Lipid chain dynamics in stratum corneum studied by spin label electron paramagnetic resonance

The lipid chain motions in stratum corneum (SC) membranes have been studied through electron paramagnetic resonance (EPR) spectroscopy of stearic acid spin-labeled at the 5th, 12th and 16th carbon atom positions of the acyl chain. Lipids have been extracted from SC with a series of chloroform/methanol mixtures, in order to compare the molecular dynamics and the thermotropic behavior in intact SC, lipid-depleted SC (containing covalently bound lipids of the corneocyte envelope) and dispersion of extracted SC lipids. The segmental motion of 5- and 12-doxylstearic acid (5- and 12-DSA) and the rotational correlation time of 16-doxylstearic acid (16-DSA) showed that the envelope lipids are more rigid and the extracted lipids are more fluid than the lipids of the intact SC over the range of temperature measured. The lower fluidity observed for the corneocyte envelope, that may be caused mainly due to lipid-protein interactions, suggests a major contribution of this lipid domain to the barrier function of SC. Changes in the activation energy for reorientational diffusion of the 16-DSA spin label showed apparent phase transitions around 54 degrees C, for the three SC samples. Some lipid reorganization may occur in SC above 54 degrees C, in agreement with results reported from studies with several other techniques. This reorganization is sensitive to the presence of the extractable intercellular lipids, being different in the lipid-depleted sample as compared to native SC and lipid dispersion. The results contribute to the understanding of alkyl chain packing and mobility in the SC membranes, which are involved in the mechanisms that control the permeability of different compounds through skin, suggesting an important involvement of the envelope in the skin barrier.     

Covalently bound omega-hydroxyacylsphingosine in the stratum corneum

Pig epidermis was heat separated, and the stratum corneum was isolated after trypsinization. Exhaustive extraction of the stratum corneum fraction with chloroform/methanol mixtures yielded 14.7% lipid on a dry weight basis. After mild saponification of the extracted residue, additional lipid could be extracted which accounted for 2.1% of the stratum corneum weight. This bound lipid proved to consist mainly (91.9%) of N-(omega-hydroxyacyl)sphingosines in which the amide-linked omega-hydroxyacids were 28 to 34 carbon atoms in length. The release of this lipid by mild alkaline hydrolysis indicates that it is bound through an ester linkage. Half of the hydroxyceramide molecules reacted in situ with acidic acetone, suggesting that half of these molecules are attached to the stratum corneum through the omega-hydroxyl function, while the other half may be linked through one of the hydroxyl groups of the sphingosine.     

Human stratum corneum lipid organization as observed by atomic force microscopy on Langmuir-Blodgett films

The barrier function of skin ultimately depends on the physical state and structural organisation of the stratum corneum extracellular lipid matrix. Ceramides, cholesterol and a broad distribution of saturated long-chain free fatty acids dominate the stratum corneum lipid composition. Additionally, smaller amounts of cholesterol sulfate and cholesteryl oleate may be present. A key feature determining skin barrier capacity is thought to be whether or not different lipid domains coexist laterally in the stratum corneum extracellular lipid matrix. In this study, the overall tendency for lipid domain formation in different mixtures of extracted human stratum corneum ceramides, cholesterol, free fatty acids, cholesterol sulfate and cholesteryl oleate were studied using atomic force microscopy (AFM) on Langmuir-Blodgett (LB) films on mica. It is shown that the saturated long-chain free fatty acid distribution of human stratum corneum prevents hydrocarbon chain segregation. Further, LB-films of human stratum corneum ceramides express a pattern of connected elongated domains with a granular domain interface. The dominating effect of both cholesterol and cholesterol sulfate is that of increased ceramide domain dispersion. This effect is counteracted by the presence of free fatty acids, which preferentially mix with ceramides and not with cholesterol. Cholesteryl oleate does not mix with other skin lipid components, supporting the hypothesis of an extra-endogenous origin. In the system composed of endogenous human ceramides and cholesterol plus 15 wt% stratum corneum distributed free fatty acids, i.e., the system mimicking most closely the lipid composition of the stratum corneum extracellular space, LB-films on mica express lateral domain formation.     

Carbonylation of cornified envelopes in the stratum corneum

Stratum corneum (SC), the outermost layer of the skin, is continuously exposed to oxidative stress via sunlight, lipid peroxidation, and is subsequently accompanied by oxidative modification. Previous studies have shown that major oxidative target proteins in the SC are keratins. However, it remains unclear to date whether cornified envelopes (CEs), protein envelopes of the corneocytes (cornified cells), would be oxidized. In this study, we first revealed oxidative modification of CEs using labeled hydrazide derivatives to detect carbonyl moieties. Carbonylation of CEs was confirmed by reaction with monoclonal antibodies against aldehyde-bound proteins, including anti-acrolein, anti-crotonaldehyde, anti-4-hydroxy-2-nonenal. The extent of carbonylation is stronger in CEs from the face, a sun-exposed area, than those from the inside of upper arm, an unexposed area. Carbonylation of CEs did not depend on their maturity, as evaluated by loss of involucrin antigenicity during maturation process, suggesting that CEs are carbonylated regardless of their maturation stage.     

ATR-FTIR spectroscopy: a chemometric approach for studying the lipid organisation of the stratum corneum

The barrier function of skin resides in the lipid components of the stratum corneum, particularly their spatial organisation. FTIR spectroscopy has already been used as a relevant tool to study this lipid organisation: IR vibration band shifts have been attributed to the variations in lipid organisation induced by temperature. Our study included a stratum corneum model, composed of the three main lipids: palmitic acid as an example of fatty acids, cholesterol and ceramide III as an example of ceramide. Different films with various ratios of these lipids were studied. In our analytical strategy, the interest of using a chemometric analysis of global data obtained from ATR-FTIR spectra to highlight the main interactions involved in the molecular organisation of lipids has been demonstrated. Two kinds of interaction between the three main lipids have been shown: a non polar interaction between the long hydrocarbon chains and a polar interaction as the hydrogen bonding between polar functional groups. By varying the lipid ratio, we have shown first that the relative importance of each interaction was modified, second, that the induced modification of organisation can be detected by chemometric analysis of the ATR-FTIR spectra. The role of each kind of lipid in the organisation has been discussed. In conclusion, associating the ATR-FTIR with chemometric treatment is a promising tool: firstly, to understand the consequence of lipid relative compositions on the structural organisation of the stratum corneum, secondly, to show the relationship between lipid organisation and percutaneous penetration data. Indeed, this methodology will be transposed to in vivo studies with IR measurements through a probe.     

FTIR spectroscopic studies of lipid dynamics in phytosphingosine ceramide models of the stratum corneum lipid matrix

IR spectroscopic studies are reported for N-stearyl-d-erythro-phytosphingosine (Cer NP) and N-stearyl-2-hydroxy-d-erythro-phytosphingosine (Cer AP) in a hydrated model of the skin lipid barrier comprised of equimolar mixtures of each ceramide with cholesterol and d₃₅-stearic acid. Examination of the methylene stretching, rocking and bending modes reveal some rotational freedom and hexagonal packing in both the ceramide and stearic acid chains. Analysis of the acid carbonyl stretch and the ceramide Amide I modes show both shift to higher frequencies, indicating weaker hydrogen bonding, in the mixed systems compared to the pure materials. For both systems, the fatty acid chain disordering temperatures are significantly increased from those of the pure acids. The observed behaviors of these phytosphingosine ceramide systems are fundamentally different from the previously reported analogous sphingosine ceramide systems. The implications of these observations for lipid organization in the stratum corneum are briefly discussed.     

Ozone potentiates vitamin E depletion by ultraviolet radiation in the murine stratum corneum

As the outermost layer of the skin, the stratum corneum is exposed to environmental oxidants. To investigate putative synergisms of environmental oxidative stressors in stratum corneum, hairless mice were exposed to ultraviolet radiation (UV) and ozone (O(3)) alone and in combination. Whereas a significant depletion of alpha-tocopherol was observed after individual exposure to either a 0.5 minimal erythemal dose of UV or 1 ppm O(3) for 2 h, the combination did not increase the effect of UV alone. However, a dose of 0.5 ppm O(3) x 2 h, which had no effect when used alone, significantly enhanced the UV-induced depletion of vitamin E. We conclude that concomitant exposure to low doses of UV and O(3) at levels near those that humans can be exposed to causes additive oxidative stress in the stratum corneum.     

Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy

Trapping membrane proteins in the confines of a crystal lattice obscures dynamic modes essential for interconversion between multiple conformations in the functional cycle. Moreover, lattice forces could conspire with detergent solubilization to stabilize a minor conformer in an ensemble thus confounding mechanistic interpretation. Spin labeling in conjunction with electron paramagnetic resonance (EPR) spectroscopy offers an exquisite window into membrane protein dynamics in the native-like environment of a lipid bilayer. Systematic application of spin labeling and EPR identifies sequence-specific secondary structures, defines their topology and their packing in the tertiary fold. Long range distance measurements (60 ?-80 ?) between pairs of spin labels enable quantitative analysis of equilibrium dynamics and triggered conformational changes. This review highlights the contribution of spin labeling to bridging structure and mechanism. Efforts to develop methods for determining structures from EPR restraints and to increase sensitivity and throughput promise to expand spin labeling applications in membrane protein structural biology.     

Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 120 mg. kg-1. day-1) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] = 1.6-5.5 microM) by infusion of L-arginine (0.2-0.6 g/kg) but not D-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an L-NAME-induced rat endothelial dysfunction model. The oral administration of L-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] = 5.7 microM). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in L-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method.     

Evidence of a precursor form of stratum corneum basic protein in rat epidermis.

The fully differentiated anucleate cells of the stratum corneum of newborn rat epidermis contain a cationic protein called stratum corneum basic protein (SCBP). This protein has a molecular weight (49 000) and an amino acid composition similar to a protein extracted from the less differentiated cell layers of the epidermis. Pulse--chase experiments with radiolabeled histidine were undertaken to test the possiblity that SCBP is derived from a preexisting protein. A protein of 52 000 daltons is rapidly but transiently labeled in extracts of the less differentiated cell layers. As the amount of label in the 52 000-dalton protein decreases, an increase in radiolabel is observed in extracts of the fully differentiated cells. This label is found in SCBP, a protein of lower molecular weight (49 000) than that initially labeled. These proteins are immunologically related and both are resistant to cyanogen bromide cleavage. They differ in apparent molecular weight on sodium dodecyl sulfate--polyacrylamide gels and in their net charge. The results are consistent with the conversion of a precursor protein into SCBP.     

Ultrastructural organization of avian stratum corneum lipids as the basis for facultative cutaneous waterproofing

The ultrastructure of naked neck epidermis from the ostrich (Struthio camelus) and ventral apterium from watered, and water-deprived, Zebra finches (Taeniopygia [Poephila] guttata castanotis) is presented. The form and distribution of the fully differentiated products of the lipid-enriched multigranular bodies are compared in biopsies post-fixed with osmium tetroxide or ruthenium tetroxide. The fine structure of ostrich epidermis suggests it is a relatively poor barrier to cutaneous water loss (CWL). The fine structure from watered, and 16-hr water-deprived Zebra finches, considered in conjunction with measurements of CWL, confirms previous reports of “facultative waterproofing,” and emphasizes the rapidity of tissue response to dehydration. The seemingly counterintuitive facts that one xerophilic avian species, the ostrich, lacks a “good barrier” to CWL, whereas another, the Zebra finch, is capable of forming a good barrier, but does not always express this capability, are discussed. An explanation of these data in comparison to mammals centers on the dual roles of the integument of homeotherms in thermoregulation and conserving body water. It is concluded that birds, whose homeothermic control depends so much on CWL, cannot possess a permanent “good barrier,” as such would compromise the heat loss mechanism. Facultative waterproofing (also documented in lizards) protects the organism against sudden reductions in water availability. In birds, and probably in snakes and lizards, facultative waterproofing involves qualititative changes in epidermal cell differentiation. Possible control mechanisms are discussed. © 1996 Wiley-Liss, Inc.     

Role of ceramide structure and its microenvironment on the conformational order of model stratum corneum lipids mixtures: an approach by FTIR spectroscopy

The aim of this study is to investigate the influence of ceramide head group architecture and free fatty acid (another main class of stratum corneum lipids) or protein (keratin), on the lamellar organization of the ceramide auto-associated in model films mimicking lipid organization within the stratum corneum. FTIR spectroscopy is a powerful technique for investigating the structure of such systems. This technique has already been used to characterize phase transitions of the SC and of related model systems. As temperature is known to modify the conformational order of lipids, we used it as a variable parameter to monitor the differences in the conformational stability of ceramides. Our study included four ceramides: ceramide 2, 3, 5 and 6 which differ by their head group architecture. Two kinds of lipid-lipid interactions were studied: non-polar and polar. We noted some structural factors which participated to the organizational behavior: insaturation of alkyl chain, alpha-hydroxyl on fatty acid moiety and sphingosine or phytosphingosine head group. There is a direct interaction of palmitic acid on alkyl chains organization and a weak interaction with polar head group in presence of keratin, both provoking a destabilization of the ceramidic orthorhombic organization.     

Light-induced switching of HAMP domain conformation and dynamics revealed by time-resolved EPR spectroscopy

HAMP domains are widely abundant signaling modules. The putative mechanism of their function comprises switching between two distinct states. To unravel these conformational transitions, we apply site-directed spin labeling and time-resolved EPR spectroscopy to the phototactic receptor/transducer complex NpSRII/NpHtrII. We characterize the kinetic coupling of NpHtrII to NpSRII along with the activation period of the transducer and follow the transient conformational signal. The observed transient shift towards a more compact state of the HAMP domain upon light-activation agrees with structure-based calculations. It thereby validates the two modeled signaling states and integrates the domain’s dynamics into the current model.     

The mechanical properties of stratum corneum: I. The effect of water and ambient temperature on the tensile properties of newborn rat stratum corneum

The tensile properties of the outermost layer of skin of neonatal rats, the stratum corneum, were investigated at a constant strain rate as a function of moisture content and ambient test temperature. The results show that the mechanical behavior of this membrane, whose primary constituent is the fibrous protein keratin, can be significantly altered by variations in both the sorbed water content and ambient temperature. In particular, a brittle to ductile transition was observed at 25°C once the hydration level exceeded 70% relative humidity. Similarly, an identical phenomenon moisture concentrations were maintained at 10 g H₂O/100 g dry protein. Differential scanning calorimetry measurements showed the presence of a molecular relaxation process which migrated from 42°C at 40% relative humidity to −18°C at 95% relative humidity. It is postulated that this relaxation process, possibly corresponding to the glass transition of the fibrous protein component of stratum corneum, is primarily respnsible for the observed behavior.     

The chemistry of phospholipid binding by the Saccharomyces cerevisiae phosphatidylinositol transfer protein Sec14p as determined by EPR spectroscopy

The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.