Molecular Cloning and Characterization of a Novel Human Classic Cadherin Homologous with Mouse Muscle Cadherin*

Yutaka ShimoyamaDagger §, Tatsuhiro Shibatapar , Masaki Kitajima**, and Setsuo HirohashiDagger par

From the Dagger  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 par  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
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Abstract
Introduction
Procedures
Results
Discussion
References

We used a novel cDNA cloning method based on the cadherin-beta -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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

cDNA Cloning-- To use beta -catenin as a probe for the first cDNA cloning procedure based on the protein-protein interaction, we constructed and purified beta -catenin-glutathione S-transferase fusion protein, as described previously (39). This protein was radiolabeled in vitro with [gamma -32P]ATP using bovine heart kinase and was used to screen a human adult brain lambda 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 lambda gt10 cDNA library using a [gamma -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-beta -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-beta -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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Molecular Cloning of Human Cadherin-15-- To identify novel classic cadherin molecules that associate with beta -catenin, a human adult brain lambda gt11 expression cDNA library was screened with a radiolabeled beta -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 beta -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.

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.

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 beta -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 beta -actin on the same membranes.

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 beta -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-beta -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 beta -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 beta -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 alpha -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-beta -catenin monoclonal antibody. L15-1 cell lysates were precipitated with normal mouse IgG (N) or the anti-beta -catenin monoclonal antibody (beta ). The precipitates were denatured, separated by 7.5% SDS-PAGE, transferred to a PVDF membrane, and stained with AuroDye forte. The cadherin-15, alpha -catenin, and beta -catenin bands are indicated by arrowheads. The faint bands below the beta -catenin band are beta -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 beta -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, beta -catenin was detected with an anti-beta -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.

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.

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-beta -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, alpha -catenin, and beta -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.


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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.


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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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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 beta -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 beta -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 beta -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 beta -catenin protein in L cells similarly, the binding strength of each cadherin subclass can be compared using transfectants that express equal amounts of beta -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 beta -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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Abstract
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Results
Discussion
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