Molecular cloning, characterization, expression and activity analysis of cathepsin L in Chinese mitten crab, Eriocheir sinensis.
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.
Li
Wei-Wei
WW
School of Life Science, East China Normal University, Shanghai, China.
Jin
Xing-Kun
XK
He
Lin
L
Jiang
Hui
H
Gong
Ya-Nan
YN
Xie
Yan-Nan
YN
Wang
Qun
Q
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003386:Brachyura / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology / Q000382:microbiology; D056668:Cathepsin L / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D007113:Immunity, Innate / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D010802:Phylogeny; D012313:RNA / Q000737:chemistry / Q000235:genetics; D019105:Random Amplified Polymorphic DNA Technique; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014733:Vibrio / Q000276:immunology; D014735:Vibrio Infections / Q000276:immunology / Q000382:microbiology
D012313:RNA; D056668:Cathepsin L