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The Lipoxygenase Gene ALOXE3 Implicated in Skin Differentiation Encodes a Hydroperoxide Isomerase

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The Lipoxygenase Gene ALOXE3 Implicated in Skin Differentiation Encodes a Hydroperoxide Isomerase The lipoxygenase gene ALOXE3 implicated in skin differentiation encodes a hydroperoxide isomerase Zheyong Yu*, Claus Schneider*, William E. Boeglin*, Lawrence J. Marnett†‡§¶, and Alan R. Brash*ʈ Departments of *Pharmacology and †Biochemistry, ‡Vanderbilt Institute of Chemical Biology, §Vanderbilt-Ingram Cancer Center, and ¶Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232 Communicated by Judith P. Klinman, University of California, Berkeley, CA, June 12, 2003 (received for review April 4, 2003) Lipoxygenase (LOX) enzymes form fatty acid hydroperoxides used in relationship to skin pathophysiology is strongly suggested by a membrane remodeling and cell signaling. Mammalian epidermal LOX recent genetic study reporting that eLOX3 or 12R-LOX are type 3 (eLOX3) is distinctive in totally lacking this typical oxygenase mutated in six families affected by nonbullous congenital ichthyo- activity. Surprisingly, genetic evidence has linked mutations in eLOX3 siform erythroderma (NCIE) (6). NCIE is a major subtype of or a colocalizing enzyme, 12R-LOX, to disruption of the normal autosomal recessive congenital ichthyosis characterized by a gen- permeability barrier of the skin [Jobard, F., Lefe`vre, C., Karaduman, A., eralized ichthyosiform (scaly skin) phenotype. Based on these Blanchet-Bardon, C., Emre, S., Weissenbach, J., O¨ zgu¨c, M., Lathrop, M., genetic findings in NCIE it was suggested that eLOX3 and 12R- Prud’homme, J. F. & Fischer, J. (2002) Hum. Mol. Genet. 11, 107–113]. LOX are both involved in skin development and that they may Herein we identify a logical link of the biochemistry to the genetics. belong to the same metabolic pathway (6). eLOX3 functions as a hydroperoxide isomerase (epoxyalcohol syn- In this article we report biochemical studies identifying a catalytic thase) by using the product of 12R-LOX as the preferred substrate. activity of eLOX3 in the conversion of HPETE substrates. The 12R-Hydroperoxyeicosatetraenoic acid (12R-HPETE) is converted to products of this reaction are specific epoxyalcohols (hepoxilins or 8R-hydroxy-11R,12R-epoxyeicosa-5Z,9E,14Z-trienoic acid, one of the hepoxilin-type products). 12R-HPETE is a particularly good sub- isomers of hepoxilin A3, and to 12-ketoeicosatetraenoic acid in a 2:1 strate, and it is converted to a product of very distinctive structure. ratio. Other hydroperoxides, including 8R-HPETE, 12S-HPETE, and Our results suggest that eLOX3, although named LOX based on its 15S-HPETE, as well as the 13S- and 13R-hydroperoxides of linoleic acid gene sequence, lacks the typical catalytic activity of this enzyme are converted less efficiently. Mass spectrometric analysis of the class and instead represents a unique type of epoxyalcohol synthase. epoxyalcohol formed from [18O]15S-HPETE showed that both hy- droperoxy oxygens are retained in the product. We propose that the Materials and Methods ferrous form of eLOX3 initiates a redox cycle, unprecedented among Expression and Purification of Human eLOX3. The cDNA for human LOX in being autocatalytic, in which the hydroperoxy substrate is eLOX3 was cloned by PCR with cDNA prepared from human isomerized to the epoxyalcohol or keto product. Our results provide keratinocytes. To prepare the eLOX3 protein with an N-terminal strong biochemical evidence for a functional linkage of 12R-LOX and (His)6 tag, the eLOX3 cDNA was subcloned into the pET3a eLOX3 and clues into skin biochemistry and the etiology of ichthyo- expression vector (Novagen) with the 5Ј sequence encoded as ATG siform diseases in humans. CAT CAC CAT CAC CAT CAC GCA, with the last codon representing the start of the wild-type enzyme. The human eLOX3 ipoxygenases (LOXs) are a family of nonheme iron- was expressed in Escherichia coli BL21 (DE3) cells (Novagen), and Lcontaining enzymes that oxygenate unsaturated fatty acids the (His)6-tagged protein was purified on nickel-nitrilotriacetic acid such as arachidonic acid to specific hydroperoxide products (1). agarose (Qiagen, Valencia, CA) according to manufacturer instruc- tions. Fractions of 0.5 ml were collected off the affinity column and These may be metabolized further to various bioactive lipid ͞ mediators including leukotrienes, lipoxins, hydroxyeicosatetra- assayed by using SDS PAGE. Fractions containing eLOX3 were enoic acids (HETEs), and hepoxilins (2). Although LOX en- pooled and dialyzed against a buffer of 50 mM Tris (pH 7.5) zymes are catalytically active with free fatty acid substrates, some containing 300 mM NaCl to remove the imidazole. will also oxygenate esterified substrates such as the phospholipid 18 or cholesterol esters. LOX metabolites play important roles in Preparation of Hydroperoxides. 15S-HPETE and [ O]15S-HPETE cell signaling or modification of membrane structures (1, 3). were prepared from arachidonic acid by using soybean LOX (Sigma There are five active LOXs found in human beings: 5-LOX, type V) (7). 12R-HPETE and 12S-HPETE were prepared from arachidonate methyl ester by the following route: (i) autoxidation 12S-LOX, 12R-LOX, 15-LOX-1, and 15-LOX-2. A sixth gene ␣ family member, epidermal LOX type 3 (eLOX3, gene symbol of arachidonate methyl ester in the presence of -tocopherol (8); (ii) isolation of a mixture of HPETE methyl esters with a 70-g ALOXE3) was described first in the mouse (4), and in humans ϫ in 2001 (5). The amino acid sequence of human eLOX3 shows open-bed silica column (3.0 25 cm) and a solvent system of 5% ethyl acetate in hexane; (iii) isolation and purification of 12R,S- the closest similarity to 12R-LOX (54% identity) and 15-LOX-2 ␮ (51%). It contains the characteristic well conserved amino acid HPETE methyl ester by RP-HPLC [Waters Symmetry C18 7- m, 1.9 ϫ 30-cm, solvent system of acetonitrile͞water (70:30 by volume) residues found in all LOXs including the putative iron-binding ͞ ligands and additional structure-determining residues. These andaflowrateof10mlmin]; (iv)resolutionof12R- and features clearly indicate that eLOX3 belongs to the LOX gene 12S-HPETE methyl esters with a Chiralpak AD-RH column, eluted with a solvent of methanol͞water (88:12 by volume) and a family. The question of the catalytic activity of eLOX3, none- ͞ Ϸ theless, has remained elusive. No enzymatic activity has been flow rate of 1 ml min; the R enantiomer eluted at 10 min, and the S enantiomer eluted at 15 min; (v) preparation of the free acids by detected by using linoleic or arachidonic acids, the prototypical ͞ ͞ C18 and C20 LOX substrates, nor with methyl arachidonate or treatment for 30 min with 0.5 M KOH in water methanol cholesteryl arachidonate (4). In the first section of Results we dichloromethane (1:1:0.05) at room temperature, followed by acid- confirm and extend these findings. Studies in humans and the mouse indicate that eLOX3 has a Abbreviations: LOX, lipoxygenase; H(P)ETE, hydro(pero)xyeicosatetraenoic acid; eLOX3, limited scope of tissue expression, being mainly confined to kera- epidermal LOX type 3; NCIE, nonbullous congenital ichthyosiform erythroderma; NDGA, tinized epithelia such as skin. From PCR evidence it seems to be nordihydroguaiaretic acid. coexpressed in tissues that express the 12R-LOX (5). A functional ʈTo whom correspondence should be addressed. E-mail: [email protected]. 9162–9167 ͉ PNAS ͉ August 5, 2003 ͉ vol. 100 ͉ no. 16 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1633612100 Downloaded by guest on September 24, 2021 ification to pH 6.0 and extraction into dichloromethane; and (vi) final purification of the free acids (12R-HPETE or 12S-HPETE) by straight-phase HPLC (Alltech Associates Econosil silica column, solvent system of hexane͞isopropanol͞acetic acid (100:1:0.1 by volume) and a flow rate of 2 ml͞min). eLOX3 Activity Assay. Incubations with the purified enzyme were conducted typically in 500 ␮l of incubation buffer (50 mM Tris͞150 mM NaCl, pH 7.5) by using 0.01–0.1 ␮M enzyme concentration in a 1-cm path length microcuvette. HPETE (5–10 ␮g) was added and incubated at room temperature for 10 min. eLOX3 activity was monitored by repetitive scanning in the range of 350–200 nm or by monitoring disappearance of the signal at 235 nm in the time-drive mode. To measure the rate of eLOX3 reaction over the substrate concentration range of 5–250 ␮M, reactions were conducted in a 2-mm path length microcuvette (0.5 ml); the decrease of absorbance at 235 nm was followed, and the rate was calculated from the initial linear part of the curve. HPLC Analysis. Products of the eLOX3 reactions with HPETE substrates were analyzed initially by RP-HPLC with a Waters Symmetry C18 5-␮m column (0.46 ϫ 25 cm) eluted at a flow rate of1ml͞min with methanol͞water͞acetic acid (80:20:0.01 by vol- ume) and UV detection at 205, 220, 235, and 270 nm with an Agilent 1100 series diode array detector. The main products were recovered from the reversed-phase solvent by the addition of water and extraction with dichloromethane. Further purification was carried out by straight-phase HPLC with an Alltech Associates Econosil silica column (0.46 ϫ 25 cm), a solvent system of hexane͞ ͞ Fig. 1. Reaction of eLOX3 with hydroperoxide substrates. (A) Overlay of UV isopropanol acetic acid (100:2:0.1 by volume), and a flow rate of ␮ ␮ ͞ spectra of reaction of eLOX3 (0.05 M) with 12R-HPETE (40 M). The sample 1ml min. was scanned from 350 to 200 nm before the addition of enzyme (t ϭ 0 min) and then immediately after mixing (Ϸ15 sec) and at reaction times of 1, 2, 3, 4, and GC-MS Analysis. Analysis of the methyl ester trimethylsilyl ether 5 min. The arrows indicate the decreasing absorbance at 235 nm and the derivatives of the products was carried out in the positive-ion increase at 285 nm during the reaction. (B) Rates were measured using 0.025 electron impact mode (70 eV) by using either a Hewlett–Packard ␮M enzyme by continuous recording of the decrease in absorbance at 235 nm.
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