title: A 12R-lipoxygenase in human skin: mechanistic evidence, molecular cloning, and expression.
abstract: 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 [10R-3H] and [10S-3H]-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 P450-catalyzed route to 12R-HETE in psoriatic scales. We cloned a lipoxygenase from human keratinocytes; the cDNA and deduced amino acid sequences share </=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.
authors.affiliation: Division of Clinical Pharmacology, Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA.
authors.forename: W E
authors.initials: WE
authors.lastname: Boeglin
authors.forename: R B
authors.initials: RB
authors.lastname: Kim
authors.forename: A R
authors.initials: AR
authors.lastname: Brash
mesh_terms: D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000378:metabolism*; D000595:Amino Acid Sequence; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011565:Psoriasis / Q000201:enzymology* / Q000473:pathology; D017421:Sequence Analysis; D012867:Skin / Q000201:enzymology* / Q000473:pathology
chemical_list: D018076:DNA, Complementary; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001092:Arachidonate 12-Lipoxygenase