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Epidermal omega-O-acylceramides (ω-O-acylCers) are essential components of a competent skin barrier. These unusual sphingolipids with ultralong N-acyl chains contain linoleic acid esterified to the terminal hydroxyl of the N-acyl, the formation of which requires the transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1). In ichthyosis with dysfunctional PNPLA1, ω-O-acylCer levels are significantly decreased, and ω-hydroxylated Cers (ω-OHCers) accumulate. Here, we explore the role of the linoleate moiety in ω-O-acylCers in the assembly of the skin lipid barrier. Ultrastructural studies of skin samples from neonatal Pnpla1+/+ and Pnpla1-/- mice showed that the linoleate moiety in ω-O-acylCers is essential for lamellar pairing in lamellar bodies, as well as for stratum corneum lipid assembly into the long periodicity lamellar phase. To further study the molecular details of ω-O-acylCer deficiency on skin barrier lipid assembly, we built in vitro lipid models composed of major stratum corneum lipid subclasses containing either ω-O-acylCer (healthy skin model), ω-OHCer (Pnpla1-/- model), or combination of the two. X-ray diffraction, infrared spectroscopy, and permeability studies indicated that ω-OHCers could not substitute for ω-O-acylCers, although in favorable conditions, they form a medium lamellar phase with a 10.8 nm-repeat distance and permeability barrier properties similar to long periodicity lamellar phase. In the absence of ω-O-acylCers, skin lipids were prone to separation into two phases with diminished barrier properties. The models combining ω-OHCers with ω-O-acylCers indicated that accumulation of ω-OHCers does not prevent ω-O-acylCer-driven lamellar stacking. These data suggest that ω-O-acylCer supplementation may be a viable therapeutic option in patients with PNPLA1 deficiency.
Graphical abstractFig. 1Molecular structures of the studied ω-OHCers and ω-O-acylCers (A), the model compositions in mol% (B; donut chart), and the compositions of the ceramide fractions in the models in mol% (B; table). ω-O-acylCers, omega-O-acylceramides; ω-OHCers, ω-hydroxylated Cers.
The ω-O-acylCers (such as N-(ω-O-linoleoyloxy)acyl sphingosine, Cer EOS according to the nomenclature system by Motta et al. (4Motta S. Monti M. Sesana S. Caputo R. Carelli S. Ghidoni R. Ceramide composition of the psoriatic scale.)) have linoleic acid ester-bound at the ω-end of their N-acyl chains, typically ultralong (up to 36 carbon atoms) (5van Smeden J. Boiten W.A. Hankemeier T. Rissmann R. Bouwstra J.A. Vreeken R.J. Combined LC/MS-platform for analysis of all major stratum corneum lipids, and the profiling of skin substitutes., 6t'Kindt R. Jorge L. Dumont E. Couturon P. David F. Sandra P. et al.Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry., 7Ceramides of pig epidermis: structure determination., 8Bowser P.A. Nugteren D. White R.J. Houtsmuller U. Prottey C. Identification, isolation and characterization of epidermal lipids containing linoleic acid.), and their synthesis requires several specific steps (9Synthesis and degradation pathways, functions, and pathology of ceramides and epidermal acylceramides.). These lipids comprise around 10 weight % (7 mol%) of the extractable stratum corneum Cers (3Masukawa Y. Narita H. Sato H. Naoe A. Kondo N. Sugai Y. et al.Comprehensive quantification of ceramide species in human stratum corneum., 6t'Kindt R. Jorge L. Dumont E. Couturon P. David F. Sandra P. et al.Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry., 10Ishikawa J. Narita H. Kondo N. Hotta M. Takagi Y. Masukawa Y. et al.Changes in the ceramide profile of Atopic Dermatitis patients., 11Janssens M. van Smeden J. Gooris G.S. Bras W. Portale G. Caspers P.J. et al.Lamellar lipid organization and ceramide composition in the stratum corneum of patients with atopic eczema.). The ω-O-acylCers are essential for the typical stratum corneum multilamellar lipid assemblies with approximately 11–13 nm repeat distance, the long periodicity lamellar phase (LPP) (12Bouwstra J.A. Cheng K. Gooris G. Weerheim A. Ponec M. The role of ceramides 1 and 2 in the stratum corneum lipid organisation., 13Bouwstra J.A. Gooris G.S. Dubbelaar F.E.R. Weerheim A.M. Ijzerman A.P. Ponec M. Role of ceramide 1 in the molecular organization of the stratum corneum lipids., 14Opálka L. Kováčik A. Pullmannová P. Maixner J. Vávrová K. Effects of omega-O-acylceramide structures and concentrations in healthy and diseased skin barrier lipid membrane models.). Due to its two cis-double bonds, ω-esterified linoleate is fluid at skin temperature (15Pham Q.D. Mojumdar E.H. Gooris G.S. Bouwstra J.A. Sparr E. Topgaard D. Solid and fluid segments within the same molecule of stratum corneum ceramide lipid.) in contrast to the N-acyl chains, which are primarily rigid and tightly packed (16Ongpipattanakul B. Francoeur M.L. Potts R.O. Polymorphism in stratum corneum lipids.). The linoleate moiety in ω-O-acyl(glucosyl)Cers is fundamental for attaching to corneocyte proteins to form the corneocyte lipid envelope (CLE) (17Takeichi T. Hirabayashi T. Miyasaka Y. Kawamoto A. Okuno Y. Taguchi S. et al.SDR9C7 catalyzes critical dehydrogenation of acylceramides for skin barrier formation., 18Wertz P.W. Madison K.C. Downing D.T. Covalently bound lipids of human stratum corneum., 19Corneocyte lipid envelope (CLE), the key structure for skin barrier function and ichthyosis pathogenesis.).The CLE and the LPP arrangement of the stratum corneum lipids are vital for permeability barrier function (20Crumrine D. Khnykin D. Krieg P. Man M.-Q. Celli A. Mauro T.M. et al.Mutations in recessive congenital ichthyoses illuminate the origin and functions of the corneocyte lipid envelope., 21Janssens M. van Smeden J. Gooris G.S. Bras W. Portale G. Caspers P.J. et al.Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients.). The complete ω-O-acylCer deficit is lethal to neonatal mice (22Jennemann R. Rabionet M. Gorgas K. Epstein S. Dalpke A. Rothermel U. et al.Loss of ceramide synthase 3 causes lethal skin barrier disruption.), while diminished ω-O-acylCer levels in humans contribute to disturbed epidermal homeostasis in skin disorders (23van Smeden J. Janssens M. Gooris G.S. Bouwstra J.A. The important role of stratum corneum lipids for the cutaneous barrier function.) such as atopic dermatitis (10Ishikawa J. Narita H. Kondo N. Hotta M. Takagi Y. Masukawa Y. et al.Changes in the ceramide profile of Atopic Dermatitis patients., 21Janssens M. van Smeden J. Gooris G.S. Bras W. Portale G. Caspers P.J. et al.Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients., 24Macheleidt O. Kaiser H.W. Sandhoff K. Deficiency of epidermal protein-bound omega-hydroxyceramides in atopic dermatitis., 25Jungersted J. Scheer H. Mempel M. Baurecht H. Cifuentes L. Høgh J. et al.Stratum corneum lipids, skin barrier function and filaggrin mutations in patients with atopic eczema.), psoriasis (4Motta S. Monti M. Sesana S. Caputo R. Carelli S. Ghidoni R. Ceramide composition of the psoriatic scale.), or ichthyoses (26Paige D. Morse-Fisher N. Harper J. Quantification of stratum corneum ceramides and lipid envelope ceramides in the hereditary ichthyoses.). In particular, several autosomal recessive congenital ichthyoses have altered ω-O-acylCer metabolism (27Oji V. Tadini G. Akiyama M. Bardon C.B. Bodemer C. Bourrat E. et al.Revised nomenclature and classification of inherited ichthyoses: results of the first ichthyosis consensus conference in soreze 2009., 28Inherited ichthyosis: non-syndromic forms.). For example, the linoleate attachment to the ω-hydroxyl group on the Cer ultra-long acyl chain requires transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1) (29Ohno Y. Kamiyama N. Nakamichi S. Kihara A. PNPLA1 is a transacylase essential for the generation of the skin barrier lipid ω-O-acylceramide.). Dysfunctional Pnpla1 gene in mice led to significantly decreased ω-O-acylCer levels and an accumulation of ω-hydroxylated Cers (ω-OHCers), diminished CLE, and impaired lamellar organization of the stratum corneum extracellular lipids (30Pichery M. Huchenq A. Sandhoff R. Severino-Freire M. Zaafouri S. Opalka L. et al.PNPLA1 defects in patients with autosomal recessive congenital ichthyosis and KO mice sustain PNPLA1 irreplaceable function in epidermal omega-O-acylceramide synthesis and skin permeability barrier., 31Grond S. Eichmann T.O. Dubrac S. Kolb D. Schmuth M. Fischer J. et al.PNPLA1 deficiency in mice and humans leads to a defect in the synthesis of omega-O-acylceramides., 32Hirabayashi T. Anjo T. Kaneko A. Senoo Y. Shibata A. Takama H. et al.PNPLA1 has a crucial role in skin barrier function by directing acylceramide biosynthesis., 33Grall A. Guaguère E. Planchais S. Grond S. Bourrat E. Hausser I. et al.PNPLA1 mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans.). Such ω-OHCers, which lack the linoleate moiety are present only in small amounts in healthy skin (6t'Kindt R. Jorge L. Dumont E. Couturon P. David F. Sandra P. et al.Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry.).Here, we explore the role of the linoleate moiety in ω-O-acylCers in the skin barrier lipid assembly. First, electron microscopy was used to examine the effect of Pnpla1 expression on epidermal lamellar lipid organization in vivo. Next, we built in vitro multilamellar lipid models composed of major stratum corneum lipid subclasses, either with ω-O-acylCer (healthy skin model) or with ω-OHCer (Pnpla1-/- model) or their combinations (model of partial PNPLA1 dysfunction and models of PNPLA1-deficient skin treated with ω-O-acylCer; Fig. 1). The ω-O-acylCers and ω-OHCers comprised sphingosine, dihydrosphingosine, and phytosphingosine as their sphingoid backbones, that is, they were mixtures of EOS/EOdS/EOP and OS/OdS/OP, respectively. Selected lipid models were stressed by assembling them at a higher temperature and reduced hydration. The lamellar organization of these models using various ω-O-acylCer/ω-OHCer concentrations was assessed by X-ray diffraction (XRD). Selected models were further examined by Fourier-transform infrared spectroscopy (FTIR) for their lipid chain order, packing, and transitions. Permeabilities of the lipid models, sandwiched in Franz cells, were probed by water loss measurements and two permeants.Materials and methodsChemicalsω-O-acylCers [Cer EOS (d18:1/h32:0/18:2), Cer EOP (t18:0/h32:0/18:2), and Cer EOdS (d18:0/h32:0/18:2)] were prepared as described previously (34Opálka L. Kováčik A. Sochorová M. Roh J. Kuneš J. Lenčo J. et al.Scalable synthesis of human ultralong chain ceramides.). ω-OHCers [Cer OS (d18:1/h32:0), Cer OP (t18:0/h32:0), and Cer OdS (d18:0/h32:0)] were prepared by a modified procedure described in Opálka et al. (34Opálka L. Kováčik A. Sochorová M. Roh J. Kuneš J. Lenčo J. et al.Scalable synthesis of human ultralong chain ceramides.) that will be published elsewhere. Cer AdS (d18:0/h24:0) was prepared according to Kováčik et al. (35Kováčik A. Vogel A. Adler J. Pullmannová P. Vávrová K. Huster D. Probing the role of ceramide hydroxylation in skin barrier lipid models by 2 H solid-state NMR spectroscopy and X-ray powder diffraction.) Cer NS (d18:1/24:0), Cer NP (t18:0/24:0), Cer AS (d18:1/h24:0), Cer AP (t18:0/h24:0), and Cer NdS (d18:0/24:0) were purchased from Avanti Polar Lipids. FFAs (lignoceric, behenic, arachidic, stearic, and palmitic acids), Chol, CholS, theophylline (TH), indomethacin (IND), gentamicin sulfate, propylene glycol, buffer components, and solvents were purchased from Merck. All solvents used were of analytical or HPLC grade. Water was purified using a Milli-Q system (Merck Millipore).Mice and electron microscopyPnpla1+/+ and Pnpla1-/- skin samples were acquired during a previously published study from neonatal mice, in which loss of Pnpla1 expression was confirmed (31Grond S. Eichmann T.O. Dubrac S. Kolb D. Schmuth M. Fischer J. et al.PNPLA1 deficiency in mice and humans leads to a defect in the synthesis of omega-O-acylceramides.). All mouse procedures were approved by medical ethics committees at the University of Graz and the Austrian Federal Ministry of Science, Research and Economy. No new mouse procedures were performed as part of the present study. Skin samples were minced into fragments 3, prefixed in half-strength Karnovsky’s fixative, rinsed three times in 0.1 mol/L cacodylate buffer, followed by postfixation in ruthenium tetroxide (Polysciences). Samples were then embedded in epoxy-Epon (Hexion) and processed for electron microscopy (36Meyer J.M. Crumrine D. Schneider H. Dick A. Schmuth M. Gruber R. et al.Unbound corneocyte lipid envelopes in 12R-lipoxygenase deficiency support a specific role in lipid-protein cross-linking.). Imaging was done on thin sections using a JEOL 100CX electron microscope at 60 V with a Gatan Bioscan camera (model 792). Images presented in Fig. 2 represent the entire samples from all three mice within the respective group. The LPP was identified in electron microscope images using the DigitalMicrograph version 3.10.0 (Gatan Inc.) and its length was determined using the density profile tool, while LPP abundance was quantified as percentage of lamellae that were part of an LPP out of the total number of lamellae captured in each image slice.Fig. 2Ultrastructure of lamellar lipids in Pnpla1+/+ and Pnpla1-/- epidermis. Images show representative lamellar bodies in the stratum granulosum and mature lipid lamellae in the stratum corneum of the indicated mice. Scale bar = 100 nm. These images have been reused in the Graphical abstract.
Preparation of stratum corneum lipid modelsThe lipid models were prepared from Cers, FFAs, Chol, and CholS as follows (Fig. 1). The Cer fraction consisted of very long Cers NS, NP, NdS, AS, AP, and AdS (in an 8.3:25.3:9.1:26.0:29.8:1.4 M ratio) mixed in different ratios with ω-O-acylCers or ω-OHCers (composed of Cers EOS/EOP/EOdS or OS/OP/OdS, respectively, in 78.0:16.4:5.6 M ratios, Fig. 1 and supplemental Table S1). Due to the unavailability of Cers based on 6-hydroxysphingosine, these Cers were (proportionally) substituted with the available Cer subclasses to maintain the same level of hydroxylation as follows: Cer AS was used in place of Cer NH, and Cer AP was used in place of Cer AH. Cer EOH (and Cer OH) were proportionally substituted with Cers EOS (OS), EOdS (OdS), and EOP (OP) in the mixtures. FFAs of even chain lengths (1.3 mol% palmitic, 3.3% stearic, 6.9% arachidic, 47.1% behenic, and 41.4% lignoceric acid) were mixed to approximate their proportions in the healthy human stratum corneum (37Groen D. Gooris G.S. Bouwstra J.A. Model membranes prepared with ceramide EOS, cholesterol and free fatty acids form a unique lamellar phase., 38Wertz P.W. Schwartzendruber D.C. Madison K.C. Downing D.T. Composition and morphology of epidermal cyst lipids.).The lipids were dissolved in CHCl3/MeOH 2:1 (v/v), mixed as shown in Fig. 1, and dried in a vacuum. Then, 1.35 mg of the lipid mixture (per sample) was dissolved in 400 μl of hexane/96% ethanol 2:1 (v/v) and sprayed using a stream of nitrogen as a carrier gas and a Linomat V (Camag) with additional y-axis movement (37Groen D. Gooris G.S. Bouwstra J.A. Model membranes prepared with ceramide EOS, cholesterol and free fatty acids form a unique lamellar phase.) on either a cover glass (22 × 22 mm) for XRD or Nuclepore polycarbonate filters (15 nm pore size, Whatman) for the permeability experiments. The sprayed area was 1 cm2, and the lipid solution flow rate was 10.2 μl/min. Thus, the films contained 1.35 mg/cm2 lipid, roughly 10-fold more than in the stratum corneum, partly compensating for the lack of tortuosity in such models. The prepared lipid films were heated above their phase transition temperatures, either at 90°C (at ambient humidity) or 70°C (at 100% relative humidity) for 10 min, and then slowly (overnight) cooled to 32°C. The membrane thickness was approximately 11 μm (39Školová B. Janůšová B. Zbytovská J. Gooris G. Bouwstra J. Slepička P. et al.Ceramides in the skin lipid membranes: length matters.). Before the experiments, the lipids were equilibrated at 32°C and 40%–50% humidity for 24 h (39Školová B. Janůšová B. Zbytovská J. Gooris G. Bouwstra J. Slepička P. et al.Ceramides in the skin lipid membranes: length matters., 40Pullmannová P. Staňková K. Pospíšilová M. Školová B. Zbytovská J. Vávrová K. Effects of sphingomyelin/ceramide ratio on the permeability and microstructure of model stratum corneum lipid membranes.). The homogeneous lipid distribution was determined by high-performance thin-layer chromatography after the CHCl3/MeOH (2:1 v/v) extraction of the membrane center and periphery (41Pullmannová P. Pavlíková L. Kováčik A. Sochorová M. Školová B. Slepička P. et al.Permeability and microstructure of model stratum corneum lipid membranes containing ceramides with long (C16) and very long (C24) acyl chains.).X-ray diffractionThe XRD of the studied lipid membranes was performed using an X’Pert PRO θ – θ powder diffractometer (PANalytical B.V., Almelo) with parafocusing Bragg-Brentano geometry using CoKα radiation (λ = 1.7903 Å, U = 35 kV, I = 40 mA). X-ray focus, type: line, length: 12 mm, width: 0.4 mm, take-off angle: 6°. I
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