Zhu Hong, Takahashi Yoshitaka, Xu Wanpeng, Kawajiri Hiroo, Murakami Takashi, Yamamoto Miyuki, Iseki Shoichi, Iwasaki Tadao, Hattori Hiroaki, Yoshimoto Tanihiro
Department of Molecular Pharmacology, Kanazawa University Graduate School of Medical Science, Kanazawa 920-8640, Japan.
J Biol Chem. 2003 Apr 11;278(15):13350-5. doi: 10.1074/jbc.M212104200. Epub 2003 Feb 3.
Oxidation of low density lipoprotein (LDL) is the key step for the development of atherosclerosis. The 12/15-lipoxygenase expressed in macrophages is capable of oxygenating linoleic acid esterified to cholesterol in the LDL particle, and thus this enzyme is presumed to initiate LDL oxidation. We recently reported that LDL receptor-related protein (LRP) was required for the enzyme-mediated LDL oxidation by macrophages and suggested the selective uptake of cholesterol ester from LDL to the plasma membrane (Xu, W., Takahashi, Y., Sakashita, T., Iwasaki, T., Hattori, H., and Yoshimoto. T. (2001) J. Biol. Chem. 276, 36454-36459). To elucidate precise mechanisms of lipoxygenase-mediated LDL oxidation, we investigated the intracellular localization of 12/15-lipoxygenase. The 12/15-lipoxygenase was predominantly detected in cytosol of resting peritoneal macrophages and of macrophage-like J774A.1 cells permanently transfected with the cDNA for the enzyme. When the cells were treated with LDL and subjected to subcellular fractionation, the 12/15-lipoxygenase was detected in the membranes with a concomitant decrease in cytosol as shown by Western blot analysis. The levels of the enzyme associated with the membrane reached maximum in 15 min after LDL addition and then decreased. However, the enzymatic activity of 12/15-lipoxygenase in the membrane fraction was very weak even after LDL treatment. This fact supports the suicide inactivation of the enzyme by the oxygenation of cholesterol ester transferred from the LDL particle to the plasma membrane. Immunohistochemical analysis using an antibody against 12/15-lipoxygenase revealed that the plasma membrane was the major site of the enzyme translocation by the LDL treatment. LDL-dependent 12/15-lipoxygenase translocation was inhibited by a blocking antibody against LRP. Furthermore, an enzyme translocation inhibitor, L655238, inhibited the LDL oxidation caused by the 12/15-lipoxygenase. We propose that cholesterol ester selectively transferred from the LDL particle to the plasma membrane via LRP is oxygenated by 12/15-lipoxygenase translocated to this membrane.
低密度脂蛋白（LDL）的氧化是动脉粥样硬化发展的关键步骤。巨噬细胞中表达的12/15-脂氧合酶能够氧化LDL颗粒中酯化到胆固醇上的亚油酸，因此推测该酶启动了LDL氧化。我们最近报道巨噬细胞介导的酶促LDL氧化需要低密度脂蛋白受体相关蛋白（LRP），并提示胆固醇酯从LDL选择性摄取到质膜（Xu，W.，Takahashi，Y.，Sakashita，T.，Iwasaki，T.，Hattori，H.，和Yoshimoto，T.（2001）J.Biol.Chem.276，36454 - 36459）。为了阐明脂氧合酶介导的LDL氧化的精确机制，我们研究了12/15-脂氧合酶的细胞内定位。在静息腹膜巨噬细胞和用该酶的cDNA永久转染的巨噬细胞样J774A.1细胞的胞质溶胶中主要检测到12/15-脂氧合酶。当细胞用LDL处理并进行亚细胞分级分离时，如蛋白质免疫印迹分析所示，在膜中检测到12/15-脂氧合酶，同时胞质溶胶中的含量降低。与膜结合的酶水平在添加LDL后15分钟达到最大值，然后下降。然而，即使在LDL处理后，膜部分中12/15-脂氧合酶的酶活性也非常弱。这一事实支持了从LDL颗粒转移到质膜的胆固醇酯的氧化导致该酶的自杀失活。使用抗12/15-脂氧合酶抗体的免疫组织化学分析表明，质膜是LDL处理后该酶易位的主要部位。抗LRP阻断抗体抑制了LDL依赖性12/15-脂氧合酶的易位。此外，一种酶易位抑制剂L655238抑制了由12/15-脂氧合酶引起的LDL氧化。我们提出，通过LRP从LDL颗粒选择性转移到质膜的胆固醇酯被易位到该膜的12/15-脂氧合酶氧化。
