A 12R-lipoxygenase in human skin: Mechanistic evidence, molecular cloning, and expression
A 12R-lipoxygenase in human skin: Mechanistic evidence, molecular cloning, and expression
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DOI：
10.1073/pnas.95.12.6744
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发表时间：
1998-06-09
影响因子：
11.1
通讯作者：
Brash, AR
中科院分区：
文献类型：
--
作者：
Boeglin, WE;Kim, RB;Brash, AR
银屑病和其他增生性皮肤病的公认特征是不寻常的花生四烯酸代谢物12 R-羟基二十碳四烯酸（12 R-HETE）在皮肤中的积累。该羟基脂肪酸在手性上与公知的12 S-脂氧合酶的产物相反，迄今为止在哺乳动物中仅已知为细胞色素P450的产物。在这里，我们提供了人银屑病组织中脂氧合酶途径至12 R-HETE的机制证据，并描述了可以解释生物合成的12 R-脂氧合酶。最初，我们证明了八氘代花生四烯酸的C-12氘在银屑病鳞屑温育中转化为12 R-HETE时的保留，表明终产物不是通过12-酮衍生物从12 S-H（P）埃特异构化形成的。其次，对[10（R）-H-3]和[10（S）-H-3]标记的花生四烯酸形成的产物的分析表明，12 R-HETE合成与花生四烯酸10-碳中前R氢的立体选择性去除有关。该结果与银屑病鳞屑中12 R-脂氧合酶催化的12 R-HETE形成相容，而与p45 O催化的12 R-HETE途径不相容。我们从人角质形成细胞中克隆了一种脂氧合酶，其cDNA和推导的氨基酸序列与其他人脂氧合酶的同源性小于或等于50%。当在Hela细胞中表达时，该酶将花生四烯酸氧化为12-HPETE，>98%的12 R构型。12 R-脂氧合酶cDNA可通过PCR在银屑病鳞屑中检测到，并且通过角质形成细胞的北方分析作为2.5-脂氧合酶mRNA检测到。这种酶的鉴定扩展了已知的R-脂氧合酶在人类中的分布，并为银屑病的潜在治疗干预提供了另一个靶点。
A recognized feature of psoriasis and other proliferative dermatoses is accumulation in the skin of the unusual arachidonic acid metabolite, 12R-hydroxyeicosatetraenoic acid (12R-HETE). This hydroxy fatty acid is opposite in chirality to the product of the well-known 12S-lipoxygenase and heretofore in mammals is known only as a product of cytochrome P450s. Here we provide mechanistic evidence for a lipoxygenase route to 12R-HETE in human psoriatic tissue and describe a 12R-lipoxygenase that can account for the biosynthesis, Initially we demonstrated retention of the C-12 deuterium of octadeuterated arachidonic acid in its conversion to 12R-HETE in incubations of psoriatic scales, indicating the end product is not formed by isomerization from 12S-H(P)ETE via the 12-keto derivative. Secondly, analysis of product formed from [10(R)-H-3] and [10(S)-H-3]-labeled arachidonic acids revealed that 12R-HETE synthesis is associated with stereospecific removal of the pro-R hydrogen from the 10-carbon of arachidonate. This result is compatible with 12R-lipoxygenase-catalyzed formation of 12R-HETE and not with a p45O-catalyzed route to 12R-HETE in psoriatic scales. We cloned a lipoxygenase from human keratinocytes; the cDNA and deduced amino acid sequences share less than or equal to 50% identity to other human lipoxygenases. This enzyme, when expressed in Hela cells, oxygenates arachidonic acid to 12-HPETE, >98% 12R in configuration. The 12R-lipoxygenase cDNA is detectable by PCR in psoriatic scales and as a 2.5-kilobase mRNA by Northern analysis of keratinocytes. Identification of this enzyme extends the known distribution of R-lipoxygenases to humans and presents an additional target for potential therapeutic interventions in psoriasis.