Ferritin M of Cynoglossus semilaevis: an iron-binding protein and a broad-spectrum antimicrobial that depends on the integrity of the ferroxidase center and nucleation center for biological activity.

Ferritin is a major intracellular iron storage protein in higher vertebrates and plays an important role in iron metabolism. In this study, we identified and analyzed the biological activity of a ferritin M subunit (CsFerM) from half-smooth tongue sole (Cynoglossus semilaevis). The open reading frame (ORF) of CsFerM is 534 bp and encodes a protein that shares 79.7-86.4% overall sequence identities with the ferritin M subunits of a number of teleosts. In silico analysis identified in CsFerM a eukaryotic ferritin domain with conserved ferroxidase diiron center and ferrihydrite nucleation center. Quantitative real time RT-PCR analysis showed that under normal physiological conditions, expression of CsFerM was highest in liver, moderate in gill, spleen, and muscle, and low in gut, heart, and brain. Following experimental challenge with bacterial pathogens, CsFerM expression was significantly upregulated in kidney, spleen, and liver in time-dependent manners. Biological activity analysis showed that recombinant CsFerM purified from Escherichia coli exhibited apparent iron-binding activity and, when present in the culture medium of six different species of fish bacterial pathogens, completely inhibited bacterial growth. In contrast, a mutant CsFerM that bears alanine substitution at two conserved residues of the ferroxidase diiron center and ferrihydrite nucleation center was abolished in both iron-binding and antimicrobial capacity. These results demonstrate that CsFerM is a biologically active iron chelator with broad-spectrum antibacterial activity, which suggests a role for CsFerM in not only iron storage but also innate immunity. These results also indicate the importance of the conserved iron uptake and mineralization sites to the function of CsFerM.