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 5

SDR9C7 catalyzes NAD+-dependent oxidation of 13-hydroxy-linoleate derivatives.

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SDR9C7 catalyzes NAD+-dependent oxidation of 13-hydroxy-linoleate deriva...
(A) Activity of recombinant SDR9C7 monitored by spectrophotometric assay of NADH formation. Top panel: SDR9C7 catalyzes oxidation of 13-HODE methyl ester (13-HODE Me) to the 13-ketone. In addition to the appearance of NADH at 340 nm, the scans in the metabolism of 13-HODE show an increase near 300 nm, which represents the edge of the UV chromophore of the 13-keto oxidation product (λmax at 279 nm); this increase in absorbance is not evident in metabolism of the epoxy-alcohol (middle panel), because its oxidation product absorbs much lower in the UV (λmax 231 nm) (10). Middle panel: SDR9C7 catalyzes oxidation of the epoxy-alcohol 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoate methyl ester (RRR-EpOH-Me) to the 13-ketone. Lower panel: the SDR9C7 R276C mutant has no detectable activity in oxidation of RRR-EpOH-Me, (as further confirmed by HPLC analysis of the incubation). (B) Reversed-phase HPLC analysis of the SDR9C7-catalyzed oxidation of RRR-EpOH-Me. The unreacted substrate is detected in the channel monitoring 205 nm, the single product mainly at 235 nm. (C) The 9,10-epoxy-11E-13-keto product was identified from its cochromatography with the authentic standard and by its characteristic UV spectrum. The HPLC analysis used an Agilent 1200 series system with an Agilent Eclipse XBD-C18 column (5 μm, 15 × 0.46 cm), a solvent of acetonitrile/water/glacial acetic acid (75/25/0.01 by volume) at a flow rate of 1 mL/min, with the diode array detector set to monitor at 205, 220, 235, and 270 nm.