Immunochemical characterization of human liver and heart ferritins with monoclonal antibodies

doi:10.1016/0167-4838(86)90147-0

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 872, Issues 1–2, 25 July 1986, Pages 61-71

Biochimica et Biophy...

https://doi.org/10.1016/0167-4838(86)90147-0 Get rights and content

Abstract

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.

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Ferritin self-assembly, structure, function, and biotechnological applications
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Citation Excerpt :
Nevertheless, non‑iron-storing serum ferritin [138], and iron-storing ferritins in liver cells [139] mainly consists of L-subunits. Simultaneously, ferritin rich in H-subunit is found in brain and heart cells, which actively use iron [139]. Although homopolymeric L-ferritin does not contain ferroxidase sites, it is still capable of notable iron accumulation under certain conditions [140].
Ferritin is a vital protein complex responsible for storing iron in almost all living organisms. It plays a crucial role in various metabolic pathways, inflammation processes, stress response, and pathogenesis of cancer and neurodegenerative diseases. In this review we discuss the role of ferritin in diseases, cellular iron regulation, its structural features, and its role in biotechnology. We also show that molecular mechanisms of ferritin self-assembly are key for a number of biotechnological and pharmaceutical applications. The assembly pathways strongly depend on the interface context of ferritin monomers and the stability of its different intermediate oligomers. To date, several schemes of self-assembly kinetics have been proposed. Here, we compare different self-assembly mechanisms and discuss the possibility of self-assembly control by switching between deadlock intermediate states.
The Ferritin-Heavy-Polypeptide-Like-17 (FTHL17) gene encodes a ferritin with low stability and no ferroxidase activity and with a partial nuclear localization
2015, Biochimica et Biophysica Acta - General Subjects
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They were collected and analyzed in SDS-PAGE 12%. The expression of FTHL17 was verified by Coomassie blue stain or by immunoblotting with anti-human FTHL17 (Sigma-Aldrich) or with monoclonal antibodies for human FTH and FTL [21]. The protein purification followed the procedure described in [26], briefly the transformed and induced E. coli were disrupted by sonication, the supernatant was added of anti-protease agents (anti protease Cocktail, Sigma Aldrich) to avoid degradation, and heated at 65 °C for 10 min and the supernatant collected.
Three functional ferritin genes have been identified so far in mammals, and they encode the cytosolic Heavy (FTH) and Light chain (FTL) and the mitochondrial ferritin. The expression of a transcript by a fourth ferritin-like gene (Ferritin-Heavy-Polypeptide-Like-17, FTHL17) on the X chromosome was reported in mouse spermatogonia and in early embryonic cells.
The intronless human FTHL17 gene encodes a protein with 64% identity to human FTH with substitution of key residues of the ferroxidase center. The gene was cloned into vectors for expression in Escherichia coli and mammalian cells, linked to a flag-tag.
The recombinant FTHL17 from E. coli purified as an assembled 24-mer ferritin devoid of ferroxidase activity and with a reduced physical stability. When transiently expressed in mammalian cells the flag-FTHL17 assembled in ferritin shells that showed reduced stability to denaturants compared with flag H and L ferritins. Immunocytochemistry with anti-flag antibody decorated the nuclei of flag-FTHL17 transfected COS cells, but not those of the cells transfected with flag-FTH or flag-FTL.
We concluded that FTHL17 encodes a ferritin-like protein without ferroxidase activity. Its restricted embryonic expression and partial nuclear localization suggest that this novel ferritin type may have functions other than iron storage.
The work confirms the presence of a fourth functional human ferritin gene with properties distinct from the canonical cytosolic ones.
Identification of a novel overlapping sequential E epitope (E') on the bovine leukaemia virus SU glycoprotein and analysis of immunological data
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Citation Excerpt :
Two other injections were performed at one-week intervals. Three days after the final administration, spleen cells were fused with P3/NS0 myeloma cells (Luzzago et al., 1986). Hybridomas were screened with an ELISA and an immunofluorescent assay.
Bovine leukaemia virus (BLV), an oncogenic C-type retrovirus, is the causative agent of enzootic bovine leucosis. Binding of BLV to its cellular receptor is mediated by the surface envelope glycoprotein subunit (SU). Previous studies have identified eight different epitopes (A through H) on the BLV SU. In this study, a new sequential epitope was identified using the monoclonal antibody 2G7 (MAb 2G7) on the C-terminal region of the BLV SU. To localise and refine the map of this epitope, a series of deleted forms in the C and N-terminal ends of the glycoprotein were made and synthesised in baculovirus and Escherichia coli expression systems. The synthetic proteins were analysed both in Western blot and MAb-capture ELISA assays. MAb 2G7 recognised a stretch of 11 amino acids, named epitope E′, corresponding to residues 189-SDWVPSVRSWA-199 (comprising the 33 amino acids signal peptide) overlapping with the E epitope of the SU. The data obtained by Enzyme-Linked Immunosorbent Assay (ELISA) revealed that the E′ epitope was hidden on whole BLV particles and that the variation in reactivity between epitope E′ and MAb 2G7 depends on the glycosylation state of SU. Similarly, the analysis of immunological data evidenced that the failure of interaction between the MAb anti-DD′ and its epitope was also due to a steric hindrance of the glycosylation. Finally, the ELISA assay analysis performed with the deleted and mutated forms of rSU evidenced that the conformational epitopes F, G and H lied into in the 34–173 amino-acids residues of N-terminal region of SU.
Structural and functional analyses of chicken liver ferritin
2011, Poultry Science
Citation Excerpt :
The 24-mer globular protein comprises H (heart or heavy) and L (liver or light) subunits with molecular masses of 21 and 19 kDa, respectively (Theil, 1987; Andrews et al., 1992; Harrison and Arosio, 1996). The H and L subunits have 50 to 60% amino acid identity and are each highly conserved within various species (H: 88–99%; L: 78–92%) but differ in functional and immunological properties (Luzzago et al., 1986; Andrews et al., 1992; Harrison and Arosio, 1996; Orino and Watanabe, 2008). The H subunit has a perfectly conserved ferroxidase domain, which is essential for iron uptake, whereas the L subunit lacks ferroxidase but uses iron nucleation on its inner surface within the ferritin molecule to incorporate iron in cooperation with H subunit (Theil, 1987; Harrison and Arosio, 1996; Orino et al., 2004; Orino and Watanabe, 2008).
Characterization of ferritins from different species has provided insight into iron regulation mechanisms and evolutionary relationships. Here, we examined chicken liver ferritin, which comprises only H subunit and has 14.8 µg of Fe/100 µg of protein. The chicken H subunit apo homopolymer showed the same iron uptake rate as bovine H subunit homopolymer expressed with a baculovirus expression system (0.31 and 0.28 mmol of Fe/min per micromole of protein for chicken and bovine H subunit, respectively). Chicken H subunit apo homopolymer showed a significantly higher biotinylated hemin-binding activity than liver holoferritin. Although bovine spleen apoferritin, which has an L (liver or light):H (heart or heavy) subunit ratio of 1:1, also shows a significantly higher biotinylated hemin-binding activity than its holoferritin, these biotinylated hemin-binding activities were markedly lower than those of both chicken holo- and apoferritins. Binding of chicken holo- and apoferritin with biotinylated hemin was strongly inhibited by hemin but not iron-free hemin, protoporphyrin IX, or Zn-protoporphyrin. These findings demonstrate that chicken ferritin comprises only an H subunit, possesses ferroxidase activity as in mammalian ferritin H subunits, and binds heme more strongly than mammalian ferritins.
Serum ferritin level changes in children with sickle cell disease on chronic blood transfusion are nonlinear and are associated with iron load and liver injury
2009, Blood
Chronic blood transfusion is increasingly indicated in patients with sickle cell disease. Measuring resulting iron overload remains a challenge. Children without viral hepatitis enrolled in 2 trials for stroke prevention were examined for iron overload (STOP and STOP2; n = 271). Most received desferrioxamine chelation. Serum ferritin (SF) changes appeared nonlinear compared with prechelation estimated transfusion iron load (TIL) or with liver iron concentrations (LICs). Averaged correlation coefficient between SF and TIL (patients/observations, 26 of 164) was r = 0.70; between SF and LIC (patients/observations, 33 of 47) was r = 0.55. In mixed models, SF was associated with LIC (P = .006), alanine transaminase (P = .025), and weight (P = .026). Most patients with SF between 750 and 1500 ng/mL had a TIL between 25 and 100 mg/kg (72.8% ± 5.9%; patients/observations, 24 of 50) or an LIC between 2.5 and 10 mg/g dry liver weight (75% ± 0%; patients/observations, 8 of 9). Most patients with SF of 3000 ng/mL or greater had a TIL of 100 mg/kg or greater (95.3% ± 6.7%; patients/observations, 7 of 16) or an LIC of 10 mg/g dry liver weight or greater (87.7% ± 4.3%; patients/observations, 11 of 18). Although SF changes are nonlinear, levels less than 1500 ng/mL indicated mostly acceptable iron overload; levels of 3000 ng/mL or greater were specific for significant iron overload and were associated with liver injury. However, to determine accurately iron overload in patients with intermediately elevated SF levels, other methods are required. These trials are registered at www.clinicaltrials.gov as #NCT00000592 and #NCT00006182.
ELISA reveals a difference in the structure of substantia nigra ferritin in Parkinson's disease and incidental Lewy body compared to control
2007, Parkinsonism and Related Disorders
Iron released from ferritin may trigger oxidative stress leading to progressive neurodegeneration of substantia nigra resulting in Parkinson's disease (PD). Change in the structure of ferritin may allow an easier efflux of iron. We compared with the use of ELISA the structure of ferritin (concentrations of H and L ferritins) in substantia nigra (SN) in ten cases of PD, six of incidental Lewy body (ILB) cases and 20 controls. SN concentration of L ferritin in ILB (50.6±11.5 ng/mg) and in PD (52.5±26.0) was lower than in control (97.9±54.9). H ferritin in PD (534.2±223.1) was higher than in ILB (336.9±87.7) and control (374.8±169.3). The decrease of L ferritin in SN in PD and ILB may suggest that the whole process of neurodegeneration starts with a higher availability of free iron, which is released from the ferritin shell.

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Regular paperImmunochemical characterization of human liver and heart ferritins with monoclonal antibodies

Abstract

J. Biol. Chem.

FEBS Lett.

Clin. Chim. Acta

Immunol. Lett.

Clin. Chim. Acta

J. Biol. Chem.

Exp. Cell Res.

Methods Enzymol.

Blood

Biochim. Biophys. Acta

FEBS Lett.

J. Biol. Chem.

Physiol. Rev.

Hum. Genet.

EMBO J.

Regular paper
Immunochemical characterization of human liver and heart ferritins with monoclonal antibodies