Perfilagem fosfoproteômica das glândulas produtoras de seda da aranha Trichonephila clavipes
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Data
2021-06-07
Autores
De-Souza, Caroline Lacerra
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Universidade Estadual Paulista (Unesp)
Resumo
Apesar do grande interesse pelas propriedades mecânico-elásticas das fibras da seda produzida pelas aranhas, visando o seu uso em aplicações biomédicas e biotecnológicas devido as suas propriedades de resistência, elasticidade e biocompatibilidade, pouco se conhece sobre os detalhes das glândulas produtoras de seda e o processo de fiação que ocorre para a produção das fibras. Dessa forma, alguns estudos têm demonstrado que as glândulas produtoras de seda apresentam diversas proteínas relacionadas com o envolvimento na secreção das proteínas da seda, transporte, regulação de atividades proteolíticas e preservação das proteínas da seda contra o estresse oxidativo e degradação, durante todo o processo de fiação; além disso, estudos também já demonstraram nas fibras de seda da teia, a presença de toxinas de ação geral e de neurotoxinas, comuns em venenos animais, sugerindo que essas toxinas/neurotoxinas contribuem para a paralisia e captura de presas. Porém, esses estudos não têm considerado a possível presença de modificações pós-traducionais (PTMs) que podem atuar sobre a atividade biológica e função dessas moléculas. A determinação de PTMs é fundamental para a elucidação de processos que controlam os eventos celulares, como crescimento, divisão e diferenciação celular, incluindo a interação entre proteínas. Portanto, no presente estudo adotamos o uso da cromatografia de afinidade com TiO2 como método de enriquecimento de fosforilação, combinados com a abordagem proteômica livre de gel - shotgun, utilizando ténicas avançadas de espectrometria de massas in tandem (µLC-ESI-micrOTOF-Q-III-CID) a fim de identificar o perfil fosfoproteômico das glândulas que secretam a seda da aranha Trichonephila clavipes. Os resultados obtidos com a análise fosfoproteômica do conjunto de glândulas produtoras de seda da ranha T. clavipes, mostrou a identificação de 1316 proteínas fosforiladas sendo que 155 foram identificadas na glândula agregada, 580 na ampulada maior, 143 na flageliforme e 438 na ampulada menor. Estas fosfoproteínas foram classificadas de acordo com a sua função geral e subdividas em sete diferentes grupos de proteínas: (i) proteínas estruturais que compõem as fibras da seda – as espidroínas; ii) proteínas relacionadas com o transporte de íons e oxigênio nas glândulas para manter a estabilidade das espidroína; (iii) proteínas relacionadas com o dobramento/conformação e modificação das espidroínas; (iv) proteínas relacionadas com a preservação das espidroínas contra o estresse oxidativo; (v) proteínas relacionadas com a preservação das características fibrilares das espidroínas no meio ambiente; (vi) proteínas housekeeping e (vii) proteínas relacionadas com a captura e pré-digestão de presas. Para o último grupo, foram identificadas 284 proteínas semelhantes a toxinas, neurotoxinas, enzimas proteolíticas, e defensinas em sua forma fosforilada. O enriquecimento funcional destas proteínas nos permitiu avaliar a contribuição de suas funções às caracteríticas fisico-químicas das proteínas estruturais da seda, bem como a interação entre os grupos funcionais das proteínas listadas envolvidas com o processo de fiação da seda; assim como também a influência sobre a atividade tóxica das toxinas. Os resultados também permitiram identificar uma série de sequências peptídicas fosforiladas que não apresentaram resultados de Identificação com proteínas depositadas nos bancos de dado e foram obtidas utilizando da abordagem de sequenciamento de novo e submetidas a um alinhamento de sequencias utilizando a ferramenta BLASTP. O ensaio de inseto-toxicidade realizado ao final do estudo, nos permitiu verificar a toxicidade do extrato bruto fosforilado das glândulas ampulada maior e agregada, que apresentaram os valores de dose letal, DL50=28,40 ng/mg de inseto (A. mellifera) para a glândula ampulada maior e DL50=23,07 ng/mg de inseto (A. mellifera) para a glândula agregada. Dessa forma, demonstramos que a combinação do uso de métodos baseados em cromatografia de afinidade, como a estratégia de enriquecimento de proteínas/peptídeos fosforilados, e o uso de uma abordagem proteômica com estudos de PTMs se mostrou uma estratégia eficiente na investigação de amostras complexas, como as glândulas produtoras de seda da teia de aranhas. Um estudo com modificações pós-traducionais das proteínas presentes nas glândulas produtoras de seda da aranha T. clavipes, como demonstrado no presente trabalho, nos possibilitou obtermos informações biológicas e químicas das características dessas moléculas, contribuindo para uma melhor compreensão sobre o perfil fosfoproteômico das glândulas analisadas.
Despite the great interest in the mechanical-elastic properties of the silk fibers produced by spiders, aiming their use in biomedical and biotechnological applications due to their properties of resistance, elasticity and biocompatibility, little is known about the details of the silk-producing glands and the spinning process that takes place for the production of the fibers. Studies have shown that the silk producing glands, present several proteins related to the involvement in the secretion of silk proteins, transport, regulation of proteolytic activities and preservation of silk proteins, against oxidative stress and degradation, throughout the spinning process; in addition, studies have also demonstrated in the silk web fibers, of general action toxins-like and neurotoxins-like common in animal venom, suggesting that these proteins contribute to paralysis and prey capture. However, these studies have not considered the possible presence of post-translational modifications (PTMs) that can act over biological activity and function of these molecules. The determination of PTMs is fundamental for the processes elucidation that control cellular events, such as cell growth, division and differentiation, including the interaction between proteins. Therefore, in the present study we adopted the use of TiO2 affinity chromatography as a phosphorylation enrichment method, combined with the gel-free proteomic approach - shotgun, using advanced techniques of in-tandem mass spectrometry (µLC-ESI-micrOTOF-Q -III-CID) in order to identify the phosphoproteomic of glands profile that secrete the spider silk Trichonephila clavipes. The results obtained with the phosphoproteomic analysis of the set of silk producing glands of the T. clavipes spider, demonstrateded the identification of 1316 phosphorylated proteins, of which 155 were identified in the aggregated gland, 580 in the major ampulate, 143 in the flagelliform and 438 in the minor ampulate. These phosphoproteins were classified according to their general function and subdivided into seven different groups of proteins: (I) structural proteins that make up the fibers of silk - the spidroins; (II) proteins related to the transport of ions and oxygen in the glands to keep the spidroins stability; (III) proteins related to the folding/shaping and modification of spidroins; (IV) proteins related to the preservation of spidroins against oxidative stress; (V) proteins related to the preservation of the fibrillar characteristics of spidroins in the environment; (VI) housekeeping proteins and (vii) proteins related to prey capture and pre-digestion. For the last group, 284 toxin, neurotoxins, proteolytic enzymes, and defensins-like proteins, in their phosphorylated form were identified. The functional enrichment of these proteins allowed us to evaluate the contribution of their functions to the physicochemical characteristics of structural silk proteins, as well as the interaction between the functional groups of the listed proteins involved with the silk spinning process; And also the influence on the toxic activity of the toxins. The results also made it possible to identify a series of phosphorylated peptide sequences that did not present identification results with proteins deposited in the data banks and were obtained using the sequencing approach de novo and subjected to a sequence alignment using the BLASTP tool. The insect-toxicity assay carried out at the end of the study allowed us to verify the toxicity of the crude phosphorylated extract of the major ampulate and aggregated glands, which showed the lethal dose values LD50 = 28.40 ng/mg of insect (A. mellifera) for the major ampulate gland and LD50 = 23.07 ng/mg of insect (A. mellifera) for the aggregated gland. Thus, we demonstrate that the combination of using methods based on affinity chromatography, such as the strategy of enriching proteins / phosphorylated peptides, and the use of a proteomic approach with studies of PTMs proved to be an efficient strategy in the investigation of complex samples, like the silk-producing glands in the spider web. A study with post-translational modifications of proteins present in the spider producing glands of the spider T. clavipes, as demonstrated in the present work, enabled us to obtain biological and chemical information on the characteristics of these molecules, contributing to a better understanding of the phosphoproteomic profile of the glands analyzed
Despite the great interest in the mechanical-elastic properties of the silk fibers produced by spiders, aiming their use in biomedical and biotechnological applications due to their properties of resistance, elasticity and biocompatibility, little is known about the details of the silk-producing glands and the spinning process that takes place for the production of the fibers. Studies have shown that the silk producing glands, present several proteins related to the involvement in the secretion of silk proteins, transport, regulation of proteolytic activities and preservation of silk proteins, against oxidative stress and degradation, throughout the spinning process; in addition, studies have also demonstrated in the silk web fibers, of general action toxins-like and neurotoxins-like common in animal venom, suggesting that these proteins contribute to paralysis and prey capture. However, these studies have not considered the possible presence of post-translational modifications (PTMs) that can act over biological activity and function of these molecules. The determination of PTMs is fundamental for the processes elucidation that control cellular events, such as cell growth, division and differentiation, including the interaction between proteins. Therefore, in the present study we adopted the use of TiO2 affinity chromatography as a phosphorylation enrichment method, combined with the gel-free proteomic approach - shotgun, using advanced techniques of in-tandem mass spectrometry (µLC-ESI-micrOTOF-Q -III-CID) in order to identify the phosphoproteomic of glands profile that secrete the spider silk Trichonephila clavipes. The results obtained with the phosphoproteomic analysis of the set of silk producing glands of the T. clavipes spider, demonstrateded the identification of 1316 phosphorylated proteins, of which 155 were identified in the aggregated gland, 580 in the major ampulate, 143 in the flagelliform and 438 in the minor ampulate. These phosphoproteins were classified according to their general function and subdivided into seven different groups of proteins: (I) structural proteins that make up the fibers of silk - the spidroins; (II) proteins related to the transport of ions and oxygen in the glands to keep the spidroins stability; (III) proteins related to the folding/shaping and modification of spidroins; (IV) proteins related to the preservation of spidroins against oxidative stress; (V) proteins related to the preservation of the fibrillar characteristics of spidroins in the environment; (VI) housekeeping proteins and (vii) proteins related to prey capture and pre-digestion. For the last group, 284 toxin, neurotoxins, proteolytic enzymes, and defensins-like proteins, in their phosphorylated form were identified. The functional enrichment of these proteins allowed us to evaluate the contribution of their functions to the physicochemical characteristics of structural silk proteins, as well as the interaction between the functional groups of the listed proteins involved with the silk spinning process; And also the influence on the toxic activity of the toxins. The results also made it possible to identify a series of phosphorylated peptide sequences that did not present identification results with proteins deposited in the data banks and were obtained using the sequencing approach de novo and subjected to a sequence alignment using the BLASTP tool. The insect-toxicity assay carried out at the end of the study allowed us to verify the toxicity of the crude phosphorylated extract of the major ampulate and aggregated glands, which showed the lethal dose values LD50 = 28.40 ng/mg of insect (A. mellifera) for the major ampulate gland and LD50 = 23.07 ng/mg of insect (A. mellifera) for the aggregated gland. Thus, we demonstrate that the combination of using methods based on affinity chromatography, such as the strategy of enriching proteins / phosphorylated peptides, and the use of a proteomic approach with studies of PTMs proved to be an efficient strategy in the investigation of complex samples, like the silk-producing glands in the spider web. A study with post-translational modifications of proteins present in the spider producing glands of the spider T. clavipes, as demonstrated in the present work, enabled us to obtain biological and chemical information on the characteristics of these molecules, contributing to a better understanding of the phosphoproteomic profile of the glands analyzed
Descrição
Palavras-chave
Fosforilação, Bioensaio de inseto-toxicidade, Glândulas de seda, Espidroínas, Proteínas semelhantes à toxinas, Abordagem proteômica, Phosphorylation, Proteomic approach, Toxin-like proteins, Silk glands, Spidroins, Insect-toxicity bioassay