Epidermis-type lipoxygenase 3 is a member of the lipoxygenase family of enzymes; in humans, it is encoded by the ALOXE3gene. This gene is located on chromosome 17 at position 13.1 where it forms a cluster with two other lipoxygenases, ALOX12B and ALOX15B. Among the human lipoxygenases, ALOXE3 is most closely related in amino acid sequence to ALOX12B. ALOXE3, ALOX12B, and ALOX15B are often classified as epidermal lipoxygenases, in distinction to the other three human lipoxygenases, because they were initially defined as being highly or even exclusively expressed and functioning in skin. The epidermis-type lipoxygenases are now regarded as a distinct subclass within the multigene family of mammalian lipoxygenases with mouse Aloxe3 being the ortholog to human ALOXE3, mouse Alox12b being the ortholog to human ALOX12B, and mouse Alox8 being the ortholog to human ALOX15B . ALOX12B and ALOXE3 in humans, Alox12b and Aloxe3 in mice, and comparable orthologs in other in other species are proposed to act sequentially in a multistep metabolic pathway that forms products that are structurally critical for creating and maintaining the skin's water barrier function.
Tissue distribution
Immunologically detected ALOXE3 and ALOX12B in humans and Aloxe3 and Alox12b in mice have a similar tissue distribution in being highly expressed in the outer, differentiated layers of the epidermis; they co-localize at the surface of keratinocytes in the stratum granulosum of mouse skin and during mouse embryogenesis appear concurrently at the onset of skin development at day 15.5. ALOXE3 mRNA in humans was also detected at low levels in the pancreas, ovary, brain, testis, placenta, and some secretory epithelia. Aloxe3 and Alox12b mRNA was detected in the tongue, forestomach, trachea, brain, testis, and adipose tissue of mice and in the spinal cord of rats.
Activity
Epidermal tissue
ALOX12B, like most of the other lipoxygenases, possesses dioxygenase activity: it catalyzes the incorporate dioxygen into a single substrate. Owing to this activity, the enzyme adds in the form of a hydroperoxyl residue to arachidonic acid at its 12th carbon thereby forming 12-hydroperoxy-5Z,8Z,10E,14Z-icosatetraenoic acid.
arachidonic acid + O2 12R-HpETE
Hydroperoxy-containing polyunsaturated fatty acids such as 12R-HETE readily breakdown through non-enzymatic transformations in which the two oxygen atoms of the hydroperoxy residue rearrange to form PUFAs containing one hydroxyl residue and one epoxide residue. This transformation may occur in tissues or during tissue preparations with 12-HpETE to form Hepoxilins, i.e. epoxyalcohols of 12-HpETE that are of the A type bond or, alternatively, B type ; these non-enxymatically formed products are a mixture of hydroxy and epoxy R,Sstereoisomers and diastereomers. In addition to arachidonic acid, ALOX12B metabolizes linoleic acid to 9-hydroperoxy-10,12-octadecadienoic acid :
LA + O2 9R-HpODE.
ALOXE3 is an atypical lipoxygenase in that under most but not all experimental conditions, it lacks the dioxygenase activity that converts PUFA to hydroperoxide metabolites; rather, it possess hepoxilin synthase activity; that is, it converts hydroperoxy-containing PUFAs to hepoxilin-like epoxyalcohol products; these products, unlike those formed by non-enzymatic transformations, are specific isomers with just one form of the chiral hydroxy and epoxy residues. ALOX3E metabolizes 12R-HpETE to 8R-hydroxy-11R,12R-epoxy-eicosatrienoic acid and metabolizes 9R-HpODE to products that contain either an epoxyalcohol or a ketone residue. It exhibits relatively weak activity in conducting this conversion on free 9R-HODE but stronger activity when 9R-HpODE is presented as its methyl ester. ALOXE3's primary function in epidermal tissue appears to be to metabolize the 9R-HpODE moiety that is not free but rather esterified to certain ceramide lipids. LA is the most abundant fatty acid in the skin epidermis, being present mainly esterified to the omega-hydroxyl residue of amide-linked omega-hydroxylated very long chain fatty acids in a unique class of ceramides termed esterified omega-hydroxyacyl-sphingosine. EOS is an intermediate component in a proposed multi-step metabolic pathway which delivers VLCFAs to the cornified lipid envelop in the skin's Stratum corneum; the presence of these wax-like, hydrophobic VLCFAs is needed to maintain the skin's integrity and functionality as a water barrier 9R,10R-trans-epoxide,13R-hydroxy-10E-octadecenoic acid, b) 9-keto-10E,12Z-octadecadienoic acid, and c)' 9R,10R-trans-epoxy-13-keto-11E''-octadecenoic acid. The ALOX12B/ALOE3-oxidized products, it is proposed, signal for their hydrolysis from EOS; this allows the multi-step metabolic pathway to proceed in delivering the VLCFAs to the cornified lipid envelop in the skin's Stratum corneum.
Other tissues
AloxE3 appears responsible for forming hepoxilins A and/or B from 12R-HpETE in the spinal fluids of rats and ALOXE3 is proposed to be responsible for the formation of these hepoxilins in various human tissues although the presence and activity of ALOXE3 in many of these hepoxilin-forming tissues has not yet been demonstrated. Spinal Aloxe3, apparently through its ability to make hepoxilins, appears responsible for the hyperalgesia which accompanies inflammation in rats. Aloxe3 appears necessary and sufficient for the differentiation of mouse 3T3-L1 fibroblast cells into adipocytes ; the function of Aloxe3 in this differentiation appears to be to its metabolism 12R-HpETE into hepoxilins A3 or B3 which directly activate Peroxisome proliferator-activated receptor gamma which in turn initiates the expression of adipocyte-differentiation genes.
Clinical significance
Congenital ichthyosiform erythrodema
Deletions of Alox12b or Aloxe3 genes by gene knockout in mice cause a congenital scaly skin disease which is characterized by a greatly reduced skin water barrier function and other features found in the autosomal recessivenonbullous Congenital ichthyosiform erythroderma disease of humans.; homozygous recessive deleterious mutations in ALOXE3 or ALOX12B are likewise causes, albeit rare, of this congenital disease in humans. ARCI refers to nonsyndromic congenital Ichthyosis including Harlequin-type ichthyosis, Lamellar ichthyosis, and Congenital ichthyosiform erythroderma. ARCI has an incidence of about 1/200,000 in European and North American populations; 40 different mutations in ALOX12B and 13 different mutations in ALOXE3 genes account for a total of about 10% of ARCI cases; these mutations are homozygous recessive, cause a total loss of ALOX12B or ALOXE3 function, and can be associated with any of the three cited forms of the disease.
In mice lacking Aloxe3 activity due to gene knockout of the Alox3 gene, levels in skin of hepoxilins A3 and B3, as well as their metabolites, trioxilins A3 and B3, are greatly reduced. Furthermore, rat Aloxe3 has been implicated in the production of hepoxilin B3 in studies that transfected its gene into cultured HEK 293 cells and similarly implicated in the inflammation-induced production of hepoxilin B3 in the spine of rats as well as the perception of pain by these animals using pharmacological inhibitor and siRNA-based gene knockdown studies. Finally, cultured human skin cells, which are rich in ALOXE3 readily convert arachidonic acid as well as 12S-hydroperoxy-eicosatetraenoic acid to Hepoxilin B3; this production, in keeping with the higher content of ALOXE3, is far greater in the skin cells isolated from subjects with psoriasis. These results suggest that ALOXE3 and its orthologs contribute greatly to or are the hepoxylin synthase activity responsible for producing bioactive hepoxilins in the skin and other ALOXE3/ortholog-rich tissues of mammals, possibly including humans.
Other possible clinical significances
The distribution of ALOXE3 and Aloxe3 suggests that these lipoxygenases may serve functions not only in the skin but also in other tissues. The studies reported in the above "Activities, Other tissues", subsection allow that the pain perception and adipocyte differentiation activities of Aloxe3 in rodents might also occur in humans.
Toxicity
Interuterine delivery of e-Lox-3 to mice at gestational day 14.5 resulted in fetal growth restriction and intrauterine death apparently due to a strongly negative effect on placental development.
