Translational regulation of ferritin synthesis in rat liver. Effects of chronic dietary iron overload.

In rats with chronic dietary iron overload, a higher amount of liver ferritin L-subunit mRNA was found mainly engaged on polysomes, whereas in control rats ferritin L-subunit mRNA molecules were largely stored in ribonucleoprotein particles. On the other hand, ferritin H-subunit mRNA was unchanged by chronic iron load and remained in the inactive cytoplasmic pool. In agreement with previous reports, in rats acutely treated with parenteral iron, only the ferritin L-subunit mRNA increased in amount, whereas both ferritin subunit mRNAs shifted to polysomes. This may indicate that, whereas in acute iron overload the hepatocyte operates a translation shift of both ferritin mRNAs to confront rapidly the abrupt entry of iron into the cell, during chronic iron overload it responds to the slow iron influx by translating a greater amount of L-subunit mRNA to synthesize isoferritins more suitable for long-term iron storage.     

Conservation in rat liver of light and heavy subunit sequences of mammalian ferritin. Presence of unique octopeptide in the light subunit

Ferritin, an iron-storage protein found in all life forms examined, is composed of varying proportions of two subunits of different molecular weight, heavy (H) and light (L). Using cDNA clones, we have determined the nucleotide sequence corresponding to the mRNA of the L-subunit of rat liver ferritin. The coding region of 546 nucleotides (182 amino acids) is flanked by 5'- and 3' -untranslated regions of approximately 130 and 150 nucleotides, respectively. The rat liver L-subunit amino acid sequence derived from the reading frame of the cDNA showed 88% and 82% homology, respectively, with the amino acid sequences of horse spleen ferritin (Heusterspreute, M., and Crichton, R. R. (1981) FEBS Lett. 129, 322-327), and human spleen ferritin (Wustefeld, C., and Crichton, R. R. (1982) FEBS Lett. 150, 43-48), thus demonstrating evolutionary conservation of the L-subunit sequence. However, a major difference between the rat and the horse and human sequences is the insertion of an octopeptide near the COOH-terminus of the rat protein resulting in a slightly longer peptide chain in this species. The reading frame and parts of the derived amino acid sequence including the octopeptide sequence were confirmed by direct amino acid sequencing of cyanogen bromide peptides from rat liver ferritin. Minor fragments of rat liver ferritin, presumably derived from the H-subunit, were also isolated after cyanogen bromide treatment. On sequencing, these H-peptides showed limited homology with regions of the L-sequence but extensive homology with published H-sequences from human liver and spleen. The H-subunit sequence did not contain the octopeptide found as part of the L-subunit sequence.     

Characterization and evolution of the expressed rat ferritin light subunit gene and its pseudogene family. Conservation of sequences within noncoding regions of ferritin genes

The iron storage protein ferritin consists of two types of subunits of different molecular weight, heavy (H) and light (L). The rat genome contains approximately 20 copies of the ferritin L-subunit gene, of which we have sequenced seven. One is an expressed ferritin gene containing three introns located between the alpha-helical domains of the L-subunit protein. The remaining six have the characteristics of processed pseudogenes. Sequence divergence suggest that these pseudogenes arose approximately 3-12 X 10(6) years ago, well within the 30 X 10(6) years of divergence of rat and mouse. By using intron probes derived from the expressed ferritin L-gene, a homologous second copy has been identified in some Fischer rats. Comparison of the 5'-untranslated region of the rat L-gene with the published sequences of this region of the human L (Santoro, C., Marone, M., Ferrone, M., Costanzo, F., Colombo, M., Minganti, C., Cortese, R., and Silengo, L. (1986) Nucleic Acids Res. 14, 2863-2876) and H (Costanzo, F., Colombo, M., Staempfli, S., Santoro, C., Marone, M., Frank, R., Delius, H., and Cortese, R. (1986) Nucleic Acids Res. 14, 721-735) genes and of a bullfrog cDNA (Didsbury, J. R., Theil, E. C., Kaufman, R. E., and Dickey, L. F. (1986) J. Biol. Chem. 261, 949-955) show a strongly conserved 28-base pair sequence, suggesting a translational regulatory function. The 5' flanking region of the rat L-gene contains sequences homologous to those in the flanking areas of the human L- and H-genes. The implications of these conserved sequences for control of ferritin expression are discussed.     

Crosslinks between intramolecular pairs of ferritin subunits: effects on both H and L subunits and on immunoreactivity of sheep spleen ferritin

Ferritin is a multisubunit protein, controlling iron storage, with a protein coat composed of 24 subunits (up to three distinct types) in different proportions depending on cell type. Little is known about the subunit interactions in ferritin protein coats composed of heterologous subunits, despite the relevance to ferritin structure and ferritin function (iron uptake and release). Synthetic crosslinking is a convenient way to probe subunit contacts. Crosslinks between subunit pairs in ferritin protein coats are also a natural post-translational modification which coincides with different iron content in ferritin from sheep spleen; ferritin from sheep spleen also contains H and L subunits. Crosslinks synthesized by the reaction of ferritin low in natural crosslinks with difluorodinitrobenzene (F2DNB) reproduced the effects of the natural crosslinks on iron uptake and release. We now extend our observations on the structural effects of natural and synthetic crosslinks to include immunoreactivity of the assembled protein, with monoclonal antibodies as a probe. We also demonstrate, for the first time, ferritin peptides involved in an apparent H- and L-subunit contact: two peptides decreased 4X in cyanogen bromide peptide maps after F2DNB crosslinking were residues L-96-138 and H-66-96; the major DNP-dipeptide was Lys-DNP-Lys. Using the structure of an all L-subunit ferritin as a model, the most likely site for the H-L DNP crosslink is L-Lys 104 (C helix) and H-Lys 67 (B helix). The B helix forms the internal subunit dimer interface, a putative site of iron core nucleation. Alteration by crosslinks of the B helix could, therefore, explain the effect of crosslinks on ferritin iron uptake, release, and iron content.     

Molecular size heterogeneity of ferritin in mouse liver

As much as 4% of the total protein in pure liver ferritin from mice with short-term parenteral iron overload produces a minor band migrating anodally to the major (alpha) band of holoferritin with non-denaturing polyacrylamide gel electrophoresis. The components in this minor band and the alpha band have been isolated to purity by preparative electrophoretic fractionation. The protein in the minor band is ferritin, since it contains ferric iron and fulfills defining criteria at the level of biochemistry, immunology and ultrastructure. Native polyacrylamide electrophoresis with pore-size-gradient gels shows that the ferritin molecules in the minor band have a slightly smaller diameter than the holoferritin in the alpha band. Isoelectric focusing reveals that the smaller ferritin has an identical number and range of charge isomers (pI 4.9-5.3) as the larger ferritin, but the relative amount of each size class within some isoferritin bands differs. The smaller ferritin molecules are structurally intact and are made from polypeptide subunits with Mr 18 000; the larger ferritin molecules have subunits with Mr 22 000. The minor species of hepatic ferritin thus has a smaller molecular size because it is made mainly from smaller subunits. No minor electrophoretic band can be detected in liver ferritin obtained from mice with normal iron levels. These results demonstrate that siderosis induces the formation of molecular size polymorphism (macroheterogeneity) in mouse liver ferritin. The new smaller hepatic ferritin could serve to redistribute excess iron into the main storage organs during the early response to iron overload, since it appears to be identical to one of the two types of serum ferritin molecules present in these siderotic mice.     

Siderosomal ferritin. The missing link between ferritin and haemosiderin?

A minor electrophoretically fast component was found in ferritin from iron-loaded rat liver in addition to a major electrophoretically slow ferritin similar to that observed in control rats. The electrophoretically fast ferritin showed immunological identity with the slow component, but on electrophoresis in SDS it gave a peptide of 17.3 kDa, in contrast with the electrophoretically slow ferritin, which gave a major band corresponding to the L-subunit (20.7 kDa). Thus the electrophoretically fast ferritin resembles that reported by Massover [(1985) Biochim. Biophys. Acta 829, 377-386] in livers of mice with short-term parenteral iron overload. The electrophoretically fast ferritin had a lower iron content (2000 Fe atoms/molecule) than the electrophoretically slow ferritin (3000 Fe atoms/molecule). Removal and re-incorporation of iron was possible without effect on the electrophoretic mobility of either ferritin species. On subcellular fractionation the electrophoretically fast ferritin was enriched in pellet fractions and was the sole soluble ferritin isolated from iron-laden secondary lysosomes (siderosomes). The amount and relative proportion of the electrophoretically fast species increased with iron loading. Haemosiderin isolated from siderosomes was found to contain a peptide reactive to anti-ferritin serum and corresponding to the 17.3 kDa peptide of the electrophoretically fast ferritin species. Unlike the electrophoretically slow ferritin, the electrophoretically fast ferritin did not become significantly radioactive in a 1 h biosynthetic labelling experiment. We conclude that the minor ferritin is not, as has been suggested for mouse liver ferritin, 'a completely new species of smaller holoferritin that represents a shift in the ferritin phenotype' in response to siderosis, but a precursor of haemosiderin, in agreement with the proposal by Richter [(1984) Lab. Invest. 50, 26-35] concerning siderosomal ferritin.     

Characterization and localization of human placental ferritin.

Ferritin has been purified from normal full-term human placentae and its antigenic and molecular characteristics compared with adult liver ferritin. Placental ferritin is composed predominantly of a single subunit type, co-migrating with a liver ferritin standard on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Comparison of dose-response curves in an immunoradiometric assay indicated some tissue-specific antigenicity for placental ferritin. This was supported by immunofluorescence studies on cryostat sections of human placentae by using antibodies to placental and spleen ferritin. Specific staining for placental ferritin was demonstrated within placental syncytiotrophoblast, particularly localized towards the microvillus plasma membrane. Ferritin has also been shown by electrophoretic and antigenic analysis to be present in protein fractions solubilized from isolated human syncytiotrophoblast microvillus plasma-membrane preparations, suggesting that ferritin may play an active role in the transfer of iron from maternal transferrin across the syncytiotrophoblast plasma membrane.     

Cleavage of high-molecular-weight kininogen by elastase and tryptase is inhibited by ferritin

Ferritin is a protein principally known for its role in iron storage. We have previously shown that ferritin can bind high-molecular-weight kininogen (HK). Upon proteolytic cleavage by the protease kallikrein, HK releases the proinflammatory peptide bradykinin (BK) and other biologically active products, such as two-chain high-molecular-weight kininogen, HKa. At inflammatory sites, HK is oxidized, which renders it a poor substrate for kallikrein. However, oxidized HK remains a good substrate for elastase and tryptase, thereby providing an alternative cleavage mechanism for HK during inflammation. Here we report that ferritin can retard the cleavage of both native HK and oxidized HK by elastase and tryptase. Initial rates of cleavage were reduced 45-75% in the presence of ferritin. Ferritin is not a substrate for elastase or tryptase and does not interfere with the ability of either protease to digest a synthetic substrate, suggesting that ferritin may impede HK cleavage through direct interaction with HK. Immunoprecipitation and solid phase binding studies reveal that ferritin and HK bind directly with a Kd of 134 nM. To test whether ferritin regulates HK cleavage in vivo, we used THP-1 cells, a human monocyte/macrophage cell line that has been used to model pulmonary inflammatory cells. We observed that ferritin impedes the cleavage of HK by secretory proteases in stimulated macrophages. Furthermore, ferritin, HK, and elastase are all present in or on alveolar macrophages in a mouse model of pulmonary inflammation. Collectively, these results implicate ferritin in the modulation of HK cleavage at sites of inflammation.     

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.     

Evidence for the presence of ferritin in plant mitochondria.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.     

Identification of a 250 kDa putative microtubule-associated protein as bovine ferritin. Evidence for a ferritin-microtubule interaction

We reported previously on the purification and partial characterization of a putative microtubule-associated protein (MAP) from bovine adrenal cortex with an approximate molecular mass of 250 kDa. The protein was expressed ubiquitously in mammalian tissues, and bound to microtubules in vitro and in vivo, but failed to promote tubulin polymerization into microtubules. In the present study, partial amino acid sequencing revealed that the protein shares an identical primary structure with the widely distributed iron storage protein, ferritin. We also found that the putative MAP and ferritin are indistinguishable from each other by electrophoretic mobility, immunological properties and morphological appearance. Moreover, the putative MAP conserves the iron storage and incorporation properties of ferritin, confirming that the two are structurally and functionally the same protein. This fact led us to investigate the interaction of ferritin with microtubules by direct electron microscopic observations. Ferritin was bound to microtubules either singly or in the form of large intermolecular aggregates. We suggest that the formation of intermolecular aggregates contributes to the intracellular stability of ferritin. The interactions between ferritin and microtubules observed in this study, in conjunction with the previous report that the administration of microtubule depolymerizing drugs increases the serum release of ferritin in rats [Ramm GA, Powell LW & Halliday JW (1996) J Gastroenterol Hepatol11, 1072-1078], support the probable role of microtubules in regulating the intracellular concentration and release of ferritin under different physiological circumstances.     

Isolation and characterization of Phycomyces blakesleeanus ferritin.

Ferritin was isolated from the fungus Phycomyces blakesleeanus and compared biochemically and immunologically with horse spleen ferritin. Phycomyces and horse spleen ferritins were shown to exhibit similar electrophoretic patterns on polyacrylamide gels. Both preparations yielded an identical single band on sodium dodecyl sulfate-containing polyacrylamide gels. Tryptic digests of Phycomyces ferritin yielded 17 ninhydrin-positive spots as compared to 26 for horse spleen ferritin tryptic digests. Phycomyces ferritin was immunologically unrelated to horse spleen ferritin.     

Formation of the ferritin iron mineral occurs in plastids.

Ferritin in plants is a nuclear-encoded, multisubunit protein found in plastids; an N-terminal transit peptide targets the protein to the plastid, but the site for formation of the ferritin Fe mineral is unknown. In biology, ferritin is required to concentrate Fe to levels needed by cells (approximately 10(-7) M), far above the solubility of the free ion (10(-18) M); the protein directs the reversible phase transition of the hydrated metal ion in solution to hydrated Fe-oxo mineral. Low phosphate characterizes the solid-phase Fe mineral in the center of ferritin of the cytosolic animal ferritin, but high phosphate is the hallmark of Fe mineral in prokaryotic ferritin and plant (pea [Pisum sativum L.] seed) ferritin. Earlier studies using x-ray absorption spectroscopy showed that high concentrations of phosphate present during ferritin mineralization in vivo altered the local structure of Fe in the ferritin mineral so that it mimicked the prokaryotic type, whether the protein was from animals or bacteria. The use of x-ray absorption spectroscopy to analyze the Fe environment in pea-seed ferritin now shows that the natural ferritin mineral in plants has an Fe-P interaction at 3.26A, similar to that of bacterial ferritin; phosphate also prevented formation of the longer Fe-Fe interactions at 3.5A found in animal ferritins or in pea-seed ferritin reconstituted without phosphate. Such results indicate that ferritin mineralization occurs in the plastid, where the phosphate content is higher; a corollary is the existence of a plastid Fe uptake system to allow the concentration of Fe in the ferritin mineral.     

Both subunits of rat liver ferritin are regulated at a translational level by iron induction.

A few hours after administering iron to rats, liver ferritin synthesis increases several fold. However, Northern blot analysis with cDNA probes for ferritin light (L) and heavy (H) subunit mRNAs failed to show an increase in total population of either messenger. Cytoplasmic distribution of ferritin messages was therefore investigated in control and iron administered rats killed at 3.5 hours. The liver post-mitochondrial supernatant was fractionated on a sucrose gradient to separate polyribosomes, monosomes, ribosomal subunits and cell sap. RNA extracted from each fraction and analyzed using Northern blotting showed that 65% of the total mRNA population for each subunit was present in the cell sap of control rats, presumably as mRNP particles since ribosomal RNA was absent from this fraction. After iron administration, these reserves of free mRNA were recruited onto the polysomes, reducing the free mRNA pool to 15% of the total. We interpret this to be due to activation of blocked ferritin messages on entry of iron into the cell.     

Computer prediction of peptide maps: assignment of polypeptides to human and mouse mitochondrial DNA genes by analysis of two-dimensional-proteolytic digest gels.

We have prepared a computer program that predicts complete and partial peptide maps from amino acid sequences. The program fragments amino acid sequences at designated cleavage sites and calculates the molecular weight and relative labeling of each peptide. These data are graphed as log molecular weight of the original protein (X-axis) vs. log molecular weight of the component peptides (Y-axis). The program is interactive, permitting adjustment of a number of graphic parameters and alteration of the position of proteins in the first dimension to accommodate aberrations in protein mobility. The program has been used to predict the V8 protease peptide maps of the 13 open reading frames (ORFs) identified in the human and the mouse mitochondrial DNA (mtDNA) sequences. The results were compared to the V8 protease peptide maps obtained for mouse and human mitochondrially synthesized proteins by two-dimensional proteolytic digest gels. A high correlation was observed between the predicted and observed peptide maps. These results suggest the assignment of several proteins to mtDNA genes.     

Oxidative damage of DNA induced by the reaction of methylglyoxal with lysine in the presence of ferritin

Methylglyoxal (MG) is an endogenous metabolite which is present in increased concentrations in diabetics and reacts with amino acids to form advanced glycation end products. In this study, we investigated whether ferritin enhances DNA cleavage by the reaction of MG with lysine. When plasmid DNA was incubated with MG and lysine in the presence of ferritin, DNA strand breakage was increased in a dose-dependent manner. The ferritin/MG/lysine system-mediated DNA cleavage was significantly inhibited by reactive oxygen species (ROS) scavengers. These results indicated that ROS might participate in the ferritin/MG/lysine system-mediated DNA cleavage. Incubation of ferritin with MG and lysine resulted in a time-dependent release of iron ions from the protein molecules. Our data suggest that DNA cleavage caused by the ferritin/MG/lysine system via the generation of ROS by the Fenton-like reaction of free iron ions released from oxidatively damaged ferritin. [BMB Reports 2013; 46(4): 225-229]     

Association of ferritin with liver cell membrane fractions.

Following intraperitoneal injection of rats with a large dose of ferric ammonium citrate containing ⁵⁹Fe, some 35 to 50% of the dose was deposited in the liver within the first 1 to 4 h. Almost all of the deposited iron could be precipitated with a ferritin antiserum from a homogenate of liver heated to 75 °C, but only half of this was precipitable when the unheated homogenate was treated with antiserum. The remainder of the ferritin iron was made available to the antiserum by treatment with deoxycholate, and was therefore presumed to be associated with membranous components of the cell.Subcellular fractionation of the liver following administration of ⁵⁹Fe-labeled ferric ammonium citrate showed that most of the radioactivity deposited within the first 4 h was equally divided between the cell sap and a light microsome (membrane-rich) fraction. Ferritin in this latter fraction was made available to antibody following deoxycholate treatment. The liver microsome fraction of the young rat contains little unavailable ferritin, but with aging there is an accumulation of ferritin in the microsomal fraction which is unavailable to antibody until the membrane is removed.It is suggested that at least part of the injected iron salt is taken up by pinocytotic vesicles and transferred to ferritin within this fraction, possibly followed by release of some of this ferritin into the cell sap.     

Cationic ferritin binding sites and surface charge densities of transformed cells

Bioelectric surface properties of the high and low tumor-producing cell lines, NCTC 2472 and NCTC 2555, respectively, were determined by cationic ferritin binding and the electrophoretic mobility of intact cells. Measurements of anionic sites were bases on the number of cationic ferritin particles per 0.01 mu 2 that were electronically tagged and counted by an image analyzer. The average particle count was 45 for the control "high" cells and 34 for the control "low" cells. The surface charge densities, expressed as electrostatic units per cm-2 x 10(-13) were 2.34 and 1.18 at 50 V (2 mA) for the "high" and "low" control cells, respectively. Enzymic cleavage of sialic acid and other carbohydrate moieties resulted in up to an 81% reduction in the charge densities and a 57% reduction of the anionic sites of the "high" cells. The electrophoretic mobility of cells with bound cationic ferritin showed that up to 50% of the exposed anionic sites fail to bind cationic ferritin. Preliminary findings on the particle size/distribution by image analysis showed wide ranges in both particle size and interparticle distances that may limit cationic ferritin binding.     

Proteolytic dissection of rat brain hexokinase: determination of the cleavage pattern during limited digestion with trypsin

Limited treatment of rat brain hexokinase (ATP: D-hexose-6-phosphotransferase; EC 2.7.1.1) with trypsin causes cleavage of the Mr 98K enzyme into three major fragments having molecular weights of 10K, 40K, and 50K, with intermediates of Mr 60K and 90K being detected. This information, in conjunction with N- and C-terminal analysis of the intact enzyme and tryptic cleavage products, has established the tryptic cleavage pattern as where T1 and T2 indicate tryptic cleavage sites; cleavage at only T1 or T2 gives rise to the 90K or 60K intermediate, respectively. Confirmation of this cleavage pattern has been provided by two-dimensional peptide mapping using Staphylococcus aureus V8 protease, and epitope mapping with two monoclonal antibodies directed against rat brain hexokinase. The epitopes recognized by one of the monoclonal antibodies is located within the 40K C-terminal fragment while the epitope for the other monoclonal antibody lies within the 50K fragment. A two-dimensional peptide mapping-immunoblotting technique has permitted a more defined localization of these epitopes to specific regions within these major tryptic cleavage fragments. Complete tryptic cleavage of the enzyme occurs with only modest (approximately 20%) loss of catalytic activity, and the cleaved enzyme retains many of the properties of intact hexokinase. Specifically, there was no effect of cleavage on the Km for Glc or the Ki for Glc-6-P, though a slight decrease in Km for ATP was consistently noted to result from cleavage. Furthermore, like the intact enzyme, cleaved hexokinase retained the ability to bind to outer mitochondrial membranes in a Glc-6-P-sensitive manner. Under nondenaturing conditions, the cleaved fragments remain associated by noncovalent forces. Thus, the cleaved enzyme sedimented at a rate comparable to intact enzyme during centrifugation on sucrose density gradients, and migrated only slightly faster when electrophoresed on gradient acrylamide gels under nondenaturing conditions.