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Ferroportin1: a new iron export molecule?
The International Journal of Biochemistry & Cell Biology 34 (2002) 103–108 www.elsevier.com/locate/ijbcb
Molecules in focus
Ferroportin1: a new iron export molecule? Nghia T.V. Le, Des R. Richardson * The Iron Metabolism and Chelation Group, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW 2050, Australia Received 5 June 2001; accepted 30 July 2001
Abstract Ferroportin1 is a newly discovered molecule that may play a role in iron export. It is expressed on the basolateral surfaces of mature enterocytes within the duodenum and in macrophages of the spleen and liver. Furthermore, this protein was found to be expressed in placental syncytiotrophoblasts and may be involved in the supply of maternal iron to the fetus. Sequence analysis of ferroportin1 predicts it has ten transmembrane domains, a reductase site and a basolateral localization signal. In addition, the ferroportin1 mRNA transcript contains an iron response element in its 5% untranslated region. This review is focused on the current state of knowledge on ferroportin1 and the medical implications of this discovery. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Iron; Absorption; Release; Export; Ferroportin1; IREG1; MTP1; Hemochromatosis
1. Introduction Iron (Fe) is an indispensable element involved in many processes vital for life, it is an important cofactor for proteins that are required for energy production, DNA synthesis and oxygen transport. In contrast, an excessive accumulation of intracellular Fe is unfavourable, since it can react with oxygen to form cytotoxic free radicals [1]. As a consequence, cellular Fe homeostasis is tightly regulated. * Corresponding author. Tel.: + 61-2-9550-3560; fax: + 612-9550-3302. E-mail address: [email protected] (D.R. Richardson).
The absorption of Fe into the body is controlled at the cellular level by duodenal enterocytes. Recently, the identification of Nramp2 (also known as the divalent metal transporter 1 or the divalent cation transporter 1) as an Fe import molecule has given important insight into the mechanism of Fe absorption (for a review see Ref. [2]). However, very little is known about the transport of absorbed Fe into the circulation. Previous studies have indirectly shown that the ceruloplasmin homologue, hephaestin, may be involved in the export of Fe from enterocytes in sex-linked anaemia (sla) mice [3]. However, hephaestin possesses only one transmembrane domain and is unlikely to be an Fe export molecule [3]. Moreover, it has been suggested that hep-
1357-2725/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 1 ) 0 0 1 0 4 - 2
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haestin may interact with an undiscovered Fe export molecule to facilitate the transfer of the metal into the circulation [3]. Recently, several groups of investigators have isolated a putative Fe export molecule within several months of each other [4– 6]. This protein was named Fe-regulated transporter 1 (IREG1) by McKie et al. [6] who were the first to identify and clone it. Subsequently, the same molecule was also named ferroportin1 [5] and metal transport protein 1 (MTP1) [4]. For simplicity, this molecule will be referred to as ferroportin1 in this review (Fig. 1).
Ferroportin1 was identified using three different techniques. Donovan et al. [5] identified ferroportin1 by positionally cloning the gene responsible for hypochromic anaemia in zebrafish. In contrast, McKie et al. [6] implemented subtractive cloning from the cDNA of hypotransferrinemic mice and Abboud et al. [4] used systemic evolution of ligands by exponential enrichment (SELEX) technologies to identify this gene. Current evidence suggests that ferroportin1 is involved in Fe export. Indeed, Zebrafish containing the weissherbst (weh) mutation lacked the ability to produce ferroportin1 and, as a conse-
Fig. 1. Schematic diagram of a possible model illustrating ferroportin1 (FPN1) and its role in Fe absorption by the duodenal enterocyte. Iron is absorbed from the lumen of the duodenum by the transport of Fe(II) via Nramp2 (also known as the divalent metal ion transporter 1 or divalent cation transporter 1). Iron(III) in the lumen is reduced by the action of the ferric reductase, duodenal cytochrome b (Dcytb). Once the Fe enters the cell it then becomes part of the poorly defined intracellular Fe pool. Iron in the pool can be used for incorporation into the iron storage protein, ferritin, or it can be released by the basolateral transporter, ferroportin1. The mechanism whereby ferroportin1 obtains Fe remains unknown, although Fe-binding chaperones and hephaestin (HPN) could play some role. It should be noted that the exact location of hephaestin remains unknown at present and its position in this figure is speculative. Chaperones could deliver either Fe(II) or Fe(III) to ferroportin1, where it is probably transported across the membrane in the Fe(II) state. Where Fe(III) interacts with ferroportin1, it could be reduced directly by the putative reductase activity of this protein. Once the Fe(II) reaches the external surface of the cell it is probably oxidized to Fe(II) by the plasma ferroxidase ceruloplasmin (Cp). The Fe(III) then binds to the serum Fe transport protein apotransferrin (see text for further details).
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Fig. 2. Schematic illustration of the structure of ferroportin1 (FPN1) showing the amino acid sequence corresponding to the ten transmembrane domains, the NADP/adenine binding site (that may confer possible reductase activity) and the PDZ target motifs (which may act as a basolateral localization signal) (see text for details).
quence, developed a hypochromic anaemia [5]. In addition, ferroportin1 expression in humans and mice has been shown to occur in areas that are critical for Fe absorption, i.e. duodenal enterocytes [4– 6]. This review will focus on the current state of knowledge regarding the role of ferroportin1 as a putative Fe exporter.
2. Structure Mouse ferroportin1 cDNA encodes a protein 570 amino acids in length with a predicted mass of 62 kDa [6]. Sequence data showed that ferroportin1 has ten putative transmembrane domains (Fig. 2) [4–6]. This protein is extremely well conserved in humans, mice and rats, showing \ 90% homology [4]. Human ferroportin1 shows little homology to the Fe importer Nramp2, although uncharacterised ferroportin1-related proteins were found in Arabidopsis and C. elegans [4 – 6]. Further analysis of the ferroportin1 protein by McKie et al. [6] showed a putative NADP/ adenine-binding site and basolateral localization signal present at its C-terminus (Fig. 2). The NADP/adenine-binding sequence of ferroportin1 comprises an IFVCGP motif that is also found in yeast ferric reductase and the neutrophil oxidoreductase gp91-phox [8]. This suggests that ferroportin1 may have a reductase activity involved in Fe transportation. Both Fe(II) and Fe(III) are known to exist in complex equilibrium within cells and the conversion between these redox states
may be important in terms of transportation and sequestration [7,9]. Indeed, the export of Fe(III) from cells may involve the conversion of Fe(III) to Fe(II) by the reductase region of ferroportin1 and this should be investigated further. The last four amino acids (TSVV) of ferroportin1 comprise a T/S-X-V/L PDZ target motif that is readily recognised by PDZ proteins. PDZ proteins are thought to be involved in the basolateral localization of proteins containing this target motif. (Fig. 2). Sequence analysis of ferroportin1 mRNA revealed the presence of an iron response element (IRE) in its 5% untranslated end [4–6]. The 5% IRE sequence of ferroportin1 is well conserved between humans, mice and zebrafish. Similar 5% IREs occur in transcripts encoding ferritin, erythroid-5-aminolevulinate synthase and mitochondrial aconitase (for review see Ref. [7]).
3. Biological function Despite ferroportin1 having a 5% IRE sequence, the regulation of its expression is currently poorly understood. IREs are sequences that form stable stem-loop structures capable of binding the iron regulatory proteins (IRP) (for review see [7]). Cells depleted of Fe contain higher levels of IRPs, which form stable complexes with the IRE [7]. When an IRE is present in the 5% end of the mRNA, formation of the IRE/IRP complex can prevent mRNA translation. However, in contrast to IRP-IRE theory, it was reported that Fe-defi-
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ciency (induced via a low Fe diet) induces ferroportin1 expression in duodenal enterocytes, while in Fe-replete mice (induced by i.m. Fe-dextran injection) lower expression was found [4]. These results suggest the presence of an IRE-independent pathway that controls the expression of ferroportin1 in enterocytes (Fig. 2). Moreover, another IRE-independent pathway was also observed in placental cells where Fe-depleted mice showed no changes in the expression of ferroportin1 [10]. In contrast to the expression of ferroportin1 in enterocytes and placental cells, the molecule was regulated in accordance to IRP-IRE theory in Kupffer cells of the liver [4]. Together these results suggest complex tissue-specific regulation of ferroportin1 that may be related to various functions of the molecule in different cell types. Studies have shown that many metal transporters are not uniquely specific for Fe. Indeed, Nramp2 has shown an ability to transport other ions, such as Zn(II), Cu(II), Co(II) and Cd(II) [11]. Thus, further studies examining the ability of ferroportin1 to transport ions other than Fe are required. In addition, while the name ‘ferroportin1’ implies Fe(II) transport, there have been no direct studies to determine whether the molecule transports either Fe(II) or Fe(III) or both. The expression of ferroportin1 in tissues that are critical in Fe absorption is further evidence for its role in this process. Ferroportin1 was expressed at the basolateral membrane of villus enterocytes within the duodenum, particularly at the tip of the villus rather than the crypt [4– 6]. However, while ferroportin1 is expressed in the duodenum [4 –6], it is not produced in either the jejunum [5,6] or ileum [5]. Other important sites of Fe metabolism where ferroportin1 expression occurs are the liver and spleen [4,5]. These tissues are involved in scavenging Fe from senescent erythrocytes. Immunostaining of these tissues revealed the expression of ferroportin1 in the cytoplasm of Kupffer cells, the cell surface of hepatocytes lining sinusoids and splenic macrophages in mice [4]. Macrophages and Kupffer cells found in the spleen or liver, respectively, readily engulf senescent erythrocytes
to release the Fe following haemoglobin degradation. Ferroportin1 is not expressed on plasma membranes of macrophages [4]. However, it may still be a vital component for Fe export. For instance, cytoplasmic ferroportin1 may be involved in the transportation of Fe into acidic cellular vesicles that may release Fe at the cell surface via exocytosis. Ferroportin1 was also readily detected at the basal surface of placental syncytiotrophoblasts [5] which interfaces with the fetal circulation (the apical surface of these cells interface with the maternal circulation). Furthermore, the expression of ferroportin1 in placental tissue is highest during the third trimester of pregnancy and is developmentally regulated [6]. This suggests a possible role for ferroportin1 in Fe transport from the mother to the developing fetus [5]. Other tissues where ferroportin1 was expressed include the large intestine [5], heart [5], skeletal muscle [4,5], kidney [6], testis [6], megakaryocytes [4] and the decidua of the pregnant uterus [4]. Iron efflux studies were conducted to further evaluate the Fe export function of ferroportin1. In these studies, Nramp2 (DMT1/DCT1) was coexpressed with ferroportin1 in order to label Xenopus oocytes with 55Fe [5,6]. Despite using the same expression system to evaluate ferroportin1mediated Fe efflux, both investigators produced slightly different results. Donovan et al. demonstrated the efflux of Fe by ferroportin1 required apotransferrin (apoTf) [5]. In contrast, McKie et al. [6] showed that Fe efflux by ferroportin1 did not require apoTf, but ceruloplasmin (Cp) instead. These contradictory results may be due to differences in the experimental protocols used in the Fe efflux assays (namely washing the cells after incubation with the 55Fe-complex used to label the cells [6] compared to not washing [5]). Obviously, if the cells are not washed after incubation with the 55Fe, non-specifically bound 55Fe bound to the external cell surface will be released after incubation with apoTf. Regardless of this, both authors were able to show that ferroportin1 may play a role in Fe export in the Xenopus expression model. Other studies showed that transient transfection of cells with a ferroportin1 construct without an
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IRE motif, demonstrated expression of the molecule at the plasma membrane [4]. In addition, these cells were found to contain higher levels of IRE-IRP binding and decreased amounts of ferritin protein [4], indirectly suggesting that the cells became Fe-deplete upon overexpression of ferroportin1. Collectively, the evidence obtained from ferroportin1 efflux and expression studies suggests the important role it plays in Fe absorption [4– 6]. However, the expression of ferroportin1 in the cytoplasm of Kupffer cells and enterocytes [4] suggests the possibility that ferroportin1 may be involved in the intracellular trafficking of Fe between the cytosol and organelles. The detection of ferroportin1 in placental syncytiotrophoblasts [5] and decidual cells of the pregnant uterus [4] suggests that ferroportin1 may play a role in the transfer of Fe from mother to embryo. Over the past few years, numerous discoveries at the molecular level have given us a better understanding of iron absorption. These novel proteins include the divalent metal transporter 1 [11], hephaestin [3], the duodenal cytochrome b ferric reductase [12] and ferroportin1. At the cellular level, Fe is absorbed at the apical membranes of villus enterocytes within the duodenum. While the uptake of Fe into enterocytes is relatively well documented, little is known about its release into the circulation. In the past, there was speculation concerning the existence of an Fe exporter at the basolateral membrane of enterocytes. The recent discoveries of ferroportin1 and hephaestin have given us an insight into Fe export from duodenal enterocytes (Fig. 1). However, the link between these molecules and the mysterious labile Fe pool remains unclear. Investigators studying copper absorption have reported the presence of intracellular copper chaperone proteins [13]. Such proteins may also exist for the intracellular trafficking of Fe and could be involved in the transfer of Fe(III) or Fe(II) from the Fe pool to ferroportin1 for export. It can be speculated that the release of Fe from its chaperone may require hephaestin. Hephaestin may interact with the Fe chaperone to facilitate Fe transfer to ferroportin1. Once transported, Fe(II) may then be released into the circulation and oxidized by Cp (Fig. 1).
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4. Possible medical applications The discovery of ferroportin1 may also be of medical benefit for the diagnosis and possible treatment of Fe overload and deficiencies. For example, the zebrafish weh mutation results in a hypochromic anaemia. It is possible that a similar condition due to the mutation of ferroportin1 exists in humans. Recently, a mutation in ferroportin1 has been associated with the development of autosomal dominant hemochromatosis [14]. A single base pair mutation at position 734 in exon 5 causes an amino acid substitution (Asp to His). This substitution may cause excessive transportation of Fe into the circulation, thus contributing to the pathology of hereditary hemochromatosis [14]. Obviously, the discovery of ferroportin1 allows the detection and possible correction of such disease states.
References [1] J.M. McCord, Iron, free radicals, and oxidative injury, Semin. Hematol. 35 (1998) 5 – 12. [2] C.N. Roy, C.A. Enns, Iron homeostasis: new tales from the crypt, Blood 96 (2000) 4020 – 4027. [3] C.D. Vulpe, Y. Kuo, T.L. Murphy, L. Cowley, C. Askwith, N. Libina, J. Gitschier, G.J. Anderson, Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse, Nat. Genet. 21 (1999) 195 – 199. [4] S. Abboud, D.J. Haile, A novel mammalian iron-regulated protein involved in intracellular iron metabolism, J. Biol. Chem. 275 (2000) 19906 – 19912. [5] A. Donovan, A. Alison, Y. Zhou, J. Shepard, S. Pratt, J. Moynihan, B.H. Paw, A. Drejer, B. Barut, A. Zapata, L. Law, C. Brugnara, S.E. Lux, G.S. Pinkus, J.L. Pinkus, P.D. Kingsley, J. Palis, M.D. Fleming, N.C. Andrews, L.I. Zon, Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter, Nature 403 (2000) 776 – 781. [6] A.T. McKie, P. Marciani, A. Rolfs, K. Brennan, K. Wehr, D. Barrow, S. Miret, A. Bomford, T.J. Peters, F. Farzaneh, M.A. Hediger, M.W. Hentze, R.J. Simpson, A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation, Mol. Cell. 5 (2000) 299 – 309. [7] D.R. Richardson, P. Ponka, The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells, Biochim. Biophys. Acta 1331 (1997) 1 – 40.
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[8] A.W. Segal, A. Abo, The biochemical basis of the NADPH oxidase of phagocytes, Trends Biochem. Sci. 18 (1993) 43 – 47. [9] T.G. St. Pierre, D.R. Richardson, E. Baker, J. Webb, A low-spin iron complex in human melanoma and rat hepatoma cells and a high-spin iron(II) complex in rat hepatoma cells, Biochim. Biophys. Acta 1135 (1992) 154 – 158. [10] L. Gambling, R. Danzeisen, S. Gair, R.G. Lea, Z. Charania, N. Solanky, K.D. Jorry, S.K.S. Srai, H.J. McArdle, Effect of iron deficiency on placental transfer of iron and expression of iron transport proteins in vivo and in vitro, Biochem. J. 356 (2001) 883 – 889. [11] H. Gunshin, B. Mackenzie, U.V. Bergers, Y. Gunshin, M.F. Romero, W.F. Boron, S. Nussberger, J.L. Gollan, M.A. Hediger, Cloning and characterization of a mam-
malian proton-coupled metal ion transporter, Nature 388 (1997) 482 – 488. [12] A.T. McKie, D. Barrows, G.O. Latunde-Dada, A. Rolfs, G. Sager, E. Mudaly, M. Mudaly, C. Richardson, D. Barlow, A. Bomford, T.J. Peters, K.B. Raja, S. Shirali, M.A. Hediger, F. Farzaneh, R.J. Simpson, An iron-regulated ferric reductase associated with the absorption of dietary iron, Science 291 (2001) 1755 – 1759. [13] M. Harrison, C. Jones, C. Dameron, Copper chaperones: function, structure and copper-binding properties, J. Biol. Inorg. Chem. 4 (1999) 145 – 153. [14] O.T. Njajou, N. Vaessen, M. Joosse, B. Berghuis, J.W.F. van Dongen, M.H. Breuning, P.J.L.M. Snijders, W.P.F. Rutten, L.A. Sandkuijl, B.A. Oostra, C.M. van Duijn, P. Heutink, A mutation on SLC11A3 is associated with autosomal dominant hemochromatosis, Nat. Genet. 28 (2001) 213 – 214.
Molecules in focus
Ferroportin1: a new iron export molecule? Nghia T.V. Le, Des R. Richardson * The Iron Metabolism and Chelation Group, The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW 2050, Australia Received 5 June 2001; accepted 30 July 2001
Abstract Ferroportin1 is a newly discovered molecule that may play a role in iron export. It is expressed on the basolateral surfaces of mature enterocytes within the duodenum and in macrophages of the spleen and liver. Furthermore, this protein was found to be expressed in placental syncytiotrophoblasts and may be involved in the supply of maternal iron to the fetus. Sequence analysis of ferroportin1 predicts it has ten transmembrane domains, a reductase site and a basolateral localization signal. In addition, the ferroportin1 mRNA transcript contains an iron response element in its 5% untranslated region. This review is focused on the current state of knowledge on ferroportin1 and the medical implications of this discovery. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Iron; Absorption; Release; Export; Ferroportin1; IREG1; MTP1; Hemochromatosis
1. Introduction Iron (Fe) is an indispensable element involved in many processes vital for life, it is an important cofactor for proteins that are required for energy production, DNA synthesis and oxygen transport. In contrast, an excessive accumulation of intracellular Fe is unfavourable, since it can react with oxygen to form cytotoxic free radicals [1]. As a consequence, cellular Fe homeostasis is tightly regulated. * Corresponding author. Tel.: + 61-2-9550-3560; fax: + 612-9550-3302. E-mail address: [email protected] (D.R. Richardson).
The absorption of Fe into the body is controlled at the cellular level by duodenal enterocytes. Recently, the identification of Nramp2 (also known as the divalent metal transporter 1 or the divalent cation transporter 1) as an Fe import molecule has given important insight into the mechanism of Fe absorption (for a review see Ref. [2]). However, very little is known about the transport of absorbed Fe into the circulation. Previous studies have indirectly shown that the ceruloplasmin homologue, hephaestin, may be involved in the export of Fe from enterocytes in sex-linked anaemia (sla) mice [3]. However, hephaestin possesses only one transmembrane domain and is unlikely to be an Fe export molecule [3]. Moreover, it has been suggested that hep-
1357-2725/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 0 1 ) 0 0 1 0 4 - 2
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haestin may interact with an undiscovered Fe export molecule to facilitate the transfer of the metal into the circulation [3]. Recently, several groups of investigators have isolated a putative Fe export molecule within several months of each other [4– 6]. This protein was named Fe-regulated transporter 1 (IREG1) by McKie et al. [6] who were the first to identify and clone it. Subsequently, the same molecule was also named ferroportin1 [5] and metal transport protein 1 (MTP1) [4]. For simplicity, this molecule will be referred to as ferroportin1 in this review (Fig. 1).
Ferroportin1 was identified using three different techniques. Donovan et al. [5] identified ferroportin1 by positionally cloning the gene responsible for hypochromic anaemia in zebrafish. In contrast, McKie et al. [6] implemented subtractive cloning from the cDNA of hypotransferrinemic mice and Abboud et al. [4] used systemic evolution of ligands by exponential enrichment (SELEX) technologies to identify this gene. Current evidence suggests that ferroportin1 is involved in Fe export. Indeed, Zebrafish containing the weissherbst (weh) mutation lacked the ability to produce ferroportin1 and, as a conse-
Fig. 1. Schematic diagram of a possible model illustrating ferroportin1 (FPN1) and its role in Fe absorption by the duodenal enterocyte. Iron is absorbed from the lumen of the duodenum by the transport of Fe(II) via Nramp2 (also known as the divalent metal ion transporter 1 or divalent cation transporter 1). Iron(III) in the lumen is reduced by the action of the ferric reductase, duodenal cytochrome b (Dcytb). Once the Fe enters the cell it then becomes part of the poorly defined intracellular Fe pool. Iron in the pool can be used for incorporation into the iron storage protein, ferritin, or it can be released by the basolateral transporter, ferroportin1. The mechanism whereby ferroportin1 obtains Fe remains unknown, although Fe-binding chaperones and hephaestin (HPN) could play some role. It should be noted that the exact location of hephaestin remains unknown at present and its position in this figure is speculative. Chaperones could deliver either Fe(II) or Fe(III) to ferroportin1, where it is probably transported across the membrane in the Fe(II) state. Where Fe(III) interacts with ferroportin1, it could be reduced directly by the putative reductase activity of this protein. Once the Fe(II) reaches the external surface of the cell it is probably oxidized to Fe(II) by the plasma ferroxidase ceruloplasmin (Cp). The Fe(III) then binds to the serum Fe transport protein apotransferrin (see text for further details).
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Fig. 2. Schematic illustration of the structure of ferroportin1 (FPN1) showing the amino acid sequence corresponding to the ten transmembrane domains, the NADP/adenine binding site (that may confer possible reductase activity) and the PDZ target motifs (which may act as a basolateral localization signal) (see text for details).
quence, developed a hypochromic anaemia [5]. In addition, ferroportin1 expression in humans and mice has been shown to occur in areas that are critical for Fe absorption, i.e. duodenal enterocytes [4– 6]. This review will focus on the current state of knowledge regarding the role of ferroportin1 as a putative Fe exporter.
2. Structure Mouse ferroportin1 cDNA encodes a protein 570 amino acids in length with a predicted mass of 62 kDa [6]. Sequence data showed that ferroportin1 has ten putative transmembrane domains (Fig. 2) [4–6]. This protein is extremely well conserved in humans, mice and rats, showing \ 90% homology [4]. Human ferroportin1 shows little homology to the Fe importer Nramp2, although uncharacterised ferroportin1-related proteins were found in Arabidopsis and C. elegans [4 – 6]. Further analysis of the ferroportin1 protein by McKie et al. [6] showed a putative NADP/ adenine-binding site and basolateral localization signal present at its C-terminus (Fig. 2). The NADP/adenine-binding sequence of ferroportin1 comprises an IFVCGP motif that is also found in yeast ferric reductase and the neutrophil oxidoreductase gp91-phox [8]. This suggests that ferroportin1 may have a reductase activity involved in Fe transportation. Both Fe(II) and Fe(III) are known to exist in complex equilibrium within cells and the conversion between these redox states
may be important in terms of transportation and sequestration [7,9]. Indeed, the export of Fe(III) from cells may involve the conversion of Fe(III) to Fe(II) by the reductase region of ferroportin1 and this should be investigated further. The last four amino acids (TSVV) of ferroportin1 comprise a T/S-X-V/L PDZ target motif that is readily recognised by PDZ proteins. PDZ proteins are thought to be involved in the basolateral localization of proteins containing this target motif. (Fig. 2). Sequence analysis of ferroportin1 mRNA revealed the presence of an iron response element (IRE) in its 5% untranslated end [4–6]. The 5% IRE sequence of ferroportin1 is well conserved between humans, mice and zebrafish. Similar 5% IREs occur in transcripts encoding ferritin, erythroid-5-aminolevulinate synthase and mitochondrial aconitase (for review see Ref. [7]).
3. Biological function Despite ferroportin1 having a 5% IRE sequence, the regulation of its expression is currently poorly understood. IREs are sequences that form stable stem-loop structures capable of binding the iron regulatory proteins (IRP) (for review see [7]). Cells depleted of Fe contain higher levels of IRPs, which form stable complexes with the IRE [7]. When an IRE is present in the 5% end of the mRNA, formation of the IRE/IRP complex can prevent mRNA translation. However, in contrast to IRP-IRE theory, it was reported that Fe-defi-
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ciency (induced via a low Fe diet) induces ferroportin1 expression in duodenal enterocytes, while in Fe-replete mice (induced by i.m. Fe-dextran injection) lower expression was found [4]. These results suggest the presence of an IRE-independent pathway that controls the expression of ferroportin1 in enterocytes (Fig. 2). Moreover, another IRE-independent pathway was also observed in placental cells where Fe-depleted mice showed no changes in the expression of ferroportin1 [10]. In contrast to the expression of ferroportin1 in enterocytes and placental cells, the molecule was regulated in accordance to IRP-IRE theory in Kupffer cells of the liver [4]. Together these results suggest complex tissue-specific regulation of ferroportin1 that may be related to various functions of the molecule in different cell types. Studies have shown that many metal transporters are not uniquely specific for Fe. Indeed, Nramp2 has shown an ability to transport other ions, such as Zn(II), Cu(II), Co(II) and Cd(II) [11]. Thus, further studies examining the ability of ferroportin1 to transport ions other than Fe are required. In addition, while the name ‘ferroportin1’ implies Fe(II) transport, there have been no direct studies to determine whether the molecule transports either Fe(II) or Fe(III) or both. The expression of ferroportin1 in tissues that are critical in Fe absorption is further evidence for its role in this process. Ferroportin1 was expressed at the basolateral membrane of villus enterocytes within the duodenum, particularly at the tip of the villus rather than the crypt [4– 6]. However, while ferroportin1 is expressed in the duodenum [4 –6], it is not produced in either the jejunum [5,6] or ileum [5]. Other important sites of Fe metabolism where ferroportin1 expression occurs are the liver and spleen [4,5]. These tissues are involved in scavenging Fe from senescent erythrocytes. Immunostaining of these tissues revealed the expression of ferroportin1 in the cytoplasm of Kupffer cells, the cell surface of hepatocytes lining sinusoids and splenic macrophages in mice [4]. Macrophages and Kupffer cells found in the spleen or liver, respectively, readily engulf senescent erythrocytes
to release the Fe following haemoglobin degradation. Ferroportin1 is not expressed on plasma membranes of macrophages [4]. However, it may still be a vital component for Fe export. For instance, cytoplasmic ferroportin1 may be involved in the transportation of Fe into acidic cellular vesicles that may release Fe at the cell surface via exocytosis. Ferroportin1 was also readily detected at the basal surface of placental syncytiotrophoblasts [5] which interfaces with the fetal circulation (the apical surface of these cells interface with the maternal circulation). Furthermore, the expression of ferroportin1 in placental tissue is highest during the third trimester of pregnancy and is developmentally regulated [6]. This suggests a possible role for ferroportin1 in Fe transport from the mother to the developing fetus [5]. Other tissues where ferroportin1 was expressed include the large intestine [5], heart [5], skeletal muscle [4,5], kidney [6], testis [6], megakaryocytes [4] and the decidua of the pregnant uterus [4]. Iron efflux studies were conducted to further evaluate the Fe export function of ferroportin1. In these studies, Nramp2 (DMT1/DCT1) was coexpressed with ferroportin1 in order to label Xenopus oocytes with 55Fe [5,6]. Despite using the same expression system to evaluate ferroportin1mediated Fe efflux, both investigators produced slightly different results. Donovan et al. demonstrated the efflux of Fe by ferroportin1 required apotransferrin (apoTf) [5]. In contrast, McKie et al. [6] showed that Fe efflux by ferroportin1 did not require apoTf, but ceruloplasmin (Cp) instead. These contradictory results may be due to differences in the experimental protocols used in the Fe efflux assays (namely washing the cells after incubation with the 55Fe-complex used to label the cells [6] compared to not washing [5]). Obviously, if the cells are not washed after incubation with the 55Fe, non-specifically bound 55Fe bound to the external cell surface will be released after incubation with apoTf. Regardless of this, both authors were able to show that ferroportin1 may play a role in Fe export in the Xenopus expression model. Other studies showed that transient transfection of cells with a ferroportin1 construct without an
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IRE motif, demonstrated expression of the molecule at the plasma membrane [4]. In addition, these cells were found to contain higher levels of IRE-IRP binding and decreased amounts of ferritin protein [4], indirectly suggesting that the cells became Fe-deplete upon overexpression of ferroportin1. Collectively, the evidence obtained from ferroportin1 efflux and expression studies suggests the important role it plays in Fe absorption [4– 6]. However, the expression of ferroportin1 in the cytoplasm of Kupffer cells and enterocytes [4] suggests the possibility that ferroportin1 may be involved in the intracellular trafficking of Fe between the cytosol and organelles. The detection of ferroportin1 in placental syncytiotrophoblasts [5] and decidual cells of the pregnant uterus [4] suggests that ferroportin1 may play a role in the transfer of Fe from mother to embryo. Over the past few years, numerous discoveries at the molecular level have given us a better understanding of iron absorption. These novel proteins include the divalent metal transporter 1 [11], hephaestin [3], the duodenal cytochrome b ferric reductase [12] and ferroportin1. At the cellular level, Fe is absorbed at the apical membranes of villus enterocytes within the duodenum. While the uptake of Fe into enterocytes is relatively well documented, little is known about its release into the circulation. In the past, there was speculation concerning the existence of an Fe exporter at the basolateral membrane of enterocytes. The recent discoveries of ferroportin1 and hephaestin have given us an insight into Fe export from duodenal enterocytes (Fig. 1). However, the link between these molecules and the mysterious labile Fe pool remains unclear. Investigators studying copper absorption have reported the presence of intracellular copper chaperone proteins [13]. Such proteins may also exist for the intracellular trafficking of Fe and could be involved in the transfer of Fe(III) or Fe(II) from the Fe pool to ferroportin1 for export. It can be speculated that the release of Fe from its chaperone may require hephaestin. Hephaestin may interact with the Fe chaperone to facilitate Fe transfer to ferroportin1. Once transported, Fe(II) may then be released into the circulation and oxidized by Cp (Fig. 1).
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4. Possible medical applications The discovery of ferroportin1 may also be of medical benefit for the diagnosis and possible treatment of Fe overload and deficiencies. For example, the zebrafish weh mutation results in a hypochromic anaemia. It is possible that a similar condition due to the mutation of ferroportin1 exists in humans. Recently, a mutation in ferroportin1 has been associated with the development of autosomal dominant hemochromatosis [14]. A single base pair mutation at position 734 in exon 5 causes an amino acid substitution (Asp to His). This substitution may cause excessive transportation of Fe into the circulation, thus contributing to the pathology of hereditary hemochromatosis [14]. Obviously, the discovery of ferroportin1 allows the detection and possible correction of such disease states.
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