The omega-hydroxyceramides of pig epidermis are attached to corneocytes solely through omega-hydroxyl groups.

The cornified outer cells of mammalian epidermis possess a monolayer of omega-hydroxyceramides that are ester-linked to the exterior of a cross-linked protein envelope. In the present study, conclusive evidence was sought on which of the ceramide hydroxyl groups are involved in the linkage to protein. This was obtained by derivatizing all free hydroxyl groups in isolated solvent-extracted porcine stratum corneum using triisopropylsilyl (TIPS) chloride in pyridine in the presence of silver nitrate. After an 18-h reaction, the tissue was recovered, rinsed, and the derivatized ceramides were then released from protein linkage by hydrolysis with 1M KOH in 95% methanol. This gave a single ceramide product that was shown by nuclear magnetic resonance to contain two triisopropyl groups. Acetylation of the product using acetic anhydride in pyridine resulted in a downfield shift of the NMR signal for the omega-methylene protons, showing that it was the omega-hydroxyl that was free in the initial reaction product, and subsequently was acetylated. These results show that all of the omega-hydroxyceramides of corneocyte lipid envelopes are attached to protein through their omega-hydroxyl groups.

Effects of Na2SO4 on hydrophobic and electrostatic interactions between amphipathic alpha-helices.

The effects of 2 molal Na2SO4 at neutral pH on hydrophobic and electrostatic interactions between amphipathic alpha-helices were investigated by circular dichroism spectroscopy. The amphipathic peptides that were studied included LEK (acetyl-LEELKKKLEELKKKLEEL-NH2) and LEE (acetyl-LEELEEELEELEEELEEL-NH2). In phosphate buffer at neutral pH, only LEK adopted a predominantly alpha-helical conformation, attributable to glu-lys+ interactions where a major contribution is evidently a hydrogen bond (Biochemistry 32: 9668-9676). Despite the presence of lys+ in the e and g' positions of the abcdefg heptad repeat, LEK exhibited mean-residue ellipticities at 222 nm ([theta]222) which were dependent on peptide concentration, indicating the presence of a coiled coil. In the presence of 2 molal Na2SO4 at 25-75 degrees C, the helical content of LEK increased, with the greatest increase observed at 75 degrees C. The value of the ellipticity ratio R ([theta]222/[theta]208) of LEK in 2 molal Na2SO4 also increased, indicating a stronger interhelical association. At 50 degrees C and 75 degrees C, LEK remained predominantly alpha-helical. In phosphate buffer at neutral pH, LEE was mainly random coil. In the presence of 2 molal Na2SO4, however, the peptide formed alpha-helices that associated to form a coiled coil. At 50 degrees C and 75 degrees C, LEE became predominantly random coil but the remaining alpha-helices were still associating. These results are consistent with the strengthening of interhelical hydrophobic interactions and the absence of screening of helix-stabilizing and helix-destabilizing electrostatic interactions in amphipathic alpha-helices by Na2SO4.

A mixture of alpha-helical and 3(10)-helical conformations for involucrin in the human epidermal corneocyte envelope provides a scaffold for the attachment of both lipids and proteins.

Involucrin plays an important role in the lipid and protein compound envelopes of mammalian epidermal corneocytes. In the present study, model peptides containing the consensus repeating units PEQQEGQLEL and LEQQEGQLEH, found in the central region of human involucrin, were studied by circular dichroism spectroscopy, molecular modeling, and energy minimization. These peptides have intrinsic alpha-helix-forming properties as indicated by their circular dichroic spectra obtained in the presence of 2,2,2-trifluoroethanol. Peptide (LEQQEGQLEH)(3) had an alpha-helix content of 100% in 100% 2, 2,2-trifluoroethanol at 0 degrees C. The energy-minimized alpha-helix showed that only 50% of the glutamate side chains may be available for the attachment of lipids. However, when a 3(10)-helix was assumed for the GQL or GQLE residues in LEQQEGQLEH, all of the glutamate side chains were arrayed on one face of the helix, and all of the glutamine side chains were arrayed on the opposite face. A similar result was obtained when the nonhelical part of PEQQEGQLEL was assumed to contain a beta-turn III, which is equivalent to a short portion of 3(10)-helix. The results of this study suggest that when the central segment of human involucrin is predominantly alpha-helical, accompanied by short 3(10)-helical segments, the protein can function as a scaffold for the attachment of both lipids and proteins.

Molecular modelling indicates that the pathological conformations of prion proteins might be beta-helical.

Creutzfeldt-Jakob disease, kuru, scrapie and bovine spongiform encephalopathy are diseases of the mammalian central nervous system that involve the conversion of a cellular protein into an insoluble extracellular isoform. Spectroscopic studies have shown that the precursor protein contains mainly alpha-helical and random-coil conformations, whereas the prion isoform is largely in the beta conformation. The pathogenic prion is resistant to denaturation and protease digestion and can promote the conversion of the precursor protein to the pathogenic form. These properties have yet to be explained in terms of the structural conformations of the proteins. In the present study, molecular modelling showed that prion proteins could adopt the beta-helical conformation, which has been established for a number of fibrous proteins and has been suggested previously as the basis of amyloid fibrils. The beta-helical conformation provides explanations for the biophysical and biochemical stability of prions, their ability to form templates for the transmission of pathological conformation, and the existence of phenotypical strains of the prion diseases.

A new 6-hydroxy-4-sphingenine-containing ceramide in human skin.

A new ceramide consisting of 6-hydroxysphingosine linked to a non-hydroxyacid was found in human epidermal lipid. This ceramide was sought because its fatty acid and sphingoid moieties are present in other combinations in human epidermal ceramides. To isolate the new ceramide, the mixture of ceramides in human epidermal lipid was first separated into fractions by thin-layer chromatography (TLC), and then each fraction was further purified by TLC after acetylation of all hydroxyl groups. TLC after acetylation revealed that one of the fractions isolated in the first TLC step contained two components, namely, the ceramide consisting of sphingosine linked to an alpha-hydroxyacid and an unknown ceramide. The new ceramide constituted about 9% of the total ceramides, and was shown by NMR spectroscopy to be N-acyl-6-hydroxysphingosine.

Fibril formation by amyloid-beta proteins may involve beta-helical protofibrils.

We have proposed that amyloid fibrils contain subunits (protofibrils) that are formed from beta-strands wound into continuous 2-3 nm-diameter beta-helices. Subsequent lateral aggregation of the beta-helices to form the widely observed 5-12 nm-diameter fibrils could be promoted by hydrophobic residues on the exterior of the postulated beta-helix. A number of short peptide fragments of the amyloid-beta (A beta) proteins, such as A beta34-42 [LMVGGVVIA], the nine-residue, carboxyl-terminal portion of A beta1-42, can also form amyloid fibrils. In the present study, it was found that a beta-helix formed from A beta34-42 accounts for features suggested by published rotational resonance solid-state NMR data, including an anomalous conformation about the Gly-37-Gly-38 region and exaggerated pleating. An analogue of A beta34-42 was synthesized in which the hydrophobic groups on the exterior of the postulated beta-helix were replaced with glutamates, giving LEVGGVEIE. The analogue was completely soluble at pH 7, but at pH 2.5 it produced 2-2.5 nm-diameter fibrils which did not associate into larger-diameter bundles. The results of this study support the proposal that amyloid fibrils are formed from beta-helical subunits.

Stabilization of amphipathic alpha-helical and beta-helical conformations in synthetic peptides in the presence and absence of ionic interactions.

The role of ionic interactions in stabilizing amphipathic alpha-helices was studied in the synthetic peptide Ac-NLEELKKKLEELKG-NH2 (NLEKG14), potentially stabilized by attraction between complementary ions in successive turns of the helix, and in the peptide Ac-NLEELEEELEELEG-NH2 (NLEG14), in which no side-chain ionic attractions are possible. At a pH below the pKa of glutamate, NLEG14 had a higher helix content than NLEKG14. At pH 3 to pH 10, the helicity of NLEKG14 did not change, whereas NLEG14 was converted to random coil at pH 7. The role of ionic interactions in stabilizing the conformation of beta-structures was studied in the synthetic peptides Ac-KLKLKLELELELG-NH2 (KLEG13) and Ac-ELELELELELELG-NH2 (ELG13). At a pH below the pKa of glutamate, ELG13 had a higher beta-content than KLEG13, as judged by their dichroic spectra, but at higher pH, ELG13 was converted to random coil, whereas KLEG13 retained a predominantly beta-conformation. At pH 7, high NaCl concentration produced a significant increase in the alpha-helix content of NLEKG14, converted NLEG14 from random coil to alpha-helix and converted ELG13 from random coil to beta-conformation. Overall, the results demonstrate that ionic attraction between side-chains plays a lesser role than hydrogen bonding and hydrophobic effects in stabilizing the alpha- and beta-conformations exhibited by these amphipathic peptides.

Beta-helical fibrils from a model peptide.

A synthetic peptide, KLEG13 (Ac-KLKLKLELELELG-NH2), composed of alternating bulky hydrophilic and hydrophobic amino acid residues formed clear, viscous dispersions of fibrils in saline solutions. The fibrils had a uniform diameter of 2 nm as measured on electron micrographs of negatively stained preparations. 13C solid-state nuclear magnetic resonance spectroscopy of the fibrils indicated the presence of a beta-conformation. Circular dichroic spectra of the dispersion of fibrils were essentially identical to the calculated spectrum of a 100% beta-helix. Space-filling CPK models of a proposed beta-helical conformation of the peptide, in which the leucine side chains form a hydrophobic core and the hydrophilic lysine and glutamate side chains extend outwards from the helix, had a diameter consistent with the observed 2-nm diameter of the fibrils. This study may have implications regarding the structure of amyloid fibrils.

Circular dichroism of model peptides emulating the amphipathic alpha-helical regions of intermediate filaments.

The a and d positions of the heptad repeats (abcdefg) found in the alpha-helical sections of intermediate-filament proteins are hydrophobic, and the remaining locations are almost exclusively hydrophilic and often charged. Two synthetic peptides that maximize these features were designed, synthesized, and investigated by circular dichroism for alpha-helix formation in water and in 50% trifluoroethanol (TFE). A 14-residue peptide, AcNLEELKKKLEELKGNH2 (NLEKG14), had mean residue ellipticities at 222 nm ([theta]222) of -18400 +/- 1000 and -37,200 +/- 1900 deg cm2 dmol(-1), in water at 2 degrees C and in 50% TFE at 2 degrees C, respectively. A longer version of NLEKG14, AcNLEELKKKLEELKQQLEELKKKLEELKQQNH2 (NLEKQ29), had [theta]222 of -43,000 +/- 2200 deg cm2 dmol(-1) in water and in 50% TFE at 2 degrees C. Using -43,000 deg cm2 dmol(-1) as [theta]222 for a 100% helix, NLEKG14 in 50% TFE at 25 degrees C was estimated to be 77% helix. This estimate was confirmed by two-dimensional 1H NMR studies of NLEKG14 in 50% TFE. Comparison with the sequences and conformations found in IF proteins indicates that the alpha-helical regions in the proteins may be exceptionally stable, but the high values for the ellipticity of alpha-helices now revealed allow for significant portions of the protein rod regions to be occupied by conformations other than alpha-helix.

Molecular modeling of vimentin filament assembly.

Intermediate-filament forming proteins are known to form rod-shaped dimers that are calculated to be 45 nm in length. Molecular modeling indicates that the dimerization is promoted by interchain hydrophobic interactions between sections of alpha helix and beta helix. Further aggregation involves the formation of tetramers in which two dimers are anti-parallel and staggered to two characteristic degrees of overlap. Modeling indicated that the degrees of stagger are dictated by the association of sections of alpha helix in 4-chain bundles, in which hydrophobic side chains are sequestered from contact with water. The staggered arrangement of two dimers produces a tetramer having sections of 2-chain rod in which hydrophobic side chains are exposed to water. Extension of the tetramer to form protofilaments may be driven by associations with the 2-chain regions that reduce aqueous exposure of the hydrophobic side chains. Exposure of hydrophobic groups may be reduced by the 2-chain regions folding back upon themselves so that the entire tetramer becomes a 4-chain conformation. This prediction is in line with electron microscope data showing that mixtures of the lower oligomers contain rods of uniform thickness ranging upwards from 45 nm in a series having incremental increases in length. Data from previous chemical crosslinking studies support this model and also the idea that the completed intermediate filaments each consist of seven 4-chain protofilaments.

Chemical cross-linking between lysine groups in vimentin oligomers is dependent on local peptide conformations.

Previous studies have shown that cytoplasmic intermediate filaments, other than the keratins, are each constructed from a single type of polypeptide chain. Studies involving chemical crosslinking between lysine groups have shown that assembly of the filaments begins with the formation of dimers in which the peptide chains are parallel and in exact register, and that these dimers further associate in antiparallel patterns having specific degrees of overlap. In the present study, molecular modeling of the conformations of vimentin molecules indicated that lysine side chains in identical positions in regions of alpha-helix in parallel chains might be unable to be linked because they are on opposite sides of the coiled coil hydrophobic core. Examination of published data on chemical crosslinking of lysines in vimentin confirmed that there were no instances of linkage within dimers between the nine pairs of identical lysines that lie more than one position within alpha-helical regions in parallel chains. Even among linkages that apparently were between dimers, only one of the 11 linkage products identified involved lysines that were both within an alpha-helical region. In 10 of the 11 identified linkages between dimers, one or both of the linked lysines were in regions of random coil conformation. These results of molecular modeling indicate that relative motion between polypeptide chains in oligomers of intermediate filament proteins is not sufficient to overcome an orientation of lysine groups that is unfavorable for their chemical linkage. This finding supports the interpretations of keratin cross-linking data indicating that parallel homodimers are the basis for keratin intermediate filament assembly.

X-ray diffraction analysis of isolated skin lipids: reconstitution of intercellular lipid domains.

Low- and wide-angle X-ray diffraction were used to determine the structural organization of lipids isolated from the stratum corneum extracellular matrix that forms the major water permeability barrier in mammalian epidermis. Hydrated pig skin ceramides gave a single low-angle reflection of about 62 angstroms and a wide-angle-reflection at 4.15 angstroms. The addition of either cholesterol or fatty acid, the other major lipid components of the skin stratum corneum extracellular matrix, modified this diffraction pattern, depending on the lipid mole ratios. In the absence of water, lipid mixtures exhibited lipid phase separation, as shown by low- and wide-angle reflections typical of a separate cholesterol phase. However, a hydrated 2:1:1 mole ratio of ceramide:cholesterol:palmitic acid (similar to that found in stratum corneum) produced a diffraction pattern with a single sharp wide-angle reflection at 4.10 angstroms and low-angle reflections which indexed as the first eight orders of a single repeat period of 130 angstroms. The repeat period and intensity distribution of the low-angle data were similar to those found in intact stratum corneum [White et al. (1988) Biochemistry 27, 3725-3732; Bouwstra et al. (1994) Biochim. Biophys. Acta 1212, 183-192]. Higher concentrations of cholesterol or palmitic acid resulted in lipid phase separations. The 130 angstrom repeat period decreased only about 3 angstroms as water was removed by incubation in low-relative humidity atmospheres. The 130 angstrom repeat period depended on the presence of a particular ceramide, N-(omega-acyloxy)-acylsphingosine, which is found only in the epidermis. In contrast, 2:1:1 mixtures of brain ceramide:cholesterol:palmitic acid gave reflections of 56 and 34 angstroms. These results indicate that a structure with dimensions similar to those of the lamellar repeating unit found in skin stratum corneum does not depend on the presence of protein but does depend on the presence of specific skin ceramides and appropriate concentrations of cholesterol and fatty acid.

Cholesterol sulfate protects Candida albicans from inhibition by sphingosine in vitro.

Sphingosine is known to have potent biological activity, including pronounced anti-microbial action in vitro against Candida albicans and some bacteria. Several sphingosine bases are present in stratum corneum at concentrations several orders of magnitude above those in other tissues. Sphingosine forms an undissociated salt with organic sulfates, however, so that the free sphingosine in the epidermis may be inactivated by the cholesterol sulfate known to be present. To investigate this hypothesis, C. albicans was grown in cultures with graded concentrations of sphingosine added in ethanol. In 1% ethanol, 0.1-100 microgram/ml sphingosine completely prevented growth of the organism for 12 h. All cultures eventually entered log-phase growth and reached limiting density at a rate inversely proportional to sphingosine concentration. When sphingosine was added, together with an equimolar amount of cholesterol sulfate, there was no delay in the onset of growth of the yeast and the rate of growth and final density were similar to control cultures. These results demonstrate that natural ratios of cholesterol sulfate neutralize the anti-microbial activity of sphingosine in vitro. In the epidermis, endogenous cholesterol sulfate is hydrolyzed by sterol sulfatase at the skin surface, where the released sphingosine may resist microbial colonization of the stratum corneum. This mechanism for liberating anti-microbial sphingosine base only at the skin surface may protect the viable epidermis against known cytotoxic effects of free sphingosine.

Free sphingosines of human skin include 6-hydroxysphingosine and unusually long-chain dihydrosphingosines.

Ceramides containing 6-hydroxysphingosine, a previously unknown long-chain base, have recently been found in human skin. The present study investigated whether human skin also contains 6-hydroxysphingosine as the free base. Human skin surface lipids were obtained by washing with ethanol. A fraction enriched in sphingoid bases was isolated by preparative thin-layer chromatography and reacted with 2,4-dinitrofluorobenzene. The resulting N-dinitrophenyl derivatives were separated by thin-layer chromatography into three components, the most polar of which accounted for 15% of the total. After acetylation of the hydroxyl groups and repurification, each component was examined by nuclear magnetic resonance spectroscopy. The spectrum of the most polar of the derivatives indicated that it was 6-hydroxysphingosine or homologues of that substance. The spectra of the other two derivatives were virtually identical to those of derivatives prepared from authentic sphingosine and dihydrosphingosine. The chain-length distributions of the skin sphingoid bases were examined by gas chromatography after conversion of the dinitrophenyl acetates to dinitrophenyl trimethylsilyl derivatives. The analysis showed that the sphingosines and 6-hydroxysphingosines ranged from 17 to 22 carbons in length, with the 18- and 20-carbon species predominating. Surprisingly, the dihydrosphingosines included species with up to 26 carbons, with the 24-, 25-, and 26-carbon species accounting for about half of the total. Examination of the sphingoid bases of pig epidermis indicated that 6-hydroxysphingosine was not present and that the major chain length in the dihydrosphingosines was the 22-carbon species.

Molecular modeling indicates that homodimers form the basis for intermediate filament assembly from human and mouse epidermal keratins.

Mammalian epidermal keratin molecules adopt rod-shaped conformations that aggregate to form cytoplasmic intermediate filaments. To investigate these keratin conformations and the basis for their patterns of molecular association, graphical methods were developed to relate known amino acid sequences to probable spacial configurations. The results support the predominantly alpha-helical conformation of keratin chains, interrupted by short non-alpha-helical linkages. However, it was found that many of the linkages have amino acid sequences typical of beta-strand conformations. Space-filling atomic models revealed that the beta-strand sequences would permit the formation of 2-chain and 4-chain cylindrical beta-helices, fully shielding the hydrophobic amino acid chains that alternate with hydrophilic residues in these sequences. Because of the locations of the beta-helical regions in human and mouse stratum corneum keratin chains, only homodimers of the keratins could interact efficiently to form 2-chain and 4-chain beta-helices. Tetramers having the directions and degrees of overlap of constituent dimers that have been identified by previous investigators are also predicted from the interactions of beta-helical motifs. Heterotetramers formed from dissimilar homodimers could combine, through additional beta-helical structures, to form higher oligomers having the dimensions seen in electron microscopic studies. Previous results from chemical crosslinking studies can be interpreted to support the concept of homodimers rather than heterodimers as the basis for keratin filament assembly.

Lipids are covalently attached to rigid corneocyte protein envelopes existing predominantly as beta-sheets: a solid-state nuclear magnetic resonance study.

C solid-state nuclear magnetic resonance at natural abundance was used to study isolated corneocyte envelopes from porcine stratum corneum. The presence of lipids covalently attached to the protein envelopes was detected by chemical shifts of methylene and methyl groups of the bound lipids. The corneocyte protein envelopes are rigid, as suggested by efficient 1H to 13C cross polarization and 13C spin-lattice relaxation studies. The chemical shift of the carbonyl carbons of the protein envelopes supports the prediction that the chemically bound lipid envelope is attached to proteins arranged predominantly in the beta-sheet conformation, allowing a dense palisade of ceramide molecules to form a water-impermeable external sheath.

Lipid organization in pig stratum corneum.

The lipid and keratin structure of pig stratum corneum has been elucidated by small- and wide-angle X-ray diffraction. The measurements were carried out as a function of hydration and temperature. In addition, the stratum corneum was measured after recrystallization of the lipids at various temperatures. The results led us to conclude that the intercellular lipids in the stratum corneum are organized in at least two different lamellar structures with repeat distances of 6 and 13.2 nm. There is an indication for the presence of a third phase with a periodicity of 9 nm. The wide-angle pattern revealed a hexagonal (0.414 nm spacing) and liquid lateral packing (approximately 0.46 nm spacing). The 0.414 nm reflection started to decrease in intensity between 60 and 66 degrees C and disappeared between 72 and 95 degrees C. Furthermore, crystalline cholesterol has been indicated by both, wide- and small-angle X-ray diffraction, while the reflections of alpha-keratin were observed in the wide-angle X-ray diffraction pattern.

6-Hydroxy-4-sphingenine in human epidermal ceramides.

The solvent-extractable lipids of human epidermal stratum corneum consist predominantly of ceramides. In addition two non-extractable ceramides are chemically bound to the stratum corneum protein. One of the bound ceramides, constituting 50% of the bound lipids, was previously shown to consist of very long chan omega-hydroxyacids in amide linkage with sphingosine. The second bound caramide, which forms 25% of the bound lipids, was shown to contain the same hydroxyacids, but the sphingoid base was neither sphingosine nor phytosphingosine. In the present study, the undefined bound ceramide was shown by NMR and chemical procedures to be the omega-hydroxyacid derivative of a new base, 6-hydroxy-4-sphingenine. In addition, a ceramide previously known to constitute 25% of the extractable human stratum corneum ceramides has been found to contain the same novel sphingoid base, amide-linked to long-chain alpha-hydroxyacids. Finally, a new acylceramide has been isolated and identified that consists of very long chain omega-hydroxyacids in amide linkage with the novel sphingolipid, with fatty acids esterified wit the terminal hydroxyl group of the hydroxyacid.

Interaction between sphingosine and cholesteryl sulfate in epidermal lipids.

Free sphingosine, a material with multiple and potent biological activities, is known to occur in high concentration in mammalian epidermis. In the present study, thin-layer chromatography showed that in lipid extracts of human and pig stratum corneum, sphingosine forms a relatively stable compound with endogenous cholesteryl sulfate. NMR spectrometry of sphingosine and its hydrochloride, sulfate, and mixtures with cholesteryl or dodecyl sulfate showed that interaction with the organic sulfates constituted simple salt formation. Under neutral or weakly acidic conditions, such salts were only slightly dissociated and migrated on thin-layer chromatograms as discrete compounds. Thin-layer chromatography revealed undissociated salt formation between several long-chain bases and organic sulfates, and showed that their interaction is stoichiometric. However, undissociated salts were not formed between long-chain bases and fatty acids or phosphatidic acid. Undissociated salt formation may therefore be specific for organic bases and sulfates. It was concluded that the free sphingosine in the stratum corneum may be present as its cholesteryl sulfate salt and in this form be unavailable for permeation into the viable epidermal cells.