©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Isolation and cDNA Cloning of Ksp-cadherin, a Novel Kidney-specific Member of the Cadherin Multigene Family (*)

(Received for publication, January 12, 1995; and in revised form, April 27, 1995)

R. Brent Thomson , Peter Igarashi , Daniel Biemesderfer , Robert Kim , Ali Abu-Alfa , Manoocher Soleimani , Peter S. Aronson (§)

From the Department of Internal Medicine, Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cadherins are recognized as the principal mediators of homotypic cellular recognition and play a demonstrated role in the morphogenic direction of tissue development. We report here the identification of a structurally unique, kidney-specific member of the cadherin multigene family (Ksp-cadherin). cDNA cloning and molecular analysis of the 130-kDa protein confirmed that it was novel and indicated that it most closely resembled members of the LI-cadherin/HPT-1 cadherin subgroup. The predicted protein possesses the definitive cadherin-specific sequence motifs LDRE, DXND, and DXD in well conserved sequential arrangement, and the characteristic cysteine residues found in the last ectodomains of almost all known cadherins. Like LI-cadherin and HPT-1, Ksp-cadherin lacks the prosequence and HAV adhesion recognition sequence typical of most classical cadherins, and possesses a truncated cytoplasmic domain (18-22 amino acids). When expressed in a transient Vaccinia/T7 expression system, Ksp-cadherin displayed the classic calcium sensitivity to trypsin proteolysis that is observed in all cadherins. Immunolocalization studies and Northern analysis indicated that expression of Ksp-cadherin was kidney-specific and limited to the basolateral membranes of renal tubular epithelial cells. In summary, we have identified and cloned a novel, kidney-specific member of the cadherin multigene family that we propose be designated Ksp-cadherin.


INTRODUCTION

The cadherin superfamily is a large and extremely diverse group of calcium-dependent, membrane-associated glycoproteins (for reviews see (1, 2, 3, 4, 5) ). Members of this group have been identified in virtually every vertebrate tissue type, and structural homologues have been identified in several invertebrate phyla (see (6) and (7) for examples). As a group, cadherins play a prominent role in the mediation of intercellular interactions (particularly homotypic cell recognition and adhesion). Specifically, cadherins have been implicated in embryogenesis(8) , tissue morphogenesis and maintenance of tissue structure(9) , cell polarization(10) , neoplastic invasiveness and metastasis(11) , and most recently, membrane transport(12) .

Vertebrate cadherins can be subdivided into several distinct groups on the basis of amino acid sequence homology, cellular localization, and proposed function (e.g. the classical cadherins, T-cadherin, LI-cadherin and HPT-1, the desmosomal cadherins, the ret proto-oncogene, and the proto-cadherins). All of the vertebrate cadherins identified to date (with the exception of T-cadherin; (13) ) possess a large N-terminal ectoplasmic domain, a single hydrophobic membrane-spanning region, and a subgroup-specific C-terminal cytoplasmic domain. The cadherin ectodomains are typically organized in a tandem series of repeats (usually four; approximately 110 amino acids each) followed by a less conserved ectodomain proximal to the plasma membrane. The distal ectodomains contain the hallmark cadherin-specific sequence motifs LDRE, DXND, and DXD in a highly conserved sequential arrangement. The final ectodomain usually contains four cysteine residues whose locations relative to the plasma membrane are extraordinarily well conserved among most cadherins.

Individual cadherins tend to be associated with characteristic tissue types (e.g. N-cadherin with neural tissue or E-cadherin with epithelial tissue), but many cadherins also have distinct secondary distributions(4) . These distributions often overlap that of other cadherins, and it has been suggested that cadherin-meditated cell recognition may be facilitated by recognition of specific combinations of cadherins, rather than by recognition of single, tissue-specific, signature molecules(3) . Moreover, it appears that cadherins can mediate a variety of context-specific functions, the exact nature of which depends on the site of expression and the stage of tissue development(5) .

In the present study, we report the identification of a novel kidney-specific member of the cadherin multigene family (which we propose be designated Ksp-cadherin).() cDNA cloning and molecular analysis of Ksp-cadherin indicate that it is a structurally unique member of the cadherin superfamily and that it most closely resembles members of the LI-cadherin/HPT-1 subgroup. Unlike most cadherins, Ksp-cadherin has a distinct organ-specific tissue distribution. Immunolocalization studies and Northern analysis indicate that expression of Ksp-cadherin is kidney-specific and confined to the basolateral membranes of the proximal tubule, the thick and thin limbs of the loop of Henle, the distal convoluted tubule, and a distinct subpopulation of cells in both the connecting tubule and the collecting duct in the adult rabbit kidney.


MATERIALS AND METHODS

Membrane Preparation

Basolateral membrane vesicles and cortical microsomes were isolated from renal cortices of male New Zealand White rabbits as described previously(14) . Membrane fractions were isolated and stored in 250 mM sucrose, 10 mM HEPES, 1 mM EDTA, 1 µM pepstatin, 1 µM leupeptin, 230 µM phenylmethanesulfonyl fluoride (PMSF), pH 7.5 at -70 °C for 4-6 weeks.

Stilbene Affinity Purification

DNDS affinity columns were prepared by nonspecific immobilization of DNDS in acrylamide beads in a manner similar to that described by Uchida and Filburn (15; see also (16) ). NADS affinity columns were prepared by covalent coupling of NADS to Affi-Gel 10 (Bio-Rad).

Membrane fractions were solubilized in membrane solubilization buffer (0.67% Triton X-100, 40 mM HEPES, 1 mM dithiothreitol (DTT), 230 µM PMSF, pH 8.1) at a protein concentration of 10 mg/ml for 1 h. The Triton X-100-soluble component was then incubated with either the DNDS or NADS affinity matrix for 60 or 30 min, respectively. The unbound material was removed and the affinity matrices were washed four times with five column volumes of wash buffer (0.1% Triton X-100, 40 mM HEPES, 1 mM DTT, 230 µM PMSF, pH 8.1). Bound proteins were eluted by incubating the DNDS or NADS affinity matrices with three column volumes of elution buffer (10 mM DNDS, 0.1% Triton X-100, 40 mM HEPES, 1 mM DTT, 230 µM PMSF, pH 8.1) for 45 or 30 min, respectively. All steps were performed at 4 °C. Protein binding to the affinity matrices was monitored by SDS-PAGE.

Monoclonal Antibody Production

The 71-kDa antigen was gel-purified from NADS affinity column eluates by SDS-PAGE. BALB/c mice were immunized intraperitoneally with 10-15 µg of antigen adsorbed onto potassium alum as described by Kashgarian et al.(17) . Hybridomas were screened by enzyme-linked immunosorbent assay (18) against rabbit renal cortical basolateral membrane vesicles, by immunoblot (19) against stilbene affinity column eluates, and by immunofluorescent localization (20) in rabbit kidney. Nine of the resulting hybridomas produced antibodies that specifically recognized the 71-kDa protein. These hybridomas were subcloned by limiting dilution and screened once more (with the same criteria) to assure continued antibody production.

All immunochemical analyses performed in this study were conducted with monoclonal antibodies isolated from the hybridoma clone C575-5I. This specific clone was selected for routine use because of its extremely high antibody titers.

Peptide Mapping

Proteins of interest were gel-purified by SDS-PAGE, electroeluted from excised gel slices into a 10 mM NHHCO, 0.02% SDS buffer(21) , concentrated to 50-60 µg of protein/ml, and stored at -70 °C. Peptide fragment maps were generated by partial digestion of the purified proteins with endoproteinase Glu-C (V8; Boehringer Mannheim) as described by Cleveland(22) .

Immunoaffinity Purification

An immunoaffinity chromatography matrix was prepared by coupling C575-5I to Protein A-Sepharose CL-4B (Pharmacia) through a rabbit anti-mouse IgG bridge as described by Schneider et al.(23) .

Triton X-100-solubilized membrane fractions (solubilized at 10 mg of protein/ml in 0.67% Triton X-100, 1 mM EDTA, 1 µM pepstatin, 1 µM leupeptin, and 230 µM PMSF in phosphate-buffered saline) were incubated with the immunoaffinity matrix for 16 h at 4 °C. The supernatant was removed and the matrix was washed twice with salt wash buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM NaEDTA, 0.1% Triton X-100, pH 7.4, 20 °C), four times with RIPA buffer (20 mM Tris-HCl, 0.5% Triton X-100, 1% deoxycholate, 0.1% SDS, 150 mM NaCl, 2.5 mM NaEDTA, pH 7.4, 20 °C), and finally twice more with salt wash buffer. Bound antigen was eluted by incubating the matrix with immunoaffinity elution buffer (50 mM glycine-HCl, 0.1% Triton X-100, pH 2.7) for 2 min at 20 °C. The eluate was immediately neutralized with 1 M Tris base. This was repeated four times, and the eluates were pooled and then concentrated to approximately 60 µl. Column yield was assessed by SDS-PAGE and immunoblotting with C575-5I.

N-terminal Protein Sequencing

The 71-kDa protein was isolated by gel purification and electroelution as described above. 16 µg of the purified 71-kDa protein were partially digested with 0.1 µg of V8 protease in a 10-20% SDS-polyacrylamide gradient gel. The resulting peptide fragments were then electroblotted onto a polyvinylidene difluoride filter. To guard against the possibility of sequencing a contaminant, a duplicate lane was probed with C575-5I to positively identify the fragment(s) derived from the protein of interest. The remainder of the blot was briefly stained with Coomassie Blue, and the appropriate band (Frag-38) was cut from the blot for N-terminal amino acid sequencing at the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University as described previously(24) .

PCR and cDNA Library Screening

The design of the gene-specific oligonucleotide PCR primer (GSP1; 5`-GAGGC(C/I)GA(C/T)AT(H)CC(N)GT(N)AA-3`) was based on the N-terminal seven amino acids of Frag-38 (sequenced above) and preferred codon usage in the rabbit(25) . Oligonucleotides were synthesized by the Yale University Department of Pathology DNA Synthesizing Service.

Each PCR (26) reaction mixture contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl, 0.001% gelatin, 400 µM each of dATP, dCTP, dGTP, and dTTP, 200 pmol of GSP1, 25 pmol of either T3ASP (5`-ACCCTCACTAAAGGGAACAA-3`) or T7ASP (5`-TCACTATAGGGCGAATTGGG-3`), 1 µl of a ZAP II rabbit kidney cortex cDNA library (Stratagene Cloning Systems), and 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer). Reaction mixtures were heated for 5 min at 97 °C and then cooled to 75 °C. AmpliTaq DNA polymerase was added, and the mixture was overlaid with mineral oil. Reaction mixtures were subjected to 40 cycles of PCR (1 min at 94 °C, 1 min at 65 °C, and 3 min at 72 °C) in a Perkin-Elmer thermal cycler. After the final cycle, the reaction mixtures were incubated at 72 °C for 15 min.

PCR products were size-fractionated on a 1.5% agarose gel and transferred to nylon filters (GeneScreen Plus; DuPont). PCR products that hybridized to the P-end-labeled internal, gene-specific oligonucleotide probes GSP2 and GSP3 (GSP2: 5`-GC(C/I)CC(C/I)GC(C/I)GG(N)TC(N)TT(Y)-3`; GSP3: 5`-GC(C/I)CC(C/I)GC(C/I)GG(N)AG(Y)TT(Y)-3`) were identified and subcloned into the PCR® II vector using the TA Cloning® system (Invitrogen Corp.).

The ZAP II rabbit kidney cortex cDNA library was screened by the plaque-lift method (27) with a P-labeled 1-kb PCR product (PCR-1). Duplicate nylon filters (Hybond-N; Amersham Corp.) were prehybridized in Church-Gilbert solution containing 20% formamide and 100 µg of denatured salmon sperm DNA/ml for 6 h at 50 °C. The filters were hybridized in the same solution containing 10 cpm/ml P-labeled PCR-1 for 48 h at 50 °C. Filters were washed in 2 SSC for 30 min at room temperature and then 0.1 SSC containing 0.5% SDS for 30 min at 68 °C. cDNA clones were recovered from positive ZAP II plaques by in vivo excision using R408 helper phage. The rescued phagemids were recovered as double-stranded DNA (in pBluescript II SK) by infection of Escherichia coli (XL1-Blue strain).

RNA Isolation and Northern Analysis

Total RNA was isolated from 0.5-1 g of rabbit tissues by a modification (28) of the acid guanidinium thiocyanate extraction method described by Chomczynski and Sacchi(29) . Poly(A) RNA was selected by oligo(dT)-cellulose chromatography(30) . 25 µg of the poly(A) RNA extracted from each tissue was electrophoresed through 1% agarose, 0.66 M formaldehyde gels and transferred to nylon filters (GeneScreen Plus; DuPont) by capillary action in 10 SSC. Northern blots were prehybridized in Church-Gilbert solution containing 50% formamide and 100 µg of denatured salmon sperm DNA/ml for 4 h at 42 °C. Blots were hybridized in the same solution containing 10 cpm/ml P-labeled PCR-1 for 24 h at 42 °C. Filters were washed in 2 SSC for 30 min at room temperature and then 0.1 SSC containing 0.5% SDS for 30 min at 68 °C.

DNA Sequencing

Plasmids containing cDNA inserts were directly sequenced by the dideoxynucleotide chain termination method (31) using modified T7 DNA polymerase (Sequenase; U. S. Biochemical Corp.) and [S]dATP (DuPont NEN). Single-stranded cDNA template was prepared from library clones by inducing transformed E. coli (XL1-Blue) to secrete pBluescript II as single-stranded DNA by infection with VCSM13 helper phage. Overlapping sequence of both strands was obtained with sequence-specific oligonucleotide primers.

Transient Expression

A T7/Vaccinia hybrid expression system (32) was used to transiently express positively identified Ksp-cadherin library clones in transfected LAP1 cells (a mouse L-cell line generously provided by Dr. J. Pouyssegur; (33) ). LAP1 cells were grown in -MEM supplemented with 10% fetal calf serum, 50 units of penicillin/ml, and 50 mg of streptomycin/ml and maintained in 5% CO/95% air at 37 °C. Cells were plated on glass coverslips or 35-mm tissue culture dishes 36-48 h prior to infection with the recombinant Vaccinia virus, vTF7-3. Cells used for infection were typically 80-90% confluent. LAP1 cells were infected with 40 plaque-forming units/cell of virus inoculum for 30 min at 37 °C. The viral inoculum was removed, and the infected cells were incubated with 5 µg of plasmid and 1.67 µg of Lipofectin (Life Technologies, Inc.) for 16-20 h at 37 °C.

Trypsin Sensitivity

LAP1 cells were grown to confluence on 35-mm tissue culture dishes, infected with Vaccinia, and then transfected with the Ksp-cadherin cDNA clone RT53 as described above. Dishes were washed with 2 ml of phosphate-buffered saline and then either 1 ml of HBS (HEPES-buffered saline: 138 mM NaCl, 4.4 mM KCl, 0.34 mM KHPO, 5.6 mM glucose, 10 mM HEPES, 0.1% trypsin, pH 7.4) containing 10 mM Ca or 1 ml of HBS containing 10 mM EDTA. Cells typically detached from the dishes in 25-20 min. 30 min after the addition of HBS, the cells were removed from the dishes, pelleted at 15,000 g for 5 min, solubilized in SDS-PAGE sample buffer, and then subjected to SDS-PAGE and Western analysis.

Data Analysis

Sequence analysis and data base searches (Entrez Release 15.0, National Center for Biotechnology Information (NCBI), Bethesda, MD) were performed using MacVector 4.1 software (Kodak International, Rochester, NY). Multiple sequence alignments were generated using Clustal Version 5(34) .


RESULTS

In preliminary studies, Soleimani et al.(16) used disulfonic stilbene affinity chromatography to identify a 71-kDa protein as a potential candidate for a structural component of the renal basolateral Na/HCO cotransporter or an associated protein. The objective of the present study was to isolate and characterize the 71-kDa protein and its cDNA clone.

Our initial step toward this objective was to develop a monoclonal antibody that recognized the 71-kDa protein both by Western analysis and immunohistochemistry. Although the 71-kDa protein was originally isolated by DNDS affinity chromatography, we found that we were unable to isolate the quantities of antigen required for monoclonal antibody production until we developed the more efficient NADS/Affi-Gel affinity matrix (see ``Materials and Methods''). The monoclonal antibody, C575-5I, was prepared by immunization of BALB/c mice with the gel-purified 71-kDa protein isolated by NADS affinity chromatography from rabbit renal cortical microsomes. Western analysis of rabbit crude cortical microsomes, NADS column eluates, and DNDS column eluates initially suggested that C575-5I reacted with several proteins in addition to the 71-kDa protein of interest (see Fig. 1). The observation of similar labeling patterns with eight other monoclonal antibodies also selected for reactivity to the 71-kDa protein (data not shown) implied that the 71-kDa protein and all the minor proteins recognized by the antibodies were, in fact, proteolytic fragments of a 130-kDa parent. This hypothesis was confirmed by the demonstration of identical Western labeling patterns in overlapping regions of V8 protease peptide fragment maps of the isolated 71-kDa and 130-kDa proteins (Fig. 2).


Figure 1: Western analysis of the 71-kDa protein. The monoclonal antibody C575-5I was used to probe a Western blot of DNDS column eluate (lane1), NADS column eluate (lane2), and solubilized basolateral membrane vesicles (lane3). The arrow indicates the position of the 71-kDa protein in each lane. Lane4 (DNDS column eluate) was probed with an irrelevant monoclonal antibody as a control.




Figure 2: Western analysis of V8 protease peptide fragment maps of the 71-kDa and 130-kDa proteins. Peptide fragment maps were generated by in situ digestion of 36 µg of the gel-purified 71-kDa protein (isolated from NADS column eluates; lane1) or the gel-purified 130-kDa protein (isolated from a C575-5I immunoaffinity column; lane2) with 240 ng of endoproteinase Glu-C (V8 protease). In lane3, V8 protease was added to the sample well in the absence of exogenous substrate. The digest was transferred to a PVDF membrane and then probed with C575-5I. The arrow indicates the position of the 38-kDa peptide fragment that was subjected to N-terminal amino acid sequencing (Frag-38; lanes1 and 2).



The 38-kDa peptide fragment (Frag-38) generated by the V8 protease digest of the 71-kDa protein (see Fig. 2) was excised and subjected to N-terminal amino acid sequencing as described above. The resulting partial amino acid sequence (EADIPVNAPAGSFLLTI) had no matches in the NCBI protein data bases.

cDNA Cloning of the 130-kDa Protein

We utilized a two-stage process to obtain a cDNA clone containing the entire coding region for the 130-kDa protein. In the first stage, anchored PCR was used to generate partial length cDNA clones from a rabbit renal cortex cDNA library. The goal was to amplify the region between the gene-specific oligonucleotide primer, GSP1 (based on Frag-38 partial amino acid sequence), and PCR primers anchored in the polylinker region of ZAP II. Since the cloning direction in ZAP II could not be predicted, we utilized both T3- and T7-based oligonucleotides as our anchored primers (in separate PCR reactions). We isolated two partial length cDNA clones using this approach (1 kb and 350 bp, respectively). Their identity was confirmed by Southern blot with internal, gene-specific oligonucleotide probes (GSP2 and GSP3) and dideoxynucleotide sequencing. Northern analysis of rabbit kidney cortex poly(A) RNA with the 1-kb PCR product (PCR-1) identified a single 3-kb transcript.

In the second stage, the 1-kb PCR product was radiolabeled and used to rescreen the kidney cortex cDNA library at high stringency. We isolated 17 partial length cDNA clones, ranging in size from 400 bp to 2.9 kb. Since our principal objective was to isolate a cDNA clone containing the entire coding region for the 130-kDa protein, we opted to screen potential clones (all those >2 kb in length) by transient expression and Western blot analysis rather than by the more tedious approach of dideoxynucleotide sequencing. One of the eight clones screened by this method (RT53; 2817 bp in length) encoded a 130-kDa protein that was recognized by Western blot analysis with C575-5I (Fig. 3).


Figure 3: Western analysis of the RT53 gene product. Positively identified cDNA library clones were transiently expressed in LAP1 cells with the T7/Vaccinia hybrid expression system. Transfected LAP1 cells were subjected to SDS-PAGE, blotted onto a PVDF membrane, and then probed with C575-5I. RT53 (lane1) was the only library clone that encoded a product recognized by C575-5I. LAP1 cells transiently transfected with a -galactosidase cDNA clone (lane2) were used as a negative control for the Western analysis.



Nucleotide and Amino Acid Sequence

RT53 contains a 5`-untranslated region (5`-UTR) of 13 nucleotides, a single long open reading frame of 2487 nucleotides, a 3`-untranslated region (3`-UTR) of 192 nucleotides, and terminates in a poly(A) tail. The poly(A) tail is preceded by a putative polyadenylation signal(35) . The proximity of the putative initiation codon to the 5`-end of RT53 prompted us to screen other library clones (by dideoxynucleotide sequencing) for more 5`-UTR sequence. Composite nucleotide sequence of RT53 and RT52 (2.4 kb in length) extended the 5`-UTR to 164 nucleotides (Fig. 4; the length of the composite sequence is 2933 nucleotides). The putative initiation codon identified above resides within a consensus sequence for initiation of translation (36) and is preceded by an upstream in-frame stop codon. The long open reading frame predicts a protein of 829 amino acids with a calculated molecular mass of 88.8 kDa. This open reading frame also encodes the 17 amino acids identified in Frag-38 (see above).


Figure 4: Nucleotide and inferred amino acid sequence of Ksp-cadherin. Cadherin-specific sequence motifs are indicated by boldtypeunderlined with a singleline. Potential N-glycosylation sites are indicated by amino acid residues highlighted with boldtype. The putative membrane-spanning region is indicated with a dashed, doubleunderline. Stop codons are indicated by nucleotide triplets highlighted with boldtype. The putative polyadenylation signal is indicated by a singleunderline. Nucleotide sequence was determined from both strands.



Hydropathy analysis (Kyte and Doolittle(37) ; window size: 20 residues) identified a 27-amino acid N-terminal hydrophobic domain, a 759-amino acid predominantly hydrophilic domain, a single putative membrane-spanning segment (21 amino acids), and a relatively short, hydrophilic C-terminal domain (22 amino acids). Signal peptide analysis using von Heijne's (38) weighted-matrix algorithm suggested that the N-terminal hydrophobic region contained a post-translationally cleaved transient leader sequence (putative cleavage at Val-18). Four potential N-linked glycosylation sites were identified in the region immediately preceding the putative membrane-spanning segment (residues 517, 602, 709, and 722; see Fig. 4).

The 130-kDa Protein Is a Member of the Cadherin Gene Family

Data base searches with the translated amino acid sequence confirmed that the protein was novel and indicated that it was a member of the cadherin gene family. The actual sequence homology of the 130-kDa protein with representative cadherins was relatively low (e.g. 20-24% identity with the exoplasmic domains of representative classical cadherins E-, B-, and M-cadherin, and 29% identity with the exoplasmic domains of LI-cadherin and HPT-1), but the overall structural organization was very similar (particularly with LI-cadherin and HPT-1). Like all previously described cadherins, the putative exoplasmic region of the 130-kDa protein is composed of a series of well defined domains (see Fig. 5). Ectodomains A, C, and D (94-113 amino acids each) contain the diagnostic cadherin-specific sequence motifs LDRE, DXND, and DXD (or modifications thereof) in well conserved sequential arrangement and ectodomain G (the premembrane ectodomain) contains the four appropriately located cysteine residues common to the last ectodomains of almost all known cadherins.


Figure 5: Amino acid sequence alignment of the corresponding protein domains of Ksp-cadherin, rat LI-cadherin, and human E-cadherin. Corresponding residues common to all sequences in each domain are boxed. Corresponding residues that specifically match those found in Ksp-cadherin are indicated by a dot above the residue. Bold residues in the exoplasmic domains indicate cadherin-specific sequence motifs.



A characteristic feature of the cadherin gene family is a calcium-modulated sensitivity to trypsin proteolysis(5, 13, 39, 40) . To determine if our putative cadherin also possessed this property, we used the T7/Vaccinia system (described above) to drive the transient expression of the 130-kDa protein in LAP1 cells and then exposed the cells to 0.1% trypsin in either the presence or absence of 10 mM calcium. The cells were subjected to SDS-PAGE and then analyzed by Western blot with mAb C575-5I. This protocol (see Fig. 6) clearly demonstrated that, like other cadherins, the 130-kDa protein possessed a marked calcium-modulated sensitivity to trypsin proteolysis. Even 30 min after the addition of trypsin, there was relatively little degradation of the 130-kDa progenitor in the presence of exogenous calcium.


Figure 6: Calcium-modulated trypsin proteolysis of Ksp-cadherin. LAP1 cells transiently expressing Ksp-cadherin were exposed to 0.1% trypsin in either the presence (lane1) or absence (lane2) of 10 mM calcium for 30 min. The transfected cells were then subjected to SDS-PAGE, transferred to a PVDF membrane, and then probed with C575-5I.



Organ and Tissue Distribution of the 130-kDa Protein

Western analysis of microsomal membranes isolated from multiple rabbit organs (Fig. 7; probed with mAb C575-5I) suggested that the 130-kDa protein had a distinct, renal-specific distribution. Antibody labeling was not observed in membrane fractions isolated from lung, gallbladder, brain, liver, stomach, spleen, skeletal muscle, or smooth muscle. Immunofluorescent labeling of semithin cryosections of rabbit kidney with C575-5I indicated that the renal distribution was limited to the basolateral membranes of the proximal tubule, the thick and thin limbs of the loop of Henle, the distal convoluted tubule, and a distinct subpopulation of cells in both the connecting tubule and the collecting duct (Fig. 8). Labeling was not observed in the glomerulus, Bowman's capsule, or non-epithelial regions of the kidney.


Figure 7: Tissue distribution of Ksp-cadherin. Microsomal membranes (100 µg) prepared from rabbit lung (lane1), gallbladder (lane2), brain (lane3), liver (lane4), stomach (lane5), spleen (lane6), skeletal muscle (lane7), smooth muscle (lane8), and kidney (lane9), were subjected to SDS-PAGE, transferred to a PVDF membrane, and then probed with C575-5I.




Figure 8: Immunofluorescent localization of Ksp-cadherin in rabbit kidney. PanelA, semithin cryosections of rabbit kidney cortex were labeled with mAb C575-5I. CD, collecting duct; DCT, distal convoluted tubule; PT, proximal tubule; arrows indicate subpopulation of cells in the collecting duct that did not immunoreact with C575-5I. PanelB, phase contrast image of panelA. Semithin cryosections (0.5 µm) were prepared and labeled with mAb C575-5I (at a concentration of 100 µg/ml) and an FITC-conjugated secondary antibody (FITC-conjugated goat anti-mouse IgG, Zymed Laboratories) as described previously (20) .



Northern analysis of a rabbit multiple organ poly(A) RNA blot (Fig. 9; probed with PCR-1) corroborated the specific renal distribution observed by Western analysis. Single 3-kb transcripts of similar intensity were observed in poly(A) RNA isolated from both the kidney cortex and the medulla. Transcript hybridization was not observed in poly(A) RNA isolated from stomach, ileum, liver, lung, brain, or skeletal muscle.


Figure 9: Tissue distribution of the Ksp-cadherin transcript. A Northern blot was prepared with 25 µg of poly(A) RNA isolated from each of the rabbit tissues listed above and then probed with PCR-1. The blot was previously probed with GAPDH to verify the relative abundance of poly(A) RNA in each of the lanes(41) .




DISCUSSION

The molecular analysis of the 71-kDa protein and its 130-kDa progenitor indicate that we have identified a new and unique member of the cadherin gene family. The inferred amino acid sequence of the 130-kDa protein includes diagnostic cadherin-specific sequence motifs in an appropriate sequential arrangement. Moreover, the expressed protein displayed the same marked calcium sensitivity to trypsin proteolysis that is observed with all cadherins. Immunolocalization studies and Northern analysis suggest that the tissue distribution of the 130-kDa protein is limited to renal epithelia. On the basis of this kidney-specific distribution, we propose that the 130-kDa protein be designated Ksp-cadherin (Kidney-specific cadherin).

Ksp-cadherin, LI-cadherin, HPT-1, and the classical cadherins all have apparent molecular masses (m; determined by SDS-PAGE) in the range of 120-140 kDa. The predicted molecular mass for Ksp-cadherin (88.8 kDa) is significantly lower than its m (130 kDa). Similar discrepancies have been reported for other cadherins (see (42, 43, 44, 45) for examples) and have been attributed to the combined effects of glycosylation and nonlinear migration of the cadherin polypeptide backbone in SDS-polyacrylamide gels.

Classical cadherins possess N-terminal signal sequences and additional prosequence peptides that are post-translationally cleaved at well conserved cleavage sites (see (46) ). Although it is likely that Ksp-cadherin possesses an N-terminal signal sequence, there is no evidence to support the existence of additional propeptides. The predicted sequence for Ksp-cadherin does not include the conserved protease cleavage site for cadherin propeptides (R-V/Q-K/R-R) and the first Ksp-cadherin LDRE-DXND couplet is located much closer to the putative signal sequence than the corresponding motifs in the classical cadherin precursors. If a prosequence region does exist, it would have to be very much shorter than that observed in other cadherins.

The exoplasmic region of a classical cadherin is typically composed of four contiguous ectodomain repeats (each containing the hallmark LDRE/DXND/DXD sequence motifs or modifications thereof), followed by a final premembrane ectodomain (see Fig. 5). The recently described solution structure of the N-terminal ectodomain of mouse E-cadherin suggests that these ectodomains form cylindrical -barrel configurations that are strung together in tandem via projecting N- and C-terminal residues(47) . The DXD, LDRE, DXND, and highly conserved PEN motifs are believed to form shared Ca-binding pockets that bind the Ca ions between tandem cadherin ectodomains.

The exoplasmic region of Ksp-cadherin is composed of seven ectodomains. Ectodomain A contains the PEN, LDRE, and DXND motifs described above and has approximately 31% amino acid identity with human E-cadherin ectodomain 1 (see Fig. 5). Ksp-cadherin ectodomains C and D contain the expected DXD, LDRE, and DXND motifs (or modifications thereof), but ectodomains B, E, and F contain only the DXD motifs. Ectodomain G contains the DXD motif and 4 cysteine residues found in the last ectodomain of almost every cadherin described to date. The presence of these key motifs in such well conserved positions and the high degree of organizational similarity observed between the ectodomains of Ksp-cadherin and E-cadherin suggest that the secondary structures of their exoplasmic domains, at least, are likely to be very similar.

The exoplasmic organization of Ksp-cadherin is particularly similar to that of LI-cadherin (see Fig. 5). Both proteins have similar ectodomain boundaries, and the first four corresponding ectodomains are 35-40% identical. Both proteins lack the prosequence and the HAV adhesion recognition sequences typical of most classical cadherins and both lack the cadherin-specific LDRE and DXND sequence motifs in their second ectodomains. The principal difference between Ksp-cadherin and LI-cadherin is that Ksp-cadherin does not possess an LDFE/DVNE sequence motif (or modification thereof) in the fifth ectodomain (E), and the LDRE/DXND motif found in ectodomain F of LI-cadherin is replaced with a DXD motif in Ksp-cadherin.

The most striking feature shared by Ksp-cadherin and LI-cadherin is a truncated cytoplasmic domain (18-22 amino acids). The cytoplasmic domains of classical cadherins are extremely well conserved (approximately 150 amino acids long) and contain sites for phosphorylation and catenin binding(48, 49) . Mutagenesis and deletion studies indicate that these sites are essential for the morphoregulatory functions of this subgroup (see (8) and (50) for examples). Cytoplasmic phosphorylation sites are not evident in Ksp-cadherin or LI-cadherin, but their cytoplasmic domains each have regions of limited similarity with the cytoplasmic domains of the classical cadherins (see Fig. 5and (51) ). Since it is not yet known if Ksp-cadherin or LI-cadherin interact with catenins or catenin-like molecules, it is difficult to assess the significance of these corresponding sequence motifs.

Despite the truncated cytoplasmic domain, LI-cadherin is capable of mediating cellular recognition and adhesion in a Ca-dependent manner similar to that described for the classical cadherins(51) . We have shown that Ksp-cadherin also interacts with calcium in a cadherin-like manner and anticipate that it, like LI-cadherin, will also be capable of mediating homotypic cellular recognition or adhesion. HPT-1 is clearly related to LI-cadherin and Ksp-cadherin. The exceptional similarity between the amino acid sequences of LI-cadherin and HPT-1 (79% sequence identity) suggests that they are probably species-specific isoforms of the same protein. Dantzig et al.(12) suggest that HPT-1 plays a key role in mediating proton-dependent peptide transport in human intestine. This raises the possibility that members of this cadherin subgroup may play a role in both the maintenance of the epithelial architecture and the mediation of vectorial solute transport.

Unlike most cadherins, Ksp-cadherin does not appear to have a secondary tissue distribution. Immunolocalization studies and Northern analysis suggest that the distribution of Ksp-cadherin is strictly limited to the basolateral membranes of renal epithelia. The immunofluorescent data indicate that the cellular distribution of Ksp-cadherin is not limited to the lateral regions of cell-cell contact, but rather that Ksp-cadherin has a much broader basolateral distribution. E-, P-, N-, K-, and T-cadherin have all been detected in vertebrate kidney(44, 52, 53, 54) . Each of these cadherins also has a distinct extrarenal tissue distribution. The stage- and segment-specific expression of E-, N-, and K-cadherin appear to play a major role in the morphogenesis and maintenance of tubuloepithelial differentiation(52, 54) . In situ hybridization studies indicate that the K-cadherin transcript is abundantly expressed in fetal rat kidney epithelial cells at the time of polarization of the nephroblastic tubules, but is absent in adult tissues(54) . N-cadherin is expressed in the human fetal kidney in the developing metanephros and maintains a proximal distribution in the adult nephron(52) . Specifically, N-cadherin was identified in the proximal tubule, the thin limb, and, to a lesser extent, the epithelial lining of Bowman's capsule. E-cadherin was identified in the expanding ureteric bud, the fetal collecting duct, the upper limb of the S-shaped bodies, and the developing distal tubule(52) . In the adult kidney, E-cadherin was identified in the epithelium lining Bowman's capsule and in all nephron segments distal to the proximal tubule. The cellular expression of N-cadherin is primarily confined to adherens junctions, whereas E-cadherin appears to be expressed over the entire cell surface in the distal segments of the nephron. The specific renal distributions of P- and T-cadherin are unknown. Given the abundance of Ksp-cadherin (as estimated by the intensity of immunofluorescent staining) and its expression along the entire length of the nephron (presumably overlapping the distributions of both N- and E-cadherin), it will be of great interest to determine what role, if any, that Ksp-cadherin plays in the orchestrated direction of tubuloepithelial differentiation in the vertebrate kidney.

In summary, we have identified a novel, kidney-specific member of the cadherin superfamily whose expression appears to be limited to the basolateral membranes of renal tubular epithelial cells. At this point, the function of Ksp-cadherin remains uncertain. Originally identified and partially purified by disulfonic stilbene affinity chromatography, it is possible that Ksp-cadherin is physically associated with a stilbene-inhibitable anion transport protein, such as the renal basolateral Na/HCO cotransporter. Future studies will be directed at elucidating the involvement of Ksp-cadherin in the development and maintenance of normal and abnormal renal structure and function.


FOOTNOTES

*
This work was supported by fellowships (to R. B. T.) from National Sciences and Engineering Research Council of Canada and the Patrick and Catherine Weldon Donaghue Medical Research Foundation and by National Institutes of Health Grant DK 17433 (to P. S. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank®/EMBL Data Bank with accession number(s) U28945[GenBank® Link].

§
To whom all correspondence should be addressed. Tel.: 203-785-7114; Fax: 203-785-7068.

The abbreviations used are: Ksp-cadherin, kidney-specific cadherin; DNDS, 4,4`-dinitrostilbene-2,2`-disulfonic acid; NADS, 4-amino-4`-nitrostilbene-2,2`-disulfonic acid; PMSF, phenylmethanesulfonyl fluoride; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; kb, kilobase pair(s); bp, base pair(s); UTR, untranslated region; FITC, fluorescein isothiocyanate.


ACKNOWLEDGEMENTS

We thank Dr. Robert Reilly for many helpful discussions and Brenda DeGray and Thecla Abbiati for expert technical assistance.


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