Synthetic dimeric Aβ(28-40) mimics the complex epitope of human anti-Aβ autoantibodies against toxic Aβ oligomers

Abstract

Covalently linked carboxyl-terminal segments of the β-amyloid peptide (Aβ) were tested for their qualification as minimal conformational epitopes of the naturally occurring human autoantibodies against β-amyloid (nAbs-Aβ). nAbs-Aβ specifically recognize the toxic oligomers of Aβ and not the monomeric or the fibrillar forms of Aβ. The synthetic dimers of Aβ(28-40) described herein mimic the toxic Aβ oligomers but are not kinetic intermediates with uncertain compositions. CD spectra identified a surprisingly rich conformational behavior of selected miniamyloids. We observed a highly cooperative conformational transition of β-sheet to α-helix upon the addition of the helix enforcing co-solvent hexafluoroisopropanol. The CD curves of dimer 9 resembled, in a completely reversible manner, the CD spectra measured during the irreversible fibrillation of the parent Aβ(1-40). Synthetic peptide epitopes with high affinities for nAbs-Aβ are needed to identify the physiological roles of nAbs-Aβ and are promising epitopes for vaccination experiments.

Keywords: Alzheimer Disease; Amyloid; Autoantibodies; Neuroimmunology; Neurological Diseases; Peptide Chemical Synthesis; Peptide Conformation; Protein Aggregation.

FIGURE 1.

The aligned arrows depict possible linkages between the Aβ peptide segments. These covalently linked oligomers are named miniamyloids (left). Head-to-head dimers and head-to-tail dimers of the C-terminal part of Aβ are the minimal structural representatives of the parallel and antiparallel contacts of fibrous Aβ, respectively (middle). Both relative orientations are observed for the C-terminal epitope of Aβ in the proposed models (right (Ref. 19)) of Aβ fibrils (Ref. 24).

FIGURE 2.

Expansions of the ¹H NMR spectra (600 MHz, 10 mm KH₂PO₄ buffer pH 7, 300 K) of peptides 1–4. Positively and negatively charged groups are indicated on the Aβ(28–40) epitope, which itself is represented by an arrow. Despite the comparable sample concentrations, nearly no detectable ¹H NMR signal was observed for 1, and only broad signals were observed for 2, whereas 3 and 4 yielded high-resolution spectra. The signal assignments for 3 are given in the supplemental data.

FIGURE 3.

Circular dichroism spectra of the peptides listed in Table 1 and supplemental Fig. S3 (10 mm KH₂PO₄ buffer, pH 7, 4 °C). The miniamyloids are divided in two groups according to their CD curves (mean residue ellipticities). a, dimeric miniamyloids, 4, 9, 11, 13, 14, 18, and tetramer 7 exhibit a minimum between 216 and 218 nm. Additionally, monomer 13 fits into this group. b, all other monomers, 3, 5, 8, 10, 12, 15, and 17, appear to be unstructured with a strong negative band below 200 nm. The dimers that fall into this group have several N-terminal charges (peptides 14 and 16). More control peptides are listed in the supplemental data. The monomeric peptides, 1 and 2, are not included because they were not soluble.

FIGURE 4.

The CD spectrum of 9 varies with pH. a, the sign inversion of the maximal mean residue ellipticity at ∼190 nm and the concomitant decrease of the signal intensity at ∼215 nm in the opposite direction identifies an isosbestic point at 205 nm upon increasing the pH from 0 to 11. b, population analysis of the CD values at 190 and 215 nm showed a sigmoidal pH dependence (right). The β-sheet appears to be formed cooperatively at approximately pH 5.8. The ¹H NMR is not significantly influenced by pH (not shown).

FIGURE 5.

Shown is the HFIP titration of miniamyloid 9 in KH₂PO₄ buffer between 0 and 40% HFIP (a) and in between 40 and 100% HFIP (b). c, a population analysis identifies different conformational states of 9. d, schematic representation of the different conformational transitions of 9 during HFIP titration. I, β-turn; II, β-sheet; III, α-helix; IV, random coil. d, snapshots from molecular dynamics simulation of 9.

FIGURE 6.

a, dot blots corresponding to the antibody affinity of different preparations of Aβ(1–40). The antibody 6E10 recognizes Aβ regardless of its aggregation state. The antibody 6F/3D binds oligomeric or fibrillar Aβ but not monomeric Aβ. nAbs-Aβ specifically bind oligomeric Aβ. b, affinity chromatography with 9a yields antibodies (nAbs-9a) that bind preparations of oligomeric Aβ with affinities similar to nAbs-Aβ. The flow-through (ft-Aβ(1–40)), which are the remaining human antibodies following isolation of nAbs-Aβ from intravenous immunoglobulins (IVIg), served as a negative control.