Coordination abilities of the 1–16 and 1–28 fragments of β-amyloid peptide towards copper(II) ions: a combined potentiometric and spectroscopic study

Abstract

Stoichiometry, stability constants and solution structures of the copper(II) complexes of the (1–16H), (1–28H), (1–16M), (1–28M), (Ac-1–16H) and (Ac-1–16M) fragments of human (H) and mouse (M) β-amyloid peptide were determined in aqueous solution in the pH range 2.5–10.5. The potentiometric and spectroscopic data (UV–Vis, CD, EPR) show that acetylation of the amino terminal group induces significant changes in the coordination properties of the (Ac-1–16H) and (Ac-1–16M) peptides compared to the (1–16H) and (1–16M) fragments, respectively. The (Ac-1–16H) peptide forms the 3N {N_Im⁶, N_Im¹³, N_Im¹⁴} complex in a wide pH range (5–8), while for the (Ac-1–16M) fragment the 2N {N_Im⁶, N_Im¹⁴} complex in the pH range 5–7 is suggested. At higher pH values sequential amide nitrogens are deprotonated and coordinated to copper(II) ions. The N-terminal amino group of the (1–16) and (1–28) fragments of human and mouse β-amyloid peptide takes part in the coordination of the metal ion, although, at pH above 9 the complexes with the 4N {N_Im, 3N⁻} coordination mode are formed. The phenolate –OH group of the Tyr¹⁰ residue of the human fragments does not coordinate to the metal ion.

Introduction

Patients with Alzheimer’s disease (AD) contain large quantities of insoluble amyloid plaques that are primarily found in brain tissue. A major component of amyloid plaque is the β-peptide (Aβ) [1], [2], a small (39–43 amino acids) polypeptide with heterogenous termini that is generated from the cleavage of a larger amyloid precursor protein (APP) [3], [4]. Amyloid deposition is likely a critical step in the neurodegenerative processes associated with AD [5]. Amyloid plaques are invariably associated with areas of nerve death, and the injection of synthetic β-peptides directly into rat brain produced cytotoxic effects [6]. It has also recently been established that soluble extracellular β-peptide is normally produced in cultured cells and human biological fluids [7], [8].

There is an emerging consensus in the literature to indicate that the homeostases of zinc, copper and iron are significantly altered in AD brain tissue [9]. Evidence for abnormal Cu homeostasis in AD includes a 2.2-fold increase in the concentration of cerebrospinal fluid (CSF) Cu [10], and an accompanying increase in ceruloplasmin in the brain and CSF of AD patients [11]. Aβ is resolubilized and extracted from postmortem AD and non-AD control brains using metal chelators. High-affinity Cu/Zn/Fe chelators markedly enhanced the resolubilization of AD deposits from postmortem AD and non-AD brain samples [12]. Aβ in vitro binds metal ions, including Zn²⁺, Cu²⁺, and Fe³⁺, inducing peptide aggregation that may be reversed by treatment with chelators such as ethylenediaminetetraacetic acid (EDTA) [13], [14]. Rats and mice do not develop amyloid [15], probably due to the three amino acid substitutions in their homologue of Aβ (Arg⁵→Gly, Tyr¹⁰→Phe, and His¹³→Arg) [16]. These changes have been shown to alter the structure and properties of the Aβ peptides [17], [18], [19] as well as the processing of APP. These sequence alternations might therefore be responsible for the virtual absence of Aβ deposits in normal or aged rodent brain [19]. In vitro it has been shown that, compared with human Aβ, rat Aβ binds Zn²⁺ and Cu²⁺ less avidly [13], that the coordination of Cu²⁺ or Fe³⁺ does not induce redox chemical reactions, and that limited reactive oxygen species are generated [20]. Through the use of synthetic peptide corresponding to the first 28 residues of human Aβ, rat Aβ, and single-residue variations, Liu et al. [21] reported a key role for His¹³ in the zinc interaction. Substitution His/Ala of the potential histidine ligands in Aβ suggests that residues His¹³ and His¹⁴ represent a critical domain for zinc interaction [22]. On the basis of nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) experiments, it has been proposed that Aβ binds Cu²⁺ via three His residues (His⁶, His¹³ and His¹⁴) and an oxygen ligand, probably Tyr¹⁰ [23].

Peptide complexes with metal ions have been extensively studied in order to mimic specific metalloprotein structures and functions [24], [25]. A prominent role of some amino acid side chains and, in particular, the imidazole moieties of histidine in the binding of metal ion has been observed in proteins and in natural [26] or synthetic peptides [27], [28]. The studies of metal ion–peptide complex formation reactions indicate that solution equilibria are rather complicated especially in the presence of coordinating side chains. For the determination of formation constants of peptide complexes potentiometry is usually used, but for the justification of potentiometric results the combined application of various spectroscopic techniques: UV–visible (UV–Vis), EPR, NMR and/or circular dichroism (CD) spectroscopies is required.

The interaction of copper(II) ions with the (1–6), (1–9), (1–10) [29] (contain one His⁶ residue) and (11–16) [30], (11–20), (11–28) [31] human (contain two His¹³ and His¹⁴ residues) and mouse (one His¹⁴ residue) fragments of β-amyloid peptide was studied. The differences of the binding mode of human and mouse fragments to copper(II) ions and stability constants for the complexes formed were determined.

The present paper reports the results of combined spectroscopic and potentiometric studies on the copper(II) complexes of the (1–16) and (1–28) human (three His⁶, His¹³, His¹⁴ residues) and mouse (two His⁶, His¹⁴ residues) fragments of β-amyloid peptide. The β-amyloid fragments studied here are: human peptide (1–16H), H-Asp–Ala–Glu–Phe–Arg–His–Asp–Ser–Gly–Tyr–Glu–Val–His–His–Gln–Lys-NH₂, DAEFRHDSGYEVHHQK-NH₂ and mouse peptide (1–16M), H-Asp–Ala–Glu–Phe–Gly–His–Asp–Ser–Gly–Phe–Glu–Val–Arg–His–Gln–Lys-NH₂, DAEFGHDSGFEVRHQK-NH₂; the fragments (1–28), human peptide (1–28H), H-Asp–Ala–Glu–Phe–Arg–His–Asp–Ser–Gly–Tyr–Glu–Val–His–His–Gln–Lys–Leu–Val–Phe–Phe–Ala–Glu–Asp–Val–Gly–Ser–Asn–Lys-NH₂, DAEFRHDSGYEVHHQKLVFFAEDVGSNK-NH₂ and mouse peptide (1–28M) H-Asp–Ala–Glu–Phe–Gly–His–Asp–Ser–Gly–Phe–Glu–Val–Arg–His–Gln–Lys–Leu–Val–Phe–Phe–Ala–Glu–Asp–Val–Gly–Ser–Asn–Lys-NH₂, DAEFGHDSGFEVRHQKLVFFAEDVGSNK-NH₂. To determine the involvement of N-terminal amino group in the metal ion coordination, the analogues with blocked N-terminal amino group (by acetylation) of the human and mouse fragments (1–16): human (Ac-1–16H), Ac-Asp–Ala–Glu–Phe–Arg–His–Asp–Ser–Gly–Tyr–Glu–Val–His–His–Gln–Lys-NH₂, Ac-DAEFRHDSGYEVHHQK-NH₂; mouse (Ac-1–16M), Ac-Asp–Ala–Glu–Phe–Gly–His–Asp–Ser–Gly–Phe–Glu–Val–Arg–His–Gln–Lys-NH₂, Ac-DAEFGHDSGFEVRHQK-NH₂ were also studied. These fragments contain the complete bonding site of Aβ and, with the exception of the C-terminal carboxylate, are representative of the full-length peptide. Therefore, the 1–16 and 1–28 fragments are considered as valid models to examine the contribution of the key histidine residues (His⁶, His¹⁴ in mouse and His⁶, His¹³, His¹⁴ in human fragments) to the Aβ–Cu²⁺ interaction. This study was performed in order to examine the difference of the binding ability of the human and mouse fragments, especially the effect of the substitutions Arg with Gly, Tyr with Phe and His with Arg residues in positions 5, 10 and 13, respectively, on the formation of complexes with Cu²⁺ ions. The (17–28) fragment of β-amyloid peptide does not contain any additional bonding site to copper(II) ions, however, the influence of these residues on the stability of the complexes formed may be estimated.