Characterization of the interaction of β-amyloid with transthyretin monomers and tetramers

Abstract

β-Amyloid (Aβ) is the main protein component of the amyloid plaques associated with Alzheimer's disease. Transthyretin (TTR) is a homotetramer that circulates in both blood and cerebrospinal fluid. Wild-type (wt) TTR amyloid deposits are linked to senile systemic amyloidosis, a common disease of aging, while several TTR mutants are linked to familial amyloid polyneuropathy. Several recent studies provide support for the hypothesis that these two amyloidogenic proteins interact, and that this interaction is biologically relevant. For example, upregulation of TTR expression in Tg2576 mice was linked to protection from the toxic effects of Aβ deposition [Stein, T. D., and Johnson, J. A. (2002) J. Neurosci. 22, 7380-7388]. We examined the interaction of Aβ with wt TTR as well as two mutants: F87M/L110M, engineered to be a stable monomer, and T119M, a naturally occurring mutant with a tetrameric stability higher than that of the wild type. On the basis of enzyme-linked immunoassays as well as cross-linking experiments, we conclude that Aβ monomers bind more to TTR monomers than to TTR tetramers. The data further suggest that TTR tetramers interact preferably with Aβ aggregates rather than Aβ monomers. Through tandem mass spectrometry analysis of cross-linked TTR-Aβ fragments, we identified the A strand, in the inner β-sheet of TTR, as well as the EF helix, as regions of TTR that are involved with Aβ association. Light scattering and electron microscopy studies demonstrate that the outcome of the TTR-Aβ interaction strongly depends on TTR quaternary structure. While TTR tetramers may modestly enhance aggregation, TTR monomers decidedly arrest Aβ aggregate growth. These data provide important new insights into the nature of TTR-Aβ interactions. Such interactions may regulate TTR-mediated protection against Aβ toxicity.

Figure 1

ELISA analysis of TTR-Aβ association. Wt TTR, T119M or M-TTR was immobilized on ELISA plates. (A) Freshly prepared Aβ(1–40) was added to each well; after 1 h incubation and washing to remove unbound material, anti-Aβ antibody 6E10 or 4G8 was used to detect bound Aβ. * p < 0.05, n = 6. (B) Freshly prepared Aβ(1–42) was added to each well; after 1 h incubation and washing to remove unbound material, anti-Aβ antibody 6E10 or 4G8 was used to detect bound Aβ. * p < 0.05, n = 4. (C) The binding of Aβ(1–40) to immobilized TTR as a function of time was measured with anti-Aβ antibody 6E10. * p < 0.05, n = 6. (D) Pre-aggregated Aβ(1–40) was added to TTR coated ELISA plates. Anti-Aβ antibody 6E10 or 4G8 was used to detect bound Aβ. * p < 0.05, n = 6. When Aβ was added to blank wells (negative controls), no absorbance was detected above empty wells (data not shown); these wells were used as “zero” absorbance.

Figure 2

Crosslinking of TTR and Aβ. Aβ was incubated with TTR for 2 hr and then crosslinked with glutaraldehyde. Samples were boiled and analyzed by SDS-PAGE. Samples were then transferred to membrane and detected with anti-TTR antibody. The gels were heavily stained in order to detect minor species. Notations: wt TTR alone (W) or with Aβ (W+A), T119M alone (T) or with Aβ (T+A), M-TTR alone (M) or with Aβ (M+A), and Aβ alone (A). (A) Aβ(1–40) cross-linked with TTR. (B) Aβ(1–42) cross-linked with TTR.

Figure 3

Crosslinking of TTR and Aβ. Aβ(1–40) was incubated with TTR for 1 day and then crosslinked with glutaraldehyde. Samples were boiled and then analyzed by SDS-PAGE and detected with Coomassie stain. Notations: wt TTR alone (W) or with Aβ (W+A), T119M alone (T) or with Aβ (T+A), M-TTR alone (M) or with Aβ (M+A), and Aβ alone (A).

Figure 4

Acid-dissociation and re-assembly of TTR with Aβ. Wt TTR was acid-dissociated into monomers and then re-assembled at neutral pH in the absence or presence of Aβ(1–40). Samples were cross-linked by glutaraldehyde and then analyzed by SDS-PAGE. Notations: wt TTR (W), acid-dissociated and re-assembled wt TTR alone (diW) or with Aβ (diW+A), T119M (T), acid-dissociated and re-assembled T119M alone (diT) or with Aβ (diT+A), M-TTR with Aβ (M+A), and Aβ alone (A).

Figure 5

Crosslinked samples used in LTQ MS/MS analysis. Aβ(1–40) was incubated with TTR for 2 hr and then crosslinked with BS³-d₀/d₄. Boxes indicate the gel bands that were excised for enzymatic digestion. (A) M-TTR and Aβ were cross-linked with 100-, or 200-fold molar excess of BS³-d₀/d₄, then the reaction was terminated after 30 or 60 min. Samples were analyzed by SDS-PAGE, transferred to membrane, and detected with anti-TTR antibody. (B) wt TTR and Aβ were cross-linked with ~2650-fold molar excess of BS³-d₀/d₄, then the reaction was terminated after 60 min. Samples were analyzed by SDS-PAGE. Dash lines indicate the center of protein bands.

Figure 6

MS/MS spectra of cross-linked peptides from trypsin in-gel digestion. (A) CID spectrum of the signal at m/z 1023.29 ([M+4H]⁴⁺) corresponding to a cross-linked product between M-TTR (residues 1–15) and Aβ (residues 17–40) cross-linked with 100-fold excess BS³-d₀/d₄, reaction time 30 min. (B) CID spectrum of the signal at m/z 1382.72 ([M+4H]⁴⁺) corresponding to a cross-linked product between wt TTR (residues 10–34) and Aβ (residues 6–28) cross-linked with 2650-fold excess BS³-d₀/d₄, reaction time 60 min.

Figure 7

MS/MS spectra of cross-linked peptides from trypsin/GluC in-solution digestion. (A) CID spectrum of the signal at m/z 983.16 ([M+4H]⁴⁺) corresponding to a cross-linked product between M-TTR (residues 10–21) and Aβ (residues 17–40). (B) CID spectrum of the signal at m/z 949.99 ([M+4H]⁴⁺) corresponding to a cross-linked product between M-TTR (residues 71–80) and Aβ (residues 17–40).

Figure 8

Normalized scattering intensity at 90° angle I_norm for Aβ alone (✕), or with 0.1 mg/mL wt TTR (▲), T119M (□) or M-TTR (♦). Scattering due to the solvent was subtracted, and results were normalized to the scattering intensity of toluene, to account for changes in laser strength and aperture, and to the mass concentration of peptide/protein.

Figure 9

TEM images of (A) Aβ alone or with (B) wt TTR, (C) T119M, or (D) M-TTR. Samples were prepared with 0.8 mg/mL Aβ and 0.1 mg/mL TTR in PBSA. Images were recorded 2 weeks after sample preparation.

Figure 10

Histogram of Aβ fibrils distribution of Aβ alone (A), wt TTR+Aβ (W+A), T119M +Aβ (T+A) and M-TTR+Aβ (M+A).

Figure 11

(A) Crystal structure of TTR monomer (pdb entry 1DVQ) with the side chains of Lys9, Lys15 and Lys76 indicated that were found to be cross-linked with the Aβ peptide. The AB loop is highlighted in yellow. Since the N-terminal 10 residues were not in the crystal structure because they are disordered, we added this strand by hand to indicate its relative position. (B) Crystal structure of TTR tetramer where the side chain of Lys15 of each monomer were shown.

Scheme 1

Fragmentation pattern of cross-linked peptides (LTQ). (A) cross-linked M-TTR+Aβ peptides, (B) cross-linked wt TTR+Aβ peptides,

Scheme 2

Fragmentation pattern of cross-linked M-TTR+Aβ peptides (Orbitrap). Aβ residues 17–40 were cross-linked to (A) M-TTR residues 10–21; and (B) M-TTR residues 71–80.