Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr;59(4):684-695.
doi: 10.1194/jlr.M082701. Epub 2018 Feb 19.

Catalytic activities of mammalian epoxide hydrolases with cis and trans fatty acid epoxides relevant to skin barrier function

Haruto Yamanashi  1 , William E Boeglin  2 , Christophe Morisseau  3 , Robert W Davis  4 , Gary A Sulikowski  4 , Bruce D Hammock  3 , Alan R Brash  5
Affiliations

Catalytic activities of mammalian epoxide hydrolases with cis and trans fatty acid epoxides relevant to skin barrier function

Haruto Yamanashi et al. J Lipid Res. 2018 Apr.

Abstract

Lipoxygenase (LOX)-catalyzed oxidation of the essential fatty acid, linoleate, represents a vital step in construction of the mammalian epidermal permeability barrier. Analysis of epidermal lipids indicates that linoleate is converted to a trihydroxy derivative by hydrolysis of an epoxy-hydroxy precursor. We evaluated different epoxide hydrolase (EH) enzymes in the hydrolysis of skin-relevant fatty acid epoxides and compared the products to those of acid-catalyzed hydrolysis. In the absence of enzyme, exposure to pH 5 or pH 6 at 37°C for 30 min hydrolyzed fatty acid allylic epoxyalcohols to four trihydroxy products. By contrast, human soluble EH [sEH (EPHX2)] and human or murine epoxide hydrolase-3 [EH3 (EPHX3)] hydrolyzed cis or trans allylic epoxides to single diastereomers, identical to the major isomers detected in epidermis. Microsomal EH [mEH (EPHX1)] was inactive with these substrates. At low substrate concentrations (<10 μM), EPHX2 hydrolyzed 14,15-epoxyeicosatrienoic acid (EET) at twice the rate of the epidermal epoxyalcohol, 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoic acid, whereas human or murine EPHX3 hydrolyzed the allylic epoxyalcohol at 31-fold and 39-fold higher rates, respectively. These data implicate the activities of EPHX2 and EPHX3 in production of the linoleate triols detected as end products of the 12R-LOX pathway in the epidermis and implicate their functioning in formation of the mammalian water permeability barrier.

Keywords: 14,15-epoxyeicosatrienoic acid; epidermis; ichthyosis; linoleic acid; lipoxygenase; trihydroxy-linoleate.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
The proposed 12R-LOX pathway in the mammalian epidermis and formation of linoleate triols. A: The 12R-LOX/eLOX3 pathway of the mammalian epidermis and proposed involvement of EH forming linoleate triol. The stepwise transformations increase the polarity of the unionized fatty acid moiety (indicated by the decreased lipid/aqueous partition coefficients, cLogP calculated by ChemDraw). B: Previously reported patterns of linoleate triols esterified in Cer-EOS as detected by LC-MS of pig and human epidermis (7). Structures of triol-1 and -3 are the same as illustrated later in Fig. 5A of the Results.
Fig. 2.
Fig. 2.
Structures of the linoleate-derived trans and cis allylic epoxyalcohols 1 and 2 and the model cis and trans fatty acid epoxides 3 and 4. The epidermal-related epoxides are trans-epoxyalcohol 1 and cis-epoxyalcohol 2. The model C18:1 epoxides are trans-epoxide 3 and cis-epoxide 4.
Fig. 3.
Fig. 3.
RP-HPLC analysis of the pH sensitivity of epoxyalcohols 1 and 2 exposed to pH 5–8. The trans-epoxyalcohol 1 and cis-epoxyalcohol 2 (30 μM) were incubated in 50 μl aliquots of 0.1 M phosphate buffers at pH 5, 6, 7, or 8 for 30 min at 37°C. The samples were then injected directly on RP-HPLC. The HPLC analysis used a Kinetex 2.6 μ 100 × 3 mm column with an isocratic solvent acetonitrile/water/glacial acetic acid (45/55/0.01, by volume) at a flow rate of 0.4 ml/min with UV detection at 205 nm. Retention times are illustrated for the starting material (epoxyalcohol), the triol hydrolysis products, and a δ-ketol formed during acid-catalyzed rearrangement (17).
Fig. 4.
Fig. 4.
Identification of the triol products of nonenzymatic hydrolysis of epoxyalcohols 1 and 2. Triol isomers were separated on LC-MS by SP-HPLC of the 1,2-acetonide PFB ester derivative and detected as the M – PFB ion at m/z 369 (7). The top chromatogram illustrates separation of eight triol isomers (A). B, C: Show that trans-epoxyalcohol 1 is hydrolyzed at pH 5 or pH 6 to a mixture of triols 1, 2, 3 and 4. D, E: The cis-epoxyalcohol 2 is hydrolyzed at pH 5 or pH 6 to a mixture of four triols, predominantly 1, and also including triols 2, 3, and 4. The HPLC analysis used a Phenomenex HILIC column (100 × 2 mm) with a solvent system of hexane/IPA (100:1, v/v) at a flow rate of 0.5 ml/min.
Fig. 5.
Fig. 5.
Structures of the triol products of nonenzymatic hydrolysis of epoxyalcohols 1 and 2. The percentage of each triol product of nonenzymatic hydrolysis is shown next to the arrows.
Fig. 6.
Fig. 6.
Amino acid sequences of EH3 (EPHX3). On top is the sequence of human EH3. Below are three constructs of mouse EH3 prepared for testing in the current study, each with a N-terminal (His)6 tag: i) truncated mouse EH3 with the rodent-specific 57 amino acid N-terminus removed; ii) truncated mouse EH3 containing a seven amino acid insert (VSVPPVK) at position 138; and iii) full-length mouse EH3. Arrowheads (with numbering of human EH3 residues) indicate the α/β-hydrolase catalytic triad (D173, D307, H337) and the two tyrosines (Y220, Y281) important for initial activation of the oxirane ring (14).
Fig. 7.
Fig. 7.
Comparison of the enzymatic hydrolysis of model cis and trans fatty acid epoxides 3 and 4. The trans-epoxide 3 and the cis-epoxide 4 were incubated in 50 μl aliquots of 0.1 M phosphate buffers at pH 8 with human sEH, human EH3, and truncated mouse EH3 containing the seven amino acid insert (see Fig. 6). The samples were then injected directly on RP-HPLC; the samples were slightly acidified by addition of 2 μl of 10-fold dilute glacial acetic acid immediately before injection on column. The HPLC analysis used a Kinetex 2.6 μ 100 × 3 mm column with an isocratic solvent, acetonitrile/water/glacial acetic acid (60/40/0.01, by volume), at a flow rate of 0.4 ml/min with UV detection at 205 nm. Retention times are illustrated for the starting material (model fatty acid epoxide) and the diol hydrolysis products.
Fig. 8.
Fig. 8.
pH profile of EH activities with trans-epoxide 3. The model trans-epoxide 3 (50 μM) was incubated in 50 μl aliquots of 0.1 M phosphate buffers at pH, 5, 6, 7, or 8 for 30 min at 37°C with human sEH and human EH3. The samples were then injected directly on RP-HPLC; the pH 7 and 8 samples were slightly acidified before injection on column. Chromatographic conditions were the same as in Fig. 7. The data points represent n = 3, ± range.
Fig. 9.
Fig. 9.
RP-HPLC analysis of the hydrolysis of epoxyalcohols 1 and 2 by EHs. The trans-epoxyalcohol 1 and the cis-epoxyalcohol 2 were incubated in 50 μl aliquots of Tris buffer (10 mM, pH 8.0) with human sEH, human EH3, two N-terminally truncated constructs of mouse EH3 (with and without the seven amino acid insert, Fig. 6), and human mEH. Chromatographic conditions were the same as in Fig. 3. Retention times are illustrated for the starting material (epoxyalcohol) and the triol hydrolysis products.
Fig. 10.
Fig. 10.
Identification of the triol products of enzymatic hydrolysis of epoxyalcohols 1 and 2. Triol isomers were separated on LC-MS by SP-HPLC of the 1,2-acetonide PFB ester derivative and detected as the M – PFB ion at m/z 369 (7). The top chromatogram illustrates separation of eight triol isomers (A). B–E: Show that human sEH, human EH3, and two constructs of mouse EH3 each hydrolyze epoxyalcohol 1 exclusively to triol 3. F, G: Show that human sEH and human EH3 hydrolyze epoxyalcohol 2 exclusively to triol 1. The HPLC analysis used a Phenomenex HILIC column (100 × 2 mm) with a solvent system of hexane/IPA (100:1, v/v) at a flow rate of 0.4 ml/min.
Fig. 11.
Fig. 11.
Comparison of the rates of hydrolysis of trans-epoxyalcohol 1 and 14,15-EET. The initial rates of transformation of trans-epoxyalcohol 1 (open squares) and 14,15-EET (open circles) are plotted against concentration of the epoxide on incubation with human sEH (A), human EH3 (B), and truncated mouse EH3 (C). Incubations were conducted in 50 μl aliquots of 0.1 M phosphate buffer at pH 8 at 37°C and terminated by addition of 50 μl acetonitrile and deuterated internal standards (d4-triol or d8-diol). The samples were analyzed using a Kinetex 2.6 μ C18 column (100 × 3 mm) with an isocratic solvent of acetonitrile/15 mM ammonium acetate pH 8.5 (25/75, by volume for triol analysis and 35/65, by volume for diol) at a flow rate of 0.4 ml/min, with LC-MS detection at m/z 329 (d0) and 333 (d4) for the triol product from trans-epoxyalcohol 1 and m/z 337 (do) and 345 (d8) for the diol product from 14.15-EET. The graph was obtained by using KaleidaGraph 4.0 (Synergy Software, Reading, PA); A, B: Data are expressed as mean ± SEM, n = 4. C: Data points represent the average of two or three determinations from two independent experiments.
See this image and copyright information in PMC

References

    1. Muñoz-Garcia A., Thomas C. P., Keeney D. S., Zheng Y., and Brash A. R.. 2014. The importance of the lipoxygenase-hepoxilin pathway in the mammalian epidermal barrier. Biochim. Biophys. Acta. 1841: 401–408. - PMC - PubMed
    1. Krieg P., and Furstenberger G.. 2014. The role of lipoxygenases in epidermis. Biochim. Biophys. Acta. 1841: 390–400. - PubMed
    1. Elias P. M., Williams M. L., Holleran W. M., Jiang Y. J., and Schmuth M.. 2008. Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism. J. Lipid Res. 49: 697–714. - PMC - PubMed
    1. Wertz P. W., and Downing D. T.. 1983. Acylglucosylceramides of pig epidermis: structure determination. J. Lipid Res. 24: 753–758. - PubMed
    1. Bowser P. A., Nugteren D. H., White R. J., Houtsmuller U. M., and Prottey C.. 1985. Identification, isolation and characterization of epidermal lipids containing linoleic acid. Biochim. Biophys. Acta. 834: 419–428. - PubMed

Publication types