Detergent-free isolation and characterization of amyloid precursor protein C99 in E. coli native lipid-nanodiscs using non-ionic polymer

Abstract

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is characterized by cognitive decline resulting from neuronal cell death. A key contributor to AD pathology is C99, a membrane-bound β-secretase-cleaved fragment of amyloid precursor protein (APP). C99 plays a central role in generating amyloid-beta (Aβ) isomers, which are directly implicated in disease progression. Understanding its structure and lipid interactions is essential for elucidating its mechanistic role in AD and guiding therapeutic development. C99 has been studied in membrane mimetics such as micelles, bicelles, and reconstituted nanodiscs. Although reconstituted nanodiscs provide a native-like lipid-bilayer environment, the use of detergents prior to reconstitution has been reported to disrupt native folding and lipid-protein interactions. In this study, we successfully isolated and purified C99 along with its associated lipids directly from E. coli cell membranes using a non-ionic pentyl-inulin polymer, avoiding the need for detergents. The purified C99-containing pentyl-inulin nanodiscs were characterized using SDS-PAGE, Western blotting, dynamic light scattering (DLS), ¹H NMR spectroscopy, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and liquid chromatography-mass spectrometry (LC-MS). Notably, we observed SDS-stable oligomers of C99. DLS and ¹H NMR confirmed the presence of large particles composed of pentyl-inulin and E. coli lipids. MALDI-TOF and LC-MS verified the molecular mass and amino acid sequence of C99, respectively. We propose that this detergent-free method for the direct isolation of C99 and native lipids using non-ionic pentyl-inulin may serve as a valuable tool for investigating the C99-secretase complex and for developing compounds aimed at inhibiting the production of amyloid-beta isomers.

Keywords: Alzheimer's disease; C99 oligomer; amyloid precursor protein (APP‐C99); detergent‐free purification; nanodisc; non‐ionic polymer.

FIGURE 1

SDS‐PAGE/Western blot analysis of recombinant C99. a) C99 expression was analyzed by SDS‐PAGE. Lane 1: Protein marker; lane 2: Non‐induced negative control; lane 3: C99 overexpressed by the autoinduction method; and lane 4: C99 overexpressed by the IPTG‐induction method. b) Detection of C99 by Western blot; wells loaded with the same samples as in Figure 1a. m, Monomer; d, Dimer; d’, Degraded dimer; t, Trimer; and o, Oligomer. The dimer and oligomer bands in lane 3 are enlarged for clarity. Samples were loaded at a normalized concentration of 0.8 OD₆₀₀ to assess protein expression levels. The protein band that appeared below the monomer is due to C99 degradation.

FIGURE 2

(a) SDS‐PAGE analysis of pentyl‐inulin‐solubilized E. coli cell membranes enriched with C99. Lanes 1 and 6 are protein markers; lane 2 (negative control) represents the supernatant of the membranes that were not treated with the polymer for solubilization; lane 3 contains the supernatant of C99 membranes that were solubilized with a pentyl‐inulin polymer (labeled with red box); lanes 4 and 5 represent pellets (insoluble fractions) from samples without and with polymer solubilization, respectively. (b) SDS‐PAGE analysis of detergent‐free extraction of C99 using Ni²⁺ affinity purification. Lanes 1 and 11 contain the protein molecular weight markers. Lanes 2–10 show eluted fractions at increasing imidazole concentrations of 20, 40, 50, 80, 120, 160, 200, 250, and 300 mM, respectively. Lane 12 corresponds to a second elution fraction using 300 mM imidazole.

FIGURE 3

(a) SEC elution profile of directly extracted C99 nanodiscs, performed on the nickel‐purified fractions. The peak represents C99‐rich pentyl‐inulin nanodiscs. (b) SDS‐PAGE analysis on the SEC fractions collected between volumes 42 to 62 mL. The fractions loaded on the gel are shown with blue arrows. c) Western blot analysis on the first four fractions (Arinaminpathy et al., ; Errey & Fiez‐Vandal, ; Levental & Lyman, ; Overington et al., 2006) of SDS‐PAGE. m, monomers; d’, dimers (most likely a dimer); t, trimers; and o, oligomers.

FIGURE 4

DLS and NMR analysis of C99 in E. coli lipid‐pentyl‐inulin nanodiscs. (a) DLS profile showing a hydrodynamic radius of ~10 nm. (b) ¹H NMR spectrum of C99‐rich pentyl‐inulin nanodiscs. The amide region (violet bar), E. coli lipids and the pentyl‐inulin polymer (highlighted by red box and green bar).

FIGURE 5

MALDI‐TOF MS spectra of C99 nanodiscs. (a). A peak corresponding to a molecular weight of 14,276 Da was observed, along with weak‐intensity peaks at higher molecular weights. The matrix used was SA. (b) After treatment with 3% Empigen, new peaks at molecular weights of 13,747.5 Da and 13,921.5 Da emerged. The matrix used was SA. (c) Following 3% Empigen treatment and analysis with the DHB matrix, the new peaks observed in Figure 5b showed increased intensity in Figure 5c, indicating improved resolution with the DHB matrix. (d) After treating C99 nanodiscs with 3% Empigen and 6 M GdnHCl (1:1) treatment, a significant suppression in the 14,269 Da peak was observed. The samples were desalted with C4 or C18 resin ZipTip 10 μL tips (Merck Millipore, USA) and mixed with SA, DHB, or CHCA matrices in a 1:1 ratio before spots on 384‐well stainless‐steel targets were prepared. Ionization was performed on a Bruker Autoflex mass spectrometer, and data were analyzed using Bruker flexAnalysis software.