An Overlooked Hepcidin-Cadmium Connection

Abstract

Hepcidin (DTHFPICIFCCGCCHRSKCGMCCKT), an iron-regulatory hormone, is a 25-amino-acid peptide with four intramolecular disulfide bonds circulating in blood. Its hormonal activity is indirect and consists of marking ferroportin-1 (an iron exporter) for degradation. Hepcidin biosynthesis involves the N-terminally extended precursors prepro-hepcidin and pro-hepcidin, processed by peptidases to the final 25-peptide form. A sequence-specific formation of disulfide bonds and export of the oxidized peptide to the bloodstream follows. In this study we considered the fact that prior to export, reduced hepcidin may function as an octathiol ligand bearing some resemblance to the N-terminal part of the α-domain of metallothioneins. Consequently, we studied its ability to bind Zn(II) and Cd(II) ions using the original peptide and a model for prohepcidin extended N-terminally with a stretch of five arginine residues (5R-hepcidin). We found that both form equivalent mononuclear complexes with two Zn(II) or Cd(II) ions saturating all eight Cys residues. The average affinity at pH 7.4, determined from pH-metric spectroscopic titrations, is 10^10.1 M^-1 for Zn(II) ions; Cd(II) ions bind with affinities of 10^15.2 M^-1 and 10^14.1 M^-1. Using mass spectrometry and 5R-hepcidin we demonstrated that hepcidin can compete for Cd(II) ions with metallothionein-2, a cellular cadmium target. This study enabled us to conclude that hepcidin binds Zn(II) and Cd(II) sufficiently strongly to participate in zinc physiology and cadmium toxicity under intracellular conditions.

Keywords: MT2A; affinity constant; cadmium; hepcidin; metallothionein; prohepcidin.

Figure 1

Titration of 12.57 μM hepcidin-25 with CdCl₂ in the presence of 0.5 mM tris(2-carboxyethyl)phosphine (TCEP) at pH 5.0. (A). The spectra featuring the CT band at 250 nm formed upon the increasing Cd(II)/hepcidin-25 ratios (color coded in the plot); the arrow marks the direction of changes; (B). The titration curve at 250 nm. The red and blue segments of the curve were used to define the straight lines of the binding-site saturation. Their crossing at 1.80 Cd(II) equivalents determines the saturation concentration.

Figure 2

The pH-metric titration of 12.2 μM hepcidin-25 with 2 eq. of CdCl₂ in the presence of 0.5 mM TCEP. (A). UV-vis spectra; (B). CD spectra, with the common color codes for pH in plot A; arrows mark the increase of the Cd(II)-S CT bands; (C). The pH dependence of CT bands at 250 nm derived from UV-vis (black dots) and CD (red dots) spectra, presented in the common relative scale; (D). Fitting of the two-sites binding model to the normalized UV-vis data at 250 nm.

Figure 3

The pH-metric titration of 15.5 μM hepcidin-25 with 2 eq. of ZnCl₂ in the presence of 0.5 mM TCEP. (A). UV-vis spectra; (B). CD spectra, with the common color codes for pH in plot A; (C). The pH dependence of A₂₃₀ in the UV-vis spectra, showing the Zn(II) binding range (red box) and the loss of solution transparency due to peptide precipitation; (D). Fitting of the one-site binding model to the CD data at 230 nm. Arrows point to pH regions pertaining to comments in plots.

Figure 4

Integrated native MS signals of Cd₂hepcidin-25 (black dots) and Cd₂5R-hepcidin (red dots) obtained from the titration of 10 μM Cd₂hepcidin-25 with 5R-hepcidin. The lines represent fits for the competition constant for the reaction Cd₂hepcidin-25 + 5R-hepcidin = hepcidin-25 + Cd₂5R-hepcidin. The MS spectra indicated only low amounts of other metallated species (see Figure S3).

Figure 5

(A). 100 μM Zn₂5R-hepcidin titrated with Cd(II) acetate in 100 mM HEPES pH 7.4. (B). The comparison of A₂₆₀ values for Cd(II) titrations of 100 μM 5R-hepcidin (black dots, data from Figure S2) and Zn₂5R-hepcidin (red dots).

Figure 6

(A). 100 μM Cd₂5R-hepcidin titrated with Zn(NO₃)₂ in 100 mM HEPES pH 7.4. Numbers in the legend denote molar excess of Zn(II) ions. (B). Titration curve at 275 nm and the fit for the competition reaction Cd₂5R-hepcidin + Zn(II) = CdZn5R-hepcidin. The 95% confidence bands are marked in red.

Figure 7

Deconvoluted Native MS spectra of 10 μM 5R-hepcidin and 20 μM Cd(II) acetate with the following concentrations of Zn₇MT2A: (A) 1 μM, (B) 3 μM, (C) 10 μM, (D) 30 μM in ammonium acetate pH 7.4.

Figure 8

Quantitation obtained by careful integration of relevant signals from native MS experiment of 10 μM 5R-hepcidin and 20 μM Cd(II) acetate titrated with Zn₇MT2A in ammonium acetate, pH 7.4. 5R-hepcidin (blue), Cd5R-hepcidin (red), Cd₂5R-hepcidin (black).