Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
Mutations associated with a congenital form of ichthyosis (NCIE) inactivate the epidermal lipoxygenases 12R-LOX and eLOX3
Introduction
Of the six lipoxygenases (LOX) in the human genome, 15-LOX-2, 12R-LOX and eLOX3 form a subgroup with preferential expression in epithelial tissues [1]. The three epithelial lipoxygenases are located as a gene cluster on human chromosome 17p13.1, and the proteins share about 50% amino acid identity. A common physiological role of these epithelial lipoxygenases was suggested in the regulation or modulation of normal proliferation and differentiation of epithelial cells and keratinocytes [1]. 12R-LOX is expressed almost exclusively in skin. Synthesis of its product, 12R-HETE, is up-regulated in psoriasis while it is almost undetectable in normal human skin [2], [3], [4], [5]. 15-LOX-2 appears to modulate the differentiation of prostate epithelial cells and acts as a negative regulator of the cell cycle; its expression tends to be lost in prostate cancer [6], [7]. Changes in lipoxygenase expression in these tissues imply an important role for these enzymes in the regulation of cellular proliferation and differentiation.
A recent genetic study links mutations in the coding sequence of eLOX3 or 12R-LOX genes to the development of an inherited skin disease, non-bullous congenital ichthyosiform erythroderma (NCIE) [8]. NCIE represents the first example in which mutations in the coding region of a LOX gene have been associated with a disease. However, the functional impact of these mutations on enzyme activities has not been defined. Since mutations in one or the other enzyme are associated with the same phenotype, the implication is that 12R-LOX and eLOX3 function in the same metabolic pathway [8]. 12R-LOX normally oxygenates arachidonic acid to the specific hydroperoxide product 12R-HPETE [9], [10]. eLOX3, however, does not catalyze a conventional lipoxygenase reaction. We recently reported that eLOX3 exhibits hydroperoxide isomerase (hepoxilin synthase) activity [11]. It preferentially transforms the 12R-LOX-derived product, 12R-HPETE, into a specific epoxyalcohol product, 8R-hydroxy-11R,12R-epoxyeicosa-5Z,9E,14Z-trienoic acid. This provides biochemical support for the concept of the two enzymes working together. We also have shown that the LOX-products 12S-HPETE and 15S-HPETE are converted to specific epoxyalcohol products of related structure, albeit with lower catalytic efficiency [11].
To explore whether the mutations reported in NCIE patients may be associated with alteration of 12R-LOX and eLOX3 functionality, in the current study we have mutated the corresponding amino acids in 12R-LOX and eLOX3. After overexpressing the wild-type and mutant proteins in E. coli and COS7 cells, we have characterized the essential catalytic properties. Our preliminary report and similar findings by another research group were presented recently [12], [13]. We also describe the further enzymatic transformation of the epoxyalcohol product of eLOX3 to a specific trihydroxy derivative, related isomers of which have been detected before in human epidermal fragments [14], [15].
Section snippets
Materials
[1-14C]arachidonic acid was purchased from PerkinElmer Life Sciences. [1-14C]15S-HPETE was prepared by reaction of [1-14C]arachidonic acid with soybean lipoxygenase type V (Sigma, St. Louis, MO). 12R-HPETE was prepared by arachidonic acid autoxidation as described previously [11]. [1-14C]12R-HPETE was prepared by reaction of [1-14C]arachidonic acid with human 12R-LOX [9].
Plasmids and site-directed mutagenesis
The cDNAs for human 12R-LOX and eLOX3 were subcloned into the pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA).
Site-directed mutagenesis and expression of 12R-LOX and eLOX3 mutants
In NCIE patients, two point mutations in 12R-LOX (L426P and H578Q) and a frameshift mutation were reported, and two point mutations (R396S and V500F) and a truncation (change arginine 234 to a premature stop codon) were found in eLOX3 [8]. The frameshift mutation and the truncation will definitely eliminate the catalytic activities. To explore whether the other naturally occurring mutations in 12R-LOX and eLOX3 may be associated with alterations of enzyme activities, we constructed all of these
Discussion
NCIE is an autosomal recessive form of ichthyosis with an incidence of about 1 in 100,000–200,000. The best characterized mutations inactivate the transglutaminase 1 gene, with resultant defects in the formation of the skin permeability barrier [22]. So far, NCIE with mutations in lipoxygenase genes has been reported in a small number of consanguineous families in the Mediterranean by Jobard et al. [8] and in additional patients from an independent study in Germany [13]. In the present study,
Acknowledgments
This work was supported by NIH grants AR-45943 and GM-53638 (to A.R.B.), by the Core Laboratories of the Vanderbilt Skin Disease Research Center Grant AR-41943, and the sequencing core of the Vanderbilt-Ingram Cancer Center Grant CA-89450.
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