The location of antigenic sites on ferritin molecules

Addison, J.M.; Treffry, Amyra; Harrison, Pauline M.

doi:10.1016/0014-5793(84)80763-2

Cited by 12 publications

(8 citation statements)

References 14 publications

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“…Amino acid sequences of dog ferritin chains were obtained by translation from the cloned canine lens ferritin chain cDNA sequences by using Swiss Protein Database (13). We selected the sequences located on the outside of the ferritin molecule based on known immunological properties of human H-and L-ferritin chains (17,18). The designed peptides were chemically synthesized, conjugated with keyhole limpet hemocyanin, and used to produce the chain-specific antiserum in immunized rabbits by Research Genetics, Inc.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Mechanism by Which Lens Epithelial Cells Limit Accumulation of Overexpressed Ferritin H-chain

Goralska

Holley

McGahan

2003

Journal of Biological Chemistry

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The primary cultures of canine lens epithelial cells were transiently transfected with cDNAs for dog ferritin H-or L-chains in order to study differential expression of these chains. By using chain-specific antibodies, we determined that at 48 h after transfection overexpression of L-chain was much higher (9-fold over control) than that of H-chain (1.7-fold). We discovered that differentially transfected cells secrete overexpressed chains as homopolymeric ferritin into the media. Fortyeight hours after transfection accumulation of H-ferritin in the media was much higher (3-fold) than that of L-ferritin. This resulted in lowering of the concentration of H-chain in the cytosol. Co-transfection of cells with both H-and L-chain cDNAs increased the intracellular levels of H-chain and eliminated secretion of Hferritin to the media. We concluded that lens epithelial cells differentially regulate concentration of both ferritin chains in the cytosol. The overexpressed L-chain accumulated in the cytosol as predominantly homopolymeric L-ferritin. This is in contrast to H-chain, which is removed to the media unless there is an L-chain available to form heteropolymeric ferritin. These data indicate that the inability of cells to more strictly control cytosolic levels of L-chain may augment its accumulation in lenses of humans with hereditary hyperferritinemia cataract syndrome, which is caused by overexpression of L-chain due to mutation in the regulatory element in the untranslated region of the mRNA of the chain.Ferritins are the highly conserved iron storage proteins found in species from bacteria to mammals. Mammalian ferritins, located predominantly in cytoplasm, are heteropolymers of 24 subunits of two types, heavy (H 1 ; 21 kDa) and light (L; 19 kDa). Despite high (ϳ50%) sequence identity and similar three-dimensional structures, the subunits are genetically different and each has a distinct and complementary role in storing iron. The H subunit has a ferroxidase center responsible for uptake and oxidation of Fe 2ϩ into ferric ions. Translocation of these ions into the core of the ferritin shell and their mineralization for long term storage is facilitated by the L subunit. The intracellular level of ferritin correlates with the size of the pool of intracellular iron, which plays the main role in the regulation of ferritin expression. The iron-controlled expression of both chains is primarily translational and involves binding of iron-regulatory proteins IRP1 and IRP2 (iron sensors) to iron-response element (IRE), a stem-loop structure present in the 5Ј-untranslated region of the mRNA (1). The expression of H-chain gene is also regulated transcriptionally by cytokines (2), hormones, oncogenes (3), and inducers of cell differentiation (4).The H and L subunit ratio of mammalian ferritins varies and is highly tissue-specific; however, mechanisms by which cells maintain the chain-specific ratio are not fully understood. Although both chains complement each other in the process of sequestering and storing iron, H-chain has ...

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Section: Methodsmentioning

confidence: 99%

Identification of a Mechanism by Which Lens Epithelial Cells Limit Accumulation of Overexpressed Ferritin H-chain

Goralska

Holley

McGahan

2003

Journal of Biological Chemistry

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“…Antigenic sites of ferritin molecules are located in continuous inter-helical regions of the polypeptide chains on the molecular surface and encompass amino acids remote in the primary structure or belonging to different subunit (Addison et al 1984). We used three different peptide fragments of peptide 87-95, 126-132, and 148-155 which are located on the surface of ferritin molecule in the loop, the CD turn, and the the DE turn, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…To elucidate the ferritin binding site (epitope) of the canine FBP, three different peptide fragments (peptide 87-95, NH 2 -KPDRDDWEN-COOH; peptide 126-132, NH 2 -DPHLCDF-COOH; and peptide 148-155: NH 2 -GDHVTNLR-COOH) were synthesized (Thermo Electron Corporation, Ulm, Germany) according to the sequence data of canine liver ferritin H subunit (GenBank accession number AB175610, peptide fragment are numbered according to amino acid numbers of the H subunit sequence) based on the previous report on antigenic sites on the ferritin molecules (Addison et al 1984). Aliquots (100 ll) of 125 lg ml )1 synthesized peptides or 1.25 lg ml )1 canine liver ferritin in 100 mM Na 2 CO 3 (pH 9.6) were added to the wells of an Immobilizer Amino plate.…”

Section: Ferritin-binding Assaymentioning

confidence: 99%

Purification and Characterization of Canine Serum Ferritin-binding Proteins

et al. 2006

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Ferritin-binding protein (FBP) is known to interact with circulating ferritins in mammals. Canine FBPs were purified from canine serum by affinity chromatography and were identified as IgM, IgG, and IgA by immunoblotting with alkaline phosphatase-labeled antibodies to canine IgM, IgG, and IgA heavy chains. Following further purification by application to a Sephacryl S-300 column, canine FBPs were separated into 81.3- and 27.7-kDa bands by sodium dodecyl sulfate-polyacryamide gel electrophoresis, and the 81.3-kDa band reacted with the anti-canine IgM heavy chain antibody. Purified canine FBP bound to canine liver ferritin, but not to canine albumin and transferrin. FBP showed greater binding to the expressed bovine ferritin H-chain homopolymer than to the expressed bovine ferritin L-chain homopolymer. The binding of FBP with canine liver ferritin was dose-dependently inhibited by anti-rat liver ferritin antibody, and the anti-ferritin antibody dissociated the bound FBP in a dose-dependent manner, even after binding FBP with liver ferritin. The canine ferritin H subunit peptide fragment with amino acid residues 148-155 (NH(2)-GDHVTNLR-COOH) in its C-terminal region was recognized by FBP. These results indicate that canine serum FBPs are autoantibodies to ferritin (IgM, IgG, and IgA) and that anti-ferritin autoantibody (IgM) recognizes the C-terminal region of ferritin H subunit.

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“…Human anti-ferritin autoantibodies (IgM, IgG and IgA) recognized different species of ferritin and both human subunits: H and L. Peptide fragment (DPHLCDF) was synthesized based on the combination of ferritin epitope analyses [33] and sequence information found commonly in amino acid sequences of H and L subunits [4]. This peptide fragment inhibited the binding between canine liver ferritin and IgM antibody binding ferritin (Supplementary Figure S1).…”

Section: Binding Analyses Of Igm Antibody Binding Ferritinmentioning

confidence: 99%

“…These antibodies are unlikely to inhibit immunocoprecipitation, and human anti-ferritin autoantibodies seem to bind ferritin over other species. In this study, a peptide fragment synthesized was common amino acid sequence (DPHLCDF: 126-132, excluding N-terminal Met) from sequence data of mammalian ferritin subunits, and expected to be the antigenic site [19,33]. This fragment significantly inhibited binding between canine liver ferritin and IgM antibody binding ferritin (Supplementary Figure S1).…”

Section: Figurementioning

confidence: 99%

Characterization Analysis of Human Anti-Ferritin Autoantibodies

et al. 2014

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Anti-ferritin autoantibodies are found in many animals. Human ferritin-binding proteins (FBPs) were partially purified from human serum by ion-exchange chromatography and immobilized metal affinity chromatography with Zn 2+ . Crude FBPs were immunocoprecipitated with canine liver ferritin followed by the addition of anti-ferritin antibodies. Immunoglobulins in the immunocoprecipitate were detected with antibodies specific for human IgG, IgM or IgA heavy chains, and immunoglobulins IgG, IgM and IgA to bind to expressed recombinant human H and L chain homopolymers were also found. A portion of human serum proteins bound to zinc ions immobilized on beads were released upon the addition of canine liver ferritin, and the released protein was identified as IgM antibody. Additionally, the released proteins recognized peptide sequence (DPHLCDF) commonly found in amino acid sequences of mammalian ferritin H and L subunits. These results suggest that human serum contains anti-ferritin autoantibodies (IgG, IgM and IgA) which bind zinc ions and preferentially bind ferritin over both the H and L subunits, and that a portion of, but not all, the IgM antibodies bound to ferritin with higher affinity than to zinc ions and may recognize the common sequence found in mammalian ferritin H and L subunits.

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The location of antigenic sites on ferritin molecules

Cited by 12 publications

References 14 publications

Identification of a Mechanism by Which Lens Epithelial Cells Limit Accumulation of Overexpressed Ferritin H-chain

Identification of a Mechanism by Which Lens Epithelial Cells Limit Accumulation of Overexpressed Ferritin H-chain

Purification and Characterization of Canine Serum Ferritin-binding Proteins

Characterization Analysis of Human Anti-Ferritin Autoantibodies

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