SDR9C7 catalyzes critical dehydrogenation of acylceramides for skin barrier formation
Takuya Takeichi, … , Alan R. Brash, Masashi Akiyama
Takuya Takeichi, … , Alan R. Brash, Masashi Akiyama
Published October 31, 2019
Citation Information: J Clin Invest. 2020;130(2):890-903. https://doi.org/10.1172/JCI130675.
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Research Article Dermatology

SDR9C7 catalyzes critical dehydrogenation of acylceramides for skin barrier formation

Abstract

The corneocyte lipid envelope, composed of covalently bound ceramides and fatty acids, is important to the integrity of the permeability barrier in the stratum corneum, and its absence is a prime structural defect in various skin diseases associated with defective skin barrier function. SDR9C7 encodes a short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7) recently found mutated in ichthyosis. In a patient with SDR9C7 mutation and a mouse Sdr9c7-KO model, we show loss of covalent binding of epidermal ceramides to protein, a structural fault in the barrier. For reasons unresolved, protein binding requires lipoxygenase-catalyzed transformations of linoleic acid (18:2) esterified in ω-O-acylceramides. In Sdr9c7–/– epidermis, quantitative liquid chromatography–mass spectometry (LC-MS) assays revealed almost complete loss of a species of ω-O-acylceramide esterified with linoleate-9,10-trans-epoxy-11E-13-ketone; other acylceramides related to the lipoxygenase pathway were in higher abundance. Recombinant SDR9C7 catalyzed NAD+-dependent dehydrogenation of linoleate 9,10-trans-epoxy-11E-13-alcohol to the corresponding 13-ketone, while ichthyosis mutants were inactive. We propose, therefore, that the critical requirement for lipoxygenases and SDR9C7 is in producing acylceramide containing the 9,10-epoxy-11E-13-ketone, a reactive moiety known for its nonenzymatic coupling to protein. This suggests a mechanism for coupling of ceramide to protein and provides important insights into skin barrier formation and pathogenesis.

Authors

Takuya Takeichi, Tetsuya Hirabayashi, Yuki Miyasaka, Akane Kawamoto, Yusuke Okuno, Shijima Taguchi, Kana Tanahashi, Chiaki Murase, Hiroyuki Takama, Kosei Tanaka, William E. Boeglin, M. Wade Calcutt, Daisuke Watanabe, Michihiro Kono, Yoshinao Muro, Junko Ishikawa, Tamio Ohno, Alan R. Brash, Masashi Akiyama

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Figure 4

Quantitative LC-MS of oxidized linoleates esterified in WT and Sdr9c7–/– (Sdr9c7 KO) epidermis.

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Quantitative LC-MS of oxidized linoleates esterified in WT and Sdr9c7–/–...
(A) Quantitation of 9-HODE, 9,10-trans-11E-13-hydroxy (Epoxy-OH), 9,10-trans-epoxy-11E-13-ketone (Epoxy-ketone), 9,10-dihydroxy-11E-13-ketone (DiOH-ketone), and 9R,10S,13R-trihydroxy-linoleate (Triol RSR). (B) The same data from A, shown on scale with the Sdr9c7 knockout levels of Triol RSR. Results are expressed in ng/mg epidermis (mean ± SEM, n = 8, P < 0.001 for all pairs of WT compared with knockout, except for DiOH-ketone [n = 4, P < 0.05]). (C) LC-MS ion chromatograms illustrating analysis of linoleate triols: top panel, blue trace shows the triols in WT; middle panel, red trace shows the 100-fold higher levels of Triol RSR in Sdr9c7–/– compared with WT; lower panel, black trace shows the d4 triol internal standard used for quantitation. (D) LC-MS ion chromatograms illustrating analysis of the Epoxy-ketone, with WT (top, blue trace), knockout (middle, red trace), and d3 epoxy-ketone internal standard (lower, black trace). Samples are analyzed with the 13-ketone derivatized to a 13-methoxime, which gives syn and anti isomers (the 2 peaks in the d0 channel); the d3 internal standard was added as a single methoxime isomer, hence only one peak appears in the lower chromatogram. Oxidized lipids were isolated for LC-MS analysis as described in the Supplemental Data.