Functional role of the putative iron ligands in the ferroxidase activity of recombinant human hephaestin
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- Vashchenko, G. & MacGillivray, R.T.A. J Biol Inorg Chem (2012) 17: 1187. doi:10.1007/s00775-012-0932-x
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Hephaestin is a multicopper ferroxidase expressed mainly in the mammalian small intestine. The ferroxidase activity of hephaestin is thought to play an important role during iron export from intestinal enterocytes and the subsequent iron loading of the blood protein transferrin, which delivers iron to the tissues. Structurally, the ectodomain of hephaestin is predicted to resemble ceruloplasmin, the soluble ferroxidase of blood. In this study, the human hephaestin ectodomain was expressed in baby hamster kidney cells and purified to electrophoretic homogeneity. Ion exchange chromatography of purified recombinant human hephaestin (rhHp) resulted in the isolation of hephaestin fractions with distinct catalytic and spectroscopic properties. The fraction of rhHp with the highest enzymatic activity also showed an enhanced molar absorptivity at 600 nm, characteristic of type 1 copper sites. Kinetic analysis revealed that rhHp possesses both high-affinity and low-affinity binding sites for ferrous iron. To investigate the role of particular residues in iron specificity of hephaestin, mutations of putative iron ligands were introduced into rhHp using site-directed mutagenesis. Kinetic analysis of ferroxidation rates of wild-type rhHp and mutants demonstrated the important roles of hephaestin residues E960 and H965 in the observed ferroxidase activity.
KeywordsMulticopper oxidaseCeruloplasminFet3pIron bindingIron oxidation
Baby hamster kidney
Dulbecco’s modified Eagle’s medium–Ham F12 nutrient mixture
Ion exchange chromatography
Recombinant human hephaestin
Mammalian hephaestins are a group of multicopper ferroxidases expressed predominantly in the small intestine [1, 2]. Localized on the basolateral surface of enterocytes, hephaestin oxidizes Fe(II) exported by the basolateral ferrous transporter ferroportin to Fe(III) (for a review see ). Through this oxidation step, iron is made bioavailable for binding to transferrin, the major iron transporter in blood that specifically binds ferric ions . The importance of this hephaestin ferroxidase function is illustrated by the sla mouse phenotype—such mice have an in-frame deletion in the heph gene resulting in a truncated form of hephaestin that shows significantly decreased ferroxidase activity compared with the wild-type protein . The subsequent impaired iron export from enterocytes into blood gives a deficiency of iron in the tissues (especially hematopoietic cells, which are the major consumers of iron). Thus, mice carrying the sla mutation develop a microcytic hypochromic anemia with iron accumulation in the intestinal epithelium . Although hephaestin is mainly expressed in the intestine, this protein has recently been found in heart, brain, and pancreas [7, 8]. In these tissues, the hephaestin ferroxidase activity may help to minimize the Fe2+ toxicity resulting from the formation of free radicals through the Haber–Weiss-Fenton series of reactions .
Putative copper ligands in hephaestin and corresponding residues in ceruloplasmin
Putative copper ligands
H304, C347, H352, M357
H276, C319, H324, L329
H656, C698, H703, M708
H637, C680, H685, M690
H1000, C1046, H1051, M1056
H975, C1021, H1026, M1031
Putative iron ligands in hephaestin and corresponding residues in ceruloplasmin
Putative copper ligands
E264, H269, S351, E652
E236, Y241, N323, E633
D616, H621, S703, D996
E597, H602, D684, E971
E960, H965, D1050, E300
E935, H940, D1025, E272
Although hephaestin has never been isolated from human tissue, we have previously expressed the recombinant hephaestin ectodomain in baby hamster kidney (BHK) cells . Recombinant human hephaestin (rhHp) produced in this expression system showed lower ferroxidase activity compared with other multicopper ferroxidases . As shown by inductively coupled plasma mass spectrometry, the copper content was 3.13 copper atoms per molecule of rhHp instead of the predicted six atoms per molecule of rhHp . The spectroscopic properties of rhHp (ε607 nm = 2,010 M−1) were consistent with an average of approximately 0.5 type 1 copper sites per molecule of hephaestin . All these observations suggested the incomplete copper loading of rhHp in this expression system. Furthermore, the use of the yeast Pichia pastoris as an expression system did not result in production of rhHp with the predicted copper content. Instead, this yeast-derived rhHp contained only 4.2 copper atoms per molecule .
In the current study, we used ion exchange chromatography (IEC) to fractionate hephaestin into several fractions of rhHp that displayed different catalytic and spectroscopic properties. By using site-directed mutagenesis, we introduced mutations of the putative Fe(II)-binding residues in rhHp. The kinetics of the ferroxidase and p-phenylenediamine oxidase activities of wild-type rhHp and mutants was investigated. These studies provide the first experimental evidence for the functional role of individual amino acid residues in iron oxidation by human hephaestin.
Materials and methods
Oligonucleotide synthesis was performed by the Nucleic Acid Protein Service (NAPS) at the University of British Columbia and Integrated DNA Technologies (San Diego, CA, USA). Enzymes for DNA manipulation were from New England Biolabs (Beverly, MA, USA). All other reagents were purchased from Sigma-Aldrich (Oakville, ON, Canada) unless otherwise noted.
A modified hephaestin complementary DNA  that had been incorporated into the pBSSK− vector (Stratagene, La Jolla, CA, USA) via NotI restriction sites was used as an initial construct for DNA manipulation. Modifications previously introduced to the hephaestin complementary DNA include replacement of the native signal peptide by the transferrin signal sequence and substitution of the predicted transmembrane domain with a 1D4 epitope at the C terminus . For the current studies, the transferrin signal sequence was replaced with the human ceruloplasmin signal sequence using a QuikChange mutagenesis kit (Stratagene, La Jolla, CA, USA) and the primers CpSignal-F and CpSignal-R (Table S1). The DNA fragment encoding the hephaestin ectodomain (with the ceruloplasmin signal peptide and 1D4 epitope at the C terminus) was subcloned into the pNUT vector  using NotI restriction sites. The resulting construct was used for expression of rhHp and as a template for subsequent site-directed mutagenesis.
Mutations of putative iron ligands in the hephaestin sequence were introduced by using the megaprimer technique  and primers E264A/H269A-R, E616A/H621A-F, E960A/H965A-F, pNUT5′, and pNUT3′ (see Table S1). For propagating DNA, Escherichia coli Mach1 cells were grown in Luria–Bertani medium supplemented with ampicillin (100 mg L−1). Plasmid DNA and PCR products were purified by using plasmid DNA miniprep kits and gel extraction kits (Epoch Biolabs, Missouri City, TX, USA). Automated DNA sequence analysis using a BigDye Terminator kit (Stratagene, La Jolla, CA, USA) and an ABI 3700 DNA sequencer (Applied Biosystems, Streetsville, ON, Canada) was used to verify the nucleotide sequences of all constructs on both strands of DNA.
Expression and purification of rhHp and mutants
BHK cells were grown to confluence in six-well tissue culture plates in Dulbecco’s modified Eagle’s medium–Ham F12 nutrient mixture (DMEM-F12) containing 5 % newborn calf serum (Invitrogen, Burlington, ON, Canada) in a humidified 5 % CO2 atmosphere. Transfection of BHK cells with recombinant DNA was performed using the FuGene 6 transfection reagent according to the manufacturer’s instructions (Roche, Indianapolis, IN, USA). One day after transfection, methotrexate was added to the medium to a final concentration of 200 mg L−1. After 14 days of growth on selective medium, colonies of methotrexate-resistant cells were expanded into flasks for further transfer into expanded surface roller bottles (1,700 cm2; Fisher Scientific, Ottawa, ON, Canada). When cells in the roller bottle reached confluence, the DMEM-F12–newborn calf serum–methotrexate medium was replaced with DMEM-F12 containing 2 % Ultroser G (BioSepra, Marlborough, MA, USA) and 10 μM CuSO4. Subsequent batches contained DMEM-F12, 1 % Ultroser G, and 10 μM CuSO4 (200 mL per roller bottle). The culture medium containing Ultroser G was collected every 2 days for subsequent recombinant protein purification.
Monoclonal antibodies to the 1D4 epitope  were coupled to Sepharose 2B by using the CNBr activation method described by Cuatrecasas . The culture medium was clarified by centrifugation and passed through the immunoaffinity column, which had been equilibrated with 20 mM tris(hydroxymethyl)aminomethane (Tris–HCl), 150 mM NaCl, pH 7.4 (storage buffer). The column was washed with ten bed volumes of the storage buffer. Elution was performed by repetitive application of 0.1 mg mL−1 1D4 peptide (N-Ac-TETSQVAPA; purchased from BioBasics, Markham, ON, Canada) diluted in storage buffer. The elution fractions were concentrated using Amicon Ultra-15 centrifugal filter units with a 30-kDa cutoff (Millipore, Billerica, MA, USA). The purity of the eluted protein in the fractions was confirmed by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by staining with 0.1 % Coomassie blue.
IEC of rhHp
Immunoaffinity-purified rhHp (1 mg) in low-salt buffer (5 mM Tris–HCl 15 mM NaCl, pH 7.4) was applied onto the column with diethylaminoethyl (DEAE)-Sepharose (bed volume 1 mL). The column was then washed with ten bed volumes of the low-salt buffer. Elution was performed by stepwise increase of the salt concentration. Each elution step involved application of 1.5 ml of 5 mM Tris–HCl, pH 7.4 buffer containing 150, 200, and 250 mM NaCl. Prior to analysis, the eluted fractions were buffer-exchanged against the storage buffer (20 mM Tris–HCl 150 mM NaCl, pH 7.4) using Amicon Ultra-15 centrifugal filter units with a 30-kDa cut off.
For N-terminal sequence analysis, 3 μg of rhHp was electrophoresed on 10 % sodium dodecyl sulfate polyacrylamide gel and then transferred to a poly(vinylidene difluoride) membrane (Bio-Rad, Mississauga, ON, Canada) in N-cyclohexyl-3-aminopropanesulfonic acid buffer according to the manufacturer’s instructions. The membrane was stained with 0.1 % Coomassie blue. The band representing rhHp was excised and analyzed with an ABI 492 Procise cLC sequencer at the Hospital for Sick Children in Toronto (http://www.sickkids.ca). UV–vis absorbance spectra of rhHp samples in 20 mM Tris–HCl, 150 mM NaCl buffer, pH 7.4 were recorded with a Varian Cary 4000 spectrophotometer (25 °C). The concentration of rhHp was determined using the experimental molar absorption coefficient for the absorbance at 280 nm (215,474 M−1 cm−1) . Inductively coupled plasma mass spectrometry using a dynamic reaction cell was performed by Applied Speciation and Consulting (Bothell, WA, USA; http://www.appliedspeciation.com) according to its established procedures.
All activity assays were performed at room temperature in 96-well plates (Corning, Corning, NY, USA). As an autooxidation control, equivalent amounts of the buffer were used instead of the protein solution. Kinetic parameters were calculated using gnuplot (http://www.gnuplot.info).
The ferroxidase activity assay was performed in 75 mM sodium acetate buffer, pH 5 with a total reaction volume of 200 μL. A defined amount of ferrous ammonium sulfate was added to each reaction mixture. After incubation with either protein solution or elution buffer, reactions were quenched with 50 μL of 4 mM ferrozine, an Fe(II)-specific chelator. The ferrozine–Fe(II) complex has a strong absorbance at 562 nm (ε562 nm = 27.9 mM−1 cm−1 ), which was measured using a microplate reader. The concentration of Fe(II) before and after incubation was determined using a calibration curve that was linear for the Fe(II) concentration range of 2–100 μM. For determination of kinetic parameters, the initial kinetic data were plotted in Eadie–Hofstee coordinates.
Oxidation of p-phenylenediamine dihydrochloride was assayed in 250 μL of 80 mM sodium acetate buffer containing 80 μM EDTA, pH 5. EDTA was added to the reaction to prevent iron-mediated oxidation of pPD [14, 23]. The rates of pPD oxidation were calculated using absorption coefficients for Bandrowski’s base (ε535 nm = 1,910 M−1 cm−1), which is the product of pPD oxidation . Because production of a single molecule of Bandrowski’s base involves oxidation of three pPD molecules , the absorbance-based oxidation rates were multiplied by 3, and the resultant catalytic rates were expressed as the number of pPD molecules oxidized by one molecule of enzyme per minute. For determination of Km and kcat, pPD oxidation rates were measured at a minimum of eight substrate concentrations ranging from 0.05 to 7.5 mM. The initial reaction rates were directly fitted to the Michaelis–Menten equation using nonlinear regression.
Expression and initial characterization of rhHp
Hephaestin ectodomain with ceruloplasmin signal peptide was expressed in BHK cells. The 1D4 epitope introduced at the C terminus of hephaestin allowed the purification of rhHp with a single immunoaffinity chromatography step. N-terminal sequence analysis of rhHp revealed the following sequence: (A/S/G)-T-R-V-Y-Y. Despite the ambiguous identity of the first amino acid, the remainder of the sequence (along with the first residue when alanine is included) corresponds to the amino acid sequence of human hephaestin. In contrast to a previous report, where hephaestin was reported to contain additional amino acids from the transferrin signal peptide , the rhHp used in this study had an authentic human hephaestin N-terminal sequence.
Fractionation of purified hephaestin by IEC
Subsequent analysis of the specific p-phenylenediamine oxidase activity showed that the fraction of hephaestin eluted at 150 mM NaCl had higher activity compared with the hephaestin sample prior to IEC as well as the other fractions eluted at higher salt concentrations (Fig. 2). We then performed a kinetic analysis of this highly enzymatically active fraction. Initial ferroxidation rates were measured at iron concentrations ranging from 2 to 10 μM. Kinetic analysis revealed the following parameters for IEC-purified rhHp: for Fe(II), Km = 4.0 ± 1.5 μM and kcat = 18 ± 3 min−1; for pPD, Km = 1.5 ± 0.2 mM and kcat = 29 ± 1 min−1. This DEAE-Sepharose fractionation of rhHp was performed in two independent experiments, and in both cases the specific activity of the rhHp fraction eluted at 150 mM (Fig. 1, diamonds in the upper-right quadrant) was significantly higher than the specific activities of unfractionated rhHp samples (Fig. 1, circles in the lower-left quadrant).
Kinetic analysis of hephaestin-catalyzed ferroxidation
Site-directed mutagenesis of putative iron ligands in rhHp
Amino acid sequence homology with ceruloplasmin predicted that hephaestin has three putative iron-binding sites in domains 2, 4, and 6 . To study the role of each binding site in the ferroxidase activity of hephaestin, we produced the following mutants: IB (E264A/H269A; D616A/H621A; E960A/H965A), IB6 (E264A/H269A; D616A/H621A), IB4 (E264A/H269A; E960A/H965A), and IB2 (D616A/H621A; E960A/H965A). All three iron-binding sites are mutated in the IB mutant, whereas mutants IB6, IB4, and IB2 have a single unaffected binding site in domains 6, 4, and 2, respectively. By mutating residues in two iron-binding sites at once (mutants IB6, IB4 and IB2), we studied the impact of the unaffected site on ferroxidase activity of rhHp.
Kinetic parameters for recombinant human hephaestin (rhHp) and mutants
rhHp and mutants
Km for pPD (mM)
Range of kcat for pPD (min−1)
Km for high-affinity oxidation of Fe(II) (μM)a
Range of kcat′ for Fe(II) (min−1)b
Ratio of kcat for pPD to kcat′ for Fe(II)
1.0 ± 0.1
4.3 ± 0.7
0.75 ± 0.11
0.41 ± 0.05
2.2 ± 0.1
0.84 ± 0.06
0.37 ± 0.01
9.5 ± 1.0
0.35 ± 0.01
10.7 ± 1.0
0.22 ± 0.01
5.5 ± 0.1
Using separation by IEC, we purified rhHp with improved catalytic and spectroscopic properties. When determined under similar conditions, the kcat for Fe2+ of rhHp (18 min−1) was higher than ferroxidation rates previously reported for hephaestin (0.74 min−1 , 2.5 min−1 ). However, IEC-fractionated rhHp gave an absorbance coefficient at 600 nm (ε600 nm = 5,500 M−1 cm−1) that indicated the presence of only one type 1 copper per molecule of hephaestin. These data disagree with the structural model of hephaestin ectodomain based upon ceruloplasmin  that predicted three type 1 copper sites. Furthermore, the total copper content of hephaestin isolated in this study is 3.5 atoms per molecule rather than the predicted six atoms. There are several possible explanations for the lower copper content of rhHp. Because the tissue culture medium was supplemented with 10 μM CuSO4, it is unlikely that the availability of copper was limiting during BHK cell growth. However, it is possible that there was incomplete copper loading of hephaestin during its biosynthesis by a heterologous expression system. It is also possible that hephaestin may contain only four copper atoms arranged as a single type 1 copper and a trinuclear cluster—this in itself would be sufficient to give ferroxidase activity as in the yeast ferroxidase Fet3p . In this case, hephaestin is most likely to retain the type 1 copper atom in domain 6, which is adjacent to the trinuclear cluster. This suggestion is supported by the presence of a functional iron-binding site in domain 6 of human hephaestin (see later). Domains 2 and 4 of hephaestin, which also contain predicted type 1 copper ligands, may still play important roles in maintaining the overall structure of the molecule or may participate in the interactions of hephaestin with other proteins.
To study the role of the predicted iron ligands in ferroxidase activity of rhHp, we produced a mutant with all three putative iron-binding sites affected (IB) and mutants that retained a single predicted site for Fe(II) binding (IB2, IB4, IB6). Mutation of the amino acid residues in all three iron-binding sites resulted in a loss of high-affinity Fe(II) oxidation along with a sevenfold increase in the ratio between pPD- and Fe(II)-oxidation rates (Table 3, column 6). Similar results were obtained for the IB2 and IB4 mutants. In contrast, the IB6 mutant showed high-affinity iron oxidation with a Km comparable to that of wild-type rhHp (Table 3, column 4); the ratios of the pPD-and Fe(II)-oxidation rates for IB6 and rhHp were also equivalent (Table 3, column 6). These results are consistent with residues E960/H965 serving as iron ligands of the high-affinity binding site in domain 6 of rhHp. In contrast, residues E264/H269 in domain 2 and D616/H621 in domain 4 appear to be dispensable for hephaestin ferroxidase activity; supporting this observation, mutations of these residues in the IB6 mutant did not result in any detectable impairment of ferroxidase function. There is a possibility that the role of residues E264/H269 and D616/H621 in iron oxidation was not revealed owing to the absence of copper at type 1 sites in domains 2 and 4 of rhHp. On the other hand, iron-binding sites in domains 2 and 4 of rhHp are not composed of a canonical set of ligands. Conserved ferrous-binding sites in domain 6 of rhHp and in domains 4 and 6 of ceruloplasmin are formed by three acidic residues and a histidine residue. In contrast, the putative sites for iron binding in domains 2 and 4 of hephaestin have one of the acidic residues substituted with a serine residue. This unusual arrangement of ligands may have a detrimental effect on iron oxidation at these sites.
In conclusion, this work advances our current understanding of the catalytic mechanism of human hephaestin, one of three human multicopper ferroxidases . The amino acid sequence homology between the ferroxidases predicts similar biochemical properties; however, any specific functions for each of the paralogs probably result from their structural differences. The current work on hephaestin has revealed substrate specificity, copper loading, and the ferroxidation mechanism in this multicopper ferroxidase. The knowledge acquired in the current study will facilitate further research focused on the structure and catalytic mechanism of iron oxidation by the ferroxidase paralogs.
We thank Grant Mauk for many fruitful discussions. This work was supported in part by a grant from the Canadian Institutes of Health Research (to R.T.A.M.).