The catalytic histidine dyad of high density lipoprotein-associated serum paraoxonase-1 (PON1) is essential for PON1-mediated inhibition of low density lipoprotein oxidation and stimulation of macrophage cholesterol efflux

Abstract

High density lipoprotein (HDL)-associated paraoxonase-1 (PON1) anti-atherogenic properties in macrophages, i.e. inhibition of cell-mediated oxidation of low density lipoprotein (LDL) and stimulation of cholesterol efflux, were studied using recombinant variants of PON1 and apoA-I expressed in Escherichia coli and reconstituted HDL (rHDL) particles composed of phosphatidylcholine/free cholesterol (PC/FC) and apoA-I. PON1 lactonase activity is stimulated by apoA-I by approximately 7-fold relative to PC/FC particles. Wild-type (WT) PON1 bound to rHDL inhibited macrophage-mediated LDL oxidation and stimulated cholesterol efflux from the cells to 2.3- and 3.2-fold greater extents, respectively, compared with WT PON1 bound to PC/FC particles without apoA-I. We also tested PON1 catalytic histidine dyad mutants (H115Q and H134Q) that are properly folded and that bind HDL in a similar mode compared with WT PON1, but that exhibit almost no lactonase activity. These could not inhibit macrophage-mediated LDL oxidation or stimulate rHDL-mediated cholesterol efflux from the cells. Furthermore, whereas HDL-bound WT PON1 induced the formation of lysophosphatidylcholine (LPC) in macrophages, the His dyad mutants did not, suggesting that the above anti-atherogenic properties of HDL-associated PON1 involve LPC release. Indeed, enrichment of macrophages with increasing concentrations of LPC resulted in inhibition of the cells' capability to oxidize LDL and in stimulation of HDL-mediated cholesterol efflux from the macrophages in an LPC dose-dependent manner. Thus, we provide the first direct indication that the anti-atherogenic properties of PON1 are related to its lipolactonase activity and propose a model in which PON1 acts as a lipolactonase to break down oxidized lipids and to generate LPC.