Cloning, Expression And Function Analysis Of Cathepsin L Gene From Lampetra Japonica

Cathepsin L （CTSL） belongs to the lysosomal cysteine protease family, which iscomposed of a signal peptide, a propeptide and a mature peptide. CTSL plays important rolesin a variety of biological processes, such as proteolysis, antigen-presenting, cell apoptosis, Tcell sorting, as well as embryonic development. In addition, the abnormal change of theexpression level of CTSL accounts for the induction of a variety of tumors, cardiovascularand kidney disease. In parasitic animals, CTSL mainly participated in nutritional intake, tissueinvasion, immune escape and tissue differentiation.In the present study, the full-length cDNA （1457bp） of Japanese lamprey CTSL（Lj-CTSL） was obtained through gene cloning,5’and3’ end of the rapid amplificationexperiments, which is composed by the5’-UTR non-coding area of104bp,3’-UTRnon-coding region of333bp, and the open reading frame of1020bp, encoding³³⁹ aminoacids. Bioinformatics analysis showed that the Lj-CTSL amino acid sequence contains a signalpeptide （Met¹-Ala18）, a precursor domain (Thr¹9-Glu¹¹⁷) and a mature domain (Ala¹¹⁸-Val³³⁹).There is a potential glycosylationsites (Asn²²⁶), six substrate binding sites (Leu186, Met¹87,Ala²⁵⁴, Leu²⁸⁰, Gly²⁸³and Ser³³⁵), and a catalytic site constituted by the cysteine (Cys¹⁴²),histidine (His²⁸²) and the asparagine （Asn306） residues. Sequence alignment showed that theamino acid sequence of the Lj-CTSL has a high homology with the CTSLs from the otherspecies, and the homology was up to81.1%with the CTSL from Xenopus laevis. Besides,Lj-CTSL contains the highly conserved motifs, ERF/WNIN and GNFD. Phylogenetic treeanalysis showed that the Lj-CTSL is closer to the CTSL from the amphibian Xenopus laevis,in accordance with the roles of creatures are evoluted from low to high. However, Lj-CTSL isalso located on its own branch,which displays its unique evolutionary status.Semi-quantitative RT-PCR showed that the Lj-CTSL is widely distributed in the lampreyheart, oral gland, kidney, intestine, liver, gills and gonads, and the expression level ofLj-CTSLwas the most in the oral gland, which suggests the Lj-CTSL plays important roles inthe nutritional intake process for the Japanese lampreys. The Lj-CTSL was subcloned into theprokaryotic expression vector pTWIN, and successfully expressed in E.coli BL21. Thesoluble recombinant protein rLj-CTSL was obtained. MTT assayes show that the rLj-CTSL inhibit the proliferations of HUVEC, ECV304and MA. In addition, rLj-CTSL suppresschicken chorioallantoic membrane vascular newborn in a dose-dependent manner.Lampreys （Lampetra japonica） belong to the Cyclostomata, which are the most primitivejawless vertebrates, lived typical migration lives between the river and the sea. The parasiticlampreys usually live in the sea, and attach the host fishes for blood. The Japanese lampreysoccupy an important position in the history of the world of zoological research due to thespecial parasitic characteristics and evolutionary status. The successful cloning, expressionand functional analysis of Lj-CTSL from Japanese lampreys provide important informationnot only on the origin and evolution of the species, but also on the clues of physiologicalfunctions of CTSL in lampreys.