Dcytb (Cybrd1) Functions As Both a Ferric and a Cupric Reductase in Vitro

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FEBS Letters 582 (2008) 1901–1906

Dcytb (Cybrd1) functions as both a ferric and a cupric reductase in vitro

Steven Wyman, Robert J. Simpson, Andrew T. McKie, Paul A. Sharp* Kings College London, Iron Metabolism Group, Nutritional Sciences Division, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom

Received 2 April 2008; revised 18 April 2008; accepted 7 May 2008

Available online 20 May 2008

Edited by Miguel De la Rosa

b homologues [13–15] and more recently the Steap family Abstract MDCK cells expressing an inducible duodenal cyto- 561 chrome b-green fluorescent protein (Dcytb-EGFP) fusion con- of metalloreductases [16,17]. Interestingly in addition to their struct were used to investigate the function of Dcytb. The ferrireductase activity, a number of the Steap proteins can also Dcytb-EGFP protein was targeted correctly to the plasma mem- reduce copper [17]. brane, and cells displayed increased ferric and cupric reductase Dcytb was identified using a subtractive cloning strategy activities, which were greatly reduced in the presence of doxycy- which also identified other important iron transport proteins cline. The data suggests that Dcytb plays a physiological role in including ferroportin [12,18]. Dcytb is a 286 amino acid, six both iron and copper uptake, through divalent metal transporter transmembrane domain, plasma membrane protein that exhib- 1 (DMT1) and copper transporter 1, respectively. In support of 59 its reductase activity in both nitro-blue tetrazolium and ferric this hypothesis, we show that Fe uptake was significantly en- nitrilotriacetic acid assays [12]. In addition, Dcytb mRNA hanced in Dcytb-EGFP expressing MDCK cells which endoge- and protein are rapidly induced in response to iron deficiency nously express DMT1. 2008 Federation of European Biochemical Societies. Pub- and hypoxia indicating that it may play a pivotal role in iron lished by Elsevier B.V. All rights reserved. metabolism [12]. Expression studies revealed Dcytb to be localised predominantly to the duodenal brush border membrane Keywords: Duodenal cytochrome b (Dcytb); Cytochrome b and it was therefore suggested that it was involved with uptake reductase 1; Reductase; Iron; Copper; Ascorbate of dietary inorganic iron, acting in concert with DMT1 [12]. However, the role of Dcytb in iron metabolism remains un- clear. There are no reports of impaired iron metabolism being associated with mutations in human Dcytb. Furthermore, genetic deletion of Dcytb in mice failed to show a pronounced 1. Introduction defect in iron acquisition and utilisation [19]. It has been suggested that this lack of phenotype may be due to the presence Iron and copper are essential for life in most living organ- of other reductases in the gut [19], e.g. Steap3 or related family isms being required for a wide variety of cellular processes, members. However, mRNA expression of the Steap metallore- ranging from DNA and neurotransmitter synthesis to haem ductases in the intestine is relatively low and none have been biosynthesis and as prosthetic groups within key enzymes re- shown to be iron-regulated [16,17]. Furthermore, while the quired for oxidative phosphorylation and the TCA cycle. sub-cellular localisation of the Steap proteins in the gut is The processes of dietary import for iron and copper in mam- not known, studies of their function and localisation in other mals share some similarities. For example, both metals are ab- tissues indicate that it is likely to be endosomal [16]. Hence sorbed in the duodenum via specific carriers which require the for these reasons it is unlikely that Steap3 or other family metal to be in the reduced state i.e. ferrous Fe(II) and cuprous members would make a major contribution to the ferric reduc- Cu(I), prior to being taken up via their respective transporters tase activity of the duodenal mucosa [16]. divalent metal transporter 1 (DMT1) [1,2] and copper trans- A further explanation for the lack of phenotype in Dcytb porter 1 (Ctr1) [3–6]. knockout mice might be the presence of other compensatory A variety of ferric reductases have been shown to aid the factors in the intestinal lumen such as ascorbate. Ascorbic acid acquisition of iron, for example the FRE family of metallore- is a normal constituent of gastric juice and other gastric secre- ductases in yeast [7,8] and the FRO protein in plants [9].In tions even in scorbutic species [20]. However, levels of ascorbic yeast, both Fre1 and Fre2 have been shown to reduce copper acid secreted into the gut lumen may vary widely between spe- as well as iron and to increase copper uptake [10,11]. In mam- cies and might be substantially higher in mice, which can gen- mals two major ferric reductase families have been described, erate ascorbate from glucose. Interestingly, we have shown duodenal cytochrome b (Dcytb) [12] and related cytochromes that ascorbate levels in the duodenal mucosa are iron-regulated, being increased in iron deficient and hypotransferrinae- mic mice and by iron deficiency in humans [21,22]. *Corresponding author. To understand the role of Dcytb in metal homeostasis, we E-mail address: [email protected] (P.A. Sharp). have characterized the functional capabilities of Dcytb in vitro. We demonstrate that Dcytb has the capacity to reduce Abbreviations: Ctr1, copper transporter 1; Dcytb, duodenal cytochrome b; DHA, dehydroascorbate; DMT1, divalent metal transporter both iron and copper complexes. These effects were markedly 1; EGFP, green fluorescent protein; NTA, nitrilotriacetic acid; PBS, increased when cells were loaded with dehydroascorbate phosphate buffered saline (DHA), which increases intracellular ascorbate. Furthermore,

0014-5793/$34.00 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.05.010 1902 S. Wyman et al. / FEBS Letters 582 (2008) 1901–1906 over-expression of Dcytb in MDCK cells, which also endoge- Fe(III)-nitrilotriacetic acid (NTA) and 200 lM 3-(2-pyridyl)-5,6-diphe- nously express DMT1, significantly increases iron uptake, indi- nyl-1,2,4-triazine-40,400-disulfonic acid sodium salt (ferrozine) or 50 lM cating that Dcytb has a role in cellular iron transport. Cu(II)-NTA and 200 lM bathocuprionedisulfonate (BCS) for the iron and copper assays, respectively. The reduction of iron was measured by the formation of the coloured Fe(II)-ferrozine complex and moni- toring the change in absorbance at 562 nm. Similarly, the formation 2. Materials and methods of the Cu(I)-BCS complex was monitored by the change in absorbance at 482 nm. Standard curves were generated to convert the absorbance value into pmoles of iron or copper reduced. All reactions were per- 2.1. Transfection and cell culture formed in the dark at 37 C unless stated. Murine Dcytb cDNA was PCR amplified from duodenal mRNA and cloned into the pEGFP-N1 (Clontech, Palo Alto, USA) to generate a green fluorescent protein (EGFP) C-terminal tagged Dcytb con- 2.5. Iron uptake assays struct. The expression of the Dcytb-EGFP protein was then placed Cells were prepared as per the reductase assay. After washing in under the control of an inducible promoter, the tetracycline responsive PBS, cells were incubated for 10 min in room temperature pH 7.5 element (TRE) of the pTRE2hyg vector (Clontech, Palo Alto, USA). Hanks Balanced Salt Solution (HBSS) [5 mM KCl, 4.2 mM KHCO3, The tagged sequence was PCR amplified and cloned into the pTRE2- 0.36 mM K2HPO4, 0.44 mM KH2PO4, 1.3 mM CaCl2 Æ 2H2O, 0.5 mM hyg vector (Clontech, Palo Alto, USA) generating a final pTRE2hyg- MgCl2 Æ 6H2O, 140 mM NaCl, 10 mM N-2-hydroxyethylpiperazine- 0 Dcytb-EGFP vector construct. Each construct was fully sequenced N -2-ethanesulphonic acid]. Cells were then incubated at room temper- 59 (Lark Technologies, Cogenics, Clinical Data Inc., Newton, USA) to ature in pH 5.5 HBSS containing 10 lM Fe(III)-NTA. After incuba- ensure no mutations were introduced during the cloning process. tion cells were washed three times with ice cold pH 7.5 HBSS and then Tetracycline-Off Madin-Darby Canine Kidney (TET-Off MDCK) lysed in 0.5 M NaOH before gamma counting. cells (Clontech, Palo Alto, USA) were transfected with the pTRE2- hyg-Dcytb-EGFP vector using lipofectamine2000 (Invitrogen, Paisley, 2.6. Data analysis UK) as per the manufacturers instructions. Cells were the cultured for Data are presented as the mean ± S.E.M. Statistical analysis was 2 weeks in Dulbeccos modified Eagles medium (Sigma–Aldrich, Gill- carried out using SPSS statistics package, and utilised one-way ANO- ingham, UK) supplemented with 10% TET system approved fetal bo- VA followed by Tukeys post hoc test or Students unpaired t-test vine serum (Clontech, Palo Alto, USA), penicillin/streptomycin where appropriate. Differences were considered significant at P < 0.05. (Sigma–Aldrich, Gillingham, UK), 1 ng/ml puromycin (Clontech, Palo Alto, USA) and 200 lg/ml hygromycin (Calbiochem, Merck KGaA, Darmstadt, Germany). Expression of Dcytb-EGFP was knocked down by addition of 3. Results and discussion 20 ng/ml doxycycline (Clontech, Palo Alto, USA) to culture medium for 4 days. Prior to either ferric or cupric reductase assays cells were Dcytb remains one of the most highly (and rapidly) iron- treated with either: 100 lM DHA (Sigma–Aldrich, Gillingham, UK) for 4 h; 100 lg/ml phloretin (Calbiochem, Merck KGaA, Darmstadt, responsive mRNA or proteins so far described. Here we have Germany) for 30 min followed by 100 lM DHA for 4 h; or 100 lM examined the functional capabilities of the Dcytb protein in DHA with 1 U/ml ascorbate oxidase (Sigma–Aldrich, Gillingham, iron reduction as well as investigating its potential physiologi- UK). cal role in reduction of copper using an inducible polarised epithelial cell system. 2.2. Confocal microscopy Dcytb-EGFP protein was expressed on the plasma mem- pTRE2hyg-Dcytb-EGFP transfected cells were cultured in Lab- TEK chambered coverglass slides (Nunc, Thermo Fisher Scientific, brane of transfected cells and expression was knocked down Loughborough, UK). Confocal microscopy was performed using a in the presence of doxycycline (Fig. 1). The expression of Leica LS2 confocal microscope (Leica Microsystems UK Ltd., Milton Dcytb-EGFP was associated with a significant increase in the Keynes, UK) and images captured using the 40· objective using an cellular capacity to reduce ferric iron (Fig. 2A) supporting excitation wavelength of 488 nm to visualise the EGFP-tagged proteins. our previous data [12]. In untransfected cells, ferric reductase activity was extremely low and could not be detected above 2.3. Western blotting the background of the assay until some 90 min after the addi- Cell monolayers were homogenised in ice cold sucrose buffer tion of iron. The low endogenous reductase activity in MDCK [250 mM sucrose (Sigma–Aldrich, Gillingham, UK), 10 mM Tris (Sig- cells is one of the features that make them a suitable vehicle for ma–Aldrich, Gillingham, UK), pH 7.5], supplemented with protease investigating Dcytb function in vitro. Ferric reductase activity inhibitors [1 mM PMSF and 1 lg/ml leupetin and pepstatin] in a Tef- lon homogeniser prior to centrifugation at 700 · g for 15 min. Protein in Dcytb-EGFP expressing MDCK cells was pH-dependent containing supernatant was denatured at 100 C for 3 min in an equal (Fig. 2B) reflecting the greater solubility of ferric iron at acid volume of Laemmli sample buffer before loading onto a 10% SDS– pH. Furthermore, the process displayed Michaelis–Menten PAGE gel. Samples were transferred to a nitrocellulose membrane be- kinetics with increasing concentrations of ferric NTA, and fore blocking overnight in ice-cold phosphate buffered saline (PBS) was susceptible to changes in the experimental temperature containing 5% fat free milk and 0.1% Tween 20. To detect Dcytb- EGFP, the membrane was probed with a murine anti-GFP (JL-8,) pri- (Fig. 2C and Table 1). mary antibody (Clontech, Palo Alto, USA). Endogenous DMT1 pro- Like iron, copper must also be reduced before it is taken up tein was detected using rabbit anti-DMT1 (NRAMP24-A) antibody across cell membranes [23]. Our data support a role for Dcytb (Alpha Diagnostics Inc., San Antonio, Texas, USA). Cross reactivity as a cupric reductase (Fig. 3A). Interestingly, unlike iron, the was detected with HRP-conjugated secondary antibodies (Dako, Ely, UK) and visualised using the SuperSignal West Femto detection sys- reduction of copper (II) did not display a strong pH-depen- tem (Pierce, Rockford, USA). dency but copper reduction was maximal at neutral pH (Fig. 3B). Cupric reductase activity was saturable with excess 2.4. Cupric and ferric reductase assays copper and enzyme kinetics varied with experimental tempera- Dcytb-EGFP transfected and untransfected TET-Off MDCK cells ture (Fig. 3C and Table 2). were plated into 24 well plates and grown for 1 week post-confluency Previous work has shown that known members of the cyto- before performing reductase assays. The cells were washed three times in PBS, pH 7.0 before incubation in a physiological buffer [25 mM chrome b561 family of reductases, including Dcytb, are ascor- MOPS, 25 mM MES, 5.4 mM KCl, 5 mM glucose, 140 mM NaCl, bate dependent enzymes [24], and our data further highlight 1.8 mM CaCl2, 800 lM MgCl2] supplemented with either 50 lM intracellular ascorbate levels as an important factor in deter- S. Wyman et al. / FEBS Letters 582 (2008) 1901–1906 1903

Fig. 1. Inducible Dcytb-EGFP expression in MDCK Tet-Oﬀ cells. (A) Confocal images of EGFP signal (green) in transfected MDCK cells in the presence or absence of 20 ng/ml doxycyline. In the absence of doxycycline prominent plasma membrane localisation of Dcytb-EGFP can be observed. (B) Western blot of protein extracted from cells treated with 20 ng/ml doxycyline compared with untreated cells. A clear band of approximately 55 kDa is visible corresponding to the expected size of the Dcytb-EGFP fusion protein.

A 60 B 1.2 * A 50 0.9 B * B 40 * C 30 0.6 D 20 * Iron Reduction * 0.3 10 E (pmol/µg cell protein) * aa Ferric Reductase Activity (pmol/µg cell protein/min) aaaa 0 0 0 30 60 90 120 150 180 3.5 5.5 6.0 6.5 7.0 7.5 Time (min) pH C 5

2 v (pmol/µg/min)

0 0 200 400 600 800 1000 [s] (µM)

Fig. 2. Dcytb functions as a ferric reductase. (A) Time course of reduction of ferric NTA in Dcytb-EGFP over-expressing- (solid line) and non- transfected (dashed line) MDCK cells. Statistical differences, *P < 0.01, between transfected and untransfected cells at each time point were determined using Students unpaired t-test. (B) Effect of extracellular pH on ferric reductase activity. Data bars that do not share common letters (uppercase – transfected cells; lowercase – untransfected cells) are significantly different from each other (P < 0.05, one-way ANOVA and Tukeys post hoc test). (C) Concentration-dependent relationships for the reduction of ferric NTA by Dcytb at 22 C() and 37 C(n). The kinetic parameters derived from Michaelis–Menten analysis of these data are shown in Table 1. Data are mean ± S.E.M. of four to six observations in each group. mining Dcytb activity (Fig. 4). We found that preloading cells rapidly reduced inside cells using glutathione or glutaredoxin with the oxidised form of ascorbate (DHA) had a profound ef- [28] to generate ascorbic acid. Our data indicate that levels fect – greatly increasing both ferric and cupric reductase activ- of intracellular ascorbate are an important determinant of ities (Fig. 4A and B). This effect was blocked by inhibiting Dcytb activity. DHA uptake using the GLUT transporter inhibitor phloretin. Interestingly, Dcytb has recently been localised to the mem- It is well known that a number of cell types, including entero- branes of mature red blood cells from scorbutic species such as cytes and renal tubular epithelial cells, are able to utilise vari- humans and guinea pigs leading to the conclusion that it may ous GLUT transporters to transport DHA [25–27]. DHA is play a role in ascorbate regeneration in some tissues [14].In 1904 S. Wyman et al. / FEBS Letters 582 (2008) 1901–1906

Table 1 Table 2 Dcytb-EGFP ferric reductase kinetics Dcytb-EGFP cupric reductase kinetics 22 C37C Statistics 22 C37C Statistics

Km (lM) 92.1 ± 19.4 74.0 ± 5.9 NS Km (lM) 15.2 ± 1.6 23.1 ± 2.5 NS Vmax 2.18 ± 0.13 4.48 ± 0.10 P < 0.05 Vmax 3.18 ± 1.61 8.34 ± 0.20 P < 0.05 (pmol/lg cell protein/min) (pmol/lg cell protein/min) Cells were prepared as described in Section 2, and incubated with Copper reduction was measured using 50 lM cupric NTA and 200 lM 50 lM ferric NTA and 200 lM ferrozine for 10 min at either 22 Cor BCS. Experimental conditions and data analysis were as described in 37 C. Reductase data was analysed using GraphPad Prism (Graph- the legend to Table 1. Pad Software Inc., San Diego, CA, USA) to generate kinetic parameters. this function to determine whether Dcytb might work in concert with DMT1, reducing ferric iron and presenting it in the vitro we found that the presence of ascorbate oxidase, which absorbable ferrous form. Over-expression of Dcytb-EGFP in abolishes all extracellular ascorbate, had no effect on the abil- MDCK cells led to a significant increase in 59Fe uptake ity of Dcytb to reduce either iron or copper (Fig. 4A and B) (Fig. 6). In contrast to previous studies with Dcytb knockout indicating that reductase activity was not secondary to either mice [19], these data provide evidence that Dcytb plays a role extracellular ascorbate regeneration by Dcytb or ascorbate in cellular iron acquisition. It remains to be determined secretion from the cell monolayer into the incubation medium. whether Dcytb plays a direct role in copper absorption. While The reduction of both iron and copper in MDCK cells was DMT1 is firmly established as the major transporter for inor- specifically dependent on Dcytb-EGFP expression and could ganic iron, the nature of the epithelial copper transporter is less be inhibited by incubation with doxycycline (Fig. 4A and B). clear. Both DMT1 and Ctr1 have been shown to transport In addition to metal–NTA complexes, more physiological copper in vitro in intestinal Caco-2 cells and renal MDCK cells forms of both iron (ferric citrate) and copper (cupric-histidine) [31–33]. However, a recent study demonstrated that Ctr1 was were also effectively reduced by Dcytb (Fig. 5A and B). Since localised to the basolateral and not the apical membrane in MDCK cells endogenously express DMT1 [29,30], we utilised these cell types [33]. Furthermore, apical copper uptake in both

AB80 1.6 * * B B A,B A,B A,B 60 * 1.2 A * 40 0.8 * c c c 20 0.4

Copper Reduction b

(pmol/µg cell protein) (pmol/µg * a a,b (pmol/µg cell protein/min) Cupric Reductase Activity 0 0 0 30 60 90 120 150 180 3.5 5.5 6.0 6.5 7.0 7.5 Time (min) pH

C 10

4 v (pmol/µg/min) 2

0 0 200 400 600 800 1000 [s] (µM)

Fig. 3. Dcytb functions as a cupric reductase. (A) Time course of reduction of cupric NTA in Dcytb-EGFP over-expressing- (solid line) and non- transfected (dashed line) MDCK cells. Statistical differences, *P < 0.01, between transfected and untransfected cells at each time point were determined using Students unpaired t-test. (B) Effect of extracellular pH on cupric reductase activity. Data bars that do not share common letters (uppercase – transfected cells; lowercase – untransfected cells) are significantly different from each other (P < 0.05, one-way ANOVA and Tukeys post hoc test). (C) Concentration-dependent relationships for the reduction of cupric NTA by Dcytb at 22 C(d) and 37 C(n). The kinetic parameters derived from Michaelis–Menten analysis of these data are shown in Table 2. Data are mean ± S.E.M. of four to six observations in each group. S. Wyman et al. / FEBS Letters 582 (2008) 1901–1906 1905

AB 1.6 2.4 B BB B 1.2 1.8

0.8 1.2 A

0.4 A 0.6 A A A,C b b C a,b

Ferric Reductase Activity a (pmol/µg cell protein/min) (pmol/µg cell protein/min) a,c baa,bb,cCupric Reductase Activity a 0 0 Untreated DHA DHA + DHA + DHA + Untreated DHA DHA + DHA + DHA + Phloretin Ascorbate Doxycycline Phloretin Ascorbate Doxycycline Oxidase Oxidase

Fig. 4. Ferric and cupric reductase activities in Dcytb-expressing cells following treatment with dehydroascorbate. (A) Ferric and (B) cupric reductase activity was determined in Dcytb-EGFP transfected cells (filled bars) compared with untransfected cells (open bars) following treatment with either: 100 lM DHA for 4 h, which generates bioavailable intracellular ascorbate; 30 min pre-treatment with 100 lg/ml phloretin (a GLUT- transporter inhibitor) followed by 100 lM DHA for 4 h; 100 lM DHA, together with ascorbate oxidase (1 U/ml) to eliminate extracellular ascorbate, for 4 h; doxycycline (20 ng/ml for 4 days) to switch off the inducible expression of Dcytb-EGFP, followed by 100 lM DHA for 4 h. Data bars that do not share common letters (uppercase – transfected cells; lowercase – untransfected cells) are significantly different from each other (P < 0.05, one-way ANOVA and Tukeys post hoc test). Data are mean ± S.E.M. of four to six observations in each group.

A 0.3 B 1 * * 0.8 * 0.2 0.6 * 0.4 0.1

0.2 Ferric Reductase Activity (pmol/µg cell protein/min) (pmol/µg cell protein/min) Cupric Reductase Activity

0 0 Iron-NTA Iron-Citrate Copper-NTA Copper-Histidine

Fig. 5. Physiological substrates for Dcytb. In addition to ferric and cupric NTA, physiological ferric (A) and cupric conjugates (B) were also reduced in Dcytb-EGFP cells (ﬁlled bars) versus untransfected cells (open bars). Data are mean ± S.E.M. of four to six observations in each group. *P < 0.01 compared with untransfected cells.

Caco-2 cells and MDCK cells appeared to occur via a non- 3 DMT1 mechanism [33]. * In conclusion, we have shown that Dcytb can function as 2.5 both a ferric and a cupric reductase in vitro. While the Steap proteins are known to possess both ferric and cupric reductase 2 * * activity [17], the ability of Dcytb to reduce copper is a previ- 1.5 ously unidentiﬁed function of the protein and may serve to * augment cellular copper absorption. This novel ﬁnding pro- 1 vides yet another link between mammalian iron and copper metabolism [23]. 0.5 * Iron uptake (normalised) Acknowledgements: This work was funded by project grants from the 0 Biotechnology and Biological Sciences Research Council. S.W. was 0 153045607590 supported by a KCL Strategic Studentship. Time (min)

Fig. 6. Uptake of 59Fe-ferric NTA is stimulated in Dcytb expressing cells. 59Fe uptake in Dcytb-EGFP expressing MDCK cells (solid line) References was signiﬁcantly higher at all time points compared with untransfected cells (dashed line). Data are mean ± S.E.M. of four to six observations [1] Fleming, M.D., Trenor, C.C., Su, M.A., Foernzler, D., Beier, in each group. Statistical diﬀerences, *P < 0.01, between transfected D.R., Dietrich, W.F., et al. (1997) Microcytic anaemia mice have and untransfected cells at each time point were determined using a mutation in Nramp2, a candidate iron transporter gene. Nat. Students unpaired t-test. Genet. 16, 383–386. 1906 S. Wyman et al. / FEBS Letters 582 (2008) 1901–1906

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