Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops

被引:120
作者
Hatters, DM
MacPhee, CE
Lawrence, LJ
Sawyer, WH
Howlett, GJ [1 ]
机构
[1] Univ Melbourne, Dept Biochem & Mol Biol, Parkville, Vic 3052, Australia
[2] Univ Oxford, Oxford Ctr Mol Sci, New Chem Lab, Oxford OX1 3QT, England
[3] Biomol Res Inst, Parkville, Vic 3052, Australia
关键词
D O I
10.1021/bi000002w
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 ail of the conserved plasma apolipoproteins, apoC-II contains amphipathic helical regions that account for the increase in ct-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation paints to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.
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页码:8276 / 8283
页数:8
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