Cathepsin C(CTSC) Contributes To The Antibacterial Immunity In Golden Pompano(Trachinotus Ovatus)

Cathepsin C(CTSC),also known as dipeptidyl peptidase I(DPPI),is a cysteine protease mainly found in lysosomes and belongs to the papain superfamily.It is mainly located in lysosomes and is widely found in viruses,bacteria,fungi,protozoa,plants and mammals.CTSC plays a very important role in the physiological and pathological processes of different tissues of the organism,including intracellular protein degradation,cell growth,neuraminidase activation and platelet factor ⅩⅢ activation,and so on.In recent years,studies on CTSC in aquatic animals have been carried out successively and found that it plays an important role in the immune response against diseases in the organism.However,the mechanism of how CTSC is involved in the antibacterial immunity of the organism in teleosts is not clear.In view of this,in this study,we cloned and identified the Cathepsin C(TroCTSC)gene of golden pompano(Trachinotus ovatus),a major deep-water net-culture fish,a major deep-water net-culture fish in the South China Sea,and conducted a preliminary study on its sequence structure and the function of antibacterial immunity.The main experimental contents and the results are as follows.TroCTSC was obtained by cloning,with a coding region length of 1368 bp,encoding 455 amino acids,a molecular weight of 50.79 kDa and a theoretical isoelectric point of 6.27.Amino acid sequence analysis revealed that amino acids 1-20 at the N terminus were signal peptide sequences;amino acids 224-450 are the cysteine protease structural domain of the papain family,which contains three active sites(Cys251,His397 and Asn419).The results of homology comparison showed that TroCTSC had high homology with other species of CTSC,ranging from 61.01%to 90.77%.Among them,the highest homology,up to 90.77%,was with the Seriola dumerili;the lowest homology was with the Xenopus laevis(62.25%).There was also high homology with Homo sapiens(60.93%)and Mus musculus(61.01%).The results of the phylogenetic tree analysis showed that TroCTSC belongs to a branch of scleractinian fishes and clustered into a branch with the Seriola dumerili belonging to the same family of Carangidae,with the closest affinity.By quantitative real-time PCR analysis of TroCTSC expression at mRNA level in immune tissues under healthy conditions and after bacterial stimulation,TroCTSC was found to be expressed in 11 tissues of healthy T.ovatus including skin,heart,muscle,stomach,spleen,intestine,head kidney,gill,liver,brain and blood,with the lowest expression in blood,which was set to 1,and the highest expression of muscle tissue was about 59.2-fold,followed by liver at about 33.5-fold.The expression of TroCTSC was significantly up-regulated in spleen,head kidney and liver after Vibrio harveyi infection in T.ovatus with the peak expression in the liver and head kidney after 9 h of infection and the peak expression in the spleen after 12 h of infection.It indicates that TroCTSC is widely present in various tissues of T.ovatus,and the high expression in immune organs suggests that TroCTSC may be involved in the antibacterial immune response of the organism.In order to investigate the mechanism of TroCTSC involvement in the antibacterial immunity of the organism,the localization of TroCTSC in the cells was firstly analyzed.The results showed that TroCTSC was mainly distributed in the cytoplasm and partially present in the lysosomes of the cytoplasm in golden pompano snout(GPS)cells.Further study revealed that stimulation by V.harveyi could induce TroCTSC to migrate from the cytoplasm to the nucleus,indicating that bacterial stimulation could activate TroCTSC to undergo nuclear translocation and thus perform its function.Based on the analysis of the active site of TroCTSC,three mutants targeting its conserved catalytic active site were successfully constructed.Based on the analysis of the active site of TroCTSC,three mutants targeting its conserved catalytic active site were successfully constructed.And the fusion proteins rTroCTSC,rTroCTSC-C251A,rTroCTSC-H397A and rTroCTSC-N419A of TroCTSC and the three mutants were induced to be expressed.In vitro enzymatic activity experiments using the CTSC-specific substrate(Gly-Phe-β-naphthylamide)revealed that the optimum pH and temperature of rTroCTSC were 5.5 and 40℃,respectively.Metal ion toxicity experiments showed that Ca2+ and Zn2+could significantly promote the activity of rTroCTSC,Fe2+ and Cu2+ could inhibit the enzymatic activity of rTroCTSC,while Mn2+ and Mg2+ had no significant inhibitory or promotional effects on the enzymatic activity of rTroCTSC.The activities of rTroCTSC-C251A,rTroCTSC-H397A and rTroCTSC-N419A were significantly lower compared to rTroCTSC,indicating that this active site is essential for TroCTSC to function.In vivo experiments were used to further analyze the effect of TroCTSC involvement in the organism’s antibacterial immunity.The results showed that when fish were infected with V.haveyi after overexpression of TroCTSC,the number of bacteria in spleen,head kidney and liver tissues was significantly lower in the overexpression group than in the control group,significantly increasing the antibacterial capacity of the organism.Consistent with this,siTroCTSC-treated fish showed significantly increased bacterial loads in spleen,head kidney and liver tissues compared to control fish.Studies on immune-related gene expression also revealed that TroCTSC overexpression significantly upregulated mRNA levels of various pro-inflammatory cytokines,including IFN-γ,TNF-α,IL-1β,IL-6 and IL-8,suggesting that TroCTSC enhances the organism’s resistance to bacterial infection.In summary,in this study,TroCTSC was cloned and characterized for antibacterial immunity in T.ovatus.The results showed that TroCTSC plays a key role in the host’s ability to regulate inflammatory responses,resist bacterial infections and enhance antimicrobial immune responses.Three active sites,Cys251,His397 and Asn419,affected the enzymatic activity of TroCTSC.The results of the study are important for further understanding the disease resistance mechanism of T.ovatus and provide a theoretical basis for the green and healthy sustainable development of T.ovatus farming.