Identification and expressional analysis of two cathepsins from half-smooth tongue sole (Cynoglossus semilaevis)

Cathepsins are a family of lysosomal proteases that play an important role in protein degradation, antigen presentation, apoptosis, and inflammation. Cathepsins are divided into three groups, i.e., cysteine protease, serine protease, and aspartic protease. Cathepsin D and cathepsin L, which are aspartic protease and cysteine protease respectively, have been identified in a number of teleosts; however, the immunological relevance of fish cathepsins is largely unknown. In this study, we cloned and analyzed the expression profiles of a cathepsin D (CsCatD) and a cathepsin L (CsCatL) homologs from half-smooth tongue sole (Cynoglossus semilaevis). CsCatD is composed of 396 amino acid residues and shares 67.6-88.4% overall sequence identities with fish and human cathepsin D. Structurally CsCatD possesses an aspartic endopeptidase domain, which contains two conserved aspartic acid residues that form the catalytic site. CsCatL is 336 residues in length and shares 64.7-90.2% overall sequence identities with fish and human cathepsin L. CsCatL has an N-terminal cathepsin propeptide inhibitor domain followed by a Papain family cysteine protease domain, the latter containing four conserved catalytic residues: Gln-133, Cys-139, His-279, and Asn-303. Recombinant CsCatL purified from Escherichia coli exhibited apparent protease activity. Quantitative real time RT-PCR analysis detected constitutive expression of CsCatD and CsCatL in multiple tissues, with the lowest level found in heart and the highest level found in liver. Experimental challenge of tongue sole with the bacterial pathogen Vibrio anguillarum and megalocytivirus caused significant inductions of both CsCatD and CsCatL expression in kidney and spleen in time-dependent manners. Immunization of the fish with a subunit vaccine also enhanced CsCatD and CsCatL expression in the first week post-vaccination. These results suggest involvement of CsCatD and CsCatL in host immune reactions to bacterial and viral infections and in the process of antigen-induced immune response.