Acrolein Modification of Apolipoprotein A‐I Alters Protein Stability and Antimicrobial Activity

Human apolipoprotein A‐I (apoA‐I) isa major component of high density lipoprotein and has an important role in cholesterolefflux. The protein also has antimicrobial properties, capable of neutralizing lipopolysaccharides (LPS) and destabilizing inner membranes of gram‐negative bacteria. Previous studies have shown that acrolein modifies the ɛ‐amino sidechains of lysine residues of apoA‐I. Acrolein is a highly reactive αβ unsaturated aldehyde and is generated mostly by cigarette smoking or during oxidation of lipids and amino acids. Acrolein‐modified apoA‐I has been detected in humana therosclerotic lesions and studies have shown that acrolein impairs lipid transport, leading to severe heart disease. The current study aims to investigate the effect of acrolein exposure on the antimicrobial activity of apoA‐I. The protein, containing 21 lysine side‐chains, was modified by acrolein and analyzed by SDS‐PAGE. At a 20‐fold molar ratio of acrolein to lysine, acrolein modification was evident by the appearance of oligomers caused by crosslinking. As the molar ratio of acrolein to protein increased, the intensity of the apoA‐Iband at 28 kDa decreased while heavily cross‐linked apoA‐I appeared with protein bands at 63, 98, and 126 kDa. Acrolein modification was further verified by western blot using an antibody (mab5F6) that specifically detects acrolein modified lysine residues in proteins. This confirmed the presence of acrolein‐modified lysine in the oligomers. To examine the structural changes of the modified protein, α‐helical content and the stability of the protein was measured using circular dichroism. The α‐helical content of acrolein modified apoA‐I was similar to unmodified protein (helical content of ~55%). However, the midpoint of guanidine‐induced denaturation increased from 0.97 (unmodified protein) to1.50 M (acrolein modified protein), indicating a significant increase in protein stability. This suggests that while modification did not alter the secondary structure, the tertiary structure was altered due to cross‐linking. To examine the binding of cross‐linked apoA‐I to phosphatidylglycerol, vesicles entrapped with calcein were used. Addition of apoA‐I to the vesicles resulted in calcein release as indicated by the increase in fluorescence intensity. 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. This shows that acrolein‐modified apoA‐I binds phosphatidylglycerol less effectively, possibly due to loss of electrostatic interaction with the anionic vesicles or by the increase in protein stability caused by crosslinking. Support or Funding Information Research reported in this presentation was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC3GM089564.