Introduction: Apolipoprotein E3 (apoE3) is an exchangeable protein that plays a significant role as an anti-atherogenic apolipoprotein. It serves as a ligand for the LDL receptor (LDLr) family of proteins that mediate cellular uptake of lipoproteins and plays a significant role in regulating plasma cholesterol homeostasis. In this study, we investigate the functional consequences of acrolein-mediated oxidative damage to human apoE3 in brain endothelial cells that are the primary constituents of the blood brain barrier. Acrolein is an α,β-unsaturated aldehyde that is an environmental pollutant and is also generated endogenously as bye-products of lipid peroxidation. It is considered an oxidative stress reagent that causes oxidative damage to cellular components, including proteins.
Hypothesis: We test the hypothesis that acrolein modification impairs the structural integrity and functional ability of apoE3.
Methods: Recombinant human apoE3 was modified with varying concentrations of acrolein, and the modification confirmed by Western Blot analysis using acrolein-Lys-specific antibodies. Modified apoE3 was reconstituted with POPC and the resultant reconstituted HDL (rHDL) was isolated by density gradient ultracentrifugation.
Results: We note that rHDL bearing acrolein-modified apoE3 does not interact with soluble LDLr in co-immunoprecipitation assays suggesting oxidative modification of essential Lys residues required for interaction with the ligand-binding domain of the LDLr. However, incubation with mouse brain cerebral cortex endothelial cells, showed internalization of acrolein-modified apoE3. Incubation with suramin, an inhibitor of the LDLr, does not abolish cellular uptake of acrolein-modified apoE3, suggesting alternative uptake pathways likely involving the scavenger receptor family of proteins. Incubation with LOX1 antibody showed a decrease in cellular uptake of acrolein-modified apoE3.
Conclusion: Taken together, this study defines acrolein-modified apoE3 as a hitherto unidentified ligand for scavenger receptors. It offers insight into the molecular basis of oxidative stress mediated damage to apolipoproteins and their role in pathogenesis of cardiovascular and cerebrovascular diseases.