PURIFICATION OF APOLIPOPROTEIN-E ATTENUATES ISOFORM-SPECIFIC BINDING TO BETA-AMYLOID

Apolipoprotein E (apoE), particularly the e4 allele, is genetically li nked to the incidence of Alzheimer's disease. In vitro, apoE has been shown to bind beta-amyloid (A beta), an amyloidogenic peptide that agg regates to form the primary component of senile plaques. In previous w ork, we demonstrated that apoE3 from tissue culture medium binds to A beta with greater avidity than apoE4 (LaDu, M. J., Falduto, M. T., Man elli, A. M., Reardon, C. A., Getz, G, S., and Frail, D. E. (1994) J. B iol. Chem. 269, 23403-23406). This is in contrast to data using purifi ed apoE isoforms as substrate for A beta (Strittmatter, W. J., Weisgra ber, 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 t his apparent discrepancy by demonstrating that the preferential bindin g of A beta to apoE3 is attenuated and even abolished with purificatio n, a process that includes delipidation and denaturation. We compared the A beta binding capacity of unpurified apoE isoforms from both tiss ue 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-polyac rylamide gel electrophoresis followed by Western immunoblotting for ei ther A beta or apoE immunoreactivity. While the level of the apoE3 . A beta complex was similar to 20-fold greater compared with the apoE4 . A beta complex in unpurified conditioned medium, apoE3 and apoE4 puri fied from this medium bound to A beta with comparable avidity. Moreove r, 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 lipop roteins exhibited little isoform specificity in binding to A beta. The se results suggest that native preparations of apoE may be a more phys iologically relevant substrate for A beta binding than purified apoE a nd further underscore the importance of subtle differences in apoE con formation to its biological activity.