From the Department of Dermatology and
** Biochemistry, Oita Medical University, Idaigaoka, Hasama, Oita
879-5593, Japan, the
Department of Biochemistry, Nagoya City
University Medical School, 1, Mizuho-cho, Mizuho-ku, Nagoya
467-8601, Japan, and the ¶ Institute of Fundamental Sciences,
Massey University, Palmerston North, New Zealand
Received for publication, December 18, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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A 450-kDa human epidermal autoantigen was
originally identified as a protein that reacted with the serum from an
individual with a subepidermal blistering disease. Molecular cloning of
this protein has now shown that it contains 5065 amino acids and has a
molecular mass of 552 kDa. As reported previously this protein, which
we call epiplakin, belongs to the plakin family, but it has some very
unusual features. Epiplakin has 13 domains that are homologous to the B
domain in the COOH-terminal region of desmoplakin. The last five of
these B domains, together with their associated linker regions, are
particularly strongly conserved. However, epiplakin lacks a coiled-coil
rod domain and an amino-terminal domain, both of which are found in all
other known members of the plakin family. Furthermore, no dimerization
motif was found in the sequence. Thus, it is likely that epiplakin
exists in vivo as a single-chain structure. Epitope mapping
experiments showed that the original patient's serum recognized a
sequence unique to epiplakin, which was not found in plectin.
Immunofluorescence staining revealed the presence of epiplakin in whole
sheets of epidermis and esophagus, in glandular cells of eccrine sweat
and parotid glands and in mucous epithelial cells in the stomach and colon.
Clarification of the basic structure of desmoplakin has been
followed by the identification of many related proteins, such as
BPAG1,1 plectin, envoplakin,
and periplakin (1-7). These proteins form a family known as the
"plakin family" (8). Almost all members of this family have a
common structure, with predicted globular amino-terminal and
COOH-terminal domains that are separated by a central rod domain. Some
homologous domain structures have been identified in both globular
domains of many plakins, while the central domain is rich in heptad
repeats and is believed to form a parallel Several years ago we described an individual with a subepidermal
blistering disease that resembled bullous pemphigoid both clinically
and pathologically (23). Immunoblot analysis revealed that the
patient's serum did not react with the 230-kDa (BPAG1) and 180-kDa
bullous pemphigoid antigens, whereas it did recognize a 450-kDa
epidermal polypeptide. This polypeptide was expressed in human
keratinocytes and in some transformed cell lines that included HeLa,
KB, and A431 cells (24). In a preliminary study, to determine the
molecular structure of this antigen, we screened a human keratinocyte
cDNA library with the patient's serum. We isolated two kinds of
cDNA: one encoding a protein that was strongly homologous to rat
plectin and another encoding a protein, with partial homology to
plectin, which appeared to be a novel and previously unidentified
protein (25).
We report here the cloning of the cDNA and determination of the
entire structure of the novel protein, which we compare with other
members of the plakin family. We also demonstrate the tissue localization of this protein, as determined by Northern blotting and
immunostaining. In our previous study, it was unclear whether the
epitope that was recognized by our patient's serum was a sequence that
is present within plectin itself. In this study, therefore, we also
identified the major epitope of the protein.
Isolation of cDNA Clones and DNA Sequencing--
A cDNA
library prepared from HeLa cells (CLONTECH) was
screened with the inserts of clones pE450-C and pE450-D, which had been
isolated by extension cloning, using the same library, after the
immunoscreening described in a previous report (25).
For isolation of the 3' end repeats, we constructed a cDNA library
from the poly(A)+ RNA of HeLa cells. cDNA was
synthesized with a cDNA synthesis kit (Life Technologies, Inc.). We
modified the procedure provided with the kit by adding trehalose to the
reaction for first-strand synthesis (26) and, as specific
primers for first-strand synthesis, we used 5'-CCAGACACAACAAGTATGCC-3'
for clone Ep115 and 5'-TAGCGCTTGACCGAGTCCATC-3' for clone Ep 4. The
cDNAs were ligated to an EcoRI adapter and then inserted
into the EcoRI site of pUC 18 (Amersham Pharmacia Biotech)
for construction of plasmid libraries. Each library was screened with
the specific probe. To confirm the size of 3' end repetitive region and
cover a small gap of the message, genomic polymerase chain reaction
(PCR) was performed using primers 5'-TCGAGAAGCAGGAAACCA-3' and
5'-CCATATGACACATAGACGAC-3'.
To obtain some 5'-upstream cDNA sequences, we performed 5'-RACE
(rapid amplification of cDNA ends) using the RACE System (Life Technologies) and total RNA from HeLa cells or the Marathon-Ready cDNAs System with adaptor-ligated double-stranded cDNA prepared from poly(A)+ RNA from HeLa cells
(CLONTECH). The products of RACE were cloned into
the pGEM-T Easy vector (Promega).
Each cDNA clone and genomic clone of interest was sequenced by a
double-strand strategy with an automatic DNA sequencer (Applied Biosystems). Internal primers were constructed for analysis of internal sequences.
Analysis of the Amino Acid Sequence--
We searched various
protein data bases, including Swiss-Prot and PIR, using the BLAST
routine available from the National Center for Biotechnology
Information (Bethesda, MD). Furthermore, we also made comparisons using
the COMPARE and DOTPLOT programs available from the University of
Wisconsin Genetics Computer Group (Madison, WI). We also used the MATCH
routine to identify regions of sequence homology among proteins.
Determinations of plausible secondary structure were made using several
predictive techniques and results are presented here only in a
preliminary form. Short regions of heptad substructure were delineated
by hand rather than by direct computer analysis since the former method
facilitated location of discontinuities in heptad phasing. Potential
interchain ionic interactions between charged residues at positions
2d'-1g, 1g'-2e, 2a'-1g, 1g'-2a, 1e'-1d, and 1d'-1e were considered as a
function of relative chain stagger and chain polarity (27, 28). The
notation 2d'-1g means that the residue at position d of the second
heptad of chain n' interacts with that in position g of the first
heptad of chain n.
Synthesis of Overlapping Peptides--
Overlapping
peptides were synthesized on derivatized cellulose membranes with Fmoc
(9-fluorenylmethoxycarbonyl) amino acids according to the protocol from
the manufacturer of the system (Auto spot robot ASP222; ABIMED
Analysen-Technik GmbH, Langenfeld, Germany) (29, 30). The ASP222
software program was used to generate the amino acid sequences of
decapeptides and the spotting schedule for each cycle of addition of an
amino acid. Peptides spanning amino acid residues 2807-3337 of
epiplakin were synthesized on cellulose membranes as a series of
decapeptides with an overlap of eight amino acids. This region includes
one of five highly conserved COOH-terminal repeats. The first cDNA
clone that we obtained by immunoscreening encoded a part of this
domain. To refine the definition of the epitope within the sequence
LVPAKDQPGRQEKMSIYQAMWKGVLRPGT, we synthesized a series of decapeptides
with an overlap of nine amino acids were synthesized on cellulose
membranes. Enzyme immunoassays of the peptides on the cellulose
membranes were performed with our patient's serum, alkaline
phosphatase-conjugated antibodies against human IgG, and
5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium. All
reagents were purchased from Bio-Rad.
Chemical Synthesis of Peptides--
The target peptides, namely
peptide A (MSIYQAMWKGVLC), peptide B (TKGFFDPNTHENC), and peptide C
(VKRYLEGTSCIAGVLVP), were synthesized by a solid-phase method (31).
Peptide chains were elongated using an automated peptide synthesizer
(model 9050; Millipore/Biosearch, Marlborough, MA) according to the
standard operating programs. Peptides were purified by high-performance liquid chromatography on a reverse-phase column (Delta PakC18; Waters)
with a Multisolvent Delivery System (600E; Waters). Peptides were
eluted with a linear gradient from water to acetonitrile in 0.1%
trifluoroacetic acid. The molecular mass and amino acid sequence of
each purified peptide were confirmed by determinations of the
matrix-assisted laser desorption ionization time of flight mass
spectrometer (Kompact MALDI II; Kratos-Shimadzu, Tokyo,
Japan) and a protein sequencer (491CLC; Applied Biosystems).
Each synthetic peptide (2 mg) was conjugated to bovine serum albumin
(BSA; 10 mg; Sigma) using
N-(4-maleimidobutyryloxy)succinimide (2 mg; Dojin, Kumamoto,
Japan) according to the manufacturer's protocol.
Enzyme-linked Immunosorbent Assay (ELISA)--
The ELISA was
performed with microplates and BSA-conjugated peptides A, B, and C. Wells of microtiter plates (Nunc A/S, Kamstrupvej, Roskilde, Denmark)
were coated with 50 ng of synthetic peptide that had been conjugated to
BSA (0.5 µg/ml in PBS) with incubation at 4 °C overnight. After
incubation with PBS that contained 0.05% Tween 20 (PBS-T) for 1 h
at room temperature to block nonspecific binding, samples of serum
(diluted 1:200 or serial 2-fold dilutions in PBS-T that contained 1%
BSA) were added to the wells. After a 1-h incubation with shaking at
room temperature, the wells were washed three times with PBS-T, and 100 µl of a solution of alkaline phosphatase-conjugated antibodies
against human IgG antibody (Bio-Rad) that had been diluted to 1:1000 in
PBS-T were added. After incubation for 1 h with shaking at room
temperature, the wells were washed three times with PBS-T and twice
with PBS. After a 1-h incubation with the substrate solution at room
temperature, absorbance (405 nm) was determined with a SOFTMAX system
(Molecular Devices Co., Sunnyvale, CA). The absorbance obtained with
each synthetic peptide was corrected by subtraction of the value due to
reaction of the antibodies with BSA by itself.
Immunoblotting--
Epidermal extracts were obtained as
described previously (23). After addition of dithiothreitol at a final
concentration of 0.01 M, the solubilized proteins were
separated by electrophoresis on an SDS-polyacrylamide gel (5%
polyacrylamide) and transferred to an ImmobilonTM polyvinylidene
difluoride membrane (Millipore), which was probed with the original
patient's serum or polyclonal antibodies against the synthetic peptide
(see below) before or after incubation with the synthetic peptide.
Production of a Glutathione S-Transferase (GST) Fusion
Protein--
The cDNA that encoded a specific region of epiplakin
was amplified by PCR with specific primers and subcloned in pGEX 4T-1 as an EcoRI fragment. The expression of the GST recombinant
protein in Escherichia coli JM 109 was induced by 1 mM isopropyl-1-thio- Production of Antibodies against the Fusion Protein and a
Synthetic Peptides--
Antibodies against a recombinant protein were
raised in rats. The purified GST recombinant protein was mixed with an
equal volume of Freund's complete or incomplete adjuvant. Initial
subcutaneous injections contained 100 µg of recombinant protein (plus
complete Freund's adjuvant) per rat. Two booster injections, each
consisting of the same amount of protein (plus Freund's incomplete
adjuvant) were given 1 and 2 weeks after the initial injection. The
rats were bled 3 weeks after the second booster injection. The inguinal lymph nodes were dissected out for production of monoclonal antibodies.
Purified peptide A (MSIYQAMWKGVLC) was conjugated with keyhole limpet
hemocyanin using N-(4-maleimidobutyryloxy)succinimide and
rabbits were immunized with the conjugate plus Freund's complete adjuvant. Antibodies (peptide A-specific antibodies) were purified by
immunoaffinity gel chromatography on a peptide-immobilized column with
absorption in 1 M NaCl (pH 7.5) and subsequent elution with 0.17 M Gly-HCl (pH 2.3) at room temperature.
Northern Dot Blot Analysis--
An RNA Master Blot (PT3004-1)
was purchased from CLONTECH and Northern dot
blotting was performed according to the protocol from
CLONTECH. The product of PCR obtained with
5'-GGCGCCAGGACCTGCTGA-3' and 5'-CACCTTGCTGAGCCGCTCCT-3' as primers and
Ep12 as template was labeled with 32P and used as the
specific probe for epiplakin mRNA. The product of PCR obtained with
5'-CCACACCACGGTGGACGA-3' and 5'-ACTCAGCAGCTGCCTCTG-3' as primers and pE
450-B as template was used as the specific probe for plectin mRNA
(25). Ubiquitin cDNA was used as a control probe.
Immunofluorescence Staining--
Human tissues were obtained at
biopsy or autopsy, embedded into OCT compound (Sakura Fine Technical
Co., Ltd., Tokyo, Japan) and frozen in liquid nitrogen. Frozen sections
(5 µm) were air-dried and incubated in PBS for 5 min and then for
1 h at room temperature in PBS that contained 100-fold diluted
specific antiserum or antiserum against GST. The sections were washed
three times in PBS and then incubated with fluorescein
isothiocyanate-conjugated (FITC-conjugated) goat antibodies against rat
(for peptide-specific antiserum) or against goat (for GST-specific
antiserum) IgG for 1 h at room temperature, with three subsequent
washes in PBS. Sections were mounted in immersion oil (Olympus) and
examined under a fluorescence microscope (UFX-DX; Nikon).
Isolation of Overlapping cDNA Clones That Encoded a Human
Epidermal Autoantigen--
We first screened the HeLa cell
For downstream cloning, we screened the HeLa cell cDNA library
using the 3' end of clone pE 450-C (Fig. 1). We isolated several almost
identical clones and a clone that included TGA (clone Ep12). From these
results, we postulated that the 3' side of the message had highly
homologous repeats of 1602 bp. Clones pE 450-C and pE 450-D both
overlapped with parts of the repetitive region. However, the
corresponding position could not be identified in this region because
of the presence of five strongly homologous repeats. We constructed a
cDNA library using a specific primer from the 3'- terminal region
of clone Ep12. The longest clone, Ep115, was 6.3 kilobases long and it
was isolated together with several shorter clones, Ep150 and Ep104. We
constructed another cDNA library using a specific primer from the
repetitive region of the 1.6-kilobase pair repeat and isolated a single
clone, designated Ep 4.
We performed genomic PCR to amplify the repetitive region and obtained
a clone that was slightly more than 9 kilobase pairs in length and
contained no introns. The gap of 760 bases in the message that had
resulted in a gap in the cDNA was, thus, covered by genomic sequence.
The Human Epidermal Autoantigen Is a Member of the Plakin
Family but Has Unusual Features--
It appeared that an open reading
frame began at the second ATG triplet, which was preceded by an
in-frame TGA triplet, because the first ATG triplet was not preceded by
a consensus sequence that would favor the initiation of translation
(32). The total length of the ORF was 15,195 bp, and the predicted
amino acid sequence (5065 amino acids) is shown in Fig.
2. The theoretical relative molecular
mass was calculated to be 552,467 Da and was in reasonable agreement
with the molecular mass of 450 kDa determined previously by
SDS-polyacrylamide gel electrophoresis. The key feature of the protein
was the presence of 13 B domains of the type first identified in the
COOH-terminal domain of desmoplakin (1). This feature alone clearly
identified epiplakin as a member of the plakin family, which includes
desmoplakin, BPAG1, and plectin. However, several unusual features were
also found, as follows. The B domains themselves could be divided into
two groups: one group with ~70% identity to the B domain in
desmoplakin (domains 3, 6, and 8-13; indicated by B in Fig.
1), and another group with ~45-50% identity to the B domain in
desmoplakin (domains 1, 2, 4, 5, and 7; indicated by B* in
Fig. 1). However, all 13 repeats were more similar to the B
domain rather than to the A or C domain that were also first recognized
in the COOH-terminal domain of desmoplakin (Table
I and Ref. 1). We found, moreover, that the linker regions (358 residues in length) that preceded domains 9-13
and followed these five B domains were almost perfectly identical (Fig.
3A). In addition, we
identified three homologous segments within each of five strongly
conserved linker regions (Fig. 3B). For example, in the
linker region (residues 2391-2748), two of the segments (residues
2391-2472 and 2473-2554) were 30% identical, with particularly
high identity over the last 63 residues. After a
glycine-proline-rich segment, a third homologous region (residues 2613-2675), which was shorter than the other two, similarly showed homology over the same region. We predicted that these quasi-repeats should contain two or three heptad-containing segments that are separated from one another by The Antigenic Epitope in Epiplakin--
To map the linear epitope
in epiplakin accurately, we tested the original patient's serum, which
had been used for cDNA screening, for reactivity against 264 consecutive cellulose-bound linear peptides of 10 amino acids in length
with an 8-amino acid overlap in the region between amino acids residues
2807 and 3337 of epiplakin (Fig. 2). This region covers one of five
strongly conserved COOH-terminal repeats and includes the sequence that
was recognized by the patient's serum (25). The patient's serum
reacted strongly with the amino acids sequence MSIYQAMWKGVL and weakly
with amino acids TKGFFDPNTH. The former sequence is unique to
epiplakin, but the latter is also found in plectin and is highly
homologous, with the exception of glutamic acid at the last position
instead of histidine (TKGFFDPNTE), to a peptide in desmoplakin. To
confirm the reactivity of the serum, we performed an ELISA and found
that the patient's serum reacted only with the former peptide (Fig.
4A). To refine the identification of the epitope, we exposed a series of decapeptides with
nine overlapping amino acids derived from the sequence
LVPAKDQPGRQEKMSIYQAMWKGVLRPGT (residues 2739-2767) to the patient's
serum in another dot blot test. We detected four strongly positive
spots, corresponding to sequences from KMSIYQAMWK to IYQAMWKGVL (Fig.
4B) and, thus, the major epitope recognized by the
patient's serum was determined to be IYQAMWK (Fig. 4C).
Western blotting showed that the patient's serum, after absorption
with the synthetic peptide MSIYQAMWKGVLC, no longer reacted with the
450-kDa epidermal autoantigen (Fig. 5,
lanes 1 and 2). These data indicated
that antibodies in the patient's serum recognized mainly the unique
epitope in epiplakin.
Distribution of Epiplakin as Revealed by Northern Dot Blots of
Transcripts--
We used a specific cDNA probe that encoded linker
regions for Northern hybridization on the RNA Master Blot. The results
are shown in Fig. 6A. The dot
blots indicated that epiplakin was widely distributed in a variety of
tissues as was plectin (Fig. 6B), but the dominant tissues
were different. The signal due to plectin transcripts was strong in the
case of muscle, heart, placenta, and spinal cord. By contrast, the
epiplakin probe reacted strongly with RNA from liver, small intestine,
colon, salivary glands, stomach, and appendix and somewhat less
strongly with RNA derived from the placenta, lung, brain, and bone
marrow.
Localization of Epiplakin in the Entire Stratified Epithelium and
Also in Simple Epithelium--
Polyclonal antibodies against the
synthetic peptide MSIYQAMWKGVLC reacted with the 450-kDa epidermal
antigen before but not after absorption with the synthetic peptide
(Fig. 5, lanes 3 and 4). Both
polyclonal and monoclonal antibodies against the recombinant protein
composed of epiplakin (residues 2443-2682) and GST also reacted with
the 450-kDa epidermal antigen (Fig. 5, lanes 5 and 7). This sequence is within one of five conserved linker
regions that is not homologous to human plectin (Fig. 2).
We performed indirect immunofluorescence staining of human skin using
the monoclonal antibodies against the recombinant protein of epiplakin.
The antibodies reacted with the entire epidermis and immunostaining was
especially prominent over basal keratinocytes (Fig.
7A). When we used polyclonal
antibodies against the synthetic peptide, which is the same epitope
recognized by the patient's serum, we obtained the similar patterns
(Fig. 7C). Using the polyclonal antiserum against the
recombinant protein of epiplakin, the epithelium of eccrine sweat
glands and hair follicles were also positive for immunostaining (Fig.
7, E and G). In the esophagus, the same antibodies reacted with all the sheets of the stratified epithelium, including the basal layer (Fig. 7I). Immunofluorescence was
apparent in acinous cells in the parotid gland and in mucous epithelial cells of the stomach and colon (Fig. 7, K, M, and
O).
In this report, we have described the cloning and sequencing of
overlapping cDNA clones and a genomic clone for a small gap region
that together correspond to the entire transcript for a novel member of
the plakin family. Epiplakin appears to be a 450-kDa human epidermal
autoantigen that is homologous to plectin and other members of the
plakin family (1-7, 25). The COOH-terminal region of plectin contains
five homologous segments known as the B domain and one other homologous
(but different) segment, known as the C domain. Both the B and C
domains contain a 39-residue quasi-repeat, which is believed to form a
stretch of The COOH-terminal domain of epiplakin includes five strongly conserved
repeats of 534 amino acid residues, each of which includes linker
regions and a single B domain. Moreover, each linker domain includes
three homologous subdomains. We identified a number of heptad-containing regions in the linker regions, but it is likely that
they form parts of The unique features of the repeated structures in epiplakin undoubtedly
contribute to the protein's function in vivo. It has been
suggested that the COOH-terminal domains of desmoplakin, plectin, and
BPAG1 all interact with intermediate filaments (11-14). Epiplakin, an
apparently novel human epidermal autoantigen, might also be expected to
bind to such filaments. However, no vimentin-binding domain, of the
type found in rat plectin (13), was detected within epiplakin; thus,
the types of intermediate filament that might act as partners might be limited.
Northern dot blots revealed that epiplakin is expressed at relatively
high levels in the brain, in particular, in the amygdala, putamen,
subthalamic nucleus, hippocampus, and occipital lobe. By contrast,
plectin was detected most prominently in spinal cord and to a lesser
extent in other neuronal tissue. Thus, epiplakin might interact with
intermediate filaments other than keratins.
In a previous report, we postulated that epiplakin might be distributed
in other organs in addition to the epidermis (25). Northern blotting
and immunofluorescence staining revealed that the stratified or simple
epithelium of the digestive system and several types of glandular
epithelium, including that of sweat and salivary glands, expressed this
protein. In our immunofluorescence study of skin, it appeared that the
protein was expressed in the entire sheet and, in some cases, more
strongly in the suprabasal layer of the epidermis. These data are
inconsistent with those in our previous study, in which we detected
immunofluorescence exclusively in basal keratinocytes (25). In the
previous study, we used polyclonal antibodies against a fusion protein
derived from a hybrid clone (pE 450-A) and encoded by a segment of 276 bp. We tried to identify the epitope recognized by these antibodies. At
maximum dilution, they recognized the sequence SGDQYNDDEIDA, which is
encoded by the 5' region of 78 bp (designated X) of the hybrid clone
and irrelevant part of the antigen.
The protein encoded by the full-length cDNA had no signal peptide
at its amino terminus and no transmembrane sequence, so it seems likely
that it is localized in the cytoplasm. The results of our
immunofluorescence study support this hypothesis. As is the case for
BPAG1, the pathological role of the autoantibodies against the
epiplakin is not known.
The presence of autoantibodies against desmoplakin, envoplakin,
periplakin and plectin has been reported in the serum of patients with
paraneoplastic pemphigus, an infrequently occurring autoimmune blistering disease (20-22). The serum of the patient whom we discussed in a previous report recognized a sequence in epiplakin that is not
found in plectin.
In conclusion, we have clarified the structure and tissue distribution
of a novel human epidermal autoantigen, which is an unusual member of
the plakin family that was originally found as an autoantigen in a
patient with a subepidermal blistering disease. The functions of this
protein and the genetic diseases in which it is involved should be
studied in further detail.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical coiled-coil
structure with a dimerization partner (9). As suggested by this model,
it has been demonstrated that desmoplakin I can form homodimers
in vitro (10). Early investigations revealed that the
COOH-terminal domains of plakins are involved in binding to
intermediate filaments (11-13). The amino-terminal domains of
desmoplakin and of BPAG1 are believed to bind to desmosomes or
hemidesmosomes. Furthermore, some splicing variants of plectin and
BPAG1 have actin- or microtubule-binding domains at their amino
termini, and it has been proposed that these domains form cross-links
between microfilaments and/or microtubules and intermediate filaments
(14-16). Studies of a few inheritable diseases that appear to involve
plectin or desmoplakin and of a BPAG1 null mouse have shown that each
plakin plays a critical role in the tissue integrity in specific
tissues (5, 17-19). Moreover, it seems likely that, in many autoimmune
blistering diseases, plakins, located in the epidermis, might be target
antigens, and these plakins are used for markers of specific diseases
(2, 20-22). However, their pathological roles remain to be clarified.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside at
32 °C in LB medium supplemented with 50 µg/ml ampicillin. After culture for 3 h with
isopropyl-1-thio-
-D-galactopyranoside, bacterial cells
were harvested and lysed by sonication in PBS, with subsequent centrifugation at 15,000 × g for 10 min. Supernatants
were incubated with 40 µl/ml glutathione-Sepharose (Amersham
Pharmacia Biotech) for 30 min at 20 °C. The glutathione-Sepharose
beads were washed with elution buffer that contained 15 mM
glutathione to elute the GST recombinant proteins. The amino acid
sequence of the protein was confirmed with the automated sequencer
after Western blotting of proteins on ImmobilonTM membranes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt11
library of random-primed cDNA using the 5' end of clone pE 450-D as
probe (Fig. 1). The longest clone,
designated clone Ep24, was isolated, and primers from the 5' end of
clone Ep24 were used for 5'-RACE to identify five overlapping clones
(clones Ep28, Ep11, Ep2, Ep79, and Ep21). The original HeLa cell
cDNA library was then screened again, and clones Ep1 and Ep5 were
obtained. In clone Ep5, the putative initiation codon ATG was preceded
by an in-frame termination codon.
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Fig. 1.
Cloning strategy for the isolation and
sequencing of the human epiplakin cDNA and schematic representation
of the deduced 552-kDa protein and comparisons with related members of
the plakin family (9, 33). The cDNAs isolated in this study
allowed identification of a 15,195-bp ORF, which corresponds to
contiguous cDNA sequences. Three previously isolated cDNAs,
pE450-A (obtained by immunoscreening), pE450-C, and pE450-D (obtained
by plaque hybridization), are also shown (25). The latter two clones
overlapped correctly with part of the five repetitive region, but their
position could not be determined. pE450-A was a hybrid clone that
contained an irrelevant region of 78 bp (box adjacent to the
bold line) at the 5'-terminal end. The gap of 760 bases in the message (shaded box) was covered by
genomic sequence. The numbers at the top are
domain numbers; numbers just above the boxes are
the amino acid residue numbers of the domains; and numbers
beneath the lines are the residue numbers of the linker
regions; the numbers under the line below the
boxes are the length of the separations between domains (in
amino acid residues). The B domains in epiplakin are 70% homologous to
the B domain in desmoplakin, and the B* domains are 46-49% homologous
to the B domain in desmoplakin (see Table I). Double-lined
linker regions and the five B domains (9, 10, 11, 12 and 13) to their
right are almost perfectly identical (see Fig. 3A). A
coiled-coil rod domain and an amino-terminal domains are shown as
open boxes. Both are found in plectin,
desmoplakin I, and BPAG1, but they are not found in epiplakin (33). The
domain structures of the COOH-terminal ends of the three plakins are
shown as boxes with letters A,
B, or C.
-turns and/or short
-strands. The
total potential number of heptad-containing segments was nine, and we
postulated that these segments should be grouped in two or three
bundles that contain antiparallel
-helices, a motif found in the
structures of many other globular proteins. We failed to identify a
coiled-coil rod domain and an amino-terminal domain, both of which are
characteristic features of all other known members of the plakin
family. Furthermore, no dimerization motif was found in the sequence,
suggesting that epiplakin probably exists in vivo as a
single-chain structure. A search for other functional protein motifs
did not reveal any transmembrane sequences or any vimentin- or
actin-binding domains (13, 16).
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Fig. 2.
The predicted sequence of 5065 amino acids of
human epiplakin, as deduced from the single ORF within the
15.2-kilobase mRNA (DNA Data Bank of Japan, accession no.
AB051895). The calculated molecular mass of the protein is
55,2467 daltons. The positions of B and B* domain borders are indicated
by horizontal arrows and numbers
(e.g.1B*, 2B*, ... 13B.). The borders of the five
COOH-terminal, strongly conserved, homologous repeats are also
indicated by vertical arrows. The sequence on the
basis of which we synthesized a series of eight amino acids overlap for
epitope mapping, residues 2807-3337, is shaded. Peptide A
(MSIYQAMWKGVL(C)) and peptide B (TKGFFDPNTHEN(C)) are indicated by
solid and dashed lines, respectively.
The sequence LVPAKDQPGRQEKMSIYQAMWKGVLRPGT, on the basis of which we
synthesized a series of decapeptides with an overlap of nine amino
acids to refine the identification of the epitope, is indicated in
bold italics. The antigenic epitope (IYQAMWK)
that was recognized by the patient's serum is shown in
double-underlining. The sequence, residues 2443-2682, on the basis of
which we produced the recombinant protein is indicated in
wavy underlining.
Comparison between segments of epiplakin and the COOH-terminal domains
A, B and C of desmoplakin
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Fig. 3.
The structure of the COOH-terminal domains of
epiplakin. A, comparison of the sequences of five
COOH-terminal, strongly conserved, homologous repeats. Each repeat
consisted of the linker regions (358 residues in length) followed by B
domains 9, 10, 11, 12, and 13 (176 residues in length). In total, 534 amino acids were almost perfectly identical. Amino acids that are
identical in five repeats are shaded. Asterisk
(*) indicates the different amino acids. B, comparison of
the sequences of three homologous segments within one of five strongly
conserved linker regions (residues 2391-2748). The sequences of three
homologous segments are compared with each other. Two of these segments
(residues 2391-2472 and 2473-2554) are 30% identical, with
particularly homology over the last 63 residues. The third homologous
region (residues 2613-2675) is shorter than the former two but,
similarly, exhibits the homology over the same region. Amino acids that
are identical in three segments are shaded.
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Fig. 4.
The Antigenic epitope in epiplakin.
A, enzyme-linked immunosorbent assays using MSIYQAMWKGVLC,
TKGFFDPNTHENC, and VKRYLEGTSCIAGVLVP (negative control). The patient's
serum reacted with the first peptide but not with the second and the
control peptide. Serum from a control patient did not react with any of
these peptides. B, a series of decapeptides with nine
overlapping amino acids from within the sequence
LVPAKDQPGRQEKMSIYQAMWKGVLRPGT (residues 2739-2767) was synthesized on
a membrane and exposed to the patient's serum. Four strongly positive
spots, corresponding to regions from KMSIYQAMWK (no. 13) to IYQAMWKGVL
(no. 16), were observed. C, the major epitope that was
recognized by the patient's serum was determined to be IYQAMWK. The
common sequence from KMSIYQAMWK (no. 13) to IYQAMWKGVL (no. 16) was
determined to be an antigenic epitope.
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Fig. 5.
Western blotting using several antisera
against the 450-kDa epidermal antigen. An extract of human
epidermis was subjected to immunoblotting analysis with the patient's
serum before (lane 1) and after (lane
2) incubation with the synthetic peptide MSIYQAMWKGVLC, and
with polyclonal antibodies against the synthetic peptide before
(lane 3) and after (lane 4)
incubation with the same synthetic peptide. Polyclonal antiserum
(lane 5) and monoclonal antibodies
(lane 7) against the GST-epiplakin fusion
protein, but not the control polyclonal antiserum (lane
6) and monoclonal antibodies (lane 8)
against GST itself, recognized the 450-kDa epidermal antigen. Two
smaller weakly stained bands in almost all lanes are artifacts due to
large amount of desmoplakin I and II in the epidermal samples.
Arrowheads indicate the position of the 450-kDa epidermal
antigen. The position of molecular size standards (in kilodaltons) are
shown on the left.
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Fig. 6.
Northern dot blots demonstrating the
expression of epiplakin in various organs. Northen analysis of
poly(A)+ RNA from various human tissues with probes that
corresponded to a specific sequence of epiplakin cDNA
(A), plectin cDNA (B), or ubiquitin cDNA
(C) as controls. In contrast to plectin, transcripts for
epiplakin were strongly expressed in liver, salivary glands, placenta,
and digestive organs, and to a lesser extent in lung, bone marrow, and
brain. The type and position of poly(A)+ RNAs and controls
dotted on the membranes are shown in the right
side.
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Fig. 7.
Presence of epiplakin in the entire epidermis
as revealed by immunofluorescence staining. Frozen sections of
human skin (A-H), of esophagus (I and
J), of salivary gland (K and L), of
stomach (M and N), and of colon (O and
P) were immunostained with monoclonal antibodies
(A), polyclonal antiserum against recombinant protein of
epiplakin (E, G, I, K,
M, and O), polyclonal antibodies against the
synthetic peptide MSIYQAMWKGVLC(C). And monoclonal antibodies against
the GST protein (B), polyclonal antiserum against GST
protein (F, H, J, L,
N, and P) or normal rabbit serum (D)
were used as negative controls. Epiplakin was detected in the entire
epidermis, in particular in suprabasal keratinocytes. The epithelium of
eccrine sweat glands and hair follicles were also immunostained. In the
esophagus, the same antibodies reacted with all the sheets of
stratified epithelium including the basal layer. Immunofluorescence was
apparent in acinous cells in the parotid gland and in mucous epithelial
cells of the stomach and colon. White arrows
indicate the positions of epidermis (B and D),
sweat glands (F), hair follicles (H), stratified
epithelium of the esophagus (J), mucous epithelial cells of
the stomach (N), and colon (P).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix that is followed by
-turns (9). Epiplakin, by
contrast, contains only B domains (13 in total), and these domains are
distributed along the sequence with relatively uniform spacing.
Furthermore, epiplakin does not appear to contain either a coiled-coil
rod domain or an amino-terminal domain. These features alone indicate that epiplakin belongs to a new category of plakins (33, 34).
-helical bundles rather than an extended coiled-coil rod. The reasons for this hypothesis are as follows. Two-stranded coiled-coil structures extend, on average, over about 11 heptads between breaks in heptad continuity, and such structures display characteristic patterns of charged residues, in particular at
positions e and g (1). By contrast,
-helices that form part of a
bundle are typically about three or four heptads long, and the e and g
positions are commonly occupied by apolar or noncharged residues. In
the amino acid sequence of epiplakin, the characteristics of the
heptad-containing regions in the linkers are rather similar to those of
a bundle of
-helices of the type commonly seen in globular proteins.
In the same context, these heptad-containing sequences in epiplakin do
not have the characteristics of a classical dimerization motif and,
thus, the probable single-chain nature of epiplakin represents another
unusual feature of this protein.
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ACKNOWLEDGEMENTS |
---|
We thank Satoko Ishihara for performing the immunofluorescence study, and we thank Mami Sakurai, Shuji Imi, and Kumiko Sakai for preparing this manuscript.
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FOOTNOTES |
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* This work was supported in part by Grants 07670954, 09670889, and 11670839 (to S. F.) and Grant 09557218 (to M. K.) from the Ministry of Education, Japan, and by a grant from the TERUMO Life Science Foundation (Kanagawa, Japan).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.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB051895.
§ To whom correspondence should be addressed. Tel.: 81-97-586-5882; Fax: 81-97-586-5889; E-mail: fujiwara@oita-med.ac.jp.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M011386200
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ABBREVIATIONS |
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The abbreviations used are: BPAG1, bullous pemphigoid antigen 1; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; ELISA, enzyme linked immunosorbent assay; GST, glutathione S-transferase; bp, base pair(s); BSA, bovine serum albumin; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline plus Tween 20.
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