Estudo de astacinas (metaloproteases) do veneno de aranhas-marrons (gênero Loxosceles) : obtenção de formas recombinantes e análises proteômicas

Orientadora : Profa. Dra. Andrea Senff RibeiroCo-orientador : Prof. Dr. Silvio Sanches VeigaTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 30/08/2012Bibliografia: fls.329-344Abstract: Brown spider bites (Loxosceles genus) generate dermonecrotic lesions with gravitational spreading and/or systemic manifestations. Loxosceles venom is a complex mixture of toxins, enriched with peptides and proteins (5-40 kDa) which show distinct biological activities. Metalloproteases have already been described in the venom of different Loxosceles species. Characterization of a metalloprotease from astacin family in the venom of L. intermedia (LALP1) was the first report of an astacin family member as component of animal venoms. Recently, astacin-like proteases were described as a family of toxins found in the venom of L. intermedia, L. laeta e L. gaucho. Biological activities of astacins from Loxosceles are unknown. The quantity of venom produced by each spider is minute and purification procedures result in low yield. Characterization of LALP1 biochemistry activity was performed after its recombinant production in a prokaryotic system as inclusion bodies, followed by refolding in vitro. However, yield of recombinant active protein was very low and it was not possible to perform biological assays. The present work aimed the production of recombinant soluble and active forms of astacins form L. intermedia venom. Although the recombinant astacins were produced in inclusion bodies in all tested conditions with different strains using pET-14b vector. LALP1 was refolded by different protocols of refolding in vitro, in quantities that would allow in vivo evaluation, though; recombinant toxins did not show proteolytic activity. LALP2 was produced as a fusion protein with SUMO and the recombinant protein was found in the soluble fraction, but there was no proteolytic activity. LALP1 and LALP2 structural predictions suggest that tags in the N-terminal position can interfere at native conformations. Results obtained herein conduct to new protocols of recombinant astacin production in E. coli, with removal of N-terminal tags, new constructions using other vectors with C-terminal tags, or even, production in eukaryotic systems. Other goal of this study was evaluation of native astacins in L. intermedia, L. laeta and L. gaucho venoms. Venom complexity was analyzed by bidimensional electrophoresis, which showed prevalence of proteins at 30-35kDa range. The subproteoma of astacinlike proteases was evaluated with 2-DE immunoblotting revealed by anti-LALP1 antibodies and 2-DE zymography assays. Subproteoma profiles suggest that there are more astacin isoforms than the ones already described. Using mass spectrometry analysis of L. intermedia venom spots, LALP2 was identified. The issues for identification of more Loxoceles astacins may be due to high mannose glycosylation, which was detected by lectin bloting with GNA lectin. Therefore, a deglycosylation protocol or a new strategy (de novo sequencing) is going to be performed for the improvement of astacin identifications

A Novel Hyaluronidase from Brown Spider (Loxosceles intermedia) Venom (Dietrich's Hyaluronidase): From Cloning to Functional Characterization

Loxoscelism is the designation given to clinical symptoms evoked by Loxosceles spider's bites. Clinical manifestations include skin necrosis with gravitational spreading and systemic disturbs. the venom contains several enzymatic toxins. Herein, we describe the cloning, expression, refolding and biological evaluation of a novel brown spider protein characterized as a hyaluronidase. Employing a venom gland cDNA library, we cloned a hyaluronidase (1200 bp cDNA) that encodes for a signal peptide and a mature protein. Amino acid alignment revealed a structural relationship with members of hyaluronidase family, such as scorpion and snake species. Recombinant hyaluronidase was expressed as N-terminal His-tag fusion protein (similar to 45 kDa) in inclusion bodies and activity was achieved using refolding. Immunoblot analysis showed that antibodies that recognize the recombinant protein cross-reacted with hyaluronidase from whole venom as well as an anti-venom serum reacted with recombinant protein. Recombinant hyaluronidase was able to degrade purified hyaluronic acid (HA) and chondroitin sulfate (CS), while dermatan sulfate (DS) and heparan sulfate (HS) were not affected. Zymograph experiments resulted in similar to 45 kDa lytic zones in hyaluronic acid (HA) and chondroitin sulfate (CS) substrates. Through in vivo experiments of dermonecrosis using rabbit skin, the recombinant hyaluronidase was shown to increase the dermonecrotic effect produced by recombinant dermonecrotic toxin from L. intermedia venom (LiRecDT1). These data support the hypothesis that hyaluronidase is a spreading factor. Recombinant hyaluronidase provides a useful tool for biotechnological ends. We propose the name Dietrich's Hyaluronidase for this enzyme, in honor of Professor Carl Peter von Dietrich, who dedicated his life to studying proteoglycans and glycosaminoglycans.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundacao Araucaria-PR (FAP)Secretaria de Estado de Ciencia, Tecnologia e Ensino Superior do Parana (SETI)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ Fed Parana, Dept Cell Biol, BR-80060000 Curitiba, Parana, BrazilUniv Fed Parana, Clin Hosp, Dept Clin Pathol, BR-80060000 Curitiba, Parana, BrazilUniv Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Ponta Grossa, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilWeb of Scienc

Phospholipase-D activity and inflammatory response induced by brown spider dermonecrotic toxin: Endothelial cell membrane phospholipids as targets for toxicity

Brown spider dermonecrotic toxins (phospholipases-D) are the most well-characterized biochemical constituents of Loxosceles spp. venom. Recombinant forms are capable of reproducing most cutaneous and systemic manifestations such as dermonecrotic lesions, hematological disorders, and renal failure. There is currently no direct confirmation for a relationship between dermonecrosis and inflammation induced by dermonecrotic toxins and their enzymatic activity. We modified a toxin isoform by site-directed mutagenesis to determine if phospholipase-D activity is directly related to these biological effects. the mutated toxin contains an alanine substitution for a histidine residue at position 12 (in the conserved catalytic domain of Loxosceles intermedia Recombinant Dermonecrotic Toxin - LiRecDT1). LiRecDT1H12A sphingomyelinase activity was drastically reduced, despite the fact that circular dichroism analysis demonstrated similar spectra for both toxin isoforms, confirming that the mutation did not change general secondary structures of the molecule or its stability. Antisera against whole venom and LiRecDT1 showed cross-reactivity to both recombinant toxins by ELISA and immunoblotting. Dermonecrosis was abolished by the mutation, and rabbit skin revealed a decreased inflammatory response to LiRecDT1H12A compared to LiRecDT1. Residual phospholipase activity was observed with increasing concentrations of LiRecDT1H12A by dermonecrosis and fluorometric measurement in vitro. Lipid arrays showed that the mutated toxin has an affinity for the same lipids LiRecDT1, and both toxins were detected on RAEC cell surfaces. Data from in vitro choline release and HPTLC analyses of LiRecDT1-treated purified phospholipids and RAEC membrane detergent-extracts corroborate with the morphological changes. These data suggest a phospholipase-D dependent mechanism of toxicity, which has no substrate specificity and thus utilizes a broad range of bioactive lipids. (C) 2010 Elsevier B.V. All rights reserved.Secretaria de Estado de CienciaTecnologia e Ensino Superior (SETI) do ParanaFundacao Araucaria-PRFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Fed Parana, Dept Cell Biol, BR-81531990 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniv Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Ponta Grossa, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilWeb of Scienc

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins

Metaloproteases do tipo astacinas são uma família de toxinas conservadas em venenos de diferentes espécies de aranhas marrons (gênero loxosceles)

Orientadora : Drª Andrea Senff RibeiroCo-Orientador: Prof. Silvio Sanches VeigaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 2009Bibliografia f.65-76Área de concentração: Biologia CelularResumoResumo: No estado do Paraná, os acidentes com aranhas marrons (gênero Loxosceles) são problema de saúde pública, em especial na cidade de Curitiba e Região Metropolitana que são consideradas áreas endêmicas para o loxoscelismo. O veneno de Loxosceles é composto por uma mistura de toxinas, na sua maioria protéica, capazes de causar lesões dermonecróticas com espalhamento gravitacional e manifestações sistêmicas como agregação plaquetária, anemia hemolítica e falência renal aguda. Nos venenos de aranhas do gênero Loxosceles foram descritas toxinas como fosfolipases-D, hialuronidades e metaloproteases, entre outras. As metaloproteases já foram descritas em venenos de diferentes espécies, como exemplo: L. intermedia, L. reclusa, L. deserta, L. rufescens, L. gaucho e L. laeta e acredita-se que tenham participações nos distúrbios hemostáticos descritos após o envenenamento, além de poderem ser um fator de espalhamento das demais toxinas do veneno devido sua ação proteolítica frente aos componentes da matriz extracelular. As espécies de maior importância clínica são: L. intermedia, L. laeta e L. gaucho, sendo a espécie L. intermedia de maior distribuição no estado do Paraná e a principal espécie relacionada com acidentes no município de Curitiba. Através de técnicas de biologia molecular, foi mostrado que uma metaloprotease presente no veneno de L. intermedia (a LALP1 - Loxosceles Astacin-like Protease) pertence à família das astacinas, diferente das metaloproteases de venenos de outros animais, como as de serpentes que pertencem à família das adamalisinas. A LALP1 foi a primeira enzima da família das astacinas descrita como constituinte de venenos animais. O presente estudo identificou, através de análise de diferentes clones de uma biblioteca de cDNA de glândulas de veneno de L. intermedia, duas novas isoformas de astacinas presentes no veneno de L. intermedia, denominadas LALP2 e LALP3. Os imunoensaios realizados com venenos das outras duas principais espécies do Paraná, L. laeta e L.gaucho, mostraram a existência de proteínas nativas relacionadas à LALP1 e que tais proteínas apresentavam atividade gelatinolítica que foi inibida por 1,10- fenantrolina (quelante de metal divalente), confirmando atividade metaloproteásica. Extrações de RNA total de glândulas de veneno de L. laeta e L. gaucho foram realizadas, os cDNAs obtidos utilizando oligonucleotídeos iniciadores para sequência da LALP1 foram sequenciados e identificou-se que esses cDNAs codificavam metaloproteases da família das astacinas, tais proteínas foram denominadas LALP4 (astacina de L. laeta) e LALP5 (astacina de L. gaucho). O alinhamento das sequências aminoacídicas de todas as LALPs e membros da família das astacinas de diferentes organismos mostraram que as astacinas de Loxosceles têm maiores identidades com as astacinas de nematódeos. Portanto, os resultados sugerem a existência de uma família gênica para astacinas no veneno de L. intermedia com expressão de diferentes isoformas (LALP1, LALP2 e LALP3),essa família gênica está presente nos venenos das três espécies estudadas (L. intermedia, L. laeta e L. gaucho) e possivelmente serão encontradas distribuídas em todo o gênero Loxosceles. Esses achados reforçam a relevância biológica das astacinas como toxinas importantes nos efeitos tóxicos dos venenos loxoscélicosAbstract: In the Parana State, accidents involving brown spiders (Loxosceles genus) are a public health problem, especially in the city of Curitiba and metropolitan region that are considered endemic areas for loxoscelism. Loxosceles venom is a mixture of toxins, mostly protein, capable of causing dermonecrotic lesions with gravitational spreading and systemic manifestations such as platelet aggregation, hemolytic anemia and acute renal failure. In Loxosceles spider venoms were described toxins as phospholipase D, hyaluronic acid, and metalloproteases, among others. Metalloproteases have been described in venoms of different species, for example: L. intermedia, L. recluse, L. deserta, L. rufescens, L. gaucho and L. laeta and is believed to be involved in hemostatic disorders described after poisoning, and can act as spreading factor for other venom toxins, due to its proteolytic action against extracellular matrix components. Species of greatest clinical importance are: L. intermedia, L. laeta and L. gaucho. In the Parana State, L. intermedia species show larger distribution and are the main species related with accidents in the city of Curitiba. By molecular biology techniques, it was demonstrated that metaloprotease from L. intermedia (LALP1 – oxosceles Astacin-like Protease) venom belongs to the astacin family, that is different from others animal venoms metalloproteases, such as snake metalloproteases that belong to the adamalysin family. LALP1 was the first astacin described as an animal venom constituent. The present study identified, by analyzing distinct clones from L. intermedia venom glands cDNA library, two new astacin isoforms present in L. intermedia venom, which were named LALP2 and LALP3. Imunoassays with L. laeta and L. gaucho venoms (two others important species in Parana) showed the existence of native proteins related to LALP1 and these proteins have gelatinolytic activity which could be inhibited by 1,10- phenanthroline (divalent metal chelator), confirming metalloprotease activity. Moreover, total RNA from L. laeta and L. gaucho venom glands were extracted, the cDNAs obtained using LALP1 primers were sequenced and the results identified that they encode metalloproteases from astacin family, named LALP4 (L. laeta astacin) and LALP5 (L. gaucho astacin). Amino acid sequences alignment with LALPs and other astacin members from different organisms showed that Loxosceles astacins have the greatest identities with nematode astacins. Therefore, the results suggest the existence of an astacin gene family in L. intermedia venom expressing different isoforms (LALP1, LALP2 and LALP3), that this gene family is present in venoms of the three species studied (L. intermedia, L. gaucho and L. laeta) and possibly will be found distributed throughout the Loxosceles genus. These findings reinforce the biological relevance of astacin toxins as important for toxic effects of loxoscelic venoms

Metaloproteases do tipo astacinas são uma família de toxinas conservadas em venenos de diferentes espécies de aranhas marrons (gênero loxosceles)

Orientadora : Drª Andrea Senff RibeiroCo-Orientador: Prof. Silvio Sanches VeigaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Biologia Celular e Molecular. Defesa: Curitiba, 2009Bibliografia f.65-76Área de concentração: Biologia CelularResumoResumo: No estado do Paraná, os acidentes com aranhas marrons (gênero Loxosceles) são problema de saúde pública, em especial na cidade de Curitiba e Região Metropolitana que são consideradas áreas endêmicas para o loxoscelismo. O veneno de Loxosceles é composto por uma mistura de toxinas, na sua maioria protéica, capazes de causar lesões dermonecróticas com espalhamento gravitacional e manifestações sistêmicas como agregação plaquetária, anemia hemolítica e falência renal aguda. Nos venenos de aranhas do gênero Loxosceles foram descritas toxinas como fosfolipases-D, hialuronidades e metaloproteases, entre outras. As metaloproteases já foram descritas em venenos de diferentes espécies, como exemplo: L. intermedia, L. reclusa, L. deserta, L. rufescens, L. gaucho e L. laeta e acredita-se que tenham participações nos distúrbios hemostáticos descritos após o envenenamento, além de poderem ser um fator de espalhamento das demais toxinas do veneno devido sua ação proteolítica frente aos componentes da matriz extracelular. As espécies de maior importância clínica são: L. intermedia, L. laeta e L. gaucho, sendo a espécie L. intermedia de maior distribuição no estado do Paraná e a principal espécie relacionada com acidentes no município de Curitiba. Através de técnicas de biologia molecular, foi mostrado que uma metaloprotease presente no veneno de L. intermedia (a LALP1 - Loxosceles Astacin-like Protease) pertence à família das astacinas, diferente das metaloproteases de venenos de outros animais, como as de serpentes que pertencem à família das adamalisinas. A LALP1 foi a primeira enzima da família das astacinas descrita como constituinte de venenos animais. O presente estudo identificou, através de análise de diferentes clones de uma biblioteca de cDNA de glândulas de veneno de L. intermedia, duas novas isoformas de astacinas presentes no veneno de L. intermedia, denominadas LALP2 e LALP3. Os imunoensaios realizados com venenos das outras duas principais espécies do Paraná, L. laeta e L.gaucho, mostraram a existência de proteínas nativas relacionadas à LALP1 e que tais proteínas apresentavam atividade gelatinolítica que foi inibida por 1,10- fenantrolina (quelante de metal divalente), confirmando atividade metaloproteásica. Extrações de RNA total de glândulas de veneno de L. laeta e L. gaucho foram realizadas, os cDNAs obtidos utilizando oligonucleotídeos iniciadores para sequência da LALP1 foram sequenciados e identificou-se que esses cDNAs codificavam metaloproteases da família das astacinas, tais proteínas foram denominadas LALP4 (astacina de L. laeta) e LALP5 (astacina de L. gaucho). O alinhamento das sequências aminoacídicas de todas as LALPs e membros da família das astacinas de diferentes organismos mostraram que as astacinas de Loxosceles têm maiores identidades com as astacinas de nematódeos. Portanto, os resultados sugerem a existência de uma família gênica para astacinas no veneno de L. intermedia com expressão de diferentes isoformas (LALP1, LALP2 e LALP3),essa família gênica está presente nos venenos das três espécies estudadas (L. intermedia, L. laeta e L. gaucho) e possivelmente serão encontradas distribuídas em todo o gênero Loxosceles. Esses achados reforçam a relevância biológica das astacinas como toxinas importantes nos efeitos tóxicos dos venenos loxoscélicosAbstract: In the Parana State, accidents involving brown spiders (Loxosceles genus) are a public health problem, especially in the city of Curitiba and metropolitan region that are considered endemic areas for loxoscelism. Loxosceles venom is a mixture of toxins, mostly protein, capable of causing dermonecrotic lesions with gravitational spreading and systemic manifestations such as platelet aggregation, hemolytic anemia and acute renal failure. In Loxosceles spider venoms were described toxins as phospholipase D, hyaluronic acid, and metalloproteases, among others. Metalloproteases have been described in venoms of different species, for example: L. intermedia, L. recluse, L. deserta, L. rufescens, L. gaucho and L. laeta and is believed to be involved in hemostatic disorders described after poisoning, and can act as spreading factor for other venom toxins, due to its proteolytic action against extracellular matrix components. Species of greatest clinical importance are: L. intermedia, L. laeta and L. gaucho. In the Parana State, L. intermedia species show larger distribution and are the main species related with accidents in the city of Curitiba. By molecular biology techniques, it was demonstrated that metaloprotease from L. intermedia (LALP1 – oxosceles Astacin-like Protease) venom belongs to the astacin family, that is different from others animal venoms metalloproteases, such as snake metalloproteases that belong to the adamalysin family. LALP1 was the first astacin described as an animal venom constituent. The present study identified, by analyzing distinct clones from L. intermedia venom glands cDNA library, two new astacin isoforms present in L. intermedia venom, which were named LALP2 and LALP3. Imunoassays with L. laeta and L. gaucho venoms (two others important species in Parana) showed the existence of native proteins related to LALP1 and these proteins have gelatinolytic activity which could be inhibited by 1,10- phenanthroline (divalent metal chelator), confirming metalloprotease activity. Moreover, total RNA from L. laeta and L. gaucho venom glands were extracted, the cDNAs obtained using LALP1 primers were sequenced and the results identified that they encode metalloproteases from astacin family, named LALP4 (L. laeta astacin) and LALP5 (L. gaucho astacin). Amino acid sequences alignment with LALPs and other astacin members from different organisms showed that Loxosceles astacins have the greatest identities with nematode astacins. Therefore, the results suggest the existence of an astacin gene family in L. intermedia venom expressing different isoforms (LALP1, LALP2 and LALP3), that this gene family is present in venoms of the three species studied (L. intermedia, L. gaucho and L. laeta) and possibly will be found distributed throughout the Loxosceles genus. These findings reinforce the biological relevance of astacin toxins as important for toxic effects of loxoscelic venoms