Abstract 411: An Anion-Exchange Chromatography Isolated Subfraction of Mouse Apolipoprotein A-I Is Unable to Activate Cellular Cholesterol Release from Mouse Peritoneal Macrophage Foam Cells
Aim: Human and mouse plasma apoA-I separate on anion exchange chromatography (6M urea) in distinct subfractions. To investigate the nature of this diversity, we analyzed the protein subfractions by IEF and Urea-PAGE, and compared their ability to generate HDL in vitro.
Methods: Human and mouse plasma HDLs were delipidated then applied to an DE52 ion-exchange column and eluted by 30-60mM Tris-HCl, pH7.80 containing 6M urea. Mouse, human, and recombinant apoA-Is were analyzed by IEF (Ettan IPGphor3) followed by CBB protein staining. Urea-PAGE analysis was performed with 14% acrylamide gel in 6M urea buffer. ApoA-Is abilities to efflux cholesterol from mouse peritoneal foam cells and to shift the size of the ACAT-accessible cholesterol pool were determined.
Results: Human plasma and recombinant human apoA-I showed two apoA-I subtypes by IEF and Urea-PAGE analysis; whereas HDL from C57BL/6 and 129SV mice contained three apoA-Is at pI=5.37, 5.31, 5.27 (mP3-1, mP2, mP3-3), and pI=5.44, 5.37, 5.32, respectively. Subfractions mP2 and mP3-3 were isolated on a DE52 column and, despite their difference in pI, were able to mediate cellular cholesterol efflux at levels similar to human plasma apoA-Is. Subfraction mP3-1, with the highest pI, eluted between mP2 and mP3-3 on DE-52 column. Interestingly, for both mouse strains, mP3-1 showed very poor cholesterol efflux and the incorporation of 14C-oleic acid to cellular CE was significantly higher when compared to mP2 and mP3-3.
Conclusion: We found that differences in the pI of anion-exchange chromatography apoA-I subfractions did not directly affect HDL generation except for mouse subtype mP3-1. mP3-1 binds strongly to the anionic DE52 resin, probably by hydrophobic interaction, and is unable to mediate cellular cholesterol efflux. Further investigation of the nature of the differences in these apoA-I subtypes will provide insights on factors that may be implicated in the biogenesis of HDL in vivo.
- © 2012 by American Heart Association, Inc.