Two novel secreted ferritins involved in immune defense of Chinese mitten crab Eriocheir sinensis

As a principal extracellular iron storage molecule, secreted ferritin plays an important role in the iron-withholding strategy of innate immunity. In this study, two novel secreted ferritins were identified from Chinese mitten crab Eriocheir sinensis (designated as EsFer-1 and EsFer-2) by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNAs of EsFer-1 and EsFer-2 were of 1278 and 1595 bp, respectively, both containing a putative iron response element (IRE) in their 5′ UTRs and multiple A + U-destabilizing elements (TATT or ATTTA) in their 3′ UTRs. The ORFs of these two crab ferritin cDNAs were of 639 and 663 bp, respectively, encoding two peptides of 212 and 220 amino acid residues each with a signal peptide and typical structures of ferritins such as four long α-helices, one short α-helix and an L-loop. EsFer-2 exhibited higher similarity with the H-ferritins from both invertebrates and vertebrates, while EsFer-1 was closer matched to L-ferritins. The eight amino acid residues identified as metal binding sites in vertebrate H-ferritins were conserved in EsFer-2 (Glu53, Tyr60, Glu87, Glu88, His91, Glu146, Glu177 and Gln178), but none of them was observed in EsFer-1. By fluorescent quantitative real-time PCR, mRNA transcripts of EsFer-1 and EsFer-2 were mainly detected in muscle, hepatopancreas and gill, and also marginally detectable in gonad, heart and hemocytes. After the crabs were challenged by bacteria Listonella anguillarum, the transcriptional levels of both EsFer-1 and EsFer-2 in hemocytes were up-regulated twice. In the first up-regulation, the mRNA relative expression levels of both EsFer-1 and EsFer-2 reached peak at 3 h post-challenge, while in the second up-regulation, they did not reach the highest point within the experiment duration. After the fungi Pichia pastoris GS115 challenge, there was only one transcriptional level peak of both the two ferritins, appearing at 6 h post-challenge. These results suggest that secreted EsFer-1 and EsFer-2 are crucial proteins in the iron-withholding defense system, and play important roles in the innate immune responses in crabs.