Clinical ApoA-IV amyloid is associated with fibrillogenic signal sequence

Abstract

Apolipoprotein A-IV amyloidosis is an uncommon form of the disease normally resulting in renal and cardiac dysfunction. ApoA-IV amyloidosis was identified in 16 patients attending the National Amyloidosis Centre and in eight clinical samples received for histology review. Unexpectedly, proteomics identified the presence of ApoA-IV signal sequence residues (p.18-43 to p.20-43) in 16/24 trypsin-digested amyloid deposits but in only 1/266 non-ApoA-IV amyloid samples examined. These additional signal residues were also detected in the cardiac sample from the Swedish patient in which ApoA-IV amyloid was first described, and in plasma from a single cardiac ApoA-IV amyloidosis patient. The most common signal-containing peptide observed in ApoA-IV amyloid, p.20-43, and to a far lesser extent the N-terminal peptide, p.21-43, were fibrillogenic in vitro at physiological pH, generating Congo red-positive fibrils. The addition of a single signal-derived alanine residue to the N-terminus has resulted in markedly increased fibrillogenesis. If this effect translates to the mature circulating protein in vivo, then the presence of signal may result in preferential deposition as amyloid, perhaps acting as seed for the main circulating native form of the protein; it may also influence other ApoA-IV-associated pathologies. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: ApoA-IV amyloidosis; ApoA-IV sequence coverage; ApoA-IV signal-containing peptide; amyloid proteomics; fibrillogenic ApoA-IV signal-containing peptide; targeted mass spectrometry in serum.

Figure 1

The ApoA‐IV Mascot score histogram for all samples (light blue bars); the histogram for ApoA‐IV clinical amyloid samples is shown as dark blue bars. The scores of the 24 clinical ApoA‐IV patients are also shown as open blue circles together with that of the original Swedish sample (open diamond); in each of these cases, ApoA‐IV was the top scoring amyloid protein. Signal sequence was detected in 17/24 samples (closed blue circles) and also in the Swedish sample (closed red circle in diamond).

Figure 2

Normalised ion chromatograms (MH₃ ³⁺) for the signal‐containing peptides p.18‐43, p.19‐43, and p.20‐43, together with the N‐terminal peptide p.21‐43 for (A) authentic standards and (B) patient 11. (C, D) MSMS spectra (MH₃ ³⁺) for the p.18‐43 standard and patient 11. The common y ion series (y3–y15) is highlighted.

Figure 3

(A) Fibrillogenesis at pH 7 and 37 °C of the signal‐containing peptides p.18‐43, p.19‐43, and p.20‐43, and the N‐terminal peptide p.21‐43 analysed at the same time. (B) Three separate experiments were performed for each peptide, and the mean (SD) of the change in ThT fluorescence emission between 0.25 and 18 h is shown. Fluorescence intensity is shown in arbitrary units (a.u.). Panels C1–C4 show the bright field Congo red staining for the p.20‐43 fibrils together with the fluorescence with and without the polarising filter, and the electron micrograph of the p.20‐43 fibrils (after 24 h of incubation).