3 research outputs found
Clonagem, análise estrutural e imunológica do alérgeno antígeno 5 do veneno da vespa Polybia paulista (Hymenoptera : Vespidae)
Polybia paulista ou popularmente “paulistinha” é uma vespa social da família Vespidae. Possui hábitos urbanos e grande ocorrência no Sudeste do Brasil, especialmente no Estado de São Paulo, onde tem causado muitos acidentes de importância médica. Na composição de seu veneno encontra-se um potente alérgeno, a proteína Antígeno 5 (Ag5) e embora sua função biológica ainda seja desconhecida, este alérgeno é responsável por importantes reações imunológicas cruzadas com o Ag5 do veneno de outros insetos sociais e com outras proteínas de eucariotos. A importância da reatividade cruzada em pacientes alérgicos ao veneno de vespas sociais é inquestionável, pois estas interações têm impacto direto sobre o diagnóstico e a seleção da melhor conduta terapêutica. Os diagnósticos de alergia são baseados na detecção de anticorpos do tipo IgE específico ao veneno por testes cutâneos ou de sangue. No entanto, respostas falso-positivas decorrentes da reatividade cruzada e respostas falso-negativas provenientes da baixa quantidade de IgE detectada, dificultam a interpretação dos resultados. A sequência completa de cDNA (621 pb) do alérgeno Ag5 do veneno da vespa P. paulista, foi clonada e a análise dos nucleotídeos revelou uma similaridade de 99% com a vespa Polybia scutellaris. Anticorpos policlonais foram produzidos contra a fração eletroforética protéica do Ag5 (25 kDA) de P. paulista e analisados imunologicamente por Western blotting. Os resultados demonstraram que os anticorpos reconheceram especificamente o alérgeno Ag5 no veneno bruto de P. paulista bem como, desenvolveram maior reação imunológica cruzada com os alérgenos Ag5 do veneno das vespas do gênero Polybia, embora não se descarte a possibilidade de ocorrência de reação cruzada com venenos de outros insetos sociais...Polybia paulista, commonly known as paulistinha, is a social wasp of the family Vespidae. This species occurs in urban areas and is frequent in the Southeastern Brazil, especially in the State of São Paulo, where it has been responsible for many accidents of medical significance. A potent allergen, the protein antigen 5 (Ag5), is an important compound of the wasp venom. Although its biological function remains unknown, this component is responsible for substantial cross-immunological reactions with the Ag5 of venoms of other social insects and with eukaryotic proteins. The importance of cross-reactivity in allergic patients to the wasp venoms is unquestionable, because these interactions present a direct impact on the diagnosis and on the selection of the therapeutic treatment. Allergenic diagnoses are based on the detection of IgE specific to the venom via cutaneous or blood tests. However, sometimes they are hampered by false-positive responses as a result of cross-reactivity and false-negative responses that can occur due to the low amount of IgE detected as consequence of the low sensitivity of the test. The fulllength cDNA (621 bp) from the venom allergen Ag5 wasp P. paulista was cloned and nucleotide analysis revealed 99% of similarity with the wasp Polybia scutellaris. Polyclonal antibodies were produced against the electrophoretic protein fraction of Ag5 (25 kDa) of P. paulista and immunologically analyzed by Western blotting. The results showed that the antibodies strongly recognized the allergen Ag5 in the venom of P. paulista and developed higher cross-immune reaction with the same allergen in wasp venoms of the genus Polybia, although the possibility of cross reaction with other insect venoms not tested in this study cannot be excluded. The model carried out for the Ag5 P. paulista revealed the... (Complete abstract click electronic access below)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES
Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis
In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/β)7 barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients. © 2013 Elsevier Ltd