From the Department of Biology and Pathology of the
Cell, INSERM Contrat Jeune Formation 96-02, Toulouse-Purpan
School of Medicine, University of Toulouse III (Institut
Fédératif de Recherche 30, INSERM-CNRS-Université P. Sabatier-Centre Hospitalier Universitaire), 31073 Toulouse, France,
¶ INSERM U346/CNRS "Human Skin and Immunity", 69437 Lyon,
France,
L'Oréal, Life Science Research, Centre Charles
Zviak, 92583 Clichy, France, and the
Department of Dermatology, University
Hospital, S-901 85 Umeå, Sweden
Received for publication, January 10, 2001, and in revised form, February 16, 2001
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ABSTRACT |
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Corneodesmosin is a putative adhesion glycoprotein located in the extracellular part of the desmosomes in the upper layers of the epidermis. Synthesized by granular keratinocytes as a 52-56-kDa protein, corneodesmosin is progressively proteolysed during corneocyte maturation. This processing is a prerequisite for desquamation. Two glycine- and serine-rich domains of the protein might take on the conformation of adhesive secondary structures similar to glycine loops.
Corneodesmosin proteolysis was further characterized. Deglycosylation
experiments and reactivity with lectins demonstrated that the
corneodesmosin carbohydrate moiety does not prevent the proteolysis.
Immunoblotting, immunohistochemistry, and immunoelectron microscopy
experiments using affinity-purified anti-peptide antibodies raised to
four of the five structural domains of corneodesmosin and a monoclonal
antibody against its fifth central domain showed that the first step in
corneodesmosin processing is the cleavage of its extremities and
probably occurs before its incorporation into desmosomes. Then the
glycine loop-related domains are cleaved, first the N-terminal and then
part of the C-terminal domain. At the epidermis surface, the multistep
proteolytic cleavage leaves intact only the central domain, which was
detected on exfoliated corneocytes and probably lacks adhesive
properties. Importantly, corneodesmosin was demonstrated to be a
preferred substrate of two serine proteases involved in desquamation,
the stratum corneum tryptic and chymotryptic enzymes.
Keratinocytes constitute the major cellular population in
epidermis, where they proliferate in the innermost basal layer. During
their transit through the spinous and granular layers toward the skin
surface, keratinocytes express a specific program of terminal
differentiation that culminates in the formation of corneocytes (1, 2).
These dead "mummified" cells accumulate and form the
outermost cornified layer of epidermis or stratum corneum, (SC)1, which plays a critical
role in the physical protection of the body. To maintain a
constant SC thickness, as observed in normal epidermis, the
continuous generation of corneocytes is balanced by cell shedding
at the external surface in the tightly regulated process of
desquamation (3).
Cohesion of the SC is largely dependent on modified desmosomes or
corneodesmosomes. In fact, at the transition between the granular layer
and the SC, profound changes are observed in desmosome morphology. The
cytoplasmic plaque, which provides anchorage for cytokeratin
intermediate filaments, is no longer visible, and a homogeneous
electron-dense plug occurs instead of the characteristic symmetrical
tri-lamellar structure of the extracellular core (4-9). Corneodesmosome degradation is of major importance in the desquamation process (6-9). In xerosis and various hyperkeratotic states, including
psoriasis, accumulation of scales is observed, and the number of
corneodesmosomes persisting over the corneocyte surface in the upper SC
is greatly increased (10-13). Several serine proteases, including the
stratum corneum chymotryptic enzyme (SCCE) and the stratum corneum
tryptic enzyme (SCTE), are thought to be involved in corneodesmosome
proteolysis (14-18). SCCE and SCTE belong to the kallikrein family, a
subfamily of serine proteases whose genes are located in a single locus
at 19q13.3-13.4 (19).
At a molecular level, the two major adhesive transmembrane components
of corneodesmosomes are desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1)
(20, 35). Desmogleins and desmocollins are glycoproteins belonging to the family of cadherins, calcium-dependent
cell adhesion molecules. Each desmosomal cadherin is known to exist in
three different isoforms encoded by three different genes. Their
expression is tissue-specific and differentiation-dependent,
Dsg1 and Dsc1 being expressed in the uppermost layers of the epidermis
(for a review see (21)).
We identified another protein, corneodesmosin (Cdsn), located in
the corneodesmosome core (9). Cdsn is synthesized in the upper spinous
and/or lower granular layers in the form of a 52-56-kDa phosphorylated
basic glycoprotein. It is exported by cytoplasmic vesicles called
keratinosomes into the extracellular space where it interacts with the
core of the desmosomes just before their transformation into
corneodesmosomes (9, 13, 22, 23). Cdsn is a 529-amino acid-long,
glycine- and serine-rich protein. These amino acids have been proposed
to form peculiar secondary structures, the so-called glycine
loop-related domains at the termini of the protein where they are
particularly frequent, i.e. amino acids 65-175 and 375-450
(24). The suggested function of these structural motifs, which are
found in other epidermal proteins such as cytokeratins and loricrin, is
to interact with identical loops on the same or neighboring proteins
(25). Some experimental evidence was obtained in favor of a role for
Cdsn in corneocyte cohesion
(26).2 During the maturation
of the SC, the protein is progressively proteolysed. We previously
proposed that the process results in the suppression of the two glycine
loop-related domains, thus eliminating the adhesive parts of the
molecule and allowing desquamation (23, 24). Until now, the enzymes
involved, and the precise steps, in this proteolysis were not known.
Here, we show that Cdsn deglycosylation is unnecessary for proteolysis
to occur in vivo, and we characterize the Cdsn cleavage steps and their sequence. Finally, we show that Cdsn is a good substrate in vitro for both SCTE and SCCE.
Antibodies--
The anti-Cdsn monoclonal antibodies (MoAbs)
G36-19 and F28-27 are part of a series of antibodies produced and
characterized in our laboratory (9, 23). DG3.10, a MoAb directed to
Dsg1 and Dsg2, and PG5.1, a MoAb directed to plakoglobin, were
purchased from Progen Biotechnik GmbH (Heidelberg, Germany). An
anti-
Antibodies to the N- and C-terminal domains of Cdsn were
elicited in rabbits by injection of synthetic peptides conjugated via a
C-terminal cysteine residue to keyhole limpet hemocyanin. The peptides
used were synthesized according to the predicted amino acid sequence
(GenBankTM accession number AF030130) as follows: peptide A,
DPCKDPTRITSPNDPC (amino acids 40-55); peptide B, SAGSFKPGTGYSQVSC
(amino acids 102-115 with an added C-terminal cysteine residue);
peptide C, GSPYHPCGSASQSPC (amino acids 409-423); and peptide D,
DGSPHPDPSAGAKPC (amino acids 472-486). Anti-peptide antibody titers
were determined by enzyme-linked immunosorbent assays (CovalAb, Lyon,
France). The antisera were affinity-purified on their corresponding
peptide coupled to agarose-activated affinity columns
(SulfoLink® kit) essentially as described by the
manufacturer (Pierce). Elution fractions obtained when the antisera
were loaded on an affinity column coupled with a peptide different from
that used for their production were used as negative controls.
Extraction of Proteins from Human Epidermis and Superficial
SC--
As previously described (23), dermo-epidermal cleavage of
breast skin (obtained from patients undergoing plastic surgery) was
performed by heat treatment, and the epidermis was sequentially extracted in a Tris-EDTA buffer containing Nonidet P-40 (TENP-40 buffer
extract), 8 M urea (TEU buffer extract), or urea and
dithiothreitol (TEUDTT buffer extract).
Superficial SC extracts were obtained from volunteers using varnish
stripping as previously reported (24). Proteins were solubilized in the
presence of 2% SDS (or 8 M urea for two-dimensional gel
electrophoresis) and 50 mM dithiothreitol, and their
concentration was measured using the Bio-Rad protein assayTM (Bio-Rad
Laboratories GmbH, Munich, Germany).
Protein Electrophoresis and Immunoblotting--
Proteins were
separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or by
two-dimensional electrophoresis in the presence of urea as previously
described (23). After electrophoresis, proteins were either stained
with Coomassie Blue or electrotransferred to reinforced nitrocellulose
membranes. The membranes were stained with either Ponceau Red or
Protogold (British BioCell International, Cardiff, UK) and probed with
antibodies as previously reported (23). The anti- Blotting with Lectins--
Proteins of the SC extracts were
separated by two-dimensional gel electrophoresis, electrotransferred to
nitrocellulose membranes, and probed with biotinylated
lectins as previously described (23).
Deglycosylation Experiments--
In SC extracts (10 µg of
protein), Nonidet P-40 and EDTA concentrations were adjusted to 1% and
20 mM, respectively. To the extracts, 2.4 units of
N-glycosidase F (EC 3.5.1.52, Oxford GlycoSystems
Ltd., Abingdon, UK) were added, and the reaction mixture was incubated
at 37 °C for 6 h. The reactions were stopped by boiling for 2 min in sample buffer. Treated and mock samples were separated by
SDS-PAGE and analyzed by immunoblotting as described above.
Deglycosylation of a TENP-40 buffer extract was used as a positive control.
Immunohistochemistry and Immunoelectron
Microscopy--
Immunohistochemistry was performed on Bouin's fixed
samples of breast skin embedded in paraffin using the
peroxidase-labeled streptavidin-biotin amplification method and
on cryosections of breast skin using indirect immunofluorescence.
G36-19, F28-27, and MOPC-21 were diluted to 2.5 µg/ml, and the
affinity-purified anti-peptide antibodies were diluted to 1/20 or 1/50.
On controls, where parallel sections were reacted in the absence of
primary antibody, no significant immunoreactivity was observed.
Immunoelectron microscopy was performed on normal skin fragments using
postembedding methods or labeling of cryosections essentially as
described previously (9, 27, 28). A mild fixation of the tissue in 3%
paraformaldehyde for 3 h was used prior to low-temperature embedding in Lowicryl K4M (Polysciences Europe GmbH, Eppelheim, Germany), whereas cryofixation in liquid propane preceded the embedding
in Lowicryl K11M (Polysciences). For ultracryosectioning, the fragments
were fixed in 2% paraformaldehyde overnight at 4 °C, soaked for 30 min in 2.3 M saccharose, and plunge-frozen in liquid
propane. Cryosections of 80 nm were obtained at Proteolysis Experiments Using a Cdsn-containing Epidermal
Extract--
Recombinant ProSCCE was produced using murine C127 cells,
purified and activated with agarose-bound trypsin as previously described (14). Epidermal extracts enriched in SCTE were obtained as
published (17). In proteolysis experiments, 50 ng of recombinant ProSCCE or SCCE, 100 ng of enriched epidermal SCTE, or an equal volume
of proteolysis buffer (10 mM sodium phosphate buffer, pH 7.2, 0.15 M NaCl) were added to the TENP-40 buffer extract
(70 µg of proteins) together with 10 times the concentrated
proteolysis buffer. The reaction mixture was incubated at 37 °C for
0-4 h. The reactions were stopped by boiling for 2 min in sample
buffer. Treated and mock samples were separated by SDS-PAGE and
analyzed by immunoblotting as described above.
Glycosylation of the 46-43- and 36-kDa Cdsn Fragments--
To
examine whether Cdsn deglycosylation is necessary for proteolysis to
occur, proteins of superficial SC extracts obtained by varnish
stripping from six different individuals were treated with
N-glycosidase F and analyzed by immunoblotting with the
anti-Cdsn MoAb G36-19 directed to amino acids 306-309. Staining of the
proteins with Protogold did not show any apparent degradation of the
proteins during incubation (data not shown). The antibody stained
several Cdsn fragments of 46-30 kDa. The glycosidase treatment induced a decrease of ~4 kDa in the apparent molecular mass of the 46-43-kDa Cdsn fragments present in all the extracts as illustrated for three
individuals in Fig. 1. The treatment also
induced a decrease in the apparent molecular mass of the 36-kDa Cdsn
fragments in some of the extracts as shown for individuals 1 and 2. However, the smaller fragments did not show any modification in their
migration rate. These results strongly suggest persistence of
N-glycosylation of the larger Cdsn fragments.
To confirm these data, proteins of the SC extracts were separated by
two-dimensional gel electrophoresis and analyzed with biotinylated
lectins and with G36-19. Concanavalin A and Pisum sativum
agglutinin strongly bound to proteins that probably correspond to
proteolysis products of desmosomal cadherins. These lectins also bound
the high but not the low molecular weight fragments of Cdsn. The other
lectins tested (wheat germ and Ricinus communis agglutinins)
did not stain any proteins (data not shown).
As a whole, these results indicate that the 46-43- and 36-kDa forms of
Cdsn are N-glycosylated, like the 52-56-kDa precursor form.
Further Characterization of Cdsn Processing--
To precisely
determine the proteolysis steps of Cdsn maturation and to examine the
fate of Cdsn fragments at the SC surface, four antibodies were
developed and affinity-purified against peptides corresponding to
different domains of the protein: serum A40-55 specifically recognizes the N terminus between the signal peptide and
the N-terminal glycine loop-related domain (amino acids 40-55), sera
B102-115 and C409-423 recognize the N- and
C-terminal glycine loop-related domains, respectively (amino acids
102-115 and 409-423, respectively), and serum D472-486
is specific to the C terminus of Cdsn (amino acids 472-486). The
specificity of the affinity-purified sera was tested against Cdsn
produced in Escherichia coli as a His-tag recombinant
protein2. All of the sera detected the protein. Moreover,
their reactivity was specifically suppressed by incubation in the
presence of the peptide used for their production (data not shown).
The antibody reactivity was then investigated by immunoblotting
analysis of sequential extracts of human breast epidermis and compared
with the reactivity of the anti-Cdsn MoAb, F28-27, directed to amino
acids 349-353 (Fig. 2A). The
epidermis was sequentially extracted in equal volumes of a
detergent-containing buffer (TENP-40 buffer extract), 8 M
urea-containing buffer (TEU buffer extract), and finally an 8 M urea- and dithiothreitol-containing buffer (TEUDTT buffer
extract). As previously described (23), F28-27 stained the 52-56-kDa
Cdsn that was mainly extracted in the presence of a detergent
(lane 1) and several Cdsn fragments of lower apparent molecular mass from 48 to 40 kDa that were partly extracted in the
presence of urea and partly in the presence of the reducing agent
(lanes 2 and 3). Serum A40-55 detected only the
52-56-kDa Cdsn. In addition to this 52-56-kDa form, the serum
B102-115 also recognized some of the fragments detected by
F28-27. Serum C409-423 stained the 52-56-kDa Cdsn and all
the fragments detected by F28-27. In addition, it stained a protein
whose molecular weight and abundance strongly suggest that it is
cytokeratin K14. The binding to this 49-kDa protein was due to
cross-reactivity as it disappeared together with the anti-Cdsn
reactivity after absorption of the purified serum on the C peptide.
Serum D472-486 detected only the 52-56-kDa Cdsn.
To characterize the molecular forms of Cdsn present at the surface of
human epidermis more precisely, superficial SC extracts from five
individuals were analyzed by immunoblotting with the purified sera
(Fig. 2B). A complex pattern of molecular species with
molecular masses extending from 46 to 30 kDa was revealed with F28-27,
as previously published (24, 26). Serum A40-55 did not
detected any protein. Serum B102-115 detected only the
higher molecular weight fragment. Serum C409-423 reacted with exactly the same fragments as F28-27. Serum D472-486 showed a very faint reaction with bands of apparent molecular mass
between 46 and 35 kDa. After a longer exposure time, MoAb F28-27 and
serum C409-423 additionally recognized identical Cdsn
forms with molecular masses down to 15 kDa (data not shown).
To determine where in the epidermis Cdsn is proteolysed,
immunohistochemical studies were performed using the affinity-purified sera on unfixed cryosections of skin as well as on sections of Bouin's
fixed skin (Fig. 3). On cryosections,
serum A40-55 showed a weak microgranular and cytoplasmic
labeling of the granular keratinocytes. This labeling was not observed
on fixed skin sections. Serum B102-115 stained not
only the granular cells with the same pattern but also the lower
SC. The labeling intensity was strong on unfixed skin cryosections but
weak on fixed skin sections. Like F28-27, sera C409-423
and D472-486 showed, on unfixed as well as on fixed skin,
an intense granular cytoplasmic staining of the granular keratinocytes
with a weaker reaction on the lower SC. In addition, and in agreement
with its reactivity against K14 on immunoblots, serum
C409-423 stained the cytoplasm of the basal
keratinocytes.
We also immunohistochemically analyzed corneocytes spontaneously
detached from human skin and collected on microscope slides. Only
one of the purified sera, namely serum C409-423, and MoAb
F28-27 labeled the desquamated corneocytes. Both antibodies showed a
granular staining of the corneocyte surface (data not shown) similar to
the previously observed labeling produced by the anti-Cdsn MoAb G36-19
on corneocytes scraped from hard palate (9).
The histological observations were confirmed and extended using
immunoelectron microscopy (Fig. 4).
Various pre- and postembedding indirect immunogold labeling methods
were performed to make sure that the observed reactivities indeed
reflected the presence or absence of Cdsn. Keratinosomes were the only
structures decorated by purified serum A40-55 (Fig.
4A). The labeling was specific and reproducible but very
faint even without chemical fixation or with use of small gold
particles and silver enhancement on non-embedded ultracryosections. In
addition to staining these secretion vesicles, purified serum
B102-115 decorated the extracellular part of both
the desmosomes in the granular layer and of the corneodesmosomes in the
lowermost part of the SC (Fig. 4, B and C).
Purified serum C409-423 labeled intermediate filaments of
the basal keratinocytes, keratinosomes in the upper spinous and
granular keratinocytes, desmosomes of the upper granular keratinocytes,
and corneodesmosomes. As previously observed with G36-19 (9, 12), the
immunolabeling persisted on corneodesmosomes up to the SC surface (Fig.
4D). The purified serum D472-486 labeled
keratinosomes and faintly decorated some desmosomes (Fig. 4,
E and F).
Cdsn Is a Preferred Substrate of Two Proteases Implicated in
Desquamation--
Two proteases present in the SC, namely SCTE and
SCCE, have been suggested to be involved in desquamation. To test
whether Cdsn actually is a substrate of these enzymes, TENP-40 buffer extracts were treated for 2 h with SCTE or with recombinant SCCE. Proteins of the extracts were then analyzed by immunoblotting with
various antibodies (Figs. 5 and
6). Staining with Protogold did not
show any apparent degradation of the major epidermal proteins during
incubation with the enzymes (data not shown).
SCTE totally degraded the 52-56-kDa Cdsn and generated Cdsn fragments
of 48 and 35 kDa that were stained by G36-19 and F28-27. Incubation in
the absence of the enzyme was without effect on the protein (Fig.
5A). Immunoblotting analysis of the same aliquots with
PG5.1, a MoAb directed to plakoglobin, demonstrated this desmosomal
plaque component to be proteolysed by SCTE and revealed the generation
of 70- and 65-kDa fragments (Fig. 5B). Immunoblotting analysis of the same aliquots with DG3.10, a MoAb directed against Dsg1
and 2, and with a serum directed against involucrin, another epidermal
protein, indicated that neither Dsgs nor involucrin are SCTE substrates
(Fig. 5B).
Incubation of the extract with SCCE induced complete degradation
of the 52-56-kDa Cdsn and generated several Cdsn fragments: a fragment
of 48 kDa that was stained by G36-19 and F28-27, a doublet of ~30 kDa
that was recognized only by G36-19 and low molecular weight fragments
that were recognized only by F28-27. Incubation with recombinant
inactive Pro-SCCE or in the absence of enzymes was without effect on
Cdsn (Fig. 6A). Immunoblotting analysis of the same aliquots
with PG5.1 demonstrated that SCCE treatment induced plakoglobin
proteolysis and generated a stable fragment of 70 kDa. Incubation of
the TENP-40 buffer extracts without enzyme did not induce plakoglobin
proteolysis. Immunoblotting analysis of the same aliquots with DG3.10
and with a serum directed against involucrin, indicated that neither
Dsg nor involucrin are substrates for SCCE (data not shown, and
Fig. 6B).
In this study, we characterized the various proteolysis
steps of human Cdsn during the late stages of terminal epidermis
differentiation. Cdsn is a 529-amino acid-long glycosylated component
of corneodesmosomes considered to have adhesive properties and to play
a major role in corneocyte cohesion. Its proteolysis seems to be a
major prerequisite for desquamation (26).
Our data indicate that the larger fragments of human Cdsn produced by
proteolysis in the SC are still glycosylated and contain N-linked oligosaccharides that comprise more than 5% of
their mass. Indeed, treatment of the fragments with
N-glycosidase F (a glycosidase specific for
N-linked sugars) induced a 2-4-kDa decrease in the apparent
molecular mass of the larger fragments (46-43 and 36 kDa).
Because the fragments reacted with biotinylated P. sativum
agglutinin and concanavalin A, they may contain
Although immunoelectron microscopy experiments performed with MoAbs
clearly indicated that Cdsn is located in corneodesmosomes of all cell
layers in the SC, the protein is usually not detected in the upper SC
when immunohistochemical methods are used (9, 12, 22). As previously
discussed (22), this is the consequence of both the poor accessibility
of the antigen in corneodesmosomes and the lower sensitivity of the
methods. This also explains why the protein was only detected in the
upper SC by anti-peptide serum C409-423 using
immunoelectron microscopy.
With MoAbs directed against the central domain of the protein, we
previously showed that Cdsn is progressively cleaved during SC
maturation. At the skin surface, this generates fragments of 15 kDa
that, as we previously reported, do not contain at least the first 200 amino acids at the N terminus of the protein. Our present results,
using anti-peptide sera, indicate that in fact, Cdsn processing begins
in the stratum spinosum and granulosum. The first proteolysis step(s)
give rise to a 48-46-kDa fragment lacking both extremities of the
protein because it is not recognized by sera directed against amino
acids 40-55 (A40-55 serum) or 472-486
(D472-486 serum), respectively, but is still endowed with
the glycine loop-related domains (Fig.
7). This step seems to occur either in
keratinosomes that were the only structures consistently stained by
serum A40-55 or very soon after Cdsn secretion. The
48-46-kDa fragment is probably the Cdsn form bound to desmosomes
because its extraction necessitates the presence of urea. Fragments of
43-40 and 36-30 kDa are not recognized by serum B102-115
directed against amino acids 102-115, indicating that the N-terminal
glycine loop-related domain is cleaved in following proteolysis steps.
Because the serum B102-115 does not stain corneodesmosomes
of the upper SC (stratum disjunctum), these steps apparently occur at
the transition between the lower SC (stratum compactum) and the stratum
disjunctum when corneocyte cohesion abruptly decreases. Cleavage of the
N-glycosylation site would explain why the smallest
fragments are not glycosylated. Cleavage of both the N-terminal glycine
loop-related domain and the carbohydrate moieties should strongly
decrease the adhesion properties of Cdsn. Finally, when most of the
Cdsn is degraded, only a central part of roughly 15 kDa, probably
devoid of adhesive properties, remains at the surface of non-cohesive
corneocytes. In agreement, detached corneocytes were not stained by
sera A40-55, B102-115, or
D472-486. According to this schema, the site(s) of Cdsn
attachment to corneocytes should be located in the central part of the
molecule.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-catenin MoAb from Transduction Laboratories (Lexington,
KY) was also used. The ascites fluid of MOPC-21 (Sigma) was used
as a negative control. An anti-involucrin antiserum was purchased from
Biomedical Technologies Inc. (Stoughton, CA).
-catenin MoAb,
G36-19, F28-27, and MOPC-21 were diluted to 0.1 µg/ml, and DG3.10 and
PG5.1 were diluted to 0.5 µg/ml. Affinity-purified anti-peptide
antibodies used were diluted to 1/250 or 1/500. Immunoreactivities were
revealed with the ECLTM Western blotting kit as described by the
manufacturer, Amersham Pharmacia Biotech.
110 °C. In all
cases, the ultrathin sections were harvested on Formvar-coated nickel
grids and subjected to immunostainings. The primary antibodies were
revealed using either a 5-nm immunogold conjugate (goat anti-rabbit or
anti-mouse, Amersham Pharmacia Biotech), or AuroProbe One, a 1-nm
colloidal gold conjugate (Amersham Pharmacia Biotech) followed by
silver enhancement (IntenSE M kit, Amersham Pharmacia Biotech) as
published (9, 22, 27) and as recommended by the manufacturer, respectively. Negative controls included sections incubated with PBS or
with non-immune rabbit serum instead of the primary antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Treatment of Cdsn fragments with
N-glycosidase F. Superficial SC obtained by
varnish stripping from three different individuals (1-3,
respectively) was solubilized in the presence of SDS and
dithiothreitol. Equal amounts of extracted proteins were either not
incubated (NI) or incubated in the absence (NT)
or presence of N-glycosidase F (PNGase). After
stopping the reactions, proteins were separated by SDS-PAGE and
immunoblotted with G36-19. The observed shifts in the apparent
molecular weight of Cdsn fragments are shown by curved
arrows. Note the interindividual variations in the pattern of Cdsn
fragments. The position of molecular mass standards (kDa) is indicated
on the left.
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Fig. 2.
Immunoblotting analysis of sequential
extracts of human epidermis and of Cdsn fragments extracted from the
outermost SC with affinity-purified anti-peptide sera directed to four
different domains of Cdsn. A, human epidermis was
serially extracted with equal volumes of buffer containing Nonidet-P40
(1), urea (2), and urea and dithiothreitol
(3). Proteins from an equal volume of each fraction were
then separated by SDS-PAGE and immunoblotted with the anti-Cdsn MoAb
F28-27 and with the affinity-purified sera A40-55,
B102-115, C409-423, and D472-486
as indicated on the top of the blots. B,
superficial SC obtained by varnish stripping from five different
individuals (1-5, respectively) was solubilized in the
presence of SDS and dithiothreitol. Equal amounts of extracted proteins
were analyzed by immunoblotting with F28-27 and with the
affinity-purified sera as indicated on the top of the blots.
The position of molecular mass standards (kDa) is indicated on the
left.
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Fig. 3.
Immunohistochemical staining of normal
human skin with affinity-purified anti-peptide sera directed to four
different domains of Cdsn. Unfixed cryosections (A and
B) and sections of Bouin's fixed samples (C and
D) of human breast skin were analyzed with the
affinity-purified sera A40-55, B102-115,
C409-423, and D472-486 by indirect
immunofluorescence and immunoperoxidase, respectively. Dashed
lines denote the dermo-epidermal junction. The various layers of
the epidermis are indicated by brackets (SB,
basal layer; SS, spinous layer; SG, granular
layer; SC, cornified layer). Bar, 50 µm.
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Fig. 4.
Immunoelectron microscopy analysis of human
skin with affinity-purified anti-peptide sera directed to four
different domains of Cdsn. Normal human skin was analyzed using
pre- and postembedding indirect immunogold labelings with the
anti-peptide antibodies. Silver enhancement of 1-nm immunogold labeling
is shown in all micrographs except in D where indirect
labeling was performed with 5-nm gold particles. A and
B correspond to ultracryosections of paraformaldehyde-fixed,
non-embedded tissue; C and E are Lowicryl
K4M-embedded, paraformaldehyde-fixed sections; D and
F are Lowicryl K11M embeddings of plunge-frozen skin.
A, serum A40-55 gives only a weak but precisely
located labeling of keratinosomes in the upper spinous layer and in the
lower granular layer. B and C, serum B102-115
labels keratinosomes in the upper spinous and granular layers and
desmosomes at the limit between the granular layer and the SC; the
labeling of corneodesmosomes does not persist beyond the lowermost SC.
D, in the SC, serum C409-423 labels
corneodesmosomes. The labeling persists throughout the SC up to the
epidermal surface. E and F, serum
D472-486 labels keratinosomes in the upper spinous and
granular layers and, infrequently, some desmosomes of the granular
layer. Arrowheads show desmosomes or corneodesmosomes;
arrows indicate keratinosomes. All bars, 200 nm.
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Fig. 5.
Effect of SCTE on epidermal proteins.
Proteins extracted from human epidermis in the presence of a detergent
(TENP-40 buffer extract) were incubated with (+) or without ( ) SCTE
for increasing periods of time as indicated on top of the
plates. After addition of Laemmli's sample buffer to stop the
reaction, proteins were immunoblotted with G36-19 and F28-27
(A) and with PG5.1 directed to plakoglobin, DG3.10 directed
to Dsg1 and 2, and a serum directed against involucrin (B).
Arrows indicate the 52-56-kDa Cdsn. Open
arrowheads show the immunodetected Cdsn fragments. The position of
molecular mass standards (kDa) is indicated on the
left.
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Fig. 6.
Effect of SCCE on epidermal proteins.
Proteins extracted from human epidermis in the presence of a detergent
(TENP-40 buffer extract) were incubated with the active enzyme (+ SCCE) or with its inactive precursor (+ ProSCCE) for
increasing periods of time as indicated on top of the
plates. After addition of Laemmli's sample buffer to stop the
reaction, proteins were immunoblotted with G36-19 and F28-27
(A) and with PG5.1 directed to plakoglobin and a serum
directed against involucrin (B). Arrows indicate
the 52-56-kDa Cdsn. Open arrowheads show the immunodetected
Cdsn fragments. The position of molecular mass standards (kDa) is
indicated on the left.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-mannose and/or
-D-glucose, like the
52-56-kDa precursor form of Cdsn. Moreover, they seem to contain
little or no galactose or
N-acetyl-D-galactosamine because they did not
react with lectins specific for these carbohydrates. We nevertheless
cannot exclude that the sugars linked to the 52-56-kDa Cdsn are
modified in the SC. The results indicate that the oligosaccharide residues do not protect Cdsn against proteolysis in the lower SC unlike
what was previously proposed (23). The sugar moiety is thought rather
to participate in stabilization or in the adhesion properties of
the protein.
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Fig. 7.
Schematic representation of Cdsn proteolysis
during the terminal differentiation of epidermis. A schema of Cdsn
proteolysis from the 52-56-kDa protein to a fragment of 15 kDa is
proposed as deduced from immunoultrastructural and biochemical data
previously obtained or presented in this paper. For details see
"Discussion." The apparent molecular mass of the various Cdsn forms
are indicated, the position of the epitopes recognized by the anti-Cdsn
anti-peptide sera (A40-55, B102-115,
C409-423, and D472-486) and MoAbs (G36-19 and
F28-27) are shown by black boxes. The glycine loop-related
domains and the N-glycosylation site of Cdsn are represented
by wavy lines and a black dot,
respectively.
After its cleavage, the N terminus of Cdsn seems to be completely degraded because, in immunoblots, the sera A40-55 and B102-115 do not detect polypeptides other than those also recognized by F28-27 MoAb. However, because serum D472-486 shows faint reactivity after immunoblotting of extracts obtained in reducing conditions, the C terminus of Cdsn could be cross-linked to the external side of the cornified cell envelopes by disulfide bounds after its cleavage. We already demonstrated that Cdsn itself is bound by covalent linkages to these corneocyte structures (9, 22, 23). However, if this were the case, the corneocytes would have been stained by the serum. Alternatively, this immunoblotting reactivity could be due to a slight cross-reactivity against an unknown molecule when it is denatured.
Until now, the proteases involved in Cdsn processing remained unknown. Here, we show that in vitro Cdsn is a preferred substrate of both SCCE and SCTE, two proteases proposed to be involved in corneodesmosome degradation and in desquamation. According to the size and immunoreactivities of the fragments generated, a SCCE-cleavage site is necessarily located between the epitopes recognized by G36-19 (amino acid position 306-309) and F28-27 (amino acid position 349-353). In agreement, the region contains several aromatic amino acids, the amino acids preferentially located (in P1 position) in SCCE cleavage sites (22). Moreover, both enzymes produce a Cdsn immunoreactive fragment of apparent molecular mass similar to that of the Cdsn form associated with desmosomes, i.e. 48-46-kDa. This suggests that SCTE and/or SCCE could be the enzyme(s) involved in the first step of Cdsn proteolysis. Because almost all fragments disappeared after a 2-4-h incubation with SCTE, this enzyme may also be involved in further processing of Cdsn.
SCCE and SCTE were proposed to be actors of a cascade of activated proteases and inactive pro-enzymes that could regulate the rate of desquamation (18). Our results suggest that SCCE and SCTE may cooperate to completely degrade Cdsn and possibly other corneodesmosomal proteins. However, whether or not the two enzymes act on Dsg1 and Dsc1 is not clear and has to be further studied. This would suppress corneocyte cohesion and induce desquamation.
CDSN (or S gene) is a highly polymorphic gene, and many of the
coding single nucleotide polymorphisms detected in CDSN induce amino
acid substitutions (29-32). Do sequence variations in Cdsn influence
the protein function? Alteration of proteolysis sites constitutes one
type of the possible dysfunction. For example, the reported C/T
transition at nucleotide position 619 leads to a
Ser202/Phe202 exchange that could alter
a potential chymotrypsin cleavage site and therefore the susceptibility
of Cdsn to SCCE. Such modifications could be involved in an
individual's susceptibility to xerosis. Similarly, Cdsn polymorphism
may also be the cause of the hyperkeratosis observed in psoriasis.
Psoriasis is a human multifactorial skin disease characterized by
T-cell infiltration, keratinocyte hyperproliferation, and epidermal
differentiation abnormalities resulting in impaired desquamation. The
involvement of Cdsn in the pathogenesis of the disease has been
suggested based on its putative function and on genetic studies
including association of various forms of psoriasis with a particular
allele of CDSN (31-34). Therefore, it appears of particular importance
to determine the primary substrate specificity of SCCE and SCTE. Such
studies, currently being performed in our laboratory, will certainly
highlight the role of Cdsn in the physiology of desquamation and
allow its involvement in the pathophysiology of psoriasis to be
precisely determined.
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ACKNOWLEDGEMENTS |
---|
We thank Profs. M. Costagliola and J.-P.
Chavoin (Service de Chirurgie Plastique, CHU Rangueil, Toulouse,
France) for providing us with normal human skin. The technical
assistance of C. Pons is gratefully acknowledged. The electron
microscopy samples were observed at the Centre de Microscopie
Electronique Appliquée
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FOOTNOTES |
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* This study was supported in part by grants from the Université Paul Sabatier-Toulouse III, from L'Oréal (Paris, France) and from INSERM.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.
§ Supported by a postdoctoral fellowship from the Société de Secours des Amis des Sciences and from the Singer Polignac Foundation.
** Recipient of a grant from the French Ministry of Research and Technology.
§§ To whom correspondence should be addressed: Laboratoire de Biologie Cellulaire et Cytologie, CHU Purpan, Place du Dr Baylac, 31059 Toulouse Cedex, France. Tel.: 33-5-61-77-23-95; Fax: 33-5-61-77-76-20; E-mail: serre.g@chu-toulouse.fr.
Published, JBC Papers in Press, February 16, 2001, DOI 10.1074/jbc.M100201200
2 N. Jonca, M. Guerrin, C. Caubet, M. Simon, and G. Serre, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: SC, stratum corneum; SCCE, stratum corneum chymotryptic enzyme; SCTE, stratum corneum tryptic enzyme; Dsc, desmocollin; Dsg, desmoglein; Cdsn, corneodesmosin; MoAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis.
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