Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops.
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.
Hatters
D M
DM
Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3052, Australia.
MacPhee
C E
CE
Lawrence
L J
LJ
Sawyer
W H
WH
Howlett
G J
GJ
D000544:Alzheimer Disease / Q000378:metabolism; D000682:Amyloid / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000737:chemistry; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C / Q000737:chemistry* / Q000648:ultrastructure; D052160:Benzothiazoles; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003224:Congo Red / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013844:Thiazoles / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry; D014461:Ultracentrifugation
D000682:Amyloid; D016229:Amyloid beta-Peptides; D053304:Apolipoprotein C-II; D001056:Apolipoproteins C; D052160:Benzothiazoles; D013844:Thiazoles; C009462:thioflavin T; D003224:Congo Red; D014364:Tryptophan