Human Heart Ferritin Research Articles

On the structure and chemistry of iron oxide cores in human heart and human spleen ferritins using graphene liquid cell electron microscopy.

Ferritin is a protein that regulates the iron ions in humans by storing them in the form of iron oxides. Despite extensive efforts to understand the ferritin iron oxide structures, it is still not clear how ferritin proteins with a distinct light (L) and heavy (H) chain subunit ratio impact the biomineralization process. In situ graphene liquid cell-transmission electron microscopy (GLC-TEM) provides an indispensable platform to study the atomic structure of ferritin mineral cores in their native liquid environment. In this study, we report differences in the iron oxide formation in human spleen ferritins (HSFs) and human heart ferritins (HHFs) using in situ GLC-TEM. Scanning transmission electron microscopy (STEM) along with selected area electron diffraction (SAED) of the mineral core and electron energy loss spectroscopy (EELS) analyses enabled the visualization of morphologies, crystal structures and the chemistry of iron oxide cores in HSFs and HHFs. Our study revealed the presence of metastable ferrihydrite (5Fe2O3·9H2O) as a dominant phase in hydrated HSFs and HHFs, while a stable hematite (α-Fe2O3) phase predominated in non-hydrated HSFs and HHFs. In addition, a higher Fe3+/Fe2+ ratio was found in HHFs in comparison with HSFs. This study provides new understanding on iron-oxide phases that exist in hydrated ferritin proteins from different human organs. Such new insights are needed to map ferritin biomineralization pathways and possible correlations with various iron-related disorders in humans.

Elucidation of Structure and Chemistry of Iron Core in Human Heart Ferritin via Graphene Liquid Cell

Elucidation of Structure and Chemistry of Iron Core in Human Heart Ferritin via Graphene Liquid Cell

Abstract 84: The L-ferritin receptor, Scara5, is expressed on breast cancer and glioma cells: A potential functional significance for cancer-associated high serum/CSF levels

Abstract Elevated levels of circulating ferritin have been associated with several neoplasms, including neuroblastoma, glioblastoma, Hodgkin's lymphoma, and breast cancer. Despite the prognostic value of serum ferritin levels, its role in cancer biology is not understood. Ferritin has long been considered exclusively as an intracellular iron storage protein. However, several lines of evidence have suggested a role for ferritin beyond iron storage, such as angiogenesis, iron delivery, and modulation of the immune response. Recently, a receptor for L-ferritin (the predominant ferritin subunit in the serum and cerebrospinal fluid) has been identified as scavenger receptor class A, member 5 (Scara5). Our preliminary data show Scara5 to be expressed in glioma, neuroblastoma, and breast cancer cells. This novel observation led us to hypothesize that Scara5 internalizes L-ferritin, and serves as a non-transferrin iron delivery mechanism in cancer cells. In support of this hypothesis, we report here that both human liver and human heart ferritins increased cellular survival of the breast cancer cell line MCF-7 in serum free conditions. Taken together, these observations suggest an active functional role for circulating ferritin in tumor biology. The elevated levels of circulating ferritin observed in some cancers may enhance tumor metabolism and growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 84.

Oxidative damage to ferritin by 5-aminolevulinic acid

5-Aminolevulinic acid (ALA), a heme precursor overproduced in various porphyric disorders, has been implicated in iron-mediated oxidative damage to biomolecules and cell structures. From previous observations of ferritin iron release by ALA, we investigated the ability of ALA to cause oxidative damage to ferritin apoprotein. Incubation of horse spleen ferritin (HoSF) with ALA caused alterations in the ferritin circular dichroism spectrum (loss of a α-helix content) and altered electrophoretic behavior. Incubation of human liver, spleen, and heart ferritins with ALA substantially decreased antibody recognition (51, 60, and 28% for liver, spleen, and heart, respectively). Incubation of apoferritin with 1–10 mM ALA produced dose-dependent decreases in tryptophan fluorescence (11–35% after 5 h), and a partial depletion of protein thiols (18% after 24 h) despite substantial removal of catalytic iron. The loss of tryptophan fluorescence was inhibited 35% by 50 mM mannitol, suggesting participation of hydroxyl radicals. The damage to apoferritin had no effect on ferroxidase activity, but produced a 61% decrease in iron uptake ability. The results suggest a local autocatalytic interaction among ALA, ferritin, and oxygen, catalyzed by endogenous iron and phosphate, that causes site-specific damage to the ferritin protein and impaired iron sequestration. These data together with previous findings that ALA overload causes iron mobilization in brain and liver of rats may help explain organ-specific toxicities and carcinogenicity of ALA in experimental animals and patients with porphyria.

Growth stimulation of ferritin of human leukemia cells in vitro.

Ferritin has been reported to inhibit the growth of some leukemia cells in serum-supplemented culture. Recently we have found that ferritin stimulates the proliferation of human acute myeloblastic leukemia cells HL-60 and erythroleukemia cells K-562-T1 in serum-free medium. In this study, we examined the effect of ferritin against 14 human leukemia cell lines using human heart ferritin in serum-depleted culture medium. Among 14 cell lines tested, 10 were stimulated to proliferate by ferritin (maximum response at 30-300 ng/ml) with 0-1% fetal calf serum (FCS). The growth of all the cell lines was significantly inhibited by ferritin in the presence of 10% FCS. These results suggest that ferritin has dual functions; it promotes the growth of leukemia cells with low concentrations of FCS, but suppresses their growth with high concentrations of FCS.

Purification of a cell growth factor from a human lung cancer cell line: Its relationship with ferritin

We have purified a cell growth factor from a human lung cancer cell line, T3M-30, which was established in a protein-free chemically defined medium. The factor, designated carcinoma-derived growth factor (CD-GF), stimulated proliferation of a variety of cells, including human leukemia cells, HL-60, and melanoma cells, SK-28. Half-maximum stimulation by the purified CD-GF was achieved at a concentration of 40 ng/ml. In the purified CD-GF, two major protein bands of 24 kDa and 22 kDa were identified on a SDS polyacrylamide gel. The partial amino acid sequences of the 24 kDa protein were determined from two peptide fragments obtained by V8 protease treatment. The partial sequences were identical to those of heavy chain of human ferritin. The activity of the purified CD-GF was coprecipitated completely with a monoclonal antibody to heavy chain of ferritin. Ferritin has been considered to inhibit cell growth. However, human heart ferritin was capable of stimulating the growth of HL-60 cells. These results suggest that CD-GF is related to ferritin and ferritin is a growth factor of HL-60 leukemia cells.

Identification and characterization of a receptor for tissue ferritin on activated rat lipocytes.

Hepatic iron overload causes lipocyte activation with resultant fibrogenesis. This study examines whether rat lipocytes express ferritin receptors, which could be involved in paracellular iron movement and in cellular regulation. Lipocytes from normal rat liver were cultured on plastic and incubated with 125I-labeled rat liver ferritin (RLF) +/- a 100-fold excess of either unlabeled RLF or human heart ferritin, human liver ferritin, human recombinant H-ferritin, a mutant human recombinant L-ferritin, or a variety of nonspecific proteins. Specific binding sites for ferritin were demonstrated by displacement of 125I-RLF by RLF (64.5 +/- 4.3%) and by other ferritins (55-60%), but not by recombinant L-ferritin. Scatchard analysis demonstrated a single class of binding sites with a Kd of 5.1 +/- 2.9 x 10(-10) M, maximum binding capacity of 4.7 +/- 1.3 x 10(-12) M, and 5,000-10,000 receptor sites/cell. Ferritin receptor expression was observed only in activated lipocytes. Internalization of RLF was observed within 15 min using FITC-RLF and confocal microscopy. This study demonstrates that (a) activated lipocytes express a specific high affinity ferritin receptor; (b) the binding appears to be dependent on the H-ferritin subunit; and (c) lipocytes internalize ferritin. Expression of ferritin receptors in activated lipocytes suggests that the receptor may either be involved in the activation cascade or may be a marker of activation.

H- and L-Rich Ferritins Suppress Antibody Production, But Not Proliferation, of Human B Lymphocytes In Vitro

AbstractThe effect of human spleen(L-rich) and heart(H-rich) ferritins on the proliferation and differentiation of human B lymphocytes was studied in comparison with that of holo- and apo-transferrins. Ferritins rich in H and L chain, as well as the transferrins, did not inhibit the proliferative response of resting and activated B cells stimulated with polyclonal B-cell mitogen. Staphylococcus aureus Cowan strain I. In contrast, the ferritins, but not the transferrins, clearly suppressed the antibody production by B blasts in T-cell-inde-pendent as well as T-cell-dependent system. Kinetic study showed that inhibitory action of ferritins on immunoglobulin (Ig) production was caused at an early stage of B-cell differentiation. The cytoplasmic Ig-containing cells decreased in proportion to the reduction of Ig secretion. The evidence that ferritin inhibited Ig synthesis of Epstein-Barr virus-transformed human B-lymphoblastoid cell line also supported the idea that the effect of ferritin was directed toward the antibody-producing B lymphocytes. The molecular analysis showed that the inhibitory effect of ferritin was regulated at the transcriptional level of the Ig generation signal. Our results suggest that H- and L-rich ferritins exert their inhibitory action on the differentiation of B cells maturing into Ig-producing cells.

H- and L-rich ferritins suppress antibody production, but not proliferation, of human B lymphocytes in vitro

The effect of human spleen(L-rich) and heart(H-rich) ferritins on the proliferation and differentiation of human B lymphocytes was studied in comparison with that of holo- and apo-transferrins. Ferritins rich in H and L chain, as well as the transferrins, did not inhibit the proliferative response of resting and activated B cells stimulated with polyclonal B-cell mitogen, Staphylococcus aureus Cowan strain I. In contrast, the ferritins, but not the transferrins, clearly suppressed the antibody production by B blasts in T-cell-independent as well as T- cell-dependent system. Kinetic study showed that inhibitory action of ferritins on immunoglobulin (Ig) production was caused at an early stage of B-cell differentiation. The cytoplasmic Ig-containing cells decreased in proportion to the reduction of Ig secretion. The evidence that ferritin inhibited Ig synthesis of Epstein-Barr virus-transformed human B-lymphoblastoid cell line also supported the idea that the effect of ferritin was directed toward the antibody-producing B lymphocytes. The molecular analysis showed that the inhibitory effect of ferritin was regulated at the transcriptional level of the Ig generation signal. Our results suggest that H- and L-rich ferritins exert their inhibitory action on the differentiation of B cells maturing into Ig-producing cells.

Neutralization of inhibitory activity derived from cells of MDS patients and ML-2 cell line by monoclonal antibodies against H and L-subunit-rich ferritins

Monoclonal antibody 2A4 against human heart ferritin and a polyclonal antibody against human placental ferritin inactivated the suppressor activity present in media conditioned by cells of patients with myelodysplastic syndrome (MDS). These antibodies also neutralized the inhibitory activity released by cells of myeloid leukemic ML-2 cell line. In contrast, a monoclonal antibody against the ferritin L subunit did not neutralize the inhibitory activity of the media conditioned either by the cells of patients with MDS or by the ML-2 cell line. Neutralization of the inhibitory activity present in supernatants of cells of MDS patients and the ML-2 cell line by monoclonal antibodies against the H subunit of ferritin strongly indicates that the inhibitory activity resides in the acidic isoferritin molecule.

Interaction of acidic isoferritins with human promyelocytic HL60 cells.

We have used undifferentiated human promyelocytic HL60 cells to study the binding of radioiodinated human ferritin in vitro. Specific binding of human heart ferritin could be demonstrated at 37 degrees C, whereas no binding of liver ferritin could be found. The uptake of labelled heart ferritin was abolished by incubation at 4 degrees C, by prior treatment of the HL60 cells with pronase and by the addition of human plasma to the medium. On the other hand, the addition of excess unlabelled human liver or rat liver ferritin had no effect on the uptake of labelled human heart ferritin. Dissociation studies showed that about 55% of the bound heart ferritin radioactivity could be released by incubation with medium alone and at least 90% with excess unlabelled heart ferritin. Over 70% of the dissociated ferritin could be precipitated with polyclonal anti-ferritin serum or trichloroacetic acid. More than two-thirds of the radioactivity which could not be released after washing in medium alone was recovered in the soluble intracellular fraction following cell lysis. Almost all of the soluble radioactivity could be precipitated with the polyclonal antiserum, indicating that very little lysosomal degradation of internalized heart ferritin had occurred. The present studies demonstrate a protein-mediated binding mechanism for acidic isoferritins on HL60 cells. These observations agree with published evidence that ferritin is often associated with cell membranes and are consistent with a possible role for the protein in the regulation of haematopoiesis or in iron transfer.

Stability of radioiodinated ferritin

Ferritin which had been radioiodinated using chloramine T exhibited marked instability on storage at 4°C. Both [ 125I]human liver and heart ferritins showed a similar rate of decline in immunoreactivity ( t 1 2 = 20–23 days) indicating that deterioration with storage was not a function of isoferritin composition. The decrease in radioactivity associated with ferritin was due not only to loss of 125I from the molecule but also to protein degradation as shown by enzyme-linked immunoassay and gel filtration. The degradation products had an M r of at least 69 000 although low M r material could be identified by gel filtration when a marked reduction in immunoreactivity had occurred. Ferritin instability was much more pronounced than when other proteins such as immunoglobulin and albumin were radioiodinated with chloramine T. These observations indicate that when performing in vivo and in vitro studies with labeled ferritin, degradation of the protein during storage should be carefully monitored and the protein repurified before use.

Immunochemical characterization of human liver and heart ferritins with monoclonal antibodies

A library of 27 murine monoclonal antibodies was obtained by using human liver and heart ferritins as immunogens. The specificity of the antibodies for the two ferritins and their subunits was studied with five different methods. The antibodies elicited by the liver ferritin bound preferentially the immunogen and were specific for the L subunit. Some antibodies elicited by the heart ferritin had characteristics similar to the anti-liver antibodies, other ones bound preferentially the heart over the liver ferritin and were specific for the H subunit. Only two antibodies were able to bind both ferritins and subunits. Some anti-H and anti-L chain antibodies were used to develop and compare four types of immunoassay to quantitate isoferritins. The results indicate the heart ferritin is immunologically more heterogeneous than liver, the H and L subunits having large immunological differences with few, if any, identical epitopes; and that that the architecture of the immunoassays have a strong influence on the crossreactivity of the antibodies with the two isoferritins, probably because H and L chains are not arranged randomly in the assembled protein.

Effect of acidic and basic isoferritins on in vitro growth of human granulocyte-monocyte progenitors

Acidic isoferritins have been previously found to be highly potent inhibitors of hematopoietic progenitors at concentrations of 10(-16) to 10(-18) mol/L, and it has been suggested that acidic isoferritin inhibitory activity plays a role in the regulation of normal hematopoiesis and also in the pathogenesis of leukemia. To characterize the ferritin species that affect the in vitro growth of human colony- forming unit-granulocyte-macrophage (CFU-GM), we tested different preparations of basic (L-subunit-rich) and acidic (H-subunit-rich) isoferritins. Three preparations of human liver (basic) ferritin did not show any effects on CFU-GM growth at concentrations up to 10(-9) mol/L, irrespective of the degree of glycosylation. Acidic isoferritins were purified both from HeLa cells and human heart. HeLa cell ferritin did not affect in vitro colony formation. One of two preparations of human heart ferritin, containing 5% glycosylated ferritin, showed a mean inhibition of 26% +/- 8% of the control at 10(-9) mol/L (P less than .02), whereas the other preparation, which contained no glycosylated ferritin, did not show any effect of CFU-GM growth. A preparation enriched for glycosylated acidic isoferritins from human heart was found to produce a mean inhibition of 32% +/- 11% of the control at 10(-9) mol/L (P less than .01), whereas another one was ineffective. A significant part of the inhibitory activity was removed by preincubation with the monoclonal antibody 2A4 directed against human heart ferritin. The present findings indicate that basic isoferritins, ie, the predominant ferritin type in human blood, have no effect on the growth of human CFU-GM, and this is in keeping with indirect clinical evidence. Inhibition of colony formation may be obtained by some preparations of acidic isoferritins that are rich in H subunits and bind to concanavalin A. The mechanism(s) responsible for this are not clear, but the effective concentrations are higher than those found in human blood both under normal conditions and in leukemia. At present, the physiologic significance of the observed inhibitory activity is uncertain.

Effect of Acidic and Basic Isoferritins on In Vitro Growth of Human Granulocyte-Monocyte Progenitors

Acidic isoferritins have been previously found to be highly potent inhibitors of hematopoietic progenitors at concentrations of 10–16 to 10–18 mol/L, and it has been suggested that acidic isoferritin inhibitory activity plays a role in the regulation of normal hematopoiesis and also in the pathogenesis of leukemia. To characterize the ferritin species that affect the in vitro growth of human colony-forming unit-granulocyte-macrophage (CFU-GM), we tested different preparations of basic (L-subunit-rich) and acidic (H-subunit-rich) isoferritins. Three preparations of human liver (basic) ferritin did not show any effects on CFU-GM growth at concentrations up to 10–9 mol/L, irrespective of the degree of glycosylation. Acidic isoferritins were purified both from HeLa cells and human heart. HeLa cell ferritin did not affect in vitro colony formation. One of two preparations of human heart ferritin, containing 5% glycosylated ferritin, showed a mean inhibition of 26% ± 8% of the control at 10–9 mol/L (P < .02), whereas the other preparation, which contained no glycosylated ferritin, did not show any effect of CFU-GM growth. A preparation enriched for glycosylated acidic isoferritins from human heart was found to produce a mean inhibition of 32% ± 11% of the control at 10–9 mol/L (P < .01), whereas another one was ineffective. A significant part of the inhibitory activity was removed by preincubation with the monoclonal antibody 2A4 directed against human heart ferritin. The present findings indicate that basic isoferritins, ie, the predominant ferritin type in human blood, have no effect on the growth of human CFU-GM, and this is in keeping with indirect clinical evidence. Inhibition of colony formation may be obtained by some preparations of acidic isoferritins that are rich in H subunits and bind to concanavalin A. The mechanism(s) responsible for this are not clear, but the effective concentrations are higher than those found in human blood both under normal conditions and in leukemia. At present, the physiologic significance of the observed inhibitory activity is uncertain.

Common Epitopes in Human Isoferritins Characterized by Murine Monoclonal Antibodies

Three monoclonal antibodies against human liver ferritin were selected to study antigenic determinants (epitopes) of human isoferritins. These monoclonal antibodies were found to form immunoprecipitin lines with ferritin in double diffusion tests (Ouchterlony), indicating multiple epitopes on a single ferritin molecule. The antibodies revealed high species specificity as well. Monoclonal antibodies MA301 and MA311 appeared to recognize different epitopes, since they did not inhibit each other in competitive enzyme-linked immunosorbent assay (ELISA). However, MA309 recognized both epitopes for MA301 and MA311 with similar competitive inhibition. These epitopes were not detectable when ferritin was treated with 8M urea (pH 2.5) and were detectable upon reconstruction by dialysis against 2 M urea (pH 7.2), suggesting that these monoclonals recognize epitopes in the tertiary structure of the ferritin molecule. As a matter of fact, these monoclonals react preferentially with intact ferritin molecule and only negligibly with subunits. Isoelectric focusing patterns of human ferritins demonstrated that liver, spleen, placenta, and hepatoma cells (Li-7) transplanted in nude mice contained basic isoferritins, whereas HeLa cells (carcinoma), Wa cells (EB virus-transformed B cells), and Raji cells (Burkitt's lymphoma) contained acidic isoferritins. Human heart ferritin displayed a somewhat intermediate pattern between liver and HeLa ferritins. In spite of the heterogeneous population of human isoferritins, the dissociation constants (Kd) of the three monoclonal antibodies to liver, HeLa, and heart isoferritins were quite similar.

Immunological Reactivity of Serum Ferritin in Patients with Malignancy

Serum ferritin has been suggested as a tumor marker in the diagnosis of certain malignancies and for following the activity or dissemination of the malignant process. Since neoplastic tissues generally contain more acidic isoferritins than their normal tissue counterparts, it has also been suggested that the specific assay of such isoferritins in serum may be of particular value in the diagnosis of malignancy. In this work, we have evaluated ferritin concentration in the serum of normal subjects and patients with acute nonlymphocytic leukemia, Hodgkin's disease, breast cancer and lung cancer by simultaneously using three different immunoassays: an immunoradiometric assay based on polyclonal antibodies against human liver (basic, L-subunit rich) ferritin, a radioimmunoassay based on polyclonad antibodies against HeLa cell (acidic, H-subunit rich) ferritin, and an immunoradiometric assay based on the monoclonal antibody 2A4 raised against human heart (acidic, H-subunit rich) ferritin. Most of the patients studied had increased values for liver-type ferritin in the absence of increased iron stores. Binding of serum ferritin to concanavalin A did not prove to be useful in distinguishing a tumor-specific basic isoferritin. The HeLa ferritin assay was found to be less specific than the heart ferritin assay in the detection of acidic isoferritins, and did not provide any advantage over the liver assay in detecting the increased levels of serum ferritin associated with malignant disease. Heart-type ferritin was found in one-fifth of normal sera and 64% of sera from patients with malignancy. Values were very low compared with those for basic ferritin, ranging from less than 0.1 to 17% of total serum ferritin (geometric mean value 1.3%) in patients with malignancy. These findings indicate that at present there is little application for serum ferritin immunoassays based on antibodies to HeLa cell or heart ferritin in the diagnosis or monitoring of malignant disease. This seems to be due to the presence in human serum of biding factors which are responsible for the rapid clearance of acidic isoferritins from the circulation. The serum concentration of basic ferritin, however, can be useful in the diagnosis and management of some malignancies, and it is possible that studies on cell isoferritins can be important in biologic monitoring of neoplastic disorders. It should also be noted that the increased levels of serum ferritin found in patients with malignancy can exert adverse effects on the host immune response and perhaps an inhibitory effect on hematopoiesis.

Use of a monoclonal antibody against human heart ferritin for evaluating acidic ferritin concentration in human serum.

Immunoassays for acidic ferritins rich in H subunits have shown that these isoferritins are predominant in some cells such as monocytes and red blood cells but have provided conflicting results about their presence in human serum. We have used an immunoradiometric assay based on a monoclonal antibody against human heart ferritin (monoclonal 2A4) for evaluating acidic ferritin concentration in human serum. This assay proved to be highly specific for acidic isoferritins having more than 60% H subunits. Heart-type ferritin was detected in only one fifth of normal sera and sera from patients with iron overload; values were very low compared with those for basic ferritin. Acidic ferritin was found in relatively high concentrations in most patients with iron deficiency anaemia. In other disease states characterized by increased serum concentrations of basic ferritin, acidic ferritin was always less than 21% of the total ferritin. Dialysis in low-ionic-strength buffer showed that both normal and pathological sera had binding factors for human heart ferritin. We conclude that: (i) human serum contains low concentrations of acidic isoferritins which, at variance with basic ferritin, do not appear to be directly related to the amount of storage iron; (ii) the findings of the present study reinforce the opinion that basic and acidic ferritins have different functional behaviours.

Assignment of human ferritin genes to chromosomes 11 and 19q13.3----19qter.

Extracts of hamster-human and mouse-human hybrids, some with translocations involving chromosome 19, have been assayed for both human spleen ferritin (rich in L subunits) and human heart ferritin (rich in H subunits). Hybrid lines retaining part of the long arm of chromosome 19 including the region 19q13.3----19qter produced human "L" type ferritin. This confirms the previous assignment of the "ferritin gene" to chromosome 19 (Caskey et al. 1983). However, lines retaining chromosome 11 were found to contain human "H" type ferritin suggesting that the gene for the "H" subunit is on this chromosome. The presence of chromosome 6 was not necessary for the expression of either "H" or "L" type human ferritin. It thus seems unlikely that the gene for idiopathic haemochromatosis is a ferritin gene.

Interaction of ferritin with serum: implications for ferritin turnover

An interaction between human and rabbit ferritins and serum was demonstrated by a coated tube binding assay, a shift in molecular size on gel filtration and by precipitation of complexes with 3.5% polyethylene glycol 6000. With polyethylene glycol and labelled ferritins, complex formation was inhibited by heating sera to 56°C for 30 min and by addition of EDTA or excess unlabelled ferritins. Human heart ferritin showed the greatest interaction with serum, followed by human spleen ferritin and least of all human plasma ferritin. Ferritins did not appear to bind to IgG or IgM in normal sera. The interaction of ‘H’ subunit-containing ferritins with serum or plasma may be partly responsible for the rapid clearance of tissue ferritins from the circulation and the absence of acidic isoferritins in the plasma of normal subjects.