The Role of Copper (II) on Kininogen Binding to Tropomyosin in the Presence of a Histidine-Proline-Rich Peptide

Abstract

The antiangiogenic activity of the H/P domain of histidine-proline-rich glycoprotein is mediated by its binding with tropomyosin, a protein exposed on endothelial cell-surface during the angiogenic switch, in presence of zinc ions. Although it is known that copper ion serum concentration is significantly increased in cancer patients, its role in the interaction of H/P domain with tropomyosin, has not yet been studied. In this paper, by using ELISA assay, we determined the modulating effect of TetraHPRG peptide, a sequence of 20 aa belonging to H/P domain, on the binding of Kininogen (HKa) with tropomyosin, both in absence and presence of copper and zinc ions. A potentiometric study was carried out to characterize the binding mode adopted by metal ions with TetraHPRG, showing the formation of complex species involving imidazole amide nitrogen atoms in metal binding. Moreover, circular dichroism showed a conformational modification of ternary systems formed by TetraHPRG, HKa and copper or zinc. Interestingly, slight pH variation influenced the HKa-TetraHPRG-tropomyosin binding. All these results indicate that both metal ions are crucial in the interaction between TetraHPRG, tropomyosin and HKa.

Keywords: angiogenesis; circular dichroism; copper; histidine–proline-rich glycoprotein; kininogen; tropomyosin; zinc.

Figure 1

Binding rate of HKa* at 80 nM in MOPS pH 7.5. Dose response effect of tetra repeat in coincubation in presence or Zn²⁺ or Cu²⁺ (10 µM). Data are the mean ± SEM of three different experiments performed in triplicate. Statistically significant differences are indicated with * = p < 0.05 and **** = p < 0.0001 vs. HKa 80 nM (One-Way ANOVA + Dunnett’s test).

Figure 2

Circular dichroism (CD) spectra of (a) copper (II) and (b) zinc (II) complexes with TetraHPRG in water (pH = 7.5), at different mol equivalent of metal to ligand molar ratio is 1:1 and 2:1; [L] = 1 × 10⁻⁵ M).

Figure 3

Species distribution diagram for the Zn²⁺ complexes with TetraHPRG at 0.5:1 (M:L) molar ratio. [Zn²⁺] = 1 × 10⁻³ M.

Figure 4

CD spectra Hka, TetraHPRG, and Hka/TetraHPRG in water (pH = 7.5), [L] = 1 × 10⁻⁵ M.

Figure 5

CD spectra of (a) copper(II) and (b) zinc(II) complexes with Hka in water (pH = 7.5), at different mol equivalent of metal (metal to ligand molar ratio is 1:1 and 2:1; [L] = 1 × 10⁻⁵ M).

Figure 6

CD difference spectra of the (a) Hka/TetraHPRG/ Cu²⁺ ternary system minus CD spectra from TetraHPRG/Cu²⁺ systems and CD spectra of the Hka/ Cu²⁺ at 1:1 metal to ligand ratios and (b) CD difference spectra of the Hka/TetraHPRG/Zn²⁺ ternary system minus normalized CD spectra from TetraHPRG/Zn²⁺ systems and CD spectra of the Hka/Zn²⁺ at 1:1 metal to ligand ratios.

Figure 7

Percentage of HKa* binding to tropomyosin at different pH. HKa* (80 nM) in MOPS pH 7.5 (a) or pH 8.0 (b), in presence or not of 25 uM of TetraHPRG and in presence or not of CuSO₄ or ZnCl₂ (10 µM). Data are the mean ± SEM of three different experiments performed in triplicate. Statistically significant differences are indicated with *** = p < 0.001 and **** = p < 0.0001 vs. HKa 80 nM (One-Way ANOVA + Dunnett’s test).

Scheme 1

ELISA assay steps in conditions of coincubation (down) and pre-treatment (up). HKa* (Kininogen activated), Me²⁺ (Cu²⁺ or Zn²⁺), Av-HRP (avidin conjugated with horseradish peroxidase), TMB (3,3-5,5-tetramethylbenzidine).

Figure 8

% Binding of HKa* to plate previously coated with tropomyosin (left group) or not (right). After tropomyosin the plate has been blocked with milk 5% and the pretreated with TetraHPRG at 25 µM before to the addition of HKa* (80 nM) in MOPS pH 7.5 in presence or not of CuSO₄ or ZnCl₂ (10 µM). Data are the mean ± SEM of three different experiments performed in triplicate. Statistically significant differences are indicated with **** = p <0.0001 vs. HKa 80 nM (One-Way ANOVA + Dunnett’s test).