Q J Med 2000; 93: 575-587
© 2000 Association of Physicians
Localization of iron transport and regulatory proteins in human cells
From the Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK
Received 5 May 2000 and in revised form 11 July 2000
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The recent discovery of HFE, the MHC-Class-I-like gene mutated in up to 90% of patients with hereditary haemochromatosis, and the gene encoding the Nramp2/divalent metal transporter-1 (DMT-1) implicated in ferrous iron transport holds promise for a greater understanding of human iron metabolism. Since the HFE protein can be crystallized as a ternary complex with the transferrin receptor and iron-saturated transferrin, and DMT-1 expression is up-regulated in hereditary haemochromatosis, these proteins are likely to interact in a common pathway for human iron homeostasis. To investigate the cellular interactions between the cognate proteins encoded by these genes, we generated a panel of rabbit and avian antisera from human HFE and DMT-1 derived peptides. The antibodies were characterized by ELISA reactions and Western immunoblotting. Immunohistochemical staining showed that DMT-1 protein localized to the brush border of human duodenum where it is predicted to serve as the principal transporter of ferrous iron from the intestinal lumen. In the human cell lines, Caco-2 (small intestinal phenotype upon differentiation) and K562 (erythroleukaemic) HFE, in the presence of iron-saturated transferrin, co-localized with transferrin receptors in an early endosome compartment using confocal immunofluorescence microscopy. This interaction may be critical in small-intestinal crypt cells which express HFE, where it may function to modulate their intrinsic iron status thereby programming iron absorption by DMT-1 in the mature enterocyte. In undifferentiated Caco-2 cells, DMT-1 localized to a discrete late endosome compartment distinct from that occupied by HFE where, in addition to brush-border iron uptake, it may function to regulate the availability of iron delivery to intracellular iron pools. Disruption of the HFE gene as a result of mutations associated with hereditary haemochromatosis may thus impair homeostatic mechanisms controlling iron absorption within the small-intestine epithelium by a direct interaction with transferrin receptors and by subsequent alteration of DMT-1 expression. Identification of the molecular interactions of HFE with DMT-1 and other key components of the iron transport pathway has implications for a mechanistic understanding of the pathophysiology of human iron storage diseases as well as the regulation of normal iron balance.
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Introduction |
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Since iron is an essential as well as a potentially toxic element, elaborate mechanisms exist to regulate body iron balance so that absorption of iron by the upper intestine (normally
1 mg/day) is finely adjusted to meet the insensible losses incurred through exfoliation of epithelia and, in females, menstruation. A limited capacity for iron storage in the form of ferritin and its proteolytic product haemosiderin exists, but when more than 1.5 g of iron accumulates in the storage compartment, iron toxicity develops, mediated by the generation of free radical species that injure cell membranes and DNA.
Hereditary haemochromatosis with associated multi-system tissue injury is the end result of disordered iron homeostasis; it reflects the long-term consequence of a qualitative disorder of iron regulation, expressed ultimately at the level of the small-intestinal mucosa in which dietary iron is absorbed surplus to requirements. In hereditary haemochromatosis, 90% of patients of North European origin are homozygous for a single missense mutation (Cys282Tyr) within the HFE gene, located on the short arm of chromosome 6.1 The frequency of homozygosity for hereditary haemochromatosis (1 in 250 in the UK)2 exceeds that of other well-known genetic disorders such as cystic fibrosis and familial hypercholesterolaemia. The protein encoded by the HFE gene appears to serve as a regulator of iron absorption: an understanding of the molecular interactions of this protein has important implications for hereditary haemochromatosis and also for its counterpart, iron-deficiency anaemia—a common affliction world-wide. The HFE protein, like its MHC Class I neighbours, forms a heterodimer with ß2-microglobulin that is expressed at the surface of many cells including duodenal crypt cells.3 The Cys282Tyr mutation disrupts this interaction and prevents cell surface expression of HFE.4 Furthermore, the wild-type protein complexes with transferrin receptors on the cell surface and appears to lower their affinity for transferrin,5 thus suggesting a mechanism by which mutated HFE might enhance the system uptake of iron into the tissues. Mature enterocytes on the villus tip, however, neither express HFE nor absorb dietary iron by this pathway, and it is unclear how HFE influences the intestinal absorption of iron.
Although for many years the physicochemical properties of iron hampered the discovery and isolation of iron transporters, since the identification of HFE, an expression cloning system using Xenopus oocytes has been used to identify a protein capable of transporting divalent transition metal ions.6 This protein, Nramp2, recently renamed divalent metal transporter-1 (DMT-1), shows sequence homology to a natural resistance-associated macrophage protein, Nramp1, which is associated with resistance to infection by intracellular parasites. At the same time, mutations in the murine Nramp2 gene were implicated as the cause of microcytic anaemia in the mk/mk mouse, which has defects in intestinal iron absorption and iron entry into developing red cells. A positional cloning approach was used to identify a homozygous missense mutation in the Nramp2 gene, G185R;7 this mutation affects a residue adjacent to that pathologically mutated in Nramp1 which is solely responsible for the variation in natural resistance to microbacterial and other infections.8 The same missense mutation in Nramp2/DMT-1 was subsequently identified as the cause of iron deficiency in the anaemic Belgrade rat that is defective in the release of iron from transferrin-cycle endosomes within erythroblasts.9 Site-directed mutagenesis of murine DMT-1 has confirmed the disabling effect of the G185R mutation on iron transport.10 A major isoform of the DMT-1 gene contains a 3' iron-response element (IRE) which may permit translational regulation of protein synthesis in relation to cellular iron status; DMT-1 expression is enhanced in iron deficiency, consistent with increased stability of the transcript. Furthermore, duodenal DMT-1 mRNA levels are increased in hereditary haemochromatosis,11 and DMT-1 protein has been immunolocalized to the duodenal brush border in iron-deficient rats.12 In mice with targeted disruption of the HFE gene, duodenal DMT-1 mRNA levels are similarly increased13 and rates of mucosal ferrous iron uptake in vitro are elevated, in keeping with DMT-1-mediated iron absorption.14
DMT-1 is therefore a candidate for iron absorption in the small intestine, and although preliminary studies have shown that HFE and DMT-1 expression are reciprocally regulated in response to changes in intracellular iron stores in Caco-2 cells,15 the nature of this interaction has yet to be characterized. Furthermore, although the peptide sequence inferred from the HFE gene was reported several years ago, very few investigators have obtained antibody of sufficient utility to provide unambiguous information about the localization and tissue expression of endogenous human HFE protein. Similarly, limited information is available about the expression of DMT-1 protein, which appears to be of key importance in iron transport. Accordingly, we report in this study the use of specific antisera directed against these components of the cellular iron pathway to investigate intracellular interactions and determine cellular localization within human cells and tissues by immunohistochemistry and confocal immunofluorescence microscopy.
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Antibody production
Two peptides derived from the human HFE sequence were conjugated to keyhole limpet haemocyanin (Immune Systems Ltd). The two peptides were designated ‘SQM’ (representing a 19-amino-acid region of the
1 domain) and ‘KQP’ (representing a 15-amino-acid sequence from the 3 domain)—these sequences were specific to HFE compared with a range of known HLA proteins. Two peptides were similarly synthesized from human Nramp2/DMT-1: ‘SQS’ (a 14-amino-acid region of extracellular domain 3) and ‘FGK’ (a 12-amino-acid region of extracellular domain 4). Four mature female rabbits received standard immunizations with each peptide conjugate, and serum was collected. In addition, a chicken was immunized with the SQS peptide of DMT-1. The peptides were additionally conjugated to bovine serum albumin (BSA) and linked to cyanogen bromide-activated sepharose (Amersham). The BSA conjugates were used to prepare immune-specific antibody by immuno-affinity chromatography; antibodies from serum were eluted in 0.1 M glycine HCl, pH 2.8, and immediately neutralized with Tris base for storage. Commercial murine monoclonal antibodies were obtained to the human transferrin receptor (Zymed); monoclonal antibodies to human CD63, a lysosomal/late endosome marker were kindly provided by Dr Paul Luzio (Department of Biochemistry, University of Cambridge).
Enzyme-linked immuno-absorbant assay (ELISA)
Plates (96 wells) were coated overnight at 4°C with bovine serum albumin-linked peptide in phosphate-buffered saline, pH 7.4, (100 µl of 10 µg/ml per well). The plates were washed three times in phosphate-buffered saline containing 0.05% v/v Tween 20 (PBST), blocked with 5% fetal calf serum in PBST for 1 h at room temperature and then incubated overnight at 4°C with serial dilutions of either pre-immune or immune serum diluted in PBST. After further rinses, the plates were incubated for 2 h at 37°C with alkaline phosphatase-linked secondary antibody conjugate (Sigma) diluted 1:1000. The plates were washed three more times successively, and substrate (paranitrophenyl pyrophosphate in diethanolamine buffer 1 mg/ml) was added at 37°C. The colour reaction was determined by measuring the optical density at 405 nm using a BioRad 550 plate reader.
Western immunoblotting
For HFE antibodies, the protein substrate used was a human liver lysate diluted 1:20 in reducing SDS sample buffer. For the rabbit DMT-1 antibodies, a lysate of Caco-2 cells was prepared and diluted in 3xreducing SDS sample buffer. The samples were denatured at 95°C for 5 min before being electrophoresed in a 10% SDS-polyacrylamide mini-gel (BioRad) using prestained polypeptide molecular weight markers (Rainbow, Amersham). The polypeptides were transferred in buffer at 4°C on to polyvinyl difluoride (Immobilon-P) membranes (Millipore). The membrane was incubated for 1 h at room temperature in a blocking solution containing 5% w/v dried milk, 0.3 M NaCl, 10 mM Tris HCl, 0.05% v/v Tween 20 at pH 7.4. This was followed by primary antibody diluted in blocking solution for 2 h at room temperature. After 3x10 min washes in PBST, the membrane was incubated with goat anti-rabbit conjugated secondary antibody. This was diluted 1:10 000, and the membrane was exposed for 1 h at room temperature. After 4x10 min washes in PBST, a chemiluminescent substrate (LumiGLO) was added for 1 min. The membrane was dried and placed in a cassette for autoradiography.
Immunohistochemistry
Paraffin sections of formaldehyde-fixed human tissue obtained at autopsy or after diagnostic biopsy were dewaxed by 3x10 minute exposures to xylene, and rehydrated in a graded series of ethanol solutions (100%, 95%, 70%) for 10 min each followed by deionized water for 5 min. The slides were gently agitated in 1% hydrogen peroxide solution in methanol (10 min at room temperature), rinsed in tap water and incubated with 0.1% trypsin solution for 5 min at 37°C. Sections were then rinsed and blocked with 10% goat serum in Tris-buffered saline, pH 7.4, for 30 min at room temperature followed by avidin and biotin blocking steps, respectively, as directed by the manufacturers (Vector Laboratories). The slides were then incubated with affinity-purified primary antibody (1:50) for 30 min at room temperature or with PBST buffer alone for control experiments. After further washes, sections were incubated with biotinylated secondary antibody (1:500 for 30 min at room temperature) followed by avidin/biotin complex and hydrogen peroxide/diaminobenzidine tetrahydrochloride substrate (Vector). Sections were briefly counterstained in Gill's haematoxylin and dehydrated through a graded alcohol series before mounting in DePeX medium. Light microscopy and photomicrography were done using a Nikon Optiphot 2 photographic microscope.
Fluorescence immunocytochemistry
Undifferentiated human Caco-2 cells were grown to subconfluence on cover slips and fixed with 4% paraformaldehyde in phosphate-buffered saline for 1 min at room temperature. Cells were permeabilized in 0.1% Triton-X100 (for 1 min at room temperature) and were then rinsed in phosphate-buffered saline. The cells were then blocked for 1 h with 10% fetal calf serum in culture medium before incubation for a further hour at room temperature with affinity-purified primary antibody or mouse monoclonal 1:50 in 5% fetal calf serum/45% medium/50% phosphate-buffered saline or no primary antibody (control). The cover slips were washed in phosphate-buffered saline three times before incubating for 1 h at room temperature with fluorescein-conjugated and/or rhodamine-conjugated secondary antibodies (Jackson Immuno Research/Sigma). An anti-mouse Texas Red secondary reagent was used for CD63 localization (Dr Paul Luzio). After three further washes, cover slips were mounted in glycerol-phosphate-buffered saline (Citifluor, Chem Labs) and examined using laser confocal microscopy (BioRad MRC 1024 ES Laser/Nikon Optiphot 2). K562 cells were air-dried on to microscope slides and fixed at -20°C in a 2:1 methanol to acetone mixture for 10 min before blocking and staining as above.
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Characterization of antisera by ELISA and Western immunoblotting
Enzyme-linked immuno-absorbant reactions obtained to the BSA-linked immunogen show the development of peptide-specific immune responses in all animals (Figure 1
). Since all the immunizations were carried out with peptides conjugated to keyhole limpet haemocyanin, the antibody responses represent immune reactions that are peptide-specific. The polyclonal antisera to human HFE and DMT-1 were further characterized by Western immunoblotting (Figure 2, panels a and b, respectively). Rabbit anti-HFE antibodies were analysed on human liver lysates and serum obtained from terminal bleeds shown in lanes 2 (SQM) and 4 (KQP) provided a single 47 kDa signal compatible with a polypeptide species inferred from the sequence of the human HFE gene. Pre-immune sera used as controls (lanes 1 and 3) gave negative signals. Anti-DMT-1 antibodies were studied using cell lysates of the human Caco-2 cell line. As shown in panel b, a single 60 kDa polypeptide species was identified giving specific immune reactions against SQS (lane 2) and FGK (lane 4) compared with pre-immune controls (lanes 1 and 3 respectively) obtained from the same animals. This is that expected from the cognate cDNA sequence of DMT-1.
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Immunohistochemistry
To investigate the utility of the antibodies in the identification of immunoactive protein species in paraffin-fixed tissue specimens, immuno-affinity rabbit antibodies were used to stain sections of human liver and human duodenum, respectively, with anti-HFE and anti-DMT-1 antibodies. The two HFE antisera identified HFE protein predominantly in the hepatic sinusoids and particularly in the Kupffer macrophages (Figure 3). Little or no staining was seen without immuno-purified HFE antisera. Also shown in this figure is staining of human duodenum using anti-DMT-1. Striking staining was observed at the microvillus membrane at the apex of the small intestinal mucosal epithelial cells. Staining was particularly strong with the anti-SQS DMT-1 antibody that was also seen with the other antibody. No staining was seen in intestinal goblet cells or in cells including macrophages within the lamina propria of the human intestinal biopsy samples examined.
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). Bottom panel shows staining of human duodenum using anti-DMT-1 antibodies; control (d)—no primary, (e) and (f) with anti-SQS and anti-FGK, respectively. DMT-1 is seen in enterocytes with a striking abundance at the brush border (
Fluorescence immunocytochemistry using confocal microscopy in cultured human cell lines
Having demonstrated histological localization of HFE in liver and DMT-1 in the intestine, we investigated whether it would be possible to identify the intracellular localization of HFE, the transferrin receptor and DMT-1 in cultured human cell lines. We investigated the localization of HFE protein in the human erythroleukaemia cell line K562 using affinity-purified antibodies. Bright staining of HFE was obtained with both HFE antibodies when the fluorescence signal was realized in green using fluoresceinated anti-rabbit conjugate (Figure 4). Similar studies using DMT-1 antibodies also generated vesicular staining pattern of DMT-1. Appropriate controls in which the antibody was pre-incubated with its related peptide in excess (50 µg/ml) showed no staining indicating specificity of the findings in these cells (data not shown). Confocal microscopy was also carried out in Caco-2 cells using rabbit antisera to HFE and DMT-1 and also chicken anti-SQS (with fluoresceinated anti-chicken secondary antibody). HFE and DMT-1 antibodies specifically identified vesicles within Caco-2 cells examined by confocal fluorescence microscopy (Figure 5).
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Having shown vesicular distribution of HFE in Caco-2 and K562 cells, co-localization experiments were conducted using different antisera detected by distinct fluorescent signals (fluorescein for HFE—green; rhodamine for the transferrin receptor—red) in these cell lines (Figure 6
). In these experiments cells were co-stained with rabbit anti-SQM and mouse anti-transferrin receptor antibodies followed by anti-rabbit fluoresceinated conjugate and anti-mouse rhodamine conjugate. In the absence of additional transferrin in the culture medium, distinct populations were identified with a low signal intensity. The transferrin receptor appeared to be in a perinuclear phase and the HFE vesicles scattered at low density throughout the cytoplasm. On inducing iron-transferrin endocytosis by the addition of 5% human serum transferrin for 24 h before fixation, many more endosomic vesicles containing transferrin receptors were identified in both cell types. During active endocytosis of iron-saturated transferrin in culture, yellow super-imposed staining of FITC and rhodamine-conjugates confirms the co-localization of HFE with the transferrin receptor in a distinct sub-population of intracellular vesicles.
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Subcellular localization of HFE and DMT-1 protein in Caco-2 cells
Caco-2 cells were stained with avian anti-SQS DMT-1 antibody and rabbit anti-SQM HFE antibodies, followed by fluorescent localization with the secondary agents, fluorescein-conjugated anti-chicken antibody and rhodamine-conjugated anti-rabbit antibody. In these experiments, strong staining of HFE with the red rhodamine dye did not co-localize with the population of DMT-1 vesicles which fluoresce green (Figure 7a). Further studies were conducted in an attempt to characterize the sub-population of vesicles staining with these iron transporter regulatory proteins in Caco-2 cells; some co-localization of the green fluorescent DMT-1 signal with the CD63 antigen (red staining representing late endosomes and lysosomes) was observed (Figure 7b). However, no such co-localization with the HFE protein in this endosomal/lysosomal compartment was demonstrated (Figure 7c). In these three experiments, no difference in staining for was observed with prior addition of transferrin to the cell preparation.
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Discussion |
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We report here the characterization of antisera for the detection of the human proteins HFE and DMT-1 in human cells and tissues. The antisera have been characterized using enzyme-linked immuno-absorbant assays and Western blotting, and detect as expected the appropriate polypeptide species present in human tissues. The antibodies have been further used in preliminary experiments to examine the tissue distribution of HFE and DMT-1 expression by immunohistochemistry. These experiments in particular have shown for the first time the localization of DMT-1 protein in humans within the microvillus membrane of the small intestine—a site at which it would be able to carry out one of its principal functions in the unidirectional uptake of free ferrous irons released by digestion and generated by the action of ferrireductases present within the brush border.
Confocal microscopy in both Caco-2 and K562 cells demonstrated that both cell types express endogenous HFE and DMT-1 protein. In particular, K562 cells can express DMT-1 protein, necessary for haem biosynthesis and iron delivery, which is deficient in the mk/mk mouse lacking functional DMT-1 transporter. Furthermore, DMT-1 protein localizes within cells in a late endosomal compartment, discrete from HFE, compatible with a transport function in the delivery of ferrous iron to ferrochelatase for haem biosynthesis and other cellular functions within the mitochondria.
Both cell types demonstrated the co-localization of HFE with transferrin receptors in endosomal vesicles in the presence of iron-saturated transferrin. It has been suggested that the HFE protein forms a high-affinity ternary complex with transferrin receptor and its iron-saturated transferrin ligand.16 Although studies have reported localization of exogenously transfected human HFE in cultured cell lines such as Hela cells,17 this study is one of the first to demonstrate co-localization of the HFE protein with transferrin receptors in cultured human cells in which endogenous HFE protein is shown to be present. This interaction, which appears to occur in intestinal Caco-2 cells and also in K562 cells, which are of haematopoietic origin, is compatible with a role in regulating the intracellular iron pool which subsequently influences the translation of DMT-1 mRNA via its 3' IRE. In hereditary haemochromatosis, mucosal epithelial cells in fact mimic the iron-deficient states with low levels of ferritin mRNA and paradoxically increased transferrin receptor expression, in keeping with inappropriate down-regulation of the intracellular iron pool.18,19 Thus a mechanism is envisaged whereby incorrect signalling of body iron status by defective HFE protein results in depletion of crypt cell iron pools and a consequent increase in DMT-1-mediated iron uptake by mature enterocytes. Clearly other steps in the pathway of iron delivery across the mucosal membrane participate, and the recently identified Ferroportin1/IREG1 protein that is involved in the egress of iron across the basolateral membrane of the enterocyte20,21 must also play a role in the net incorporation of iron from the intestinal lumen into the body.
Our results indicate that we have generated a panel of rabbit and avian antisera to the HFE and DMT-1 protein which can be used further to characterize the molecular defect in hereditary haemochromatosis and the physiological interactions that serve to regulate body iron balance. The antibodies have been used to demonstrate the interactions of endogenous HFE and DMT-1 with other intracellular transport proteins by confocal microscopy in cultured human cell lines.
The finding that HFE in the presence of transferrin interacts with transferrin receptors in early endosomes in these cell lines indicates that it is present at a site at which it may regulate the entry of iron into cells. This may be critical in small-intestinal crypt cells which express HFE that may function to modulate their iron status, thus determining their absorptive activity on maturation to enterocytes at the villus tips. DMT-1 protein is highly expressed on the microvillus apical membrane of human duodenal epithelial cells and in K562 and Caco-2 cells. DMT-1 protein localized to a late endosome compartment that is discrete from the vesicular compartment containing the HFE protein. Here DMT-1 may facilitate the transport of iron into an intracellular iron pool as well as its unidirectional uptake across the brush-border membrane. Disruption of the HFE gene may thus disrupt homeostatic mechanisms operating within the small-intestinal epithelium that control iron absorption by its direct interaction with transferrin receptors and by the subsequent alteration of DMT-1 expression. We envisage that identification of the molecular interactions of HFE with DMT-1 and other key components of iron homeostasis, including the newly-identified and homologous transferrin receptor 2,22 will lead to a fuller mechanistic understanding of the pathophysiology of hereditary haemochromatosis and other iron storage diseases as well as the means by which iron balance is finely regulated in health.
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Acknowledgments |
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We thank Mrs Joan Grantham and Mr George Lee for secretarial and technical assistance, respectively. This work is supported by a Clinical Training Fellowship award from the Wellcome Trust, the European Union EU Biomed BMH4-CT95-0994, the Grocers' Charity, the Sackler Foundation and the Children's Liver Disease Foundation.
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Address correspondence to Dr W.J.H. Griffiths, Department of Medicine, PO Box 157, Level 5, Addenbrooke's Hospital, Cambridge CB2 2QQ
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References |
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