Gene Cloning, Expression And Bioinformatic Analysis Of Cathepsin D From Lampetra Japonica

Cathepsin D (CTSD), which belongs to the aspartic proteinase superfamily in thelysosome of eukaryotic cells, usually participates in the process of protein degradation.However, recent studies have shown that CTSD also plays important roles in a variety ofphysiological conditions, such as cell proliferation, cell apoptosis, cell aging, tissuehomeostasis, and so on. In addition, the expression level of CTSD has also been changedduring multiple pathological progresses, such as Alzheimer’s disease, atherosclerosis,congenital myopathy and cancer.Adult lampreys usually live semi-parasitic lives in seawater which attach to the host fishesto suck the flesh and blood for a long time. Due to the key position which exsited betweenvertebrates and invertebrates, lampreys are of great value to study. And they are ideal animalmodels to illuminate the origin, evolution, embryonic development and organ differentiationof vertebrates. Thus, lampreys have gained much more attention during the process ofzoological research. Recently, most studies have been focused on CTSD from the othervertebrates, while little work has been done on the CTSD of jawless vertebrates.In the present study, the full-length cDNA (1790bp) of lamprey CTSD (lampreycathepsin D, L-CTSD) was obtained through gene cloning,5’and3’ end of the rapidamplification, which is composed by the5’-UTR of124bp,3’-UTR of472bp, and the openreading frame of1194bp, encoding396amino acids. Bioinformatics analysis showed that theamino acid sequence of L-CTSD contains a signal peptide (Met1-Ala20), a precursor domain(Thr21-Ala48) and a mature domain (Gln76-Val396). In addition, the amino acids of L-CTSDpossess a potential glycosylationsites (Leu53). Sequence alignment showed that the sequenceof L-CTSD shares a high homology with the CTSDs from the other species, and the homologywas up to70%with the CTSD from Lates calcarifer. Besides, phylogenetic tree analysisshowed that L-CTSD is closer to the CTSD from fishes, in accordance with the creaturesevoluted from low to high. However, L-CTSD is also located on its own branch, whichdisplays its unique evolutionary status.Through Real Time quantitative PCR, we also found that the L-CTSD was widelyexpressed in the buccal gland, gill, heart, liver, intestine, kidney and supernatural myeloidbody of lampreys. After stimulated with E.coli or S.aureus, the relative expression level of L-CTSD was up-regulated in the above tissues, which suggested that L-CTSD may playimportant roles in the process of nutritional intake and immune escape. Furthermore, theprokaryotic expression vectors pColdⅠ-CTSD was constructed and successfully expressed inE.coli BL21. The purified recombinant L-CTSD (rL-CTSD) was obtained through his-tagbind column.In conclusion, the detection of L-CTSD gene in Lampetra japonica, the most ancientjawless vertebrate still alive, provides us much more clues to better understand thephysiological functions of CTSD in lampreys, as well asthe origin and evolution of thevertebrates.