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.     

Identification of a mechanism by which lens epithelial cells limit accumulation of overexpressed ferritin H-chain

The primary cultures of canine lens epithelial cells were transiently transfected with cDNAs for dog ferritin H- or L-chains in order to study differential expression of these chains. By using chain-specific antibodies, we determined that at 48 h after transfection overexpression of L-chain was much higher (9-fold over control) than that of H-chain (1.7-fold). We discovered that differentially transfected cells secrete overexpressed chains as homopolymeric ferritin into the media. Forty-eight hours after transfection accumulation of H-ferritin in the media was much higher (3-fold) than that of L-ferritin. This resulted in lowering of the concentration of H-chain in the cytosol. Co-transfection of cells with both H- and L-chain cDNAs increased the intracellular levels of H-chain and eliminated secretion of H-ferritin to the media. We concluded that lens epithelial cells differentially regulate concentration of both ferritin chains in the cytosol. The overexpressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin. These data indicate that the inability of cells to more strictly control cytosolic levels of L-chain may augment its accumulation in lenses of humans with hereditary hyperferritinemia cataract syndrome, which is caused by overexpression of L-chain due to mutation in the regulatory element in the untranslated region of the mRNA of the chain.     

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.     

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.     

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.     

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.     

A fibroin secretion-deficient silkworm mutant, Nd-sD, provides an efficient system for producing recombinant proteins

The silkworm Nd-s(D) mutant is silk fibroin-secretion deficient. In the mutant, a disulfide linkage between the heavy (H) and light (L) chains, which is essential for the intracellular transport and secretion of fibroin, is not formed because of a partial deletion of the L-chain gene. To utilize the inactivity of the mutant L-chain, we investigated the possibility of using the Nd-s(D) mutant for the efficient production of recombinant proteins in the silkworm. A germ line transformation of the mutant with a normal L-chain-GFP fusion gene was performed. In the transgenic mutant, normal development of the posterior silk gland (PSG) was restored and it formed a normal cocoon. The biochemical analysis showed that the transgenic silkworms expressed the introduced gene in PSG cells, produced a large amount of the recombinant protein, secreted it into the PSG lumen, and used it to construct the cocoon. The molar ratio of silk proteins, H-chain:L-chain-GFP:fibrohexamerin, in the lumen and cocoon in the transgenic silkworm was 6:6:1, and the final product of the fusion gene formed about 10% of the cocoon silk. This indicates that the transgenic mutant silkworm possesses the capacity to produce and secrete the recombinant proteins in a molar ratio equal to that of the fibroin H-chain, contributing around half molecules of the total PSG silk proteins.     

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.     

Influence of site-directed modifications on the formation of iron cores in ferritin

The structure and crystal chemical properties of iron cores of reconstituted recombinant human ferritins and their site-directed variants have been studied by transmission electron microscopy and electron diffraction. The kinetics of Fe uptake have been compared spectrophotometrically. Recombinant L and H-chain ferritins, and recombinant H-chain variants incorporating modifications in the threefold (Asp131----His or Glu134----Ala) and fourfold (Leu169----Arg) channels, at the partially buried ferroxidase sites (Glu62,His65----Lys,Gly), a putative nucleation site on the inner surface (Glu61,Glu64,Glu67----Ala), and both the ferroxidase and nucleation sites (Glu62,His65----Lys,Gly and Glu61,Glu64,Glu67----Ala), were investigated. An additional H-chain variant, incorporating substitution of the last ten C-terminal residues for those of the L-chain protein, was also studied. Most of the proteins assimilated iron to give discrete electron-dense cores of the Fe(III) hydrated oxide, ferrihydrite (Fe2O3.nH2O). No differences were observed for variants modified in the three- or fourfold channels compared with the unmodified H-chain ferritin. The recombinant L-chain ferritin and H-chain variant depleted of the ferroxidase site, however, showed markedly reduced uptake kinetics and comprised cores of increased diameter and regularity. Depletion of the inner surface Glu residues, whilst maintaining the ferroxidase site, resulted in a partially reduced rate of Fe uptake and iron cores of wider particle size distribution. Modification of both ferroxidase and inner surface Glu residues resulted in complete inhibition of iron uptake and deposition. No cores were observed by electron microscopy although negative staining showed that the protein shell was intact. The general requirement of an appropriate spatial charge density across the cavity surface rather than specific amino acid residues could explain how, in spite of an almost complete lack of identity between the amino acid sequences of bacterioferritin and mammalian ferritins, ferrihydrite is deposited within the cavity of both proteins under similar reconstitution conditions.     

Translational control of immunoglobulin synthesis. I. Repression of heavy chain synthesis

When myeloma cells are incubated at 25 °C the secretion of myeloma protein ceases within 20 minutes. The synthesis of heavy and light chains and the assembly into the completed 7 S immunoglobulin continue at over 40% of the synthetic rate at 37 °C, resulting in an increasing intracellular concentration of myeloma protein with time. When myeloma cells containing an increased myeloma protein pool were re-incubated at 37 °C, there was an initially decreased synthesis of H-chain² relative to L-chain or total protein. Whereas L-chain synthesis returned to the pre-25 °C synthetic rate within 15 minutes, the synthesis of H-chain required over 60 minutes to return to the pre-incubation rate.Myeloma cells maintained in exponential growth contain a larger intracellular pool of H₂L₂ than cells in late stationary phase. When both populations of cells were incubated at 25 °C and the synthesis of H and L-chain protein measured, a reduced synthesis of H-chain was again observed. Exponentially growing cells showed an 80% reduction of H-chain synthesis after 100 minutes at 25 °C. Stationary cells, with the reduced intracellular level of H₂L₂, required 210 minutes to effect an equivalent reduction of H-chain synthesis.The opposite effect on myeloma protein synthesis was observed following depletion of the H₂L₂ pool. The intracellular H₂L₂ pool was reduced by allowing secretion in the absence of protein synthesis. When protein synthesis was allowed to continue following the depletion, a stimulation of myeloma protein synthesis relative to total protein synthesis was observed.These experiments suggest a close relation between the intracellular level of H₂L₂ and the production of H-chain. From the rapidity of the repression and de-repression of H-chain synthesis, a regulation at the translational level is suggested.     

Oxidative modification of ferritin induced by hydrogen peroxide

Excess free iron generates oxidative stress that may contribute to the pathogenesis of various causes of neurodegenerative diseases. In this study, we assessed the modification of ferritin induced by H(2)O(2). When ferritin was incubated with H(2)O(2), the degradation of ferritin L-chain increased with the H(2)O(2) concentration whereas ferritin H-chain was remained. Free radical scavengers, azide, thiourea, and N-acetyl-(L)-cysteine suppressed the H(2)O(2)-mediated ferritin modification. The iron specific chelator, deferoxamine, effectively prevented H(2)O(2)-mediated ferritin degradation in modified ferritin. The release of iron ions from ferritin was increased in H(2)O(2) concentration-dependent manner. The present results suggest that free radicals may play a role in the modification and iron releasing of ferritin by H(2)O(2). It is assumed that oxidative damage of ferritin by H(2)O(2) may induce the increase of iron content in cells and subsequently lead to the deleterious condition.     

Predominant synthesis of fibroin heavy and light chains on the membrane-bound polysomes prepared from the posterior silk gland of the silkworm, Bombyx mori

Membrane-bound polysomes were prepared from the posterior silk gland of the silkworm, Bombyx mori, on the fourth to fifth day in the fifth larval instar. The polysomes, when supplemented with a soluble fraction from the posterior silk gland, exhibited the elongation reaction of the growing polypeptide-chains, but the initiation reaction of polypeptide synthesis was not demonstrated in this system. The predominant products synthesized on the membrane-bound polysomes were fibroin heavy chain (H-chain) and light chain (L-chain), while polypeptides of heterogeneous size classes were synthesized on the 105,000 X g-sedimentable polysomes. A substantial fraction of the fibroin L-chain synthesized was bound to the H-chain by disulfide bond. Most of the newly synthesized fibroin H- and L-chains on the membrane-bound polysomes were proved to be present within microsomal membrane vesicles because of their insensitivity to digestion with proteases in the absence of Triton X-100.     

Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA

A procedure is described for the large-scale purification of light (L) and heavy (H) chain mRNAs from plasmacytomas produced in mice. Intact RNA is selectively precipitated in high yield from frozen tumors homogenized in 3 M LiCl and 6 M urea. L and H-chain mRNAs were purified by oligo(dT)-cellulose chromatography and either sucrose gradient centrifugation in conditions preventing aggregation or by means of high-resolution preparative gel electrophoresis under non-denaturing conditions. gamma 2a and alpha H-chain mRNAs sedimented as major components at 15.5 S and 16.5 S respectively, when L-chain mRNAs sedimented as 12-S species. H-chain mRNAs isolated by continuous elution during preparative gel electrophoresis were completely separated from both L-chain mRNA and residual 18-S rRNA, and migrated as single components of 1900 +/- 50 nucleotides on analytical denaturing gels. The partially purified H-chain mRNAs were translated into major components of molecular weights of 56,000 (gamma 2a) and 60,000 (alpha) in an mRNA-dependent rabbit reticulocyte lysate, whereas L-chain mRNAs yielded polypeptides of molecular weights of 25,000 (gamma) and 27,000 (chi). Up to 95% of the translation products directed by the purified mRNAs were immunoprecipitated using specific antisera. The purity of L and H-chain mRNAs was assessed by hybridization of corresponding cDNAs with excess recombinant plasmid DNA. The results indicated a minimum purity of 47% (gamma 2a), 62% (alpha), for H-chain mRNAs and 60% (chi), for L-chain mRNAs.     

mu-1,2-peroxo diferric complex formation in horse spleen ferritin. A mixed H/L-subunit heteropolymer

Previous kinetics studies with homopolymer ferritins (bullfrog M-chain, human H-chain and Escherichia coli bacterial ferritins) have established that a mu-1,2-peroxo diferric intermediate is formed during Fe(II) oxidation by O2 at the ferroxidase site of the protein. The present study was undertaken to determine whether such an intermediate is formed also during iron oxidation in horse spleen ferritin (HoSF), a naturally occurring heteropolymer ferritin of H and L-subunits (approximately 3.3 H-chains/HoSF), and to assess its role in the formation of the mineral core. Multi-wavelength stopped-flow spectrophotometry of the oxidative deposition of iron in HoSF demonstrated that a transient peroxo complex (lambda(max) approximately 650 nm) is produced in this protein as for other ferritins. The peroxo complex in HoSF is formed about fourfold slower than in human H-chain (HuHF) and decays more slowly (approximately threefold) as well, at an iron level of two Fe(II)/H-chain. However, as found for HuHF, a second intermediate is formed in HoSF as a decay product of the peroxo complex. Only one-third of the expected peroxo complex forms at the ferroxidase centers of HoSF when two Fe(II)/H-subunits are added to the protein, dropping to only approximately 14% when 20 Fe(II)/H-chain are added, indicating a declining role of the peroxo complex in iron deposition. In contrast to HuHF, HoSF does not enzymatically regenerate the observable peroxo complex. The kinetics of mineralization in HoSF are modeled satisfactorily by a mechanism in which the ferroxidase site rapidly produces an incipient core from a single turnover of iron, upon which subsequent Fe(II) is oxidized autocatalytically to build the Fe(O)OH(s) mineral core. This model supports a role for the L-chain in iron mineralization and helps to explain the widespread occurrence of heteropolymer ferritins in tissues of vertebrates.     

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.     

Clearance rates of biotinylated ferritins in canine: A possible physiological role of canine serum ferritin as an iron transporter

To elucidate the physiological role of canine serum ferritin, we measured clearance rates of biotinylated ferritins in beagle. Biotinylated canine tissue ferritins were cleared rapidly from circulation. The clearance time (T_1/2) of liver ferritin (H/L subunit ratio=0.43) was 6.8 to 11.8 min, and that of heart ferritin (H/L=3.69) was 9.3 to 25.0 min. T_1/2 of biotinylated canine liver ferritin was independent of iron content, whereas canine heart apoferritin (T_1/2=31.2 and 32.7 min) was more slowly removed from circulation than the holoferritin. On the other hand, biotinylated recombinant bovine H-chain ferritin homopolymer show a much slower rate of removal (T_1/2=153.8 and 155.0 min) compared with the L-chain ferritin homopolymer (T_1/2=26.4 and 31.3 min). The rapid clearance of canine tissue ferritin suggests that serum ferritin is an iron transporter in canines.     

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.     

Mutational analysis of the channel and loop sequences of human ferritin H-chain.

Human ferritin H-chain mutants were obtained by engineering the recombinant protein expressed by Escherichia coli. The mutagenesis were directed to the C-terminal sequence forming the hydrophobic channel, to the hydrophilic channel and to the loop sequence. The mutants were analysed for extent of expression, for stability, for capacity to incorporate iron and for kinetics of iron uptake and iron oxidation. Of the 22 mutants analysed only two with deletions of single residues in the loop sequence and one with deletion of the last 28 amino acid residues did not assemble into ferritin-like proteins. The other mutants assembled correctly and showed similar chemical/physical properties to the wild-type; they included duplication of an 18-amino acid-residue stretch, deletion of the last 22 and the last seven residues and various mutations of single amino acid residues. Two mutants with extensive alteration in the C-terminal sequence had a diminished thermostability associated with incapability to incorporate iron though they still catalysed iron oxidation. The mutants with alterations of the sequence around the hydrophilic channel showed diminished iron uptake and oxidation kinetics, together with a slightly larger apparent molecular size. The results indicate (i) that two of the sequences are important for ferritin assembly/stability, (ii) that the presence of the hydrophobic channel is essential for formation of the iron core and (iii) that the sites of iron interaction and the path of iron penetration into ferritin remain unidentified.