RESUMO
Trypsin-like serine proteases are involved in large number of processes, especially in digestive degradation and immune responses. Here, we identify the characterization of a trypsin-like serine protease in planarian, Djtry, which interestingly has the incompletely conserved catalytic triad (K, D, and S). Phylogenetic analysis suggests that Djtry is an ancient type of trypsin-like serine proteases. The spatial and temporal expression patterns of Djtry are shown during regenerating and embryonic development by whole-mount in situ hybridization. Djtry is found to display a tissue specific expression pattern, with a predominant expression detected in whole gut region of intact and regenerating planarian. While the tissue- and stage-specific expression patterns during the embryonic development imply the roles of Djtry involve in yolk degradation and gut formation. Quantitative real-time PCR was carried out to analyze the function of this protease in vivo after planarians were stimulated to a bacterial challenge and food. The results showed that Djtry increased after a bacterial challenge and was basically stable for food. Therefore, the trypsin-like serine protease might be involved in the innate defense reactions against bacterial infection.
Assuntos
Proteínas de Helminto/genética , Planárias/enzimologia , Tripsina/genética , Animais , Sistema Digestório/embriologia , Sistema Digestório/enzimologia , Escherichia coli/fisiologia , Regulação Enzimológica da Expressão Gênica , Proteínas de Helminto/metabolismo , Interações Hospedeiro-Patógeno/genética , Imunidade Inata/genética , Larva/enzimologia , Dados de Sequência Molecular , Filogenia , Planárias/embriologia , Planárias/imunologia , Planárias/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Tripsina/metabolismoRESUMO
The cDNA Djtry, encoding a planarian trypsin, was identified from the cDNA library of Dugesia japonica. Multiple alignment analysis showed that the Tryps_SPc domain contained the incompletely conserved catalytic triad in which the first amino acid His was substituted by Lys. Phylogenetic analysis indicateed that Djtry protein falls at the base of other animal trypsins. The Djtry cDNA was cloned into a bacterial vector pET-28a and was transferred into E. coli BL21. The His-tagged Djtry fusion protein expression was induced by IPTG. SDS-PAGE analysis revealed that the Djtry was expressed as inclusion bodies in E. coli BL21 with the estimated molecular weight of approximately 26 kDa. Western blotting with His-tag antibody showed that the antibody was reacted with the fusion protein after refolding. Compared to bovine trypsin using BAEE as special substrate of trypsin, the enzyme activity of Djtry was measured. These results indicate that Djtry represents the archetype of animal trypsins, and this type of mutational trypsin Djtry still performs the trypsin nature with slightly weaker activity.