Self-association of Human Apolipoprotein E3 and E4 in the Presence and Absence of Phospholipid

Abstract

Human apolipoprotein E (apoE) exists as three main isoforms, differing by single amino acid substitutions, with the apoE4 isoform strongly linked to the incidence of late onset Alzheimer's disease. We have expressed and purified apoE3 and apoE4 from Escherichia coli and compared their hydrodynamic properties by gel permeation liquid chromatography, capillary electrophoresis, circular dichroism, and sedimentation methods. Sedimentation velocity experiments, employing a new method for determining the size distribution of polydisperse macromolecules in solution (Schuck, P. (2000) Biophys. J. 78, 1606 -1619), provide direct evidence for the heterogeneous solution structures of apoE3 and apoE4. In a lipid-free environment, apoE3 and apoE4 exist as a slow equilibrium mixture of monomer, tetramer, octamer, and a small proportion of higher oligomers. Both sedimentation velocity and equilibrium experiments indicate that apoE4 has a greater propensity to self-associate. We also demonstrate that apoE3 and apoE4 oligomers dissociate significantly in the presence of dihexanoylphosphatidylcholine micelles (20 mM) and to a lesser extent at submicellar concentrations (4 mM). The ␣-helical content for both isoforms was almost identical (50%) in the presence and absence of dihexanoylphosphatidylcholine. These results reveal that apoE oligomers undergo phospholipid-induced dissociation to folded monomers, suggesting the monomeric form prevails on the lipoprotein surface in vivo.

Examination of Lipid-bound Conformation of Apolipoprotein E4 by Pyrene Excimer Fluorescence

“…Excimer emission formation is indicative of spatial proximity between positions 61 and 255 in lipid-free apoE4. However, apoE has been indicated to exist in solution as a tetramer by protein-protein interactions via its CT domain (18,21,22,43), leading us to examine whether the excimer emission originates from intermolecular spatial proximity between these specified sites. Pyr-R61C/E255C/apoE4 was mixed with a 10-fold molar excess of WT apoE4 under denaturing conditions, followed by slow refolding as described under "Experimental Procedures."…”

“…2). Taking the sum of fluorescence intensities of all lipoprotein-bound fractions as 100%, the percentage distributions of pyr-R61C/E255C/apoE4 among the various lipoprotein fractions were calculated to be as follows: 38% in VLDL (fractions 5-9); 25% in intermediate density lipoprotein/LDL (fractions 11-15); 37% in HDL (fractions [17][18][19][20][21]. This distribution profile is largely consistent with that reported previously (24), indicating that the distinctive binding preference of apoE4 is retained in pyr-R61C/E255C/apoE4.…”

Structural differences between apolipoprotein E3 and E4 as measured by 19F NMR

“…[1][2][3][4][5] Because oligomerization could make interpretation of the NMR spectra difficult, we investigated the monomeric form of the protein. Figure 3 shows that the NMR spectra for the monomeric apoE3 is still broad and poorly resolved suggesting that the nature of the spectrum is not an issue related to oligomerization.…”

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

“…This suggests that the CED-4/CED-9DNDC complex may exist as either a 2 : 1 or 2 : 2 CED-4/CED-9DNDC complex. Therefore, to confirm the quaternary structure of the CED-4/CED-9DNDC complex, sedimentation equilibrium experiments, which are independent of molecular shape estimates and therefore give a more robust measure of molecular mass, 26 were also conducted at an initial protein concentration of 1.0 mg/ml ( Supplementary Figure 1d-f). The nonlinear least-squares best-fit yielded a molar mass of 18073.8 kDa, in excellent agreement with the theoretical heterotetrameric mass of a 2 : 2 CED-4/CED-9DNDC complex (175 kDa).…”