Abstract 396: Acrolein Modification Weakens the Antimicrobial Activity of Apolipoprotein A1

Abstract

Human apolipoprotein A-I (apoA-I) has been show to exhibit antimicrobial activity by neutralizing lipopolysaccharides and destabilizing inner membranes of gram-negative bacteria. Previous studies showed that acrolein, a highly reactive αβ unsaturated aldehyde generated in cigarette smoking, modifies ε-amino side chains of lysine residues in apoA-I. The current study investigated the effect of acrolein exposure on the structure and antimicrobial activity of apoA-I. Incubation of apoA-I with acrolein using a 1:20 molar ratio, acrolein modification was evident by the appearance of apoA-I oligomers due to intermolecular crosslinking. Increase of the acrolein to protein ratio resulted in heavily cross-linked apoA-I, with protein bands appearing at 63, 98, and 126 kDa. The presence of acrolein-modified lysines in the oligomers was verified through Western blot analysis using mab5F6 antibody that specifically detects acrolein modified lysine residues in proteins. The structural changes of modified apoA-I was analyzed using circular dichroism. The α-helical content of acrolein-modified apoA-I was not significantly different from the unmodified protein. However, the midpoint of guanidine-induced denaturation increased from 0.97 to 1.50 M guanidine upon modification, indicating a significant increase in protein stability. This suggests that while modification did not alter the secondary structure, the protein fold was altered due to cross-linking. To measure the effect of acrolein modification on the interaction with bacterial membranes, binding experiments were performed with phosphatidylglycerol entrapped with calcein. This showed that the percentage of calcein released by apoA-I decreased from 87.5 ± 2.3 % to 4.7 ± 0.13 % when the protein was modified by acrolein. Thus acrolein-modified apoA-I binds phosphatidylglycerol less effectively, possibly due to loss of electrostatic interactions with anionic phospholipid vesicles. In addition, binding of apoA-I to lipopolysaccharides was significantly weaker when the protein was modified by acrolein. These results suggest that apoA-I modification by acrolein results in a protein with a decreased ability to bind to bacterial membranes and is thus less potent as an antimicrobial protein.