Sonia Levi

Structure, function, and evolution of ferritins

The ferritins of animals and plants and the bacterioferritins (BFRs) have a common iron-storage function in spite of differences in cytological location and biosynthetic regulation. The plant ferritins and BFRs are more similar to the H chains of mammals than to mammalian L chains, with respect to primary structure and conservation of ferroxidase center residues. Hence they probably arose from a common H-type ancestor. The recent discovery in E. coli of a second type of iron-storage protein (FTN) resembling ferritin H chains raises the question of what the relative roles of these two proteins are in this organism. Mammalian L ferritins lack ferroxidase centers and form a distinct group. Comparison of the three-dimensional structures of mammalian and invertebrate ferritins, as well as computer modeling of plant ferritins and of BFR, indicate a well conserved molecular framework. The characterisation of numerous ferritin homopolymer variants has allowed the identification of some of the residues involved in iron uptake and an investigation of some of the functional differences between mammalian H and L chains.

Iron detoxifying activity of ferritin: Effects of H and L human apoferritins on lipid peroxidation in vitro

Three recombinant human apoferritin variants were added to ferrous iron and the amount of lipid peroxidation produced by hydrogen peroxide was studied. The H‐apoferritin had the strongest inhibitory effect on lipid peroxidation, probably due to its ferroxidase activity. The L‐apoferritin inhibited lipid peroxidation slowly and only at neutral pH. The H‐mutant 91, deleted of the last 22 C‐terminal amino acids, and which is not able to form an iron core, had minimal effects on iron lipid peroxidation. It was concluded that both ferro‐oxidase and iron mineralization activities are necessary for ferritin iron detoxifying action.

Characterization of human ferritin H chain synthetized in Escherichia coli

We have inserted the coding region of the cDNA for human ferritin H chain into the expression vector pEMBLex2. The plasmid obtained is able to direct the synthesis of the ferritin H chain in Escherichia coli up to a concentration of 15% of total soluble proteins. All expressed subunits are found correctly assembled in the complete ferritin molecule, which can be easily purified. We have shown that the ferritin synthesized in E. coli has an Mr, electrophoretic mobility, and thermal stability similar to natural human isoferritins and is recognized by monoclonal antibodies specific for the H, but not by those for the L human ferritin chains.

Characteristics of a ferritin-binding protein present in human serum

Summary. The ferritin present in human serum differs from the ferritins found in tissues and other body fluids in having negligible proportions of H subunits. This has been related to the possible presence of binding factors which would form complexes with H‐subunit containing ferritins and thereby determine their rapid clearance and/or interference with immunoassays (serum inhibition’). In this work we have tried to identify and characterize these binding factors. Dotting and blotting experiments demonstrated an interaction between tissue ferritins and human serum. This was stronger with human heart and recombinant H‐type ferritin obtained from E. coli than with human liver ferritin. The serum binder appeared to be a glycoprotein migrating in the beta‐2 region and with a molecular weight of about 200 000 and pi between 4 and 5. Two different approaches to purification of the ferritin‐binding protein yielded enriched fractions containing also the complement proteins C3 and C4, the plasma protease inhibitor alpha‐2‐macroglobulin, and immunoglobulins. These in vitro findings may have physiological relevance.

Characteristics of ferritins in human milk secretions: similarities to serum and tissue isoferritins.

The ferritins present in the first day human colostrum and mature milk were compared with serum and tissue ferritins on the basis of their iron content, immunological reactivity and glycosylation. Both had a low iron content. The degree of glycosylation and immunochemical properties of colostrum ferritin showed strong similarities with serum ferritin. The isoelectric points and immunochemical properties of milk ferritin were similar to heart ferritin. The concentration of colostrum ferritin was more significantly correlated to body iron stores than milk ferritin.

Regulation of ferritin synthesis in malignant and non-malignant lymphoid cells

The different amounts of H-rich and L-rich isoferritins found in malignant and non malignant lymphoid cells are accompanied by proportional variations in the relative quantity of messenger RNAs for the H and L subunits of ferritin. The correlation between levels of messenger RNAs and proteins suggests that the amount of messenger RNA plays an important role in ferritin biosynthesis in these cells. The enhanced expression of ferritin messenger RNAs in some neoplastic cells is not caused by gross alterations in the structure of ferritin genes.

Properties of ferritin from the earthworm Octolasium complanatum

Abstract The isolation and characterization of a protein with a high iron content from a common Italian earthworm ( Octolasium complanatum ) is described. This protein represents the first example of a purified ferritin from annelids. O. complanatum ferritin migrates on polyacrylamide gel electrophoresis with a mobility similar to that of horse spleen ferritin, and has hydrodynamic properties, i.e., elution volume and sedimentation velocity, which likewise correspond to those of horse ferritins. It can be resolved in a family of isoferritins with isoelectric points ranging from 6 to 7; accordingly, SDS gel electrophoresis reveals the presence of two subunit types of M r 19700 and 20500, which are present in roughly equal amounts. O. complanatum ferritin crystallizes from poly(ethylene glycol) and is immunologically unreactive with antisera against human or horse ferritin. Apoferritin can be prepared by means of reducing agents, just like mammalian apoferritins. It catalyses iron oxidation and incorporation inside the protein shell under conditions that are known to lead to ferritin reconstitution. The spectroscopic properties of O. complanatum apoferritin indicate that the aromatic residues are immobilized in a hydrophobic environment, as in the case of mammalian apoferritins. Moreover, the presence of dichroic bands in the region 320–400 nm suggests that an extrinsic chromophore copurifies with earthworm ferritin.

Human ferritin H-chains can be obtained in non-assembled stable forms which have ferroxidase activity

We found conditions to obtain the Leu‐169 → Arg mutant of human ferritin H chain in a stable and non‐assembled state. The protein obtained is an oligomer of subunits with a high degree of structured conformation, and when concentrated it re‐assembles into ferritin cages. Functional studies showed that (i) it promotes iron oxidation like the assembled ferritin, but at slower rate, (ii) it is readily precipitated by the oxidised iron unless apotransferrin or L‐chain ferritin are added to sequester Fe(III). The results confirm that ferroxidase activity is located within the H‐chain, and indicate that the cages of the fully assembled ferritins are important not only in maintaining iron in a soluble form, but also in eliciting the activity of the ferroxidase centres.

H and L ferritins in myocardium in iron overload

Abstract Ferritin is an iron-containing protein composed of 2 subunits (H and L) with largely different primary structures and immunochemical properties. 1 The normal L-subunit-rich, basic isoferritin is prevalent in the liver and spleen, whereas the H-subunit-rich, acidic isoferritin predominates in the heart, pancreas and kidneys.2 A variation in heart isoferritin was reported in iron-overloaded patients,2 and the absence of the more acidic isoferritin was hypothesized to be specific for hereditary hemochromatosis.3 These observations prompted us to perform immunohistochemical investigations on H and L ferritins in endomyocardial biopsy samples of patients with primary or secondary iron overload, using monoclonal antibodies to H and L. This study attempted to assess the pattern of heart isoferritins and the factors influencing their occurrence in iron accumulation.

Measurement of ferritin-bearing lymphocytes in man. Preliminary studies on the use of monoclonal antibodies specific for the L and H subunits of ferritin.

We used the monoclonal antibodies LFO3 (specific for the L subunit of ferritin) and 2A4 (specific for the H subunit) in an indirect immunofluorescence test for enumerating ferritin-bearing lymphocytes (FBL). In 13 normal subjects, the geometric mean value of FBL was 4 % (range 0–13 %) with the monoclonal antibody LFO3, and 3 % (range 0–8 %) with the monoclonal antibody 2A4. Values in 5 subjects with transfusional iron overload and increased plasma L-type ferritin concentration were 5 % (4–7 %) and 3 % (2–4 %), respectively, which is similar to those in normal subjects. Thirteen patients with malignant disease had normal to increased values for plasma ferritin; the circulating protein was largely of L-type with undetectable or very low concentrations of H-type ferritin. In the same patients, the percentage of FBL was greater with the monoclonal antibody 2A4 (geometric mean value 8 %; range 3–12 %) than with the monoclonal antibody LFO3 (geometric mean value 3 %; range, 1–7 %). It is concluded that acidic and basic isoferritins can be differently expressed on the surface of peripheral blood lymphocytes, and that the monoclonal 2A4 could be particularly useful in the measurement of FBL in patients with malignancy.