The Protein Journal

, Volume 37, Issue 2, pp 113–121 | Cite as

The C19S Substitution Enhances the Stability of Hepcidin While Conserving Its Biological Activity

  • Edina Pandur
  • Zsuzsanna Fekete
  • Kitti Tamási
  • László Grama
  • Edit Varga
  • Katalin Sipos


Hepcidin, the key hormone of iron homeostasis is responsible for lowering the serum iron level through its interaction with iron exporter ferroportin. Thus, hepcidin agonists provide a promising opportunity in the treatment of iron disorders caused by lacking or decreased hepcidin expression. We investigated the importance of each of the eight highly conserved cysteines for the biological activity of hepcidin. Eight cysteine mutants were created with site directed mutagenesis. The binding ability of these hepcidin mutants to the hepcidin receptor ferroportin was determined using bacterial two-hybrid system and WRL68 human hepatic cells. The biological activity of hepcidin mutants was determined by western blot analysis of ferroportin internalization and ferroportin ubiquitination. To investigate the effect of mutant hepcidins on the iron metabolism of the WRL68 cells, total intracellular iron content was measured with a colorimetric assay. The stability of M6 hepcidin mutant was determined using ELISA technique. Our data revealed that serine substitution of the sixth cysteine (M6) yielded a biologically active but significantly more stable peptide than the original hormone. This result may provide a promising hepcidin agonist worth testing in animal models.


Hepcidin Cysteine Hepcidin agonist 



Carbenicillin, tetracycline, chloramphenicol, kanamycin




Dulbecco’s modified Eagle’s medium


Enzyme-linked immunosorbent assay


Ferric-ammonium citrate


Fetal bovine serum




Ferritin heavy chain


Hepcidin antimicrobial peptide




Human hemochromatosis protein


Hereditary hemochromatosis


Human fibrosarcoma cell line


Immunoglobulin G


Juvenile hemochromatosis


Phosphate buffer saline


Bait plasmid


Polymerase chain reaction


Target plasmid


Polycythemia vera


Sodium dodecyl sulfate polyacrylamide gel electrophoresis


Tris–HCl, EDTA, NaCl, Trition-X, Glycerol




Human hepatic cell line



The project has been supported by the European Union, co-financed by the European Social Fund [EFOP-3.6.1-16-2016-00004], by University of Pécs, Medical School Research foundation, PTE KA [2015/30019/KA to E.P.] and by University of Pécs, in the frame of Pharmaceutic Talent Center program [PST 480132].


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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pharmaceutical Biology, Faculty of PharmacyUniversity of PécsPécsHungary
  2. 2.Department of Medical Biology, Medical SchoolUniversity of PécsPécsHungary
  3. 3.Department of Biophysics, Medical SchoolUniversity of PécsPécsHungary

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