Purification of Apolipoprotein E Attenuates Isoform-specific Binding to β-Amyloid

Abstract

Apolipoprotein E (apoE), particularly the e4 allele, is genetically linked to the incidence of Alzheimer's disease. In vitro, apoE has been shown to bind beta-amyloid (A beta), an amyloidogenic peptide that aggregates to form the primary component of senile plaques. In previous work, we demonstrated that apoE3 from tissue culture medium binds to A beta with greater avidity than apoE4 (LaDu, M. J., Falduto, M. T., Manelli, A. M., Reardon, C. A., Getz, G. S., and Frail, D. E. (1994) J. Biol. Chem. 269, 23403-23406). This is in contrast to data using purified apoE isoforms as substrate for A beta (Strittmatter, W. J., Weisgraber, K. H., Huang, D. Y., Dong, L.-M., Salvesen, G. S., Pericak-Vance, M., Schmechel, D., Saunders, A. M., Goldgaber, D., and Roses, A. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8098-8102). Here we resolve this apparent discrepancy by demonstrating that the preferential binding of A beta to apoE3 is attenuated and even abolished with purification, a process that includes delipidation and denaturation. We compared the A beta binding capacity of unpurified apoE isoforms from both tissue culture medium and intact human very low density lipoproteins with that of apoE purified from these two sources. The interaction of human A beta-(1-40)-peptide and apoE was analyzed by nonreducing SDS-polyacrylamide gel electrophoresis followed by Western immunoblotting for either A beta or apoE immunoreactivity. While the level of the apoE3.A beta complex was approximately 20-fold greater compared with the apoE4.A beta complex in unpurified conditioned medium, apoE3 and apoE4 purified from this medium bound to A beta with comparable avidity. Moreover, using endogenous apoE on very low density lipoproteins from plasma of apoE3/3 and apoE4/4 homozygotes, apoE3 was again a better substrate for A beta than apoE4. However, apoE purified from these plasma lipoproteins exhibited little isoform specificity in binding to A beta. These results suggest that native preparations of apoE may be a more physiologically relevant substrate for A beta binding than purified apoE and further underscore the importance of subtle differences in apoE conformation to its biological activity.

Production and characterization of astrocyte-derived human apolipoprotein E isoforms from immortalized astrocytes and their interactions with amyloid-β

“…After a longer exposure time (5 min), an apparent apoE3-Ah complex running at~40 kDa could be detected, likely representing an SDSstable apoE-Ah complex (Fig. 6B) that has previously been described (Aleshkov et al, 1997;LaDu et al, 1994LaDu et al, , 1995Yang et al, 1997). A faint band likely representing an apoE4/Ah complex could be seen at even longer exposure times (data not shown).…”

“…Some in vitro studies do show differential effects of apoE isoforms on Ah fibrillogenesis (E4 N E3) with some studies demonstrating that apoE inhibits and others showing apoE enhances Ah fibril formation (Castano et al, 1995;Evans et al, 1994;Ma et al, 1994). Importantly, while lipidated apoE has been used in some Ah binding and association studies (LaDu et al, 1994(LaDu et al, , 1995Tokuda et al, 2000), lipidated apoE has not been extensively utilized in studies examining the effect of apoE on Ah clearance and fibrillogenesis. The apoE particles derived from astrocytes should provide a novel tool to assist in studying the effects of apoE on Ah fibrillogenesis and clearance using more physiological reagents.…”

“…(DeMattos et al, 2001a), whereas the ratio of apoE/cholesterol with the immortalized astrocytes is~3-5:1, similar to that seen with primary astrocytes. The apoprotein/lipid ratio of lipoprotein particles can markedly influence properties such as receptor binding (Narita et al, 2002) and interactions with other molecules such as Ah (LaDu et al, 1994(LaDu et al, , 1995Tokuda et al, 2000). Importantly, two recent studies demonstrate that if apoE is poorly lipidated in the CNS due to the absence of the protein ATP-binding cassette transporter A-1 (ABCA1), apoE particles are rapidly metabolized in the brain resulting in levels that are 2% of normal in the CSF and b20% of normal in the cortex (Hirsch-Reinshagen et al, 2004;Wahrle et al, 2004).…”

Interaction between human amphipathic apolipoproteins and amyloid β‐peptide: surface plasmon resonance studies

“…Apparently, the AI3-binding site of apoE is localized in lipid-binding sites. Indeed, delipidation of apoE increases the 'avidity' of apoE-A[3 binding in comparison with lipid-bound apoE [31], indicating the involvement of lipid-binding sites of apoE in the interaction with AI3. Moreover, it was shown that the C-terminal fragment of apoE 244-272 was critical for formation of apoE-AI3 complexes and the C-terminal domain of apoE was co-purified with A[3 from senile plaques [5,32].…”

Nascent Astrocyte Particles Differ from Lipoproteins in CSF

“…Although the mechanism for these associations is still unknown, two general observations may be relevant for these associations. First, apoE and apoE-containing lipoproteins have been shown to interact with /3-amybid (A/3) both in vitro (Strittmatter et al, 1993a,b;LaDu et al, 1994LaDu et al, , 1995 and in vivo in the brains of AD patients (Namba et al, 1991;Naslund eta!., 1995) and after head trauma (Nicoll et al, 1995). Furthermore, apoE can influence the rate of A/3 fibril formation in vitro (Evans et a!., 1994;Wood eta!., 1996).…”