Cloning, Expression And Functional Analysis Of Cynoglossus Semilaevis Thioredoxin

Thioredoxin (Trx) is a small redox protein existing ubiquitously in all living organisms and plays an important role in multiple cellular processes, including transcriptional regulation and immune response. To date very few studies have been carried out to examine the function of piscine Trx. In this study, we identified and analyzed the function of a Trx homologue, CsTrx1, from half-smooth tongue sole (Cynoglossus semilaevis). The deduced amino acid sequence of CsTrxl is composed of107residues and shares54.1-60.8%overall identities with the Trx of other teleosts. CsTrxl contains the highly conserved CXXC motif, which in mammals is known to be the active site, in the form of CQPC. Expression of CsTrxl as determined by quantitative real-time reverse transcriptase PCR was highest in liver and upregulated in time-dependent manners by bacterial infection and by exposure to iron, copper, and hydrogen peroxide. Purified recombinant CsTrxl (rCsTrxl) exhibited insulin disulfide reductase activity and antioxidant activity, both which, however, were lost when the two cysteine residues in the CQPC motif were mutated to serine. Further analysis showed that rCsTrxl was able to stimulate the proliferation of head kidney leukocytes, upregulate the expression of immune relevant genes, and enhance the resistance of leukocytes against bacterial infection. Taken together, these results indicate that CsTrxl is a biologically active reductase and an antioxidant that requires the CXXC motif for activity and that CsTrxl possesses cytokine-like immunoregulatory property. These results suggest a role for CsTrxl in protecting cells against oxidative stress caused by oxidant exposure and pathogen infection.