Interaction between human prion protein and amyloid-beta (Abeta) oligomers: role OF N-terminal residues

Abstract

Soluble oligomers of Abeta42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrP(C), on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128-1132). Here we characterized the interaction between synthetic Abeta42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met(129) and Val(129) polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with Abeta oligomers. It was also found that in addition to the previously identified approximately 95-110 segment, the second region of critical importance for the interaction with Abeta42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23-27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for Abeta42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well.

FIGURE 1.

Atomic force microscopy images of different types of Aβ42 aggregates used in the present study. A, oligomers at low magnification; B, the same oligomers at higher magnification; C, amyloid fibrils; D, amyloid fibrils fragmented by sonication. The scale bars correspond to 1 μm.

FIGURE 2.

Binding of different forms of Aβ42 to full-length huPrP as probed by surface plasmon resonance. A, sensorgrams for soluble Aβ42 oligomers at different concentrations. The concentrations (in nm) are indicated as a number at each curve. The arrow indicates the end of the association phase (i.e. the point at which ligand-containing buffer was replaced with ligand-free buffer). RU, resonance units. B, sensorgrams for Aβ42 monomers, soluble oligomers, long fibrils, and fibrils fragmented by sonication (2 μm in each case). huPrP (Met¹²⁹ polymorphic form) was immobilized on the surface of a CM5 sensor chip at a density corresponding to ∼4000 resonance units.

FIGURE 3.

The effect of deletions in huPrP on Aβ42 binding as probed by surface plasmon resonance. A, schematic diagram of huPrP deletion variants used in this study. The unstructured N-terminal domain and the folded C-terminal domain are depicted in gray and black, respectively. The deleted regions are indicated as dashed bars. The disulphide bond between Cys¹⁷⁹ and Cys²¹⁴ is indicated by S-S. B and C, representative sensorgrams for binding of Aβ42 oligomers to deletion variants of huPrP (Met¹²⁹ polymorphic form). Prion protein variants were immobilized on the surface of a CM5 sensor chip at a density corresponding to ∼4000 resonance units (RU) (4000 ± 300), and the Aβ42 oligomers were injected at a concentration of 2 μm.

FIGURE 4.

Binding of different forms of Aβ42 to full-length huPrP as probed by EPR spectroscopy. A, EPR spectrum for free huPrP labeled at residue 30 (30R huPrP) shows three relatively sharp features indicating high mobility of the nitroxide label. B, the spectrum of the protein in the presence of a saturating concentration of Aβ42 oligomers is characteristic of highly immobilized nitroxide label, indicating binding of huPrP to the oligomers. C–E, no huPrP binding was detected to Aβ42 monomer (C), Aβ42 amyloid fibrils (D), or fibrils fragmented by sonication (E). The concentration of 30R huPrP (Met¹²⁹) was 2 μm, and the concentration of Aβ42 was 100 μm in each case. For visualization purposes, spectra are scaled to the same vertical size.

FIGURE 5.

Determination of apparent equilibrium binding parameters for the interaction between Aβ42 oligomers and huPrP. A, representative EPR spectra for 30R huPrP at varying concentrations of Aβ42 oligomers. The concentration of 30R huPrP was 2 μm, and the concentration of Aβ42 is shown at each spectrum. The intensity of the low field sharp feature (labeled a) is a measure of the amount of free huPrP; the broad feature b arises from 30R huPrP bound to Aβ42 oligomers. For visualization purposes, spectra are scaled to the same vertical size. B, binding isotherm for Aβ42 oligomer interaction with 30R huPrP. θ represents the fraction of bound 30R huPrP as determined from relative intensity of the low field sharp feature in EPR spectra (see “Results”). The equilibrium binding isotherm was analyzed according to Equation 3 (see “Experimental Procedures”), yielding the apparent dissociation constant, K_d, of 71 nm and the number of Aβ42 monomers per binding site of 21.

FIGURE 6.

The effect of deletions in huPrP on Aβ42 binding as probed by EPR spectroscopy. A, EPR spectra for different deletion variants of huPrP labeled at position 30 (30R huPrP) in the presence of Aβ42 oligomers. B, EPR spectra for huPrP labeled at position 113 (113R huPrP) alone (top) and in the presence of Aβ42 oligomers (middle). The bottom spectrum represents Δ23–89 113R huPrP in the presence of Aβ42 oligomers. The lack of immobilized component in this spectrum indicates that the deletion of residues 23–89 completely abolishes huPrP binding to Aβ42 oligomers. In each case, the concentration of the prion protein and Aβ42 was 2 and 100 μm, respectively. For visualization purposes, spectra are scaled to the same vertical size.