Abstract
BackgroundIron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit). Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron.MethodsSequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR) fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas). The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA.ResultsDolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98) ; L: 98–100%). The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species.ConclusionThese results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed.
Highlights
Iron-storage protein, ferritin plays a central role in iron metabolism
Preparation of total RNA and genomic DNA from blood samples Blood samples were obtained from the tail veins of apparently healthy individuals of six different species of dolphin (P. crassidens, L. obliquidens, G. griseus, G. macrorhynchus, T. truncatus, and D. leucas) kept at Yokohama Hakkeijima Sea Paradise (Yokohama, Japan)
Sequence analysis of dolphin ferritin H and L subunits Two or more cDNA (~4 clones) clones were sequenced for each ferritin subunit with the exception of the L subunits of L. obliquidens and T. truncatus, for which only one clone each was sequenced
Summary
Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit). It functions to segregate iron in a non-toxic form to prevent iron-catalyzed reactive oxygen species production [3,4,5,6,7]. Mammalian tissue ferritins consist of two functionally different subunits, termed as H (heavy chain, heart-type) and L (light chain, liver-type), and a central cavity accommodating up to 4,500 iron atoms [1,8]. The H subunit plays a crucial role in incorporating iron through its ferroxidase activity [10,11]. The L subunit lacks ferroxidase activity, but is involved in iron nucleation to allow more iron to be sequestered [8,1113]
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