Expression And Functional Analysis Of Ferritin Gene In Amphioxus Branchiostoma Belcheri

Amphioxus or lancelet, a protochordate, has long been regarded as a model animal for gaining understanding of the origin of vertebrates. Its genetic information on gene sequence and expression pattern has been widely used for interspecies comparative genome studies and developmental homology analysis.Ferritin plays a key role in cellular iron metabolism including iron storage and detoxification, which has been identified in a wide range of organisms including bacteria, fungi, plants and animals. However, little information is available regarding ferritin in the protochordates to date.The cDNA (GenBank accession number: AAQ21039) obtained from the gut cDNA library of B. belcheri is 1038 bp long, and its longest open reading frame (ORF) was 519 bp, corresponding to a deduced protein of 172 amino acids with a predicted molecular mass of approximately 20 kDa. The 5'-untranslated region (UTR) was 116 bp long, and the 3'-UTR 403 bp long with a polyadenylation signal AATAAA and a polyadenylation tail . The initiation codon (ATG) was assigned on the basis that there is no ATG in the 5'-UTR 116 nucleotides and the DNA surrounding the initiation codon AT G has a purine at positions of both -3 and +4, which is in accordance with the Kozak consensus sequence. Therefore, cDNA encodes a full-length sequence protein. Analysis of the 5'-UTR demonstrated the presence of a putative iron-responsive element (IRE) in the 5'UTR of BbFRT 73 bp upstream of the start codon ATG, which includes a typical CAGUGN loop and a bulged Cys. The predicted secondary structure of BbFRT IRE mRNA was a typical stem loop structure like that identified in most ferritin mRNAs.Sequence comparisons showed that BbFRT was more identical to H-chains (68%) of vertebrate ferritins than to the L-chains (46-51%). It also shared 72-77% identity to invertebrate ferritins documented so far. Phylogenetic analysis revealed two separate clusters of vertebrate and invertebrate ferritins . The new ferritin BbFRT was grouped together with the invertebrate ferritin cluster.An expression vector including the entire open reading frame of BbFRT and a 5'additional tag of pET28a is constructed and transformed into E. coli. Recombinant protein is expressed and purified. Rabbit antisera against the purified BbFRT were obtained. Western blotting analysis exhibited that the rabbit antisera reacted with B. belcheri humoral fluids, producing a ~20 kDa band, corresponding to the molecular mass predicted by BbFRT cDNA. These denoted that the rabbit antisera prepared display a conspicuous antigen-specific reactivity.Both in situ hybridization histochemistry and immunohistochemical staining revealed that BbFRT was ubiquitously expressed in B. belcheri.In addition, BbFRT expression was up-regulated by 1.6-fold and 1.5-fold, respectively, following exposure to LPS and iron at both transcriptional and translational levels. These suggest that BbFRT seems a protein with a dual function functioning in both immune response and iron metabolism.