From hexamer to amyloid: Marginal stability of apolipoprotein SAA2.2 leads to in vitro fibril formation at physiological temperature

Abstract

Serum amyloid A (SAA) is a major acute phase reactant and a small apolipoprotein of high density lipoproteins (HDL) in the serum. In cases of prolonged inflammation, SAA may form amyloid fibrils, leading to the disease of amyloid A (AA) amyloidosis. Recently, we have shown that murine SAA2.2, a non-amyloidogenic isoform in vivo, forms a hexamer in vitro containing a putative central channel. It is reported herein that upon thermal denaturation, hexameric SAA2.2 irreversibly dissociates to a misfolded monomer at physiological temperature, formation of which coincides with a significant loss of α-helical and gain of β-sheet structure. When SAA2.2 is incubated for several days at 37°C, sedimentation analytical ultracentrifugation reveals the presence of soluble high molecular weight aggregates, which upon further incubation undergo subsequent self-assembly into amyloid fibrils. Limited proteolysis experiments suggest that the in vitro amyloidogenecity of SAA2.2 is related to structural alteration in its N-terminus. Our observation that SAA2.2 can form amyloid fibrils in vitro at physiological temperatures suggests that SAA2.2's inability to cause amyloidosis may be related to other factors, such as the stabilization of hexameric SAA2.2 (possibly through ligand binding), and/or the slow kinetics of aberrant misfolding and self-assembly.

Keywords:

• Serum amyloid A
• amyloidosis
• misfolding
• apolipoprotein
• inflammation
• aggregation

Abbreviations
AA	=	amyloid A
CD	=	circular dichroism
EM	=	electron microscopy
GCL	=	glutaraldehyde cross-linking
HDL	=	high density lipoprotein
RT	=	room temperature
SVAU	=	sedimentation velocity analytical ultracentrifugation
ThT	=	thioflavin T
Tm	=	transition midpoint temperature
λmax	=	wavelength of maximum fluorescence

Abbreviations
AA	=	amyloid A
CD	=	circular dichroism
EM	=	electron microscopy
GCL	=	glutaraldehyde cross-linking
HDL	=	high density lipoprotein
RT	=	room temperature
SVAU	=	sedimentation velocity analytical ultracentrifugation
ThT	=	thioflavin T
Tm	=	transition midpoint temperature
λmax	=	wavelength of maximum fluorescence