Abstract 103: Mass Spectral Analysis of Oxidative Stress Modification of Apolipoprotein E by Acrolein
Apolipoprotein E (apoE), an anti-atherogenic apolipoprotein, plays a significant role in the metabolism of lipoproteins. It lowers plasma lipid levels by acting as a ligand for the low-density lipoprotein receptor (LDLr) family of proteins. ApoE mediates this function via essential lysine residues that interact with the LDLr. Preliminary studies from our lab showed that rats exposed to environmental tobacco smoke displayed oxidative modification of apoE by acrolein (a highly reactive unsaturated aldehyde) and dissociation of lipoprotein-bound apoE; however, the details regarding the modification were not determined at the molecular level. The objectives of this project are to identify the specific sites of modification and the effect of modification on the structure and function of recombinant rat apoE. Acrolein modification was verified by Western blot analysis using an acrolein-lysine specific antibody. Matrix Assisted Laser Desorption Ionization-Time of Flight/Time of Flight Mass Spectrometry (MALDI-TOF/TOF MS) analysis identified Lys64, 67, 68, 138 & 234 as likely modification sites by acrolein. Guanidine HCl-induced denaturation studies indicated that the overall fold of the modified protein was significantly altered as revealed by circular dichroism spectroscopy. Modified apoE also demonstrated a decrease in heparin binding affinity and lipid binding ability. The LDLr binding ability of modified apoE was significantly impaired. Loss of LDLr binding ability is attributed to acrolein modification of Lys138, an essential residue located on helix 4, that is directly involved in interaction with the LDLr as seen for human apoE3. Overall, we conclude that oxidative modification by acrolein disrupts the structural and functional integrity of apoE, which is likely to affect its role in maintaining plasma cholesterol homeostasis. Our data provide a molecular basis for the potential role of oxidative stress- (due to environmental factors or aging) mediated modification of apoE in altering lipoprotein metabolism, with direct implications in cardiovascular disease.
- © 2013 by American Heart Association, Inc.