Acidic pH Is Required for the Multilamellar Assembly of Skin Barrier Lipids In Vitro

Lipid membrane remodeling belongs to the most fundamental processes in body. The skin barrier lipids, which are ceramide dominant and highly rigid, must attain an unusual multilamellar nanostructure with long periodicity restrict water loss prevent entry of potentially harmful environmental factors. Our data suggest that acid mantle, apart from regulating enzyme activities keeping away pathogens, may also be a prerequisite for assembly lipids. Atomic force microscopy on monolayers composed synthetic or human stratum corneum lipids showed multilayer formation (approximately 10-nm step height) acidic but not neutral environment. X-ray diffraction, Fourier transform infrared spectroscopy, permeability studies markedly altered lipid increased at pH compared pH. These findings consistent organization transepidermal under conditions such as inadequate acidification, example, neonates, elderly, patients atopic dermatitis. protective barriers rely environment far more than tissues. mantle uppermost epidermal layer, (SC), keeps microbes, activates enzymes synthesize biochemically (Hannun Obeid, 2018Hannun Y.A. Obeid L.M. Sphingolipids their metabolism physiology disease [published correction appears Nat Rev Mol Cell Biol. 2018;19:673.Nat 2018; 19: 175-191Crossref PubMed Scopus (658) Google Scholar; Nicolaides, 1974Nicolaides N. Skin lipids: biochemical uniqueness.Science. 1974; 186: 19-26Crossref (445) Scholar), inhibits serine proteases would compromise SC integrity and/or cohesion, prevents release proinflammatory cytokines (Elias, 2017Elias P.M. how, why clinical importance acidification.Exp Dermatol. 2017; 26: 999-1003Crossref (38) Fluhr Elias, 2002Fluhr J.W. Elias Stratum pH: Function ‘Acid Mantle’.Exogenous 2002; 1: 163-175Crossref (147) Scholar). essential role ∼5.5 synthesis major ceramides (Cers), precursors (mainly glucosylceramides [GlcCers]) has been well-recognized (Breiden Sandhoff, 2014Breiden B. Sandhoff K. sphingolipid cutaneous formation.Biochim Biophys Acta. 2014; 1841: 441-452Crossref (116) However, how released into extracellular space unique architecture, is resist factors, known. Membrane living cells largely protein regulated (McMahon Gallop, 2005McMahon H.T. Gallop J.L. curvature mechanisms dynamic cell remodelling.Nature. 2005; 438: 590-596Crossref (1533) In contrast, assemble typical phase (LPP) (repeat distance [d] = 12–13 nm) (Bouwstra et al., 1992Bouwstra J.A. Gooris G.S. Salomons-de Vries M.A. van der Spek Bras W. Structure function temperature hydration: wide-angle diffraction study.Int J Pharm. 1992; 84: 205-216Crossref (229) Madison 1987Madison K.C. Swartzendruber D.C. Wertz P.W. Downing D.T. Presence intact intercellular lamellae upper layers corneum.J Invest 1987; 88: 714-718Crossref (330) Scholar)) population forming short (d 5–6 vitro absence proteins. molecular physiological fully understood because (i) extremely structurally heterogeneous (over 1,000 species, mostly commercially unavailable), (ii) have complex polymorphism, (iii) require high-temperature annealing vitro. Considering necessity during assembly, it remarkable described decades ordered rigid structures Mendelsohn 2006Mendelsohn R. Flach C.R. Moore D.J. Determination conformation permeation via IR microscopy, imaging.Biochim 2006; 1758: 923-933Crossref (172) Pham 2017Pham Q.D. Topgaard D. Sparr E. Tracking solvents through atomically resolved measurements mobility corneum.Proc Natl Acad Sci USA. 114: E112-E121Crossref (27) We recently shown very different dynamics Cer sphingosine acyl chains: whereas lignoceroyl chain all-trans state, substantial fraction chains fluid state (Engberg 2020Engberg O. Kováčik A. Pullmannová P. Juhaščik M. Opálka L. Huster al.The [NS] show structure challenge our understanding barrier.Angew Chem Int Ed Engl. 2020; 59: 17383-17387Crossref (6) mobile ability these reorganize correct structure. To fill this crucial gap origin barrier, we studied behavior multilayers either isolated 5.5 7.4. aqueous subphase reasonably mimic one leaflet (two-dimensional arrangement) granulosum‒SC interface without assuming whether come lamellar bodies (Feingold 2014Feingold K.R. Role maintenance barrier.Biochim 280-294Crossref (224) Scholar) suggested cubic (Narangifard 2018Narangifard den Hollander Wennberg C.L. Lundborg Lindahl Iwai I. al.Human takes place transition analyzed by cryo-electron EM-simulation.Exp Res. 366: 139-151Crossref (14) Importantly, monolayer undergo two-dimensional‒three-dimensional (Lee, 2008Lee K.Y. Collapse Langmuir monolayers.Annu Phys Chem. 2008; 771-791Crossref (180) if choose do so, similar what expected barrier. For monolayer‒multilayer occurs naturally was reproduced lung surfactant (Wang 2005Wang Cai Galla H.J. He H. Monolayer–multilayer transitions model: reflection–absorption spectroscopy atomic microscopy.Eur J. 34: 243-254Crossref (59) nanostructures were further (XRD) (FTIR), functional consequences probed measuring films SC. study first comprising (5.5) near-neutral (7.4) (Figure 1a b Supplementary Figure S1). equimolar mixtures sphingolipids (nonhydroxyacyl sphingosine, is, NS subclass, nonhydroxyacyl GlcCer various ratios roughly mimicking polar head change postsecretory GlcCer-to-Cer processing) free fatty acids (FFAs; C16:0–C24:0), cholesterol (Chol). selected values relevant those encountered secreted progressively locally acidified activate Cers (GlcCers sphingomyelin) (Behne 2003Behne M.J. Barry N.P. Hanson K.M. Aronchik Clegg R.W. Gratton al.Neonatal development gradient: localization leading emergence optimal function.J 2003; 120: 998-1006Abstract Full Text PDF Breiden At pH, FFAs COOH form, deprotonation palmitic mixed Chol (Mimeault Bonenfant, 2002Mimeault Bonenfant FTIR spectroscopic analyses influences behaviors interactions.Talanta. 56: 395-405Crossref (30) When concentration exceeded GlcCer, occupied greater mean areas (calculated using individual components) 1c). Such positive deviation reported GlcCer‒Cer explained dipolar repulsion (Maggio, 2004Maggio Favorable unfavorable lateral interactions ceramide, glycosphingolipids gangliosides monolayers.Chem Lipids. 2004; 132: 209-224Crossref (50) apparent followed additivity rule except slight negative 50:50 sample. film compression moduli did linearly ratio (Supplementary S2). Thus, properties responsive changes they encounter keratinization (in agreement previous models) 1998Bouwstra Dubbelaar F.E. Weerheim A.M. Ponec sulfate, affect organization.J 1998; 3: 69-74Abstract (96) Kitson 1994Kitson Thewalt Lafleur Bloom A model approach barrier.Biochemistry. 1994; 33: 6707-6715Crossref (118) better understand pH-dependent behavior, transferred air‒liquid mica observed surface ambient humidity 1d S3). had two domains average height 0.9–1.3 nm, higher domain occupying 48–62% sample area [upper row] S3), results (Skolová 2013Skolová Janůšová Zbytovská G. Bouwstra Slepička al.Ceramides membranes: length matters.Langmuir. 2013; 29: 15624-15633Crossref (63) high content behaved concerning difference heights percent areas, although became continuous [bottom dramatically changed images. Bright islets protruded 10–14 nm bottom layer dendritic 1e f). This likely corresponds four stacked so-called medium ∼ 11 found Cer/FFAs/Chol (Pullmannová 2019Pullmannová Ermakova Maixner al.Long phases membranes.J 2019; 60: 963-971Abstract (8) 10–21% area. stabilized ionized FFAs, FFA protonation 5.5, collapse rearrange multilayers. finding stability liposomes only (Hatfield Fung, 1995Hatfield R.M. Fung L.W. Molecular system: large unilamellar vesicles.Biophys 1995; 68: 196-207Abstract (36) driving mechanism hydrophobic mismatch between makes hairpin energetically Putatively, drive where extended clustered chains, 2020Narangifard Han al.Molecular reorganization situ [e-pub ahead print].J (accessed 16 November 2020)https://doi.org/10.1016/j.jid.2020.07.040Abstract (7) multilayered annealed buffers 7.4 1g). Most packed orthorhombic subcell indicated scissoring vibration doublet arises vibrational coupling tightly 1g, part i). Deuteration resulted CH2 CD2 singlets ii), suggesting good mixing deuterated (because causing does occur C-H C-D bonds). With both deuterated, broad iii) unionized cluster significantly diluted unlabeled presence disturb coupling). prefers splayed-chain (extended) associated Engberg Scholar, 2012Iwai Svensson S. Ofverstedt L.G. Anwar organized bilayers molecules sphingoid moiety.J 2012; 2215-2225Abstract (162) Školová 2014Školová Hudská Palát Roh al.Different packing (C16) (C24) acyls lipids.J 118: 10460-10470Crossref (49) Scholar. all partially samples mixture acidification induces clustering key event assembly. reproduce physiologically setting, purified them chromatographically remove contaminants, spread subphases 2a b). strikingly less condensed (compression <100 mN/m) mentioned earlier 385 513 mN/m, respectively). acylCers containing linoleate tails (Pham 2018Pham Mojumdar E.H. Solid segments within same molecule lipid.Q Biophys. 51: e7Crossref (9) Wang Klauda, 2019Wang Klauda J.B. Am Soc. 141: 16930-16943Crossref (17) plateau isotherm 22‒23 mN/m transition. pronounced absent revealed 1.8-nm 2c, right). 45% decorated small, ∼1 peaks (<1% area). (∼2 discontinuous contained bright, ∼11 islands left). stable least 2 weeks. intriguingly close repeat [Bouwstra Scholar] [Iwai Narangifard Scholar]). Notably, experiment Chol, sulfate 1:1:1:0.13 molar neither isotherms nor (data shown), pointing important full spectrum (likely FFAs) event. Temperature seems 21 °C report (Norlén 2007Norlén Gil I.P. Simonsen Descouts Human Langmuir–Blodgett films.J Struct 2007; 158: 386-400Crossref (32) (we used 32 study). arrangement sculpted corneocyte envelope, covalently bound corneocytes (Candi 2005Candi Schmidt Melino cornified envelope: death skin.Nat 6: 328-340Crossref (1221) Crumrine 2019Crumrine Khnykin Krieg Man M.Q. Celli Mauro T.M. al.Mutations recessive congenital ichthyoses illuminate functions envelope.J 139: 760-768Abstract (26) clearly nucleation requires (or microdomains) enough being protonated. support probe nature environments, XRD 2d‒g). sprayed glass slides buffer checked ionization resistant hydration. Indeed, 70 (above melting transition) required evidenced decrease intensity carbonyl stretching increase COO– 2d). (partial) decreased order methylene symmetric wavenumbers 2e) relative proportion demonstrated 730 cm–1 components rocking (Mendelsohn 2f). LPP 13.53–13.72 5.56–5.60 nm). region reflections corresponded distances planes 0.41 0.37 characteristic 2g, red line). 1991Bouwstra Structural investigations small-angle scattering.J 1991; 97: 1005-1012Crossref (460) vapor S4; LPP, d 13.29–13.31 nm; minor phase, 5.60–5.65 separated 3.42 diminished vapor, peak q 1.7 nm–1 3.7 appeared. relate form liquid-ordered having 14–18 carbon above (Ouimet Lafleur, 2004Ouimet Hydrophobic match saturated liquid phases.Langmuir. 20: 7474-7481Crossref (29) pig sharp resemble native architecture all. Although traces could found, 1.35 dominated diffractograms, other sufficiently permit assignment black vivo immature animals elevated improved acidification:(i)Incomplete no periodic seen newborn rats yet begun 2004Fluhr Mao-Qiang Brown B.E. Hachem J.P. Moskowitz D.G. Demerjian al.Functional neonatal rat 123: 140-151Abstract (86) Acidification, becomes evident postnatal day 3, correlated mature compacted Scholar).(ii)In tape-stripped mice, exposure (but 5.5) persistence immature, (Mauro 1998Mauro T. Holleran W.M. Grayson Gao W.N. Kriehuber al.Barrier recovery impeded independent ionic effects: implications processing Arch Dermatol Res 1998;290:405].Arch 290: 215-222Crossref (232) treated phosphate rough and, occasionally, vesicle-like (Van Hal 1996Van D.A. Jeremiasse Junginger H.E. Spies F. hydrated corneum: freeze-fracture electron study.J 1996; 106: 89-95Abstract (112) Daily application superbase tetramethylguanidine mice unprocessed (immature) membranes (Hachem 2005Hachem Uchida Y. Rogiers V. al.Sustained activity prolonged leads degradation profound alterations integrity.J 125: 510-520Abstract (230) Scholar).(iii)Acidification polyhydroxy accelerated maturation 2010Hachem Roelandt Schürer Pu X