RESUMO
Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, function in immune response in vertebrates; however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of cathepsin L (catL) in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full-length catL cDNA (1274 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catL cDNA contained a 978 bp open reading frame (ORF) that encoded a putative 325 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate sequences revealed conserved gene structure and enzyme active sites common among papain-like cysteine proteases, and high percent identity among other invertebrate cathepsins. CatL mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in hepatotpancreas, gill, stomach, and hemocytes, and (b) responsive in hemocytes to a Vibrio anguillarum challenge, the catL expression level and enzyme activity both with peak exposure observed 8 h post-injection. Collectively, data demonstrate the successful isolation of catL from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.
Assuntos
Braquiúros/enzimologia , Braquiúros/genética , Catepsina L/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Braquiúros/imunologia , Braquiúros/microbiologia , Catepsina L/biossíntese , Catepsina L/imunologia , Clonagem Molecular , Cricetinae , Cricetulus , Imunidade Inata/genética , Imunidade Inata/imunologia , Dados de Sequência Molecular , Filogenia , RNA/química , RNA/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Vibrio/imunologia , Vibrioses/imunologia , Vibrioses/microbiologiaRESUMO
Cathepsins, a superfamily of hydrolytic enzymes produced and enclosed within lysosomes, function in immune response in vertebrates; however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of cathepsin C (catC) in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full-length catC cDNA (1481 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a hepatopancreatic cDNA library. The catC cDNA contained a 1284 bp open reading frame (ORF) that encoded a putative 427 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate cathepsins sequences revealed high percent identity. CatC mRNA expression in E. sinensis was responsive in hemocytes to a Vibrio anguillarum challenge, with peak exposure observed 6 h post-injection. Collectively, data demonstrate the successful isolation of catC from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.
