Nature Genetics 37, 1264 - 1269 (2005)
Published online: 16 October 2005; | doi:10.1038/ng1658
Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cellsRobert S Ohgami1, 2, Dean R Campagna1, Eric L Greer1, Brendan Antiochos1, Alice McDonald3, Jing Chen4, 7, John J Sharp5, 7, Yuko Fujiwara6, Jane E Barker5
& Mark D Fleming11 Department of Pathology Children's Hospital and Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 02115, USA. 2 Medical Scientist Training Program, Harvard Medical School, Tosteson Medical Education Center, Room 168, 260 Longwood Avenue, Boston, Massachusetts 02115, USA. 3 Millennium Pharmaceuticals, 45-2 Sidney Street, Cambridge, Massachusetts 01239, USA. 4 Division of Hematology, Brigham and Women's Hospital, 1 Blackfan Circle, Boston, Massachusetts 02115, USA. 5 The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA. 6 Division of Hematology and Howard Hughes Medical Institute, Children's Hospital, 1 Blackfan Circle, Boston, Massachusetts 02115, USA. 7 Present addresses: Winship Cancer Institute, Emory University, 1365-C, Clifton Rd. N.E., Atlanta, Georgia 30322, USA (J.C.); Center for Comparative Medicine 600D, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA (J.J.S.).
Correspondence should be addressed to Mark D Fleming mark.fleming@childrens.harvard.edu The reduction of iron is an essential step in the transferrin (Tf) cycle, which is the dominant pathway for iron uptake by red blood cell precursors. A deficiency in iron acquisition by red blood cells leads to hypochromic, microcytic anemia. Using a positional cloning strategy, we identified a gene, six-transmembrane epithelial antigen of the prostate 3 (Steap3), responsible for the iron deficiency anemia in the mouse mutant nm1054. Steap3 is expressed highly in hematopoietic tissues, colocalizes with the Tf cycle endosome and facilitates Tf-bound iron uptake. Steap3 shares homology with F420H2:NADP+ oxidoreductases found in archaea and bacteria, as well as with the yeast FRE family of metalloreductases. Overexpression of Steap3 stimulates the reduction of iron, and mice lacking Steap3 are deficient in erythroid ferrireductase activity. Taken together, these findings indicate that Steap3 is an endosomal ferrireductase required for efficient Tf-dependent iron uptake in erythroid cells.
Red blood cell (RBC) precursors are uniquely dependent on the Tf cycle to acquire iron to synthesize heme in amounts sufficient for hemoglobin production1. In the Tf cycle, iron bound to Tf binds to the Tf receptor (Tfr1), the complex is taken up by receptor-mediated endocytosis, and iron is released from Tf by endosomal acidification to be delivered to the cytoplasm by the divalent metal transporter 1 (Dmt1)2,
3,
4. Tf carries ferric iron (Fe3+), whereas Dmt1 is selective for ferrous iron (Fe2+)4. Therefore, iron must be reduced in the Tf cycle endosome. Despite functional evidence of such an activity, the molecular identity of the reductase that carries out this activity is unknown. An ascorbate-dependent b-type cytochrome ferrireductase, Dcytb (Cybrd1), expressed predominantly in the duodenum, was recently described5. But Dcytb is not expressed highly in erythroid precursors, and Dcytb-null mice have normal iron metabolism and normal hematologic parameters6. Such data argue against the possibility that Dcytb has a key role in the Tf cycle. Several studies have shown the existence of an NAD(P)H-dependent, cell-surface or endosomal ferrireductase activity in many cell types, including erythroid cells7,
8,
9. Here we show that the major erythroid ferrireductase of the Tf cycle endosome is Steap3, which is predicted to be a flavin-NADPH-dependent, membrane-bound oxidoreductase.
Previously, we described the phenotype of a mouse mutant, nm1054, with microcytic, hypochromic anemia10. To identify the gene underlying the anemia, we took a positional cloning approach. We mapped nm1054 to a 0.5-cM interval on mouse chromosome 1 in 542 affected (CBA/J-nm1054  CAST/Ei) F2 intercross mice10 and defined a minimal physical interval for the locus between microsatellite markers D1Mit27 and D1Mit471 (Supplementary Fig. 1 online). Three contiguous markers, D1Mit54, D1Mit191 and D1Mit389, could not be amplified in affected mice, suggesting that the nm1054 mutation might be a large genomic deletion. Serial analysis of genomic PCR amplicons across the region confirmed this deletion and defined it to a region of  400 kb that contained all or part of six genes (Fig. 1a). RT-PCR, Southern-blot and northern-blot analyses showed that each of these genes was absent in homozygous mutant mice (data not shown). We developed a BAC contig of the deletion and created transgenic lines from four overlapping BAC clones that spanned the entire deletion without disrupting any of the genes (Fig. 1a). Two transgenic lines derived from RPCI-22 BAC clone 11D19, which contains two genes, corrected the anemia (Fig. 1b). The first, located in the deletion interval, is six-transmembrane epithelial antigen of the prostate 3 (Steap3, also known as pHyde11,
12,
13, TSAP6 (refs. 14,15) and Dudulin2). The second, which lies outside the deletion, is complement component 1, q subcomponent 2-like (C1ql2). Two partial BAC 11D19 insertions containing C1ql2 alone failed to complement the anemia, suggesting that Steap3 was the gene on BAC 11D19 underlying the anemia.
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Because the nm1054-associated anemia is intrinsic to the hematopoietic compartment and manifests as a Tf-dependent iron uptake defect10, we expected the underlying gene to be expressed in hematopoietic tissues and the protein to be present in a physiologically relevant cellular location. In situ hybridization analysis of mouse embryos and adult tissues, as well as quantitative real-time PCR analysis of human tissues, showed that Steap3 was highly expressed in fetal liver (the site of midgestational hematopoiesis), adult bone marrow, placenta, liver, skeletal muscle and pancreas (Fig. 2). This pattern of distribution supports the possibility that Steap3 has a role in iron metabolism, particularly in cells, such as erythroid precursors, that are highly dependent on Tf for iron acquisition. Furthermore, we found that a stably expressed, epitope-tagged form of Steap3 partially colocalized with Tf, Tfr1 and Dmt1 in endosomes (Fig. 3). In transiently transfected cells expressing high levels of Steap3, the protein could be detected on the cell surface by immunofluorescence (data not shown), like Tfr1 and Dmt1. This colocalization with three other key components of Tf-dependent iron uptake places Steap3 in a subcellular compartment equipped for high-affinity, high-avidity iron transport.
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To assess further whether Steap3 underlay the nm1054-associated anemia, we attempted to correct somatically the hematopoietic defect in vivo. We retrovirally transduced mutant fetal liver hematopoietic cells with a wild-type copy of Steap3 and transplanted them into lethally irradiated wild-type mice. Expression of Steap3 rescued the anemia, as indicated by an increase in the mean cell volume and cellular hemoglobin content of the reticulocytes. Furthermore, because the proportion of protoporphyrin IX complexed with zinc (ZnPP) increases in states of iron deficiency16, the decreased ZnPP:heme ratio in the complemented mutant reflects specific correction of the intraerythroid iron deficiency (Fig. 4). Taken together, these data indicate that Steap3 rescues the hematopoietic defect in nm1054.
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To expand on the Steap3 complementation result, we created a Steap3 targeted deletion allele (Supplementary Fig. 2 online). Phenotypic analysis of (129S4/SvJae-Steap3+/- 129S6/SvEvTac-nm1054/+) F2 intercross progeny showed that the nm1054-associated anemia was allelic with the Steap3 targeted deletion and that homozygosity with respect to the Steap3 null allele recapitulated the homozygous nm1054 anemia phenotype (Fig. 5a–e and Supplementary Tables 1 and 2 online). Furthermore, Steap3-/- reticulocytes, like nm1054/nm1054 reticulocytes10, have a defect in Tf-dependent iron uptake. Steap3-/- reticulocytes were more than three times less efficient in acquiring iron from Tf compared with reticulocytes from wild-type littermate controls (Fig. 5f). After reincubation with media containing an excess of apo-Tf to capture released iron, they lost a substantial fraction of iron initially taken up into the cell (17.8  3.3%), whereas control cells lost almost none (-1.6 3.7%). Taken together, these data conclusively demonstrate that the nm1054-associated anemia is due to loss of Steap3, that nm1054 is a deletion allele of Steap3 (Steap3nm1054) and that mutation of Steap3 results in a defect in Tf-mediated iron uptake.
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Sequence analysis predicts Steap3 to have an N-terminal oxidoreductase domain and a C-terminal transmembrane region (Fig. 6a). The oxidoreductase domain is predicted to be an unusual flavin-NAD(P)H binding structure (PFAM 03807.8)17 homologous to one found in archaea and bacteria that uses NADPH and the modified flavin F420 as cofactors18,
19. This domain is not present in yeast, nematodes or fruit flies, and only two other structurally similar mammalian proteins, Steap4 (also called Tiarp or Tnfaip9; ref. 20) and Steap2 (also called STAMP1; ref. 21), have homologous domains (Fig. 6b). The C-terminal region of the protein is predicted to have six transmembrane helices that are very distantly related to the yeast FRE family of b-type cytochrome metalloreductases, which are essential for iron and copper uptake18,
22 (Fig. 6b). In particular, two of four histidine residues implicated in binding heme in the FRE proteins23 are conserved in all four Steap family members, including Steap24,
25, which lacks the N-terminal oxidoreductase domain (Fig. 6b). Like the Steap proteins, the yeast FRE proteins also contain a predicted flavin-NAD(P)H-binding domain, but this domain is structurally distinct and is located C-terminal, rather than N-terminal, to the predicted transmembrane spans (Fig. 6a).
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Steap3 was initially described as a putative tumor suppressor capable of inhibiting tumor cells through a caspase-3–dependent pathway11,
12,
13,
15. It was later reported to interact with and facilitate secretion of the proinflammatory translationally controlled tumor protein (TCTP, also called histamine-releasing factor, HRF)14. Steap4 has been characterized as a cell surface protein induced by tumor necrosis factor-alpha (TNF- ) in an adipogenic cell line20. STEAP and STEAP2 are highly expressed in the prostate and circumstantially implicated in prostate cancer metastasis21,
24,
25. But no specific enzymatic function has been attributed to this family of proteins.
The functional defect in Steap3nm1054/nm1054 and Steap3-/- reticulocytes, as well as the structural homology to oxidoreductases in archaea and the yeast FRE proteins, suggested that Steap3 might function as a ferrireductase. To address this hypothesis, we measured ferrireductase activity in Steap3nm1054/nm1054 and Steap3-/- reticulocytes4,
26. Reticulocytes of both homozygous mutant genotypes were similarly deficient in ferrireductase activity relative to controls (Fig. 6c,d). To determine whether Steap3 overexpression could stimulate ferrireductase activity in vitro, we transiently transfected HEK 293T cells with Steap3 and found that ferrireductase activity was increased (Fig. 6e). Mutation of either of two residues implicated in NAD(P)H binding (S58I and R59L) completely abrogated this activity (Fig. 6e). We observed a similar loss of function in Steap3 mutants in which either of the two putative heme-binding histidines was changed to leucine (H316L or H409L; Fig. 6e). In each case, the mutations had no effect on protein expression or localization (data not shown). These results demonstrate the importance of these conserved motifs for reductase activity and argue against the possibility that Steap3 had an indirect effect in inducing another ferrireductase protein.
Steap3 has structural features distinct from those of other known ferrireductases. In eukaryotes, the F420H2:NADP+ oxidoreductase domain is unique to members of the Steap family (Supplementary Fig. 3 online). Prior studies of the Steap proteins predict a cytoplasmic orientation for the oxidoreductase domain20,
21,
24. In such a configuration, this domain would have access to the cytoplasmic pool of NAD(P)H as a source of electrons for the reduction of iron (Fig. 6f). In the case of the yeast FRE proteins, the transfer of electrons from NADPH across the membrane is believed to occur sequentially, first through FAD and then through two heme molecules27. These heme moieties are coordinated by four histidine residues in the transmembrane segments22. In Steap3 and its mammalian homologs, only two of these histidine residues are conserved, predicting one intramembrane heme molecule (Fig. 6a,b). Computational modeling (using TMpred) of the transmembrane domains of Steap proteins places the single heme more centrally in the membrane, whereas in the yeast FRE proteins, the heme groups are arranged in tandem, at opposite poles of the transmembrane helices (Fig. 6a,b,f)18.
The modeling and in vitro ferrireductase activity of Steap3 predict that Steap3 binds NAD(P)H and a flavin derivative, as well as one heme, and that the reduction of iron is accomplished by sequential transfer of electrons from the NAD(P)H oxidoreductase domain to an intramembrane heme cofactor and, ultimately, to Fe3+ in the endosome. It is not known whether 'free' Fe3+ is the substrate of this reaction or whether reduction facilitates release of iron from Tf9.
Taken together, these data formally define the role of iron reduction in the Tf cycle and support the hypothesis that Steap3 is an endosomal ferrireductase crucial for Tf-Tfr1–mediated iron uptake in erythroid cells. Because Steap3 is found in abundance in other tissues that express high levels of Tfr1, including placenta, the absence of pathologic phenotypes of iron metabolism attributable to these tissues suggests that there is functional overlap in the ferrireduction system. As the other Steap2 and Steap4 are highly homologous to Steap3 and contain oxidoreductase domains, the requirement for an iron reductase in other organs could potentially be met by other family members.
Methods
Genetic and physical mapping of nm1054.
All animal work was reviewed and approved by the Animal Care and Use Committee at Children's Hospital Boston. We obtained, genotyped and analyzed 542 affected (CBA/J-nm1054 CAST/Ei) F2 intercross mice as previously described10. We used closely linked microsatellite markers D1Mit27 and D1Mit217, located centromerically and telomerically of the deletion, respectively, to detect the CBA/J-derived Steap3nm1054 allele. We constructed a redundant overlapping contig of BAC clones by screening the CITB strain 129S4/SvJae library (Invitrogen) by PCR and the RPCI-22 strain 129S6/SvEvTac library with gene-specific probes.
Steap3 RNA expression analysis.
We carried out in situ hybridization on frozen sections of mouse tissues using 35S-labeled RNA probes (coding sequence nucleotides 124–519) as previously described28. We carried out real-time PCR in triplicate on the Human MTC cDNA Panel I and Human Immune System MTC Panel cDNA sets (BD Biosciences) using the SYBR Green PCR Master Mix (Stratagene) and the ABI Prism 7700 Sequence Detection System Mx3000P Real-Time PCR System in accordance with the manufacturer's instructions. We generated standard curves using 1, 0.2, 0.1, 0.05, 0.02 and 0.01 ng of total cDNA from human liver. We designed primers (Supplementary Table 3 online) using the real-time PCR primer design software from GeneScript. We carried out dissociation curve analysis, gel electrophoresis and sequencing of PCR products to validate and determine the specificity of the assays. The experiment was repeated three times with similar results.
Transient and stable expression of Steap3.
We cloned the mouse Steap3 cDNA into pCDNA6-myc-HisA (Invitrogen) to create pCDNA6-Steap3-Myc-His and introduced mutations using the QuickChange II site-directed mutagenesis kit (Stratagene). We subcloned the C-terminally Myc-tagged Steap3 from pCDNA6-Steap3-Myc-His into the retroviral vector pMSCVneoEB to generate pMSCV-Steap3-Myc. We maintained tissue culture cells in Dulbecco's modified Eagle medium (Gibco-BRL) supplemented with 10% fetal bovine serum (Invitrogen) and penicillin-streptomycin. We obtained stable expression cell lines by transfecting HEK 293T or COS-7 cells with linearized pCDNA6-Steap3-Myc-His using Geneporter2 (Gene Therapy Systems) and culturing with blasticidin antibiotic selection. We chose clones on the basis of antibiotic resistance, as well as by protein expression determined by western-blot and immunofluorescence analyses.
Subcellular localization.
We plated HEK 293T clones stably expressing low levels of the Steap3-Myc-His protein on poly-D-lysine–coated coverslips (Becton-Dickinson) and incubated them in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 10  g ml-1 human holo-Tf (Sigma) for 24 h before carrying out immunofluorescence analysis. For Tf labeling, we incubated cells with 30 g ml-1 Alexafluor 488-labeled human holo-Tf (Molecular Probes) for 30 min before washing them three times with phosphate-buffered saline. We then fixed cells and treated them with a Cy3-labeled mouse monoclonal antibody to Myc 9E10 (Sigma) before viewing them by confocal microscopy. For Tf receptor colocalization studies, we stained cells with fluorescein isothiocyanate–conjugated antibody to TfR (clone #M-A712, BD Pharmingen). We transfected a 293T clone stably expressing Myc-tagged Steap3 with a C-terminally Flag-tagged DMT1 in pCS2. We stained cells as previously described29.
Retroviral complementation.
We carried out retroviral production, infection and transplantation using modified established procedures30. We collected retroviral supernatants from HEK 293T cells 48 h after transfection with pMSCVneoEB retrovirus constructs and Ecopac (Cell Genesys). We obtained C57BL/6J-nm/nm Gpi1b/b [N19] fetal liver hematopoietic cells at embryonic days 14.5–16.5. We lysed mature erythrocytes in 150 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA (pH 7.4); incubated the nucleated fetal liver cells for 24 h in RPMI medium supplemented with murine IL-3 (6 U ml-1; R&D Systems), recombinant murine stem cell factor (SCF, 5 U ml-1; Stem Cell Technologies), recombinant murine IL-6 (10,000 U ml-1; R&D Systems), 10% fetal bovine serum and 100 U ml-1 penicillin-streptomycin; and transduced them twice at 24-h intervals with retroviral supernatants. After retroviral transduction, we transplanted 2 106 nucleated fetal liver cells by retro-orbital injection into lethally irradiated C57BL/6J-Gpi1a/a mice (Jackson Labs). We collected peripheral blood for phenotypic and chimerism (Gpi1b donor versus Gpi1a recipient) analysis 4 weeks after transplantation as previously described10.
Gene targeting, allelism testing and phenotyping.
We created BAC transgenics by injecting pronuclei of C57BL/6 blastocysts with circular BAC DNA prepared with the NucleoBond BAC Maxi Kit (BD Biosciences). We carried out embryonic stem cell (J1 line; 129S4/SvJae derivation) gene targeting, culture and blastocyst injections using standard techniques. We bred chimeric Steap3+/- males to 129S6/SvEvTac-nm1054/+ [N5] (Steap3nm1054/+) females and intercrossed compound heterozygous (Steap3nm1054/-) and heterozygous (Steap3nm1054/+ and Steap3+/-) F1 mice to produce F2 litters, which we phenotyped as previously described10. We determined routine genotypes and phenotypes using a PCR assay for the targeted and wild-type alleles and using a peripheral blood smear. Statistical analysis was done using StatView 5.0.1 (SAS Institute Inc.)
Iron uptake and iron reductase experiments.
We carried out iron uptake experiments on reticulocyte-rich RBCs as previously described10. Reductase assays used HEK 293T cells in 12-well dishes. We transfected cells with 1 g of pCDNA6-Steap3-Myc-His or an antisense construct using Geneporter2 (Gene Therapy Systems) in accordance with the manufacturer's instructions. For the assay, we incubated transfected cells in phosphate-buffered saline supplemented with 5.6 mM glucose containing 50 M Fe3+-nitrilotriacetic acid (Fe-NTA; 50 M ferric chloride + 100 M nitrilotriacetic acid) and 200 M ferrozine5. After incubation at 37 °C for 60 min, the Fe2+-ferrozine complex was detected by measuring the absorbance at 562 nm and converted to Fe2+ generated by using the extinction coefficient 27.9 mM-1cm-1. Pilot experiments determined the time- and temperature-dependence of the assay. For reticulocyte experiments, we induced reticulocytosis by bleeding10 and incubated washed reticulocyte-rich RBCs in Hank's balanced salt solution containing 50 M Fe3+-nitrilotriacetic acid and 200 M ferrozine for 30 min. Iron uptake and ferrireductase activity were normalized to RNA content10.
URLs.
TMpred is available at http://www.ch.embnet.org/software/TMPRED_form.html. The RPCI-22 strain 129S6/SvEvTac library is available at http://bacpac.chori.org/home.htm. MultAlign is available at http://prodes.toulouse.inra.fr/multalin/multalin.html.
Accession codes.
GenBank: Dudulin2, AY029586.1.
Note: Supplementary information is available on the Nature Genetics website.
Received 16 June 2005; Accepted 31 August 2005; Published online: 16 October 2005.
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Acknowledgments
We thank L. Lee, H. Gunshin, N. Andrews, E. Neufeld and members of the laboratories of N. Andrews and E. Neufeld for ongoing support and criticism and N. Stokes and T. Borjeson for technical support. This work was supported by the Pew Biomedical Scholars Program (M.D.F) and grants from the US National Institutes of Health (M.D.F. and J.E.B.). Transgenic core facilities were supported by a grant from the US National Institutes of Health.
Competing interests statement:
The authors declare that they have no competing financial interests. |
