Biochemical Characterization and Functional Analysis of Two
Type II Classic Cadherins, Cadherin-6 and -14, and Comparison with
E-cadherin*
Yutaka
Shimoyama
§¶,
Hiroshi
Takeda
,
Shouko
Yoshihara
,
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 300-2635, the § Departments of Surgery and Clinical
Research, National Okura Hospital, 2-10-1 Okura, Setagaya-ku,
Tokyo 157-8535, the
Department of Surgery, School of Medicine,
Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, and
the ** Pathology Division, National Cancer Center Research Institute,
5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
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ABSTRACT |
Classic cadherins can be grouped based on their
deduced primary structures. Among them the type I cadherins have been
well characterized; however, little is known about non-type I
cadherins. In this study we characterized two human type II cadherins,
cadherin-6 and cadherin-14, using a cDNA transfection system. They
were each detected as two bands electrophoretically, were expressed on
the external cell surface at cell-cell contact sites, and were
associated with caten- ins. Direct sequencing of the N-terminal
amino acids showed that the two bands of cadherin-14 corresponded to
precursor and mature forms, whereas the two bands of cadherin-6 both
had the N-terminal sequence of the mature form. Unlike type I
cadherins, both cadherin-6 and -14 were not protected from trypsin
degradation by Ca2+. We evaluated their adhesive
functions by a long term cell aggregation method. The results suggest
that both cadherin-6 and -14 have cell-cell binding strengths virtually
equivalent to that of E-cadherin and that their binding specificities
are distinct from that of E-cadherin. Cadherin-6 and -14 interacted
with each other in an incomplete manner. They have a QAI tripeptide in
the first extracellular subdomain instead of the HAV motif that is
characteristic of type I cadherins and is intimately involved in the
adhesive function. The QAI tripeptide, however, appeared not to be
involved in the adhesive functions of cadherin-6 and -14.
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INTRODUCTION |
Cadherin was originally identified as a cell-cell adhesion
molecule that functions in a Ca2+-dependent and
homophilic manner and that is involved in various morphogenetic events
during development (1). The first cadherins to be identified are known
as the classic cadherins. In the last decade, numerous molecules that
share the extracellular subdomain (EC)1 structure (cadherin
repeat) of the classic cadherins have been discovered in both
vertebrates and invertebrates, and they are now considered to
constitute a large gene family, the cadherin superfamily. Besides the
classic cadherins this family includes truncated type cadherins,
desmosomal cadherins, protocadherins and protocadherin-related
proteins, and HPT/LI-cadherins (2). The biological functions of most of
these non-classic cadherins remain elusive.
Classic cadherins share a common primary structure that consists of a
signal peptide and a prosequence, which are both removed by
intracellular proteolytic processing, five cadherin repeats, a
transmembrane domain, and a highly conserved cytoplasmic domain, which
is essential for association with catenins, the ensuing linkage to the
cytoskeleton, and full functioning as a
Ca2+-dependent cell-cell adhesion molecule
(3-5). Full cDNA cloning of 11 human classic cadherin molecules
has been accomplished so far as follows: E-, N-, and P-cadherin,
cadherin-4 (R-cadherin), -5, -6, -8, -11 (OB-cadherin), -12, -14, and
-15 (M-cadherin) (6-14). Suzuki (2) proposed that the classic
cadherins were divided into two subgroups, type I and type II, on the
basis of their overall sequence similarities and the conservation of
several motifs and aromatic amino acid residues in their extracellular domains. 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 are classified as type II. Although cadherin-5 and -15 (M-cadherin) have been proposed to be type II and I, respectively (2),
we think that they do not clearly belong to either subgroup (14).
The type I classic cadherins, especially E- and N-cadherin, have been
well characterized both functionally and structurally. By contrast,
characterization of the non-type I classic cadherins has just begun,
and it is still not clear whether they behave as do E- and N-cadherin.
It has been suggested that some of them have weaker cell-cell binding
strengths than the type I cadherins because they are expressed in
loosely associated cells (15). In fact, Suzuki and colleagues (2, 16)
reported that cadherin-5 and -8 appear to lack strong cell-cell
adhesion activity. Some of the type II cadherins, however, were
reported to mediate definite cell-cell adhesion (11, 17). Thus, the
relation of the molecular characteristics and functions of these
non-type I classic cadherins to their roles in various tissues is intriguing.
We recently cloned two novel human type II cadherins, cadherin-6 and
-14 (12, 13). In the present study we analyzed the two molecules in
detail using an L fibroblast cDNA transfection system, and we
compared them with E-cadherin. Friedlander et al. (18) and
Steinberg and Takeichi (19) showed that the expression level of a
cadherin can influence cadherin-mediated cell sorting, suggesting that
it is important to check the relative expression level of each
cadherin. However, a method for quantifying the expression of
individual cadherins on the cell surface has not been established, so
it is difficult to compare their binding strengths precisely. In this
context, we have paid particular attention to the expression level of
-catenin protein by our cadherin transfectants in order to
semi-quantify the expression of cadherins and to evaluate their binding
strengths and specificities. In this report we describe and discuss the
functional characteristics of cadherin-6 and -14 together with their
biochemical properties.
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EXPERIMENTAL PROCEDURES |
Expression Vector Construction and Transfection--
To express
human cadherin-6 and -14 in mouse L fibroblasts, the expression vectors
pBAT6H and pBAT14H were constructed by replacing the mouse E-cadherin
cDNA of pBATEM2 (20) with cDNA fragments covering the entire
open reading frames of cadherin-6 and -14, respectively. Transfection
of the expression vectors into L cells was performed using
LipofectAMINE reagent (Life Technologies, Inc.) together with pSTneoB,
which carries the neomycin resistance gene (21), according to the
manufacturer's instructions. The transfected cells were selected in
Dulbecco's modified Eagle's medium (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-6 and -14 expression and maintained under the same conditions.
Mouse E-cadherin transfectants were also obtained using pBATEM2
together with pSTneoB or pMAM2-BSD (Kaken Pharma Co.) and used as
controls. When pMAM2-BSD was used, clones of the transfected cells were
maintained in the presence of 5 µg/ml blasticidin S hydrochloride
(Kaken Pharma Co.) instead of G418. Mouse L fibroblasts (LTK
) were
supplied by the Riken Gene Bank.
We also used a newly reconstructed expression vector, designated pBAX,
to prepare cadherin expression vectors and establish cadherin
transfectants. pBAX was constructed by replacing the promoter region of
pcDNA3 (Invitrogen Corp.) with that of pBATEM2 and reconstructing
the multicloning sites. pBAX with cadherin cDNAs induced the
expression of cadherin proteins in L cells at similar levels to pBATEM2
and its derivatives (data not shown).
To analyze the proteolytic processing of cadherin-6 and the functional
significance of the QAI tripeptide in the first EC (EC1) of cadherin-6
and -14, amino acid substitutions were introduced into cadherin-6 and
-14 by site-directed mutagenesis using the QuikChange Site-directed
Mutagenesis Kit (Stratagene). The mutant molecules were expressed in L
cells as described above.
Antibodies--
To produce a cadherin-6-specific antibody we
generated a cadherin-6 fusion protein. A cadherin-6 cDNA fragment
corresponding to EC5, a region with low homology to the other classic
cadherins (12-14), and containing KpnI and
HindIII sites at the 5'- and 3'-ends, respectively, was
amplified by polymerase chain reaction, digested with KpnI
and HindIII, and ligated to a prokaryotic expression vector,
pRSET B (Invitrogen Corp.) that had been cleaved with the same enzymes.
The resultant plasmid was introduced into an Escherichia
coli strain, BL21(DE3)pLysS (Novagen), and expression of the
fusion protein was induced by 1 mM
isopropyl-
-D-thiogalactoside. The protein was purified
from bacterial lysates using a metal affinity resin (Talon,
CLONTECH). A polyclonal anti-cadherin-6 antibody
was then raised by immunizing rabbits with the fusion protein and was
affinity purified using the same protein. A polyclonal anti-cadherin-14
antibody was raised by immunizing rabbits with a synthetic peptide,
corresponding to the 15 C-terminal amino acids, conjugated to keyhole
limpet hemocyanin, and affinity purified using the same peptide.
An anti-
-catenin monoclonal antibody (Transduction Laboratories) was
used to detect
-catenin by immunoblotting and to immunoprecipitate cadherin-catenin complexes.
Short Term Cell Aggregation--
Dispersed cell suspensions were
obtained by treating cells with 10 mM Hepes-buffered
Ca2+- and Mg2+-free Hanks' solution (HCMF)
containing 0.01% trypsin (crystallized porcine pancreas; Sigma) and 5 mM CaCl2 at 37 °C for 15 min at 80 rpm. Then
they were washed three times with HCMF and suspended at 105
cell particles/ml HCMF containing 1% bovine serum albumin. The cell
suspensions were added to a 24-well plastic plate (0.5 ml/well; Ultra
Low Cluster; Corning Costar Corp.) with or without 5 mM CaCl2, and the cell particles were allowed to aggregate at
37 °C for 60 min at 80 rpm.
Long Term Cell Aggregation--
Completely dispersed cell
suspensions were obtained by treating the cells with phosphate-buffered
saline containing 0.05% trypsin and 0.02% EDTA at 37 °C for 15 min. Then the cells were washed twice with DMEM supplemented with 10%
calf serum and were resuspended in DMEM supplemented with 10% calf
serum and 70 units/ml DNase I (Takara) at a cell density of 2 × 105 cells/ml. The cell suspensions were added to a 24-well
plastic plate (105 cells in 0.5 ml per well; Ultra Low
Cluster; Corning Costar Corp.) and allowed to aggregate at 37 °C for
48 h at 100 rpm in a humidified atmosphere comprising 5%
CO2, 95% air. To examine the heterotypic interactions
between the cadherin subclasses, mixed cell-type aggregation
experiments were performed using two transfectant cell lines
expressing different cadherins; one line was labeled with 40 µg/ml 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (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
cell lines (5 × 104 in 0.25 ml each) were mixed and
allowed to aggregate for 12 h as described above.
Immunoblotting and Immunocytochemistry--
Immunoblot analysis
was carried out as described previously (4) except that the antigens
were detected using the ECL system (Amersham Pharmacia Biotech). To
quantify the amount of
-catenin protein in the cells, the density of
each band of
-catenin was determined by densitometry and normalized
for those of the parent L cells and the LE-5 transfectant cell line,
which expresses mouse E-cadherin at a high level.
L transfectants cultured on plastic dishes were fixed with 4%
paraformaldehyde and 2% sucrose in 10 mM Hepes (pH 7.4),
150 mM NaCl, 2 mM CaCl2 (HNC, a
buffer containing 10 mM Hepes (pH 7.4), 150 mM
NaCl, 2 mM CaCl2) at 4 °C for 10 min and
then with methanol at 4 °C for 30 min. The dishes were heated at
90 °C for 10 min in HNC buffer containing 1% 2-mercaptoethanol and
then rinsed extensively with water and finally with HNC. After
treatment with 5% bovine serum albumin in HNC for 1 h, the cells
were incubated with the affinity purified anti-cadherin-6 or -14 antibody at 4 °C overnight. They were then incubated with
biotinylated anti-rabbit IgG (Vector Laboratories) at room temperature
for 1 h, followed by 1-h incubation with fluorescein
isothiocyanate-conjugated streptavidin (Vector Laboratories). Finally,
after extensive rinsing with HNC, they were mounted with Perma Fluor
(Shandon Lipshaw) and examined with a Zeiss Axiophot microscope.
Other Biochemical Procedures--
Immunoprecipitation,
N-terminal amino acid sequencing, and determination of the trypsin
sensitivity was performed as described previously (14). Exposure of
cadherin molecules on the surface of the transfected L cells was also
examined as described previously (14).
The N-linked oligosaccharides of cadherin-6 were removed by
enzymatic deglycosylation. L6-33 cells were lysed with 50 mM Tris-HCl (pH 8.6), 1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin. The cell lysate
was clarified by centrifugation, heated at 100 °C for 5 min,
incubated with various amounts of glycopeptidase F
(peptide:N-glycosidase F; Takara) at 37 °C for 18 h,
and then analyzed by immunoblotting with the anti-cadherin-6 polyclonal antibody.
 |
RESULTS |
Expression of Cadherin-6 and -14 by L Cells--
L cells, which
are mouse lung fibroblasts deficient in cadherin activity (22), were
transfected with pSTneoB and either pBAT6H or pBAT14H. Over 30 transfectant clones of each cadherin were isolated after G418 selection
and screened for cadherin expression. Concomitantly, transfectant
clones of cells transfected with pSTneoB, pBAX, or pMAM2-BSD, the
control clones, were isolated; they were indistinguishable from the
parent L cells based on the tests used in this study (data not shown).
The transfectant clones designated L6-33 and L14-4 showed the highest
levels of cadherin-6 and -14 expression, respectively, in immunoblots
(Fig. 1). Two bands of protein of
approximately 125 and 120 kDa were labeled by the anti-cadherin-6
polyclonal antibody (Fig. 1A), and two bands of protein of
approximately 112 and 105 kDa were labeled by the anti-cadherin-14
polyclonal antibody (Fig. 1B). As reported previously (23),
these cadherin transfectants exhibited elevated levels of
-catenin
protein (Fig. 1C) as well as
-catenin protein (data not
shown), suggesting that both cadherin-6 and -14 interact with catenins,
form cadherin-catenin complexes, and stabilize catenins, as do other
classic cadherins (24). The association of cadherin-6 and -14 molecules
with catenins was confirmed by immunoprecipitation experiments using
the anti-
-catenin monoclonal antibody. The antibody precipitated
cadherin-6 and -14 molecules, which showed the same mobilities and band
patterns on sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) as in the immunoblotting samples (Fig. 1, A and
B), as well as
- and
-catenins (Fig.
2). To determine the difference between the two forms of cadherin-6 and -14, each band stained with Coomassie Blue was cut from the polyvinylidene difluoride (PVDF) membrane (Millipore), and the N-terminal amino acid sequence was
determined.

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Fig. 1.
Immunoblot analysis of cadherin
transfectants. Cell lysates of L, L6-33, L14-4, L6PRE-2, L6HAV-6,
L14HAV-6, LE-1, and LE-5 cells (20 µg protein/lane) were separated by
7.5% SDS-PAGE and transferred to PVDF membranes. A,
cadherin-6 was detected with an anti-cadherin-6 polyclonal antibody.
The two main bands of cadherin-6 are indicated by arrows.
The lower bands in the L6-33 sample probably
indicate degradation of cadherin-6. B, cadherin-14 was
detected with an anti-cadherin-14 polyclonal antibody. The two main
bands of cadherin-14 are indicated by arrows. The
lower bands in the L14-4 sample probably indicate
degradation of cadherin-14. C, -catenin was detected with
an anti- -catenin monoclonal antibody. The lower bands in
the LE-5 sample probably indicate degradation of
-catenin. Bars on the left indicate the
mobilities of molecular mass markers (200, 116, and 97.4 kDa in
A, 200, 116, 97.4, and 66.2 kDa in B, and 200, 116, 97.4, 66.2, and 45 kDa in C).
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Fig. 2.
Immunoprecipitation of cadherin-6 and -14 with an anti- -catenin monoclonal
antibody. L6-33 or L14-4 cell lysate was incubated 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 Kit (Amersham Pharmacia Biotech). The cadherin-6 and -14 and -
and -catenin bands are indicated by arrows and
arrowheads, respectively. The faint bands below
the -catenin bands are -catenin degradation products, which was
confirmed by immunoblotting (data not shown), and the intense lower
bands are derived from immunoglobulins. Bars on the
left indicate the mobilities of molecular mass markers (200, 116, 97.4, 66.2, and 45 kDa).
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The sequence of the larger (112 kDa) cadherin-14 molecule was
Thr-Ala-His-His-Ser-Ser-Ile-Lys-Val-Met-Arg, indicating that it is a
precursor form of cadherin-14 that is cleaved between Gly-24 and Thr-25
by signal peptidases. This site agrees with possible cleavage sites of
eukaryotic signal peptides proposed by Nielsen et al. (25).
The N-terminal sequence of the shorter cadherin-14 molecule (105 kDa)
was Gly-Trp-Val-Trp-Asn-Gln-Phe-Phe-Val-Leu-Glu-Glu, indicating that it
is a mature form of cadherin-14 that is cleaved just after a pair of
basic amino acids (Lys-52, Arg-53) by endopeptidases. By contrast, the
two cadherin-6 peptides (125 and 120 kDa) had the same N-terminal
sequence, Ser-Trp-Met-Trp-Asn- Gln-Phe-Phe-Leu-Leu-Glu-Glu, indicating
that they both correspond to a putative mature form of cadherin-6 (12)
that is proteolytically processed.
We wondered whether one of the two cadherin-6 peptides was an
N-terminally blocked non-mature form, and this result was due to
contamination of this band by the mature form, because the two bands
were contiguous (Figs. 1A and 2). We repeated the analysis and obtained the same result. Next we prepared a mutant cadherin-6 molecule with a single amino acid substitution of Arg-53 to Gly, which
would not be cleaved to the mature form of cadherin-6 and which would
correspond to a precursor form of cadherin-6. To do this we replaced an
A with a G at nucleotide position 276, expressed the mutant in L cells,
and compared the gene product with the two molecular forms of
cadherin-6 by immunoblotting (Fig. 3). The immunoblot analysis showed that the mutant cadherin-6 expressed in
an L cell transfectant clone, designated L6PRE-2, also resulted in two
protein bands on SDS-PAGE, with higher molecular masses than the
wild-type cadherin-6 molecule expressed by L6-33 cells. These findings
show that the larger wild-type cadherin-6 is not a precursor form,
unlike the larger cadherin-14 protein, and suggest that both bands of
cadherin-6 were mature forms.

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Fig. 3.
Immunoblot analysis of wild-type and mutant
cadherin-6. Cell lysates of L6-33, L6PRE-2, and L6HAV-6 cells were
separated by 7.5% SDS-PAGE, transferred to PVDF membranes, and probed
with an anti-cadherin-6 polyclonal antibody. The amount of protein in
each lane was not adjusted in this blot. The lower bands in
all the lanes probably indicate degradation of cadherin-6.
Bars on the left indicate the mobilities of
molecular mass markers (200, 116, 97.4, and 66.2 kDa).
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We previously reported that the deduced cadherin-6 protein sequence
contains five possible N-linked glycosylation sites (12). To
examine the influence of glycosylation on the mobility of cadherin-6, N-linked oligosaccharides were removed enzymatically. Fig.
4 shows the N-deglycosylation
effect of various concentrations of glycopeptidase F on cadherin-6; the
molecular masses of the pair of cadherin-6 bands were reduced stepwise
as the enzyme concentration increased; however, the two-band pattern
never changed. We also used tunicamycin, an inhibitor of
N-linked oligosaccharide synthesis, but the two-band pattern
remained unchanged (data not shown).

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Fig. 4.
N-Deglycosylation effect of
glycopeptidase F on cadherin-6. L6-33 cell lysate was treated with
glycopeptidase F at 0 (lane 1), 0.005 (lane 2),
0.02 (lane 3), 0.05 (lane 4), 0.2 (lane
5), 0.5 (lane 6), 2.0 (lane 7), 5.0 (lane 8), and 50 milliunits/ml (lane 9) at
37 °C for 18 h and then analyzed by immunoblotting with an
anti-cadherin-6 polyclonal antibody. Bars on the
left indicate the mobilities of molecular mass markers (200, 116, and 97.4 kDa).
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The subcellular distributions of cadherin-6 and -14 in the transfectant
clones L6-33 and L14-4, respectively, were examined immunocytochemically (Fig. 5). As
expected, both cadherin-6 and -14 were concentrated at cell-cell
contact sites. Furthermore, exposure of cadherin-6 and -14 molecules on
the external cell surface was confirmed by extracellular biotin
labeling of the cadherins (Fig. 6).
Unexpectedly, this experiment showed that not only the mature forms of
cadherin-6 and -14 but also the precursor form of cadherin-14 were
exposed on the cell surface.

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Fig. 5.
Immunocytochemical detection of cadherin-6
and -14. Cadherin-6 expressed in L6-33 and cadherin-14 expressed
in L14-4 cells were detected by immunofluorescent staining with an
anti-cadherin-6 polyclonal antibody and an anti-cadherin-14 polyclonal
antibody, respectively. Both cadherins were concentrated at cell-cell
contact sites. Scale bar, 100 µm.
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Fig. 6.
Exposure of cadherin-6 and -14 on cell
surfaces. L6-33 and L14-4 cell membrane proteins were biotinylated
extracellularly, immunoprecipitated with an anti- -catenin monoclonal
antibody, separated by 7.5% SDS-PAGE, and electroblotted onto PVDF
membranes. Then the immunoprecipitates were stained with AuroDye Forte
Kit to detect all of the proteins (A) or with
diaminobenzidine using the avidin-biotin-peroxidase complex (Elite ABC;
Vector Laboratories) to detect proteins exposed on the cell surface
(B). The cadherin-6, cadherin-14, -catenin, and
-catenin bands are indicated by arrowheads. Both forms of
cadherin-6 and -14 were biotinylated, indicating that all four proteins
were exposed on the external cell surface. Note that the catenins were
not biotinylated, demonstrating the reliability of this experiment.
Bars on the left indicate the mobilities of
molecular mass markers (200, 116, and 97.4 kDa).
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Cell-Cell Binding Activities and Biochemical Properties of
Cadherin-6 and -14--
The exposure of cadherin-6 and -14 at
cell-cell contact sites, as shown in Figs. 5 and 6, suggests that these
molecules function as intercellular connectors, as do type I cadherins.
We first examined their cell-cell binding activities using a
conventional method, the short term cell aggregation assay. We also
examined the up-regulation of
-catenin protein expression in L cells
with introduced cadherins (Fig. 1C). Assuming that the
different cadherins interact with
-catenin and contribute to its
preservation in a similar manner, the binding strengths of the
cadherins can be compared using transfectants that express equal
amounts of
-catenin protein. Both the L6-33 and L14-4 cells,
however, exhibited only weak Ca2+-dependent
cell-cell aggregation in this assay system as compared with LE-1 cells
and other E-cadherin transfectants (data not shown) that express almost
the same amount of
-catenin (Table
I).
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Table I
Short-term cell aggregation of cadherin transfectants
Cell suspensions obtained after treatment with trypsin in the presence
of Ca2+ were plated into 24-well plastic plates without
(Ca( )) or with (Ca(+)) 5 mM CaCl2 in triplicate
and allowed to aggregate at 37 °C for 60 min at 80 rpm. The extent
of cell aggregation is represented by the aggregation index
(n0-n60)/n0,
where n60 and n0 are the mean
total numbers of particles after incubation for 60 min and at the start
of incubation, respectively. Values are the means of at least three
separate experiments.
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We recently reported that the susceptibility of cadherin-15 to trypsin
digestion in the presence of Ca+ causes a low aggregation
rate in the short term assay (14). We therefore examined the trypsin
sensitivities of cadherin-6 and -14 (Fig.
7). Both molecular forms of cadherin-6
were digested by trypsin treatment, even in the presence of
Ca2+, whereas most of the mature form of cadherin-14 was
digested by trypsin but a small fragment remained regardless of the
presence of Ca2+. The mature form of cadherin-14 appeared
to be slightly more stable in the presence of Ca2+ than in
the absence of Ca2+, and partially digested molecules were
detected only in the presence of Ca2+. Interestingly, a
fragment of the precursor form of cadherin-14 was detected after the
trypsin treatment. These results suggest that the low
Ca2+-dependent cell-cell aggregation rates
described above were due to the digestion of cadherin-6 and -14 by
trypsin and thus that the conventional aggregation assay cannot be used
to evaluate the cell-cell adhesion activities of these cadherins
correctly.

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Fig. 7.
Trypsin sensitivity of cadherin-6 and
-14. L6-33 and L14-4 cells were treated with HCMF containing 5 mM CaCl2 (N), or 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 an anti-cadherin-6 polyclonal
antibody and an anti-cadherin-14 polyclonal antibody, respectively.
Whole cell lysates derived from the same number of cells were loaded
onto each lane. A possible precursor form of cadherin-14, which was
reduced in molecular mass by trypsin treatment, was detected
(arrow). A partially digested fragment of cadherin-14 was
also detected (arrowhead). Bars on the
left indicate the mobilities of molecular mass markers (200, 116, 97.4, and 66.2 kDa).
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After treatment with trypsin and EDTA to destroy most of the
E-cadherin, cadherin-6, and cadherin-14 molecules expressed by the
transfected L cells, the expression levels of these cadherins returned
to the initial levels within 3 h (data not shown). Therefore, we
repeated the cell aggregation assay using a 48-h aggregation period to
compare the cell-cell binding activity of each cadherin. The influence
of trypsin pretreatment to disperse the cells was considered to be
negligible in these assays. The results are shown in Fig.
8. After 48-h incubations L6-33 and L14-4
cells formed aggregates that were almost indistinguishable in both size
and cell-cell adhesiveness from LE-1 aggregates, whereas virtually no
parent L cell aggregation was observed under the same conditions (Fig.
8). Therefore, both cadherin-6 and -14 do function as cell-cell connectors. Taking account of the almost equivalent level of
-catenin protein expressed in these three transfectant cell lines
(Fig. 1 and Table I) and assuming that these lines express almost
equivalent numbers of cadherin molecules per cell, these results
suggest that the cell-cell binding strengths of these three cadherins are virtually the same. Our observations that LE-5 cells with a higher
-catenin expression level (Fig. 1 and Table I) formed larger and
tighter aggregates (Fig. 8) and that many transfectant lines that
expressed
-catenin at lower levels than L6-33, L14-4, and LE-1 cells
formed smaller aggregates (data not shown) support the above idea. In
addition, L6PRE-2 cells, which expressed a mutant cadherin-6 molecule
corresponding to its precursor form (Fig. 3), did not show any
significant aggregation (Fig. 8). This result indicates that, as has
been demonstrated for E-cadherin (26), cadherin-6 requires the removal
of the precursor segment by proteolytic processing for its cell-cell
binding activity.

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Fig. 8.
Long term cell aggregation of cadherin
transfectants. L (A), L6-33 (B), L14-4
(C), LE-1 (D), LE-5 (E), L6PRE-2
(F), L6HAV-6 (G), and L14HAV-6 (H)
cells were trypsinized completely in the presence of EDTA to obtain
single cells, which were suspended in DMEM supplemented with 10% calf
serum and 70 units/ml DNase I. The cells (105 cells in 0.5 ml per well) were plated in a 24-well plastic plate and allowed to
aggregate at 37 °C for 48 h at 100 rpm in a CO2
incubator. Then phase-contrast micrographs were taken. Scale
bar, 200 µm.
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We next performed mixed cell aggregation experiments to determine the
cell-cell binding specificities of cadherin-6 and -14. First, L6-33
cells or L14-4 cells were mixed with L cells and allowed to aggregate;
both the L6-33 cells and L14-4 cells formed aggregates that excluded
the parent L cells (data not shown), indicating that cadherin-6 and -14 mediate cell-cell adhesion in a homophilic manner, as do other classic
cadherins (1, 14). Next, one cell line was labeled with
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate
(DiI) reagent, mixed with another unlabeled line, and allowed to
aggregate for 12 h. Representative results are shown in Fig.
9. When DiI-labeled and unlabeled cells
of the same cell line were mixed, randomly intermixed aggregates were
formed (Fig. 9, A and B). When LE-1 cells were
mixed with L6-33 or L14-4 cells, the LE-1 cells aggregated separately
from the L6-33 or L14-4 cells, and chimeric aggregates were not found
(Fig. 9, C and D). Interestingly, when L6-33 and
L14-4 cells were mixed, chimeric aggregates composed of clusters of
each cell type were formed (Fig. 9, E and F). We
performed this set of experiments repeatedly and always observed
similarly intermixed aggregates. These findings indicate that
cadherin-6 and -14 possess binding specificities distinct from that of
E-cadherin and that they partially interact with each other.

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Fig. 9.
Mixed cell aggregation of cadherin
transfectants. DiI-labeled L6-33 cells (5 × 104)
were mixed with an equal number of unlabeled L6-33 (A and
B), LE-1 (C and D), or L14-4 cells
(E and F) in DMEM supplemented with 10% calf
serum and 70 units/ml DNase I and were allowed to aggregate, as
described in the legend to Fig. 8, for 12 h. Phase-contrast
(A, C, and E) and fluorescence
(B, D, and F) micrographs of the same
fields are shown. DiI-labeled and unlabeled L6-33 cells formed randomly
mixed aggregates (A and B). When DiI-labeled
L6-33 cells were mixed with unlabeled LE-1 cells, mixed cell aggregates
were not seen (C and D), whereas L6-33 and L14-4
cells formed chimeric aggregates composed of clusters of each cell type
(E and F). Scale bar, 100 µm.
|
|
The HAV motif in the EC1 of type I classic cadherins is intimately
involved in their adhesive functions (27-29), whereas the HAV motif is
replaced by a QAI tripeptide in cadherin-6 and in cadherin-14. To
determine the significance of the QAI tripeptide to the adhesive
functions of cadherin-6 and -14, mutant cadherin-6 and -14 cDNAs
that encoded HAV instead of the QAI tripeptide were constructed by
replacing the A at nucleotide positions 507 and 511 (12) with C and G,
respectively, in cadherin-6 and by replacing the A at nucleotide
positions 700 and 704 (13) with C and G, respectively, in cadherin-14.
These mutated cadherin-6 and -14 cDNAs were introduced into L cells
using the pBAX expression vector, and transfectant lines stably
expressing each mutant molecule were cloned. The L6HAV-6 and L14HAV-6
lines, which showed the highest expression of the mutant cadherin-6 and
-14 molecules, respectively, were used for further analyses. These
mutant molecules appeared to be indistinguishable from the respective
wild-type molecules except that the mutant cadherin-6 molecule migrated more slowly than the wild-type cadherin-6 on SDS-PAGE (Fig. 3). The
mutant cadherin-14 molecule showed almost the same mobility as that of
the wild-type cadherin-14 on SDS-PAGE (data not shown). Compared with
L6-33 and L14-4 cells, L6HAV-6 and L14HAV-6 cells showed lower
expression of
-catenin protein (Fig. 1 and Table I), and they formed
smaller aggregates in long term cell aggregation experiments (Fig.
8).
L6HAV-6 and L14HAV-6 cells were tested in mixed cell aggregation
experiments to examine the effect of the conversion of the QAI
tripeptide to HAV on the adhesive functions of cadherin-6 and
cadherin-14. Representative results are shown in Fig.
10, and the interactions between
different cadherins are summarized in Table
II. L6HAV-6 and L14HAV-6 cells formed
almost randomly mixed aggregates with L6-33 and L14-4 cells,
respectively (Fig. 10, A, B, E, and
F). When L6HAV-6 and L14-4 cells, as well as L14HAV-6 and
L6-33 cells, were mixed chimeric aggregates composed of clusters of
each cell type were formed (Fig. 10, C, D,
G, and H). Moreover, L6HAV-6 and L14HAV-6 cells never formed
chimeric aggregates with LE-1 cells (Table II). Thus, the conversion of
QAI to HAV did not appear to affect the binding specificities of
cadherin-6 and -14 in this series of experiments. We also examined the
effects of synthetic peptides, including the QAI tripeptide (LRAQAINRRT for cadherin-6 and LHAQAIDRRT for cadherin-14), on cell aggregation of
cadherin-6 and cadherin-14 transfectants, but we did not observe any inhibitory effects (data not shown).

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Fig. 10.
Effect of a mutation of QAI to HAV in
cadherin-6 and -14 on mixed cell aggregation. Equal numbers
(5 × 104) of DiI-labeled L6HAV-6 and unlabeled L6-33
cells (A and B), DiI-labeled L6HAV-6 and
unlabeled L14-4 cells (C and D), DiI-labeled
L14HAV-6 and unlabeled L14-4 cells (E and F), or
DiI-labeled L14HAV-6 and unlabeled L6-33 cells (G and
H) were mixed and allowed to aggregate as in Fig. 9.
Phase-contrast (A, C, E, and
G) and fluorescence (B, D,
F, and H) micrographs of the same fields are
shown. Scale bar, 100 µm.
|
|
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Table II
Summary of interactions between cadherins
This table is based on the results of mixed cell aggregation
experiments.
|
|
 |
DISCUSSION |
Many members of the cadherin superfamily have been discovered in
the last decade; over 20 classic cadherins have been identified in
various vertebrates. To our knowledge, in humans 11 classic cadherins
have been characterized so far by full cDNA cloning and sequencing.
The classic cadherins can be divided into subtypes (type I, type II,
and others) on the basis of their deduced amino acid sequences (2, 14).
Among the classic cadherins, E- and N-cadherin, both of which are type
I cadherins, have been investigated most thoroughly and have been well
characterized; however, little is known about the other, especially the
non-type I, cadherins. We therefore planned to analyze the molecular
nature of cadherin-6 and -14, both of which are classified as type II
classic cadherins.
Cadherin-6 and -14 cDNAs were introduced into mouse L fibroblasts,
and transfectant clones stably expressing each cadherin were isolated.
Immunoprecipitation and immunoblot experiments showed that these
cadherins interacted with and stabilized catenins, as does E-cadherin.
On SDS-PAGE cadherin-6 and -14 were seen as two bands, 125 and 120 kDa,
and 112 and 105 kDa proteins, respectively. It is widely thought that
transcribed cadherin molecules are cleaved by signal peptidases on the
endoplasmic reticulum membranes and that the resultant precursor forms
undergo further proteolytic processing by endoproteases to generate the
functional mature forms (26). Indeed, a faint band is often observed
just above the main band of a cadherin on immunoblotting, which may
correspond to the precursor form (30). Here, we demonstrated that the
larger of the two cadherin-14 proteins seen on SDS-PAGE was indeed a precursor that had been cleaved just after the signal peptide and that
at least part of the precursor was exposed on the cell surface similar
to the mature form. The significance of the cadherin-14 precursor on
the cell surface remains unclear at present. We also detected two
cadherin-6-positive bands; however, direct sequencing of each protein
showed that they had the same N terminus as the putative mature form of
cadherin-6; both were expressed on the external cell surface, and they
had similar trypsin sensitivities and detergent
solubilities.2 Interestingly,
cadherin-6 expressed in various human cell lines also shows the same
two-band pattern.2
The phosphorylation of a protein often alters its mobility on SDS-PAGE.
We therefore examined, by potato acid phosphatase treatment (31) and
immunoblot analysis using antibodies against phosphoamino acid residues
(32), whether the two forms of cadherin-6 are phosphorylated and
non-phosphorylated forms of the mature cadherin-6 protein, but
phosphorylation of cadherin-6 does not seem to alter its
electrophoretic mobility.2 It is also unlikely that there
is post-translational modification of the C-terminal region, because
anti-pan cadherin rabbit serum (Sigma) raised against the C-terminal
sequence of chicken N-cadherin detects both bands on
immunoblots.2
It is well known that cadherins undergo sugar modifications (33, 34)
and that cadherin-6 has five consensus sites for N-linked
glycosylation (12). Therefore, we suspected that the two molecular
forms of cadherin-6 might represent different stages of glycosylation.
However, the two bands were not replaced by a single band after
treatment with glycopeptidase F or tunicamycin, although these
experiments could not rule out the above possibility completely. Note
that in the N-deglycosylation experiment using glycopeptidase F, the relative molecular mass of cadherin-6 became smaller in a stepwise manner as the enzyme concentration increased (Fig. 4). For example, the upper band of cadherin-6 is clearly seen in
the 0.5 milliunit/ml glycopeptidase F sample, but this band becomes
faint at 2 milliunits/ml glycopeptidase F and is absent at 5 milliunits/ml glycopeptidase F; the cadherin-6 molecule corresponding
to the upper band at 0.5 milliunit/ml glycopeptidase F appears to be
migrating at the same rate as the lower band with the advance of
deglycosylation (Fig. 4). If this is the case, the difference between
the two molecular forms of mature cadherin-6 may result from one
N-linked oligosaccharide chain that is resistant to
experimental deglycosylation. At present, this issue and the difference
in adhesive function between the two forms remains to be resolved.
At an early stage of cadherin research the resistance of cadherin to
trypsin degradation in the presence of Ca2+ was considered
a hallmark of the cadherin family (1). However, the present study has
demonstrated clearly that both cadherin-6 and -14 were not fully
protected from trypsin by Ca2+. We reported recently that
cadherin-15, a human homologue of mouse M-cadherin, is also sensitive
to trypsin in the presence of Ca2+ (14) and Tanihara
et al. (16) described similar trypsin sensitivity for
cadherin-5. Thus, protection from trypsin degradation by
Ca2+ may not be a common feature of cadherin family
members, particularly for non-type I classic cadherins.
Cell-cell adhesion activities of cadherins have been evaluated mainly
by short term cell aggregation assays, in which cells dispersed by
trypsin treatment in the presence of Ca2+ were studied
(35). However, we have found that this assay system does not always
accurately evaluate the cell-cell adhesive functions of cadherins.
Therefore, we used a long term cell aggregation assay in this study.
Another important point to be considered when evaluating cadherin
function is how individual cadherin molecules are quantified.
Therefore, we measured the expression level of
-catenin protein in
the cadherin transfectants used in this study. We assumed that in the L
fibroblast cDNA transfection system each classic cadherin subclass
interacts with and stabilizes
-catenin in a similar manner and that,
therefore, the binding strength and specificity of each cadherin
subclass can be compared using transfectants that express equal amounts
of
-catenin protein. Thus, we used cadherin-6, cadherin-14, and
E-cadherin transfectants that expressed almost the same amount of
-catenin protein to evaluate the adhesive functions of these
cadherins. In the long term cell aggregation experiments, all of the
transfectants formed aggregates that were similar in both size and
cell-cell adhesiveness, suggesting that the cell-cell binding strengths
of cadherin-6, cadherin-14, and E-cadherin are virtually equivalent.
Moreover, the mixed cell aggregation experiments showed that both
cadherin-6 and -14 had binding specificities distinct from that of
E-cadherin. Interestingly, these experiments also showed that
cadherin-6 and -14 can interact with each other in an incomplete
manner. Similar heterophilic interactions between cadherins from the
same species have been described for N-cadherin and R-cadherin
(cadherin-4) (36, 37), chick B-cadherin and liver cell adhesion
molecule (L-CAM) (38), and chick cadherin-6B and -7 (17). It is
possible that heterophilic interactions between cadherins of different subclasses are responsible for the interaction of adjacent tissues in vivo, because in the mixed cell aggregation experiments
referred to above chimeric aggregates composed of clusters of each cell type were observed, except for the combination of chick B-cadherin and
L-CAM. Much more work is necessary, however, to elucidate the
significance of these interactions.
It is widely accepted that the HAV tripeptide motif, which resides in
EC1 of type I classic cadherins, and its flanking amino acids is
intimately involved in the adhesive function and binding specificity
(27-29). In non-type I classic cadherins, however, the HAV motif is
replaced by other tripeptides (14), but it is not known whether these
tripeptides are involved in the binding functions of the cadherins.
Both cadherin-6 and -14 have a QAI tripeptide instead of the HAV motif
(12, 13), prompting us to investigate whether the QAI motif is involved
in the binding specificities of cadherin-6 and -14, especially the
partial interaction between cadherin-6 and -14. We constructed mutant
cadherin-6 and -14 molecules that had the HAV motif instead of QAI, but
this mutation had no effect on the binding specificities of cadherin-6 and -14. We also examined the effects of synthetic peptides, including the QAI tripeptide, on cell aggregation of cadherin-6 and cadherin-14 transfectants, but they did not show any inhibitory effects. Thus, we
did not observe any evidence that the QAI tripeptide and its flanking
amino acids are involved in the adhesive functions of cadherin-6 and
-14 and the region that is responsible for the adhesive functions of
the non-type I classic cadherins remains to be identified. More
detailed studies including structural analyses will provide answers to
this question.
 |
ACKNOWLEDGEMENT |
We thank Dr. M. Takeichi for providing the
pBATEM2 and pSTneoB expression vectors.
 |
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.
¶
To whom correspondence should be addressed: Dept. of Surgery,
National Okura Hospital, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan. Tel: 81-3-3416-0181; Fax: 81-3-3416-2222.
2
Y. Shimoyama, H. Takeda, S. Yoshihara, M. Kitajima, and S. Hirohashi, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
EC, extracellular
subdomain;
DMEM, Dulbecco's modified Eagle's medium;
PAGE, polyacrylamide gel electrophoresis;
PVDF, polyvinylidene difluoride;
DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate.
 |
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