Rosenblat Mira, Gaidukov Leonid, Khersonsky Olga, Vaya Jacob, Oren Roni, Tawfik Dan S, Aviram Michael
Lipid Research Laboratory, Technion Faculty of Medicine, Haifa 31096, Israel.
J Biol Chem. 2006 Mar 17;281(11):7657-65. doi: 10.1074/jbc.M512595200. Epub 2006 Jan 10.
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.
利用在大肠杆菌中表达的对氧磷酶 -1（PON1）和载脂蛋白A -I（apoA -I）的重组变体以及由磷脂酰胆碱/游离胆固醇（PC/FC）和apoA -I组成的重组高密度脂蛋白（rHDL）颗粒，研究了与高密度脂蛋白（HDL）相关的PON1在巨噬细胞中的抗动脉粥样硬化特性，即抑制细胞介导的低密度脂蛋白（LDL）氧化和刺激胆固醇流出。相对于PC/FC颗粒，apoA -I可将PON1内酯酶活性刺激约7倍。与结合不含apoA -I的PC/FC颗粒的野生型（WT）PON1相比，结合到rHDL上的WT PON1分别将巨噬细胞介导的LDL氧化抑制程度和从细胞中刺激胆固醇流出的程度提高到2.3倍和3.2倍。我们还测试了PON1催化组氨酸二元突变体（H115Q和H134Q），它们折叠正确，与WT PON1以类似方式结合HDL，但几乎没有内酯酶活性。这些突变体不能抑制巨噬细胞介导的LDL氧化，也不能刺激rHDL介导的细胞胆固醇流出。此外，虽然与HDL结合的WT PON1可诱导巨噬细胞中溶血磷脂酰胆碱（LPC）的形成，但组氨酸二元突变体则不能，这表明HDL相关的PON1的上述抗动脉粥样硬化特性涉及LPC释放。事实上，用浓度不断增加的LPC富集巨噬细胞会导致细胞氧化LDL的能力受到抑制，并以LPC剂量依赖的方式刺激巨噬细胞中HDL介导的胆固醇流出。因此，我们首次直接表明PON1的抗动脉粥样硬化特性与其脂内酯酶活性有关，并提出了一个模型，其中PON1作为脂内酯酶分解氧化脂质并生成LPC。
