The lipoxygenase gene ALOXE3 implicated in skin differentiation encodes a hydroperoxide isomerase

Abstract

Herein we identify a logical link of the biochemistry to the genetics. eLOX3 functions as a hydroperoxide isomerase (epoxyalcohol synthase) by using the product of 12R-LOX as the preferred substrate. 12R-Hydroperoxyeicosatetraenoic acid (12R-HPETE) is converted to 8R-hydroxy-11R,12R-epoxyeicosa-5Z,9E,14Z-trienoic acid, one of the isomers of hepoxilin A 3, and to 12-ketoeicosatetraenoic acid in a 2:1 ratio. Other hydroperoxides, including 8R-HPETE, 12S-HPETE, and 15S-HPETE, as well as the 13S-and 13R-hydroperoxides of linoleic acid are converted less efficiently. Mass spectrometric analysis of the epoxyalcohol formed from [ 18 O]15S-HPETE showed that both hydroperoxy oxygens are retained in the product. We propose that the ferrous form of eLOX3 initiates a redox cycle, unprecedented among LOX in being autocatalytic, in which the hydroperoxy substrate is isomerized to the epoxyalcohol or keto product. Our results provide strong biochemical evidence for a functional linkage of 12R-LOX and eLOX3 and clues into skin biochemistry and the etiology of ichthyosiform diseases in humans. L ipoxygenases (LOXs) are a family of nonheme ironcontaining enzymes that oxygenate unsaturated fatty acids such as arachidonic acid to specific hydroperoxide products (1). These may be metabolized further to various bioactive lipid mediators including leukotrienes, lipoxins, hydroxyeicosatetraenoic acids (HETEs), and hepoxilins (2). Although LOX enzymes are catalytically active with free fatty acid substrates, some will also oxygenate esterified substrates such as the phospholipid or cholesterol esters. LOX metabolites play important roles in cell signaling or modification of membrane structures (1, 3).There are five active LOXs found in human beings: 5-LOX, 12S-LOX, 12R-LOX, 15-LOX-1, and 15-LOX-2. A sixth gene family member, epidermal LOX type 3 (eLOX3, gene symbol ALOXE3) was described first in the mouse (4), and in humans in 2001 (5). The amino acid sequence of human eLOX3 shows the closest similarity to 12R-LOX (54% identity) and 15-LOX-2 (51%). It contains the characteristic well conserved amino acid residues found in all LOXs including the putative iron-binding ligands and additional structure-determining residues. These features clearly indicate that eLOX3 belongs to the LOX gene family. The question of the catalytic activity of eLOX3, nonetheless, has remained elusive. No enzymatic activity has been detected by using linoleic or arachidonic acids, the prototypical C18 and C20 LOX substrates, nor with methyl arachidonate or cholesteryl arachidonate (4). In the first section of Results we confirm and extend these findings.Studies in humans and the mouse indicate that eLOX3 has a limited scope of tissue expression, being mainly confined to keratinized epithelia such as skin. From PCR evidence it seems to be coexpressed in tissues that express the 12R-LOX (5). A functional relationship to skin pathophysiology is strongly suggested by a recent genetic study reporting that eLOX3 or 12R-LOX are mutated in six families affected by nonbul...

Hepoxilin A3 (HXA3) synthase deficiency is causative of a novel ichthyosis form

“…The antibody raised against rat 12S-LOX (HXA 3 synthase) recognized also human epidermal 12S-LOX (HXA 3 synthase). As mutated eLOX3 is still expressed at the protein level in cells [7,8], these findings suggest to us that the patient in the case study has a different ichthyosis form than that reported for NCIE patients, who showed deficiency of 12R-LOX or eLOX3 due to gene mutations [5][6][7].…”

“…These authors also postulated that product of one of these enzymes (12R-LOX) may serve as substrate for the other (eLOX3), thus functioning in the same metabolic pathway [4]. Yu et al [6,7] showed that eLOX3, which does not exhibit any oxygenase activity, operates as an epoxy alcohol synthase utilizing preferably the product from 12R-LOX, termed 12R-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12R-HpETE) as substrate, thus providing for the first time a clear-cut evidence for a functional linkage of proteins 12R-LOX and eLOX3. Both proteins 12R-LOX and eLOX3 are coexpressed in tissues, and mutations in any of the two causative genes can lead to NCIE [4].…”

Complicating the role of p53 in aging

“…For example, the deleted region contains a cluster of 3 genes involved in lipid oxygenation, two of which are epidermal lipoxygenases that are expressed primarily in the mouse skin (Boeglin et al ., 1998;Jisaka et al ., 1997;Heidt et al ., 2000). Lipoxygenases have been shown to play important biological roles in mediating adipocyte and skin differentiation, inflammatory responses, and are also expressed in human hair roots (Baer & Green, 1993;Shillabeer et al ., 1998;Furstenberger et al ., 2002;Kantarci & Van Dyke, 2003;Yu et al ., 2003;Yu et al ., 2005). Correlating to this expression pattern, we do see skin phenotypes that include reduction in dermal thickness, subcutaneous adipose layer, hair re-growth potential and skin wound healing in the p53+/m mice, indicating that the loss of these genes may be responsible for this phenotype.…”

Harlequin ichthyosis and other autosomal recessive congenital ichthyoses: The underlying genetic defects and pathomechanisms

“…Mutations in the lipid transporter protein, ABCA12 cause defective lipid secretion into lamellar granules which then become expelled from the apical surface of keratinocytes [6] as mentioned above and lead to either LI [5] and HI [6,18] phentoypes. Lipoxygenase-3 and 12(R)-lipoxygenase are non-heme iron-containing dioxygenases expressed in the epidermis, and their exact functions are unknown [19,20]. They may be associated with lipid metabolism in the lamellar granule contents and/or intercellular lipid layers in the epidermis.…”