Identificação e caracterização molecular do antígeno 5 do veneno da vespa neotropical Apoica pallens (Hymenoptera, Vespidae)
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Data
2019
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Universidade Estadual Paulista (Unesp)
Resumo
O antígeno 5 (Ag 5) é uma das proteínas mais comuns no veneno de vespas sociais. Embora sua função biológica seja desconhecida, o Ag 5 possui grande importância clínica por ser um dos componentes do veneno com maior potencial de causar reações de hipersensibilidade em pessoas alérgicas. O correto diagnóstico e o tratamento de casos de alergia envolvem o uso de alérgenos purificados para a identificação da espécie responsável pela sensibilização primária, o que, por sua vez, requer extensos trabalhos de caracterização molecular das proteínas do veneno. No Brasil, no entanto, há uma carência de dados sobre os alérgenos de espécies nativas de relevância clínica. Neste trabalho, foram utilizadas estratégias proteômicas e bioinformáticas para identificar e caracterizar o Ag 5 (Apo p 5) da vespa social Apoica
pallens. A proteína foi identificada por SDS-PAGE e purificada por cromatografia líquida de troca catiônica, seguido de digestões in gel e em solução com enzimas proteolíticas. Os peptídeos gerados foram identificados por espectrometria de massas
do tipo MALDI-ToF e ESI-QToF e comparados com sequências de Ag 5 de referência até a obtenção da estrutura primária completa do Apo p 5. Ferramentas in silico permitiram modelar a estrutura 3D do Apo p 5 e predizer possíveis modificações pós traducionais. Como resultado, obteve-se uma proteína com 207 resíduos de aminoácidos, massa teórica de 23 kDa e alta identidade com o Ag 5 de espécies da subfamília Polistinae. Ensaios de immunoblot confirmaram a alergenicidade da proteína, reconhecida pelo IgE de soros de pacientes alérgicos ao veneno de vespas. A estrutura 3D revelou uma proteína com 28 % de α-hélices e 17,1 % de folhas-β, também com alta similaridade com o Ag 5 de himenópteros de elevada relevância clínica. Os dados obtidos com este trabalho fornecem as bases para a produção
heteróloga da proteína e seu emprego em sistemas de diagnóstico de alergia ao veneno de A. pallens no Brasil.
Antigen 5 (Ag 5) is a protein commonly found in the venom of social wasps. Although its biological function is unknown, it has a high clinical relevance, since it’s one major elicitors of hypersensitivity reactions in sensitized patients. The reliable diagnostic and immunotherapy of venom allergy often requires the use of purified allergens to identify the species responsible for the primary sensitization. The rational design of panels of individuals components requires previous extensive molecular characterization of the venom allergens. In Brazil, however, there’s a lack of data concerning the allergenic arsenal on venom of most native wasps. In this work we used proteomic and bioinformatic approaches to identify and characterize the venom Ag 5 (Apo p 5) of the social neotropical wasp Apoica pallens. The protein was identified by SDS-PAGE and purified by cation exchange liquid chromatography, followed by in gel and in solution digestion with proteolytic enzymes. The peptides generated were identified by MALDI ToF and ESI-QToF mass spectrometry and compared to an Ag 5 reference sequence in order to obtain the complete primary structure of Apo p 5. In silico tools allowed the molecular modelling of the 3D structure of the protein and the prediction of post translational modifications. Immunoblot assays confirmed the allergenicity of the protein, recognized by IgE from sera of patients allergic to wasp’s venom. As a result, we acquired a protein with 207 amino acid residues, theoretical mass of 23 kDa and high identity with Ag 5 of species within the subfamily Polistinae. The 3D structure revealed a protein with 28 % of α-helices and 17,9 % of β-sheets, also highly similar to the described structure of the venom Ag 5 from other taxonomically related clinically relevant hymenopterans. The data obtained here provides the basis for the heterologous production of Apo p 5 and further use employment in routine diagnosis of A. pallens-caused allergy in Brazil.
Antigen 5 (Ag 5) is a protein commonly found in the venom of social wasps. Although its biological function is unknown, it has a high clinical relevance, since it’s one major elicitors of hypersensitivity reactions in sensitized patients. The reliable diagnostic and immunotherapy of venom allergy often requires the use of purified allergens to identify the species responsible for the primary sensitization. The rational design of panels of individuals components requires previous extensive molecular characterization of the venom allergens. In Brazil, however, there’s a lack of data concerning the allergenic arsenal on venom of most native wasps. In this work we used proteomic and bioinformatic approaches to identify and characterize the venom Ag 5 (Apo p 5) of the social neotropical wasp Apoica pallens. The protein was identified by SDS-PAGE and purified by cation exchange liquid chromatography, followed by in gel and in solution digestion with proteolytic enzymes. The peptides generated were identified by MALDI ToF and ESI-QToF mass spectrometry and compared to an Ag 5 reference sequence in order to obtain the complete primary structure of Apo p 5. In silico tools allowed the molecular modelling of the 3D structure of the protein and the prediction of post translational modifications. Immunoblot assays confirmed the allergenicity of the protein, recognized by IgE from sera of patients allergic to wasp’s venom. As a result, we acquired a protein with 207 amino acid residues, theoretical mass of 23 kDa and high identity with Ag 5 of species within the subfamily Polistinae. The 3D structure revealed a protein with 28 % of α-helices and 17,9 % of β-sheets, also highly similar to the described structure of the venom Ag 5 from other taxonomically related clinically relevant hymenopterans. The data obtained here provides the basis for the heterologous production of Apo p 5 and further use employment in routine diagnosis of A. pallens-caused allergy in Brazil.
Descrição
Palavras-chave
Bioquímica, Proteínas, Hymenoptera, Alergia, Antígeno 5, Proteômica, Antigen 5, Allergy, Proteomics