Biochemical Society Transactions

Metal Metabolism: Transport, Development and Neurodegeneration

The role of Dcytb in iron metabolism: an update

Andrew T. McKie

Abstract

Dcytb (duodenal cytochrome b) is an iron-regulated ferric reductase highly expressed in duodenal enterocytes. Its location and strong regulation by iron has indicated it plays an important role in iron absorption. Expression of Dcytb in cells (Caco-2 and MDCK) was found to increase both ferric reductase activity and stimulate uptake of 59Fe. An additional increase in cupric reductase activity was found in MDCK (Madin–Darby canine kidney) cells expressing Dcytb. Expression and purification of Dcytb in insect cells reveals that Dcytb is a di-haem protein and that the haems are reducible by ascorbate, indicating that ascorbate is the likely intracelluar electron donor. Studies underway in Dcytb-knockout mice reveal that Dcytb is the only iron-regulated ferric reductase in the duodenal mucosa and that loss of Dcytb affects iron absorption.

  • Cybrd1
  • duodenal cytochrome b (Dcytb)
  • duodenum
  • ferric reductase
  • haem
  • iron metabolism

Introduction

A variety of ferric reductases have been shown to aid the acquisition of iron, e.g. the FRE family of metalloreductases in yeast [1,2] and the FRO protein in plants [3]. In yeast, both Fre1 and Fre2 have been shown to reduce copper as well as iron and to increase copper uptake [4,5]. In mammals, two major ferric reductase families have been described, Dcytb (duodenal cytochrome b) [6] and related cytochrome b561 homologues [79] and the Steap family of metalloreductases [10], which, in addition to their ferrireductase activity, can also reduce copper.

Dcytb (also known as Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush-border membrane. It has ferric reductase activity and is thought to play a physiological role in dietary iron absorption. On the basis of sequence homology, Dcytb is a member of the cytochrome b561 family. Dcytb was identified using a subtractive cloning strategy which also identified other important iron transport proteins such as DMT1 (divalent metal transporter 1) and ferroportin [6,11]. Dcytb is a plasma membrane protein containing 286 amino acids with six transmembrane domains that exhibits ferric reductase activity in both NBT (Nitro Blue Tetrazolium) and ferric nitrilotriacetic acid assays. In addition, Dcytb mRNA and protein are rapidly induced in response to iron deficiency and hypoxia, indicating a key role in iron metabolism [6]. Expression studies revealed Dcytb to be localized predominantly to the duodenal brush-border membrane, and it was therefore suggested that it was involved with absorption of dietary inorganic iron, acting in concert with DMT1 [6]. However, the requirement of Dcytb for iron metabolism has since been questioned, with generation of the Dcytb-knockout mouse which did not display a pronounced defect in iron acquisition [12]. This lack of phenotype has led to suggestions that Dcytb may have other physiological roles such as in uptake of copper [13] or in ascorbate metabolism [7]. Indeed, Dcytb may have a broader role in cellular redox mechanisms, as it is expressed in many tissues. In humans, it is highly expressed in erythrocyte membranes, where it may contribute to reduction of extracellular dehydroascorbate. In mice, in contrast, the protein is only expressed in erythroblasts and is absent from mature erythrocytes [7].

To understand the role of Dcytb in metal homoeostasis, the functional capabilities of Dcytb were characterized both in vitro and in vivo. Overexpression of Dcytb in MDCK (Madin–Darby canine kidney) or Caco-2 cells, which also endogenously express DMT1, significantly increases iron uptake, indicating that Dcytb plays a role in iron transport [14,15]. Dcytb has the capacity to reduce both iron and copper complexes and that activity was markedly increased when cells were loaded with DHA (dehydroascorbate), which increases intracellular ascorbate [15].

Dcytb expression in cells stimulates iron uptake

The functional capabilities of the Dcytb protein in iron reduction and iron uptake were examined using an inducible cell system in MDCK cells and Caco-2 cells [14,15].

In the inducible system, plasma membrane expression of the Dcytb–EGFP (enhanced green fluorescent protein) fusion protein in MDCK cells was confirmed by confocal microscopy and Western blotting, with expression strongly inhibited in the presence of doxycycline [15]. In MDCK cells, the expression of Dcytb–EGFP was associated with a significant increase in the cellular capacity to reduce ferric iron. Expression of Dcytb–EGFP in MDCK cells led to a significant increase in 59Fe uptake, providing evidence that Dcytb does play a role in iron acquisition. Similar results revealing increased ferric reductase activity and associated increased uptake of 59Fe were obtained in Caco-2 cells expressing an untagged Dcytb [14]. Like iron, copper must also be reduced before it is taken up across the cell membrane. In MDCK cells, expression of Dcytb–EGFP also stimulated cupric reductase activity. Hence it is possible that Dcytb may also reduce copper in the intestine, delivering Cu(I) to Ctr1 for uptake from the gut lumen.

Previous work has shown that known members of the cytochrome b561 family of reductases, including Dcytb, are ascorbate-dependent enzymes [16], and our data further highlight intracellular ascorbate levels as an important factor in determining Dcytb activity. Pre-loading cells with the oxidized form of ascorbate (DHA) has a profound effect, greatly increasing both ferric and cupric reductase activities in MDCK cells and ferric reductase activity in Caco-2 cells. In MDCK cells, this effect was blocked by inhibiting DHA uptake using the GLUT (glucose transporter) inhibitor phloretin [15].

Dcytb has been localized to the membranes of mature red blood cells from scorbutic species such as humans and guinea-pigs, raising the possibility that it may play a role in ascorbate regeneration in some tissues [7]. In MDCK cells, the presence of ascorbate oxidase, which abolishes all extracellular ascorbate, had no effect on the ability of Dcytb to reduce either iron or copper, indicating that reductase activity was not secondary to either extracellular ascorbate regeneration by Dcytb or ascorbate secretion from the cell monolayer into the incubation medium [15].

Biochemical analysis of purified Dcytb

To obtain a better understanding of biochemical aspects of Dcytb, a His6-tagged Dcytb construct was expressed in insect cells (Sf9) using a baculovirus expression system [17]. Dcytb was then purified from Sf9 cells using metal-affinity chromatography. Haem content analyses of purified His6-tagged Dcytb derived from Sf9 cell preparations contained an average of ∼1.7 haem b per Dcytb monomer, indicating that Dcytb is indeed a di-haem protein. In addition, the Dcytb EPR spectrum at 12 K displayed similar features to those of cytochrome b561 recorded at 15 K [18]. The presence of two haems with bis-histidine co-ordination was also supported by the site-directed mutagenesis studies, in which the replacement of any of the identified four Dcytb histidine residues led to a loss of most of the haem spectrum. The equivalent histidine residues are conserved in the cytochrome b561 sequence and have been shown to be required for Fe3+ reductase activity [19]. Replacement of the non-conserved His33 had negligible effect on haem binding, as was observed for mutation of the equivalent histidine in cytochrome b561. In a static reduction experiment, 67% of the haem was reduced by ascorbate. This is the first report of the isolation of Dcytb, and the data provide direct evidence that the cytochrome contains two haem groups per molecule and identify four critical histidine residues predicted to be involved in haem binding. In these respects, and in spectroscopic properties, Dcytb is similar to other cytochromes of the b561 family. The midpoint reduction potentials of the haems show that they are reducible by ascorbate, indicating that this is a likely intracellular electron donor.

Studies in Dcytb-knockout mice

To investigate the role of Dcytb in vivo, we investigated duodenal ferric reductase activity and iron absorption in Dcytb-knockout mice [20]. Dcytb-knockout mice were fed on a iron-deficient diet for 3–6 weeks or 0.5 atm (1 atm=101.325 kPa) hypoxia for 3 days. Duodenal ferric reductase activity was measured in Dcytb-knockout mice by NBT (1 mM) or ferrozine (0.446 mM) assays. Duodenal iron absorption was measured by injecting radioactive 59Fe into tied duodenal segments [21]. The data show that Dcytb is the primary iron-regulated ferric reductase in the duodenum. Iron uptake into the mucosa from the diet tends to be lower in Dcytb-knockout mice; however, there is more efficient transfer from mucosa to the body. Thus there may be compensatory changes in transfer of iron via Ireg1 as a result of changes in body iron stores. Body non-haem iron stores, and expression and localization of other genes such as those encoding DMT1 and Ireg1 in Dcytb-knockout and transgenic mice are currently under investigation. This demonstrates that loss of Dcytb affects iron absorption.

Conclusions

Our data strongly suggest that Dcytb plays a role in dietary iron absorption in the duodenal mucosa by direct reduction of ferric iron which enables uptake via DMT1. This role may be particularly important in iron deficiency and in scorbutic species such as humans. The physiological role of Dcytb in other tissues such as red blood cells of scorbutic species remains to be determined.

Acknowledgments

This work was funded by grants from the EU (European Union) Framework Programme 5, MRC (Medical Research Council) and BBSRC (Biotechnology and Biological Sciences Research Council).

Footnotes

  • Metal Metabolism: Transport, Development and Neurodegeneration: A Biochemical Society Focused Meeting held at Imperial College London, U.K., 9–10 July 2008. Organized and Edited by David Allsop (Lancaster, U.K.) and Harry McArdle (Rowett Research Institute, Aberdeen, U.K.).

Abbreviations: Dcytb, duodenal cytochrome b; DHA, dehydroascorbate; DMT1, divalent metal transporter 1; EGFP, enhanced green fluorescent protein; MDCK, Madin–Darby canine kidney; NBT, Nitro Blue Tetrazolium

References

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