From the Laboratory of Skin Biology, NIAMS, National Institutes of
Health, Bethesda, Maryland 20892-2752
An important component of barrier function in
human epidermis is contributed by ceramides that are bound by ester
linkages to undefined proteins of the cornified cell envelope (CE). In this paper, we have examined the protein targets for the ceramide attachment. By partial saponification of isolated foreskin epidermal CEs followed by limited proteolysis, we have recovered several lipopeptides. Biochemical and mass spectroscopic characterization revealed that all contained near stoichiometric amounts of ceramides of
masses ranging from about 690 to 890 atomic mass units, of which six
quantitatively major species were common. The array of ceramides was
similar to that obtained from pig skin, the composition of which is
known, thereby providing strong indirect data for their fatty acid and
sphingosine compositions. The recovered peptides accounted for about
20% of the total foreskin CE ceramides. By amino acid sequencing,
about 35% of the peptides were derived from ancestral
glutamine-glutamate-rich regions of involucrin, an important CE
structural protein. Another 18% derived from rod domain sequences of
periplakin and envoplakin, which are also known or suspected CE
proteins. Other peptides were too short for unequivocal identification.
Together, these data indicate that involucrin, envoplakin, periplakin,
and possibly other structural proteins serve as substrates for the
attachment of ceramides by ester linkages to the CE for barrier
function in human epidermis.
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INTRODUCTION |
Mammalian epidermis lies at the interface with the environment,
where it plays an essential role in providing a physical, chemical, and
water barrier for the organism (1-4). Cornified keratinocytes, which
constitute the major cell type of the epidermis, have evolved an
elaborate barrier system. Part of this is contributed by the cornified
cell envelope (CE),1 which is
a 15-20-nm thick layer on the periphery of the corneocyte, and
consists of two components. An
15-nm-thick layer of several defined
structural proteins is deposited on the intracellular surface of the
cell membrane in the upper spinous and granular cells of the living
epidermis (5-8). These proteins become cross-linked together by
disulfide bonds and
N
(
-glutamyl)lysine or
N1,N8-bis(
-glutamyl)spermidine
isopeptide bonds formed by the action of transglutaminases (5-9). This
process appears to begin with the cross-linking of certain early
protein components, such as involucrin and envoplakins, at or near to
the site of desmosomes (10-13), which together form a scaffold (12,
14) for the subsequent stages of addition of elafin, small proline-rich
proteins, and much larger amounts of loricrin (10-12, 15-17). At a
late stage of protein envelope assembly, perhaps in upper granular
cells, the lipid envelope component is assembled (reviewed in Refs.
2-4). The phospholipid-rich cellular plasma membrane is rebuilt as a 5-nm-thick layer of ceramide lipids, which subsequently become covalently attached to the protein envelope on the extracellular surface (18-21). The lipids are synthesized, packaged into lamellar bodies, and extruded from the granular cells into the intercellular space (reviewed in Refs. 2-4 and 22). A minor but important component
are the acylglucosyl-ceramides, of which about two-thirds are converted
to
-hydroxyceramides that become covalently attached by ester
linkages to the protein envelope to complete CE assembly. Many other
lipids, such as cholesterol esters and free fatty acids, function to
form the lamellae in the intercellular milieu and contribute in a major
way to the barrier against desiccation (2-4, 17-23). One idea is that
the ceramides that are ester-linked to the outer surface of the CE
contribute a hydrophobic surface to the corneocyte that has important
consequences for water barrier function by associations with and
perhaps organization of intercellular lipids (2).
We have been interested in the structure of the CE and have devised
models on the nature and sequences of the proteins involved in its
formation (10-12). However, rather less is known about how the lipid
envelope becomes attached to the protein envelope. Specifically, although a model has been suggested (2, 21, 24), the nature of the
protein substrate for lipid addition is not yet known. In this study,
we explored this question. Partial removal of ester-linked lipids from
isolated foreskin epidermal CEs by saponification has allowed the
isolation of lipopeptides consisting of ceramides attached to certain
glutamines and/or glutamic acid residues of involucrin, as well as
other likely CE proteins.
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MATERIALS AND METHODS |
Preparation of CEs--
"Mature" CEs were prepared from the
stratum corneum tissue of freshly-prepared foreskin epidermis as
described previously (10-12). They were resuspended in a buffer of 0.1 M N-ethylmorpholine acetate (pH 8.3) and
digested with trypsin (Sigma, sequencing grade, 1% by weight, for
2 h at 37 °C) to remove contaminating adherent proteins (12)
and pelleted at 14,000 × g.
Saponification--
Aliquots of CEs containing known amounts of
protein were resuspended in 1 M KOH in 95% methanol for
15-120 min at 45 °C, washed with methanol, and dried. Potential
ceramide-peptide adducts were reacted similarly to hydrolyze the ester
linkages (generally a 2-h reaction). The saponified lipids were
recovered by extraction into chloroform/methanol (1:2 v/v) and
dried.
Protein Chemistry Procedures--
Amino acid analysis of
hydrolyzed samples (5.7 N HCl at 110 °C for 24 h
in vacuo) was used to routinely measure protein amounts. Partially or completely saponified CEs were resuspended (1 mg of CE
protein/ml) in buffer and redigested with trypsin or proteinase K (Life
Technologies, Inc.; 1% by weight) for up to 3 h at 37 °C. The
solubilized material was collected following removal of the CE remnants
by pelleting and dried. Peptides were fractionated on a C18
high performance liquid chromatography (HPLC) column as before (10,
11). To recover potential lipopeptide adducts, a Phenomenex
C4 HPLC column (200 × 2.5 mm) was used at a flow rate
of 0.22 ml/min with 10% acetonitrile containing 0.08% trifluoroacetic acid and with a linear gradient of up to 15% (v/v) isopropanol in
acetonitrile and collected into 0.11-ml fractions. Aliquots of selected
peptide peaks were subjected to amino acid analysis or attached to a
solid support (Sequelon-AA, Millipore) for sequencing as described
previously (11, 12). In addition, aliquots (5-20 pmol) were removed
for mass measurements by mass spectroscopy both before and after
saponification, using a matrix assisted laser desorption ionization
procedure. Following an initial scan from 600 atomic mass units (below
which reliable data could not be obtained), scans were repeated in the
vicinity of the major mass species.
Ceramide Assay--
Aliquots of potential lipopeptides were
saponified for 2 h as described above. The released ceramides were
then extracted with chloroform/methanol (95:5) and quantified using the
diacylglycerol assay reagent kit and [
-32P]ATP
(Amersham Pharmacia Biotech) as described (25). This reaction specifically labels an hydroxyl of the sphingosine moiety of a ceramide
only. High performance thin layer chromatography silica gel plates
(Merck) were developed with chloroform:acetone:methanol:acetic acid:water 10:4:3:2:1. N-stearoyl-D-sphingosine
(C18 ceramide) (Sigma) was used as quantitation standard.
The dried plates were then exposed for up to 6 days. Bands were
quantitated by scanning densitometry of several exposures of the x-ray
films.
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RESULTS AND DISCUSSION |
Recovery of Potential Lipopeptides from Foreskin Epidermal
CEs--
Previously, we have demonstrated that saponification of
foreskin epidermal CEs, a mild alkaline hydrolysis procedure that is
likely to cleave ester bonds, exposes for proteolytic attack a number
of structural proteins of the protein portion of the CE (11, 12). Our
immunogold electron microscopy analyses and proteolytic digestion
experiments suggested that in intact CEs, the ester-linked lipids mask
access to structural proteins corresponding to the inner or earlier
stages of CE assembly (12). These proteins were predominantly
involucrin, envoplakin, and desmoplakin (12), but they may also consist
of other desmosomal and calcium-binding proteins (12, 13). In this
paper, we have addressed the questions of the nature of the possible
protein substrates to which these lipids are covalently attached,
their sites of attachment, and the properties of the attached lipids.
We reasoned that by reducing the time of an initial saponification
reaction to only a few minutes, followed by limited proteolysis, it
should be possible to isolate peptides with ester-linked lipid adducts.
Such adducts could be identified by HPLC fractionation on a
C4 HPLC column monitored at 220 nm. Free hydrophilic
peptides typically found in CE proteins should not be retarded, and
retarded free lipids cannot be detected at 220 nm. We performed a
number of trial experiments titrating the degree of saponification (in
the range of 10-30 min) and subsequent proteolysis with trypsin or
proteinase K singly or in combination. Although trypsin released
several retarded peaks resolvable on the C4 column, a
reproducibly better yield of 10 arbitrarily numbered peaks was obtained
by proteinase K digestion alone (Fig. 1),
even though a significant portion of the peptide material released would have originated from the cytoplasmic (loricrin-rich) face of the
CE, where lipids are largely absent (10-12). Optimized conditions were
obtained for 15 min of saponification and 10-60 min of proteinase K
digestion. Of these, peaks 5-7 were stable for
1 h of digestion, peaks 3 and 4 became well resolved only after 20-30 min of digestion, peaks 8-10 were lost after
20 min of digestion, and peaks 1 and 2 were rapidly lost in a time- and shape-dependent manner.
The 15-min saponification procedure alone did not result in a
significant release of peptide material. In a typical experiment with
15 min of saponification followed by digestion with proteinase K, we released 5.1% (10 min), 12.7% (30 min), 15.8% (1 h) and 19.5% (3 h,
complete digestion, cf. 20% in Ref. 11) of the protein mass
of the CEs used. Of this, the total amount of retarded peptide material
recovered in Fig. 1 represents
0.4% (10-min digestion) or 0.25%
(60-min digestion) of the 220-nm absorbing material eluted from the
HPLC column, which corresponds to 0.02-0.05% of the total protein
mass of the CEs, or
250 ng/mg of CEs. None of the retarded peptide
fractions contained the isodipeptide cross-link, indicating that
different regions of CE proteins were used for lipid attachments and
for cross-linking.

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Fig. 1.
Separation by HPLC on a
C4 column of proteinase K peptides following 15 min of partial saponification. The profiles shown denote different
times of proteinase K digestion. The 10 arbitrarily numbered peaks of
material used for further analyses are indicated.
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The material from each peak was recovered from the pooled epidermis of
20-30 foreskins. By amino acid analysis, each contained protein, and
based on the composition of the simplest peptides (see below), the
yields were 20-100 pmol. After a 2-h resaponification reaction,
followed by chromatography on the same HPLC column, the 220-nm
absorbing peptides of each peak eluted at the column wash, and there
was no detectable retarded material. These data suggest the presence of
ester-linked hydrophobic lipopeptides.
Retarded Peptide Peaks Contain Near-Stoichiometric Amounts of
Ceramides--
Earlier data have demonstrated that a major class of
lipid molecules ester-linked to the CE proteins are ceramides (19-24). To ascertain whether the retarded peptide-containing species identified in Fig. 1 contained ceramides, we used an established method that specifically incorporates a phosphate group onto an hydroxyl group of
the sphingosine moiety of a ceramide (25). Using 10-40-pmol aliquots
(based on peptide content), the data of Fig.
2 demonstrate that each peak contains
ceramides. Following densitometric scans of the bands, and in relation
to the reaction with the C18 ceramide standard, we
calculated that the peaks contain 0.6 mol (peaks 4 and 10), 0.7 mol
(peak 2), 0.8 mol (peaks 1, 5, 6, and 9), or up to 0.9 mol (peaks 3, 7, and 8) of ceramide/mol of peptide. The somewhat lower stoichiometry and
the multiplicity of species may indicate degradation or oxidation of
the ceramides. Alternatively, because most peptide peaks contained
multiple ceramide bands that migrated significantly faster than the
C18 ceramide standard (Fig. 2), the bands may represent
species with variably-sized sphingoid and/or fatty acid chains that
were considerably longer than those of the C18 ceramide
standard. Fig. 2 also shows the reaction of 50 pmol of ceramides
recovered from a 2-h saponification reaction of human foreskin CEs and
pig skin CEs (a kind gift from Dr. P. Wertz). These broad bands suggest
the presence of multiple unresolved ceramide species of similar size to
those of the 10 recovered peaks.

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Fig. 2.
Ceramide assay using the diacylglycerol
reaction. Upper panel, autoradiographs of reactions with the
C18 standard (amounts shown), as well as 50 pmol of
total ceramides recovered from human foreskin epidermis
(HFS) or pig skin (PS). Lower panel,
autoradiographs of reactions with 10 pmol of each of the 10 peaks of
Fig. 1. Multiple exposures were developed in order to quantitate the
amounts of ceramide in relation to the C18 standard. Note
that in many peaks, the labeled products migrated faster than the
standard but at rates similar to those of HFS and PS, suggestive of the
presence of longer ceramides.
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Following a 2-h saponification reaction, we calculated that the total
yield of human foreskin CE ceramides was 2750 pmol/mg of CE protein.
Assuming that the CE is about 15 nm thick and its density is about 1 g/cm3 (26), 1 mg of protein would occupy about 7 × 1016 nm2 of CE surface. This means that there
is about 1 molecule of ceramide/40 nm2 of CE. Based on the
predicted size of these ceramides (Refs. 19-21 and see below), this
means that the entire CE surface is effectively coated by a
monomolecular layer of ceramides (19, 20).
Finally, we could account for 15-20% of this total amount of
ceramides in separate experiments by summation of the yields recovered
from the 10 peaks of Fig. 1. Another
70% of the total ceramides was
present in the CE pellet following the 15-min limited saponification
and 60-min proteinase K digestion procedures.
Six Retarded Peaks Contain Identifiable Involucrin or
Involucrin-like Sequences--
Aliquots (5-20 pmol) of each peak were
used for amino acid sequencing. As Table
I shows, peaks 3-8 contained peptides of sequences that are either identical (peaks 4, 6, and 8) or very similar
(peaks 3, 5, and 7) to human involucrin. In peaks 3, 5, and 7, a Gln
residue in human involucrin was sequenced as a Glu residue. There are
two possible explanations for this. The first is that these Gln
residues may have participated in the initial step of a cross-linking
reaction with transglutaminases but failed to complete the transfer of
the enzyme-substrate complex to an acceptor substrate amine, such as a
Lys residue: the net result would be deamidation of the Gln residue
(27-29). We think that this is a relatively rare event because we have
found only a few cases of such modifications in the sequences of many
cross-linked peptides characterized to
date.2 Moreover, such
hydrophilic peptides would not have been retarded by the C4
column. A second, more plausible possibility is that a lipid adduct was
linked through an ester bond to the Gln residue, which, following
hydrolysis (saponification) of the ester bond, would generate a Glu
residue. Such an hydrolysis would likely occur during the amino acid
sequencing chemistry reactions. Thus, these modified residues identify
the likely target position of lipid attachment. However, in peptides 4, 6, and 7, in which there was an exact match with involucrin, the lipid
was likely attached to an existing Glu residue, but the residue
position of modification could not be ascertained.
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Table I
Sequences of peptides resolved on C4 column of Fig. 1
Single letter amino acid code is used. The letters in boldface
delineate the possible site of attachment of ester-lined ceramides.
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In total, in different experiments, the identifiable involucrin-like
sequences accounted for about 35% of the total retarded peptide
material (molar amount) at the earlier times of proteolysis. These were
attached through seven identifiable sequences of involucrin, most of
which were located in the first 140 amino acid residues of the protein,
in sequences thought to constitute the ancestral portion (30, 31). An
exception is peptide 6, which is located in the more modern (still
evolving) parts of the protein. Therefore, it could be argued that the
attachment of ceramides to involucrin is an evolutionarily ancient
aspect of barrier function in mammalian epidermis.
Identification of Other CE Proteins as Substrates of Lipid
Attachment--
Peaks 1, 2, 9, and 10, which were sensitive to the
extent of proteolysis (Fig. 1), possessed complex amino acid
compositions, suggesting the presence of multiple short peptide
species. The material of peaks 9 and 10 accounted for <10% of the
retarded material at the earliest time of digestion and was not
explored further. Next, we pooled material from the mobile peaks 1 and 2 from the 10-, 20-, and 30-min digests, which accounted for about 50%
(molar basis of peptide material) of the total retarded material in
separate experiments (Fig. 1). Because these were poorly resolved by
the C4 HPLC column, we removed the ceramide adducts by
saponification for 2 h and rechromatographed the released peptide
material on a C18 HPLC column (Fig.
3). Of this, 65-70% eluted at <8 min
and could not be further resolved because it consisted of single amino acids or di- or tripeptides. Several peaks resolved at >10 min (
30% of total) were recovered for amino acid sequencing (Table II). Four matched with various nonhuman
proteins, but two coincided exactly with the novel CE protein
periplakin.3,4 Two
others were highly homologous to envoplakin (32) and periplakin by
having a Glu to Gln residue substitution as seen for involucrin. Another peptide may have derived from envoplakin or desmoplakin (33).
Each of these is a structural protein located at the cell periphery of
keratinocytes, and they either have been established as (desmoplakin
and envoplakin; Refs. 11-13) or are thought to be (periplakin) protein
components of the CE. These data suggest that periplakin and envoplakin
may each constitute 6-10% of the substrates for ceramide attachment.
The other six retarded 2-4-residue peptides (about 6% of the total)
commonly occur in these CE proteins, as well as in loricrin or
involucrin (Table II). The remainder of the retarded material was
composed of numerous other peptides of low abundances (Fig. 3; Table
II).

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Fig. 3.
Separation by HPLC on a
C18 column of unresolved peptides of peaks 1 and
2 of Fig. 1 following saponification. Each peak resolved after 10 min was subjected to sequencing, and those with three or more residues
are listed in Table II.
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Mass Spectroscopic Analyses of Ceramide-Peptide
Adducts--
Aliquots of 5-10 pmol of each ceramide-peptide peak from
Fig. 1 were used for mass spectroscopic analyses. Each generated complex profiles with multiple mass species, of which only the regions
containing the quantitatively major species are shown in Fig.
4 (left). Peaks 1, 2, 9, and
10 (data for peak 9 not shown) yielded profiles with broad peaks to
which masses could not be assigned, presumably because of the presence
of multiple short peptides (Tables I and II). In the cases of peaks 1 and 2, there also were minor mass components in the range of 1250-1750
atomic mass units (data not shown). However, peaks 3-8 all yielded
profiles with species of well defined masses. Some peaks contained
multiple species with masses differing by 2-4 atomic mass units.
Notably, many peaks were separated from each other by 12-17 atomic
mass units, most commonly 14 atomic mass units, suggestive of species differing by units of a methylene (CH2) group and perhaps
of species differing by double bonds and/or hydroxyl groups. The most
complex profile was generated for peak 8, which consisted of two
similar sets of species.

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Fig. 4.
Mass spectroscopy of peaks before (left
panels) and after (right panels) saponification.
Following initial scans to identify the size ranges of significant
masses, spectra were performed over narrower mass ranges to generate
these deconvoluted spectra. Data for peak 9 are not shown. The masses
of ceramide-peptide (left) or ceramide (right)
peaks are indicated by the vertical annotations. The masses
of likely peptide species are indicated by horizontal
annotations. For peaks 1 and 2, the arrows
(right) indicate masses of 701 and 746 atomic mass units,
which correspond to the masses of the peptides EELEAL and EQQTLQ (see
Table II). amu, atomic mass units.
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Next, we treated each peak by saponification for 2 h to hydrolyze
the ester linkage between the ceramide and peptide moieties and
repeated the mass measurements (Fig. 4, right). In all
cases, we obtained well defined mass species ranging from 664 to 888 atomic mass units, including peaks 1, 2, 9, and 10. Many of the peaks
were also separated by an average of 14 atomic mass units. In each of
the cases of peaks 3-8, an additional major species, representing
approximately one-half of the total mass present in the samples was
obtained that corresponded very closely (±1 atomic mass unit) with the
expected mass of the peptide as deduced from the amino acid sequencing
analyses (Table I). Peak 8 material generated a simpler profile after
saponification, and it generated two peptide peaks, establishing the
presence of two ceramide-peptide adducts as implicated from the
sequencing data (Table I). In the case of peaks 1, 2, 9, and 10 (data
for peak 9 not shown), most of the poorly resolved ceramide-peptide
adducts (Fig. 4, left) also contained a limited array of
ceramides of well defined masses after saponification
(right). Thus, these ceramides had been attached to a
complex array of single amino acids or small peptides that themselves
could not be resolved in this study by chromatography (Fig. 3 and Table
II) or mass spectroscopy. However, in the cases of peaks 1 and 2, peptides of 887 and 962 atomic mass units were present (Fig. 4), which
correspond to the masses expected for the peptides of sequence EQQLLQQ
and EQEEAEAR and are probably derived from envoplakin and periplakin,
respectively, recovered as described in Fig. 3 and Table II. Similarly,
minor peaks of masses 702 and 736 atomic mass units were recovered
(Fig. 4A, arrows) that correspond to two other peptides of
sequences EELEAL and EQQTLQ, respectively (Table II). Peaks 6 and 10 contained other species (Fig. 4B, asterisks) that were also
about 14 atomic mass units apart but were 5-7 atomic mass units
different in size from the major ceramides. Because they were minor
components, it is not known whether they are ceramides or some other
lipid material that was ester-linked to the peptides.
Interestingly, there was a trend of increasing size of ceramide species
from peak 1 to peak 10 (Fig. 4 and Table
III), which is consistent with the notion
that the later peaks were more retarded by the C4 HPLC
column because of the presence of ceramides having somewhat longer
hydrophobic chains. A striking observation from the present work was
that ceramide species of the same masses were present in several of the
10 peaks (Fig. 4 and Table III), including in particular masses of 750, 776, 778, and 804 atomic mass units. These data suggest that there were
common higher abundance ceramides that were used for attachment to
multiple sites on involucrin and the other CE proteins.
Unfortunately, because only limited amounts of newborn foreskin
epidermis was available to us, we were unable to acquire sufficient material to perform additional analyses to ascertain the exact fatty
acid and sphingosine composition of the ceramides identified in this
study. Nevertheless, we have performed two additional analyses. In the
first, we determined the masses of ceramides recovered from pig skin
(Fig. 5). The data show the presence of many ceramide species, and interestingly, components of masses of about
736, 750, 776, 778, 804, and 832 atomic mass units accounted for the
majority. The chemical natures of the
-hydroxy fatty acids and
sphingosines of these ceramides and their relative abundances in both
human and pig skin have been determined (34-36) and are listed in
Table IV. In a second analysis, we
calculated the relative amounts of the different ceramide masses
identified in this study, based on the amount of each peak of Fig. 1
and the masses of the array of ceramides identified within each peak.
Together, the comparisons of the pig and human data reveal remarkable
consistency in especially the most abundant ceramides of masses 736, 750, 776, 778, 804, and 832 atomic mass units. These analyses thereby provide robust indirect data on the likely chemistry of the
-hydroxy fatty acid and sphingosine components of the human ceramides recovered in the present work.

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Fig. 5.
Mass spectroscopy of ceramide lipids of pig
skin. The data were collected as for Fig. 4 using a sample kindly
supplied by Dr. Philip Wertz.
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Conclusions--
We have demonstrated that a family of ceramides
within the range of 680-880 atomic mass units having fatty acid and
sphingosine chains of varying size are covalently attached by way of
ester bonds to involucrin as well as other known proteins of the human foreskin epidermal CE. This report confirms earlier work indicating that ceramides of this size range are esterified to proteins of human
cornified epidermis (34, 35). Moreover, our work identifies for the
first time some of the protein targets and residue positions of this
attachment. However, the present experiments can account for only about
20% of the total ceramides of human foreskin epidermal CEs.
Nevertheless, the analyses of Table III suggest that the recovered ceramides are representative of the total. On the other hand, we cannot
exclude the possibility that a significant proportion of the ceramides
are attached to other proteins of the CE.
Furthermore, these data are consistent with current models of the
mechanism of assembly of the CE (7, 8, 10-13). Involucrin, desmoplakin, envoplakin, and periplakin have been identified as some of
the earliest components assembled by transglutaminase cross-linking
onto the protein CE (11-14) and are presumably located physically very
close to the plasma membrane, near where keratin intermediate filaments
interact in desmosomes. This complex is thought to function as a
scaffold to which large amounts of loricrin and small proline-rich
proteins are attached later on the cytoplasmic side. At some advanced
stage of CE assembly, the plasma membrane is dispersed, leaving a
cross-linked layer of protein on the exterior of the cell (7-13).
Subsequently or concurrently, attachment of ceramides to these proteins
is thought to occur from the external surface of the corneocyte (1-4,
18-23). Thus, the identification of involucrin, periplakin, and
envoplakin as at least three of the protein targets of ceramide
attachment is spatially and temporally consistent with existing data
and models.
In addition, these data offer significant support to an earlier
hypothesis that predicted that ceramides might be linked to a
Glu/Gln-rich protein target (20, 23). Human involucrin contains about
40% of these residues (29). The other peptides described here likewise
resided in Glu/Gln-rich regions of the proteins (Tables I and
II).
Future experiments now will focus on the mechanisms by which ceramides
are delivered and become attached to the CE proteins. The linkage of
the ceramides to Glu residues by simple ester bonds could involve acyl
transferases. The linkage to Gln residues potentially may involve
transglutaminases because they are capable of transferring an activated
protein-bound Gln side chain to an alcohol acceptor, resulting in an
ester bond (37).
We thank Drs. Henry Fales, Normal Gershfeld,
and Ken Parker for advice with the mass spectroscopy; Philip Wertz for
the gift of pig skin ceramides; and Zoltan Nemes for many useful
comments.