Directed selection of a conformational antibody domain that prevents mature amyloid fibril formation by stabilizing Abeta protofibrils

Abstract

The formation of amyloid fibrils is a common biochemical characteristic that occurs in Alzheimer's disease and several other amyloidoses. The unifying structural feature of amyloid fibrils is their specific type of beta-sheet conformation that differentiates these fibrils from the products of normal protein folding reactions. Here we describe the generation of an antibody domain, termed B10, that recognizes an amyloid-specific and conformationally defined epitope. This antibody domain was selected by phage-display from a recombinant library of camelid antibody domains. Surface plasmon resonance, immunoblots, and immunohistochemistry show that this antibody domain distinguishes Abeta amyloid fibrils from disaggregated Abeta peptide as well as from specific Abeta oligomers. The antibody domain possesses functional activity in preventing the formation of mature amyloid fibrils by stabilizing Abeta protofibrils. These data suggest possible applications of B10 in the detection of amyloid fibrils or in the modulation of their formation.

Fig. 1.

Conformational sensitivity of B10AP. (a) B10 homology model based on the coordinate file 1ZVH.pdb (45). Complementarity determining regions 1–3 are shown in green (CDR1), red (CDR2), and blue (CDR3). (b) Surface plasmon resonance sensorgrams of 100 nM 22C4 (blue) or B10AP (red) to biosensors covered with fibrils or disaggregated Aβ(1–40). (Lower) The blue curve is scaled to 1/10th of its original value. (c) B10AP staining of disaggregated and fibrillar Aβ(1–40) blotted onto nitrocellulose, quantitated by densitometry. Standard error of mean from three experiments is shown. (d and e) Hippocampal sections from an Alzheimer brain tissue (d) and a control case without clinical signs of dementia (e). B10AP staining is visualized with “Permanent Red” chromogene. Alzheimer tissue shows B10AP-positive plaques (>10 μm, arrow heads). (f) Cell culture-derived amyloid plaques (46) stained with anti-Aβ (1–16), B10AP and Hoechst 33342 (cell nuclei). See SI Text for material descriptions (d–f).

Fig. 2.

The B10-epitope is common to different amyloid fibrils. (a) B10AP staining of blotted Aβ(1–40) or Aβ(1–42) amyloid fibrils formed in vitro, or amyloid fibrils extracted from the tissue of one AA amyloid case and two AL amyloid cases (AL1 and AL2). (b) B10AP staining intensity of blotted AL1 fibrils after preincubation of B10AP with Aβ(1–40) fibrils at different wt/wt ratio (AL1 vs. Aβ). Similar data were obtained also for AL2 and AA amyloid (not presented). Error bars show standard error of mean (n = 6). (c and d) Cerium phosphate staining at the lateral side of Aβ(1–40) fibrils decorated with B10AP (c) or without B10AP (d). Cerium phosphate leads to a black precipitate.

Fig. 3.

B10AP interacts weakly with Aβ(1–40) oligomers. (a) TEM image of Aβ(1–40) oligomers. (b) B10AP staining intensity of Aβ(1–40) amyloid fibrils and oligomers. (c) Far-UV CD spectrum of 58 μM oligomers. A single ellipticity minimum at 217 nm is highly characteristic for β-sheet conformation. (d) ATR-FTIR spectrum of 2 mg/ml amyloid fibrils in pure water (pH 7.0) and 1 mg/ml Aβ(1–40) oligomers (arrowhead shows peak at 1,693 cm⁻¹). CD and ATR-FTIR spectroscopy were carried out as described (44). (e and f) Optical absorption of CR (e) and fluorescence emission of ThT (f) in the presence of Aβ(1–40) fibrils, oligomers, and only buffer.

Fig. 4.

B10AP prevents mature fibril formation by stabilization of Aβ(1–40) protofibrils. (a) Kinetics of fibril formation monitored with ThT fluorescence at 490 nm (blue), B10AP immunoblots (green) and TEM from aliquots drawn at indicated time points. Data fit with a sigmoidal model (47). Incubation conditions: 1 mg/ml Aβ(1–40) in 50 mM Hepes (pH 7.4), 50 mM NaCl at 37°C. (b) Aβ(1–40) fibril formation in the presence of absence of B10AP monitored online with ThT (37°C) and TEM. (c) ATR-FTIR spectra of B10AP and B10AP-stabilized Aβ protofibrils (1:10 B10AP:Aβ molar ratio). Arrowhead shows additional peak at 1,624 cm⁻¹. (d) x-ray diffraction image of fully hydrated B10AP-Aβ protofibril complex. (e and f) ThT fluorescence emission (e) and CR absorption spectra (f) of Aβ(1–40) amyloid fibrils, fragmented amyloid fibrils, B10AP, B10AP-stabilized protofibrils, B10AP-decorated mature fibrils and dye in Hepes buffer. (g) TEM images of mature fibrils before (Left) and after fragmentation by 1 x freezing (Right). (h) Digestion of 50 μM freshly dissolved, disaggregated Aβ(1–40), B10AP-stabilized protofibrils and mature Aβ(1–40) amyloid fibrils with 62.5 ng/ml proteinase K in 50 mM Hepes (pH 7.4), 50 mM NaCl, 37°C. Gel electrophoresis shows the disappearance of the Aβ(1–40) band. (i) Total sample (T), soluble (S) and pellet fractions (P) of preformed Aβ(1–40) amyloid fibrils with or without B10AP after incubation in 50 mM Hepes (pH 7.4), 50 mM NaCl (37°C) for 1 week and centrifugation (513.000 × g, 30 min). Aβ and B10AP lanes are cut out from a Coomassie-stained denaturing gel.