Molecular Identification, Functional Characterization, And Mechanism Of NKEF And PBEF Genes In Tetraodon Nigroviridis

This article focused on the molecular identification, functional characterization, and mechanism of NKEF and PBEF genes in fish.In part one, based on the bioinformatics method such as synteny comparative analysis approach, we successfully identified and cloned the two highly conserved genes, NKEF-A and NKEF-B, in Tetraodon nigroviridis. The gene expression profiling of both normal and LPS-stimulated were also detected. The highly homologous and conserved active sites implied the conserved antioxidant activity of NKEF. Utilizing the soluble recombinant protein which expressed in E.coli, we analyzed the antioxidant function and mechanism of TnNKEF in cell level, in vitro molecular level, E.coli and zebrafish models. The results showed that TnNKEF exhibited antioxidant function which may partially due to the peroxidase activity. However, both the reduced and oxidized form of TnNKEF possessed a catalase-like activity, which may be related to the tertiary structure of NKEF.In part two, we cloned and analyzed the TnPBEF gene for the first time. Meanwhile, both the gene and protein expression profilings in normal condition were detected. To determine whether the vertebrates PBEF contributes to the biosynthesis of NAD and its associated activities, we reconstructed an extra pyridine nucleotide cycle III (PNC III) salvage pathway by transforming the fish PBEF gene into E.coli which lacks such pathway and measured the antioxidant activity of cells. Results clearly demonstrated that the reconstructed NAD salvage pathway could significantly increase the biosynthesis of NAD and enhance the cellular antioxidant activity. It indicated that PBEF can improve the cellular antioxidant activity by regulation of the NAD biosynthesis and the NAmPRTase-like activity of this cytokine is functionally conserved through evolution.So the antioxidant function of NKEF and PBEF were all related to the biosynthesis of NAD. Transforming the TnPBEF gene into the cells can significantly increase the cellular NAD and NADP concentrations, which can provide more reducing power for the peroxidase activity of NKEF. Such novel antioxidant pathway nearly existed in vertebrate and possessed certain theoretical significance and applicable value.