Abstract
The plasma form of platelet-activating factor (PAF) acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) inactivates potent lipid messengers such as PAF and modified phospholipids generated in settings of oxidant stress. In humans, PAF-AH circulates in blood in fully active form and associates with high and low density lipoproteins (HDL and LDL). Several studies suggest that the location of PAF-AH affects both the catalytic efficiency and the function of the enzyme in vivo. The distribution of PAF-AH among lipoproteins varies widely among mammals. Here, we report that mouse and human PAF-AHs associate with human HDL particles of different density. We made use of this observation in the development of a binding assay to identify domains required for association of human PAF-AH with human HDL. Sequence comparisons among species combined with domain-swapping and site-directed mutagenesis studies led us to the identification of C-terminal residues necessary for the association of human PAF-AH with human HDL. Interestingly, the region identified is not conserved among PAF-AHs, suggesting that PAF-AH interacts with HDL particles in a manner that is unique to each species. These findings contribute to our understanding of the mechanisms responsible for association of human PAF-AH with HDL and may facilitate future studies aimed at precisely determining the function of PAF-AH in each lipoprotein particle.
Highlights
platelet-activating factor (PAF), oxidatively fragmented glycerophospholipids, esterified F2-isoprostanes, and phospholipid hydroperoxides [2,3,4,5]
We previously reported that PAF-AH can migrate among lipoproteins [12], found that the location of the enzyme impacts its catalytic activity [13], and presented evidence supporting a model wherein HDL may act as a transport system to distribute PAF-AH among LDL particles [14]
We found that mouse and human PAF-AHs associated with Pefabloc-treated murine HDL (p-mHDL) particles of the same density
Summary
Materials—[3H-acetyl]PAF was purchased from Amersham Biosciences (Piscataway, NJ) and unlabeled PAF was from Avanti Polar Lipids (Alabaster, AL). HDL particles were isolated as previ- cated, we utilized “modified” KBr density gradients designed to ously described [12] and were treated with Pefabloc according to improve resolution in the heavy density region. These gradients the instructions provided by the manufacturer. PAF-AH associated exclusively tively, and expressed the recombinant proteins in the Esche- with HDL particles, in agreement with a previous report
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