THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 276, No. 16, Issue of April 20, pp. 13340 –13347, 2001
Printed in U.S.A.

Epiplakin, a Novel Member of the Plakin Family OriginallyIdentified as a 450-kDa Human Epidermal Autoantigen
STRUCTURE AND TISSUE LOCALIZATION*
Received for publication, December 18, 2000, and in revised form, January 17, 2001
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M011386200

Sakuhei Fujiwara‡§, Naoko Takeo‡, Yuichiro Otani‡, David A. D. Parry¶,
Mitoshi Kunimatsu储, Rui Lu储, Makoto Sasaki储, Noritaka Matsuo**, Mohammed Khaleduzzaman**,and Hidekatsu Yoshioka**
From the ‡Department of Dermatology and **Biochemistry, Oita Medical University, Idaigaoka, Hasama, Oita 879-5593,
Japan, the 储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

A 450-kDa human epidermal autoantigen was originally identified as a protein that reacted with the serumfrom an individual with a subepidermal blistering disease. Molecular cloning of this protein has now shownthat it contains 5065 amino acids and has a molecularmass 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 aminoterminal domain, both of which are found in all otherknown members of the plakin family. Furthermore, no
dimerization motif was found in the sequence. Thus, it islikely that epiplakin exists in vivo as a single-chain
structure. Epitope mapping experiments showed thatthe 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 domainsthat are separated by a central rod domain. Some homologous* 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 thisarticle were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to
the DDBJ/GenBank娂/EBI Data Bank with accession number
AB051895.
§ To whom correspondence should be addressed. Tel.: 81-97-5865882; Fax: 81-97-586-5889; E-mail: fujiwara@oita-med.ac.jp.
1
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.

domain structures have been identified in both globular domains of many plakins, while the central domain is rich inheptad repeats and is believed to form a parallel ␣-helicalcoiled-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 arebelieved to bind to desmosomes or hemidesmosomes. Furthermore, some splicing variants of plectin and BPAG1 have actinor 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 nullmouse have shown that each plakin plays a critical role in thetissue 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, andthese plakins are used for markers of specific diseases (2,20 –22). However, their pathological roles remain to beclarified.
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 230kDa (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 sometransformed 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 comparewith 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.

13340

This paper is available on line at http://www.jbc.org

Epiplakin, a 450-kDa Human Epidermal Autoantigen

13341

FIG. 1. Cloning strategy for the isolation and sequencing of the human epiplakin cDNA and schematic representation of thededuced 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 byimmunoscreening), pE450-C, and pE450-D (obtained by plaque hybridization), are also shown (25). The latter two clones overlapped correctly withpart 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 beneaththe 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 theirright are almost perfectly identical (see Fig. 3A). A coiled-coil rod domain and an amino-terminal domains are shown as open boxes. Both are foundin plectin, desmoplakin I, and BPAG1, but they are not found in epiplakin (33). The domain structures of the COOH-terminal ends of the threeplakins are shown as boxes with letters A, B, or C.
EXPERIMENTAL PROCEDURES

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 ina previous report (25).
For isolation of the 3⬘ end repeats, we constructed a cDNA libraryfrom the poly(A)⫹ RNA of HeLa cells. cDNA was synthesized with acDNA synthesis kit (Life Technologies, Inc.). We modified the procedureprovided with the kit by adding trehalose to the reaction for first-strand
synthesis (26) and, as specific primers for first-strand synthesis, weused 5⬘-CCAGACACAACAAGTATGCC-3⬘ for clone Ep115 and 5⬘-TAGCGCTTGACCGAGTCCATC-3⬘ for clone Ep 4. The cDNAs were ligatedto 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 of3⬘ 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 (LifeTechnologies) and total RNA from HeLa cells or the Marathon-Ready
cDNAs System with adaptor-ligated double-stranded cDNA preparedfrom 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 adouble-strand strategy with an automatic DNA sequencer (Applied
Biosystems). Internal primers were constructed for analysis of internalsequences.
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 ina preliminary form. Short regions of heptad substructure were delin-

eated 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 afunction 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 ofchain 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 thespotting schedule for each cycle of addition of an amino acid. Peptides
spanning amino acid residues 2807–3337 of epiplakin were synthesizedon cellulose membranes as a series of decapeptides with an overlap ofeight 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 nineamino 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-phasemethod (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 toacetonitrile in 0.1% trifluoroacetic acid. The molecular mass and aminoacid sequence of each purified peptide were confirmed by determinations of the matrix-assisted laser desorption ionization time of flight13342

Epiplakin, a 450-kDa Human Epidermal Autoantigen
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 ␮g/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) wereadded to the wells. After a 1-h incubation with shaking at room temperature, the wells were washed three times with PBS-T, and 100 ␮l ofa 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 witheach synthetic peptide was corrected by subtraction of the value due toreaction of the antibodies with BSA by itself.
Immunoblotting—Epidermal extracts were obtained as described
previously (23). After addition of dithiothreitol at a final concentrationof 0.01 M, the solubilized proteins were separated by electrophoresis on
an SDS-polyacrylamide gel (5% polyacrylamide) and transferred to an
Immobilon娂 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 withthe 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-␤-D-galactopyranoside at 32 °C in LB medium supplemented with 50 ␮g/ml ampicillin. After culture for 3 h with isopropyl-1-thio-␤-D-galactopyranoside,bacterial cells were harvested and lysed by sonication in PBS, withsubsequent centrifugation at 15,000 ⫻ g for 10 min. Supernatants wereincubated with 40 ␮l/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 elutethe GST recombinant proteins. The amino acid sequence of the protein
was confirmed with the automated sequencer after Western blotting ofproteins on Immobilon娂 membranes.
Production of Antibodies against the Fusion Protein and a Synthetic
Peptides—Antibodies against a recombinant protein were raised inrats. The purified GST recombinant protein was mixed with an equalvolume of Freund’s complete or incomplete adjuvant. Initial subcutaneous injections contained 100 ␮g of recombinant protein (plus complete Freund’s adjuvant) per rat. Two booster injections, each consistingof 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

FIG. 2. The predicted sequence of 5065 amino acids of human
epiplakin, as deduced from the single ORF within the 15.2kilobase 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 byhorizontal arrows and numbers (e.g.1B*, 2B*, . . . 13B.). The borders of
the five COOH-terminal, strongly conserved, homologous repeats arealso 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)) andpeptide B (TKGFFDPNTHEN(C)) are indicated by solid and dashedlines, 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 indouble-underlining. The sequence, residues 2443–2682, on the basis ofwhich we produced the recombinant protein is indicated in wavy
underlining.

Epiplakin, a 450-kDa Human Epidermal Autoantigen

13343

TABLE I
Comparison between segments of epiplakin and the COOH-terminal domains A, B and C of desmoplakin
All 13 domains, with the exception of domains 1, 2 and 4, consisted of 176 amino acid residues. These other three domains consisted of 178(domain 1), 171 (domain 2), and 173 (domain 4) residues, respectively. An asterisk indicated B domains of epiplakin that are approximately
46 – 49% homologous to the B domain in desmoplakin.
Desmoplakin
Epiplakin

Domain no.
1
2
3
4
5
6
7
8
9
10
11
12
13

A

Segment
27–204
271–441596–771916–1088
1232–1407
1557–1732
1883–2058
2215–2390
2749–2924
3283–3458
3817–3992
4351–4526
4885–5060

Identities
64
66
86
78
68
83
77
81
83
83
83
82
83

B

%Ident
36
39
49
45
39
47
44
46
47
47
47
47
47

adjuvant. Antibodies (peptide A-specific antibodies) were purified byimmunoaffinity 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
32
P 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 asa control probe.
Immunofluorescence Staining—Human tissues were obtained at biopsy or autopsy, embedded into OCT compound (Sakura Fine TechnicalCo., Ltd., Tokyo, Japan) and frozen in liquid nitrogen. Frozen sections(5 ␮m) 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 specificantiserum or antiserum against GST. The sections were washed threetimes in PBS and then incubated with fluorescein isothiocyanate-conjugated (FITC-conjugated) goat antibodies against rat (for peptidespecific 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 afluorescence microscope (UFX-DX; Nikon).
RESULTS

Isolation of Overlapping cDNA Clones That Encoded a Human Epidermal Autoantigen—We first screened the HeLa cell
␭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 andEp5 were obtained. In clone Ep5, the putative initiation codon
ATG was preceded by an in-frame termination codon.
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⬘ sideof 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 offive strongly homologous repeats. We constructed a cDNA library using a specific primer from the 3⬘- terminal region ofclone Ep12. The longest clone, Ep115, was 6.3 kilobases longand it was isolated together with several shorter clones, Ep150

Identities
81
79
122
83
82
123
87
123
124
124
124
123
124

C

%Ident
46*
46*
69
48*
47*
70
49*
70
70
70
70
70
70

Identities
52
46
57
52
52
57
52
55
56
56
56
55
55

%Ident
30
27
32
30
30
32
30
31
32
32
32
31
31

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 regionand obtained a clone that was slightly more than 9 kilobase
pairs in length and contained no introns. The gap of 760 basesin 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 byan in-frame TGA triplet, because the first ATG triplet was notpreceded by a consensus sequence that would favor the initiation oftranslation (32). The total length of the ORF was 15,195 bp, and thepredicted amino acid sequence (5065 amino acids) is shown in Fig.
2. The theoretical relative molecular mass was calculated to be552,467 Da and was in reasonable agreement with the molecularmass of 450 kDa determined previously by SDS-polyacrylamide gelelectrophoresis. 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 domainrather than to the A or C domain that were also first recognizedin the COOH-terminal domain of desmoplakin (Table I and Ref.
1). We found, moreover, that the linker regions (358 residues inlength) 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 fivestrongly conserved linker regions (Fig. 3B). For example, in thelinker region (residues 2391–2748), two of the segments (residues
2391–2472 and 2473–2554) were 30% identical, with particularlyhigh identity over the last 63 residues. After a glycine-prolinerich 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 segmentsthat are separated from one another by ␤-turns and/or short␤-strands. The total potential number of heptad-containing
segments was nine, and we postulated that these segments

13344

Epiplakin, a 450-kDa Human Epidermal Autoantigen

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 thelinker 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 fivestrongly conserved linker regions (residues 2391–2748). The sequencesof three homologous segments are compared with each other. Two ofthese 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 thatare identical in three segments are shaded.

should be grouped in two or three bundles that contain antiparallel ␣-helices, a motif found in the structures of many otherglobular 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 singlechain structure. A search for other functional protein motifs did
not reveal any transmembrane sequences or any vimentin- or
actin-binding domains (13, 16).
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

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 andthe control peptide. Serum from a control patient did not react with anyof these peptides. B, a series of decapeptides with nine overlapping
amino acids from within the sequence LVPAKDQPGRQEKMSIYQAMWKGVLRPGT (residues 2739 –2767) was synthesized on a membraneand 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 thepatient’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.

against 264 consecutive cellulose-bound linear peptides of 10
amino acids in length with an 8-amino acid overlap in theregion 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 wasrecognized 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 thepatient’s serum reacted only with the former peptide (Fig. 4A).
To refine the identification of the epitope, we exposed a seriesof 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Epiplakin, a 450-kDa Human Epidermal Autoantigen

13345

FIG. 5. Western blotting using several antisera against the 450kDa epidermal antigen. An extract of human epidermis was subjected
to immunoblotting analysis with the patient’s serum before (lane 1) andafter (lane 2) incubation with the synthetic peptide MSIYQAMWKGVLC,
and with polyclonal antibodies against the synthetic peptide before (lane3) 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 inalmost all lanes are artifacts due to large amount of desmoplakin I and II
in the epidermal samples. Arrowheads indicate the position of the 450kDa epidermal antigen. The position of molecular size standards (inkilodaltons) are shown on the left.

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, nolonger reacted with the 450-kDa epidermal autoantigen (Fig. 5,
lanes 1 and 2). These data indicated that antibodies in thepatient’s serum recognized mainly the unique epitope in
epiplakin.
Distribution of Epiplakin as Revealed by Northern Dot Blotsof 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 indicatedthat 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 andsomewhat less strongly with RNA derived from the placenta,
lung, brain, and bone marrow.
Localization of Epiplakin in the Entire Stratified Epitheliumand 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 theentire epidermis and immunostaining was especially prominent over basal keratinocytes (Fig. 7A). When we used polyclonal antibodies against the synthetic peptide, which is thesame 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

FIG. 6. Northern dot blots demonstrating the expression of
epiplakin in various organs. Northen analysis of poly(A)⫹ RNA fromvarious human tissues with probes that corresponded to a specificsequence of epiplakin cDNA (A), plectin cDNA (B), or ubiquitin cDNA(C) as controls. In contrast to plectin, transcripts for epiplakin werestrongly expressed in liver, salivary glands, placenta, and digestiveorgans, 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.

was apparent in acinous cells in the parotid gland and inmucous epithelial cells of the stomach and colon (Fig. 7, K, M,
and O).
DISCUSSION

In this report, we have described the cloning and sequencingof overlapping cDNA clones and a genomic clone for a small gap
region that together correspond to the entire transcript for anovel member of the plakin family. Epiplakin appears to be a
450-kDa human epidermal autoantigen that is homologous toplectin 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 (butdifferent) segment, known as the C domain. Both the B and C
domains contain a 39-residue quasi-repeat, which is believed toform a stretch of ␣-helix that is followed by ␤-turns (9). Epiplakin, by contrast, contains only B domains (13 in total), andthese 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 aminoterminal domain. These features alone indicate that epiplakinbelongs to a new category of plakins (33, 34).
The COOH-terminal domain of epiplakin includes fivestrongly conserved repeats of 534 amino acid residues, each ofwhich 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

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Epiplakin, a 450-kDa Human Epidermal Autoantigen

FIG. 7. Presence of epiplakin in the entire epidermis as revealed by immunofluorescence staining. Frozen sections of humanskin (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 withmonoclonal 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, inparticular 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).

regions, but it is likely that they form parts of ␣-helical bundles
rather than an extended coiled-coil rod. The reasons for thishypothesis are as follows. Two-stranded coiled-coil structures
extend, on average, over about 11 heptads between breaks inheptad 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 theheptad-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 aclassical dimerization motif and, thus, the probable singlechain nature of epiplakin represents another unusual featureof this protein.
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 typefound 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 atrelatively high levels in the brain, in particular, in the amygdala, putamen, subthalamic nucleus, hippocampus, and occipital lobe. By contrast, plectin was detected most prominently inspinal 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 systemand several types of glandular epithelium, including that ofsweat 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, morestrongly 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 serumof patients with paraneoplastic pemphigus, an infrequently
occurring autoimmune blistering disease (20 –22). The serum of
the patient whom we discussed in a previous report recognizeda 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 isan unusual member of the plakin family that was originallyfound 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.
Acknowledgments—We thank Satoko Ishihara for performing theimmunofluorescence study, and we thank Mami Sakurai, Shuji Imi,
and Kumiko Sakai for preparing this manuscript.
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