Functional assembly of recombinant human ferritin subunits in Pichia pastoris

Ferritin is an iron storage protein found in most living organisms as a natural assembled macromolecule. For studying the functional ability of the ferritin assembly, human H- and L-ferritins were expressed and purified from Pichia pastoris strain GS115. The recombinant H- and L-ferritins showed a globular form with transmission electron microscopy. The rate of iron uptake for H-ferritin was significantly faster than that for the L-ferritin in vitro. By gel permeation chromatography analysis, recombinant ferritins were confirmed as multimeric subunits with high molecular weight and it was indicated that assembled subunits were able to store iron in vivo.     

Improved coexpression and multiassembly properties of recombinant human ferritins subunit in Escherichia coli

Human heavy chain (H-) and light chain (L-) ferritins were amplified from a human cDNA library. Each ferritin gene was inserted downstream of the T7 promoter of bacterial expression vectors, and two types of coexpression vectors were constructed. The expression levels of recombinant ferritins ranged about 26-36% of whole-cell protein. Hferritin exhibited a lower expression ratio compared with L-ferritin, by a coexpression system. However, the coexpression of HL-ferritins was significantly increased above the expression ratio of H-ferritin by cultivation without IPTG induction overnight. Purified recombinant H-, L-, HL-, and LHferritins were shown to be homo- and heteropolymeric high molecular complexes and it was indicated that their assembled subunits would be able to work functionally in the cell. Thus, these results indicate an improvement in the expression strategy of H-ferritin for heteropolymeric production and studies of ferritin assembly in Escherichia coli.     

Enhanced expression and functional characterization of the human ferritin H- and L-chain genes in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

Enhanced expression of the human ferritin H- and L-chain genes (hfH and hfL) was achieved in Saccharomyces cerevisiae by modifying the N-terminal region of the structural genes. The yeast episomal vector YEp352 with the galactokinase1 (GAL1) promoter was used to construct expression plasmids. The expression of each gene was examined using SDS-PAGE and Western blot analysis. Iron uptake was examined and the cellular iron concentration was increased in S. cerevisiae expressing hfH. When cultured cells were incubated with 14.3 mM Fe(2+), the recombinant yeast expressing hfH had a cellular iron concentration 1.5 times greater than that of the control strain. The relationship between the iron taken up by the cells and the expressed proteins was examined. Iron-binding H-chain ferritin (H-ferritin) was seen in the recombinant S. cerevisiae incubated with iron, while small amounts of iron-binding L-chain ferritin (L-ferritin) were observed. Combined, these observations demonstrate that human H-ferritin has a function in iron storage in S. cerevisiae, while L-ferritin does not.     

L-Ferritin Binding to Scara5: A New Iron Traffic Pathway Potentially Implicated in Retinopathy

Iron is essential in the retina because the heme-containing enzyme guanylate cyclase modulates phototransduction in rods and cones. Transferrin endocytosis is the classical pathway for obtaining iron from the blood circulation in the retina. However, the iron storage protein ferritin has been also recently proposed as an iron carrier. In this study, the presence of Scara5 and its binding to L-ferritin was investigated in the retina. Our results showed that Scara5, the specific receptor for L-ferritin, was expressed in mouse and human retinas in many cell types, including endothelial cells. Furthermore, we showed that intravenously injected ferritin crossed the blood retinal barrier through L-ferritin binding to Scara5 in endothelial cells. Thus, suggesting the existence of a new pathway for iron delivery and trafficking in the retina. In a murine model of photoreceptor degeneration, Scara5 was downregulated, pointing out this receptor as a potential player implicated in retinopathy and also as a possible therapeutic target.     

Biochemical,Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin–Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin–ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.     

Functional roles of the ferritin receptors of human liver, hepatoma, lymphoid and erythroid cells.

Ferritin receptors are present on the membranes of many normal and malignant cells. The binding specificity of these receptors for H and L subunits was examined using recombinant human ferritin homopolymers. At least two different types of ferritin receptors were found, one derived from normal rat, pig, and human liver which shows similar binding of H- and L-ferritin. The second receptor type, specific for the H-chain ferritin, has been identified on membranes of hepatic and other transformed cells, and of normal lymphoblasts and erythroid precursors. These two receptor types may have different metabolic functions: the hepatic receptor acting as a scavenger for circulating ferritin and possibly for iron exchange between hepatocytes and macrophages; the H-ferritin receptor having a regulatory role which is not directly related to iron metabolism. The expression of the H-ferritin receptor is closely related to the activation and proliferation state of the cells. Addition of H-ferritin to the culture medium of cells expressing the H-ferritin receptor resulted in inhibition of cell proliferation and of colony formation.     

Expression and purification of intact and functional soybean (Glycine max) seed ferritin complex in Escherichia coli

Soybean seed ferritin is essential for human iron supplementation and iron deficiency anemia prevention because it contains abundant bioavailable iron and is frequently consumed in the human diet. However, it is poorly understood in regards its several properties, such as iron mineralization, subunit assembly, and protein folding. To address these issues, we decided to prepare the soybean seed ferritin complex via a recombinant DNA approach. In this paper, we report a rapid and simple Escherichia coli expression system to produce the soybean seed ferritin complex. In this system, two subunits of soybean seed ferritin, H-2 and H-1, were encoded in a single plasmid, and optimal expression was achieved by additionally coexpressing a team of molecular chaperones, trigger factor and GroEL-GroES. The His-tagged ferritin complex was purified by Ni2+ affinity chromatography, and an intact ferritin complex was obtained following His-tagged enterokinase (His-EK) digestion. The purified ferritin complex synthesized in E. coli demonstrated some reported features of its native counterpart from soybean seed, including an apparent molecular weight, multimeric assembly, and iron uptake activity. We believe that the strategy described in this paper may be of general utility in producing other recombinant plant ferritins built up from two types of subunits.     

A Novel Approach for the Synthesis of Human Heteropolymer Ferritins of Different H to L Subunit Ratios

Mammalian ferritins are predominantly heteropolymeric species consisting of 24 structurally similar, but functionally different subunit types, named H and L, that co-assemble in different proportions. Despite their discovery more than 8 decades ago, recombinant human heteropolymer ferritins have never been synthesized, owing to the lack of a good expression system. Here, we describe for the first time a unique approach that uses a novel plasmid design that enables the synthesis of these complex ferritin nanostructures. Our study reveals an original system that can be easily tuned by altering the concentrations of two inducers, allowing the synthesis of a full spectrum of heteropolymer ferritins, from H-rich to L-rich ferritins and any combinations in-between (isoferritins). The H to L subunit composition of purified ferritin heteropolymers was analyzed by SDS-PAGE and capillary gel electrophoresis, and their iron handling properties characterized by light absorption spectroscopy. Our novel approach allows future investigations of the structural and functional differences of isoferritin populations, which remain largely obscure. This is particularly exciting since a change in the ferritin H- to L-subunit ratio could potentially lead to new iron core morphologies for various applications in bio-nanotechnologies.     

Anaerobic iron deposition into horse spleen, recombinant human heavy and light and bacteria ferritins by large oxidants

Large-molecule oxidants oxidize Fe(II) to form Fe(III) cores in the interior of ferritins at rates comparable to or faster than the iron deposition reaction using O(2) as oxidant. Iron deposition into horse spleen ferritin (HoSF) occurs using ferricyanide ion, 2,6-dichlorophenol-indophenol, and several redox proteins: cytochrome c, stellacyanin, and ceruloplasmin. Cytochrome c also loads iron into recombinant human H-chain (rHF), human L-chain (rLF), and A. vinelandii bacterioferritin (AvBF). The enzymatic activities of ferritins were monitored anaerobically using stopped-flow kinetic spectrophotometry. The reactions exhibit saturation kinetics with respect to the large oxidant concentrations, giving apparent Michaelis constants for cytochrome c as oxidant: K(m)=39.6 microM for HoSF and 6.9 microM for AvBF. Comparison of the kinetic parameters with that of iron deposition by O(2) shows that large oxidants load iron into HoSF and AvBF more effectively than O(2) and may use a mechanism different than the ferroxidase center. Large oxidants did not deposit iron as efficiently with rHF and rLF. The results suggest that the heme groups in AvBF and the protein redox centers present in heteropolymers may assist in anaerobic iron deposition by large oxidants. The physiological relevance of iron deposition by large molecules, including protein oxidants is discussed.     

A mutation in the HFE gene is associated with altered brain iron profiles and increased oxidative stress in mice

Because of the increasing evidence that H63D HFE polymorphism appears in higher frequency in neurodegenerative diseases, we evaluated the neurological consequences of H63D HFE in vivo using mice that carry H67D HFE (homologous to human H63D). Although total brain iron concentration did not change significantly in the H67D mice, brain iron management proteins expressions were altered significantly. The 6-month-old H67D mice had increased HFE and H-ferritin expression. At 12 months, H67D mice had increased H- and L-ferritin but decreased transferrin expression suggesting increased iron storage and decreased iron mobilization. Increased L-ferritin positive microglia in H67D mice suggests that microglia increase iron storage to maintain brain iron homeostasis. The 6-month-old H67D mice had increased levels of GFAP, increased oxidatively modified protein levels, and increased cystine/glutamate antiporter (xCT) and hemeoxygenase-1 (HO-1) expression indicating increased metabolic and oxidative stress. By 12 months, there was no longer increased astrogliosis or oxidative stress. The decrease in oxidative stress at 12 months could be related to an adaptive response by nuclear factor E2-related factor 2 (Nrf2) that regulates antioxidant enzymes expression and is increased in the H67D mice. These findings demonstrate that the H63D HFE impacts brain iron homeostasis, and promotes an environment of oxidative stress and induction of adaptive mechanisms. These data, along with literature reports on humans with HFE mutations provide the evidence to overturn the traditional paradigm that the brain is protected from HFE mutations. The H67D knock-in mouse can be used as a model to evaluate how the H63D HFE mutation contributes to neurodegenerative diseases.     

Characterization of the H- and L-subunit ratios of ferritins by sodium dodecyl sulfate-capillary gel electrophoresis

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was used to characterize the H- and L-subunit ratios of several mammalian ferritins and one bacterioferritin. Traditionally, SDS-PAGE has been used to characterize the H- and L-subunit ratios in ferritin; however, this technique is relatively slow and requires staining, destaining, and scanning before the data can be processed. In addition, the H- and L-subunits of ferritin are fairly close in molecular weight (approximately 21,000 and approximately 20,000, respectively) and are often difficult to resolve in SDS-PAGE slab gels. In contrast, SDS-CGE requires no staining or destaining procedures and the peak quantitation is superior to SDS-PAGE. SDS-CGE is effective in quickly resolving the H- and L-subunits of ferritins from horse spleen, human liver, recombinant human H and L homopolymers, and mixtures of the two- and the single-subunit of a bacterioferritin from Escherichia coli. The technique has also proven useful in assaying the quality of the protein sample from both commercial and recombinant sources. Significant amounts of low-molecular-weight degradation products were detected in all commercial sources of horse spleen ferritin. Most commercial horse spleen ferritins lacked intact H-subunits under denaturing conditions.     

The universal mechanism for iron translocation to the ferroxidase site in ferritin, which is mediated by the well conserved transit site

Ferritins are ubiquitous iron storage proteins. Recently, we identified a novel metal-binding site, transit site, in the crystal structure of phytoferritin. To elucidate the function of the transit site in ferritin from other species, we prepared transit-site-deficient mutants of human H ferritin, E140A and E140Q, and their iron oxidation kinetics was analyzed. The initial velocities of iron oxidization were reduced in the variants, especially in E140Q. The crystal structure of E140Q showed that the side chain of the mutated Gln140 was fixed by a hydrogen bond, whereas that of native Glu140 was flexible. These results suggest that the conserved transit site also has a function to assist with the metal ion sequestration to the ferroxidase site in ferritins from vertebrates.     

Mitochondrial ferritin

A novel ferritin type specifically targeted to mitochondria has been recently found in human and mouse. It is structurally and functionally similar to the cytosolic ferritins, well-characterized molecules found in most living systems which are designed to store and detoxify cellular iron. Cytosolic ferritins in mammals are ubiquitous while mitochondrial ferritin expression is restricted mainly to the testis, neuronal cells and islets of Langherans. In addition, it is abundant in the iron-loaded mitochondria of erythroblasts of patients with sideroblastic anaemia. The characterization of recombinant and transfected mitochondrial ferritin indicated that this protein has a role in protecting mitochondria from iron-induced damage. These data suggest that it is an interesting tool to study the iron metabolism in this organelle. In addition, it may be useful for the diagnosis of myelodysplastic syndromes and in protecting mitochondria from the toxic effects of excess iron.     

Human mitochondrial ferritin expressed in HeLa cells incorporates iron and affects cellular iron metabolism

Mitochondrial ferritin (MtF) is a newly identified ferritin encoded by an intronless gene on chromosome 5q23.1. The mature recombinant MtF has a ferroxidase center and binds iron in vitro similarly to H-ferritin. To explore the structural and functional aspects of MtF, we expressed the following forms in HeLa cells: the MtF precursor (approximately 28 kDa), a mutant MtF precursor with a mutated ferroxidase center, a truncated MtF lacking the approximately 6-kDa mitochondrial leader sequence, and a chimeric H-ferritin with this leader sequence. The experiments show that all constructs with the leader sequence were processed into approximately 22-kDa subunits that assembled into multimeric shells electrophoretically distinct from the cytosolic ferritins. Mature MtF was found in the matrix of mitochondria, where it is a homopolymer. The wild type MtF and the mitochondrially targeted H-ferritin both incorporated the (55)Fe label in vivo. The mutant MtF with an inactivated ferroxidase center did not take up iron, nor did the truncated MtF expressed transiently in cytoplasm. Increased levels of MtF both in transient and in stable transfectants resulted in a greater retention of iron as MtF in mitochondria, a decrease in the levels of cytosolic ferritins, and up-regulation of transferrin receptor. Neither effect occurred with the mutant MtF with the inactivated ferroxidase center. Our results indicate that exogenous iron is as available to mitochondrial ferritin as it is to cytosolic ferritins and that the level of MtF expression may have profound consequences for cellular iron homeostasis.     

Mitochondrial ferritin expression in adult mouse tissues.

Mitochondrial ferritin (FtMt) is a novel ferritin type specifically targeted to mitochondria. It is highly expressed in the human testis and in sideroblasts from patients with sideroblastic anemia, but other organs have not been studied. To study its expression in the main organs of the mouse, we first used RT-PCR and then produced recombinant mouse FtMt and specific antibodies. Immunohistochemistry analyses confirmed that FtMt is highly expressed in mouse testis, particularly in spermatocytes and interstitial Leydig cells. The protein was also identified in other organs including heart, brain, spinal cord, kidney, and pancreatic islet of Langerhans but not in liver and splenocytes, which have iron storage function and express high levels of cytosolic ferritins. Results indicate that the primary function of ferritin FtMt is not involved in storing cellular or body iron, but its association with cell types characterized by high metabolic activity and oxygen consumption suggests a role in protecting mitochondria from iron-dependent oxidative damage.     

Ferritin: a novel mechanism for delivery of iron to the brain and other organs

Traditionally, transferrin has been considered the primary mechanism for cellular iron delivery, despite suggestive evidence for additional iron delivery mechanisms. In this study we examined ferritin, considered an iron storage protein, as a possible delivery protein. Ferritin consists of H- and L-subunits, and we demonstrated iron uptake by ferritin into multiple organs and that the uptake of iron is greater when the iron is delivered via H-ferritin compared with L-ferritin. The delivery of iron via H-ferritin but not L-ferritin was significantly decreased in mice with compromised iron storage compared with control, indicating that a feedback mechanism exists for H-ferritin iron delivery. To further evaluate the mechanism of ferritin iron delivery into the brain, we used a cell culture model of the blood-brain barrier to demonstrate that ferritin is transported across endothelial cells. There are receptors that prefer H-ferritin on the endothelial cells in culture and on rat brain microvasculature. These studies identify H-ferritin as an iron transport protein and suggest the presence of an H-ferritin receptor for mediating iron delivery. The relative amount of iron that could be delivered via H-ferritin could make this protein a predominant player in cellular iron delivery. blood-brain barrier; iron transport; H-ferritin     

Multiple pathways for mineral core formation in mammalian apoferritin. The role of hydrogen peroxide

Human ferritins sequester and store iron as a stable FeOOH((s)) mineral core within a protein shell assembled from 24 subunits of two types, H and L. Core mineralization in recombinant H- and L-subunit homopolymer and heteropolymer ferritins and several site-directed H-subunit variants was investigated to determine the iron oxidation/hydrolysis chemistry as a function of iron flux into the protein. Stopped-flow absorption spectrometry, UV spectrometry, and electrode oximetry revealed that the mineral core forms by at least three pathways, not two as previously thought. They correspond to the ferroxidase, mineral surface, and the Fe(II) + H2O2 detoxification reactions, respectively: [see reactions]. The H-subunit catalyzed ferroxidase reaction 1 occurs at all levels of iron loading of the protein but decreases with increasing iron added (48-800 Fe(II)/protein). Reaction 2 is the dominant reaction at 800 Fe(II)/protein, whereas reaction 3 occurs largely at intermediate iron loadings of 100-500 Fe(II)/protein. Some of the H2O2 produced in reaction 1 is consumed in the detoxification reaction 3; the 2/1 Fe(II)/H2O2 stoichiometry of reaction 3 minimizes hydroxyl radical production during mineralization. Human L-chain ferritin and H-chain variants lacking functional nucleation and/or ferroxidase sites deposit their iron largely through the mineral surface reaction 2. H2O2 is shown to be an intermediate product of dioxygen reduction in L-chain as well as in H-chain and H-chain variant ferritins.