The monomers and oligomers of ferritin and apoferritin: association and dissociation.

We have reinvestigated the association and dissociation of ferritin and apoferritin in phosphate buffer (pH 7.2, I = 0.05). When oligomer-enriched solutions of horse spleen ferritin were mixed with more concentrated, but unenriched solutions of horse spleen apoferritin, there was dissociation of the ferritin oligomers, as determined by polyacrylamide gel electrophoresis and from iron/protein ratios. Some evidence was also obtained for association of monomers in the mixture of ferritin and apoferritin after pelleting and redissolution of pellets in minimal volumes of the phosphate buffer. Monomer-enriched, biosynthetically labeled rat liver ferritin was pelleted, redissolved in minimal volumes of phosphate buffer, and separated by polyacrylamide gel electrophoresis; the fractions were isolated and counted. The results revealed that an association of monomers of the rat liver ferritin had taken place which doubled the concentration of dimers. However, our results also indicate that association by concentration was limited to a fraction of monomers.     

Functional characterization of artemin, a ferritin homolog synthesized in Artemia embryos during encystment and diapause

Oviparously developing embryos of the crustacean Artemia franciscana encyst and enter diapause, exhibiting a level of stress tolerance seldom seen in metazoans. The extraordinary stress resistance of encysted Artemia embryos is thought to depend in part on the regulated synthesis of artemin, a ferritin superfamily member. The objective of this study was to better understand artemin function, and to this end the protein was synthesized in Escherichia coli and purified to apparent homogeneity. Purified artemin consisted of oligomers approximately 700 kDa in molecular mass that dissociated into monomers and a small number of dimers upon SDS/PAGE. Artemin inhibited heat-induced aggregation of citrate synthase in vitro, an activity characteristic of molecular chaperones and shown here to be shared by apoferritin and ferritin. This is the first report that apoferritin/ferritin may protect cells from stress other than by iron sequestration. Stably transfected mammalian cells synthesizing artemin were more resistant to heat and H(2)O(2) than were cells transfected with vector only, actions also shared by molecular chaperones such as the small heat shock proteins. The data indicate that artemin is a structurally modified ferritin arising either from a common ancestor gene or by duplication of the ferritin gene. Divergence, including acquisition of a C-terminal peptide extension and ferroxidase center modification, eliminated iron sequestration, but chaperone activity was retained. Therefore, because artemin accumulates abundantly during development, it has the potential to protect embryos from stress during encystment and diapause without adversely affecting iron metabolism.     

Studies on iron uptake and micelle formation in ferritin and apoferritin

Summary Iron uptake and micelle formation in ferritin and apoferritin have been followed both spectrophotometrically and by means of sedimentation velocity experiments. Information was thus obtained on the molecular weight distribution of the reconstitution product.To achieve incorporation native ferritin (whole ferritin as purified from horse spleen), native apoferritin (apoferritin prepared by fractionation of ferritin preparations) and reduced apoferritin (apoferritin prepared by reduction of ferritin by dithionite or ascorbic acid) have been incubated with ferrous salts in the presence of oxidizing agents under different experimental conditions.Although some iron is incorporated in native ferritin, full saturation is not achieved and the molecular weight distribution of the incubated products remains heterogeneous.Native and reduced apoferritin show a similar iron incorporation, but the reconstitution products markedly differ in terms of their iron distribution.Ferritin reconstituted from native apoferritin has a broad molecular weight distribution, while that reconstituted from reduced apoferritin is characterized by a narrow, homogeneous molecular weight distribution. However treatment of apoferritin with reducing or oxidizing agents prior to the incubation alters the characteristics of the iron distribution without changing the iron incorporation properties.These results point to a role of the protein moiety not only in iron oxidation, but also in micelle formation.     

The consequences of hydroxyl radical formation on the stoichiometry and kinetics of ferrous iron oxidation by human apoferritin

Despite previous detection of hydroxyl radical formation during iron deposition into ferritin, no reports exist in the literature concerning how it might affect ferritin function. In the present study, hydroxyl radical formation during Fe(II) oxidation by apoferritin was found to be contingent on the "ferroxidase" activity (i.e., H subunit composition) exhibited by apoferritin. Hydroxyl radical formation was found to affect both the stoichiometry and kinetics of Fe(II) oxidation by apoferritin. The stoichiometry of Fe(II) oxidation by apoferritin in an unbuffered solution of 50 mM NaCl, pH 7.0, was approximately 3.1 Fe(II)/O(2) at all iron-to-protein ratios tested. The addition of HEPES as an alternate reactant for the hydroxyl radical resulted in a stoichiometry of about 2 Fe(II)/O(2) at all iron-to-protein ratios. HEPES functioned to protect apoferritin from oxidative modification, for its omission from reaction mixtures containing Fe(II) and apoferritin resulted in alterations to the ferritin consistent with oxidative damage. The kinetic parameters for the reaction of recombinant human H apoferritin with Fe(II) in HEPES buffer (100 mM) were: K(m) = 60 microM, k(cat) = 10 s(-1), and k(cat)/K(m) = 1.7 x 10(5) M(-1) x (-1). Collectively, these results contradict the "crystal growth model" for iron deposition into ferritin and, while our data would seem to imply that the ferroxidase activity of ferritin is adequate in facilitating Fe(II) oxidation at all stages of iron deposition into ferritin, it is important to note that these data were obtained in vitro using nonphysiologic conditions. The possibility that these findings may have physiological significance is discussed.     

Subunit dimers in sheep spleen apoferritin. The effect on iron storage

Ferritin with high and low iron content, 2000 and 790 iron atoms/molecule, was isolated from the spleens of copper-poisoned and control lambs, respectively. Differences in the iron content in vivo were reflected in the properties of the apoferritin protein shells, since the apoprotein from the low iron ferritin took up iron relatively more slowly (0.52 +/- 0.09) and released it more rapidly (1.68 +/- 0.06) in vitro. Although the two types of apoferritin were indistinguishable in terms of surface charge (pI range 4.98-5.43) and in consisting of both heavy and light subunits, the subunit interactions differed markedly; 40-50% of the subunits of low iron ferritin were in dimers stable to reduction and carboxylmethylation, 4% mercaptoethanol, 8% sodium dodecyl sulfate, and 100 degrees C for 30 min, 70% formic acid, and 30% methanol. Subunit dimers were also observed in liver ferritin from mouse and neonatal pig and were enriched in a low iron fraction of horse spleen ferritin. Based on cyanogen bromide fragmentation and NH2-terminal analysis, the natural and chemically cross-linked subunit dimers had two peptides in common; natural subunit dimers also appeared to have a second region cross-linked, suggesting the possibility of both intra- and intersubunit links in the natural dimers. In sheep spleen ferritin, both heavy and light subunits appeared to participate in subunit dimerization. Natural subunit dimers were enriched in low iron ferritin fractions of all ferritin preparations tested (linear correlation = 0.94) and can explain, at least in part, the previously observed effects of iron core size on the apoferritin shell. Whether the subunit cross-links represent part of the subunit assembly process subsequently cleaved by iron (or copper) or whether the cross-links form after iron core formation in vivo has yet to determined. In either case, it is clear that such post-translational variations can affect iron uptake and release and emphasize the importance of the protein shell in determining the iron storage properties of ferritin.     

Stoichiometry of Fe(II) oxidation during ceruloplasmin-catalyzed loading of ferritin.

Ceruloplasmin catalyzed the incorporation of iron into apoferritin with a stoichiometry of 3.8 Fe(II)/O2. This value remained the same when ferritin containing varying amounts of iron was used. Contrary to the "crystal growth" model for ferritin formation, no iron incorporation into holoferritin was observed in the absence of ceruloplasmin. Fe(II)/O2 ratios close to 2 were obtained for iron incorporation into apo- and holoferritin in Hepes buffer, in the absence of ceruloplasmin, indicating the formation of reduced oxygen species. Sequential loading of ferritin in this buffer resulted in increasing oxidation of the protein as measured by carbonyl formation. Sequential loading of ferritin using ceruloplasmin did not result in protein oxidation and a maximum of about 2300 atoms of iron were incorporated into rat liver ferritin. This corresponded to the maximum amount of iron found in rat liver ferritin in vivo after injection with iron. These results provide evidence for ceruloplasmin as an effective catalyst for the incorporation of iron into both apo- and holoferritin. The possibility that these findings may have physiological significance is discussed.     

Apolipoprotein B binds ferritin by hemin-mediated binding: evidence of direct binding of apolipoprotein B and ferritin to hemin

Apolipoprotein B (apoB) is known to be a ferritin-binding protein. Here we show that apoB binds to ferritin through hemin-mediated binding. Human apoB bound to bovine spleen, horse spleen, and canine liver ferritins, but did not bind to bovine apoferritin, even after incorporation of iron into it. Incubation of apoferritin with hemin resulted in apoB binding with apoferritin at the same level as with holoferritin. In contrast, hemin inhibited binding of apoB to ferritin. Bovine spleen apoferritin bound biotinylated hemin, and hemin inhibited the binding between the apoferritin and biotinylated hemin, suggesting that ferritin binds hemin directly. ApoB and LDL containing apoB bound biotinylated hemin, and their bindings were also inhibited by hemin, but not protoporphyrin IX. These data demonstrate that binding of apoB to ferritin is mediated through ferritin’s binding to hemin, and also that apoB binds hemin directly.     

The incorporation of iron into apoferritin as mediated by ceruloplasmin

Ceruloplasmin, a copper ferroxidase, promotes the incorporation of Fe(III) into the iron storage protein, apoferritin. The product formed is identical to ferritin as judged by polyacrylamide electrophoresis and iron/protein measurements. Of several proteins examined, only apoferritin accumulates the Fe(III) produced by ceruloplasmin. When ceruloplasmin was replaced by tyrosinase, which we have shown to have ferroxidase activity, no iron incorporation into apoferritin was observed. It is proposed that Fe(III) is transferred directly and specifically to apoferritin. These data support a more specific role for ceruloplasmin in iron metabolism than has previously been proposed.     

Effect of actinomycin D, cycloheximide, and acute blood loss on ferritin synthesis in rat liver

1. The mechanism of the stimulation of ferritin synthesis by iron in vivo has been studied in rat liver. Ferritin synthesis and turnover was measured by [(14)C]leucine incorporation. 2. Actinomycin D had no inhibitory effect, after administration of iron, on [(14)C]leucine incorporation into ferritin but appeared to augment the effect of iron on ferritin synthesis. 3. Cycloheximide completely abolished the stimulation by iron of [(14)C]leucine into ferritin and was subsequently utilized to show that iron acts in vivo by translational induction of apoferritin synthesis, rather than by stabilization of apoferritin or its precursors. 4. This conclusion was confirmed by showing that 2 days after acute bleeding, when iron was in the process of being removed from hepatic ferritin stores, ferritin synthesis was decreased whereas breakdown rates were unchanged.     

Interactions and aggregation of apoferritin molecules in solution: effects of added electrolytes

We have studied the structure of the protein species and the protein-protein interactions in solutions containing two apoferritin molecular forms, monomers and dimers, in the presence of Na(+) and Cd(2+) ions. We used chromatographic, and static and dynamic light scattering techniques, and atomic force microscopy (AFM). Size-exclusion chromatography was used to isolate these two protein fractions. The sizes and shapes of the monomers and dimers were determined by dynamic light scattering and AFM. Although the monomer is an apparent sphere with a diameter corresponding to previous x-ray crystallography determinations, the dimer shape corresponds to two, bound monomer spheres. Static light scattering was applied to characterize the interactions between solute molecules of monomers and dimers in terms of the second osmotic virial coefficients. The results for the monomers indicate that Na(+) ions cause strong intermolecular repulsion even at concentrations higher than 0.15 M, contrary to the predictions of the commonly applied Derjaguin-Landau-Verwey-Overbeek theory. We argue that the reason for such behavior is hydration force due to the formation of a water shell around the protein molecules with the help of the sodium ions. The addition of even small amounts of Cd(2+) changes the repulsive interactions to attractive but does not lead to oligomer formation, at least at the protein concentrations used. Thus, the two ions provide examples of strong specificity of their interactions with the protein molecules. In solutions of the apoferritin dimer, the molecules attract even in the presence of Na(+) only, indicating a change in the surface of the apoferritin molecule. In view of the strong repulsion between the monomers, this indicates that the dimers and higher oligomers form only after partial denaturation of some of the apoferritin monomers. These observations suggest that aggregation and self-assembly of protein molecules or molecular subunits may be driven by forces other than those responsible for crystallization and other phase transitions in the protein solution.     

In vitro loading of apoferritin.

This study compared the effect of loading apoferritin either with ferrous ammonium sulfate in various buffers or with ceruloplasmin and chelated ferrous iron. It was shown that loading of apoferritin with ferrous ammonium sulfate was dependent on buffer and pH, and was directly related to the rate of iron autoxidation. The ceruloplasmin-dependent loading of apoferritin, however, was unaffected by these factors. Isoelectric focusing and amino acid analysis of the differently loaded ferritins showed that ferrous ammonium sulfate loading of apoferritin resulted in the depletion of the basic amino acids, lysine and histidine, probably as a result of protein oxidation. No significant differences in amino acid composition was noted for ceruloplasmin-loaded ferritin. Furthermore, ferritin loaded with ferrous ammonium sulfate released more iron than either native or ceruloplasmin-loaded ferritin when either paraquat or EDTA was used as an iron mobilizing agent. We suggest that the loading of apoferritin with ferrous ammonium sulfate occurred as a result of iron autoxidation and may result in oxidation of amino acids and loss of integrity of the protein, and that ceruloplasmin may act as a catalyst for the incorporation of iron into apoferritin in a manner more closely related to that occurring in vivo.     

Hydrogen ion interactions of horse spleen ferritin and apoferritin.

The interactions of horse spleen ferritin and its derivative apoferritin with H+ ions were studied by potentiometric and spectrophotometric titration; to aid in data analysis, heats of ionization over a limited pH range and amide content were also determined. Per apoferritin subunit, all tyrosine and cysteine side chains, two of the nine lysine side chains and at least three of the six histidine side chains were found not to titrate; a preliminary but self-consistent analysis of the titration data is proposed. The titration curve of ferritin was identical with that of apoferritin in the pH range 5.5 to 3. In addition, under the conditions used, the reactivities of ferritin histidines to bromoacetate and of ferritin lysines to formaldehyde were identical with those in apoferritin. Above pH 8, a time-dependent titration of the ferritin core occurs which prevents comparison of the titration curves of the two proteins in this region. However, in the pH regions 5.5 to 7.5, two extra groups per subunit titrate reversibly in ferritin relative to apoferritin. Moreover, although the isoionic points of ferritin and apoferritin are identical in water, the isoionic point of ferritin is 0.5 pH unit lower than that of apoferritin in 0.16 to 1 M KCl. The different effects of KCl and NaCl on the two proteins indicate the presence of cation binding sites in ferritin that are absent in apoferritin and possibly also the presence of anion binding sites in apoferritin that are occupied in ferritin by anions of the core. The difference between the isoionic points of the two proteins in KCl has been interpreted to indicate the presence of approximately 2 phosphate residues per ferritin subunit which serve as cation binding sites and which are negatively charged at the isoionic point in KCl. These phosphates may also represent the additional residues that titrate in ferritin between pH 5.5 and 7.5, or may interact with positively charged residues on the inner surface of the ferritin shell, or both.     

The antigenic structure of ferritin. 1. Isolation of ferritin subunits and their immunochemical characteristics]

A method for the isolation of monomers of ferritin subunits has been developed. The procedure comprises dissociation of ferritin by treatment with thioglycolic acid in the presence of phosphate ions and subsequent gel-permeation chromatography. Ferritin and a number of its structural analogues (apoferritin, carboxymethylated ferritin, H- and L-subunits of ferritin) have been immunochemically characterized. The immunoreactivity of ferritin is shown to vary along with the degree of denaturation. Isolation of monomers of H- and L-subunits results in appearance of new antigenic sites. These "hidden" antigenic determinants are presumed to be localized in the regions of intersubunit contacts and intracapsular surface of the ferritin molecule and are responsible for the differences in immunochemical properties of its H- and L-subunits.     

Model system oxidations supporting the crystal growth model of ferritin iron uptake

Fe²⁺ is oxidized and taken up by ferritin or ápoferritin in the presence of dioxygen. Iodate causes Fe²⁺ oxidation and uptake by ferritin, but not by apoferritin. Synthetic iron polymer facilitates Fe²⁺ oxidation by either dioxygen or iodate. Nitrilotriacetic acid or iminodiacetic acid facilitate oxidation of Fe²⁺ by oxygen but not by iodate. These results support the crystal growth model of ferritin iron uptake, with iron polymer serving as a model for the ferritin core and aminocarboxylic acids mimicking the metal-binding sites of apoferritin.     

Biosynthesis of ferritin in rat hepatoma cells and rat livers. II. Binding of iron by ferritin protein.

Ferritin and its protein subunits in rat hepatoma cell clone M-5123-C1 were biosynthetically labeled with [14C]leucine and 59Fe. Radioimmunoassays of ferritin/apoferritin and of protein subunits in the free polyribosome, membrane-bound polyribosome, smooth membrane, and cytosol fractions were done with ferritin-specific and subunit-specific rabbit IgG antibodies at various time intervals after pulsing. Much more 59Fe was bound by ferritin/apoferritin than by subunits in all of the cell fractions. Binding of iron to subunits may have been a random process. When hepatoma cells were simultaneously pulse-labeled with 59Fe and [14C]leucine, uptake of much of the 59Fe by ferritin occurred relatively early, in comparison to incorporation of [14C]leucine, in all of the cell fractions examined. Thus, 59Fe was readily incorporated into pre-existing ferritin. We conclude that most, if not nearly all, of the iron is incorporated after assembly of protein subunits.     

pH-dependent structures of ferritin and apoferritin in solution: disassembly and reassembly

The pH-dependent structures of the ferritin shell (apoferritin, 24-mer) and the ferrihydrite core, under physiological conditions that permit enzymatic activity, were investigated by synchrotron small-angle X-ray scattering (SAXS). The solution structure of apoferritin was found to be nearly identical to the crystal structure. The shell thickness and hollow core volumes were estimated. The intact hollow spherical apoferritin was stable over a wide pH range, 3.40-10.0, and the ferrihydrite core was stable over the pH range 2.10-10.0. The apoferritin subunits underwent aggregation below pH 0.80, whereas the ferrihydrite cores aggregated below pH 2.10 as a result of the disassembly of the ferritin shell under the strongly acidic conditions. As the pH decreased from 3.40 to 0.80, apoferritin underwent stepwise disassembly by first forming a hollow sphere with two holes, then a headset-shaped structure, and, finally, rodlike oligomers. As the pH was increased from pH 1.96, the disassembled rodlike oligomers recovered only to the headset-shaped structure, and the disassembled headset-shaped intermediates recovered only to the hollow spherical structure with two hole defects. The apoferritin hole defects that formed during the disassembly process did not heal as the pH was increased to neutral or slightly basic conditions. The pH-induced apoferritin disassembly and reassembly processes were not fully reversible, although they were pseudoreversible over a limited pH range, between 10.0 and 2.66.     

Identification of catalytic residues involved in iron uptake by L-chain ferritins

When either horse spleen apoferritin (containing more than 90% of L chains) or recombinant horse L apoferritin are modified with glycineamide or taurine in the presence of a water-soluble carbodiimide, a total of 11 to 12 carboxyl groups per subunit are modified, and iron incorporation is effectively abolished. In contrast, when horse spleen ferritin (containing on average 2500 atoms per molecule) is modified under similar conditions, seven to eight carboxyl groups are modified. When apoferritin is prepared from this modified ferritin, it retains full iron incorporation activity. Apoferritin in which seven to eight carboxyls per subunit have been modified by glycineamide can subsequently be modified by taurine; a total of three to four carboxyl groups are modified accompanied by total loss of iron incorporation. Additional studies confirm that three carboxyl groups per subunit are protected from modification by glycineamide by Cr(III) inhibition of iron incorporation. Using tandem mass spectroscopy we have looked for taurine-labelled peptides in tryptic digests of succinylated apoferritins after taurine modification. In the sample where the residues involved in iron uptake have been modified with taurine, we have identified the peptide: This corresponds to residues 53–59 of the L subunit, where it is part of a region of the B-helix which is directed towards the inside of the apoferritin protein shell. The same peptide was identified using classical protein sequencing techniques after (1,2-³H)-taurine modification. We conclude that in L-chain apoferritins the Glu residues at positions 53, 56 and 57 are involved in the mechanism of iron incorporation. Glu 53 and 56 are conserved in L but not in H ferritins, and are located in close proximity to each other within the three-dimensional structure. There is ample room for rotation of Glu 57 to join with the other two to form an iron-binding site. This may represent a site of iron incorporation (most probably involving nucleation) unique to L-chain ferritins, and may explain the predominant L-chain involvement in conditions of iron overload.     

Control of crystal forms of apoferritin by site-directed mutagenesis

Surface charges of protein molecules are not only important to biological functions but also crucial to the molecular assembly responsible for crystallization. Appropriate alteration in the surface charge distribution of a protein molecule induces new molecular alignment in the proper direction in the crystal and, hence, controls the crystal form. Apoferritin molecules are known to crystallize in two- and three-dimensional forms in the presence of cadmium ions, which bridge neighboring protein molecules. Here we report a controlled transformation of the apoferritin 2-D crystal by site-directed mutagenesis. In mutant apoferritin, two amino acid residues binding a cadmium-ion through their negative charge, were replaced by one type of nonionic amino acid residues. The amino acid residues, Asp-84 and Gln-86 in the sequence of recombinant (i.e., wild-type) horse L -apoferritin, were replaced by Ser. The wild-type apoferritin yielded a hexagonal lattice 2-D crystal in the presence of cadmium ions. In contrast, the mutant apoferritin yielded two types of oblique crystals independent of the presence of cadmium ions. Image reconstruction of electron micrographs of the mutant crystals made clear that the mutant apoferritin molecules oriented themselves with the 2-fold symmetry axis perpendicular to the crystal plane in both crystals, while the wild-type apoferritin molecules oriented themselves with the 3-fold symmetry axis perpendicular to the crystal plane. The changes of crystal forms and molecular orientation in the 2-D crystals were well explained by a change of the electrostatic interactions induced by the mutagenesis. © 1995 Wiley-Liss, Inc.     

Isolation, purification and characterization of bovine spleen ferritin

Ferritin was isolated from bovine spleen and used to prepare apoferritin and reconstituted ferritin. The mol. wt of bovine ferritin was 464,000 with monomer subunits about 18,000-19,500. Gel electrophoresis showed three bands each for ferritin, apoferritin and reconstituted ferritin; all stained for protein and carbohydrate. Only apoferritin failed to stain for iron. Bovine ferritin had higher concentrations of proline, threonine, and valine than equine or human ferritin. The iron:protein ratio of bovine ferritin was 0.161 and of equine ferritin was 0.192. After iron uptake by the apoferritins the iron:protein ratios were 0.186 and 0.278 for the bovine and equine ferritins, respectively.     

Structure, surface charge, and self-assembly of the S-layer lattice from Bacillus coagulans E38-66.

In freeze-etched preparations, whole cells from Bacillus coagulans E38-66 exhibited an oblique S-layer lattice (a = 9.4 nm; b = 7.4 nm; gamma = 80 degrees). The three-dimensional structure of the crystalline array was characterized by optical and computer image analysis. The lattice showed two distinctly shaped types of pores. In vitro self-assembly of isolated subunits yielded flat sheets and open-ended cylinders composed of two back-to-back monolayers. Unlike whole cells, in vitro self-assembly products were capable of binding polycationized ferritin (pI, approximately 11). This showed that only the inner S-layer face adhering to the peptidoglycan-containing layer in whole cells was net negatively charged. S-layer monomers and/or oligomers were capable of generating a closed monolayer with oblique symmetry on poly-L-lysine-coated supports. The monolayer had a typical crazy paving appearance, with numerous crystal boundaries. The handedness of the oblique lattice and ability to bind polycationized ferritin revealed that the subunits had bound with the outer, not net negatively charged face to the poly-L-lysine-coated supports. Carbodiimide-activated carboxyl groups on either cell wall fragments or self-assembly products could covalently bind high-molecular-weight nucleophiles such as ferritin. This confirmed the location of negatively charged carboxyl groups on the outermost surface of both S-layer faces. The difference in pH optimum for carbodiimide activation indicated a preponderance of alpha- and beta-carboxyl groups on the inner S-layer face and a preponderance of beta- and gamma-carboxyl groups on the outer S-layer face.