Isolation of amyloidogenic aggregates of transthyretin by ceramic hydroxyapatite chromatography

Abstract

Amyloidogenic proteins can lapse into unstable monomeric precursors which misfold and self-assemble into toxic oligomers and amyloid aggregates leading to intracellular deposition of insoluble amyloid fibrils, the hallmark of amyloidosis and major neurodegenerative diseases. The assembly pathway in amyloid diseases presents strong similarities regardless of the culprit protein and highly purified structures are required for its characterization. Herein, we have successfully separated tetrameric form from aggregated versions of the acidic amyloidogenic protein transthyretin (TTR), by a single step purification using ceramic hydroxyapatite (CHT) media. Native TTR elution from CHT media is phosphate-dependent, achieved at approximately 30 mM sodium phosphate. Adoption of a mild acetate environment favoured conversion of native TTR to a heterogeneous mixture with particles of 10–500 nm diameter and characteristics mimicking amyloid cascade components. We best explored the metal affinity and cation exchange dual CHT separation mechanism by generating a novel purification protocol that allowed for binding of TTR mixture at high salt presence, and distinct elution of tetramer and aggregate via application of a phosphate desalting gradient. We deduce that tetrameric version binds to the calcium site of CHT whereas amyloid aggregation uses both the calcium and phosphate binding sites of CHT, making it a suitable target for mixed-mode purification. The presence of granular aggregates was confirmed via transmission electron microscopy. Collectively, the data highlight the strength of hydroxyapatite as purification media in the rapidly expanding amyloidosis medical research field.