Abstract
Hepcidin-25 was identified as the main iron regulator in the human body, and it by binds to the sole iron-exporter ferroportin. Studies showed that the N-terminus of hepcidin is responsible for this interaction, the same N-terminus that encompasses a small copper(II)-binding site known as the ATCUN (amino-terminal Cu(II)- and Ni(II)-binding) motif. Interestingly, this copper-binding property is largely ignored in most papers dealing with hepcidin-25. In this context, detailed investigations of the complex formed between hepcidin-25 and copper could reveal insight into its biological role. The present work focuses on metal-bound hepcidin-25 that can be considered the biologically active form. The first part is devoted to the reversed-phase chromatographic separation of copper-bound and copper-free hepcidin-25 achieved by applying basic mobile phases containing 0.1% ammonia. Further, mass spectrometry (tandem mass spectrometry (MS/MS), high-resolution mass spectrometry (HRMS)) and nuclear magnetic resonance (NMR) spectroscopy were employed to characterize the copper-peptide. Lastly, a three-dimensional (3D) model of hepcidin-25 with bound copper(II) is presented. The identification of metal complexes and potential isoforms and isomers, from which the latter usually are left undetected by mass spectrometry, led to the conclusion that complementary analytical methods are needed to characterize a peptide calibrant or reference material comprehensively. Quantitative nuclear magnetic resonance (qNMR), inductively-coupled plasma mass spectrometry (ICP-MS), ion-mobility spectrometry (IMS) and chiral amino acid analysis (AAA) should be considered among others.
Highlights
11..22..AATTCCUUNNMMoottiiff TThheeAATTCCUUNN mmoottiiff hhaass bbeeeenn ssttuuddiieedd ffoorr mmoorree tthhaann 5500 yyeeaarrss [[1100––1133]]
Plonka et al employed potentiometric titration and ultraviolet-visible light (UV-Vis) spectroscopy, which are widely recognized as the standard procedure for the calculation of stability constants of metal complexes [18,25], and characterized the flexible N-terminal hexapeptide of hepcidin-25 (DTHFPI) as the strongest ATCUN ligand ever reported with a dissociation constant of 10−14.66 M, which is even higher than the affinity of albumin for copper (KD = 10−12 M) [26]
Such extraordinary affinity of the N-terminus of hepcidin-25 for copper is supported by reports, which showed that the conserved presence of aspartic acid (D) as residue 1 in the ATCUN motif increases the basicity of the nitrogen atoms involved in the metal complex, and the copper binding is enhanced [28]
Summary
The interaction of human Hep-25 with metals was explored by other groups. Such extraordinary affinity of the N-terminus of hepcidin-25 for copper is supported by reports, which showed that the conserved presence of aspartic acid (D) as residue 1 in the ATCUN motif increases the basicity of the nitrogen atoms involved in the metal complex, and the copper binding is enhanced [28] Considering this high affinity and the concentration of Cu2+ in blood, it is reasonable to assume that a significant fraction of hepcidin-25 is present in the copper-bound form under physiological conditions. Publications regarding N-terminus of Hep-25 reported this motif to be unstructured in contrast to the rest of the molecule which is highly rigid due to four intramolecular disulfide bridges [5,6] This suggests that the N-terminal fragment could serve as a model for metal binding and structural studies, the results of which should be transferable to full-length hepcidin-25 (Figure 1). A 3D model of the copper complex of trout hepcidin-25 was proposed [9], based on the outdated structural template [6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
