From the Kekulé-Institut für Organische
Chemie und Biochemie, Universität Bonn, Gerhard-Domagk-Strasse 1, D-53121 Bonn, Germany, the § Department of Dermatology,
School of Medicine, University of California and the Dermatology
Service and Research Unit (190), Veterans Affairs Medical Center, San
Francisco, California 94121, and the
Departments of Neurology
and Psychiatry, School of Medicine, University of North Carolina,
Chapel Hill, North Carolina, 27599
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ABSTRACT |
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The epidermal permeability barrier is maintained
by extracellular lipid membranes within the interstices of the stratum
corneum. Ceramides, the major components of these multilayered
membranes, derive in large part from hydrolysis of glucosylceramides
mediated by stratum corneum Sphingolipid activator proteins
(SAPs1 or saposins) are
nonenzymatic glycoprotein cofactors required for lysosomal degradation of various glycosphingolipids by exohydrolases (1, 2). Proteolytic cleavage of SAP precursor (pSAP) in acidic compartments releases four
homologous polypeptides (i.e. SAP-A, SAP-B, SAP-C, and
SAP-D), thought to function either by direct activation of their
respective enzyme or as biological detergents that lift substrates out
of the membrane plane (3-5). One of the activator proteins, SAP-C, has
been shown to stimulate lysosomal glucosylceramide (GlcCer) degradation
by In terrestrial mammals, the permeability barrier that restricts excess
transepidermal water loss is maintained by unique arrays of lamellar
membranes localized in the interstices of the outermost stratum corneum
layer of the epidermis. These lamellar arrays derive from the extruded
lipid contents of lamellar bodies after fusion of these organelles with
the apical plasma membrane of the outermost granular cell. The
initially secreted lipids are processed by a set of co-localized lipid
hydrolases into a more hydrophobic mixture, enriched in ceramides
(Cer). The resultant Cer, comprising a family of compounds with complex
chemical structures, together with free fatty acids and cholesterol are
the dominant constituents of the stratum corneum (SC) lamellar
membranes (11). Two of these species, which possess an The postsecretory processing of lamellar body-derived GlcCers to
ceramides by Animals--
Generation of SAP-deficient mice through targeted
gene disruption has been described previously (20). Newborn knockout
mice (homozygous, n = 9; heterozygous,
n = 8) as well as newborn wild-type littermates
(n = 7) were studied. Genotyping was performed by polymerase chain reaction. The primers used had the following sequence:
forward primer, 5'-GCC CAC AGC GGT GAG TGC-3', matching on the
pSAP genomic DNA on exon 2/intron 2; reverse primer,
5'-CAG CAA GTT CCC AGC TTC GG-3', matching on exon 3. With those
primers a band of 1300 bp appeared in the pSAP Preparation of Epidermis--
Whole skin was removed at autopsy
from newborn mice. Epidermis was separated from dermis after floating
the skin on Dispase (Boehringer Mannheim, grade II) diluted 1:1 in
Hanks' buffer at 4 °C overnight.
Lipid Analysis--
Tissue samples were homogenized,
lyophilized, and weighed. Epidermal lipids were extracted for 24 h
at 37 °C in each of three solvent mixtures
(chloroform/methanol/water 1:2:0.5 (v/v/v); chloroform/methanol 1:1
(v/v), and chloroform/methanol 2:1 (v/v). Total lipid extracts were
applied to thin-layer Silica Gel 60 plates (Merck Darmstadt, Germany). All plates were washed with chloroform/methanol (9:1, v/v)
before sample application. For separation of glucosylceramides, the
chromatograms were developed with chloroform/methanol/water (70:30:5,
v/v/v). Ceramides were resolved twice using chloroform/methanol/acetic acid (190:9:1, v/v/v) as developing solvent. For quantitative analytical TLC determination, increasing amounts of standard lipids (N-stearoyl-sphingosine (kind gift of Beiersdorf AG,
Hamburg, Germany), sphingomyelin (Sigma), and glucosylceramide
(purified from Gaucher spleen in our laboratory)) were applied. After
development, plates were air-dried, sprayed with 8% (w/v)
H3PO4 containing 10% (w/v) CuSO4,
and charred at 180 °C for 10 min, and lipids were quantitated by
photodensitometry (Shimadzu, Kyoto, Japan).
Recovery and Analysis of Covalently Bound Lipids--
After
sequential extraction of epidermal samples as described above, pellets
were extracted three times for 5 min at room temperature with methanol
followed by two additional extractions for 2 h with 95% methanol
at 60 °C. The final fraction was checked for lipid content.
Covalently bound lipids were released by incubation with 1 ml of 1 M KOH in 95% methanol for 2 h at 60 °C. Lipids were subsequently recovered by adding 2 ml of water and 2 ml of chloroform. The chloroform layer was removed, and the aqueous phase was
extracted once more with 2 ml of chloroform. The organic phases were
combined and evaporated under a stream of nitrogen. Lipids were applied
to prewashed (chloroform/methanol 9:1, v/v) thin-layer Silica Gel 60 plates (Merck) and separated using 2× chloroform/methanol/acetic acid
(190:9:1, v/v/v) as developing solvent. Quantification of lipids was
performed as described above. For quantification of fatty acids,
increasing amounts of palmitic acid (Fluka, Buchs, Switzerland) were
applied to the plates.
MALDI Mass Spectrometry of Lipids--
Aliquots of lipid
extracts were separated by TLC techniques as described above.
Individual sphingolipids were scraped from plates and redissolved in
chloroform/methanol 1:1 (v/v). Approximately 50-200 pmol of lipid were
mixed with 20 µg of 2,5-dihydroxybenzoic acid (ICN Biochemicals)
directly on the target. Mass spectrometric analysis was performed on a
TofSpec E (MicroMass, Manchester, UK) mass spectrometer operating
at an acceleration voltage of 20 kV with a 337-nm nitrogen laser.
External calibration was performed using PEG-1000 (Sigma).
Light and Electron Microscopy--
Skin samples were taken at
autopsy, either immediately or 24 h after birth, and processed for
light and electron microscopy. For light microscopy, samples were fixed
by immersion in phosphate-buffered paraformaldehyde (4%), embedded in
paraffin, sectioned (5 µm), stained with hematoxylin and eosin, and
photographed with a Zeiss Axioplan microscope. For electron microscopy,
samples were minced to <0.5 mm, fixed overnight in HEPES buffer (0.16 M, pH 7.3) containing glutaraldehyde/formalin (1.25:0.5%)
and 0.1 mM CaCl2, and postfixed in both
ruthenium tetroxide, as described previously (22), and 2% aqueous
osmium tetroxide, each containing 1.5% potassium ferrocyanide. After
fixation, all samples were dehydrated in graded ethanol solutions and
embedded in an Epon/epoxy mixture. Ultrathin sections were examined,
with or without further contrasting with lead citrate, in an electron
microscope (Zeiss 10A, Carl Zeiss, Thornwood, NY) operated at 60 kV.
Statistical Analysis--
Statistical evaluation of data was
performed using a two-tailed Student's t test.
Epidermal Sphingolipids in pSAP Knockout Mice--
To address the
question of whether the pSAP product SAP-C is involved in the
processing of epidermal GlcCer to Cer and therefore important for
epidermal barrier formation, we first determined the GlcCer and Cer
levels in the epidermis of pSAP knockout mice. After extraction,
sphingolipids were separated by TLC and quantitated by
photodensitometry. Aliquots of lipid extracts taken in parallel were
used to isolate individual sphingolipids by TLC for MALDI mass
spectrometric analysis. Although we had proposed likely compositions for sphingolipid structures, we could not distinguish differences in
fatty acid and sphingoid chain length distribution from masses of
intact molecular ions. In this work, structural data are presented in
accordance with the epidermal Cer terminology proposed by Motta et al. (23) and modified by Robson et al. (24).
Briefly, the Cer structures are denoted by the composition of the
sphingoid base (either sphingosine (S) or phytosphingosine (P)) and the N-acyl fatty acid by the presence of
Distinct differences in the epidermal lipid composition were evident
between pSAP-deficent and wild-type animals. First, the content of each
of the three distinct GlcCer fractions that were readily separated by
TLC (see mass spectrometric analysis for structural assignments below)
was significantly elevated in the epidermis of pSAP
The storage of GlcCers was accompanied with a decrease in the level of
epidermal ceramides. Ceramides were separated into five fractions (Fig.
1B) and later identified by MALDI mass spectrometric analysis (see below). With the exception of Cer(C16-AS), all of the
other epidermal ceramides were modestly decreased in the SAP
We next identified each of the GlcCer and Cer fractions separated by
TLC. Using MALDI mass spectrometric analysis, the most hydrophobic
glycolipid, GlcCer(EOS), was found to contain
MALDI mass spectrometric analysis of ceramides revealed sphingoid and
fatty acid compositions similar to those found in their corresponding
glucosylceramide precursors as well. With regard to the ceramide
moiety, GlcCer(EOS) appeared to correspond to Cer(EOS) having the same
fatty acid pattern, again with a majority being C32:0 (M + Na, 1062.9 atomic mass units) and C34:1 (M + Na, 1088.8 atomic mass units) (Fig.
2E). Cer(NS) contained different non-hydroxy fatty acids
with chain length between C24 and C26 (Fig. 2F). Cer(NP)
appeared to be hydroxylated in the sphingoid backbone because it
comigrated with synthetic phytoceramide on TLC chromatograms. The
molecular masses indeed demonstrated an additional hydroxyl group in
Cer(NP) together with various degrees of saturation of long chain fatty
acids (Fig. 2G). Cer(C24,26-AS) and Cer(C16-AS) both were
identified as ceramides containing
Processing of sphingomyelin to Cer by acidic sphingomyelinase (SMase)
could also contribute to the pool of Cer available for barrier
formation within the epidermis. A significant in vivo activation of acidic SMase by SAPs could not be concluded from lipid
profiles of brain and liver from pSAP knockout mice (20). However, in
the epidermis the sphingomyelin level was increased 1.5-fold over that
in wild-type mice (p < 0.02, data not shown).
Covalently Bound Lipids in pSAP Knockout Mice--
Terminally
differentiated keratinocytes (corneocytes) are coated by a monolayer of
Morphological Alterations in the Epidermis of pSAP Knockout
Mice--
Newborn pSAP knockout mice displayed an ichthyotic skin
phenotype including a red and wrinkled appearance previously observed in a more dramatic fashion in the Alterations in Epidermal Ultrastructure--
To further delineate
the basis for the cutaneous abnormalities induced by the deficiency of
pSAP in the epidermis, micrographs from pSAP knockout, heterozygous,
and wild-type littermates were compared (Fig.
6). The pSAP-deficient epidermis ( In this work, we isolated lipids from murine epidermis and for the
first time presented a complete mass spectrometric characterization of
both epidermal ceramides and corresponding glucosylceramides. Our data
on the ceramides are in good agreement with previous structural
characterization of murine Cer(EOS), Cer(NS), and Cer(C16-AS) (26).
Because we exclusively obtained intact molecular ions (by MALDI), the
fatty acid and sphingosine chain length distributions are not
discernable. However, the sphingoid bases from human and pig epidermal
ceramides are known to have limited chain length heterogeneities
between C16 and C22 (27, 30), consistent with the substrate specificity
of epidermal serine palmitoyltransferase (31). We therefore proposed
likely compositions of individual sphingolipids. The structural
assignment of Cer(NP), referred to here as phytoceramide, is based both
on its mass spectrum and its comigration on TLC plates with synthetic
phytoceramide. We found similar sphingoid and fatty acid compositions
of ceramides and corresponding glucosylceramides. For example,
GlcCer(EOS) and Cer(EOS) both exhibited mainly C32:0 and C34:1 Processing of epidermal GlcCers to ceramides by the action of acidic
Despite the massive accumulation of GlcCers, epidermal Cer levels were
only moderately reduced in newborn pSAP In principle, It is well known that a monolayer of Because we detected small amounts of bound GlcCer(OS) in both pSAP +/ Finally, morphological alterations in the epidermis of pSAP -glucocerebrosidase (
-GlcCerase).
Prosaposin (pSAP) is a large precursor protein that is proteolytically
cleaved to form four distinct sphingolipid activator proteins, which
stimulate enzymatic hydrolysis of sphingolipids, including
glucosylceramide. Recently, pSAP has been eliminated in a mouse model
using targeted deletion and homologous recombination. In addition to
the extracutaneous findings noted previously, our present data indicate
that pSAP deficiency in the epidermis has significant consequences
including: 1) an accumulation of epidermal glucosylceramides together
with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope;
3) a thickened stratum lucidum with evidence of scaling; and 4) a
striking abnormality in lamellar membrane maturation within the
interstices of the stratum corneum. Together, these results demonstrate
that the production of pSAP, and presumably mature sphingolipid
activator protein generation, is required for normal epidermal barrier
formation and function. Moreover, detection of significant amounts of
covalently bound
-OH-GlcCer in pSAP-deficient epidermis suggests
that deglucosylation to
-OH-Cer is not a requisite step prior to
covalent attachment of lipid to cornified envelope proteins.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-glucocerebrosidase (
-GlcCerase) (6, 7). A deficiency of SAP-C
causes a variant form of Gaucher disease (8-10).
-hydroxy
moiety on the N-acyl fatty acid, are covalently bound to
structural proteins of the cornified cell envelope forming a
hydrophobic surface on terminally differentiated keratinocytes (12,
13).
-GlcCerase is essential for maintenance of permeability
barrier homeostasis (14-17). Because SAP-C stimulates lysosomal GlcCer
degradation in vivo, it seems reasonable to predict that
expression of pSAP would also be important for epidermal barrier
formation. Decreased levels of pSAP have been detected in atopic skin,
suggesting a role for SAPs in epidermal function (18). Furthermore, a
20-week pSAP-deficient human fetus, with a phenotype resembling acute
neuronopathic (type 2) Gaucher disease, displayed extensive cutaneous
lysosomal storage deposits and neutral glycosphingolipid accumulation
in dermal fibroblasts (19). A SAP precursor-deficient mouse (pSAP
/
) has recently been created by targeted gene disruption and
homologous recombination (20). Because of the deficiency of the
complete precursor protein, none of the four SAPs are generated in any
cell type. Initial characterization of these mice exhibited two
distinct phenotypes, i.e. neonatally fatal and later onset.
The pathology of the latter is complex, including severe
hypomyelination and storage of multiple sphingolipids in the brain (20,
21), as well as an ichthyosiform dermatosis. Thus, in the present work,
we focused on the physiologic function of pSAP in the context of
cutaneous permeability barrier formation and investigated the effects
of pSAP deficiency on epidermal lipid composition and structure.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
mice, a band of
806 bp appeared in pSAP +/+ mice, and both bands appeared in pSAP +/
mice.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-hydroxy group (A),
-hydroxy group (O), or no-hydroxy group (N) and whether the
position is further acylated (i.e. esterified (E)).
/
versus wild-type littermates (Fig.
1, A and C). In the
absence of pSAP, GlcCer(EOS) accumulated 5.2-fold (p < 10
8, n = 9), whereas the more polar
(i.e., lower Rf value) GlcCer A/B and GlcCer(C16-AS)
accumulated 2.1-fold (p < 10
4) and
2.2-fold (p < 0.02), respectively. In heterozygous
mice, which demonstrate nearly 50% reduction in the expression of pSAP protein (20), only the GlcCer(EOS) fraction showed significant accumulation (1.4-fold, p < 0.03), whereas the other
GlcCer fractions remained unchanged. These results suggest not only
that the processing of epidermal GlcCers by acidic
-GlcCerase
requires the production of pSAP, and presumably mature SAP generation,
but that the hydrolysis of the more hydrophobic epidermal GlcCers
(e.g. GlcCer(EOS)) is more dependent upon the presence of
SAP-C.
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Fig. 1.
Epidermal sphingolipid composition of pSAP
knockout mice. One-dimensional TLC separations of polar epidermal
lipids (panel A; PE, phosphatidylethalolamin; PC,
phosphatidylcholine; PS, phosphatidylserine, SM,
sphingomyelin) and epidermal ceramides (panel B; X1 and
X2, not identified) are shown. Densitometric quantitations
of GlcCer (panel C) and Cer (panel D) are given.
C and D, data are presented as the mean ± S.E. (n 7). Note the accumulation of GlcCers,
especially of GlcCer(EOS), in the homozygous knockout mice. GlcCer A/B
is a complex mixture of GlcCer(NS), GlcCer(NP), and GlcCer(C24,26-AS).
The lipid contents of either heterozygous or homozygous mice differ
significantly from wild-type levels at: ***, p
10
5; **, p
0.01; and *,
p
0.05. n.s., not significant.
/
epidermis in comparison with wild-type littermates (by approximately 20%) (Fig. 1D). In contrast, the least hydrophobic
ceramide, Cer(C16-AS), accumulated 1.3-fold over control
(p < 0.05). Moreover, the Cer levels of heterozygous
mouse epidermis were not significantly different from normal
littermates (Fig. 1D). The diminished levels of epidermal
Cer in conjunction with the preferential accumulation of hydrophobic
GlcCer (noted above) strongly suggest a role for either pSAP itself or
a mature SAP, presumably SAP-C, in the hydrolytic processing of
GlcCer-to-Cer in epidermis.
-OH fatty acids of
various chain lengths (C28-C36) and degrees of saturation with a
majority being C32:0 (M + Na, 1224.7 atomic mass units) and C34:1 (M + Na, 1250.6 atomic mass units) (Fig. 2A). The
-OH moiety of this
GlcCer fraction is known to be acylated in the epidermis with linoleic
acid, as shown here and previously (25). GlcCer A/B (Fig.
2B) comprised a complex mixture of GlcCers (i.e. GlcCer(NS) GlcCer(NP), and GlcCer(C24,26-AS)).
Non-hydroxy fatty acids with dominant chain length of C24:0, C25:0, and
C26:0 were attached to either sphingosine or phytosphingosine. The less abundant
-hydroxy fatty acids in GlcCer A/B showed a similar pattern
with C24:0-OH (M + Na, 850.4 atomic mass units), C25:0-OH (M + Na,
864.4 atomic mass units), and C26:0-OH (M + Na, 878.4 atomic mass
units). The presence of significant amounts of C25 and other odd-chain
fatty acids in epidermal ceramides has also previously been reported
(26, 27). Because of the differences in molecular polarity, it seems
likely that the upper part of the double band, GlcCer A/B, represents
GlcCers with non-hydroxy fatty acids, whereas the lower part
corresponds to the GlcCers containing
-hydroxylated fatty acids. The
most hydrophilic glycolipid, GlcCer(C16-AS), was identified as a
homogenous GlcCer containing mainly a C16:0-OH fatty acid (M + Na,
738.5 atomic mass units, Fig. 2C).
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Fig. 2.
MALDI mass spectrometry of epidermal
sphingolipids. Lipids were isolated from either wild-type or pSAP
knockout mice by TLC and analyzed in the positive ion mode. All
indicated masses represent sodium adducts of intact molecules. The
likely compositions of the most prominent peaks are indicated. The
fatty acids are indicated first followed by the sphingoid bases. The
proposed chemical structures of individual sphingolipids are shown. The
lipid backbones of GlcCer A/B are Cer(NS), Cer(NP), and
Cer(C24,26-AS).
-hydroxy fatty acids attached to
sphingosine. In Cer(C24,26-AS) C24:0-OH (M + Na, 688.5 atomic mass
units), C25:0-OH (M + Na, 702.6 atomic mass units), and C26:0-OH (M + Na, 716.6 atomic mass units) fatty acids were found (Fig.
2H). Taken together, Cer(NS), (NP), and (C24,26-AS)
comprised the lipid backbones of GlcCer A/B. The most hydrophilic
ceramide, Cer(C16-AS), possessed mainly a C16:0-OH fatty acid and
therefore corresponds to GlcCer(C16-AS) (Fig. 2D).
-OH ceramides, ester-linked to proteins of the outer surface of the
cornified cell envelope. This lipid-bound envelope is thought to be
essential for either corneocyte cohesion and/or organization of
extracellular lipid lamellae (28, 29). We characterized these lipids
obtained after exhaustive prior extraction of epidermal samples
followed by alkaline hydrolysis (12). To ascertain that the recovered
lipids were indeed covalently attached, each sample was checked to
ensure that there was no residual extractable lipids before undergoing
base hydrolysis (Fig. 3A,
lanes 1, 3, and 5). A mixture of long
chain fatty acids, Cer(OS), and
-OH fatty acids were released from
the wild-type epidermis (Fig. 3A, lane 2). In
addition to these lipids, substantial amounts of GlcCer(OS) were
covalently attached to the cornified cell envelope in pSAP
/
mice
(Fig. 3A, lane 4). All three
-OH species had
the same fatty acid chain length distribution with a majority being
C32:1, C32:0, and C34:1 as demonstrated by mass spectrometry (Fig.
4). Furthermore, the levels of bound
Cer(OS) and
-OH fatty acids in homozygous knockout mice were
significantly decreased to 80% (p = 0.05) and 48%
(p = 0.002) of normal control, respectively. Comparably
low levels of covalently bound GlcCer(OS) could also be detected in
pSAP +/
as well as in wild-type epidermis. In summary, these data
show that in pSAP
/
mice there is a switch from a hydrophobic to a
more hydrophilic lipid-bound envelope.
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Fig. 3.
Covalently bound lipids in wild-type and pSAP
knockout mice. Ester-linked epidermal lipids were released by
alkaline hydrolysis and analyzed by TLC. A, representative
chromatographical separation of recovered lipids from either wild-type
or pSAP knockout mice (lanes 2, 4, and
6). Note that the samples were devoid of unbound lipids
before undergoing alkaline hydrolysis (lanes 1,
3, and 5). X, not identified.
B, individual lipid levels were quantified by densitometric
analysis. Data are presented as the mean ± S.E.
(n 7). The lipid contents of either heterozygous or
homozygous mice differ significantly from wild-type levels at:
***, p
10
5; **,
p
0.01; *, p
0.05. n.s., not significant.
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Fig. 4.
Mass spectrometry of ester-linked epidermal
lipids. Bound lipids were released by alkaline hydrolysis and
isolated using TLC. Indicated molecular masses represent positively
charged sodium adducts of intact molecules. The likely compositions of
the most prominent peaks are indicated. The fatty acids are indicated
first, followed by the sphingoid bases. The proposed chemical
structures of individual lipids are shown. *, unidentified impurity
extracted from silica gel.
GlcCerase-deficient Gaucher mice (15, 17). These findings are consistent with a defect in the
epidermal permeability barrier and suggest underlying defects in the
critical membrane domains in the stratum corneum. Therefore, to
ascertain the significance of pSAP deficiency in the epidermis, we
first analyzed epidermal sections at the light microscopic level (Fig.
5). In the homozygous knockout mice,
morphological alterations were limited to the stratum corneum and the
stratum lucidum, which represents the interface between the upper
stratum granulosum and the lower stratum corneum. The thicker stratum lucidum in the knockout mice in comparison with the wild-type mice
could be the result of an accumulation of unprocessed GlcCer. Furthermore, large parts of the stratum corneum were absent in sections
of the knockout animals, possibly because of reduced mechanical
stability resulting from swelling of intercellular domains.
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Fig. 5.
Light micrographs of skin sections of pSAP
knockout mice in comparison with wild-type mice. Whole skin was
removed from the abdomen of either wild-type or homozygous knockout
mice, and paraffin-embedded thin sections were stained using
hematoxylin and eosin. SL, stratum lucidum. Bars,
20 µm.
/
)
displayed a striking abnormality in lamellar membrane maturation within the interstices of the lower-to-middle stratum corneum (Fig. 6, A and B) when compared with that of normal (Fig.
6C) littermates. The distinctive membrane unit structures
seen in the normal stratum corneum (Fig. 6C) were replaced
by arrays of foreshortened lamellae in loose, discontinuous aggregates
in the pSAP-deficient stratum corneum (Fig. 6, A and
B, arrows), resembling those seen with
-GlcCerase deficiency (14, 15). Although normal quantities of
lamellar bodies were present and secreted normally in pSAP-deficient epidermis (Fig. 6E, arrowheads), their internal
structure appeared to be altered from those in heterozygous or normal
littermates (Fig. 6D and inset); individual
organelles often appeared enlarged and engorged with non-lamellar,
electron-dense material, which in turn disrupts the internal contents
normally seen in wild-type epidermis. Moreover, the secreted lamellar
body-derived contents in the intercellular spaces at the stratum
corneum-stratum granulosum interface in pSAP
/
animals retained a
compact, spherical pattern (Fig. 6A, asterisks) rather than
progressively unfurling into linear configurations, as occured in the
heterozygous and wild-type stratum corneum. These studies indicate that
a profound deficiency of pSAP does not disrupt production, trafficking,
or secretion of lamellar body contents into the intercellular domains.
Instead, pSAP deficiency results in major abnormalities in the
following: (a) the internal organization of lamellar body
contents; (b) dispersal after secretion; and (c)
extracellular processing of extruded lamellar body contents.
Heterozygous animals, although expressing approximately 50% of the
pSAP protein levels (20), did not display abnormalities in the contents
or maturation of the lamellar body, suggesting that sufficient pSAP is
expressed to sustain the extracellular hydrolytic activity required to
generate mature lamellar membrane organization.
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Fig. 6.
Ultrastructure of pSAP knockout epidermis in
comparison with wild-type littermates. In the lower to mid-SC from
pSAP-deficient ( /
) mouse epidermis, conventional osmium tetroxide
staining reveals extruded lamellar body contents to be highly
disorganized (A, open arrows), with persistence of
osmiophilic material in the interstices out to at least the fourth to
fifth SC cell layers (A, stars), an area routinely
undeposited by osmophilic material in normal wild-type SC tissues (not
shown). Moreover, extruded lamellar body contents retain a spherical
pattern in the interstices (A, asterisks), suggesting that
linearization of lamellar body-derived lamellae is delayed or
defective. Ruthenium staining (22) further reveals that the
pSAP-deficient interstices (B, between arrows) lack the
regular, compact pattern of lamellar membranes observed in normal,
pSAP-replete SC (C, between open arrows).
Although significant quantities of lamellar bodies are formed in
pSAP-deficient epidermis (E, arrowheads), the contents have
a more condensed appearance when compared with normal wild-type
littermates (D, arrowheads; and inset,
asterisk shows an enlarged image of a lamellar body from
normal epidermis). Bars represent 0.25 (A-E) and
0.125 µm (D, inset) with final image
magnifications (all are ×103) of: A, 46.6; B,
68.8; C, 62.5; D, 64.0; E, 44.8;
D, inset, 120. sg, stratum
granulosum.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-OH
fatty acids, strongly suggesting a precursor/product relationship
between these two related epidermal lipids.
-GlcCerase is well known to be essential for cutaneous barrier
formation and homoeostasis (14-17). In pSAP
/
mice, an accumulation of all GlcCers was evident, demonstrating that optimal epidermal
-GlcCerase activity in vivo requires
stimulation by SAPs (i.e. SAP-C). Furthermore, the
importance of SAP-C mediation is underscored by the observation that
degradation of the most hydrophobic GlcCer, GlcCer (EOS), was found
to be most impaired in the absence of SAPs. SAP-C is thought to
stimulate
-GlcCerase by an allosteric mechanism, resulting in
trimeric complex formation between the water-soluble enzyme, the
activator protein, and the membrane-associated glycolipid (1, 7).
In vitro data have demonstrated the ability of SAP-C to form
complexes with
-GlcCerase in the presence of phosphatidylserine
(32, 33). However, in vivo binding of SAP-C to GlcCers may
also contribute to the stimulation of GlcCer hydrolysis. Indeed, SAP-C
has recently been shown to solubilize membrane-bound GlcCers presumably
facilitating their processing by water-soluble enzyme (7).
/
mice compared with
wild-type littermates. One explanation for the near normal levels of
Cer might be increased sphingomyelin (SM)-to-Cer hydrolysis. However,
the increased SM content in the pSAP
/
mouse epidermis reported
here is also of consequence. In normal epidermis, the levels of both SM
and GlcCer are high in nucleated cells but these lipids are nearly
absent from the stratum corneum (34). Previous results with
-GlcCerase null allele animals also suggest that the majority of
stratum corneum ceramides are derived from the hydrolysis of GlcCer
precursors (15). The importance of stratum corneum SMase for barrier
homoeostasis has only recently been demonstrated2,3.
Thus, a portion of the Cer in stratum corneum normally may derive from
SM, or this pathway could be up-regulated in the face of a
-GlcCerase blockade. Moreover, although SAP-C has been shown to
stimulate acidic SMase activity in vitro (35, 36), the role
of SAPs in SM hydrolysis in vivo has not been adequately addressed. SMase activity is normal in extracts from dermal fibroblasts isolated from a patient with pSAP deficiency (8). However, the
increased epidermal SM content in the absence of pSAP expression described here suggests that SAP(s) may have a role in the regulation of epidermal SMase activity. Whether increased epidermal SM contributes to the stratum corneum pathology remains to be determined.
-GlcCerase can degrade GlcCer without SAP-C.
Furthermore, an excess of
-GlcCerase activity is present in normal epidermis, as Gaucher heterozygous animals with approximately 50% of
normal activity do not exhibit SC or epidermal pathology (15).
Therefore, it seems likely that during embryonic development substantial amounts of ceramides are provided by slow hydrolysis of
GlcCer without any loss by desquamation starting after birth. Finally,
ceramides could also derive directly from de novo synthesis under pathological conditions or even physiologically. The most polar
ceramide, Cer(C16-AS), was found to be elevated in the absence of pSAP,
suggesting a compensatory capacity of sphingolipid biosynthetic pathways in keratinocytes.
-OH-Cer is covalently attached
to the surface of corneocytes connecting proteins of the cornified cell
envelope to hydrophobic intercellular lipid lamellae (12). Recently,
some structural proteins within the epidermis have been shown to be
esterified at Glu or Gln residues with Cer(OS) (13). Although little is
known about the attachment of lipids to proteins of the epidermal
cornified cell envelope, it was assumed that free Cer(EOS) are the
direct precursors of bound Cer(OS). We have analyzed covalently bound
epidermal lipids in pSAP knockout mice. Most importantly, in pSAP
/
mice a novel lipid was recovered and identified as GlcCer(OS), which
had the same fatty acid chain length distribution as found in the bound Cer(OS) and in
-OH fatty acids as well. Obviously, these lipids are
metabolically related to each other. It has been an open question whether Cer(OS) is bound to amino acid side chains either via the
-hydroxyl group or via C1 of sphingoid bases. Because GlcCer(OS) cannot be bound through C1, where the glucose moiety is located, this
finding provides evidence that at least a portion of all three
-OH
species are attached to the cornified cell envelope via the terminal
-hydroxyl group.
and wild-type mice, this phenomenon appears to be physiologically important. In our model, lamellar body-derived GlcCer(EOS) is, in part,
utilized by an enzyme that exchanges esterified linoleic acid against
the side chain of a glutamate residue. Resulting bound GlcCer(OS) would
subsequently be deglycosylated to bound Cer(OS) by SAP-C-assisted
-GlcCerase and then hydrolyzed to bound
-OH fatty acid and free
sphingosine by SAP-D-assisted ceramidase. This metabolic relationship
is supported by identical fatty acid chain length distribution in all
three
-OH species. Stimulation of lipid-bound envelope-associated
processing of lipids by hydrolytic enzymes would mechanistically work
by an allosteric enzyme activation rather than by solubilization of
lipids, which are covalently attached and therefore cannot be lifted by
an activator protein. On the other hand, it cannot be excluded that
free Cer(EOS) would also be transferred to proteins of the cornified
cell envelope by an enzyme, which utilizes either Cer(EOS) or
GlcCer(EOS). In pSAP
/
mice, levels of bound
-OH fatty acid and
Cer(OS) were significantly decreased to 48 and 80% of control,
respectively. These data support the concept that bound GlcCer(OS) is
the likely precursor of bound Cer(OS) and
-OH fatty acid. Formation
of the latter is more severely impaired than formation of Cer(OS)
because, in addition to reduced substrate concentration due to SAP-C
deficiency, ceramidase is missing its physiological activators, SAP-D
(37) and SAP-C.4 However, it needs to be
clarified in future studies whether covalently attached Cer(OS) and
GlcCer(OS) are indeed substrates for ceramidase and
-GlcCerase, respectively.
/
mice
were significant and reminiscent of the alterations observed in
-GlcCerase-deficient Gaucher epidermis. Electron microscopy revealed
a striking abnormality in SC lamellar membrane organization. Moreover,
light micrographs revealed a partial loss of stratum corneum in pSAP
/
mice, which may reflect decreased SC coherence. Thus, the
accumulation of the more hydrophilic glucosylated Cer species, rather
than Cer, within the extractable SC lipid and/or the lipid-bound
envelope is likely responsible for the observed alterations in
epidermal structure. However, since pSAP also exists as a surface
component of neuronal cells (38), other potential functions for this
protein in the epidermis, beyond its precursor role, still need to be explored.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 284) and the National Institutes of Health (AR39448 and AR19098).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Beiersdorf AG, Unnastrasse 48, D-20245 Hamburg, Germany.
** To whom correspondence should be addressed. Tel.: 49-228-73-5346; Fax: 49-228-737778; E-mail: Sandhoff{at}uni-bonn.de.
2 E. Proksch and J. M. Jensen, personal communication.
3 W. M. Holleran and P. M. Elias, unpublished observation.
4 T. Linke and K. Sandhoff, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
SAP, sphingolipid
activator protein;
pSAP, prosaposin;
-GlcCerase,
-glucocerebrosidase;
Cer, ceramide;
GlcCer, glucosylceramide;
N, non-hydroxy fatty acid;
A,
-hydroxy fatty acid;
O,
-hydroxy fatty
acid;
S, sphingosine;
P, phytosphingosine;
E, esterified;
MALDI, matrix-assisted laser desorption ionization;
SC, stratum corneum;
SM, sphingomyelin;
SMase, sphingomyelinase.
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REFERENCES |
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