Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

Bernardes, Carolina P.; Santos-Filho, Norival A.; Costa, Tássia R.; Gomes, Mário S.R.; Torres, Fernanda Silva; Costa, Júnia de Oliveira; Borges, Márcia Helena; Richardson, Michael; Santos, Daniel Moreira dos; Pimenta, Adriano Monteiro de Castro; Homsi-Brandeburgo, Maria Inês; Soares, Andreimar M.; Oliveira, Fábio de

doi:10.1016/j.toxicon.2007.11.017

Cited by 70 publications

(57 citation statements)

References 44 publications

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“…Pain and tissue inflammation are hallmarks of viperid snake bites; other common symptoms include blisters, necrosis, and hemorrhage (13, 31, 32). B. moojeni venom contains a complex mixture of metalloproteinases (33,34), serine proteases (35), L-amino acid oxidases (36), acidic PLA2s (37), and Lys49 myotoxins (38), all of which likely contribute to the envenomation symptoms. Here we show that a specific venom component, a Lys49 myotoxin, can directly elicit pain and inflammation as well as mechanical and heat hypersensitivity.…”

Section: Distinct Fiber Types Contribute To Bomotx-evoked Acute Andmentioning

confidence: 99%

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release

Zhang

Medzihradszky

Sánchez

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X 2 and/or P2X 3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.Lys49 myotoxin | ATP release | pannexin | pain | purinergic receptor V enoms from spiders, snakes, cone snails, and scorpions have evolved to produce a vast pharmacopoeia of toxins that modulate receptor or channel function as a means of producing shock, hemolysis, paralysis, and pain (1, 2). As such, venoms from these animals represent a rich potential source of potent and selective toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. For example, toxins can directly target ion channels expressed by nociceptive sensory neurons to elicit or inhibit pain. Well-validated examples include toxins that target transient receptor potential (TRP) ion channels, acid-sensing ion channels (ASICs), and Na v channels (3-8). However, the "toxinome" is incredibly diverse, and there are many painproducing venoms for which the responsible toxins have not yet been identified.In addition to targeting receptors and channels directly, algogenic toxins could also promote the synthesis and/or release of metabolites, transmitters, or second messengers that activate primary afferent nociceptors. Although this could occur through indiscriminate actions of venom lipases and proteases that release cellular contents through tissue damage and cell death, more specific mechanisms may also exist. Their identification could reveal important new pathways by which exogenous or endogenous pain-producing agents mediate their effects.To identify novel pain-producing toxins, we have exploited cultured sensory neurons as a facile in vitro platform for unbiased screening of crude venoms, optimizing conditions to reduce nonspecific cell damage so as to reveal toxins having specific cellular effects. With this approach, we have identified a toxin (BomoTx) from the Brazilian lancehead pit viper (Bothrops moojeni) f...

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Section: Distinct Fiber Types Contribute To Bomotx-evoked Acute Andmentioning

confidence: 99%

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release

Zhang

Medzihradszky

Sánchez

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Several studies show that proteolytic enzymes of bothropic venoms are also able to increase the plasma CK level (14)(15). Results obtained in our laboratories showed that the crude venom of B. moojeni provoked a greater proteolytic activity than other venoms, whereas the crude venom of B. alternatus produced the lowest level of such activity (data not shown).…”

Section: Discussionmentioning

confidence: 62%

“…B. moojeni venom contains a variety of enzymes including acidic phospholipase A 2 , basic phospholipase A 2 , metalloproteinases, serine proteinases, l-amino acid oxidase and myotoxin phospholipase A 2 which can contribute to the biological actions (edema, hemorrhage, necrosis, and anticoagulant and platelet-aggregating activities) of this venom (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

Section: Introductionmentioning

confidence: 99%

Biological characterization of a myotoxin phosphoplipase A2 homologue purified from the venom of the snake Bothrops moojeni

Queiroz¹,

Mamede²,

Fonseca³

et al. 2011

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

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Abstract:A myotoxin phospholipase A 2 homologue, BmooMtx, was isolated from the venom of Bothrops moojeni by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. SDS-PAGE showed the enzyme to be a monomer with a molecular weight of 16,500. BmooMtx induced release of creatine kinase and morphological analyses indicated that it provoked an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 hours after injection. Anti-BmooMTx antibodies partially neutralized the myotoxic activity of BmooMtx and crude B. moojeni venom, as judged by determination of plasma creatine kinase levels and histological evaluation of skeletal muscle in mice. Anti-BmooMTx antibodies were effective in reducing the plasma creatine kinase levels of crude B. alternatus and B. leucurus venoms, evidencing immunological cross-reactivity between BmooMTx and other bothropic venoms. Intraplantar (i.pl.) injection of BmooMtx (1 to 15 μg/animal) caused a doseand time-dependent hyperalgesia and edematogenic responses. Dexamethasone (0.4 mg/kg), meloxicam (2 mg/kg) and promethazine (5 mg/kg) markedly reduced the hyperalgesia. Our data suggest that these drugs may likely serve as complementary therapies in cases of accidents with Bothrops moojeni, provided that such pharmacological treatments are administered immediately after the incident.

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“…Este espectro foi analisado utilizando a técnica de bioinformática em MALDI TOF, com o programa Mascot Search Results (Matrix Science), o qual revelou score significativo (score de 80 e p<0.05) com a metaloprotease não hemorrágica BmooMPα-I, proveniente também da peçonha da serpente do gênero Bothrops moojeni (BERNADES et al, 2008 (WATANABE et al, 2003). Desta maneira, pode-se afirmar, de acordo com a classificação das metaloproteases apresentadas por FOX; SERRANO, (2010), que BthMP é uma metaloprotease da classe P-I.…”

Section: Resultsunclassified

Purificação e avaliação da interferência de BthMP (uma metaloprotease da peçonha de Bothrops moojeni) na coagulação sanguínea

et al. 2016

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In the present was work reported the purification of the metalloprotease BthMP, from the venom of Bothrops moojeni. For purification of the protease was used ion exchange chromatography (DEAE-Sepharose) and molecular exclusion , the product of these processes was a protein band with high purity, visualized on SDS-PAGE 14%, referred the BthMP. This protease when analyzed on MALDI-TOF revealed the molecular weight of the native form of 23.050 Da and 23.872 Da in the reduced form, and from the peptide fragments obtained by Peptide Mass Fingerprinting (PMF) in MS (MALDI-TOF/TOF) was observed high similarity with BmooMPα-I metalloprotease. In enzymatic terms, BthMP showed proteolytic activity on azocasein using PMSF and benzamidine, while this activity was inhibited in the presence of EDTA; 1,10-Phenanthroline and β-mercaptoethanol, it is therefore a zinc-dependent metalloprotease of the class P-I. To still with this purpose, we contemplate its enzymatic specificity of the Aα and Bβ chains of fibrinogen and also the consumption of fibrinogen in vivo. It was also found its action on the components of the coagulation cascade, due to prolongation of prothrombin time and partial thromboplastin time. Thus, the sharp fibrinogenolytic activity and the high consumption of fibrinogen in vivo are results that indicate the anticoagulant action of BthMP; furthermore, their ability to interfere with the coagulation cascade suggested that this protease is promising for future studies that might indicate a new antithrombotic agent model. K E Y W O R D SCoagulation cascade, Metalloproteinase, Venom snake R E S U M ONeste trabalho relatamos a purificação da metaloprotease BthMP, proveniente da peçonha da serpente Bothrops moojeni. Para a purificação desta protease, utilizaram-se os passos cromatográficos de troca iônica (DEAE-Sepharose) e de exclusão molecular (Sephadex G-75), sendo o produto desses processos uma banda proteica com elevado grau de pureza, visualizada em SDS-PAGE a 14%, denominada BthMP. Esta, por sua vez, quando analisada em MALDI-TOF revelou a massa molecular nativa de 23.050 Da e 23.872 Da na forma reduzida, e a partir dos fragmentos peptídicos obtidos por Peptide Mass Fingerprinting (PMF) em MS (MALDI-TOF/TOF) indicou alta similaridade com a metaloprotease BmooMPα-I. Em termos enzimáticos, BthMP mostrou atividade proteolítica sobre azocaseína e frente ao PMSF e benzamidina, enquanto que esta atividade foi inibida na presença de EDTA, 1,10-fenantrolina e β-mercaptoetanol, sendo portanto uma metaloprotease zinco dependente da classe P-I. Ainda com este propósito, verificou-se sua especificidade enzimática sobre as cadeias Aα e Bβ do fibrinogênio, e também o consumo de fibrinogênio in vivo. Foi constatado ainda sua ação em componentes da cascata de coagulação, devido ao prolongamento do Tempo de Protrombina (TP) e do Tempo de Tromboplastina Parcial ativada (TTPa). Desta forma, a acentuada atividade fibrinogenolítica e o alto consumo de fibrinogênio in vivo são resultados que indicam a ação anticoagulante da BthMP; al...

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Isolation and structural characterization of a new fibrin(ogen)olytic metalloproteinase from Bothrops moojeni snake venom

Cited by 70 publications

References 44 publications

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release

Biological characterization of a myotoxin phosphoplipase A2 homologue purified from the venom of the snake Bothrops moojeni

Purificação e avaliação da interferência de BthMP (uma metaloprotease da peçonha de Bothrops moojeni) na coagulação sanguínea

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