Análise do dimorfismo sexual em venenos da aranha acanthoscurria juruenicola por peptidômica quantitativa
Data
2018-06-28
Autores
Almeida, Erika Sayuri Nishiduka Costa De 
Orientadores
Tashima, Alexandre Keiji
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Os aracnídeos estão entre os grupos de maior sucesso evolutivo da história do
planeta, e um dos fatores de maior importância para isso é a produção de um veneno
farmacologicamente complexo, composto por uma coleção de proteínas e peptídeos
biologicamente ativos e altamente específicos. Apesar dos peptídeos constituírem
grande parte da composição dos venenos, estudos peptidômicos ainda são escassos,
consequentemente as identidades e atividades biológicas de muitos desses peptídeos
permanecem desconhecidas. Além disso, em diversas espécies de aranhas é
observado dimorfismo sexual, o que pode ocasionar diferenças nas composições de
venenos de machos e fêmeas. O objetivo deste trabalho foi caracterizar e comparar
quantitativamente o perfil peptídico do veneno de espécimes machos e fêmeas da
aranha Acanthoscurria juruenicola. Peptídeos nativos foram fracionados por extração
em fase sólida e analisados por espectrometria de massas, assim como seus
fragmentos gerados pela digestão isolada por 5 enzimas distintas (tripsina,
quimotripsina, termolisina, GluC
e Aspn).
Os dados espectrais foram submetidos a
análise de novo, busca em banco de dados de transcriptoma de glândula e
sobreposição das clivagens utilizando ferramentas de bioinformática. Dentre 11
sequências maduras de toxinas completamente mapeadas, 8 eram novos peptídeos
ricos em cisteínas, apresentando entre 3 a 5 ligações dissulfeto. Buscas em bancos
de sequências de A. geniculata e Araneae resultaram em mais 24 proteínas
homólogas identificadas. Observamos dimorfismo sexual no veneno: a concentração
total de toxinas nos venenos das fêmeas foi aproximadamente 5 vezes superior à dos
machos e apresentaram peptídeos com significativa expressão diferencial. Uma das
toxinas de maior expressão em fêmeas, a U2TRTXAj1b,
de massa molecular 4,9
kDa e 3 ligações dissulfeto, foi fracionada por cromatografia de filtração em gel. Essa
toxina apresentou atividade citotóxica contra aproximadamente 44% das células de
melanoma da linhagem A375 em ensaios de MTT em concentração de 3,8 μM. Esses
resultados podem evidenciar diferenças em toxicidade e comportamento biológico de
acordo com o gênero do animal, além de embasar a potencial aplicação
biotecnológica desses peptídeos na área farmacológica.
Arachnids are among the most successful evolutionary groups in planet history. This is in part due to the production of a pharmacologically complex venom consisting in a collection of biologically active and highly specific proteins and peptides. Although venoms consist mostly of peptides, peptidomics studies are still scarce, so that their identities and biological activities remain unknown. Besides that, sexual dimorphism has been observed in many spider species, which may entail differences in male and female venom compositions. This study aims at characterizing and comparing the peptidic venom profile of male and female specimens of the Brazilian spider specie Acanthoscurria juruenicola in a quantitative aproach. Native peptides were fractionated by solid phase extraction, followed by mass spectrometry analysis (LCMS/ MS), as well as the fragments generated by the digestion isolated by 5 different enzymes (trypsin, chymotrypsin, thermolysin, GluC and AspN). Spectral data were submitted to de novo analysis, gland transcriptomics databank search and cleavages overlapping by bioinformatics tools. Among 11 completely sequenced mature toxins, 8 were new cysteine rich peptides, ranging from 3 to 5 disulfide bridges. Further, searches against A. geniculata e Araneae databanks resulted in 24 homologous identified proteins. We observed sexual dimorphism: the concentration of toxins in the venoms of the females was about 5 times higher than the male and presented differentially expressed peptides. One of the most expressed peptide in females, U2TRTXAj1b, presented molecular mass of 4,9 kDa and 3 disulfide bridges, was obtained by gel filtration chromatography. This toxin presented a cytotoxic potential against 44% of melanoma A375 cells by MTT method, on 3.8 μM concentration. These results may evidence differences in toxicity and biological behavior, according to the animal´s genus, in addition to supporting the potential biotechnological application of these peptides in pharmacological field.
Arachnids are among the most successful evolutionary groups in planet history. This is in part due to the production of a pharmacologically complex venom consisting in a collection of biologically active and highly specific proteins and peptides. Although venoms consist mostly of peptides, peptidomics studies are still scarce, so that their identities and biological activities remain unknown. Besides that, sexual dimorphism has been observed in many spider species, which may entail differences in male and female venom compositions. This study aims at characterizing and comparing the peptidic venom profile of male and female specimens of the Brazilian spider specie Acanthoscurria juruenicola in a quantitative aproach. Native peptides were fractionated by solid phase extraction, followed by mass spectrometry analysis (LCMS/ MS), as well as the fragments generated by the digestion isolated by 5 different enzymes (trypsin, chymotrypsin, thermolysin, GluC and AspN). Spectral data were submitted to de novo analysis, gland transcriptomics databank search and cleavages overlapping by bioinformatics tools. Among 11 completely sequenced mature toxins, 8 were new cysteine rich peptides, ranging from 3 to 5 disulfide bridges. Further, searches against A. geniculata e Araneae databanks resulted in 24 homologous identified proteins. We observed sexual dimorphism: the concentration of toxins in the venoms of the females was about 5 times higher than the male and presented differentially expressed peptides. One of the most expressed peptide in females, U2TRTXAj1b, presented molecular mass of 4,9 kDa and 3 disulfide bridges, was obtained by gel filtration chromatography. This toxin presented a cytotoxic potential against 44% of melanoma A375 cells by MTT method, on 3.8 μM concentration. These results may evidence differences in toxicity and biological behavior, according to the animal´s genus, in addition to supporting the potential biotechnological application of these peptides in pharmacological field.