Functional analysis of a novel enzyme of the phospholipase D type involved in the endocannabinoid

参与内源性大麻素的新型磷脂酶 D 型酶的功能分析

基本信息

批准号：
17590251
负责人：
UEDA Natsuo
金额：
$ 2.24万
依托单位：
Kagawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Anandamide (N-arachidonoylethanolamine), which was found as an endogenous ligand for cannabinoid receptors (an endocannabinoid), is formed from membrane glycerophospholipids by two-step enzyme reactions in animal tissues. However, the responsible enzymes remained poorly characterized, and it has been difficult to study the anandamide biosynthesis by molecular biological approaches. Recently we succeeded for the first time in cDNA cloning and functional expression of a novel mammalian enzyme of the phospholipase D type (NAPE-PLD) that generates anandamide and other N-acylethanolamines from their corresponding N-acylphosphatidylethanolamines (NAPEs) (Okamoto et al. J. Biol. Chem. 279, 5298-305, 2004). In the present study, we principally characterized recombinant rat NAPE-PLD.The recombinant NAPE-PLD was expressed in Escherichia coli as a GST-fusion protein together with molecular chaperone. The enzyme was then solubilized with CHAPS, followed by purification to apparent homogeneity by glutathione affinity chromatography and hydroxyapatite chromatography. The purified enzyme was highly active with NAPEs, and did not discriminate various N-acyl species with C_4-C_<20>. In contrast, the enzyme was almost inactive with major membrane glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol). These results suggested the ability of NAPE-PLD to specifically degrade different NAPEs without damaging other membrane phospholipids. The purified enzyme was remarkably activated in a dose-dependent manner by millimolar concentrations of Mg^<2+> as well as Ca^<2+>. Atomic absorption spectrometry exhibited the presence of catalytically important zinc in NAPE-PLD. In addition, site-directed mutagenesis studies revealed that Asp-147, His-185, His-187, Asp-189, His-190, His-253, Asp-284, and His-321 of NAPE-PLD, that are highly conserved within the metallo-□-lactamase family, play crucial roles in the catalytic activity.

Anandamide（N-氨基甲酰乙醇胺）被发现是一种用于大麻素受体（内源配体）的内源配体，是由动物组织中的两步酶反应由膜甘油磷脂形成的。但是，负责的酶的特征仍然很差，并且很难通过分子生物学方法研究anandamide的生物合成。 Recently we succeeded for the first time in cDNA cloning and functional expression of a novel mammalian enzyme of the phospholipase D type (NAPE-PLD) that generates anandamide and other N-acylethanolamines from their corresponding N-acylphosphatidylethanolamines (NAPEs) (Okamoto et al. J. Biol. Chem. 279, 5298-305, 2004).在本研究中，我们主要表征了重组大鼠NAPE-PLD。重组NAPE-PLD在大肠杆菌中表达为GST融合蛋白与分子链条一起表达。然后将酶溶于谷物，然后通过谷胱甘肽亲和色谱和羟基磷灰石色谱法纯化为明显的均匀性。纯化的酶在NAPE上高度活跃，并且没有用C_4-C_ <20>区分各种N-酰基物种。相比之下，该酶几乎没有主要的膜甘油磷脂（磷脂酰胆碱，磷脂酰硫代苯胺，磷脂酰氧基和磷脂酰肌醇）。这些结果表明，Nape-PLD在不损害其他膜磷脂的情况下特异性降解不同的NAP的能力。纯化的酶通过毫米浓度的Mg^<2+>以及Ca^<2+>以剂量依赖性方式显着激活。原子滥用光谱法在NAPE-PLD中表现出催化重要的锌。此外，以网站为导向的诱变研究表明，NAPE-PLD的ASP-147，HIS-185，HIS-187，ASP-189，HIS-190，HIS-253，ASP-284和HIS-321在Metallo-□-lactamase家族中高度保守，在该猫科中发挥了至关重要的作用。