Copper(II) complexes of neuropeptide gamma with point mutations (S8,16A) products of metal-catalyzed oxidation

https://doi.org/10.1016/j.jinorgbio.2013.08.011Get rights and content

Highlights

  • The acid-base properties of the neuropeptide gamma with point mutations (S8,16A) were determined.

  • The potentiometric measurements were performed in 2.5 – 10.5 pH range.

  • The stoichiometry and stability constants for the copper(II) complexes are determined.

  • For the complexes at pH 7.4 the products of copper(II)-catalyzed oxidation are given.

Abstract

To obtain the information about the influence of the serine residues (S8,S16) on the acid–base properties of the neuropeptide gamma, the peptide with point mutations (S8,16A) and its N-acetyl derivative were synthesized. Any additional deprotonations were not observed. It means that the presence of serine residues is necessary in the amino acid sequence of the neuropeptide gamma to have its acid–base properties. The stability constants, stoichiometry and solution structures of copper(II) complexes of the neuropeptide gamma mutants D1AGH4GQIA8H9KRH12KTDA16FVGLM21-NH2 (S8,16A) 2ANPG and its N-acetyl derivative Ac-2ANPG were determined in aqueous solution. The equilibrium and structural properties of copper(II) complexes have been characterized by pH-metric, spectroscopic (UV–visible, CD, EPR) and mass spectrometric (MS) methods. At physiological pH 7.4 the 2ANPG forms the CuH2L and CuHL complexes in equilibrium with 3N {NH2,βCOO-D1,2NIm} and 4N {NH2,N,2NIm} binding sites, respectively. The exchange Ser on Ala residues does not alter the coordination mode of the peptide. To elucidate the products of the copper(II)-catalyzed oxidation of 2ANPG and Ac-2ANPG the liquid chromatography–mass spectrometry method (LC–MS) and the Cu(II)/H2O2 as a model oxidizing system were employed. For solutions containing a 1:4 peptide–hydrogen peroxide molar ratio oxidation of the methionine residue to methionine sulphoxide was observed. For the 1:1:4 Cu(II)–2ANPG–H2O2 system oxidation of two His residues and cleavage of the G3single bondH4 peptide bond was observed, while for the 1:1:4 Cu(II)–Ac-2ANPG–H2O2 system oxidation of three histidine residues to 2-oxohistidines was also observed.

Graphical abstract

The neuropeptide gamma with point mutations (S8,16A) and its N-acetyl derivative were studied. The potentiometric measurements were performed in 2.5–10.5 pH range, spectroscopic and mass spectrometric study of Cu(II) binding to the neuropeptide gamma with point mutations and its acetyl derivative were carried out. The products of the copper(II)-catalyzed oxidation are determined.

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Introduction

Tachykinin peptide neurotransmitters form a large functional group of signaling peptides in mammals, amphibia, mollusks, and invertebrates [1]. Mammalian tachykinins, previously named as neurokinins, consist of a family of three primary functional peptides: substance P, neurokinin A and neurokinin B [1], [2]. To this family of peptides recently classified peptides: neuropeptide K [3], neuropeptide gamma [4] as well as hemokinin-1 [5] and endokinins A–D [6]. Tachykinins comprise a group of neuropeptides that share a common carboxy-terminal amino acid sequence, Phe-X-Gly-Leu-Met-NH2, where X is a hydrophobic residue that is either an aromatic or a betabranched aliphate [2].

Tachykinins act through binding on specific receptors, designated as tachykinin NK1, NK2 and NK3 receptors, each exhibiting a characteristic rank-order of affinity [7]. Tachykinins are widely distributed in the central nervous system and have roles as neurotransmitters and/or neuromodulators [2] and they are implicated in many physiological and pathological processes [8]. Peptides belong to tachykinin family formed amyloid-like fibrils in vitro in the presence of heparin, and these amyloids were found to be nontoxic in neuronal cells [9]. Recent studies have reported that these neuropeptides may modulate the toxic aggregation pathways of amyloid beta associated with Alzheimer's disease and a specific interaction is formed between tachykinins and amyloid beta [10].

Neuropeptide gamma is a 21-amino-acid peptide containing sequence as follows: Asp1-Ala-Gly-His4-Gly-Gln-Ile-Ser-His9-Lys-Arg-His12-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met21-NH2 [11]. Neuropeptide gamma is involved in many biological responses such as broncoconstriction, vasodepression, increase in heart rate, stimulation of salivary secretion and antidispogenic action [12], [13], [14]. Several reports have indicated that this peptide act on the hypothalamic–pituitary–gonadal axis to regulate functions related to reproduction and modulate the regulation of growth hormone secretion [12], [13]. Neuropeptide gamma has been also identified as the most potent contractile tachykinin in human isolated bronchus and an important mediator of inflammatory lung disorders, e.g., asthma [11].

Copper is an essential trace element to human health [15]. Cu is a cofactor and/or a structural component of a number of enzymes. Copper-dependent enzymes are involved in redox reactions and participate in important biochemical pathways including energy metabolism (e.g., cytochrome c oxidase), antioxidative defense (e.g., Cu/Zn superoxide dismutase) and iron metabolism (e.g., ceruloplasmin) [16]. As transition metal, copper can exist in a variety of oxidation states, a reduced Cu(I) state and oxidized Cu(II) state [15], [17].

Under aerobic conditions, this redox property enables copper to catalyze the production of hydroxyl radicals via the Fenton and Haber–Weiss reactions. The hydroxyl radical is reactive with most types of macromolecules, resulting in damage to lipids, proteins, and nucleic acids [18]. The hydroxyl radical has a very short lifetime (10 9 s) and is reactive with practically any biological molecules near the site of its formation [19]. It has been suggested that oxidative damage to biological molecules is a determinant factor in a number of diseases, such as cardiovascular disease, cancer, Parkinson's disease, inflammation and rheumatoid arthritis [20], [21].

The present paper reports the results of combined spectroscopic and potentiometric studies on the copper(II) complexes of the modified neuropeptide gamma (2ANPG) with point mutations (S8,16A) D1AGH4GQIA8H9KRH12KTDA16FVGLM21-NH2 (2ANPG). To determine the involvement of N-terminal amino group in the metal ion coordination, the analog with blocked N-terminal amino group (by acetylation) of the neuropeptide gamma with point mutations (S8,16A): Ac-D1AGH4GQIA8H9KRH12KTDA16FVGLM21-NH2 (Ac-2ANPG) was also studied.

Our earlier studies on the acid–base properties and coordination abilities towards copper(II) of the neuropeptide gamma were performed at pHs 2.5–7.4. At higher pH the protons from the neuropeptide gamma were released and good fitting of the experimental data to calculate at a pH higher than 7.4 was impossible [22]. Moreover, the acid–base properties and coordination abilities towards copper(II) of the neurokinin A [23], neuropeptide gamma and its fragments [22], [24], [25], and neuropeptide K fragments [26] (all peptides contain the neurokinin sequence in their C-terminal), clearly indicate the presence of additional deprotonation of the ligands studied. The CID MS/MS analysis (collision-induced dissociation tandem mass spectrometry analysis) of the neurokinin A [23] indicates that Ser5 may be particularly responsible for this additional deprotonation. For the neurokinin A with point mutation (S5A) and its N-acetyl derivative the additional deprotonation was not detected. It suggests that the presence of the serine residue in the tachykinin peptides with the C-terminal sequence of neurokinin A is necessary to have additional deprotonation [27].

We would like to demonstrate the influence of the mutations (S8,16A) especially on the acid–base properties of the neuropeptide gamma and its derivative and their complexation abilities towards copper(II) ions. We would like to know if the presence of the serine residues in the neuropeptide gamma sequence beside the histidine residues is necessary to have the labile protons.

The copper(II) complexes were studied by the combined application of potentiometric equilibrium, spectroscopic UV–visible (UV–VIS), CD, EPR and mass spectrometric (MS) methods. The present paper also presents the products of the copper(II)-catalyzed oxidation on the base of binding sites of the peptides to copper(II) ions at pH 7.4. We demonstrate the relationship between the binding sites of copper(II) ions and products of oxidation for the ligands studied.

Section snippets

Synthesis of peptides

Syntheses of peptides 2ANPG and Ac-2ANPG were performed on a polystyrene/polyethylene glycol copolymer resin (TentaGel R RAM Resin, substitution 0.18 mmol/g) using Fmoc strategy with continuous-flow methodology [28], [29], [30]. Acetylation of the N-terminal amino group was performed on the resin using 1 M acetylimidazole in dimethylformamide (DMF). Both peptides were cleaved from the resin and deprotected by 2 h shaking in a mixture containing 94% of trifluoroacetic acid (TFA), 2.5% of H2O, 2% of

Protonation constants

Global formation (log β) and protonation (log K) constants of the neuropeptide gamma with two point mutations (S8,16A) (2ANPG) and its N-acetyl derivative (Ac-2ANPG) have been determined by potentiometric titrations, and the data are given in Table 1. These peptides in the investigated 2.5  10.5 pH range have 8 and 7 protonation sites, respectively. The 2ANPG molecule contains two lysyl (K10, K13), three histydyl (H4, H9, H12), two aspartic acids (D1, D15) residues and N-terminal amine group. The

Conclusions

The neuropeptide gamma, because of the additional deprotonations at pH above 7.4 was pH-metric investigated in the pH 2.5–7.4 range. The exchange of the Ser (S8,S16) residues on the Ala makes possible the measurements in whole of the 2.5–10.5 pH range. It means that Ser residues in the amino acid sequence of the neuropeptide gamma play an important role in its acid–base properties. It is likely that these Ser residues take place in the hydrogen bonds and in the formation of labile protons which

Acknowledgment

Financial support from the National Science Centre (NCN) for Scientific Grant 2011/01/N/ST5/02563 has been gratefully acknowledged.

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