Oxidation of organic and biogenic amines by recombinant human hephaestin expressed in Pichia pastoris

Hephaestin is a multicopper ferroxidase involved in iron absorption in the small intestine. Expressed mainly on the basolateral surface of duodenal enterocytes, hephaestin facilitates the export of iron from the intestinal epithelium into blood by oxidizing Fe(2+) into Fe(3+), the only form of iron bound by the plasma protein transferrin. Structurally, the human hephaestin ectodomain is predicted to resemble ceruloplasmin, the major multicopper oxidase in blood. In addition to its ferroxidase activity, ceruloplasmin was reported to oxidize a wide range of organic compounds including a group of physiologically relevant substrates (biogenic amines). To study oxidation of organic substrates, the human hephaestin ectodomain was expressed in Pichia pastoris. The purified recombinant hephaestin has an average copper content of 4.2 copper atoms per molecule. The K(m) for Fe(2+) of hephaestin was determined to be 3.2μM which is consistent with the K(m) values for other multicopper ferroxidases. In addition, the K(m) values of hephaestin for such organic substrates as p-phenylenediamine and o-dianisidine are close to values determined for ceruloplasmin. However, in contrast to ceruloplasmin, hephaestin was incapable of direct oxidation of adrenaline and dopamine implying a difference in biological substrate specificities between these two homologous ferroxidases.     

Hephaestin--a ferroxidase of cellular iron export

Hephaestin is a transmembrane copper-dependent ferroxidase necessary for effective iron transport from intestinal enterocytes into the circulation. Hephaestin is mutated in sex-linked anemia (sla) mice. The initial uptake of iron from the diet in these animals is normal, but the basolateral export of iron from enterocytes is defective, resulting in iron deficiency and microcytic hypochromic anemia. In addition to the small intestine, hephaestin is expressed to a lesser extent in colon, spleen, placenta and kidney but its role in these tissues remains unknown. So far, hephaestin has not been linked to a human disease.     

The C-Terminal Regions Have an Important Role in the Activity of the Ferroxidase Center and the Stability of Chlorobium tepidum Ferritin

The recombinant Chlorobium tepidum ferritin (rCtFtn) is able to oxidize iron using ferroxidase activity but its ferroxidase activity is intermediate between the H-chain human ferritin and the L-chain human ferritin. The rCtFtn has an unusual C-terminal region composed of 12 histidine residues, as well as aspartate and glutamate residues. These residues act as potential metal ion ligands, and the rCtFtn homology model predicts that this region projects inside the protein cage. The rCtFtn also lacks a conserved Tyr residue in position 19. In order to know if those differences are responsible for the altered ferroxidase properties of rCtFtn, we introduced by site-directed mutagenesis a stop codon at position 166 and a Tyr residue replaced Ala19 in the gene of rCtFtn (rCtFtn 166). The rCtFtn166 keeps the canonical sequence considered important for the activity of this family of proteins. Therefore, we expected that rCtFtn 166 would possess similar properties to those described for this protein family. The rCtFtn 166 is able to bind, oxidize and store iron; and its activity is inhibit by Zn(II) as was described for other ferritins. However, the rCtFtn 166 possesses a decrease ferroxidase activity and protein stability compared with the wild type rCtFtn. The analysis of the Ala19Tyr rCtFtn shows that this change does not affect the kinetic of iron oxidation. Therefore, these results indicate that the C-terminal regions have an important role in the activity of the ferroxidase center and the stability of rCtFtn.     

Copper deficiency increases iron absorption in the rat

Release of iron from enterocytes and hepatocytes is thought to require the copper-dependent ferroxidase activity of hephaestin (Hp) and ceruloplasmin (Cp), respectively. In swine, copper deficiency (CD) impairs iron absorption, but whether this occurs in rats is unclear. By feeding a diet deficient in copper, CD was produced, as evidenced by the loss of copper-dependent plasma ferroxidase I activity, and in enterocytes, CD reduced copper levels and copper-dependent oxidase activity. Hematocrit was reduced, and liver iron was doubled. CD reduced duodenal mucosal iron and ferritin, whereas CD increased iron absorption. Duodenal mucosal DMT1-IRE and ferroportin1 expression remained constant with CD. When absorption in CD rats was compared with that seen normally and in iron-deficient anemic animals, strong correlations were found among mucosal iron, ferritin, and iron absorption, suggesting that the level of iron absorption was appropriate given that the erythroid and stores stimulators of iron absorption are opposed in CD. Because CD reduced the activity of Cp, as evidenced by copper-dependent plasma ferroxidase I activity and hepatocyte iron accumulation, but iron absorption increased, it is unlikely that the ferroxidase activity of Hp is important and suggests another function for this protein in the export of iron from the enterocyte during iron absorption. Also, the copper-dependent ferroxidase activity of Cp does not appear important for iron efflux from macrophages, because Kupffer cells of the liver and nonheme iron levels of the spleen were normal during copper deficiency, suggesting another role for Cp in these cells.     

An X-ray structural study of human ceruloplasmin in relation to ferroxidase activity

The role of ceruloplasmin as a ferroxidase in the blood, mediating the release of iron from cells and its subsequent incorporation into serum transferrin, has long been the subject of speculation and debate. However, a recent X-ray crystal structure determination of human ceruloplasmin at a resolution of around 3.0?Å, in conjunction with studies associating mutations in the ceruloplasmin gene with systemic haemosiderosis in humans, has added considerable weight to the argument in favour of a ferroxidase role for this enzyme. Further X-ray studies have now been undertaken involving the binding of the cations Co(II), Fe(II), Fe(III), and Cu(II) to ceruloplasmin. These results give insights into a mechanism for ferroxidase activity in ceruloplasmin. The residues and sites involved in ferroxidation are similar to those proposed for the heavy chains of human ferritin. The nature of the ferroxidase activity of human ceruloplasmin is described in terms of its three-dimensional molecular structure.     

Analysis of the human hephaestin gene and protein: comparative modelling of the N-terminus ecto-domain based upon ceruloplasmin

Hephaestin was implicated in mammalian iron homeostasis following its identification as the defective gene in murine sex-linked anaemia. It is a member of the family of copper oxidases that includes mammalian ceruloplasmin, factors V and VIII, yeast fet3 and fet5 and bacterial ascorbate oxidase. Hephaestin is different from ceruloplasmin, a soluble ferroxidase, in having a membrane-spanning region towards the C-terminus. Here we report the gene structure, spanning approximately 100 kb, of the human homologue of mouse hephaestin. The sequence was assembled from the cDNA clones and the chromosome X genomic sequence data available at the Sanger Centre. It has an open reading frame that encodes a protein of 1158 residues, 85% identical with the murine homologue. A model of the N-terminal ecto-domain has been built based on the known three-dimensional structure of human ceruloplasmin. The overall tertiary structure for the hephaestin and the putative residues involved in binding copper and iron appear to be highly conserved between these proteins, which suggests they share the same fold and a conserved function.     

The role of cysteine residues in the oxidation of ferritin

We have shown that ferritin is oxidized during iron loading using its own ferroxidase activity and that this oxidation results in its aggregation (Welch et al., Free Radic. Biol. Med. 31:999-1006; 2001). In this study we determined the role of cysteine residues in the oxidation of ferritin. Loading iron into recombinant human ferritin by its own ferroxidase activity decreased its conjugation by a cysteine specific spin label, indicating that cysteine residues were altered during iron loading. Using LC/MS, we demonstrated that tryptic peptides of ferritin that contained cysteine residues were susceptible to modification as a result of iron loading. To assess the role of cysteine residues in the oxidation of ferritin, we used site-directed mutagenesis to engineer variants of human ferritin H chain homomers where the cysteines were substituted with other amino acids. The cysteine at position 90, which is located at the end of the BC-loop, appeared to be critical for the formation of ferritin aggregates during iron loading. We also provide evidence that dityrosine moieties are formed during iron loading into ferritin by its own ferroxidase activity and that the dityrosine formation is dependent upon the oxidation of cysteine residues, especially cysteine 90. In conclusion, cysteine residues play an integral role in the oxidation of ferritin and are essential for the formation of ferritin aggregates.     

Ferritin M of Cynoglossus semilaevis: an iron-binding protein and a broad-spectrum antimicrobial that depends on the integrity of the ferroxidase center and nucleation center for biological activity

Ferritin is a major intracellular iron storage protein in higher vertebrates and plays an important role in iron metabolism. In this study, we identified and analyzed the biological activity of a ferritin M subunit (CsFerM) from half-smooth tongue sole (Cynoglossus semilaevis). The open reading frame (ORF) of CsFerM is 534?bp and encodes a protein that shares 79.7-86.4% overall sequence identities with the ferritin M subunits of a number of teleosts. In silico analysis identified in CsFerM a eukaryotic ferritin domain with conserved ferroxidase diiron center and ferrihydrite nucleation center. Quantitative real time RT-PCR analysis showed that under normal physiological conditions, expression of CsFerM was highest in liver, moderate in gill, spleen, and muscle, and low in gut, heart, and brain. Following experimental challenge with bacterial pathogens, CsFerM expression was significantly upregulated in kidney, spleen, and liver in time-dependent manners. Biological activity analysis showed that recombinant CsFerM purified from Escherichia coli exhibited apparent iron-binding activity and, when present in the culture medium of six different species of fish bacterial pathogens, completely inhibited bacterial growth. In contrast, a mutant CsFerM that bears alanine substitution at two conserved residues of the ferroxidase diiron center and ferrihydrite nucleation center was abolished in both iron-binding and antimicrobial capacity. These results demonstrate that CsFerM is a biologically active iron chelator with broad-spectrum antibacterial activity, which suggests a role for CsFerM in not only iron storage but also innate immunity. These results also indicate the importance of the conserved iron uptake and mineralization sites to the function of CsFerM.     

Ferritin structure from Mycobacterium tuberculosis: comparative study with homologues identifies extended C-terminus involved in ferroxidase activity

Ferritins are recognized as key players in the iron storage and detoxification processes. Iron acquisition in the case of pathogenic bacteria has long been established as an important virulence mechanism. Here, we report a 3.0 Å crystal structure of a ferritin, annotated as Bacterioferritin B (BfrB), from Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis that continues to be one of the world''s deadliest diseases. Similar to the other members of ferritin family, the Mtb BfrB subunit exhibits the characteristic fold of a four-helical bundle that possesses the ferroxidase catalytic centre. We compare the structure of Mtb BfrB with representatives of the ferritin family belonging to the archaea, eubacteria and eukarya. Unlike most other ferritins, Mtb BfrB has an extended C-terminus. To dissect the role of this extended C-terminus, truncated Mtb BfrB was purified and biochemical studies implicate this region in ferroxidase activity and iron release in addition to providing stability to the protein. Functionally important regions in a protein of known 3D-structure can be determined by estimating the degree of conservation of the amino-acid sites with its close homologues. Based on the comparative studies, we identify the slowly evolving conserved sites as well as the rapidly evolving variable sites and analyze their role in relation to structure and function of Mtb BfrB. Further, electrostatic computations demonstrate that although the electrostatic environment of catalytic residues is preserved within the family, extensive variability is exhibited by residues defining the channels and pores, in all likelihood keeping up with the diverse functions executed by these ferritins in varied environments.     

The putative "nucleation site" in human H-chain ferritin is not required for mineralization of the iron core

It is widely believed that the putative nucleation site (Glu61, Glu64, and Glu67) in mammalian H-chain ferritin plays an important role in mineral core formation in this protein. Studies of nucleation site variant A2 (E61A/E64A/E67A) of H-chain ferritin have traditionally shown impaired iron oxidation activity and mineralization. However, recent measurements have suggested that the previously observed impairment may be due to disruption of the ferroxidase site of the protein since Glu61 is a shared ligand of the ferroxidase and nucleation sites of the protein. This study employed a new nucleation site variant A1 (E64A/E67A) which retains the ferroxidase site ligand Glu61. The data (O(2) uptake, iron binding, and conventional and stopped-flow kinetics measurements) show that variant A1 retains a completely functional ferroxidase site and has iron oxidation and mineralization properties similar to those of the wild-type human H-chain protein. Thus, in contrast to previously published literature, this study demonstrates that the putative "nucleation site" does not play an important role in iron uptake or mineralization in H-chain ferritin.     

Decreased hephaestin expression and activity leads to decreased iron efflux from differentiated Caco2 cells

Iron is transported across intestinal brush border cells into the circulation in at least two distinct steps. Iron can enter the enterocyte via the apical surface through several paths. However, iron egress from the basolateral side of enterocytes converges on a single export pathway requiring the iron transporter, ferroportin1, and hephaestin, a ferroxidase. Copper deficiency leads to reduced hephaestin protein expression and activity in mouse enterocytes and intestinal cell lines. We tested the effect of copper deficiency on differentiated Caco2 cells grown in transwells and found decreased hephaestin protein expression and activity as well as reduced ferroportin1 protein levels. Furthermore, the decrease in hephaestin levels correlates with a decrease of ⁵⁵Fe release from the basolateral side of Caco2 cells. Presence of ceruloplasmin, apo‐transferrin or holo‐transferrin did not significantly alter the results observed. Repletion of copper in Caco2 cells leads to reconstitution of hephaestin protein expression, activity, and transepithelial iron transport. J. Cell. Biochem. 107: 803–808, 2009. © 2009 Wiley‐Liss, Inc.     

Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO(4)) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a cellular compartment in close proximity but not overlapping with the basolateral surface. Surface biotinylation studies indicate that hephaestin in the peri-basolateral location is accessible to the extra-cellular environment. These results support the hypothesis that hephaestin is involved in iron mobilization of iron from the intestine to circulation.     

The Amyloid Precursor Protein (APP) Does Not Have a Ferroxidase Site in Its E2 Domain

The ubiquitous 24-meric iron-storage protein ferritin and multicopper oxidases such as ceruloplasmin or hephaestin catalyze oxidation of Fe(II) to Fe(III), using molecular oxygen as oxidant. The ferroxidase activity of these proteins is essential for cellular iron homeostasis. It has been reported that the amyloid precursor protein (APP) also has ferroxidase activity. The activity is assigned to a ferroxidase site in the E2 domain of APP. A synthetic 22-residue peptide that carries the putative ferroxidase site of E2 domain (FD1 peptide) has been claimed to encompass the same activity. We previously tested the ferroxidase activity of the synthetic FD1 peptide but we did not observe any activity above the background oxidation of Fe(II) by molecular oxygen. Here we used isothermal titration calorimetry to study Zn(II) and Fe(II) binding to the natural E2 domain of APP, and we employed the transferrin assay and oxygen consumption measurements to test the ferroxidase activity of the E2 domain. We found that this domain neither in the presence nor in the absence of the E1 domain binds Fe(II) and it is not able to catalyze the oxidation of Fe(II). Binding of Cu(II) to the E2 domain did not induce ferroxidase activity contrary to the presence of redox active Cu(II) centers in ceruloplasmin or hephaestin. Thus, we conclude that E2 or E1 domains of APP do not have ferroxidase activity and that the potential involvement of APP as a ferroxidase in the pathology of Alzheimer’s disease must be re-evaluated.     

The involvement of a multicopper oxidase in iron uptake by the green algae Chlamydomonas reinhardtii

In the unicellular green algae Chlamydomonas reinhardtii, high-affinity uptake of iron (Fe) requires an Fe(3+)-chelate reductase and an Fe transporter. Neither of these proteins nor their corresponding genes have been isolated. We previously identified, by analysis of differentially expressed plasma membrane proteins, an approximately 150-kD protein whose synthesis was induced under conditions of Fe-deficient growth. Based on homology of internal peptide sequences to the multicopper oxidase hephaestin, this protein was proposed to be a ferroxidase. A nucleotide sequence to the full-length cDNA clone for this ferroxidase-like protein has been obtained. Analysis of the primary amino acid sequence revealed a putative transmembrane domain near the amino terminus of the protein and signature sequences for two multicopper oxidase I motifs and one multicopper oxidase II motif. The ferroxidase-like gene was transcribed under conditions of Fe deficiency. Consistent with the role of a copper (Cu)-containing protein in Fe homeostasis, growth of cells in Cu-depleted media eliminated high-affinity Fe uptake, and Cu-deficient cells that were grown in optimal Fe showed greatly reduced Fe accumulation compared with control, Cu-sufficient cells. Reapplication of Cu resulted in the recovery of Fe transport activity. Together, these results were consistent with the participation of a ferroxidase in high-affinity Fe uptake in C. reinhardtii.     

Expression of soluble,active, fluorescently tagged hephaestin in COS and CHO cell lines

Hephaestin (Hp) is a trans-membrane protein, which plays a critical role in intestinal iron absorption. Hp was originally identified as the gene responsible for the phenotype of sex-linked anaemia in the sla mouse. The mutation in the sla protein causes accumulation of dietary iron in duodenal cells, causing severe microcytic hypochromic anaemia. Although mucosal uptake of dietary iron is normal, export from the duodenum is inhibited. Hp is homologous to ceruloplasmin (Cp), a member of the family of multi copper ferroxidases (MCFs) and possesses ferroxidase activity that facilitates iron release from the duodenum and load onto the serum iron transport protein transferrin. In the present study, attempts were made to produce biologically active recombinant mouse hephaestin as a secretory form tagged with green fluorescent protein (GFP), Hpsec-GFP. Plasmid expressing Hpsec-GFP was constructed and transfected into COS and CHO cells. The GFP aided the monitoring expression in real time to select the best conditions to maximise expression and provided a tag for purifying and analysing Hpsec-GFP. The protein had detectable oxidase activity as shown by in-gel and solution-based assays. The methods described here can provide the basis for further work to probe the interaction of hephaestin with other proteins using complementary fluorescent tags on target proteins that would facilitate the fluorescence resonance energy transfer measurements, for example with transferrin or colocalisation studies, and help to discover more about hephaestin works at the molecular level.     

Multicopper oxidase-1 orthologs from diverse insect species have ascorbate oxidase activity

Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism.     

Cloning and characterization of Chlorobium tepidum ferritin

The Chlorobium tepidum ferritin (CtFtn) gene was synthesized and cloned into a pET3a expression vector (Novagen). CtFtn was expressed in Escherichia coli and purified to electrophoretic homogeneity. Sequence analysis indicates that all the conserved amino acids required to form the Fe²⁺ oxidizing ferroxidase center are present. Ftn is highly conserved from bacteria to humans, each subunit folds into a 4-helical bundle (helices A-D), with a long loop connecting helices B and C, plus a fifth short E-helix at the C-terminus. Calculations based on the secondary structure of CtFtn predict that each of these helices forms. However, the sequence of CtFtn shows a much longer C-terminus with a significant number of polar amino acids. Size-exclusion chromatography shows that CtFtn elutes at a size consistent with a 24-subunit protein cage. Incubation of CtFtn with Fe²⁺ produced an increase in the absorbance at 310 nm consistent with the incorporation of iron inside CtFtn. Assays monitoring ferroxidase activity showed that CtFtn possesses ferroxidase activity but it is less active than human H-chain ferritin. Additionally, the iron loading capacity of CtFtn is significantly reduced compared to proteins from other organisms. We propose that the unique extended C-terminus in CtFtn causes the decreased iron loading in CtFtn and possibly influences the slower rate of iron oxidation at the ferroxidase center.     

The crystal structure of the Dps2 from Deinococcus radiodurans reveals an unusual pore profile with a non-specific metal binding site

The crystal structure of recombinant Dps2 (DRB0092, DNA protecting protein under starved conditions) from the Gram-positive, radiation-resistant bacterium Deinococcus radiodurans has been determined in its apo and iron loaded states. Like other members of the Dps family, the bacterial DrDps2 assembles as a spherical dodecamer with an outer shell diameter of 90 A and an interior diameter of 40 A. A total of five iron sites were located in the iron loaded structure, representing the first stages of iron biomineralisation. Each subunit contains a mononuclear iron ferroxidase centre coordinated by residues highly conserved amongst the Dps family of proteins. In the structures presented, a distinct iron site is observed 6.1 A from the ferroxidase centre with a unique ligand configuration of mono coordination by the protein and no bridging ligand to the ferroxidase centre. A non-specific metallic binding site, suspected to play a regulative role in iron uptake/release from the cage, was found in a pocket located near to the external edge of the C-terminal 3-fold channel.     

Iron imports. III. Transfer of iron from the mucosa into circulation

Transfer of iron from the mucosa is a critical step in dietary iron assimilation that is tightly regulated to ensure the appropriate amount of iron is absorbed to meet the body's demands. Too much iron is highly toxic, and failure to properly control intestinal iron export causes iron overload associated with hereditary forms of hemochromatosis. One form of genetic iron overload, ferroportin disease, originates due to defects in ferroportin, the membrane iron exporter. Ferroportin acts in conjunction with the intestinal ferroxidase hephaestin to mediate release of iron from the enterocyte. How iron is then acquired by transferrin and released into circulation remains an unknown step in this process.