Advertisement

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
Article

Abstract

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.

Keywords

Hepcidin Cysteine Hepcidin agonist 

Abbreviations

CTCK

Carbenicillin, tetracycline, chloramphenicol, kanamycin

DFO

Desferrioxamine

DMEM

Dulbecco’s modified Eagle’s medium

ELISA

Enzyme-linked immunosorbent assay

FAC

Ferric-ammonium citrate

FBS

Fetal bovine serum

Fp

Ferroportin

FTH

Ferritin heavy chain

HAMP

Hepcidin antimicrobial peptide

HEPC

Hepcidin

HFE

Human hemochromatosis protein

HH

Hereditary hemochromatosis

HT-1080

Human fibrosarcoma cell line

IgG

Immunoglobulin G

JH

Juvenile hemochromatosis

PBS

Phosphate buffer saline

pBT

Bait plasmid

PCR

Polymerase chain reaction

pTRG

Target plasmid

PV

Polycythemia vera

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TENTG

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

ubiq

Ubiquitin

WRL68

Human hepatic cell line

Notes

Acknowledgements

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].

References

  1. 1.
    Krause A, Neitz S, Schulz A et al (2000) LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 480:147–150CrossRefGoogle Scholar
  2. 2.
    Park CH, Valore EV, Waring AJ, Ganz T (2001) Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 276:7806–7810CrossRefGoogle Scholar
  3. 3.
    Pigeon C, Ilyin G, Courselaud B et al (2001) A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem 276:7811–7819CrossRefGoogle Scholar
  4. 4.
    Nicolas G, Bennoun M, Devaux I et al (2001) Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci USA 98:8780–8785CrossRefGoogle Scholar
  5. 5.
    Kroot JJC, Tjalsma H, Fleming RE, Swinkels DW (2011) Hepcidin in human iron disorders: diagnostic implications. Clin Chem 57:1650–1669CrossRefGoogle Scholar
  6. 6.
    Scamuffa N, Basak A, Lalou C et al (2008) Regulation of prohepcidin processing and activity by the subtilisin-like proprotein convertases Furin, PC5, PACE4 and PC7. Gut 57:1573–1582CrossRefGoogle Scholar
  7. 7.
    Valore EV, Ganz T (2008) Posttranslational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin. Blood Cells Mol Dis 40:132–138CrossRefGoogle Scholar
  8. 8.
    Schranz M, Bakry R, Creus M et al (2009) Activation and inactivation of the iron hormone hepcidin: Biochemical characterization of prohepcidin cleavage and sequential degradation to N-terminally truncated hepcidin isoforms. Blood Cells Mol Dis 43:169–179CrossRefGoogle Scholar
  9. 9.
    Jordan JB, Poppe L, Haniu M et al (2009) Hepcidin revisited, disulfide connectivity, dynamics, and structure. J Biol Chem 284:24155–24167CrossRefGoogle Scholar
  10. 10.
    Donovan A, Lima CA, Pinkus JL et al (2005) The iron exporter ferroportin/Slc40a1 is essential for iron homeostasis. Cell Metab 1:191–200CrossRefGoogle Scholar
  11. 11.
    Nemeth E (2008) Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science 306:2090–2093CrossRefGoogle Scholar
  12. 12.
    De Domenico I, McVey Ward D, Langelier C et al (2007) The molecular mechanism of hepcidin-mediated ferroportin down-regulation. Mol Biol Cell 18:2569–2578CrossRefGoogle Scholar
  13. 13.
    Nemeth E, Preza GC, Jung CL et al (2006) The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study. Blood 107:328–333CrossRefGoogle Scholar
  14. 14.
    Chaston T, Chung B, Mascarenhas M et al (2008) Evidence for differential effects of hepcidin in macrophages and intestinal epithelial cells. Gut 57:374–382CrossRefGoogle Scholar
  15. 15.
    Mena NP, Esparza A, Tapia V et al (2007) Hepcidin inhibits apical iron uptake in intestinal cells. Am J Physiol Gastrointest Liver Physiol 294:G192-G198Google Scholar
  16. 16.
    Brasse-Lagnel C, Karim Z, Letterton P et al (2011) Intestinal DMT1 contransporter is down-regulated by hepcidin via proteasome internalization and degradation. Gastroenterology 140:1261–1271CrossRefGoogle Scholar
  17. 17.
    Clark RJ, Tan CC, Preza GC et al (2011) Understanding the structure/activity relationships of the iron regulatory peptide hepcidin. Chem Biol 18:336–343CrossRefGoogle Scholar
  18. 18.
    Preza GC, Ruchala P, Pinon R et al (2011) Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload. J Clin Invest 121:4880–4888CrossRefGoogle Scholar
  19. 19.
    Fernandes A, Preza GC, Phung Y et al (2009) The molecular basis of hepcidin-resistant hereditary hemochromatosis. Blood 114:437–443CrossRefGoogle Scholar
  20. 20.
    Pandur E, Nagy J, Poór VS, Sarnyai A et al (2008) Alpha-1 antitrypsin binds preprohepcidin intracellularly and prohepcidin in the serum. FEBS J 276:2012–2021CrossRefGoogle Scholar
  21. 21.
    Arezes J, Nemeth E (2015) Hepcidin and iron disorders: new biology and clinical approaches. Int J Lab Hematol 37:92–98CrossRefGoogle Scholar
  22. 22.
    Riemer J, Hoepken HH, Czerwinska H et al (2004) Colorimetric ferrozine-based assay for the quantitation of iron in cultured cells. Anal Biochem 331:370–375CrossRefGoogle Scholar
  23. 23.
    Sebastiani G, Wilkinson N, Pantopoulos K (2016) Pharmacological targeting of the hepcidin/ferroportin axis. Front Pharmacol 7:1–11CrossRefGoogle Scholar
  24. 24.
    Ganz T (2013) Systemic iron homeostasis. Physiol Rev 93:1721–1741CrossRefGoogle Scholar
  25. 25.
    Powell LW, Seckington RC, Deugnier Y(2016) Haemochromatosis Lancet 388:706–716CrossRefGoogle Scholar
  26. 26.
    Pietrangelo A (2010) Hereditary hemochromatosis: pathogenesis, diagnosis, and treatment. Gastroenterology 139:393–408CrossRefGoogle Scholar
  27. 27.
    Nemeth E, Ramos E, Ruchala P et al (2012) Minihepcidins prevent iron overload in a hepcidin-deficient mouse model of severe hemochromatosis. Blood 120:3829–3836CrossRefGoogle Scholar
  28. 28.
    Casu C, Oikonomidou PR, Chen H et al (2016) Minihepcidin peptides as disease modifiers in mice affected by β-thalassemia and polycythemia vera. Blood 128:265–276CrossRefGoogle Scholar
  29. 29.
    Chua K, Fung E, Micewicz ED et al (2015) Small cyclic agonists of iron regulatory hormone hepcidin. Bioorg Med Chem Lett 25:4961–4969CrossRefGoogle Scholar
  30. 30.
    Roetto A, Daraio F, Porporato P et al (2004) Screening hepcidin for mutations in juvenile hemochromatosis: identification of a new mutation (C70R). Blood 103:2407–2409CrossRefGoogle Scholar
  31. 31.
    Delatycki MB, Allen KJ, Gow P et al (2004) A homozygous HAMP mutation in a multiply consanguineous family with pseudo-dominant juvenile hemochromatosis. Clin Genet 65:378–383CrossRefGoogle Scholar
  32. 32.
    De Gobbi M, Caruso R, Daraio F et al (2003) Diagnosis of juvenile hemochromatosis in an 11-year-old child combining genetic analysis and non-invasive liver iron quantitation. Eur J Pediatr 162:96–99Google Scholar

Copyright information

© 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

Personalised recommendations