagulant disorders by this venom. Venom (0.5 mg/kg)-induced hemorrhagic activity was slightly reduced by VPL (1 mM) alone or combined with antivenom (antivenom:venom ratio 1:3 ‘v/w’) in rats, with antivenom alone producing no protective action on this parameter. In conclusion, VPL does not inhibit other major enzymatic groups of L. m. rhombeata venom, with its high PLA2 antagonize activity efficaciously preventing the venom-induced coagulation disturbances.

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Section: Introductionmentioning

confidence: 99%

Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom

et al. 2022

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“…Few components have been purified from L. muta rhombeata venom until now, including a L-amino acid oxidase, PLA 2 , PLB, hyaluronidase, serine protease and BPPs [ 9 , 10 , 12 , 13 ]. Although there are studies about these components, little is known about some mechanisms induced by these proteins and peptides, especially in Lachesis envenomation, since its venom is difficult to obtain and is hard to keep bushmasters in captivity [ 5 , 31 ].…”

Section: Discussionmentioning

confidence: 99%

“…Among the components identified in Lachesis muta rhombeata venom (LmrV), are a hyaluronidase [ 9 ], phospholipases A 2 (PLA 2 ) [ 10 , 11 ], phospholipase B (PLB) [ 9 ], L-aminoacid oxidase (LAAO) [ 12 ], serine protease [ 9 ] and bradykinin-potentiating peptides (BPPs) [ 13 ]. Although two snake venom metalloproteases (SVMPs) have been identified in L. muta muta venom (LmmV), this is the first study with metalloprotease from LmrV.…”

Section: Introductionmentioning

confidence: 99%

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

Cordeiro

Coutinho

Wiezel

et al. 2018

J Venom Anim Toxins Incl Trop Dis

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BackgroundLachesis 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 resultsLmr-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.ConclusionThe 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.

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“…The serine proteinases from these venoms are usually involved in hydrolysis of coagulation factors and may act in blood pressure reduction [14-16] whereas metalloproteinases present hemorrhagic actions [17, 18]. On the other hand, PLA 2 s are myotoxic enzymes that cause extensive local damage in the bite site [19, 20] and LAAO present high cytotoxic action [21].…”

Section: Introductionmentioning

confidence: 99%

“…However, there is a certain difficulty in maintain Lachesis snakes in captivity due to the peculiar characteristics of their natural habitat [22] and this hinders the study of their venoms. Few components have been isolated from LmrV, including LAAO [21], PLA 2 [19], serine proteinases [23-25] and hyaluronidase [13]. Hyaluronidase was the last isolated component to be reported about 3 years ago.…”

Section: Introductionmentioning

confidence: 99%

Subproteome of Lachesis muta rhombeata venom and preliminary studies on LmrSP-4, a novel snake venom serine proteinase

Wiezel

Bordon

Silva

et al. 2019

J. Venom. Anim. Toxins incl. Trop. Dis

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Background: Lachesis muta rhombeata is one of the venomous snakes of medical importance in Brazil whose envenoming is characterized by local and systemic effects which may produce even shock and death. Its venom is mainly comprised of serine and metalloproteinases, phospholipases A 2 and bradykinin-potentiating peptides. Based on a previously reported fractionation of L. m. rhombeata venom (LmrV), we decided to perform a subproteome analysis of its major fraction and investigated a novel component present in this venom. Methods: LmrV was fractionated through molecular exclusion chromatography and the main fraction (S5) was submitted to fibrinogenolytic activity assay and fractionated by reversed-phase chromatography. The N-terminal sequences of the subfractions eluted from reversed-phase chromatography were determined by automated Edman degradation. Enzyme activity of LmrSP-4 was evaluated upon chromogenic substrates for thrombin (S-2238), plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) and upon fibrinogen. All assays were carried out in the presence or absence of possible inhibitors. The fluorescence resonance energy transfer substrate Abz-KLRSSKQ-EDDnp was used to determine the optimal conditions for LmrSP-4 activity. Molecular mass of LmrSP-4 was determined by MALDI-TOF and digested peptides after trypsin and Glu-C treatments were analyzed by high resolution MS/MS using different fragmentation modes. Results: Fraction S5 showed strong proteolytic activity upon fibrinogen. Its fractionation by reversed-phase chromatography gave rise to 6 main fractions (S5C1-S5C6). S5C1-S5C5 fractions correspond to serine proteinases whereas S5C6 represents a C-type lectin. S5C4 (named LmrSP-4) had its N-terminal determined by Edman degradation up to the 53 rd amino acid residue and was chosen for characterization studies. LmrSP-4 is a fibrinogenolytic serine proteinase with high activity against S-2302, being inhibited by PMSF and benzamidine, but not by 1,10-phenantroline. In addition, this enzyme exhibited maximum activity within the pH range from neutral to basic and between 40 and 50 °C. About 68% of the LmrSP-4 primary structure was covered, and its molecular mass is 28,190 Da. Conclusions: Novel serine proteinase isoforms and a lectin were identified in LmrV. Additionally, a kallikrein-like serine proteinase that might be useful as molecular tool for investigating bradykinin-involving process was isolated and partially characterized.

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A New Phospholipase A<sub>2</sub> from Lachesis <i>muta rhombeata</i>: Purification, Biochemical and Comparative Characterization with Crotoxin B

Cited by 11 publications

References 0 publications

Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom

Action of Varespladib (LY-315920), a Phospholipase A2 Inhibitor, on the Enzymatic, Coagulant and Haemorrhagic Activities of Lachesis muta rhombeata (South-American Bushmaster) Venom

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

Subproteome of Lachesis muta rhombeata venom and preliminary studies on LmrSP-4, a novel snake venom serine proteinase

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