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Cathepsin L of the sea cucumber Apostichopus japonicus-molecular characterization and transcriptional response to Vibrio splendidus infection

Yang, Jingwen, Liu, Huihui, Zheng, Gang, Xiang, Xiaowei, Lv, Zhenming, Wang, Tianming
Fish & shellfish immunology 2016 v.49 pp. 387-395
Apostichopus japonicus, Strongylocentrotus, Vibrio splendidus, active sites, cathepsin L, complementary DNA, cysteine, enzyme activity, expressed sequence tags, gene expression regulation, immune response, innate immunity, invertebrates, isoelectric point, messenger RNA, molecular weight, open reading frames, pathogens, phylogeny, quantitative polymerase chain reaction, rapid amplification of cDNA ends, transcription (genetics)
Cathepsin L, a lysosomal endopeptidase, has been noted for its involvement in the innate immune response in invertebrates. Here, the cathepsin L cDNA of the sea cucumber Apostichopus japonicus (AjCatL) is identified from an EST library and then cloned by the rapid amplification of the cDNA ends (RACE) PCR. The full-length cDNA is 1678 bp long containing an open reading frame (ORF) of 1002 bp, an 80 bp 5′ UTR and a 599 bp 3′ UTR. The cDNA encodes 333 amino acid residues with a predicted molecular mass of 37.07 kDa and a theoretical isoelectric point (pI) of 5.01. The full-length AjCatL contains three active sites of eukaryotic thiol (cysteine) protease at positions 133–144, 278–288 and 295–314. Analysis of the predicted tertiary structure of prepro-CatL (17–333 aa) and mature-CatL (116–333 aa) reveals that the propeptide region (17–115 aa) blocks access to the substrate-binding cleft. Phylogenetic analysis shows that the AjCatL is clustered together with two other CatLs from Strongylocentrotus purpuratus. The enzymatic activity of AjCatL was verified using a substrate hydrolyzing assay with recombinant mAjCatL. Further analysis of real time-PCR demonstrates that the expression of AjCatL mRNA is significantly up-regulated in the coelomocytes in cases of infection with the common bacterial pathogen, Vibrio splendidus. This suggests that the AjCatL is likely to be involved in the immune response.