Paloma Sirigatti Knittel
Caracterizando o perfil enzimático de extratos de tentáculos da água-viva do Atlântico Sul Olindias sambaquiensis (Hydrozoa)
Dissertação apresentada ao Programa de Pós-Graduação em Ciências-Toxinologia do Instituto Butantan, para obtenção do título de Mestre em Ciências.
São Paulo
2016
Paloma Sirigatti Knittel
Caracterizando o perfil enzimático de extratos de tentáculos da água-viva do Atlântico Sul Olindias sambaquiensis (Hydrozoa)
Dissertação apresentada ao Programa de Pós-Graduação em Ciências-Toxinologia do Instituto Butantan, para obtenção do título de Mestre em Ciências.
Orientadora: Ana Maria Moura da Silva
São Paulo
2016
FICHA CATALOGRÁFICA
Knittel, Paloma Sirigatti
Caracterizando o perfil enzimático de extratos de tentáculos da água-viva do Atlântico Sul Olindias sambaquiensis (Hydrozoa) / Paloma Sirigatti Knittel; Orientador Ana Maria Moura da Silva. - - São Paulo, 2016.
Dissertação (Mestrado) – Instituto Butantan. Programa de Pós-Graduação em Ciências-Toxinologia. Linha de Pesquisa: Bioprospecção e desenvolvimento.
Título em inglês: Characterising the enzymatic profile of tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Hydrozoa)
1. Toxinas animais. 2. Venenos. 3. Enzimas proteolíticas. 4. Phospholipase A2. 5. Cnidaria. I. Orientador: Silva, Ana Maria Moura da. II. Programa de Pós-Graduação em Ciências-Toxinologia. Instituto Butantan. III.Título.
CDD 615.9
Elaborada com instruções fornecidas pela Biblioteca do Instituto Butantan
AUTORIZAÇÃO PARA REPRODUÇÃO DE TRABALHO
Eu, Paloma Sirigatti Knittel, aluna de mestrado do Programa de Pós- graduação em Ciências-Toxinologia do Instituto Butantan, através deste autorizo a divulgação de minha dissertação por mídia impressa eletrônica ou qualquer outra, somente após defesa e publicação de trabalho em revista científica, assim como reprodução parcial ou total desta dissertação após publicação, para fins acadêmicos desde que citada a fonte.
São Paulo, de de
...... Aluno(a)
De acordo: ...... Orientador(a):
PARECER DE COMISSÕES INSTITUCIONAIS REGULATÓRIAS
DECLARAÇÃO DE ISENÇÃO
AGRADECIMENTOS
Agradeço à minha orientadora, Ana Maria Moura da Silva, pelas oportunidades e ensinamentos ao longo da elaboração desse trabalho.
Ao Dr. Paul Long e ao Dr. Antonio Carlos Marques, pelas contribuições valiosas durante o projeto.
Ao Dr. Gabriel Padilla pelo auxílio no isolamento dos nematocistos.
Aos colaboradores do Centro de Biologia Marinha da USP pelo suporte na realização das coletas.
À Dra. Adriana Rios, Dr. André Zaharenko e Dr. Daniel Pimenta pelas sugestões no exame de qualificação.
Aos colegas do laboratório de Imunopatologia do Instituto Butantan, principalmente aos meus companheiros de laboratório, que tornaram essa experiência mais leve e divertida. Em especial ao Dr. José Antonio Portes Junior, pelo incentivo, paciência e amizade e à Ma. Michelle Teixeira de Almeida pelo companheirismo e amizade no laboratório e na vida.
Ao Programa de Pós Graduação em Ciências-Toxinologia do Instituto Butantan e à Kimie e Rosana pela paciência e pelo suporte.
Agradeço imensamente à minha família, em especial à minha mãe Noêmia, pelo amor, dedicação, incentivo e por ser minha grande inspiração, a principal responsável por todas minhas realizações.
Ao meu namorado, Igor, por me apoiar, incentivar e por estar presente mesmo nos momentos mais difíceis.
Ao apoio financeiro da FAPESP através dos projetos 2013/25593-4 e 2014/26058-8 e CNPq através do projeto número 304025/2014-3.
Finalmente, agradeço a todos aqueles que de alguma maneira contribuíram para a realização desse trabalho.
Muito Obrigada!!!
RESUMO
Knittel, Paloma Sirigatti. Caracterizando o perfil enzimático de extratos de tentáculos da água-viva do Atlântico Sul Olindias sambaquiensis (Hydrozoa). 2016. 38 f. Dissertação (Ciências-Toxinologia). Instituto Butantan, São Paulo, 2016.
As águas-vivas ou medusas estão entre os animais peçonhentos marinhos mais populares. Acidentes envolvendo medusas são comuns e sua toxicidade para humanos pode variar de sintomas leves até envenenamentos fatais em alguns casos. Os venenos de medusas são de importância biotecnológica, apresentando toxinas com atividade antimicrobiana, analgésica e antitumoral. Além disso, enzimas proteolíticas também já foram descritas. Recentemente o perfil proteômico de toxinas putativas dos nematocistos isolados da hidromedusa Olindias sambaquiensis foi caracterizado, identificando a presença de proteases e fosfolipases. Nesse trabalho nós apresentamos os resultados de um estudo que confirma a presença de atividade enzimática no extrato de tentáculos de O. sambaquiensis. Os espécimes foram coletados na costa de São Sebastião, SP, Brasil. O procedimento para obtenção do extrato de tentáculos foi padronizado, rendendo grande quantidade de proteínas de diferentes massas moleculares. O extrato foi avaliado quanto a presença de atividade de metaloproteases, serino proteases e fosfolipases A2, utilizando substratos genéricos para essas enzimas e também substratos específicos para as metaloproteases do tipo ADAMs e MMPs. O extrato apresentou atividade sobre todos os substratos, confirmando evidências proteômicas anteriores. Além disso, a atividade proteolítica também foi detectada por ensaios de zimografia em caseína e gelatina. Quando comparamos essas atividades com venenos de serpentes, que são abundantes em proteases e fosfolipases, a atividade de metaloproteases obtida foi bem menor que os altos índices demonstrados pelo veneno de Bothrops jararaca, entretanto, as atividades observadas para serino proteases e fosfolipases A2 foram comparáveis à atividade observada em venenos de serpentes Bothrops, conhecidas pela sua forte atividade anticoagulante e miotóxica devido a presença de serino proteases e fosfolipases A2, respectivamente.
Esses resultados validam as propriedades funcionais das proteínas caracterizadas previamente pelo proteoma e fornecem um melhor entendimento do arsenal tóxico desse animal peçonhento pouco explorado, indicando o papel das serino proteases e fosfolipases A2 na ação do veneno.
Palavras-chave: Toxinas animais, Venenos, Enzimas proteolíticas, Fosfolipase A2, Cnidaria.
ABSTRACT
Knittel, Paloma Sirigatti. Characterising the enzymatic profile of tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Hydrozoa). 2016. 38 p. Master thesis (Science-Toxinology). Instituto Butantan, São Paulo, 2016.
Jellyfishes are amongst the most familiar of venomous marine animals. Jellyfish encounters with humans are common, although subsequent envenomation has varying toxicity ranging from usually mild symptoms to sometimes lethal consequences. Jellyfish venoms are of biotechnological importance, with toxins displaying antimicrobial, analgesic and anti-tumor activities. Furthermore, proteolytic enzymes have also been described. Recently, the proteomic profile of putative toxins isolated from nematocysts of the hydromedusae Olindias sambaquiensis was reported, which included protease and phospholipase enzymes. Here we present the results of a study to experimentally confirm enzymatic activity in extract of O. sambaquiensis tentacles. Specimens of O. sambaquiensis were collected at the coast of São Sebastião, SP, Brazil. A procedure to generate tentacle extract was standardised, yielding high quantities of proteins of different molecular masses. The extract was assayed for the presence of metalloproteases, serine proteases and phospholipase A2 activities using substrates that cover a wide range of each class of these enzymes and also specific substrates for metalloproteases ADAMs and MMPs. The extract showed activity towards all substrates confirming previous proteomic evidence for the presence of these enzymes. In addition, proteolytic activity was also detected by zymography assays on casein and gelatin. However, when comparing these activities to snake venoms, which are rich in proteases and phospholipases, the metalloprotease activity detected was lower than the high levels observed in
Bothrops jararaca venom, but levels of serine protease and phospholipase A2 enzyme activity was comparable to the one observed in venoms of Bothrops snakes, known for its strong anti-coagulant and myotoxic activities due to serine proteases and phospholipases A2 molecules, respectively. These data validate the functional properties of proteins characterised previously by proteomics and provide a better
understanding of the toxin arsenal of this underexplored venomous animal, indicating the role of active serine proteases and phospholipases A2 in the action of the venom.
Keywords: Animal toxins, Venoms, Proteolytic Enzymes, Phospholipase A2, Cnidaria.
SUMÁRIO
1. INTRODUÇÃO ...... 13
2. OBJETIVOS ...... 18
3. ARTIGO SUBMETIDO À PUBLICAÇÃO NA REVISTA TOXICON: Characterising the enzymatic profile of tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Hydrozoa) ...... 19
ABSTRACT ...... 19
1. Introduction ...... 20
2. Materials and methods ...... 20 2.1. Obtaining the extract of Olindias sambaquiensis tentacles ...... 20 2.2. Proteolytic activity ...... 21 2.2.1. Metalloproteases ...... 21 2.2.2. Serine proteases ...... 22 2.2.3. Zymography ...... 22 2.3. Phospholipase activity ...... 22
3. Results ...... 22 3.1. Obtaining the extract of Olindias sambaquiensis tentacles ...... 22 3.2. Proteolytic activity using peptide substrates ...... 23 3.2.1 Proteolytic activity using macromolecular substrates ...... 23 3.3. Phospholipase activity ...... 24
4. Discussion ...... 25
References ...... 27
4. CONCLUSÃO ...... 30
REFERÊNCIAS ...... 31
1. INTRODUÇÃO
Para a humanidade provavelmente não há animal mais assustador e fascinante do que aqueles que são venenosos. Os venenos (ou peçonhas) de animais são misturas complexas de proteínas e peptídeos que conjuntamente com sais e outros componentes orgânicos são liberados para as vítimas como meio de defesa e/ou são utilizados para imobilizar suas presas, facilitando assim a caça, e em alguns casos, na digestão dos tecidos dos animais predados (HODGSON, 2012). A diversidade de animais que são venenosos é impressionante, compreendendo invertebrados marinhos onde se incluem os cnidários, como medusas e anêmonas-do-mar, gastrópodes, cefalópodes e equinodermos, peixes marinhos e de água doce, além dos animais terrestres onde se destacam os anfíbios, répteis, especialmente as serpentes, uma grande diversidade de artrópodes, mais notadamente escorpiões, aranhas e insetos, e ainda espécies de mamíferos, incluindo musaranho e o ornitorrinco (WONG; BELOV, 2012). O arsenal tóxico que compõe os venenos de animais é igualmente impressionante. Por exemplo, estima-se a existência de cerca de 700 gastrópodes marinhos, principalmente do gênero Conus, onde cada espécie seria responsável pela síntese de aproximadamente 1.000 peptídeos ativos, gerando 700.000 potenciais conotoxinas (OLIVERA; TEICHERT, 2007). Essas toxinas vêm sendo muito estudadas sob o ponto de vista farmacológico e biotecnológico, diferentes compostos têm sido descobertos, o que tem derivado o desenvolvimento de novas drogas. Por exemplo, o derivado sintético da ω-conotoxina, SNX-111 (comercializado como Ziconotida ou Prialt®), foi aprovado em 2004 para o uso clínico nos Estados Unidos da América pelo Food and Drug Administration como o primeiro analgésico não opiáceo (VER DONCK et al., 2013). As águas-vivas, ou medusas, são os animais marinhos peçonhentos mais populares e estão presentes em um grande número de praias. Pertencem ao filo dos Cnidários, um filo extremamente diverso que inclui, em sua maioria, organismos marinhos como anêmonas, águas-vivas, corais, entre outros. O filo dos Cnidários é dividido em seis classes, Anthozoa, Cubozoa, Scyphozoa, Hydrozoa, Myxozoa e Staurozoa (VAN ITEN et al., 2014; OKAMURA et al., 2015). Enquanto os membros
13 da classe Anthozoa não apresentam em seu ciclo de vida o estágio medusoide, sendo animais de vida séssil e forma polipoide, os membros das classes Cubozoa, Scyphozoa, Hydrozoa e Staurozoa podem apresentar ambos os estágios, medusoide e polipoide, em seu ciclo de vida (MARQUES; COLLINS, 2004). A mais nova classe agregada ao filo, Myxozoa, compreende cerca de 2.000 espécies de parasitas microscópicos obrigatórios, foram inclusos no filo dos Cnidários em 2015, após análises genômicas e transcriptômicas (CHANG et al., 2015). A estrutura mais característica do filo Cnidaria é o cnidocisto, uma das organelas mais complexas existentes, presente em células denominadas cnidócitos. Já se tem conhecimento de cerca de 30 tipos de cnidocistos (FAUTIN, 2009), estes são agrupados em três grupos. O primeiro compreende os nematocistos, estruturas formadas por uma cápsula de formato fusiforme e paredes duplas que se invagina formando um longo túbulo altamente espiralado. Esse túbulo eversível, quando o nematocisto entra em contato com a presa ou recebe algum estímulo, se evagina realizando o transporte das toxinas. Além disso, os nematocistos podem conter espinhos que facilitam a penetração e fixação na presa (SALLEO et al., 1996). Os nematocistos estão presentes predominantemente na região oral e tentáculos, e são responsáveis pela captura de presas, defesa e adesão. Os outros grupos, espirocistos e pticocistos, são responsáveis principalmente pela adesão e estão presentes apenas nos membros da classe Anthozoa (KASS-SIMON; SCAPPATICCI JUNIOR, 2002). A eficácia dessas organelas para a predação está relacionada ao seu conteúdo de toxinas conjuntamente com seu aparato mecânico. São descritas cerca de 13.500 espécies de cnidários no mundo enquanto no Brasil, até o momento, se tem conhecimento de aproximadamente 550 espécies (DALY et al., 2007; SILVEIRA; MORANDINI, 2011; MORANDINI et al., 2014). Considerando o Brasil com suas dimensões continentais, sua ampla costa e a grande biodiversidade que apresenta, é possível que esse número esteja subestimado, o que gera a necessidade de maiores estudos acerca desses animais no Brasil. A importância no estudo desses animais está, não somente pelo potencial biotecnológico de seus venenos, mas também em grande parte devido ao grande número de acidentes envolvendo seres humanos. A toxicidade dos venenos de cnidários para os seres humanos pode variar desde sintomas leves até envenenamentos fatais em alguns casos (LUMLEY et al., 1988; HADDAD JUNIOR
14 et al., 2002). Sendo que os quadros de envenenamentos graves ou fatais são mais frequentemente associados a membros das classes Cubozoa e Scyphozoa. Nas praias brasileiras, são comuns os acidentes ocasionados por medusas. As principais espécies relacionadas a esses acidentes são Chiropsalmus quadrumanus e Tamoya haplonema, pertencentes à classe Cubozoa, e Physalia physalis, também conhecida como “caravela-portuguesa”, e Olindias sambaquiensis, que fazem parte da classe Hydrozoa (HADDAD JUNIOR, 2003). A espécie O. sambaquiensis, chamada popularmente de “reloginho” faz parte da classe dos hidrozoários, família Olindiidae. Apesar de seu tamanho pequeno, não ultrapassando os 10 centímetros de diâmetro, comparada aos outros membros da classe são consideradas grandes hidromedusas. Possuem três séries diferentes de tentáculos marginais, presentes em grande número. Os primários, inseridos acima da margem da umbrela, possuem coloração avermelhada bastante característica. À segunda série, pertencem os tentáculos secundários, longos filamentos, extremamente finos que emergem da margem da umbrela e a terceira série de tentáculos é composta por pequenos tentáculos de formato claviforme (VANNUCCI, 1951). Espécimes de O. sambaquiensis são facilmente reconhecidos pela sua coloração característica, além de um manúbrio de coloração amarela intensa, possuem quatro canais radiais, cuja tonalidade varia do rosa ao vermelho vivo. Essa espécie é endêmica da região ocidental do Atlântico Sul, sendo bastante comum desde o litoral norte do Estado de São Paulo (Brasil) até a Argentina. Ocorre durante o ano todo, sendo predominante no inverno (VANNUCCI, 1951; OLIVEIRA et al., 2016). Os acidentes ocasionados por O. sambaquiensis desenvolvem um quadro de dor intensa e possuem um padrão de lesão cutânea característico com erupção pápulo-eritematosa podendo evoluir para bolhas ou necrose (HADDAD JUNIOR et al., 2010; MOSOVICH; YOUNG, 2012). Ao longo das décadas, diversos estudos voltaram sua atenção para o veneno dos membros das classes Anthozoa, Cubozoa e Scyphozoa, descrevendo diferentes mecanismos de ação, como a atividade hemolítica (HESSINGER; LENHOFF, 1973; LONG; BURNETT, 1989; GUSMANI et al., 1997; HELMHOLZ et al., 2007; LI et al., 2013), formação de poros em membranas (MACEK, 1992; WANG et al., 2013), ação em canais iônicos (LASSEN et al., 2012), atividade neurotóxica (CARNEIRO et al.,
15
2011), atividade fosfolipásica (GUSMANI et al, 1997), entre outros. Além disso, enzimas proteolíticas também já foram descritas (CALTON; BURNETT, 1982, 1983; LEE et al., 2011). Os extratos da espécie australiana Chironex fleckeri, da classe Cubozoa, têm recebido bastante atenção devido à alta gravidade dos acidentes que induzem. Os acidentes com C. fleckeri induzem distúrbios cardiovasculares, aumento de potássio circulante, na maioria dos casos podendo levar a morte (BAXTER; MARR, 1969; ENDEAN; HENDERSON, 1969; ENDEAN, 1987; LUMLEY et al., 1988; ENDEAN et al., 1993; WINTER et al., 2007; YANAGIHARA; SHOHET, 2012; JOUIAEI et al., 2015; PONCE et al., 2015). Embora grandemente estudada em aspectos como filogenia, morfologia, reprodução e distribuição, a classe Hydrozoa ainda carece de estudos acerca do seu conteúdo tóxico. Apesar de possuir cerca de 3.000 espécies, comparando-se com a quantidade de estudos existentes para as outras classes, o veneno dos hidrozoários tem recebido pouca atenção, provavelmente devido ao tamanho reduzido dos seus representantes e à dificuldade de obtenção de grandes quantidades de veneno (STILLWAY; LANE, 1971; TAMKUN; HESSINGER, 1981; SHER; ZLOTKIN, 2009; BOUCHARD; ANDERSON, 2014; HADDAD JUNIOR et al., 2014; GARCÍA- ARREDONDO et al., 2015). Conjuntamente com estudos de caracterização dos compostos nocivos para os seres humanos, alguns compostos com interesse biotecnológico também vêm sendo encontrados no veneno de Cnidários. U m composto com ação analgésica foi encontrado no veneno da anêmona do mar Bunodosoma cangicum (ZAHARENKO et al., 2011). Um composto antimicrobiano foi descrito a partir da água-viva da espécie Aurelia aurita (SHENKAREV et al., 2012), enquanto frações com atividade antitumoral em potencial (antiproliferativa e inibidores de adesão célula-matriz) e analgésicos foram descritos em extratos da água-viva Pelagia noctiluca (AYED et al., 2012a,b). Além dos estudos de atividade biológica, estudos de caracterização molecular vêm recebendo especial atenção nos últimos anos. Análises proteômicas de componentes do veneno de algumas espécies já foram realizadas, dentre elas destaca-se, na classe Anthozoa, a análise da fração neurotóxica do veneno da anêmona Bunodosoma cangicum (ZAHARENKO et al., 2008) e a análise da
16 secreção da anêmona Stichodactyla duerdeni (CASSOLI et al., 2013). O perfil proteômico do veneno das água-vivas das espécies C. fleckeri, classe Cubozoa, e Stomolophus meleagris, classe Scyphozoa, foi caracterizado em 2012 e 2014, respectivamente (BRINKMAN et al., 2012; LI et al., 2014). Na classe Hydrozoa, Balasubramanian e col. (2012) realizaram a caracterização dos componentes dos nematocistos de Hydra magnipapillat, Além disso, recentemente o perfil proteômico de toxinas putativas dos nematocistos isolados da espécie O. sambaquiensis foi caracterizado. Nesse proteoma, destaca-se a presença de enzimas comummente presentes em venenos de animais terrestres, como as fosfolipases A2 e as enzimas proteolíticas, como as metaloproteases e serinoproteases (WESTON et al., 2013). Essas enzimas são de importância fundamental na toxicidade de venenos de animais como aranhas e serpentes. As fosfolipases A2 podem representar os componentes principais de alguns dos venenos mais tóxicos de serpentes, agindo nas vítimas com alta atividade neurotóxica (GUTIÉRREZ; LOMONTE, 2013). As proteases também são abundantes em venenos, principalmente no veneno de serpentes da família Viperidae, onde ocupam um papel relevante nos distúrbios de coagulação, citotoxicidade e lesões locais induzidas por esses venenos (MARKLAND, 1998; MOURA-DA-SILVA et al., 2007). Dessa forma, neste trabalho tivemos como objetivo a obtenção de extratos de tentáculos de O. sambaquiensis em condições de atividade biológica conservada para verificarmos a importância das atividades funcionais das metaloproteases, serinoproteases e fosfolipases A2 nos venenos e envenenamentos provocados por esses animais. Os resultados aqui apresentados estão na forma de manuscrito submetido à publicação na revista Toxicon.
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2. OBJETIVOS
Caracterizar o perfil enzimático de extratos de tentáculos da hidromedusa da espécie Olindias sambaquiensis.
Os objetivos específicos desse trabalho são:
Obter os extratos de tentáculos de Olindias sambaquiensis em forma solúvel, com atividade preservada e em quantidade suficiente para realização dos ensaios enzimáticos;
Avaliar a atividade de metaloproteases, serinoproteases e fosfolipases A2 nos extratos de tentáculos de Olindias sambaquiensis;
Correlacionar a possível relevância das atividades enzimáticas para os envenenamentos ocasionados por essa espécie.
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3. ARTIGO SUBMETIDO À PUBLICAÇÃO NA REVISTA TOXICON:
Characterising the enzymatic profile of tentacle extracts from the South Atlantic jellyfish Olindias sambaquiensis (Hydrozoa)
Paloma S. Knittela, Paul F. Longb,c, Antonio C. Marquesd, Michelle T. Almeidaa, Gabriel Padillae, Ana M. Moura-da-Silvaa* a Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil b Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, Stamford Street, London SE1 9NH, United Kingdom c Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brazil d Instituto de Biociências and Centro de Biologia Marinha, Universidade de São Paulo, São Paulo, SP, Brazil e Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil
*Corresponding author: Ana M. Moura da Silva Phone: #55-11-2627.9779 e-mail: [email protected] Keywords: Jellyfish Venom Enzyme Phospholipase Proteolytic activity Olindias sambaquiensis
ABSTRACT
Jellyfish are amongst the most familiar of venomous marine animals. Jellyfish encounters with humans are common, although subsequent envenomation has varying toxicity ranging from usually mild symptoms to sometimes lethal consequences. Jellyfish venoms are of biotechnological importance, with toxins displaying antimicrobial, analgesic and anti-tumor activities. Although proteolytic enzymes have also been described, detailed characterisation of these proteins is scant. Recently, the proteomic profile of putative toxins isolated from nematocysts of the hydromedusae Olindias sambaquiensis was reported, which included protease and phospholipase enzymes. Here we present the results of a study to experimentally confirm enzymatic activity in extract of O. sambaquiensis tentacles. Specimens of O. sambaquiensis were collected at the coast of São Sebastião, SP, Brazil. A procedure to generate tentacle extract was standardised, yielding high quantities of proteins of different molecular masses. Extract was assayed for the presence of metalloproteases, serine proteases and phospholipase A2 activities using substrates that cover a wide range of each class of these enzymes and also specific substrates for ADAMs and MMPs. The extract showed activity towards all substrates confirming previous proteomic evidence for the presence of these enzymes. In addition, proteolytic activity was also detected by zymography assays on casein and gelatin. However, when comparing these activities to snake venoms, which are rich in proteases and phospholipases, the metalloprotease activity detected was lower than the high levels observed in Bothrops jararaca venom, but levels of serine protease and phospholipase A2 enzyme activity was comparable to the one observed in venoms of Bothrops snakes, known for its strong anti-coagulant and myotoxic activities due to serine proteases and phospholipases A2, respectively. These data validate the functional properties of proteins characterised previously by proteomics and provide a better understanding of the toxin arsenal of this underexplored venomous animal, indicating the role of active serine proteases and phospholipases A2 in the action of the venom. 19
1. Introduction This demersal species is endemic to the southwestern Atlantic, fairly common from the The phylum Cnidaria comprises about north coast of the state of São Paulo (Brazil) to 13,500 species, ~85 % living in marine habitats the province of Buenos Aires (Argentina) (Okamura et al., 2015; Morandini et al., 2016). (Vannucci, 1951; Oliveira et al., 2016). The Cnidaria is both the largest and oldest phylum species has already been associated with of toxic animals (Ozbek et al., 2009; Van Iten accidents with swimmers and fishermen, in et al., 2013; Van Iten et al., 2014). The phylum which the victim developed an intense painful is divided into six classes, viz., Staurozoa, condition, and a skin patter of the lesions of Cubozoa, Scyphozoa, Hydrozoa, Myxozoa, short, round marks, or sometimes with short and Anthozoa (Van Iten et al., 2014; Okamura welt lines, the lesions may develop into blisters et al., 2015). The cnidarian lineage is defined or necrosis (Haddad Junior et al., 2010; by the presence of cnida, an organelle present Resgalla Junior et al., 2011; Mosovich and in specialized stinging cells called cnidocytes Young, 2012). To date, despite the abundance (Marques and Collins, 2004; Morandini et al., of this species and the large number of 2016). These cells are all over the epidermis, accidents caused in Brazil, few studies usually concentrated on the oral region and reported effects of O. sambaquiensis in tentacles, and sometimes also present on the humans (Kokelj et al., 1993; Mianzan et al., gastrodermis. The main type of cnidae, the 2001; Haddad Junior et al., 2002; Haddad nematocysts, are used for capturing prey, Junior et al., 2010; Mosovich and Young, defense, and adhesion (Kass-Simon and 2012), and even rarer studies focused on the Scappaticci Jr, 2002). The efficiency of the biochemical characterisation of O. nematocysts for predation depends on its toxic sambaquiensis toxic components (Haddad content (Salleo et al., 1996). Junior et al., 2014). This is probably related to Cnidarian toxins are responsible for difficulties in obtaining sufficient quantities of accidents all over the world, varying from mild proteins isolated from nematocysts, in which to severe, even fatal, cases (Lumley et al., biological activity is preserved. This is the 1988; Haddad Junior et al., 2002). In recent reason why most of the knowledge on protein studies, jellyfish toxins have shown neurotoxic composition of O. sambaquiensis venom is still (Carneiro et al., 2011; Lassen et al., 2012), derived from proteomic analysis, in which myotoxic (Endean, 1987; Yanagihara and functional indicatives are not present. Shohet, 2012) and pore forming cytolytic The proteomic profile of putative toxins biological activities (Long and Burnett, 1989; isolated from O. sambaquiensis nematocysts Li et al., 2013; Wang et al., 2013; Haddad was featured recently, highlighting enzymes Junior et al., 2014). Enzymes such as commonly present in other animals’ venoms, proteases (Calton and Burnett, 1983; Gusmani such as phospholipases A2 and proteolytic et al., 1997; Lee et al., 2011) and enzymes (Weston et al., 2013). These phospholipases (Gusmani et al., 1997) have enzymes play a major role in the toxicity of also been reported. Nevertheless toxicity and venoms of terrestrial animals, such as spiders harmful effects to humans, recent studies have and snakes. Phospholipase A2 may represent also identified compounds with the main component of some of the most toxic biotechnological interest in Cnidarian venoms. snake’s venom, causing high neurotoxic and A compound with analgesic action was found myotoxic activities on the victims (Gutiérrez in the venom of the sea anemone and Lomonte, 2013). Proteases are also Bunodosoma cangicum (Zaharenko et al., abundant in venoms, especially in the venom 2011). An antimicrobial compound was of Viperidae snakes, in which they play an described from Aurelia aurita (Shenkarev et al., important role in coagulation disorders, 2012), while fractions with potential antitumor cytotoxicity, and local lesions (Markland, 1998; (antiproliferative and cell-matrix adhesion Moura-da-Silva et al., 2007). In this study, we inhibitors) and analgesics were disclosed in present the results of an analysis that aimed to Pelagia noctiluca extracts (Ayed et al., 2012a; experimentally confirm the presence of activity Ayed et al., 2012b). of proteins identified by proteomic of O. The jellyfishes of O. sambaquiensis sambaquiensis, and infer their functional Müller, 1861 is assigned to the class relevance by comparison to the activity of Hydrozoa, family Olindiidae. It occurs all year similar components present on snake venoms. long, being prevalent in winter in the coast of the State of São Paulo (Vannucci, 1951), 2. Materials and methods because water temperature is an important factor for their abundance, often associated 2.1. Obtaining the extract of Olindias with cold water (< 20 °C) (Nagata et al., 2014). sambaquiensis tentacles 20
An expedition to collect specimens of (Bradford, 1976) and an analysis by SDS- hydromedusae O. sambaquiensis was held on PAGE 12.5 % under reducing or non-reducing September 23th 2014 in the São Sebastião conditions (Laemmli, 1970) was performed. Channel, northern coast of São Paulo, along the Enseada and Cigarras beaches 2.2. Proteolytic activity (23°41'28.5"S 45°21'25.6"W) at an average depth of 10 m. Animals were caught by six 2.2.1. Metalloproteases towings of 20 min each using trawling nets of shrimp fisheries, with a 25 mm mesh. Metalloprotease activity was assessed Specimens of O. sambaquiensis (Fig. 1) were using FRET (Fluorescence Resonance Energy morphologically identified based on previous Transfer) substrates: descriptions, considering the presence of four Abz-AGLA-EDDnp (GenOne) was radial canals of orange color, pleated gonads used as a broad substract for along canals, long marginal tentacles, present metalloproteases, which is currently used for in large numbers and diameter not exceeding assaying snake venom metalloproteases 10 cm (Vannucci, 1951). (SVMP) activity. Abz-AGLA-EDDnp was used a final concentration of 200 µM in 50 µL and incubated with 50 µL of O. sambaquiensis tentacular extract (10 µg) diluted in 10 mM CaCl2, 150 mM NaCl and 0.05 % Brij 35, containing 50 mM sodium acetate solution for pH 5.0 and 50 mM Tris solutions for pH 7.5 and 9.0 as described by Kuniyoshi et al. (2012), with modifications. Reactions were monitored on SpectraMax® M2 fluorimeter (Molecular Devices) at λEM 420 nm and λEX 320 nm in kinetic mode for 10 min with read range 1 min, at different temperatures (4, 25 e 37 °C). As a positive control we used Jararhagin, a snake venom metalloprotease abundant in Bothrops jararaca venom, at a concentration of 0.25 µg of Jararhagin/100 µL reaction. The specific activity of three independent experiments was calculated in RFU/min/µg. Mca-PLAQAV-Dpa-RSSSR-NH2 (R&D Systems) is an ADAM (A Disintegrin And Metalloprotease) specific substrate and was
Fig. 1. Representative specimen of Olindias dissolved to 10 µM in buffer (25 mM Tris sambaquiensis jellyfish F. Müller, 1861 (courtesy of Dr. containing 2.5 mM of ZnCl2 and 0.005 % Brij Alvaro Migotto from the University of São Paulo). 35, pH 9.0). As a positive control we used the recombinant enzyme ADAM-17 (R&D Animals were transported alive in Systems) at a concentration of 0.01 µg ADAM- buckets with local sea water to the laboratory 17/100 µL reaction. Mca-PLGL-Dpa-AR-NH2 of the Center for Marine Biology/USP - São (R&D Systems) is a substrate directed to Sebastião/SP, where tentacles were excised MMPs (Matrix Metalloproteases) that was used and aliquoted in microtubes with seawater, at 10 µM in buffer (50 mM Tris containing 10 forming pools. Microtubes were transported on mM CaCl2, 150 mM NaCl and 0.05 % Brij 35, dry ice to the Immunopathology Laboratory of pH 7.4). As a positive control recombinant the Instituto Butantan, and stored at -80 °C enzymes MMP-2 and MMP-9 (R&D Systems) until preparation of the extracts. The tentacles at a concentration of 0.01 µg protein/100 µL of contained in a microtube were centrifuged for 3 reaction were used. As samples, we used the min at 3,000 g at 4 °C to remove sea water O. sambaquiensis extract in concentration of used in the storage, tentacles were then gently 10 µg protein/100 µL reaction. For these homogenized in a tissue homogenizer (Potter) substrates, the increase in fluorescence in 2 mL Sodium acetate 10 mM solution, pH intensity was monitored at SpectraMax® M2 5.0. Homogenate was centrifuged for 5 min at fluorimeter (Molecular Devices) at λEM 405 nm 15,000 g at 4 °C, the pellet containing λEX 320 nm in kinetic mode for 10 min with fragments and cell debris was removed, and read range of 1 min. The specific activity of the protein concentration in the supernatant MMPs and ADAMs was calculated from three was evaluated using standard protocol independent experiments in U/mg using the 21 conversion factor (pmol/RFU) derived from the 2.3. Phospholipase activity standard curve of 7-amino-4-Methylcoumarin (Sigma). The presence of metalloprotease The presence of phospholipase A2 activity in B. jararaca venom was also activity in O. sambaquiensis tentacular extract assessed on all substrates. This venom was was determined as described by Culma et al. tested at concentration of 10 µg venom/100 µL (2014) with modifications. The extract or the reaction for MMPs substrate, and 1 µg viperid venom (B. jararaca or B. jararacussu), venom/100 µL reaction for broad in concentration of 5 µg/20 µL were incubated metalloproteases and ADAMs substrates. with 20 µL of specific chromogenic synthetic substrate 4-nitro-3-[octanoyloxy] benzoic acid 2.2.2. Serine proteases (Enzo® Life Sciences) in a final concentration of 320 µM in sodium acetate buffer 10 mM, pH The presence of serine protease 5.0 containing 10 mM CaCl2, 100 mM NaCl, or activity was determined by the method 10 mM Tris, pHs 7.5 or 9.0, containing 10 mM described by Zhu et al. (2005) adapted to 96- CaCl2, 100 mM NaCl, a total final volume of well microplates. The tests were performed 240 µL. The plates were incubated for 40 min using 20 µL of the chromogenic synthetic at 4, 25 or 37 °C and hydrolysis values were substrate Benzoyl-arginyl-p-nitroanilide (L- determined according to DO at 425 nm in a BAPNA) (Sigma-Aldrich®) at a final spectrophotometer SpectraMax® M2 concentration of 500 µM, incubated with 20 µL (Molecular Devices). As positive control was of samples (5 µg O. sambaquiensis tentacular used Crotoxin B (CB) subunit isolated from extract, 5 µg B. jararaca venom or 0.5 µg Crotalus durissus terrificus venom with proved porcine pancreas trypsin (Sigma-Aldrich), used phospholipase activity, at a concentration of as control) in 200 µL of sodium acetate buffer 1.5 µg CB/240 µL reaction. 50 mM, pH 5.0 or 50 mM Tris-HCl for pH 7.5 The phospholipase activity was and 9. The reactions were incubated at expressed by increase in absorbance at 425 different temperatures (4, 25 or 37 °C) for 40 nm caused by the cleavage of the substrate min and proteolysis of the substrate was and consequent release of the chromophore measured spectrophotometrically SpectraMax® product (3-hydroxy-4-nitrobenzoic acid) from M2 (Molecular Devices) at 405 nm. The serine three independent experiments. protease activity was expressed by increase in absorbance at 405 nm caused by increased 3. Results chromophore p-nitroalinida after cleavage by a specific enzyme. 3.1. Obtaining the extract of Olindias sambaquiensis tentacles 2.2.3. Zymography A total of 40 hydromedusae of O. Zymography in gel using casein and sambaquiensis, with 4-8 cm in diameter, were gelatin as substrates (Heussen and Dowdle, captured in the collect, in six towings. 1980; Vandooren et al., 2013) was used to Tentacles were excised and subjected estimate the molecular masses of proteases to different extraction approaches. Initially we and their action on macromolecular substrates. tested a methodology similar to that described Samples were separated by SDS-PAGE in by Weston et al., (2013), which is summed up Laemmli system, in a polyacrylamide gel 12.5 in two stages, first the isolation of nematocysts % containing casein or 15 % containing gelatin present in the tentacles by centrifugation in 2 mg/mL as substrates. Samples (20 µg) of gradient of sugar and finally the extraction of tentacular extract of O. sambaquiensis were proteins present in nematocysts through the applied to the gel and after electrophoresis, membrane disruption by sonication. However, gels were washed twice for 20 min with Triton with the use of this method the amount of X-100, 2.5 % for removal SDS and protein obtained was not sufficient to perform renaturation of the proteins. Then gels were the tests and proteins precipitated during the incubated for 17 h at 37 °C with 20 mM Tris sonication process probably due to containing 0.5 mM CaCl2, pH 7.4, allowing denaturation (not shown). The best protocol, enzymatic digestion of casein or gelatin. which resulted in good yields of biologically Finally, gels were stained with solution 0.125 active enzymes consisted basically in % Coomassie Blue R-250, containing 45 % submitting homogenized tentacle tissue to low methanol and 10 % acetic acid for 1 h and osmolarity (2 mL – 10 mM Sodium acetate pH destained with 40 % solution of ethanol, 5.0) and soluble proteins were recovered by containing 10 % acetic acid until hydrolysis centrifugation at 15,000 g, 4 °C , for 5 min as bands become visible. described in Material and Methods. 22
Protein concentration in tentacular testing against serine protease substrates had extracts ranged from 2 to 6 mg protein/mL in a activity comparable to the levels detected for total volume of 2 mL in each extraction, B. jararaca venom in which serine proteases resulting good yields, sufficient for conducting are abundant and very important for the the biological assays. SDS-PAGE analysis expression of pro- and anticoagulant properties verified the presence of a great complexity of of this venom. protein bands of different molecular masses, predominantly high molecular masses (Fig. 2).
Fig. 2. Electrophoretic profile of the extract of O. sambaquiensis tentacles. Samples (15 µg) were subjected to SDS-PAGE 12.5 % under non-reducing (A) or reducing (B) conditions. Protein bands were stained with Comassie blue R-250. Fig. 3. Proteolytic activity at different pHs and temperatures. Extracts (10 µg) were incubated with 200 µM Abz-A-G-L-A-EDDnp as metalloprotease substrate (A) 3.2. Proteolytic activity using peptide and 5 µg of extracts were incubated with 500 µM L- substrates BAPNA for serine protease (B). For metalloproteases, hydrolysis was monitored on SpectraMax® M2 fluorimeter at λEM 420 nm and λEX 320 nm in kinetic mode for 10 min In order to confirm and quantify the with read range 1 min and for serinoproteases, proteolysis presence of activity of the main enzymes of the substrate was measured at 405 nm. described in the proteome, the O. sambaquiensis tentacular extract was tested 3.2.1. Proteolytic activity using macromolecular against broad substrates for metalloproteases substrates and serine proteases at different pHs and temperatures. The maximal activity was The evaluation of presence of achieved using pH 7.5 buffer at 37 °C (Fig. 3) proteolytic activity on macromolecular for both substrates. substrates resulted in degradation of casein Then, the extract was tested against and gelatin as a hydrolysis band by O. selected substrates for metalloproteases sambaquiensis sample (Fig. 4), in casein gel (MMPs and ADAMs). Specific proteolytic (A) with mobility correspondent to activity of tentacular extract of O. approximately 30 kDa, another band of sambaquiensis against all specific synthetic approximately 36 kDa, and two weaker bands substrates for metalloproteases (broad, MMPs between 45 and 60 kDa. In gelatin gel (B) and ADAMs) and serine proteases is shown in there were two bands between 20 and 24 kDa Table 1. The tentacular extract of O. as well as a higher molecular mass band of sambaquiensis showed activity against all approximately 50 kDa. tested substrates, confirming previous Olindias sambaquiensis tentacular proteomic evidences of these enzymes in the extract was also tested for fibrinolytic activity extract. The results were compared to the on agarose plates containing fibrin, similar to proteolytic activity of B. jararaca venom, which snake venom tests (Baldo et al., 2008). Control was also tested. Proteolytic activity of the samples of jararhagin and B. jararaca venom extract of O. sambaquiensis tentacles was low showed fibinolytic activity in all tests, with halos against substrates for metalloproteases, but of 112 mm2 and 156 mm2, respectively 23
(average of three independent experiments). extract showed phospholipase A2 activity Olindias sambaquiensis extracts (20, 30 and higher than the one observed in B. jararaca 40 µg/well), however, did not induce fibrin venom and about half of the activity presented hydrolysis in any of the experiments, and by B. jararacussu venom (Fig. 5B), known as formed no halo (data not shown). one with the highest phospholipase A2 activity amongst Bothrops snakes.
Fig. 4. Zymography of O. sambaquiensis tentacular extract. Extracts (20 µg) were submitted under non- reducing condition to SDS-PAGE 12.5 % containing casein (A) or SDS-PAGE 15 % containing gelatin (B) as substrate. The gels were incubated for 17 hours at 37 °C with 20 mM Tris containing 0.5 mM CaCl2, pH 7.4, allowing enzymatic digestion of substrates. The gel was stained with Coomassie Blue R-250. Arrows indicate hydrolysis bands . 3.3. Phospholipase activity
Activity of phospholipase A2, another important family of enzymes detected in O. sambaquiensis’ proteome, tested positive at different temperatures and pHs, with the Fig. 5. Phospholipase A2 activity. (A) Extracts (5 µg) were incubated with 320 µM substrate in different pHs and highest activity achieved in tests using pH 7.5 temperatures. Hydrolysis was monitored on SpectraMax® and 37 °C (Fig. 5A). Comparisons of extract M2 fluorimeter (Molecular Devices) at 425 nm. (B) activity with activity of viperid venom (B. Comparison of phospholipase A2 activity of O. jararaca and B. jararacussu) indicated that sambaquiensis tentacular extract and snake venom (B. jararacussu). Activity was accessed at pH 7.5, 37 ºC. 24
Results are expresses as mean ± sd of three independent greater activity observed (pH 7.5, 37 °C) was experiments. in highly different temperature conditions in relation to the natural habitat of O. 4. Discussion sambaquiensis and its preys. Similar patterns were also observed in other studies of enzyme Marine animal venoms have received kinetics for other cnidarians (Calton and special attention in recent years, and the Burnett, 1983; Gusmani et al., 1997), but search for components with therapeutic plausible explanations are still lacking. potential has grown. However, studying Metalloproteases SVMPs are a family cnidarian venoms has some limitations, mainly of toxins playing an important role in because their venom is not contained in pathologies derived from Viperid glands, like in snakes, but in organelles envenomations, and they were the most present all over the animals. This makes the abundant toxins detected in the O. obtainment of pure venom laborious and sambaquiensis proteome (Weston et al. 2013). constrained to low yields. A first attempt to Catalytic actions of SVMPs are related to its solve this issue, following protocol by Weston core activities, such as induction of et al. (Weston et al., 2013), failed to obtain hemorrhage, apoptosis of endothelial cells, sufficient amounts of protein to proceed on and part of its pro-inflammatory action (Moura- enzymatic assays. Therefore, we separate da-Silva et al., 2007). Kinetic tests with the nematocysts from tissue by homogenization, a tentacular extract of O. sambaquiensis technique widely used proved to be efficient presented lower activities of metalloproteases (Endean and Henderson, 1969; Tamkun and when compared to B. jararaca venom (Table Hessinger, 1981), and together with the 1). This pattern supports the absence of homogenization method, an approach bleeding in accidents involving O. targeting the release of toxins by nematocysts sambaquiensis, suggesting this enzyme has a (extrusion) was also applied. minor role in the envenomation frame, despite The nematocyst discharge mechanism being the most detected in the proteome. is not fully elucidated (Picken and Skaer, 1966; Metalloproteases MMPs and ADAMs are Thurm et al., 2004; Tibballs, 2006; Anderson enzymes of great biotechnological interest and Bouchard, 2009). Large amounts of because of their role in several diseases. solutes inside the nematocyst cause an MMPs are enzymes related to the degradation osmotic pressure higher than 140 bar (Picken of extracellular matrix components of the and Skaer, 1966). We used this osmotic connective tissue, are involved in tissue hypothesis to stimulate the release of remodeling and act during embryogenesis and nematocyst’s toxins by using distilled water development, as well as in wound healing, (Hessinger and Lenhoff, 1973; Gusmani et al., besides being also involved in diseases such 1997; Ayed et al., 2012b), but this as arthritis and cancer (Blundell, 1994). methodology resulted in denatured proteins of ADAMs is a family of transmembrane proteins obtained as a precipitate. Marine animal toxins with proteolytic function (metalloprotease are labile molecules, and this instability makes domain) and cell adhesion by binding to them easily precipitable. Toxins within the integrins (disintegrin domain) (Yang et al., nematocysts were suggested to be in an 2006), associated with certain human diseases inactive and stable condition, with enzymes such as asthma, cardiac hypertrophy, becoming active and labile after extrusion rheumatoid arthritis, inflammation, and (Hessinger, 1988). Several studies reported Alzheimer's disease. Tentacular extracts of O. gradual loss of activity of toxins in cnidarians, sambaquiensis also showed low activity on even with all care being taken (Baxter and MMPs and ADAMs substrates, and this Marr, 1969; Calton and Burnett, 1983; Endean, residual activity could be related to 1987; Othman and Burnett, 1990; Endean et contamination of adjacent tissue or venom al., 1993; Carrette and Seymour, 2004; Winter metalloproteases sharing structural and et al., 2007; Sher and Zlotkin, 2009). We functional domains with ADAMs and MMPs adapted previous methods performing the (Bode et al., 1993; Stöcker and Bode, 1995). extraction under low salt solution (10 mM Therefore, the activity against all sodium acetate, pH 5.0). This approach metalloproteases substrates can be related to resulted in significant protein yields with toxins the similarity between these enzymes and a with preserved activity, as shown by the possible shared affinity for substrates. enzymatic assays. Broad synthetic substrates Serine proteases comprise a class of were used to verify the presence of proteases proteolytic enzymes whose main characteristic and fosfolipase A2 in the tentacular extracts at is the catalytic triad serine, histidine, and different pHs and temperatures. Curiously, the aspartic acid. They are present in several 25 physiological activities such as general protein of 26-28 kDa, hydrolysis predominant band on digestion, but also specific activities such as casein zymography gel, and also present in the activation of coagulation cascade proteins gelatin gel, are probably belonging to serine (Bode et al., 1989; Whitcomb and Lowe, 2007), protease family, corroborating its presence in and in the immune system of insects and the proteome. Proteolysis of substrates with plants (Gorman et al., 2000). Tentacular important biological function was also tested extract of O. sambaquiensis was active against with fibrin as a macromolecular substrate in serine protease substrate, similar to that fibrin-plate assays, a method capable to observed for the B. jararaca venom, in which characterize different venoms acting in the this enzyme is about 10 % of the venom hemostatic system of the prey, but no action composition. The action of serine proteases in was observed. several components of coagulation cascade, Phospholipase is a superfamily of leads to a typical and frequent consumption enzymes acting on cellular membranes, syndrome in patients bitten by Bothrops specifically catalyzing the cleavage of snakes presenting unclotable blood when phospholipids into fatty acids and admitted to the hospital care (Serrano and lysophospholipids (Kini, 1997). They are Maroun, 2005). These symptoms were never abundant in nature, found in high reported for O. sambaquiensis accidents, what concentrations and are highly toxic in snake, can be credited to the low concentration of bee and wasp venoms (Harris, 1985). They these enzymes in the nematocysts, or to their participate in prey digestion, but also exhibit a nonspecificity to coagulation cascade broad spectrum of action, including hemolytic, substrates. One possible substrate that is often neurotoxic, cardiotoxic, aggregating platelet, related to serine proteases is the complement anticoagulant, edematogenic, myotoxic, system, and its activation by serine proteases bactericidal, and pro-inflammatory activities lead to a number of pro-inflammatory (Kini and Evans, 1989). The presence of mediators that could explain part of the phospholipase A2 in cnidarian venoms is symptoms observed in jellyfish patients (Sim possibly involved with the inflammatory and Tsiftsoglou, 2004). It is also reasonable reaction at the bite site, increasing hemolytic that proteolytic enzymes are important to activity (Helmholz et al., 2007). Tentacular digest preys. Once we are dealing with extracts of O. sambaquiensis had a significant tentacular extracts, the proteins recovered activity of this enzyme compared to the venom include structural components of the of B. jararacussu, for which the phospholipase nematocyst capsule, plus components of A2 is the most abundant compound (Sousa et adjacent tissues. The proteolytic activity found al., 2013), and showed greater activity than the in cnidarian venom may be related to one presented to the B. jararaca venom. A contamination by tissue surrounding the putative action of these enzymes is nematocysts, not necessarily an activity in responsible for lesions of accidents caused by crude venom (Tamkun and Hessinger, 1981; O. sambaquiensis, in which 80 % of the Calton and Burnett, 1982). Furthermore, Moran accidents resulted in edematogenic lesions et al., (2012) published a study which (Haddad Junior et al., 2010). This suggests demonstrates the presence of a neurotoxin into phospholipases A2 is the main enzyme ectodermal gland cells of sea anemone responsible for envenomation symptoms by O. Nematostella vectensis rather than sambaquiensis, especially because nematocysts. haemorrhagic lesions, typical of proteolytic The efficacy of the extract in enzymes, were not reported in these cases. hydrolyzing macromolecular substrates was The presence of phospholipase A2 activity in carried out using the macromolecular jellyfish venom, evidencing the importance of substrates casein and gelatin, selected these enzymes for cnidarians in general, was because they allow detection of proteases already reported (Stillway and Lane, 1971; (Heussen and Dowdle, 1980; Snoek-van Hessinger and Lenhoff, 1973; Gusmani et al., Beurden and Von den Hoff, 2005). The protein 1997; Helmholz et al., 2007). Carneiro et al. family responsible for the proteolysis was (Carneiro et al., 2011) observed that the inferred in the zymography by comparing phospholipase activity resulted in venom different molecular masses corresponding to fractions of Phyllorhiza punctata was similar to the hydrolysis bands with molecular masses of the phospholipase activity found in Crotalus proteins obtained in the proteome of O. durissus terrificus’ venom. sambaquiensis (Weston et al., 2013). This study was a pioner to carry out an Proteomics identified metalloproteases with enzymatic characterisation of tentacular molecular mass of ~50-60 kDa, corroborated in extracts of O. sambaquiensis, whose the zymography. Proteins with molecular mass populations are associated with coastal 26 accidents with bathers in the South-West BnP1, a novel P-I metalloproteinase from Bothrops Atlantic. The study was made possible by neuwiedi venom: Biological effects benchmarking relatively to jararhagin, a P-IIISVMP. Toxicon 51, 54-65. developing a new method for obtaining proteins from tentacles of O. sambaquiensis, Baxter, E.H., Marr, A.G., 1969. Sea wasp (Chironex preserving the integrity of proteins, providing a fleckeri) venom: lethal, haemolytic and dermonecrotic significant income and whose enzymatic properties. Toxicon 7, 195-210. activity is not impaired. We highlight presence Blundell, T.L., 1994. Metalloproteinase superfamilies and of proteases and phospholipases A2. drug design. Nat Struct Biol 1, 73-75. Comparing the in vitro activity observed in this study with the symptoms observed in accidents Bode, W., Gomis-Rüth, F.X., Stöckler, W., 1993. Astacins, serralysins, snake venom and matrix metalloproteinases by O. sambaquiensis, it is concluded that exhibit identical zinc-binding environments serine proteases and phospholipases A2 are (HEXXHXXGXXH and Met-turn) and topologies and possibly involved in the O. sambaquiensis should be grouped into a common family, the 'metzincins'. envenomation. Finally, the characterisation of FEBS Lett 331, 134-140. the enzymatic activity of metalloproteases, Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., serine proteases and phospholipase A2 in O. Hofsteenge, J., 1989. The refined 1.9 A crystal structure of sambaquiensis tentacular extract, both human alpha-thrombin: interaction with D-Phe-Pro-Arg macromolecular substrates as for synthetic chloromethylketone and significance of the Tyr-Pro-Pro- Trp insertion segment. EMBO J 8, 3467-3475. substrates gave a better understanding of the toxic venom arsenal that little explored specie, Bradford, M.M., 1976. A rapid and sensitive method for the and therefore an additional tool in the search quantitation of microgram quantities of protein utilizing the for new compounds with biotechnological principle of protein-dye binding. Anal Biochem 72, 248- 254. potential is now avaliable. Calton, G.J., Burnett, J.W., 1982. Partial purification and Acknowledgements characterization of the acid protease of sea nettle (Chrysaora quinquecirrha) nematocyst venom. Comp Biochem Physiol B 72, 93-97. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo Calton, G.J., Burnett, J.W., 1983. Partial purification and (FAPESP) grant number 2013/25593-4, PSK characterization of the alkaline protease of sea nettle and 2014/26058-8, AMMS and Conselho (Chrysaora quinquecirrha) nematocyst venom. Comparative Biochemistry and Physiology Part C: Nacional de Desenvolvimento Científico e Comparative Pharmacology 74, 361-364. Tecnológico (CNPq) grant number 304025/2014-3, AMMS. 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4. CONCLUSÃO
O extrato de tentáculos de O. sambaquiensis foi obtido com sucesso e o método demonstrou-se eficaz, apresentando um bom rendimento e proteínas com integridade e atividade preservada. Corroborando com o que foi descrito pelo proteoma, o extrato apresentou atividade sobre substratos específicos para metaloproteases, serinoproteases e fosfolipases A2, sendo que estas últimas apresentaram atividade enzimática comparável à atividade encontrada em venenos de viperídeos. Além disso, comparando a atividade enzimática observada in vitro com os sintomas apresentados pelos pacientes nos envenenamentos por essa espécie, é possível admitir que possivelmente as serinoproteases e fosfolipases A2 possuem papel fundamental nos quadros de envenenamento por O. sambaquiensis.
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