Human Apolipoprotein C-II Forms Twisted Amyloid Ribbons and Closed Loops

Abstract

Human apolipoprotein C-II (apoC-II) self-associates in solution to form aggregates with the characteristics of amyloid including red-green birefringence in the presence of Congo Red under cross-polarized light, increased fluorescence in the presence of thioflavin T, and a fibrous structure when examined by electron microscopy. ApoC-II was expressed and purified from Escherichia coli and rapidly exchanged from 5 M guanidine hydrochloride into 100 mM sodium phosphate, pH 7.4, to a final concentration of 0.3 mg/mL. This apoC-II was initially soluble, eluting as low molecular weight species in gel filtration experiments using Sephadex G-50. Circular dichroism (CD) spectroscopy indicated predominantly unordered structure. Upon incubation for 24 h, apoC-II self-associated into high molecular weight aggregates as indicated by elution in the void volume of a Sephadex G-50 column, by rapid sedimentation in an analytical ultracentrifuge, and by increased light scattering. CD spectroscopy indicated an increase in beta-sheet content, while fluorescence emission spectroscopy of the single tryptophan revealed a blue shift and an increase in maximum intensity, suggesting repositioning of the tryptophan into a less polar environment. Electron microscopy of apoC-II aggregates revealed a novel looped-ribbon morphology (width 12 nm) and several isolated closed loops. Like all of the conserved plasma apolipoproteins, apoC-II contains amphipathic helical regions that account for the increase in alpha-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation points to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.

The Molecular Chaperone, α-Crystallin, Inhibits Amyloid Formation by Apolipoprotein C-II

“…Freshly prepared apoC-II gave a spectrum characteristic of an unordered protein conformation (Fig. 3, solid line) and is similar to the spectra previously reported for monomeric apoC-II (5,15,28). In contrast, the spectrum for apoC-II incubated for 5 days in the absence of ␣-crystallin showed more negative molar ellipticities between 200 and 250 nm (Fig.…”

“…In a lipid-free environment, apoC-II slowly aggregates into fibers with all the hallmarks of amyloid including the binding to thioflavin T, the binding to Congo Red with red/green birefringence under cross-polarized light, and increased ␤-structure as measured by circular dichroism spectroscopy (5). The amyloid fibrils formed by apoC-II have a helical twisted ribbon morphology under physiological conditions of pH, salt concentrations, and temperature with no indication of amorphous aggregates (5).…”

“…The amyloid fibrils formed by apoC-II have a helical twisted ribbon morphology under physiological conditions of pH, salt concentrations, and temperature with no indication of amorphous aggregates (5). In vitro amyloid formation by apoC-II can be compared with the in vivo deposition of amyloid involving other apolipoproteins such as apoA-I (6, 7), apoA-II (8,9), apoE (10), and apolipoprotein-like proteins, ␣-synuclein (11) and serum amyloid A (12).…”

“…In vitro amyloid formation by apoC-II can be compared with the in vivo deposition of amyloid involving other apolipoproteins such as apoA-I (6, 7), apoA-II (8,9), apoE (10), and apolipoprotein-like proteins, ␣-synuclein (11) and serum amyloid A (12). A significant clue to the prevalence of apolipoproteins in amyloid formation is provided by the observation that many apolipoproteins have limited conformational stability or secondary structure in the absence of lipid (5). Destabilized conformations in many proteins promote amyloid formation (13,14).…”

Apolipoproteins and amyloid fibril formation in atherosclerosis

“…In addition to the evidence that apolipoproteins contribute to amyloid disease indirectly, several apolipoproteins form amyloid in their own right (Higuchi et al, 1991b;Westermark et al, 1995;Wisniewski et al, 1995;Genschel et al, 1998;Hatters et al, 2000;Lange et al, 2000). There is no clear understanding of the molecular basis of the prominent role of apolipoproteins in many amyloidoses; however the low conformational stability of this family of proteins and the possibility of aberrant lipid interactions are proposed as potential factors involved .…”