Structure and expression of genes involved in transport and storage of iron in red-blooded and hemoglobin-less antarctic notothenioids Rosaria Scudiero a , Francesca Trinchella a , Marilisa Riggio a , Elio Parisi b, ⁎ a Department of Biological Sciences, University “Federico II”, Napoli, Italy b CNR Institute of Protein Biochemistry, via P. Castellino 111, 80131 Napoli, Italy Received 11 January 2007; received in revised form 3 March 2007; accepted 12 March 2007 Available online 21 March 2007 Abstract Antarctic notothenioids are characterized by a drastic reduction of the hemoglobin content, a condition that reaches its extreme in icefish that, following a gene deletion event, are completely devoid of hemoglobin. To answer the question on what type of adaptive changes occurred in icefish to prevent accumulation of potentially dangerous ferrous iron, we investigated the genes of four proteins known to play a key role in iron metabolism. For this purpose, we cloned and sequenced the cDNAs encoding ceruloplasmin, transferrin, ferritin and divalent metal transporter 1. While the inferred amino acid sequences of transferrin from different Antarctic fish species showed a high level of similarity with the homologous proteins from other species, ceruloplasmin sequence featured amino acid substitutions affecting a copper binding site. Another peculiarity was the presence in subunit H of the icefish ferritin of the two sets of sites involved in iron oxidation and iron mineralization, which in mammals are located on two distinct ferritin subunits. Significant differences in the expression levels of the four genes were found between hemoglobinless and red-blooded notothenioids. An increased expression of ceruloplasmin mRNA in icefish was interpreted as a compensatory mechanism to prevent accumulation of ferrous iron in hemoglobinless fish. In icefish, the amounts of ferritin H-chain mRNA measured in liver, blood and head kidney were lower than in the same organs of the red-blooded fish. In the spleen of both fishes, the expression levels of ferritin H-chain were significantly lower than in the spleen of a “pink-blooded” notothenioid with an intermediate hemoglobin content. Finally, the amount of divalent metal transporter mRNA measured in the head-kidney was lower in the icefish than in the same organ of its red-blooded counterpart. These results indicate that the loss of hemoglobin in icefish is accompanied by remodulation of the iron metabolism. © 2007 Elsevier B.V. All rights reserved. Keywords: Ceruloplasmin; Divalent metal transporter; Ferritin; Iron metabolism; mRNA expression; Transferrin 1. Introduction Antarctica is one of the coldest areas of the planet, but about 30 million years ago this continent and the surrounding waters were warmer than they are today. The movements of the Earth's land masses pushed Antarctica to a southern position causing its progressive cooling, whilst the opening of Drake's passage created a powerful water stream, known as the Antarctic Polar Front, that prevented mixing of Antarctic waters with waters of the more temperate ocean (Scher and Martin, 2006). Fossil evidence indicates that, before the cooling, different groups of fish thrived in Antarctica, but the mutated environmental conditions allowed the survival of few of these groups (Eastman, 1993). Today, the Antarctic marine habitat is dominated by the notothenioid suborder, comprising numerous species, representing about 95% of the Antarctic fish fauna. The evolutionary success of notothenioids seems to be linked to the development of peculiar adaptations that conferred advantages in coping with the severe environmental conditions. Most Antarctic fish keep their body temperature close to the water temperature without freezing thanks to the presence of powerful anti-freeze glycopeptides (DeVries, 1986). In addition, they achieved a decrease in blood viscosity through the reduction of the hematocrit (and consequently of the hemoglobin content) (Verde et al., 2006); a condition that reaches its extreme in the members of the Channichthyidae family (also known as icefish) Gene 397 (2007) 1 – 11 www.elsevier.com/locate/gene Abbreviations: bp, base pair(s); cDNA, DNA complementary to RNA; Cp, ceruloplasmin; DMT1, divalent metal transporter 1; Ft, ferritin; Hb, hemoglobin; PCR, polymerase chain reaction; Tf, transferrin; UTR, untranslated region(s). ⁎ Corresponding author. Tel.: +39 081 6132279; fax: +39 081 6132248. E-mail address: e.parisi@ibp.cnr.it (E. Parisi). 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.03.003