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. 1998 Feb 15;330(Pt 1):315–320. doi: 10.1042/bj3300315

Transient overexpression of human H- and L-ferritin chains in COS cells.

B Corsi 1, F Perrone 1, M Bourgeois 1, C Beaumont 1, M C Panzeri 1, A Cozzi 1, R Sangregorio 1, P Santambrogio 1, A Albertini 1, P Arosio 1, S Levi 1
PMCID: PMC1219142  PMID: 9461525

Abstract

The understanding of the in vitro mechanisms of ferritin iron incorporation has greatly increased in recent years with the studies of recombinant and mutant ferritins. However, little is known about how this protein functions in vivo, mainly because of the lack of cellular models in which ferritin expression can be modulated independently from iron. To this aim, primate fibroblastoid COS-7 cells were transiently transfected with cDNAs for human ferritin H- and L-chains under simian virus 40 promoter and analysed within 66 h. Ferritin accumulation reached levels 300-500-fold higher than background, with about 40% of the cells being transfected. Thus ferritin concentration in individual cells was increased up to 1000-fold over controls with no evident signs of toxicity. The exogenous ferritin subunits were correctly assembled into homopolymers, but did not affect either the size or the subunit composition of the endogenous heteropolymeric fraction of ferritin, which remained essentially unchanged in the transfected and non-transfected cells. After 18 h of incubation with [59Fe]ferric-nitrilotriacetate, cellular iron incorporation was similar in the transfected and non-transfected cells and most of the protein-bound radioactivity was associated with ferritin heteropolymers, while H- and L-homopolymers remained iron-free. Cell co-transfection with cDNAs for H- and L-chains produced ferritin heteropolymers that also did not increase cellular iron incorporation. It is concluded that transient transfection of COS cells induces a high level of expression of ferritin subunits that do not co-assemble with the endogenous ferritins and have no evident activity in iron incorporation/metabolism.

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Selected References

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  1. Arosio P., Yokota M., Drysdale J. W. Characterization of serum ferritin in iron overload: possible identity to natural apoferritin. Br J Haematol. 1977 Jun;36(2):199–207. doi: 10.1111/j.1365-2141.1977.tb00640.x. [DOI] [PubMed] [Google Scholar]
  2. Bauminger E. R., Treffry A., Hudson A. J., Hechel D., Hodson N. W., Andrews S. C., Levi S., Nowik I., Arosio P., Guest J. R. Iron incorporation into ferritins: evidence for the transfer of monomeric Fe(III) between ferritin molecules and for the formation of an unusual mineral in the ferritin of Escherichia coli. Biochem J. 1994 Sep 15;302(Pt 3):813–820. doi: 10.1042/bj3020813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beaumont C., Leneuve P., Devaux I., Scoazec J. Y., Berthier M., Loiseau M. N., Grandchamp B., Bonneau D. Mutation in the iron responsive element of the L ferritin mRNA in a family with dominant hyperferritinaemia and cataract. Nat Genet. 1995 Dec;11(4):444–446. doi: 10.1038/ng1295-444. [DOI] [PubMed] [Google Scholar]
  4. Cham B. E., Roeser H. P., Nikles A. C. Cytosolic ferritin and lipid-associated ferritin are metabolically different in guinea-pig livers. Biochem J. 1989 Nov 1;263(3):989–992. doi: 10.1042/bj2630989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Girelli D., Corrocher R., Bisceglia L., Olivieri O., De Franceschi L., Zelante L., Gasparini P. Molecular basis for the recently described hereditary hyperferritinemia-cataract syndrome: a mutation in the iron-responsive element of ferritin L-subunit gene (the "Verona mutation") Blood. 1995 Dec 1;86(11):4050–4053. [PubMed] [Google Scholar]
  6. Harrison P. M., Arosio P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim Biophys Acta. 1996 Jul 31;1275(3):161–203. doi: 10.1016/0005-2728(96)00022-9. [DOI] [PubMed] [Google Scholar]
  7. Hentze M. W., Kühn L. C. Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8175–8182. doi: 10.1073/pnas.93.16.8175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jordan M., Schallhorn A., Wurm F. M. Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res. 1996 Feb 15;24(4):596–601. doi: 10.1093/nar/24.4.596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Keller G. A., Tokuyasu K. T., Dutton A. H., Singer S. J. An improved procedure for immunoelectron microscopy: ultrathin plastic embedding of immunolabeled ultrathin frozen sections. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5744–5747. doi: 10.1073/pnas.81.18.5744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Klausner R. D., Rouault T. A., Harford J. B. Regulating the fate of mRNA: the control of cellular iron metabolism. Cell. 1993 Jan 15;72(1):19–28. doi: 10.1016/0092-8674(93)90046-s. [DOI] [PubMed] [Google Scholar]
  11. Lawson D. M., Artymiuk P. J., Yewdall S. J., Smith J. M., Livingstone J. C., Treffry A., Luzzago A., Levi S., Arosio P., Cesareni G. Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts. Nature. 1991 Feb 7;349(6309):541–544. doi: 10.1038/349541a0. [DOI] [PubMed] [Google Scholar]
  12. Levi S., Luzzago A., Cesareni G., Cozzi A., Franceschinelli F., Albertini A., Arosio P. Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site. A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants. J Biol Chem. 1988 Dec 5;263(34):18086–18092. [PubMed] [Google Scholar]
  13. Levi S., Salfeld J., Franceschinelli F., Cozzi A., Dorner M. H., Arosio P. Expression and structural and functional properties of human ferritin L-chain from Escherichia coli. Biochemistry. 1989 Jun 13;28(12):5179–5184. doi: 10.1021/bi00438a040. [DOI] [PubMed] [Google Scholar]
  14. Levi S., Santambrogio P., Corsi B., Cozzi A., Arosio P. Evidence that residues exposed on the three-fold channels have active roles in the mechanism of ferritin iron incorporation. Biochem J. 1996 Jul 15;317(Pt 2):467–473. doi: 10.1042/bj3170467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Levi S., Santambrogio P., Cozzi A., Rovida E., Corsi B., Tamborini E., Spada S., Albertini A., Arosio P. The role of the L-chain in ferritin iron incorporation. Studies of homo and heteropolymers. J Mol Biol. 1994 May 20;238(5):649–654. doi: 10.1006/jmbi.1994.1325. [DOI] [PubMed] [Google Scholar]
  16. Levi S., Yewdall S. J., Harrison P. M., Santambrogio P., Cozzi A., Rovida E., Albertini A., Arosio P. Evidence of H- and L-chains have co-operative roles in the iron-uptake mechanism of human ferritin. Biochem J. 1992 Dec 1;288(Pt 2):591–596. doi: 10.1042/bj2880591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Luzzago A., Arosio P., Iacobello C., Ruggeri G., Capucci L., Brocchi E., De Simone F., Gamba D., Gabri E., Levi S. Immunochemical characterization of human liver and heart ferritins with monoclonal antibodies. Biochim Biophys Acta. 1986 Jul 25;872(1-2):61–71. doi: 10.1016/0167-4838(86)90147-0. [DOI] [PubMed] [Google Scholar]
  18. Miller L. L., Miller S. C., Torti S. V., Tsuji Y., Torti F. M. Iron-independent induction of ferritin H chain by tumor necrosis factor. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4946–4950. doi: 10.1073/pnas.88.11.4946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Otsuka S., Maruyama H., Listowsky I. Structure, assembly, conformation, and immunological properties of the two subunit classes of ferritin. Biochemistry. 1981 Sep 1;20(18):5226–5232. doi: 10.1021/bi00521a020. [DOI] [PubMed] [Google Scholar]
  20. Picard V., Renaudie F., Porcher C., Hentze M. W., Grandchamp B., Beaumont C. Overexpression of the ferritin H subunit in cultured erythroid cells changes the intracellular iron distribution. Blood. 1996 Mar 1;87(5):2057–2064. [PubMed] [Google Scholar]
  21. Rucker P., Torti F. M., Torti S. V. Role of H and L subunits in mouse ferritin. J Biol Chem. 1996 Dec 27;271(52):33352–33357. doi: 10.1074/jbc.271.52.33352. [DOI] [PubMed] [Google Scholar]
  22. Santambrogio P., Levi S., Arosio P., Palagi L., Vecchio G., Lawson D. M., Yewdall S. J., Artymiuk P. J., Harrison P. M., Jappelli R. Evidence that a salt bridge in the light chain contributes to the physical stability difference between heavy and light human ferritins. J Biol Chem. 1992 Jul 15;267(20):14077–14083. [PubMed] [Google Scholar]
  23. Santambrogio P., Levi S., Cozzi A., Corsi B., Arosio P. Evidence that the specificity of iron incorporation into homopolymers of human ferritin L- and H-chains is conferred by the nucleation and ferroxidase centres. Biochem J. 1996 Feb 15;314(Pt 1):139–144. doi: 10.1042/bj3140139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Santambrogio P., Levi S., Cozzi A., Rovida E., Albertini A., Arosio P. Production and characterization of recombinant heteropolymers of human ferritin H and L chains. J Biol Chem. 1993 Jun 15;268(17):12744–12748. [PubMed] [Google Scholar]
  25. Sun S., Arosio P., Levi S., Chasteen N. D. Ferroxidase kinetics of human liver apoferritin, recombinant H-chain apoferritin, and site-directed mutants. Biochemistry. 1993 Sep 14;32(36):9362–9369. doi: 10.1021/bi00087a015. [DOI] [PubMed] [Google Scholar]
  26. Treffry A., Zhao Z., Quail M. A., Guest J. R., Harrison P. M. Iron(II) oxidation by H chain ferritin: evidence from site-directed mutagenesis that a transient blue species is formed at the dinuclear iron center. Biochemistry. 1995 Nov 21;34(46):15204–15213. doi: 10.1021/bi00046a028. [DOI] [PubMed] [Google Scholar]
  27. Villa A., Podini P., Panzeri M. C., Söling H. D., Volpe P., Meldolesi J. The endoplasmic-sarcoplasmic reticulum of smooth muscle: immunocytochemistry of vas deferens fibers reveals specialized subcompartments differently equipped for the control of Ca2+ homeostasis. J Cell Biol. 1993 Jun;121(5):1041–1051. doi: 10.1083/jcb.121.5.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]

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