Preliminary Studies On Antioxidant Activity Of Peroxiredoxin Ⅳ And Its Interactive Protein In Large Yellow Croaker Pseudosciaena Crocea

Peroxiredoxin IV in large yellow croaker Pseudosciaena crocea, named as Lyc-PrxIV is a typical 2-Cys peroxidase with an N-terminal signal peptide. PrxIV proteins are mainly localized in the endoplasmic reticulum and extracelluar space, orchestrating multiple biological processes including antioxidant response, regulation of hydrogen peroxide mediated signal transduction, cell proliferation, heme synthesis, and protection of cells against injury and oxidative stress. To investigate the in vitro antioxidant activity of Lyc-PrxIV, we constructed a recombinant plasmid pCMV-Lyc-PrxIV to transiently transfect the HEK-293 T cells. The expression of Lyc-PrxIV in HEK-293 T cells was analysed by Western blot. The antioxidant activity of Lyc-PrxIV was evaluated by quantifing the amount of intracellular hydrogen peroxide. The results showed that the Lyc-PrxIV was correctly expressed in HEK-293 T cells transfected with pCMV-Lyc-PrxIV. The concentration of hydrogen peroxide in the cells transfected with pCMV-Lyc-PrxIV was considerably lower than that in cells transfected with pCMV at 6 h, 12 h and 24 h after transfection. These results indicate that the Lyc-PrxIV can disintegrate the hydrogen peroxide in cells, thus participating in the control of intracellular oxidation reduction status.To further determine the role of Lyc-PrxIV in host immune response and disease resistance, the yeast two-hybrid system as well as PCR amplification was used to obtain the Gateway○R joint restructuring reaction sequence of Lyc-PrxIV gene fragments. Ater BP, LR reorganization, a dual purpose fragment of yeast bait plasmid, termed as pDESTTM32-Lyc-PrxIV, was successfully constructed. The expression of pDESTTM32-Lyc-PrxIV was confirmed and no self-activation was detected at 25 mM 3-amino-1, 2, 4-triazole(3AT). We performed yeast two-hybrid screening experiments using the yeast strain MaV203 transformed with pDESTTM32-Lyc-PrxIV and a large yellow croaker spleen cDNA library. 15 positive clones of prey protein were screened by yeast two-hybrid system, and then they were identified by squence analysis. Two prey proteins were obtained, including large yellow croaker Peroxiredoxin II(Lyc-PrxII) and large yellow croaker Sulfiredoxin(Lyc-Srx). Furthermore, the validity of interactive proteins was confirmed by co-immunoprecipitation method in HEK-293 T. According to the structure and function of Lyc-PrxIV, we hypothesized that Lyc-PrxIV can form a decamer(pentamer of dimers) from the initial homologous dimers when Lyc-PrxIV and Lyc-PrxII interated together. As Lyc-PrxIV and Lyc-Srx belong to important endogenous enzymes in biological antioxidants, we thought that the interaction between PrxIV and Srx may occur during the redox reaction.Our previous study showed that Lyc-PrxIV plays an important role in the defense against bacterial infection in large yellow croaker. To further investigate the regulatory mechanism of PrxIV, 5’ flanking regulatory region sequence of Lyc-PrxIV was obtained using genome walker technology. Putative promoter region of Lyc-PrxIV was analyzed by bioinformatic methods. To verify the activity of 5’ flanking regulatory region sequence, pEGFP-Lyc-PrxIV-pro and pGL-3-Basic-Lyc-PrxIV-pro recombinant plasmids were constructed and then transfected into EPC cells respectively. Our results showed that the 5’ flanking regulatory region sequence of Lyc-PrxIV can activate the expression of EGFP, as indicated in the green fluorescence and luciferase reporter gene, thus exhibiting its promoter activity. In order to determine the Lyc-PrxIV promoter response to immune stimulation, the recombinant plasmid pGL-3-Basic-Lyc-PrxIV-pro and internal control plasmid were co-transfected to EPC cells and luciferase reporter assays were performed after stimulation with 10 μg/mL Poly(I:C), 100 ng/ mL LPS, 10 μg/mL LTA and 10 μg/mL PGN. The result demonstrated that the activity of the Lyc-PrxIV promoter was not affected by the immune stimulatants. By constructing deletion mutants of the promoter, the change in Lyc-PrxIV promoter activity was found, we predicted that the most weak region(-82/+145) of Lyc-PrxIV promoter can have basic transcriptional activity sites, and the most powerful region(-757/+145) of Lyc-PrxIV promoter contains positive regulatory regions.