Molecular Cloning and Characterization of a Novel Human Classic
Cadherin Homologous with Mouse Muscle Cadherin*
Yutaka
Shimoyama
§¶,
Tatsuhiro
Shibata
,
Masaki
Kitajima**, and
Setsuo
Hirohashi
From the
Hirohashi Cell Configuration Project,
Exploratory Research for Advanced Technology, Japan Science and
Technology Corporation, Tsukuba Research Consortium,
5-9-4 Tokodai, Tsukuba 300-26, the § Departments of
Surgery and Clinical Research, National Okura Hospital,
2-10-1 Okura, Setagaya-ku, Tokyo 157, the
Pathology
Division, National Cancer Center Research Institute,
5-1-1 Tsukiji, Chuo-ku, Tokyo 105, and the ** Department of
Surgery, School of Medicine, Keio University, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160, Japan
 |
ABSTRACT |
We used a novel cDNA cloning method based on
the cadherin-
-catenin protein interaction and identified a new human
classic-type cadherin, which we named cadherin-15, from adult brain and
skeletal muscle cDNA libraries. Sequence analysis revealed that
this cadherin was closely related to mouse muscle cadherin and seemed
to be its human counterpart. However, its deduced amino acid sequence differed from that of mouse muscle cadherin in that it had an extra
31-amino acid sequence at its C terminus that has been found neither in
mouse muscle cadherin nor in any other known classic cadherin. Analysis
of cadherin-15 protein expressed in L fibroblasts showed that it was
cleaved proteolytically, expressed on the cell surfaces as a mature
form of about 124-kDa, and functioned as a cell-cell adhesion molecule
in a homophilic and specific manner, but Ca2+ did not
protect it against degradation by trypsin. Our findings also suggest
that cadherin-15 mediates cell-cell adhesion with a binding strength
comparable to that of E-cadherin.
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INTRODUCTION |
It is generally accepted that various molecules that have the
extracellular subdomain (EC)1
structure of the classic cadherins in common constitute a large gene
family, the cadherin superfamily. This superfamily includes the classic
cadherins (1), truncated-type cadherins, which lack the characteristic
cytoplasmic domain of the classic cadherins (2-4), desmosomal
cadherins, which are localized in desmosomes (5-7), protocadherins,
which have more than five extracellular subdomains (8-9), and
molecules showing high similarities to rat LI-cadherin (10-12).
Each classic cadherin comprises a signal sequence and a precursor
region, which are both cleaved by intracellular proteolytic processing,
five cadherin extracellular subdomain repeats, a transmembrane domain
and a characteristic cytoplasmic domain, which is highly conserved
among the subclasses and is indispensable for association with
catenins, the ensuing linkage to the cytoskeleton and full functioning
as a Ca2+-dependent cell-cell adhesion molecule
(13-15). It is now understood that these cadherins play essential
roles in various morphogenetic events in multicellular organisms (1).
The first classic cadherins to be identified were E-, N-, and
P-cadherins, as a result of the establishment of their respective
blocking antibodies (16-23), and then V-cadherin was identified using
a blocking monoclonal antibody (24). Thereafter, several cadherins were
identified (25-28) by determining their cross-reactivities with
antibodies raised against conserved peptide sequences or
cross-hybridization with cDNA fragments of known cadherins. Over
the past few years, the existence of more classic cadherin molecules
has been demonstrated by PCR-based cDNA cloning methods (3,
29-34).
Full cDNA cloning of nine independent human classic cadherin
molecules has been reported, i.e. E-, N-, and P-cadherins
and cadherin-4, -5, -6, -8, -11 (OB-cadherin), and -12 (3, 29, 30, 31,
35-37). We are interested in how many classic cadherin molecules
actually exist in humans and how these molecules function and cooperate
in the development and maintenance of the integrity of the human body,
as well as in various pathogenic states such as cancers. In an attempt
to find novel human classic cadherin molecules that have not been
identified by the aforementioned methods, we devised a new strategy
based on the cadherin-catenin interaction. Our technique is a protein
interaction cloning method using
-catenin, which binds directly to
the cytoplasmic domains of classic cadherins (13, 38). By using this
method, we found two novel human classic cadherins, which we named
cadherin-14 and -15. The former is a novel type II classic cadherin
expressed widely in the central nervous system, and recently, we
reported its cDNA cloning (39), and the latter closely resembles
mouse M (muscle)-cadherin and appears to be its human counterpart.
However, this molecule has an additional peptide sequence at its C
terminus not possessed by M-cadherin. In this report, we describe
molecular cloning and functional and biochemical analyses of this human cadherin-15 molecule.
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EXPERIMENTAL PROCEDURES |
cDNA Cloning--
To use
-catenin as a probe for the
first cDNA cloning procedure based on the protein-protein
interaction, we constructed and purified
-catenin-glutathione
S-transferase fusion protein, as described previously (39).
This protein was radiolabeled in vitro with
[
-32P]ATP using bovine heart kinase and was used to
screen a human adult brain
gt11 expression cDNA library
(CLONTECH). Positive clones were plaque-purified
and sequenced.
To isolate a cadherin-15 cDNA covering the entire open reading
frame, we screened a human adult skeletal muscle
gt10 cDNA library using a [
-32P]dCTP-labeled PCR probe
corresponding to the 227-bp nucleotide sequence at the 5'-end of the
cDNA clone yielded by the first cloning procedure. Positive clones
were purified by several rounds of rescreening and subjected to the
following sequence analysis.
DNA Sequence Analysis--
The cDNA inserts were excised
from the purified phage DNAs by EcoRI digestion and
subcloned into the EcoRI site of the pBluescript II SK(
)
or (+) phagemid vector, and overlapping subclones were prepared by the
stepwise deletion method (40). The cDNA sequences on both strands
were determined by an ABI PRISM 377 DNA sequencer (Perkin-Elmer) using
a Dye Primer Cycle Sequencing Kit (Perkin-Elmer). To identify the
5'-end of cadherin-15 mRNA, 5' RACE (41) was performed using human
adult skeletal muscle poly(A)+ RNA and the 5' RACE System
(Life Technologies, Inc.), according to the manufacturer's
instructions. The nucleotide and amino acid sequences were analyzed
using the GeneWorks software package (IntelliGenetics) and the BLAST
and FASTA programs.
RNA Blot Analysis--
Poly(A)+ RNAs of cultured
cells were purified using the QuickPrep Micro mRNA Purification Kit
(Amersham Pharmacia Biotech), and poly(A)+ RNAs of normal
human tissue were prepared in the same manner from specimens obtained
during surgery or autopsy or purchased from
CLONTECH. RNA blottings were performed as described
previously (35). Premade filters (human brain multiple tissue Northern blots II and III and human muscle multiple tissue Northern blot) purchased from CLONTECH were also used. To avoid
cross-hybridization with cadherins of other subclasses, a 143-bp
nucleotide sequence (positions 1669-1811 in Fig. 1) located within the
EC5, where homologies with other cadherin subclasses are below 30%
(Table I), was chosen, amplified by a PCR-labeling procedure (42), and
used as a probe.
Expression Vector Construction and Transfection--
To express
human cadherin-15 in mouse L fibroblasts, an expression vector,
pBAT15H, was constructed by replacing the mouse E-cadherin cDNA of
pBATEM2 (43) with a cDNA fragment of cadherin-15 containing the
entire open reading frame. Transfection of pBAT15H into L cells was
performed using LipofectAMINE reagent (Life Technologies, Inc.)
together with pSTneoB carring the neomycin resistance gene (44),
according to the manufacturer's instructions. The transfected cells
were selected in DMEM supplemented with 10% calf serum in the presence
of 400 µg/ml G418 in a humidified atmosphere comprising 5%
CO2, 95% air at 37 °C for about 2 weeks. Then, the
G418-resistant colonies were isolated, screened for cadherin-15
expression by RNA blotting, and maintained under the above condition.
Mouse E-cadherin transfectants were also obtained using pBATEM2 and pSTneoB and used as controls. Mouse L fibroblasts (LTK
) were supplied
by the Riken GenBank.
Immunoprecipitation and N-terminal Peptide Sequence
Determination--
Cells were lysed in 1% Nonidet P-40, 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml
leupeptin, 100 kallikrein inhibitor units/ml aprotinin, 10 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM CaCl2 with gentle pipetting on ice. The cell
lysate was clarified by centrifugation at 6,200 × g at
4 °C for 10 min twice and preadsorbed with an anti-mouse IgG
antibody coupled to Sepharose 4B (Organon Teknika Corporation) at
4 °C for 1 h followed by removal of the beads by centrifugation
and passing through a filter. The resulting lysate was incubated with
an anti-
-catenin monoclonal antibody (Transduction Laboratories) at
4 °C for 1 h and then with the anti-mouse IgG antibody coupled
to Sepharose 4B at 4 °C for 1 h. The beads were washed five
times with the lysis buffer and then three times with distilled water.
The bound materials were eluted from the beads with 1 M
acetic acid, lyophilized, redissolved in Laemmli's sample buffer (45),
heat-denatured, separated by 7.5% SDS-PAGE, electroblotted onto PVDF
membranes (Millipore), and visualized by staining with Coomassie
Brilliant Blue R-250 (Sigma) or AuroDye forte (Amersham Pharmacia
Biotech). The cadherin-15 bands were cut out from some membranes, and
the N-terminal amino acid sequence was determined using the HP G1005
Protein Sequencing System (Hewlett-Packard).
Cell Aggregation--
Short term cell aggregation experiments
were performed as described previously (14) with some minor
modifications. Briefly, dispersed cell suspensions were obtained by
treating cells with HCMF (HCMF, 10 mM HEPES-buffered
Ca2+,Mg2+-free Hanks' solution) containing
0.01% trypsin and 5 mM CaCl2 at 37 °C for
15 min at 80 rpm. Fifty thousand cells suspended in 0.5 ml of HCMF with
or without 5 mM CaCl2 containing 1% bovine serum albumin were placed in each well of a 24-well plastic plate (Ultra Low Cluster, Costar) and allowed to aggregate at 37 °C for 60 min at 80 rpm. The extent of cell aggregation was represented by the
index (n0
n60)/n0, where
n60 and n0 were the total
numbers of particles after incubation for 60 min and at the start of
incubation, respectively.
Long term cell aggregation experiments were performed as follows.
Single cell suspensions were obtained by treating cells with
phosphate-buffered saline containing 0.05% trypsin and 0.02% EDTA at
37 °C for 15 min, washed twice with DMEM supplemented with 10% calf
serum, and resuspended in DMEM supplemented with 10% calf serum and 70 units/ml DNase I (Takara) at a cell density of 2 × 105 cells/ml. One-hundred thousand cells (0.5 ml) were
placed in each well of a 24-well plastic plate (Ultra Low Cluster,
Costar) and allowed to aggregate at 37 °C for 24 h at 100 rpm
in a humidified atmosphere comprising 5% CO2, 95% air.
For mixed cell aggregation experiments using two cell lines, one line
was labeled with 40 µg/ml DiI (Molecular Probes) in DMEM supplemented
with 10% calf serum for 1 h, and the other was unlabeled; the
cells were suspended, as described above, and equal numbers of cells of
the two lines were mixed and allowed to aggregate for 12 h, as
described above.
Other Biochemical Procedures--
Determination of the trypsin
sensitivity and detergent solubility of cadherin-15 and immunoblot
analysis were performed as described previously (14). The ECCD-2
monoclonal antibody (46) and a commercially available polyclonal
anti-mouse M-cadherin antibody (M-cadherin (1), Santa Cruz
Biotechnology) were used to detect mouse E-cadherin and cadherin-15,
respectively, by immunoblotting. Exposition of cadherin-15 molecules on
the surfaces of cadherin-15-transfected L cells was examined by
labeling membrane proteins of the transfectants with a
membrane-impermeable reagent, EZ-Link Sulfo-NHS-Biotin (Pierce), as
described by Nelson and co-workers (47), immunoprecipitating the
biotinylated cadherin-15 proteins with catenins using an
anti-
-catenin monoclonal antibody (Transduction Laboratories),
separation by 7.5% SDS-PAGE, and transfer to PVDF membranes, as
described above. Then, the immunoprecipitates were stained with AuroDye
forte (Amersham Pharmacia Biotech) to detect all the components or with
diaminobenzidine using the avidin-biotin-peroxidase complex (Elite ABC,
Vector Laboratories) to detect components exposed on the external cell surfaces.
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RESULTS |
Molecular Cloning of Human Cadherin-15--
To identify novel
classic cadherin molecules that associate with
-catenin, a human
adult brain
gt11 expression cDNA library was screened with a
radiolabeled
-catenin fusion protein. Approximately 106
recombinants were screened, and 23 positive clones were isolated. DNA
sequence analysis of these clones disclosed 11 clones of N-cadherin, 1 of cadherin-11, 8 of adenomatous polyposis coli tumor suppressor protein, which also associates with
-catenin (48, 49), and 3 of
novel proteins, 2 of which showed high sequence similarities to each
other and to known cadherin molecules. One of the two cadherin-related
molecules was named cadherin-14 and was reported recently by our group
(39), and the other, designated cadherin-15, was analyzed in detail in
this study. This clone contained a 1656-bp cDNA insert, which
showed the highest homology with mouse M-cadherin (30). However,
comparison of its nucleotide sequence with that of mouse M-cadherin
revealed that it lacked the part encoding the translation initiation
codon, signal peptide, precursor region, and EC1-3.
As preliminary RNA blot analysis showed that this clone was expressed
strongly in skeletal muscle (data not shown), a human adult skeletal
muscle library was screened using a PCR probe located at the 5'-end of
the cDNA clone yielded by the first cloning procedure. About
1.7 × 105 phages were screened, and five positive
clones were isolated. A clone containing the longest cDNA insert
was selected, and the cDNA was subjected to sequence analysis. This
clone comprised 2833 bp with a poly(A) tail and covered the entire open
reading frame. To identify the 5'-end of the full mRNA, 5' RACE was
performed, and an additional 24-nucleotide sequence was obtained. The
combined nucleotide and deduced amino acid sequences are shown in Fig. 1. The open reading frame begins with an
ATG codon at positions 78-80, terminates at a TGA codon at positions
2520-2522, and consists of 2442 nucleotides encoding 814 amino acids.
The nucleotide sequence of the former cDNA clone isolated from a
brain library differed from this sequence (Fig. 1) at three points as
follows: A at position 1827, A at position 2048, and T at position 2818 were replaced by C, G, and G, respectively. These replacements altered
Lys at amino acid position 584 to Gln in the amino acid sequence but did not affect the open reading frame.

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Fig. 1.
Nucleotide and deduced amino acid sequences
of human cadherin-15. The 5'-terminal 24-nucleotide sequence shown
was determined by 5' RACE. The stop codon is denoted by an
asterisk; the amino acid sequence is shown in
one-letter code; the putative signal peptide and
transmembrane region are indicated by underlining; the
N-terminal peptide sequence determined by direct sequencing is
boxed; the proteolytic cleavage site is indicated by a
solid triangle, and the solid circles indicate
possible N-linked glycosylation sites. In comparison with
the amino acid sequence of mouse M-cadherin, cadherin-15 has an
additional 31-amino acid chain (marked by a dotted
underline) at the C terminus. The nucleotide sequence data has
been submitted to the DDBJ/EMBL/GenBankTM data bases with
the accession number D83542.
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Sequence Analysis of Cadherin-15--
The deduced amino acid
sequence consists of two hydrophobic sequences corresponding to the
signal peptide and transmembrane domain, a long extracellular domain
containing five cadherin extracellular subdomain repeats, and a
relatively short cytoplasmic domain, which are structural
characteristics of the classic cadherins. The cleavage site of the
signal peptide was deduced according Nielsen et al. (50). We
expected this protein to undergo further proteolytic cleavage at amino
acid positions 45-46 (51) and to be expressed on cell surfaces as a
mature and functional protein, 769 amino acids long with 4 consensus
sites for N-linked glycosylation (52). The proteolytic
cleavage and exposition on the cell surfaces were confirmed, as
described below.
A search for homologies with known classic cadherins revealed that
human cadherin-15 resembled mouse M-cadherin most closely, and the
homologies of cadherin-15 with mouse M-cadherin and the other known
human classic cadherins are summarized in Table
I. The putative mature protein of human
cadherin-15 shows 83% homology with that of mouse M-cadherin but much
lower homologies with those of the human classic cadherins reported so
far, suggesting that cadherin-15 is a human counterpart of mouse
M-cadherin. However, alignment of human cadherin-15 and mouse
M-cadherin revealed marked differences between the two molecules.
Cadherin-15 has an additional 31-amino acid stretch at its C terminus,
which has not been found in any other classic cadherin. The fact that
both the cDNA clones isolated from brain and skeletal muscle
libraries encode this sequence indicates that this is not due to a
cloning artifact or individual variations.
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Table I
Homologies of the separate regions and putative mature proteins of
human cadherin-15 and other classic cadherins
The extracellular domains are divided into five subregions, according
to Tanihara et al. (3), and mM (mouse M-cadherin), E, N, P,
4, 5, 6, 8, 11, 12 and 14 represent the percentage of homologies of
human cadherin-15 with mouse M-cadherin (30), human E-cadherin (37),
N-cadherin (36), P-cadherin (35), cadherin-4 (3), -5 (29), -6 (33), -8, -11, -12 (3) and -14 (39), respectively. EC1-5, extracellular
subdomains 1-5; TM, transmembrane domain; CP, cytoplasmic domain.
Homologies over 50% are highlighted in bold.
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Cadherin-15 Expression in Human Tissues--
Fig.
2 shows cadherin-15 expression in various
normal human tissues. Skeletal muscle showed intense expression of a
transcript of about 2.9 kb and faint expression of two of about 5.8 and
6.7 kb. Expression of cadherin-15 transcripts was faint in the brain and very faint in the placenta, prostate, spinal cord, and thyroid. Next, we performed RNA blotting of several muscle and brain tissues (Fig. 3). As shown in Fig. 3A,
of the muscle tissues examined only skeletal muscle expressed
cadherin-15; no cadherin-15 transcripts were detected in smooth or
cardiac muscle. In various brain sections, cadherin-15 transcripts were
detected only in the cerebellum (Fig. 3B), suggesting that
the faint expression in the whole brain (lanes 2 and
13 in Figs. 2 and 3B, respectively) derived from
the cerebellum.

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Fig. 2.
RNA blot analysis of cadherin-15 in normal
human tissues. Poly(A)+ RNAs (2 µg/lane) from 19 human tissues (lane 1, adrenal gland; lane 2,
brain; lane 3, heart; lane 4, kidney; lane
5, liver; lane 6, lung; lane 7, mammary
gland; lane 8, pancreas; lane 9, placenta;
lane 10, prostate; lane 11, salivary gland;
lane 12, skeletal muscle; lane 13, small
intestine; lane 14, spinal cord; lane 15, spleen;
lane 16, stomach; lane 17, testis; lane
18, thymus; lane 19, thyroid) were separated
electrophoretically on 1% agarose/formaldehyde gels, transferred to
nitrocellulose filters, and hybridized with the specific 143-bp PCR
probe. The positions of the RNA size markers (9.5, 7.5, 4.4, 2.4, and
1.35 kb) are indicated on the left, and the lower
panel shows hybridized -actin on the same filters.
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Fig. 3.
RNA blot analysis of cadherin-15 in various
muscle (A) and brain (B) tissues. A,
poly(A)+ RNAs (2 µg/lane) from eight human muscle tissues
(lanes 1, skeletal muscle; lane 2, uterus;
lane 3, colon; lane 4, small intestine;
lane 5, bladder; lane 6, heart; lane
7, stomach; lane 8, prostate) were separated
electrophoretically on a 1.2% agarose/formaldehyde gel, transferred to
a nylon membrane, and hybridized with the specific 143-bp PCR probe.
B, poly(A)+ RNAs (2 µg/lane) from 16 different
human brain sections (lanes 1, cerebellum; lane
2, cerebral cortex; lane 3, medulla; lane 4,
spinal cord; lane 5, occipital pole; lane 6,
frontal lobe; lane 7, temporal lobe; lane 8,
putamen; lane 9, amygdala; lane 10, caudate
nucleus; lane 11, corpus callosum; lane 12,
hippocampus; lane 13, whole brain; lane 14,
substantia nigra; lane 15, subthalamic nucleus; lane
16, thalamus) were separated electrophoretically on 1.2%
agarose/formaldehyde gels, transferred to nylon membranes, and
hybridized with the specific 143-bp PCR probe. The positions of the RNA
size markers (9.5, 7.5, 4.4, 2.4, and 1.35 kb) are indicated on the
left, and the lower panels show hybridized
-actin on the same membranes.
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Cadherin-15 Transfection and Expression in Mouse L
Fibroblasts--
To analyze the molecular nature and functional
characteristics of cadherin-15, cadherin-15 cDNA placed downstream
from the chicken
-actin promoter was introduced into L cells, which
are mouse fibroblasts deficient in cadherin activity (53). Over 30 G418-resistant colonies were isolated and screened for cadherin-15 expression by RNA blotting (data not shown). In this study, a transfectant clone designated L15-1, which showed the highest level of
cadherin-15 expression, was used for further analysis.
Cadherin-15 Protein Expressed in L Cells--
To detect the
cadherin-15 protein, the cadherin-catenin complex was
immunoprecipitated from L15-1 cells with the anti-
-catenin monoclonal antibody. Cadherin-15 protein was detected as a single band
of approximately 124 kDa (Fig. 4). This
band stained with Coomassie Blue was cut out and subjected to
N-terminal amino acid sequencing. The sequence was
Ala-Trp-Val-Ile-Pro-Pro-Ile-Ser-Val-Ser-Glu-Asn (Fig. 1), which agreed
with that expected in the light of the sequences of other classic
cadherins (51). Cadherin-15 molecule expression in L15-1 was also
detected by immunoblotting with a polyclonal anti-mouse M-cadherin
antibody, which cross-reacted weakly with human cadherin-15 (Fig.
5A). Transfection of classic cadherin cDNAs into L cells is known to be accompanied by
up-regulation of catenin proteins, probably because association of
catenins with cadherins retards their turnover (54). Consistent with this observation, L15-1 cells expressed far more
-catenin protein than the parent L cells (Fig. 5B). As a control for further
functional analysis, a mouse E-cadherin-transfectant, LE-1 cells, which
express similar amounts of
-catenin protein, was selected, and the
results are also shown in Fig. 5B. Assuming that cadherin-15
and E-cadherin associate with catenins and are processed and degraded
in the same manner, L15-1 cells can be considered to express virtually the same number of cadherin molecules per cell as LE-1 cells. These two
transfectants expressed similar amounts of
-catenin protein, and
they both expressed more than the parent L cells (data not shown).

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Fig. 4.
Immunoprecipitation from L15-1 cells with an
anti- -catenin monoclonal antibody. L15-1 cell lysates were
precipitated with normal mouse IgG (N) or the
anti- -catenin monoclonal antibody ( ). The precipitates were
denatured, separated by 7.5% SDS-PAGE, transferred to a PVDF membrane,
and stained with AuroDye forte. The cadherin-15, -catenin, and
-catenin bands are indicated by arrowheads. The faint
bands below the -catenin band are -catenin degradation
products, confirmed by immunoblotting (data not shown), and the intense
lower bands are derived from immunoglobulins. The positions of the
protein size markers (200, 116, 97.4, 66.2, and 45 kDa) are indicated
on the left.
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Fig. 5.
Immunoblot analysis of cadherin-15
(A) and -catenin (B). Cell lysates in
1× sample buffer (20 µg·protein/lane) were separated by 7.5%
SDS-PAGE and transferred to PVDF membranes. A, cadherin-15
was detected with a polyclonal anti-mouse M-cadherin antibody
(L, parent L cells; 15, L15-1 cells). The
cadherin-15 bands are indicated by arrowheads. The lower two
bands are probably its degradation products. The nonspecific
reactivities of the antibody are also visible in this figure.
B, -catenin was detected with an anti- -catenin
monoclonal antibody (L, parent L cells; 15, L15-1
cells; E, LE-1 cells). The positions of the protein size
markers (200, 116, 97.4, 66.2, and 45 kDa) are indicated on the
left.
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Cell-Cell Binding Activity and Biochemical Properties of
Cadherin-15--
First, short term cell aggregation experiments were
performed to examine whether cadherin-15 functions as a cell-cell
adhesion molecule. Unexpectedly, L15-1 cells showed only weak
Ca2+-dependent aggregation, whereas LE-1 cells
aggregated strongly in the presence of Ca2+ under the same
conditions (Table II). There are three
possible explanations for this low
Ca2+-dependent aggregation rate of L15-1 cells
as follows: first, cadherin-15 molecules were not exposed on the cell
surfaces; second, unlike E-cadherin, cadherin-15 was not protected by
Ca2+ against trypsin treatment and was degraded during cell
suspension preparation; and third, the cell-cell binding activity of
cadherin-15 was much weaker than that of E-cadherin.
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Table II
Short term aggregation of L cells and transfectants
Cell suspensions obtained after treatment with trypsin in the presence
of Ca2+ were placed in a 24-well plastic plate without (Ca( ))
or with (Ca(+)) 5 mM CaCl2 and allowed to aggregate
at 37 °C for 60 min at 80 rpm. The extent of cell aggregation was
represented by the aggregation index
(n0-n60)/n0,
where n60 and n0 are the total
numbers of particles after incubation for 60 min and at the start of
incubation, respectively.
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As shown in Fig. 6, cadherin-15 molecules
expressed in L15-1 cells were labeled extracellularly with
sulfo-NHS-biotin, as were E-cadherin molecules. Cadherin-15 also showed
a detergent solubility comparable to that of E-cadherin in LE-1 cells
(Fig. 7); a considerable amount of
cadherin-15 could not be extracted with Nonidet P-40. Next, we compared
the trypsin sensitivities of cadherin-15 and E-cadherin. As shown in
Fig. 8, E-cadherin expressed in LE-1
cells showed the characteristic resistance to trypsin treatment in the
presence of Ca2+ that has been documented to be a key
property of the classic cadherins (54). Cadherin-15 expressed in L15-1
cells, however, was not fully protected by Ca2+ against
trypsin; most of the cadherin-15 appeared to be degraded by trypsin in
the presence of Ca2+ (Fig. 8). Interestingly, even in the
absence of Ca2+, a few cadherin-15 molecules, a similar
number to those after trypsin treatment in the presence of
Ca2+ (Fig. 8), remained intact, suggesting that the
cadherin-15 molecules that were resistant to trypsin irrespective of
the presence or absence of Ca2+ might not have been exposed
on the cell surfaces. Taking these findings together, we conclude that
the majority of the cadherin-15 molecules were exposed on the external
cell surfaces and linked to the cytoskeletal system via catenins, in a
similar manner to E-cadherin, and that the low
Ca2+-dependent aggregation rate of L15-1 cells
in the short term cell aggregation experiments was attributable to the
susceptibility of cadherin-15 to trypsin in the presence of
Ca2+.

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Fig. 6.
Exposition of cadherin-15 on L15-1 cell
surfaces. LE-1 and L15-1 cell membrane proteins were biotinylated,
immunoprecipitated with the anti- -catenin monoclonal antibody,
separated by 7.5% SDS-PAGE, and electroblotted onto PVDF membranes.
Then the immunoprecipitates were stained with AuroDye forte to detect
all the components (A) or with diaminobenzidine using the
avidin-biotin-peroxidase complex to detect transmembrane components
exposed on the cell surfaces (B). The E-cadherin,
cadherin-15, -catenin, and -catenin bands are indicated by
arrowheads. Note that the catenins are not biotinylated,
showing this experiment is reliable. The positions of the protein size
markers (200, 116, 97.4, and 66.2 kDa) are indicated on the
left.
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Fig. 7.
Detergent solubility of cadherin-15. The
detergent solubilities of E-cadherin and cadherin-15 expressed in L
cells were examined as described previously (14). Detergent-soluble
(S) and -insoluble (I) fractions of LE-1 and
L15-1 cells were analyzed by immunoblotting using the monoclonal ECCD-2
(LE-1) and polyclonal anti-mouse M-cadherin
(L15-1) antibodies, respectively. Arrowheads
indicate E-cadherin and cadherin-15.
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Fig. 8.
Trypsin sensitivity of cadherin-15. LE-1
and L15-1 cells were treated with HCMF containing 5 mM
CaCl2 (N), 0.01% trypsin and 1 mM
EGTA (TE), or 0.01% trypsin and 5 mM
CaCl2 (TC) at 37 °C for 15 min and were
analyzed by immunoblotting with the monoclonal ECCD-2 (LE-1)
and polyclonal anti-mouse M-cadherin (L15-1) antibodies,
respectively. Whole cell lysates derived from the same numbers of cells
were loaded onto each lane. Arrowheads indicate E-cadherin
and cadherin-15.
|
|
Therefore, we conducted long term cell aggregation experiments in an
attempt to establish whether cadherin-15 really does act as a cell-cell
adhesion molecule. The influence of trypsin treatment was considered
negligible in this assay, because cadherin-15 protein expression in
L15-1 cells recovered to its initial level within 3 h of the
trypsin and EDTA treatment described under "Experimental Procedures" (data not shown). After incubation for 24 h, L15-1 cells formed definite aggregates almost identical in size and cell-cell
adhesiveness to the LE-1 aggregates, whereas virtually no parent L cell
aggregation under the same conditions was observed (Fig.
9), demonstrating that cadherin-15 really
does function as a cell-cell adhesion molecule. Assuming that L15-1 and
LE-1 cells express equivalent numbers of cadherin molecules per cell, as discussed above, we conclude that the cell-cell binding strengths of
cadherin-15 and E-cadherin are virtually the same.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 9.
Long term aggregation of L15-1 cells. L,
LE-1, and L15-1 cells were trypsinized completely in the presence of
EDTA to produce single cell suspensions and suspended in DMEM
supplemented with 10% calf serum and 70 units/ml DNase I. One-hundred
thousand cells (0.5 ml) were placed in each well of a 24-well plastic
plate and allowed to aggregate at 37 °C for 24 h at 100 rpm in
a CO2 incubator, and phase contrast micrographs of unfixed
aggregates were taken. Scale bars, 100 µm.
|
|
To determine the cell-cell binding specificity of cadherin-15, equal
numbers of DiI-labeled L15-1 cells and unlabeled L, LE-1, or L15-1
cells were mixed and allowed to aggregate for 12 h. As can be seen
in Fig. 10, L15-1 cells did not
interact with the parent L cells, indicating that cadherin-15 mediates
cell-cell adhesion in a homophilic manner, as do the other classic
cadherins (55). When L15-1 cells were mixed with LE-1 cells, each
transfectant aggregated separately, and chimeric aggregates were never
found (Fig. 10). In contrast, when DiI-labeled and unlabeled L15-1
cells were mixed, aggregates containing random labeled and unlabeled cells were formed (Fig. 10). L15-1 cells did not interact with P-cadherin, cadherin-6, or cadherin-14 transfectants in another series
of mixed cell aggregation experiments (data not shown). These results
indicate that the cell-cell binding specificity of cadherin-15 is
unique and distinct from those of the other known classic
cadherins.

View larger version (64K):
[in this window]
[in a new window]
|
Fig. 10.
Mixed aggregation of L15-1 cells. Equal
numbers of DiI-labeled L15-1 cells and unlabeled L, LE-1, or L15-1
cells were mixed in DMEM supplemented with 10% calf serum and 70 units/ml DNase I and allowed to aggregate, as described in the legend
to Fig. 9, for 12 h. The resulting aggregates were fixed with
formaldehyde, mounted, and photographed. The upper panels
show phase contrast micrographs of individual mixed cell aggregates,
and the corresponding fluorescence micrographs in the same fields are
shown in the lower panels. Note that although DiI-labeled
and unlabeled L15-1 cells formed randomly mixed aggregates, L15-1 cells
did not form aggregates with unlabeled L or LE-1 cells (shown by
arrowheads). Scale bars, 100 µm.
|
|
 |
DISCUSSION |
Many molecules classified as cadherin superfamily members have
been identified in the past few years. Full cDNA cloning of nine
human classic cadherin molecules has been accomplished so far (3, 29,
30, 31, 35-37), but exactly how many members belong to this family is
unknown. We employed a novel cDNA cloning method based on the
protein interaction between classic cadherins and
-catenin and
identified two novel human classic cadherin molecules, cadherin-14 (39)
and cadherin-15, from a human adult brain cDNA library. Therefore,
this method is considered useful for searching for new members of the
classic cadherin family as well as for unknown molecules that associate
with
-catenin, which is a multifunctional protein involved in both
the cadherin cell adhesion and receptor-mediated intercellular signal
transduction systems (56). We have tested this method on only one
library, but it is possible that applying this method to other
expression cDNA libraries derived from different sources will lead
to the discovery of more novel molecules that interact with
-catenin.
Our cDNA sequence analysis of cadherin-15 revealed that, of the
known cadherins, cadherin-15 showed a very close resemblance to mouse
M-cadherin, which was first identified in muscle cells and is
considered to be involved in the fusion of myoblasts to myotubes (30).
Mouse M-cadherin has been reported to be expressed in skeletal muscle
and cerebellum but not in cardiac or smooth muscle (30, 57-59), an
expression pattern in complete agreement with that of cadherin-15 that
we observed. These two findings suggest strongly that cadherin-15 is a
human homologue of mouse M-cadherin. However, the two molecules differ
markedly, cadherin-15 has a unique 31-amino acid sequence at its C
terminus that has been found neither in mouse M-cadherin nor in the
other known classic cadherins. Data base searches revealed no peptide
sequences similar to this sequence, and what properties its addition
confers on the function and molecular nature of cadherin-15 are
unknown. The linkage with catenins and the cell-cell binding function
of cadherin-15, at least, did not appear to be affected by this
sequence, and no cytoplasmic components that interacted with
cadherin-15 other than catenins were detected in repeated
immunoprecipitation experiments. Molecular biological approaches,
including site-directed mutagenesis, should clarify the significance of
this sequence.
The classic cadherins have been proposed to be divided into two
subgroups, types I and II, on the basis of their overall sequence similarities and conservation of several motifs and aromatic amino acid
residues in their extracellular domains (3, 29). The human classic
cadherins, E-, N-, and P-cadherin and cadherin-4, have been classified
as type I and cadherin-6, -8, -11, -12, and -14 as type II. Although
cadherin-5 was initially reported to be a type II cadherin (29), it was
later found not to resemble the type II cadherins very closely (33,
39). Thus, it would appear that cadherin-5 cannot be classified as
either type I or II. Cadherin-15 is more similar to type I than type II
cadherins (Table I). However, it should be noted that there is an
important difference between cadherin-15 and the other four type I
cadherins. All the type I cadherins have the HAV tripeptide motif in
EC1 which, together with its flanking amino acids, is intimately
involved in the adhesive function and binding specificities of these
cadherins (60-62) but has been replaced by the FAL tripeptide at amino
acid position 123-125 in cadherin-15 (Fig. 1). Incidentally, the type II cadherins cadherin-6, -8, -11, -12, and -14 have QAI or QAD instead
of the HAV motif, and cadherin-5 has VIV at the corresponding position.
Cadherin-15 also exhibited a definitive biochemical difference from
type I cadherins as follows: Ca2+ did not protect it
against trypsin, suggesting the structures of the extracellular domains
of cadherin-15 and type I cadherins differ. Similar trypsin sensitivity
has been reported only for cadherin-5 (63). Moreover, the LDRE motif
found in EC4 of the other human classic cadherins has been replaced by
LSPA in cadherin-15. Therefore, we propose that cadherin-15 and
cadherin-5 cannot be classified as either type I or II cadherins.
As there is no appropriate method for quantifying cadherins at present,
the binding strength of each cadherin subclass cannot be evaluated
precisely. In a cDNA transfection system using L fibroblasts, which
lack cadherin activity and express very little catenin at the protein
level but large amounts at the RNA level, ectopic cadherin expression
induces accumulation of catenin proteins, probably because they are
stabilized by association with cadherins. In this study, we paid
particular attention to the expression level of
-catenin protein,
which associates directly with the cytoplasmic domain of classic
cadherins (13, 38), in transfectants. Assuming that any classic
cadherin subclass influences the preservation of
-catenin protein in
L cells similarly, the binding strength of each cadherin subclass can
be compared using transfectants that express equal amounts of
-catenin protein. Therefore, we compared the aggregation of a
cadherin-15 transfectant with that of an E-cadherin transfectant
expressing almost the same amounts of
-catenin protein. The sizes
and cell-cell adhesiveness of the aggregates were indistinguishable. On
the assumption stated above, it is conceivable that the cell-cell
binding strengths of cadherin-15 and E-cadherin are virtually
equivalent. Finally, our mixed cell aggregation assays showed that
cadherin-15 mediates cell-cell adhesion in a homophilic manner and
exhibits cell-cell binding specificity, i.e. it did not
interact with the molecules expressed on L cells, mouse E-cadherin,
human P-cadherin, cadherin-6, or cadherin-14.
In conclusion, we have isolated a full-length human cadherin-15
cDNA using a novel cDNA cloning method and characterized the cadherin-15 molecule using an L fibroblast cDNA transfection
system. We hope that the technique we have developed and results of
this study will be useful for further investigations into cell-cell interactions.
 |
ACKNOWLEDGEMENT |
We thank Dr. M. Takeichi for providing
pBATEM2, pSTneoB expression vectors, and ECCD-2 monoclonal antibody.
 |
FOOTNOTES |
*
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(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D83542.
¶
To whom correspondence should be addressed: Dept. of Surgery,
National Okura Hospital, 2-10-1 Okura, Setagaya-ku, Tokyo 157, Japan. Tel.: 81-3-3416-0181; Fax: 81-3-3416-2222.
1
The abbreviations used are: EC, extracellular
subdomain; PCR, polymerase chain reaction; M-cadherin, muscle cadherin;
RACE, rapid amplification of cDNA ends; PAGE, polyacrylamide gel
electrophoresis; PVDF, polyvinylidene difluoride; DiI,
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; bp,
base pair; DMEM, Dulbecco's modified Eagle's medium.
 |
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