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Cellular binding analysis of recombinant hybrid heteropolymer of camel hepcidin and human ferritin H chain. The unexpected human H-ferritin binding to J774 murine macrophage cells

Abstract

Ferritin is a molecule with enormous potentiality in biotechnology that have been already used to encapsulate molecules, as contrast in magnetic resonance imaging and to carry epitopes. We proposed to use it to carry another key protein of iron metabolism, hepcidin that is a small hormone peptide that control systemic iron homeostasis. In this work, we purified the previously produced camel hepcidin and human H-ferritin heteropolymer (HepcH-FTH) and to monitor its binding capability toward J744 cell line in presence or absence of ferric ammonium citrate. Fused camel hepcidin and human H-ferritin monomer (HepcH) as well as the assembled HepcH-FTH heteropolymer (ratio 1:5) was easily purified by a one-step purification using size exclusion chromatography. SDS-PAGE electrophoresis of HepcH, purified from soluble and insoluble fractions, showed a single band of 24 kDa with an estimated purity of at least 90%. The purification yields of HepcH from the soluble and insoluble fractions was, respectively, of about 6.80 and 2 mg/L of bacterial culture. Time curse cellular binding assays of HepcH-FTH revealed its great potential to bind the J774 cells after 15 min of incubation. Furthermore, HepcH-FTH was able to degrade ferroportin, the unique hepcidin receptor, even after 30 min of incubation with J774 cells treated with 100 µM ferric ammonium citrate. In conclusion, we proposed ferritin as a peptide carrier to promote the association of the hybrid HepcH-FTH nanoparticle with a particular type of cell for therapeutic or diagnostic.

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Abbreviations

PAGE:

Polyacrylamide gel electrophoresis

HepcH:

Camel hepcidin-human ferritin H chain (monomer)

HepcH-FTH:

Camel hepcidin-human ferritin H assembled with human ferritin H chain (heteropolymer)

FAC:

Ferric ammonium citrate

FPN1:

Ferroportin

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Acknowledgments

MB was supported by a scholarship (1 month) from’Consorzio Interuniversitario Biotecnologie’ (CIB), Trieste–Italy and the Doctoral School of the National Institute of Applied Sciences and Technology (INSAT), University of Carthage, Tunis–Tunisia. Special thanks for the technical help of Dr. Paola Ruzzenenti at the Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, Brescia, Italy.

Author information

MB carried out the experiments, performed the data analysis and wrote the manuscript; MP, FC, MA, AG and MB contributed to the experiments; MP, PA and MNM supervised the study, participated in its design and data analysis and revised the manuscript. All authors read and approved the final manuscript.

Correspondence to Mohamed Boumaiza.

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Fig. S1 Cellular binding analysis of HepcH-FTH toward J774 cells treated and non-treated with FAC. (A) J774 cells treated with 100 µM FAC. Lane 1 and 2, untreated cells; Lane 3, cells treated with 0.5 µM FTH for 2 h; Lane 4 and 5, cells treated with 0.5 µM HepcD-FTH for 30 min; Lane 6, cells treated with 0.5 µM FTH for 2 h; Lane 7, cells treated with 0.5 µM HepcD-FTH for 2 h. (B) Cells non-treated with FAC. Lane 1, non-treated cells; lane 2, cells treated with 0.5 µM FTH for 30 min; Lane 3 and 4, cells treated with 0.5 µM HepcD-FTH during 30 min; Lane 5, cells treated with 0.5 µM FTH for 2 h; Lane 6 and 7, cells treated with 0.5 µM HepcD-FTH for 2h. (C) Western blot analysis of the J774 cells treated with 0.5 µM HepcH-FTH heteropolymer and FTH, using anti-FTH antibodies (rH02). Lane 1, non-treated cells; Lane 2, cells treated with 6 M guanidine; Lane 3, cells treated with 0.5 µM FTH for 30′; Lane 4, cells treated with 0.5 µM HepcH-FTH for 30′; Lane 5, cells treated with human hepcidin for 30 min; Lane 6, cells treated with FTH for 2 h; Lane 7, cells treated with HepcH-FTH for 2 h; Lane 8, cells treated with human hepcidin for 2 h. (D) Cells treated and non-treated with FAC and incubated in presence of FTH for 30 min and 2 h. NT, non-treated cells (CRT). (TIFF 142 kb)

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Boumaiza, M., Poli, M., Carmona, F. et al. Cellular binding analysis of recombinant hybrid heteropolymer of camel hepcidin and human ferritin H chain. The unexpected human H-ferritin binding to J774 murine macrophage cells. Mol Biol Rep 47, 1265–1273 (2020). https://doi.org/10.1007/s11033-019-05234-3

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Keywords

  • Camel hepcidin
  • Human H-ferritin
  • Heteropolymer, FAC
  • Ferroportin
  • J774 cells