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.     

Expression and structural and functional properties of human ferritin L-chain from Escherichia coli

The human ferritin L-chain cDNA was cloned into a vector for overproduction in Escherichia coli, under the regulation of a lambda promoter. The plasmid obtained contains the full L-chain coding region modified at the first two codons. It is able to direct the synthesis of the L-chain which can constitute up to 15% of the total soluble protein of bacterial extract. The L-chains assemble to form a ferritin homopolymer with electrophoretic mobility, molecular weight, thermal stability, spectroscopic, and immunological properties analogous to natural ferritin from human liver (95% L-chain). This recombinant L-ferritin is able to incorporate and retain iron in solution at physiological pH values. At variance with the H-ferritin, the L form does not uptake iron at acidic pH values and does not show detectable ferroxidase activity. It is concluded that ferritin L-chain lacks the ferroxidase site present in the H-chain and that the two chains may have specialized functions in intracellular iron metabolism.     

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.     

Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity

Transfectant HeLa cells were generated that expressed human ferritin H-chain wild type and an H-chain mutant with inactivated ferroxidase activity under the control of the tetracycline-responsive promoter (Tet-off). The clones accumulated exogenous ferritins up to levels 14-16-fold over background, half of which were as H-chain homopolymers. This had no evident effect in the mutant ferritin clone, whereas it induced an iron-deficient phenotype in the H-ferritin wild type clone, manifested by approximately 5-fold increase of IRPs activity, approximately 2.5-fold increase of transferrin receptor, approximately 1.8-fold increase in iron-transferrin iron uptake, and approximately 50% reduction of labile iron pool. Overexpression of the H-ferritin, but not of the mutant ferritin, strongly reduced cell growth and increased resistance to H(2)O(2) toxicity, effects that were reverted by prolonged incubation in iron-supplemented medium. The results show that in HeLa cells H-ferritin regulates the metabolic iron pool with a mechanism dependent on the functionality of the ferroxidase centers, and this affects, in opposite directions, cellular growth and resistance to oxidative damage. This, and the finding that also in vivo H-chain homopolymers are much less efficient than the H/L heteropolymers in taking up iron, indicate that functional activity of H-ferritin in HeLa cells is that predicted from the in vitro data.     

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     

Active human ferritin H/L-hybrid and sequence effect on folding efficiency in Escherichia coli

In Escherichia coli, the recombinant human L-chain ferritin was synthesized in the form of inclusion bodies under the control of T7 promoter system. We developed a recombinant ferritin H/L-hybrid by a direct gene fusion between H- and L-chain subunits. Surprisingly, the presence of heavy-chain polypeptide at the amino terminus of light chain significantly increased the cytoplasmic solubility of the recombinant ferritin hybrid, i.e., more than 80% of synthesized ferritin hybrid was soluble in intracellular region regardless of the changes in cell growth and gene expression conditions such as type of inducer, growth media, culture scale, etc. The soluble ferritin H/L-hybrid was biologically active with the iron storage capacity (295mol Fe(+3) per mol H/L-hybrid) equivalent to ferritin standard. Different types of hybrid mutants were also developed using various H-chain derivatives. Comparison of the intracellular solubilities of the synthesized hybrid mutants showed that the N-terminus four helices of heavy subunit were of crucial importance in maintaining the high solubility in E. coli cytoplasm. Consequently, the increased solubility of the ferritin hybrid seems to be related to such H-chain sequence that forms ferroxidase center and promotes effective intra-molecular interaction with L-chain domain of H/L-hybrid for enhancing the folding efficiency.     

Evidence that a salt bridge in the light chain contributes to the physical stability difference between heavy and light human ferritins.

Human ferritin, a multimeric iron storage protein, is composed by various proportions of two subunit types: the H- and L-chains. The biological functions of these two genic products have not been clarified, although differences in reactivity with iron have been shown. Starting from the hypothesis that the high stability typical of ferritin is an important property which may be relevant for its iron storage function, we studied ferritin homopolymers of H- and L-chains in different denaturing conditions. In addition we analyzed 13 H-chain variants with alterations in regions conserved within mammalian H-chains. In all the denaturation experiments H-chain ferritin showed lower stability than L-chain ferritin. The difference was greater in guanidine HCl denaturation experiments, where the end products are fully unfolded peptides, than in acidic denaturation experiments, where the end products are peptides with properties analogous to "molten globule." The study on H-chain variants showed: (i) ferritin stability was not affected by alterations of regions exposed to the inner or outer surface of the shell and not involved in intra- or inter-chain interactions; (ii) stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels; (iii) stability was increased by the substitution of 2 amino acids inside the four-helix bundle with those of the homologous L-chain. One of the residues is involved in a salt bridge in the L-chain, and we concluded that the stability difference between H- and L-ferritins is to a large extent due to the stabilizing effect of this salt bridge on the L-subunit fold.     

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.     

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.     

High level of ferritin light chain mRNA in lens

Ferritin is of particular interest with regard to cataract because (i) cataract occurs in individuals with hereditary hyperferritinemia cataract syndrome (HHCS), a condition in which ferritin light chain (L-ferritin) protein is overexpressed systemically, and (ii) ferritin is an important regulator of oxidative stress, a primary factor in the etiology of aging-related cataract. From gene array analysis two novel observations were made with respect to ferritin gene expression: first, lenses from guinea pigs and humans have disproportionately high levels of L-ferritin mRNA relative to the amounts of ferritin protein present, and second, L-ferritin message increased markedly in lenses from guinea pigs with hereditary nuclear cataract. The human lens L-ferritin sequence was identical to previous data from human liver; the guinea pig sequence was 86% identical to the human sequence at the amino acid level. Despite mRNA levels similar to those of major lens crystallins, lens ferritin was undetectable by Western blot techniques.     

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.     

Ferritin,iron homeostasis,and oxidative damage

Ferritin is one of the major proteins of iron metabolism. It is almost ubiquitous and tightly regulated by the metal. Biochemical and structural properties of the ferritins are largely conserved from bacteria to man, although the role in the regulation of iron trafficking varies in the different organisms. Recent studies have clarified some of the major aspects of the reaction between iron and ferritin, which results in the formation of the iron core and production of hydrogen peroxide. The characterization of cellular models in which ferritin expression is modulated has shown that the ferroxidase catalytic site on the H-chain has a central role in regulating iron availability. In turn, this has secondary effects on a number of cellular activities, which include proliferation and resistance to oxidative damage. Moreover, the response to apoptotic stimuli is affected by H-ferritin expression. Altered ferritin L-chain expression has been found in at least two types of genetic disorders, although its role in the determination of the pathology has not been fully clarified. The recent discovery of a new ferritin specific for the mitochondria, which is functionally similar to the H-ferritin, opens new perspectives in the study of the relationships between iron, oxidative damage and free radicals.     

A mutational analysis of the epitopes of recombinant human H-ferritin.

Murine monoclonal antibodies were elicited by the recombinant human H-ferritin overexpressed in Escherichia coli. They had a specificity analogous to that of the antibodies elicited by natural human H-chain, and all of them showed low additivity in binding the recombinant ferritin. Four antibodies of each group were challenged with four H-ferritin mutants overexpressed in E. coli, altered in different accessible areas of the molecule. They consisted of deletions of the first 13 and last 22 amino acids, a duplication of an 18 amino acid sequence in the loop region, and a substitution of a 5 amino acid stretch in the three-fold symmetry axis region. Double diffusion, immunodot analyses and inhibition plots indicated that: (1) all the mutants were recognized by at least one antibody; (2) the deletion of the N-terminus and the duplication in the loop region had the strongest effect on antibody binding; and (3) epitope boundaries of the various antibodies could not be recognized. The antibodies were tested with H-containing ferritins from rat and hen hearts, and showed low or absent reactivities despite their high structural homology with human ferritin. Comparison of the amino acid sequences of human, mouse, rat and hen H-chains, together with mutational data, suggested that; (i) ferritin epitopes are large, probably encompassing a large portion of the subunit surface and (ii) Thr-5 and Cys-90 have a role in H-ferritin immunogenicity.     

Characterization of autoantibodies to ferritin in canine serum

Ferritin-binding proteins circulating in mammalian blood are thought to be involved in the clearance of ferritin. The present study characterizes canine serum autoantibodies (IgM and IgA) that react with ferritin. Canine IgM and IgA bound to bovine spleen ferritin as well as canine liver ferritin. To examine the specificity of canine IgM and IgA to ferritin H and L subunits, we used canine heart ferritin and canine liver ferritin with H/L subunit ratios of 3.69 and 0.43, respectively. Canine IgM and IgA recognized both of the H- and L-subunit-rich isoferritins, showing that their binding activities to ferritin depend on the H-subunit content. Recombinant bovine H-chain ferritin homopolymer expressed in a baculovirus expression system bound more with IgM and IgA than the recombinant L-chain homopolymer expressed under the same conditions. These results suggest that canine IgM and IgA recognize H-subunit-rich isoferritins, and that H-subunit-rich isoferritins are cleared from the circulation more rapidly than L-subunit-rich isoferritins.     

Development of myelin oligodendrocyte glycoprotein autoreactive transgenic B lymphocytes: receptor editing in vivo after encounter of a self-antigen distinct from myelin oligodendrocyte glycoprotein

We explored mechanisms involved in B cell self-tolerance against brain autoantigens in a double-transgenic mouse model carrying the Ig H-chain (introduced by gene replacement) and/or the L-chain kappa (conventional transgenic) of the mAb 8.18C5, specific for the myelin oligodendrocyte glycoprotein (MOG). Previously, we demonstrated that B cells expressing solely the MOG-specific Ig H-chain differentiate without tolerogenic censure. We show now that double-transgenic (THkappa(mog)) B cells expressing transgenic Ig H- and L-chains are subjected to receptor editing. We show that in adult mice carrying both MOG-specific Ig H- and L-chains, the frequency of MOG-binding B cells is not higher than in mice expressing solely the transgenic Ig H-chain. In fact, in THkappa(mog) double-transgenic mice, the transgenic kappa(mog) L-chain was commonly replaced by endogenous L-chains, i.e., by receptor editing. In rearrangement-deficient RAG-2(-) mice, differentiation of THkappa(mog) B cells is blocked at an immature stage (defined by the B220(low)IgM(low)IgD(-) phenotype), reflecting interaction of the autoreactive B cells with a local self-determinant. The tolerogenic structure in the bone marrow is not classical MOG, because back-crossing THkappa(mog) mice into a MOG-deficient genetic background does not lead to an increase in the proportion of MOG-binding B cells. We propose that an as yet undefined self-Ag distinct from MOG cross-reacts with the THkappa(mog) B cell receptor and induces editing of the transgenic kappa(mog) L-chain in early immature B cells without affecting the pathogenic potential of the remaining MOG-specific B cells. This phenomenon represents a particular form of chain-specific split tolerance.     

Over-expression of natural and variant human H-chain ferritins in E. coli

The natural human H-chain ferritin was expressed in E. coli using a multi-copy expression vector containing the lambda pL promoter. A variant H-ferritin, having an altered N-terminus, was also produced. These proteins are overproduced (greater than 30% of the soluble protein), correctly assembled into its 24-subunit shell, and able to bind iron. The identity of the products was confirmed using an antibody specific for H-ferritin.     

Hydrophobic interaction of P25, containing Asn-linked oligosaccharide chains, with the H-L complex of silk fibroin produced by Bombyx mori.

Fibroin light (L-) chain and P25 are low molecular weight protein components of silk fibroin which are secreted from the posterior silk gland cells of the silkworm, Bombyx mori. The primary structure of L-chain was determined previously by cDNA cloning and peptide analysis, but that of P25 has only been deduced from its genomic sequence. Our previous studies with specific antibodies against L-chain and P25 have shown that L-chain and H-chain are linked by disulfide bond(s) but P25 is not covalently linked to H-chain. Here, we present evidence that P25 associates with the H-L complex primarily by hydrophobic interactions and that P25 is a glycoprotein containing Asn-linked oligosaccharide chains. From the analysis of three fibroin-secretion-deficient 'naked pupa' mutant breeds [Nd(2), Nd-s and Nd-sD], it is suggested that P25 interacts with H-chain in the absence of H-L linkage but its content of oligosaccharide is reduced when the H-L linkage is not formed. From these results, models are presented implying that the H-L complex and P25 are associated to form a higher-order complex of specific conformation during the processes of intracellular transport and secretion, and that the Asn-linked glycosylation of P25 is partially altered under such conditions.     

Defining metal ion inhibitor interactions with recombinant human H- and L-chain ferritins and site-directed variants: an isothermal titration calorimetry study

Zinc and terbium, inhibitors of iron incorporation in the ferritins, have been used for many years as probes of structure-function relationships in these proteins. Isothermal titration calorimetric and kinetic measurements of Zn(II) and Tb(III) binding and inhibition of Fe(II) oxidation were used to identify and characterize thermodynamically ( n, K, Delta H degrees, Delta S degrees, and Delta G degrees ) the functionally important binding sites for these metal ions in recombinant human H-chain, L-chain, and H-chain site-directed variant ferritins. The data reveal at least two classes of binding sites for both Zn(II) and Tb(III) in human H-chain ferritin: one strong, corresponding to binding of one metal ion in each of the eight three-fold channels, and the other weak, involving binding at the ferroxidase and nucleation sites of the protein as well as at other weak unidentified binding sites. Zn(II) and Tb(III) binding to recombinant L-chain ferritin showed similar stoichiometries for the strong binding sites within the channels, but fewer weaker binding sites when compared to the H-chain protein. The kinetics and binding data indicate that the binding of Zn(II) and Tb(III) in the three-fold channels, which is the main pathway of iron(II) entry in ferritin, blocks the access of most of the iron to the ferroxidase sites on the interior of the protein, accounting for the strong inhibition by these metal ions of the oxidative deposition of iron in ferritin.