The N-terminus of hepcidin is essential for its interaction with ferroportin: structure-function study

Abstract

Hepcidin is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid (aa) peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in mice by the induction of hypoferremia. Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when 5 N-terminal aa's were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin and did not interfere with ferroportin internalization by native hepcidin. Deletion of 2 C-terminal aa's did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not affect peptide activity in vitro. However, these peptides were less active in vivo, likely because of their decreased stability in circulation. G71D and K83R, substitutions previously described in humans, did not affect hepcidin activity. Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations.

Figure 1.

Schematic representation of hepcidin derivatives. Hatched circles represent deleted residues. Solid light gray circles represent substitutions. The numbering system refers to hep25, so that residues 1 to 25 correspond to residues 60 to 84 of the prepropeptide. In conformity with the original reports of these mutations, G71D and K83R refer to the prepropeptide numbering system.

Figure 2.

Representative CD spectra of parent hep25 peptide and disulfide bond variants. Hep25 (solid lines) was compared to hepcidin C13A/C14A (A), hepcidin C7A/C23A (B), hepcidin C11A/C19A (C), and hepcidin 1SS (del9-12 [FCCG], del19-22 [CGMC], C13A/C14A) (D).

Figure 3.

Relative activity of hepcidin derivatives. (A) Flow cytometry measuring degradation of ferroportin-GFP. (B) Ferritin ELISA measuring cellular iron retention. For both panels, the values were expressed as a fraction of hep25 activity. Peptides were added to cells for 24 hours at 1 μM concentration, except for hep3 and hep6, which were added at 4 μM. 25 = hep25; 24 to 20 = del1(D) to del1-5(DTHFP); 3 = hep3 (DTH); 6 = hep6 (DTHFPI); 26 = hep26(+A); dKT = del24-25(KT); AA1 = C7A/C13A; AA3 = C11A/C19A; AA4 = C13A/C14A; 1SS = [del9-12(FCCG), del19-22(CGMC), C13A/C14A]; zebra = zebra fish. The activities corresponding to the unmodified, naturally occurring active forms of hepcidin are shown as black bars.

Figure 4.

Activity of hepcidin derivatives in vivo. Diluent or peptides (50 μg/mouse) were injected in mice, and serum iron levels were determined after 2 hours. P values (Student t test) above bars denote statistically significant difference in comparison to PBS-injected mice. 25 = hep25; 20 = hep20 [del1-5(DTHFP)]; dKT = del24-25(KT); AA1 = C7A/C13A; AA3 = C11A/C19A; AA4 = C13A/C14A; 1SS = [del9-12(FCCG), del19-22(CGMC), C13A/C14A]. The activity corresponding to the naturally occurring active form of hepcidin is shown as a black bar. PBS and hep25 were each tested in 10 mice, and 4 to 6 mice were used for each of the other peptides.