Research Article

An Iron-Regulated Ferric Reductase Associated with the Absorption of Dietary Iron

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Science  02 Mar 2001:
Vol. 291, Issue 5509, pp. 1755-1759
DOI: 10.1126/science.1057206

Abstract

The ability of intestinal mucosa to absorb dietary ferric iron is attributed to the presence of a brush-border membrane reductase activity that displays adaptive responses to iron status. We have isolated a complementary DNA, Dcytb (for duodenal cytochrome b), which encoded a putative plasma membrane di-heme protein in mouse duodenal mucosa. Dcytb shared between 45 and 50% similarity to the cytochrome b561 family of plasma membrane reductases, was highly expressed in the brush-border membrane of duodenal enterocytes, and induced ferric reductase activity when expressed in Xenopus oocytes and cultured cells. Duodenal expression levels of Dcytb messenger RNA and protein were regulated by changes in physiological modulators of iron absorption. Thus, Dcytb provides an important element in the iron absorption pathway.

Iron is essential for a large number of biological processes ranging from O2 transport to DNA synthesis and electron transport. Therefore, iron acquisition is a fundamental requirement in almost all living organisms. At physiological pH and in the presence of oxygen, iron exists predominantly in the highly insoluble ferric Fe(III) form, whereas iron transport systems take up the ferrous Fe(II) ion, which is very unstable and quickly oxidizes to ferric iron. To overcome this problem, specialized transmembrane electron transport systems have evolved, known collectively as ferric or ferric-chelate reductases. These redox systems use intracellular reducing cofactors to reduce ferric Fe(III) to the ferrous Fe(II) form at the extracellular surface, thus allowing the cell to take up the ferrous iron. A number of genes encoding plasma membrane ferric reductases have been cloned and characterized in yeast (1), plants (2), and bacteria (3), but none have so far been described in mammalian systems.

In mammals, iron is taken up by the proximal small intestinal epithelium, primarily the duodenum, where it is known that ferrous iron is more efficiently absorbed than ferric iron. A divalent cation transporter (DCT1), also known as Nramp2 and DMT1 (4–6), has now been shown to be responsible for the uptake of ferrous iron from the lumen into the mucosa. However, because most dietary nonheme iron is in form of ferric iron complexes, these must be reduced to yield ferrous ions before iron can be successfully transported by DCT1. A brush-border surface ferric reductase enzymic activity has been demonstrated, both in the duodenal mucosa itself and in cultured intestinal cells (7, 8). The enzyme has been partially purified and is associated with heme containing b-type cytochrome (9). By using a subtractive cloning strategy designed to identify intestinal genes involved in iron absorption, we isolated a previously unidentified gene encoding a cytochrome b–like molecule, which we named Dcytb (for duodenal cytochrome b) (Fig. 1) (10, 11).

Figure 1

Multiple sequence alignment of the mouse and human Dcytb with the NH2-terminus of the rabbit hemoprotein cytochrome b558 (p30) and seven cytochrome b561 sequences from different species. The sequence data for the cytochromes were obtained from the SwissProt database, and their alignment with Dcytb was determined with the program PileUp (GCG, Madison, Wisconsin). Sequences are shown in single-letter code (28) with the six predicted transmembrane domains (TM1 through TM6) highlighted in blue. The four conserved His residues proposed as heme ligands are highlighted in red, and the regions of b561 thought to be related to substrate binding or recognition are boxed.

Dcytb Is a Homolog of Cytochrome b561

A BLAST search (12) of the SwissProt database revealed that the predicted protein sequence of Dcytb was most similar to sheep cytochrome b561 (accession number p49447) [41% identical, 54% similar (over a 218–amino acid region)]. Weak homology was also found to mitochondrial cytochrome b from various species. Interestingly, no significant homology was found between Dcytb and previously described ferric reductases from plant or yeast origin. The NH2-terminal region of Dcytb was virtually identical to that of a hemoprotein or cytochrome b558, called p30 (SwissProt accession number G546819), isolated from rabbit neutrophil plasma membranes (13). No full-length protein or cDNA sequence for p30 has been reported. p30 is known to be 30 kD in size, but the physiological function of the molecule is not known. An alignment of Dcytb and p30 revealed that there was only one change in 20 residues (Fig. 1), a Gly (GGN) to Val (GUN) at position 6. Therefore, p30 is either an isoform or the rabbit homolog of Dcytb. The sizes of b561, p30, and Dcytb proteins are similar, and both b561 and p30 are thought to be di-heme proteins and reside in the plasma membrane. Cytochrome b561 functions as a transmembrane electron shuttle between the cytoplasm, where ascorbate acts as a reducing cofactor, and the inside of chromaffin granules, where the electron is accepted by semidehydroascorbic acid (14). It has been suggested that His pairs 48 and 118 and 84 and 157 of cytochrome b561 are potential heme ligands (14). An alignment of Dcytb with b561 sequences from a number of species revealed that these His residues were conserved in Dcytb, suggesting that Dcytb may also bind two heme groups (Fig. 1). Cytochrome b561 and Dcytb share a similar membrane topology, with six predicted transmembrane spanning regions (Fig. 1). Putative binding sites for the cytochrome b561 substrates [ascorbic acid and semidehydroascorbic acid (14)] were found to be partially conserved in Dcytb (Fig. 1, boxed regions), suggesting that Dcytb might react with one or more of these compounds.

Regulation of Dcytb by Iron Status

Iron-deficiency anemia and chronic anemia caused by ineffective erythropoiesis are common disorders of iron metabolism affecting Western populations and are potent stimulators of iron absorption. Homozygous hpx mice (which lack circulating transferrin) develop chronic anemia because of a failure to mobilize iron to the erythron (15). Compared to heterozygotes, homozygous hpx mice have an expanded erythron and greatly increased iron absorption (16). By using Northern and Western blotting (17, 18), we confirmed that Dcytb protein and mRNA levels were highly up-regulated in the duodenal mucosa from homozygous hpx mice, as compared with the heterozygotes (Fig. 2, A and D), indicating that chronic anemia up-regulated Dcytb levels. Iron deficiency induced by feeding mice an iron-deficient diet also strongly increased Dcytb mRNA levels and protein expression in duodenal extracts (Fig. 2, B and C) (17). Three major transcripts of ∼1, ∼4, and >5 kb in size were detectable by Northern blot analysis (Fig. 2C, arrows), indicative of alternate splicing or the presence of unprocessed pre-mRNA species. Relative expression of Dcytb mRNA in other tissues from mice fed a normal diet was lower (Fig. 2C). We did not test the effect of iron deficiency in all other tissues; however, there was no detectable effect of iron deficiency on mRNA levels in ileal extracts (Fig. 2D).

Figure 2

Expression and regulation of Dcytb in mouse tissues. (A) Western blot analysis of Dcytb protein levels in duodenal extracts from three individual hpx mice (right three lanes), compared with three individual heterozygotes (left three lanes). (B) Western blot analysis of duodenal extracts from two individual iron-replete mice (+Fe), two individual iron-deficient mice (–Fe), one individual mouse fed a normal diet (Norm), and one mouse fed a normal diet but exposed to 24 hours of hypoxia (Hypo). Ten micrograms of protein was loaded in each lane in (A) and (B). (C) Northern blot analysis showing Dcytb mRNA expression in the following mouse tissues: Pla, placenta; +Fe, iron-replete duodenum; –Fe, iron-deficient duodenum; Bra, whole brain; He, heart; Kid, kidney; Lu, lung; Sk, skeletal muscle; Tes, testis; and NL, neonatal liver. Arrows indicate the three main transcripts of Dcytb, and the positions of the 18S and 28S ribosomal RNA bands are indicated. Lower panel shows the ethidium bromide–stained RNA gel before blotting. (D) Reverse transcription–PCR analysis of the effect of hypoxia, hypotransferrinemia, and iron deficiency on Dcytb, gp91-phox, and GAPDH mRNA expression in mouse intestine. Lanes 1 through 4 show duodenal extracts from mice fed a normal diet: lane 1, no hypoxia; lanes 2, 3, and 4, mice exposed to 6, 24, and 72 hours of hypoxia, respectively. Lanes 5 through 8 show ileal extracts from mice fed a normal diet: lane 5, no hypoxia; lanes 6, 7, and 8, mice exposed to 6, 24, and 72 hours of hypoxia, respectively. Lanes 9 and 10 show duodenal extracts from homozygous and heterozygous hpx mice, respectively. Lanes 11 and 12 show duodenal extracts from mice fed an iron-replete diet and an iron-deficient diet, respectively. Lanes 13 and 14 show ileal extracts from mice fed an iron-replete diet and an iron-deficient diet, respectively.

Exposure to hypoxia (0.5 atm) for 1 to 3 days is another stimulator of iron absorption (and ferric reductase), which acts independently of body iron stores (19). Dcytb protein and mRNA levels both increased in the hypoxic mouse duodenum (Fig. 2, B and D). The effect of hypoxia on Dcytb expression was less marked in the ileum (Fig. 2D), as is the case for intestinal ferric reductase activity (7). As a control, we analyzed how levels of another mammalian membrane reductase, the neutrophil oxidoreductase gp91-phox, was regulated in the intestine by changing iron status (Fig. 2D). gp91-phox is not thought to reduce ferric iron complexes, despite homology to the yeast ferric reductases FRE1 and FRE2 (20). In contrast to Dcytb mRNA, which showed a marked gradient of expression between normal duodenal and ileal tissues, gp91-phox mRNA levels were constant. In addition, there was no increase in duodenal expression of gp91-phoxmRNA in mice exposed to hypoxia (Fig. 2D), in hypotransferrinemic mice (Fig. 2D), or in mice with iron deficiency (Fig. 2D). Thus, the expression and regulation of Dcytb by iron status in the intestine is similar to the ferric reductase activity measured in intestinal fragments (7), as would be expected for a protein involved in iron acquisition.

Dcytb Is Located in the Brush Border and Functions as a Ferric Reductase

We next investigated whether Dcytb protein was present in an appropriate subcellular location to have a role in reduction of dietary ferric iron (18). In the duodenum of iron-deficient mice, Dcytb mRNA was mostly localized in the mature enterocytes of the upper villus region, with the crypt cells being negative for Dcytb (Fig. 3A). The upper villus region, populated largely by mature enterocytes, is the site of the highest iron-transporting activity (21). Dcytb protein was highly expressed in brush-border membrane vesicles prepared from the duodenal mucosa but not from those prepared from ileal mucosa (Fig. 3B). This is consistent with the decreased reductase activity associated with this region of the intestine (7). Immunostaining for Dcytb mouse duodenal mucosa (Fig. 3C) provided further evidence that Dcytb was present in the brush-border membrane, although some cytoplasmic staining was observed within enterocytes. No staining was present in the crypts, and expression in the brush border of enterocytes was visible along the entire length of villi from the crypt-villus junction to the villus tip. Staining was more intense in sections from mice fed an iron-deficient diet. This staining pattern is similar to that observed with DCT1 in iron-deficient rat duodenum (22). Thus, Dcytb was in the region of the intestine most active in the absorption of dietary iron (duodenum) and at the appropriate subcellular location (brush-border membrane) to have a role in reduction of dietary iron.

Figure 3

Dcytb localizes to the brush-border membrane. (A) In situ hybridization with sense and antisense probes for Dcytb on sections from a wild-type CD-1 mouse fed an iron-deficient diet for 4 weeks. The right panel shows the antisense image at a higher power magnification. (B) Western blot analysis of brush-border membrane vesicles. Expression of Dcytb in brush-border membrane vesicles prepared from the duodenum of two individual mice fed a normal diet (left two lanes) and in vesicles prepared from the ileum of two individual mice fed a normal diet (right two lanes); 10 μg of protein were loaded in each lane. Arrow indicates Dcytb protein. (C) Low-power (×10) immunofluorescence image of mouse duodenal mucosa [from an iron-replete (+Fe) (left) and an iron-deficient (–Fe) animal (middle)] stained with a Dcytb antibody. Box represents the area of the higher power image (right) from the iron-deficient animal (×40). Nuclei are stained with propidium iodide (red), and Dcytb is visualized with a FITC-labeled secondary antibody (green).

To establish whether expression of Dcytb itself was capable of reducing ferric complexes, we microinjected Xenopus oocytes with Dcytb cRNA (23). A five- to sixfold increase in ferric reductase activity was observed in oocytes injected with Dcytb cRNA, as compared to either water- or mock-injected oocytes (Fig. 4A). We also transfected cell lines derived from intestinal cells (HuTu-80 and CaCo-2) with Dcytb (23). We measured ferric reductase activity in whole cells and in membrane-enriched fractions, using many substrates known to react with the reductase found in the duodenal mucosa. In intact HuTu-80 cells incubated with MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; thiazole blue], there was a significant (P < 0.001) increase in MTT reductase activity in cells transfected with Dcytb at all time points (Fig. 4B). Treatment with Dcytb antibody significantly (P < 0.001) reduced MTT reductase in transfected cells after 60 min and also inhibited reductase activity in untransfected cells after incubation for 45 (P < 0.01) and 60 min (P < 0.05). In CaCo-2 cells, ferric reductase activity was measured in a plasma membrane plus microsome and cytosol fraction by using ferric iron [Fe(III)NTA2] (NTA, nitrilotriacetic acid) as the substrate (Fig. 4C). Ferric reductase activity was significantly (P < 0.001) higher in membranes prepared from cells transfected with Dcytb, as compared to untransfected cells (Fig. 4C). Treatment of transfected cells with Dcytb antibody blocked the increase in ferric reductase activity seen on transfection (P < 0.05).

Figure 4

Dcytb expression induces ferric reductase activity. (A) Reductase activity inXenopus oocytes injected with Dcytb cRNA, water, and an unrelated cRNA. The formation of ferrous iron was calculated by measuring the optical density of the oocyte incubation buffer at 562 nm (23). Results are shown as the means of at least five individual oocytes incubated in ND96. (B) MTT reductase activity in HuTu-80 cells transfected with Dcytb (15 to 60 min). Data are presented as the means (six data points) ± SEM. (C) Ferric reductase activity in CaCo-2 cells transfected with Dcytb. Data are the means of three experiments ± SEM. Reductase activity was measured after a 5-min incubation period. In (B) and (C), T represents cells transfected with Dcytb, UT represents untransfected cells, and T/Ab are transfected cells treated with antibody to Dcytb. Error bars in (A) to (C) indicate SEM. (D) Duodenal slices from a normal littermate (top) and hypotransferrinemic (hpx) mice after incubation with NBT to localize reductase activity, demonstrating that NBT reductase is induced in a similar fashion to Dcytb and ferrireductase activity. (E) NBT reductase in hypotransferrinemic mouse duodenum is blocked by preincubation with antiserum to Dcytb. Control tissues (left and right) were incubated along with tissue slices incubated with serum (anti-Dcytb and preimmune).

Nitroblue tetrazolium (NBT) is an electron acceptor that produces a water-insoluble, intensely blue reduced product, enabling localization of redox reactions (23). NBT reductase was increased in hypotransferrinemic mouse duodenal mucosa (Fig. 4D) and in iron-deficient mice (24), paralleling previously described changes in ferrireductase activity (7). This activity was blocked by preincubation of tissue slices with a polyclonal antibody to Dcytb (Fig. 4E) but not with preimmune serum. The antibody was also found to inhibit the reduction of Fe(III)/NTA (1:2) by mouse duodenal fragments by up to 60% of the control incubation (no serum present), 61.1 ± 1.6; 1% preimmune serum present during incubation, 58.2 ± 5.1; and 1% anti-Dcytb serum present during incubation, 23.3 ± 1.9 (results are mean ± SEM, n = 3; units are pmol/min/mg of tissue;P < 0.003). These results indicate that Dcytb is responsible for the iron-regulated ferric reductase activity observed in the duodenum.

Conclusions

We identified a mammalian plasma membrane b-type cytochrome with ferric reductase activity from the duodenal mucosa. Unlike previously described ferric reductases from plants (2) and yeast (1, 25, 26), Dcytb appears to lack any conventional NADH (reduced form of nicotinamide adenine dinucleotide), NADPH (reduced form of nicotinamide adenine dinucleotide phosphate), or flavin binding motifs that would allow these cofactors to act as intracellular electron donors. The lack of sequence homology with yeast and plant sequences indicates that Dcytb evolved as a ferric reductase independently. Cytochrome b561 receives an electron from ascorbate (27) and does not appear to require other components. Dcytb may also use ascorbate or, like gp91-phox, associate with several other proteins to form an active complex. Dcytb was highly expressed in the brush-border membrane of duodenal enterocytes and was capable of reduction of ferric iron complexes in both Xenopus oocytes and cultured cells. Antibody-blocking experiments support the notion that Dcytb is responsible for the physiological reductase activity present in the duodenal mucosa. Dcytb mRNA and protein levels were up-regulated by several independent stimulators of iron absorption, including chronic anemia, iron deficiency, and hypoxia. The lack of a definable iron-responsive element within the mRNA sequence of Dcytb is unusual for a protein of iron metabolism and points to iron-dependent regulation by other mechanisms, including transcription. The isolation of Dcytb provides an important clue as to how dietary ferric iron is absorbed, and it identifies an iron-regulated mechanism by which ferrous iron could be supplied to the divalent cation transporter DCT1/Nramp2. Such a mechanism would be particularly important in iron deficiency.

  • * To whom correspondence should be addressed. E-mail: andrew.t.mckie{at}kcl.ac.uk

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