The English (H6R) Mutation of the Alzheimer's Disease Amyloid-β Peptide Modulates Its Zinc-Induced Aggregation

Abstract

The coordination of zinc ions by histidine residues of amyloid-beta peptide (Aβ) plays a critical role in the zinc-induced Aβ aggregation implicated in Alzheimer's disease (AD) pathogenesis. The histidine to arginine substitution at position 6 of the Aβ sequence (H6R, English mutation) leads to an early onset of AD. Herein, we studied the effects of zinc ions on the aggregation of the Aβ42 peptide and its isoform carrying the H6R mutation (H6R-Aβ42) by circular dichroism spectroscopy, dynamic light scattering, turbidimetric and sedimentation methods, and bis-ANS and thioflavin T fluorescence assays. Zinc ions triggered the occurrence of amorphous aggregates for both Aβ42 and H6R-Aβ42 peptides but with distinct optical properties. The structural difference of the formed Aβ42 and H6R-Aβ42 zinc-induced amorphous aggregates was also supported by the results of the bis-ANS assay. Moreover, while the Aβ42 peptide demonstrated an increase in the random coil and β-sheet content upon complexing with zinc ions, the H6R-Aβ42 peptide showed no appreciable structural changes under the same conditions. These observations were ascribed to the impact of H6R mutation on a mode of zinc/peptide binding. The presented findings further advance the understanding of the pathological role of the H6R mutation and the role of H6 residue in the zinc-induced Aβ aggregation.

Keywords: English mutation; aggregation; amyloid-β; zinc.

Figure 1

Dependence of turbidity (optical density at 405 nm, OD₄₀₅) of Aβ42 and H6R-Aβ42 preparations on the incubation time after the addition of Zn²⁺ at different Zn²⁺/peptide molar ratios. Panel (A)—Aβ42; panel (B)—H6R-Aβ42. Curves 1, 2, 3, 4, and 5 correspond to the Zn²⁺/peptide molar ratios of 0, 1, 2, 3, and 4, respectively. Peptides in buffer H (10 mM HEPES, pH 6.8, 50 mM NaCl); peptide concentration—25 µM. Data points are means of three measurements; relative standard deviations for any of the points did not exceed 15%. The insert in panel (A) shows the turbidity at the 30th min of incubation as a function of the Zn²⁺/peptide molar ratio. Curves I and II—Aβ42 and H6R-Aβ42 preparations, respectively.

Figure 2

The characteristic diameter (D) of Zn²⁺-induced Aβ aggregates, measured after 30-min incubation as a function of the Zn²⁺/peptide molar ratio. Curves 1 and 2—Aβ42 and H6R-Aβ42 preparations, respectively. Peptides in buffer H (10 mM HEPES, pH 6.8, 50 mM NaCl); peptide concentration—25 µM. The means and standard deviations for three measurements are shown.

Figure 3

Circular dichroism spectra (A,B) and fractions of the secondary structure components drawn from the spectra with CDNN software (C,D). Peptides Aβ42 (A,C) and H2R-Aβ42 (B,D). CD spectra were collected in 20 min of incubation either in the absence or in the presence of Zn²⁺. Each CD spectrum is an average of three separate measurements. “Beta-Sheet” is a sum of parallel and antiparallel β-sheet components. The Zn²⁺ concentrations are indicated by color: Black—no Zn²⁺ added, blue and red—50 and 100 µM Zn²⁺, respectively. Peptides in buffer H (10 mM HEPES, pH 6.8, 50 mM NaCl); peptide concentration—25 µM.

Figure 4

Dependencies of fluorescence of bis-ANS/Aβ complexes, F, on the Zn²⁺/peptide molar ratio. The fluorescence was measured 30 min after the addition of Zn²⁺. Curves 1 and 2—Aβ42 and H6R-Aβ42, respectively. Peptides in buffer H (10 mM HEPES, pH 6.8, 50 mM NaCl); peptide concentration—25 µM. Bis-ANS/peptide molar ratio—1:10. The means and standard deviations for triplicate measurements are shown.