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.