Cloning, Expression And Functional Characterization Of The Ferritin M Of Paralichthys Olivaceus

In this study, two-dimensional electrophoresis(2-DE) combined with mass spectrometry(MALDI-TOF MS/MS) were conducted to examine flounder proteins altered in expression at 12 h post Edwardsiella tarda infection. Twenty differentially expressed proteins from spleen of flounder were identified, and one of the upreguleted protein ferritin was deep researched.Ferritin is an evolutionarily conserved protein that plays a vital role in maintaining iron homeostasis. In this study, we identified a ferritin M(PoFerM) from Japanese flounder(Paralichthys olivaceus) and analyzed its biological property. PoFerM is composed of 176 amino acid residues and contains the conserved ferroxidase site and the ferrihydrite nucleation center typical of M ferritins.Expression of PoFerM occurred in multiple tissues and was most abundant in blood. Bacterial infection upregulated PoFerM expression in head kidney, spleen, and liver in a time-dependent manner. Recombinant PoFerM(rPoFerM) purified from Escherichia coli exhibited iron-chelating activity and inhibited bacterial growth, whereas rPoFerMM, the mutant protein that bears alanine substitution at two conserved residues of the ferroxidase center and the ferrihydrite nucleation center, failed to do so.Oxidative protection analysis showed that rPoFerM, but not rPoFerMM, was able to alleviate the deleterious effect caused by hydroxyl radicals through Fenton-type reactions on plasmid DNA and primary flounder cells. Furthermore, E. coli expressing rPoFerM, but not rPoFerMM, exhibited significantly increased survival capacity upon H2O2 exposure. Together these results indicate that PoFerM is an iron chelator with antimicrobial and antioxidative properties, all which depend on the conserved ferroxidase center and the ferrihydrite nucleation site.