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
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
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DOI：
10.1074/jbc.m512595200
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发表时间：
2006-03-17
影响因子：
4.8
通讯作者：
Aviram, M
中科院分区：
文献类型：
--
作者：
Rosenblat, M;Gaidukov, L;Aviram, M
利用在大肠杆菌中表达的高密度脂蛋白相关的对氧磷酯酶-1(PON1)的重组变异体和由磷脂酰胆碱/游离胆固醇(PC/Fc)和载脂蛋白A-I组成的重组高密度脂蛋白(RHDL)颗粒，研究了其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)氧化和刺激胆固醇外流。与PC/FC颗粒相比，apoA-I对PON1内酯酶活性的刺激作用类似于7倍。与rHDL结合的野生型(WT)PON1抑制巨噬细胞介导的低密度脂蛋白氧化和刺激细胞胆固醇外流的程度分别是与不含apoA-I的PC/FC颗粒结合的WT PON1的2.3和3.2倍。我们还测试了PON1催化组氨酸二联体突变体(H115Q和H134Q)，它们被正确折叠，与WT PON1以类似的方式结合高密度脂蛋白，但几乎没有乳糖酶活性。它们不能抑制巨噬细胞介导的低密度脂蛋白氧化，也不能刺激r高密度脂蛋白介导的胆固醇外流。此外，尽管高密度脂蛋白结合的WT PON1诱导巨噬细胞形成溶血磷脂酰胆碱(LPC)，但His二联突变体不能，这表明上述抗动脉粥样硬化的特性与高密度脂蛋白相关的PON1释放LPC有关。事实上，随着LPC浓度的增加，巨噬细胞的浓缩导致细胞氧化低密度脂蛋白的能力受到抑制，并以LPC剂量依赖的方式刺激高密度脂蛋白介导的胆固醇从巨噬细胞流出。因此，我们首次提出PON1的抗动脉粥样硬化作用与其脂酶活性有关，并提出了PON1作为脂酶分解氧化脂质并生成LPC的模型。
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 similar to 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.