Identiifcation And Expression Analysis Of Cymoglossus Semilaevis Cathepsin B、D And L

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(including cathepsin B、L), serine protease, and aspartic protease(including cathepsin D). Cathepsin B、D、L have been identified in many other speices,i.e., mammals, inserts and shellfish; However, very little is known about the function of cathepsin B、D、 L in fish. In this study, we cloned and analyzed the expression profiles of a cathepsin B(CsCatB), a cathepsin D (CsCatD) and a cathepsin L (CsCatL) homologs from half-smooth tongue sole(Cynoglossus semilaevis).CsCatB is composed of322amino acid residues and shares70-81.3%overall sequence identities with its counterpart in teleosts and humans. CsCatB possesses typical cathepsin B structural features including the propeptide region and the papain family cysteine protease domain, the latter containing the four catalytic residues (Q101, C107, H277, and N297) that are conserved in lower and higher vertebrates. Quantitative real time RT-PCR analysis showed that CsCatB expression occurred in multiple tissues and was positively regulated by bacterial infection and by immunization with a subunit vaccine. Recombinant CsCatB purified from Escherichia coli exhibited apparent protease activity, which was optimal at35°and pH5.5. In contrast, a mutant CsCatB bearing glutamic acid substitution at H277was dramatically reduced in proteolytic activityCsCatD is composed of396amino acid residues and shares67.6e88.4%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 is336residues in length and shares64.7e90.2%overall sequence identitieswith fish and human cathepsinL. 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.xThese results indicate that CsCatB and CsCatD are biologically active proteases and are likely to be involved in host immune reactions during bacterial infection and vaccination. In addition to the above function CsCatD and CsCatL are also suggested to be involved in immune response to viral infections.