On the Aggregation of Apolipoprotein A-I

Abstract

In vivo, apolipoprotein A-I (ApoA-I) is commonly found together with lipids in so-called lipoprotein particles. The protein has also been associated with several diseases-such as atherosclerosis and amyloidosis-where insoluble aggregates containing ApoA-I are deposited in various organs or arteries. The deposited ApoA-I has been found in the form of amyloid fibrils, suggesting that amyloid formation may be involved in the development of these diseases. In the present study we investigated ApoA-I aggregation into amyloid fibrils and other aggregate morphologies. We studied the aggregation of wildtype ApoA-I as well as a disease-associated mutant, ApoA-I K107Δ, under different solution conditions. The aggregation was followed using thioflavin T fluorescence intensity. For selected samples the aggregates formed were characterized in terms of size, secondary structure content, and morphology using circular dichroism spectroscopy, dynamic light scattering, atomic force microscopy and cryo transmission electron microscopy. We find that ApoA-I may form globular protein-only condensates, in which the α-helical conformation of the protein is retained. The protein in its unmodified form appears resistant to amyloid formation; however, the conversion into amyloid fibrils rich in β-sheet is facilitated by oxidation or mutation. In particular, the K107Δ mutant shows higher amyloid formation propensity, and the end state appears to be a co-existence of β-sheet rich amyloid fibrils and α-helix-rich condensates.

Keywords: aggregation; apolipoprotein A-I; condensates; plaques.

Figure 1

(A) Possible forms of aggregated ApoA-I, with α-helical ApoA-I in globular condensates or β-sheet rich amyloid fibril structure, both of which may be in equilibrium with the monomer. (B) Number-based size distribution derived using dynamic light scattering (DLS) for an ApoA-I sample directly after isolation by size-exclusion chromatography (SEC; t = 0) and after 8 days incubation at 37 °C. (C) AFM image of the same sample after 8 days.

Figure 2

Time evolution of ThT fluorescence and CD spectra at pH 6.0. ThT fluorescence intensity versus time measured in duplicates in the presence of 14 µM ApoA-I wt (A), 5 and 14 µM oxidized ApoA-I wt (C) and 18 µM K107Δ ApoA-I (E). CD spectra recorded for 11 µM wt (B), 5 µM oxidized wt (D) and 18 µM K107Δ (F) at time zero (0 h) or at a later stage (4 or 6 days) as indicated in each panel.

Figure 3

Time evolution of ThT fluorescence and CD spectra of the ApoA-I mutant K107Δ in phosphate buffer at pH 6.0. (A) The ThT fluorescence intensity over time for 10 and 20 µM protein in triplicates. The initial decrease in ThT fluorescence signal is due to temperature stabilization. (B) The observed CD spectrum for the initial state (t = 0, dotted line) and at a later stage (4 days, red line). (C) The observed CD spectra after separation of the soluble (blue) and insoluble fractions (green). The 4 days sample from panel (B) (red) is included for comparison. The two spectra of the supernatant and the pellet were summed (yellow), to compare their superposition to the spectrum of the non-separated sample. (D) Zoom-in on the pellet sample spectrum in (C), to observe this weak spectrum more easily.

Figure 4

Cryo-TEM images of oxidized (A) wildtype ApoA-I, aggregated under agitation, and (B,C) K107Δ ApoA-I. Both samples were aggregated in buffer with pH 6.0. For (B,C), the co-existence of larger, more compact aggregates (B), as well as more fibrillar structures (C) and single fibrils (Figure S7C,D) could be observed. The scale bar in each image is 200 nm (note the difference in magnification of the images).