(Received for publication, January 12, 1995; and in revised form, April 27, 1995)
From the
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.
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).(
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.
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.
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 Na
Each PCR (26) reaction mixture contained 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl
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
The
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
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).
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 (
Figure 3:
Western analysis of the
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.
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.
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.
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) .
Figure 9:
Tissue distribution of the Ksp-cadherin
transcript. A Northern blot was prepared with 25 µg of
poly(A)
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
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
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
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
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank®/EMBL Data Bank with accession number(s)
U28945[GenBank® Link].
We thank Dr. Robert Reilly for many helpful
discussions and Brenda DeGray and Thecla Abbiati for expert technical
assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
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.
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).
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.
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) .
EDTA, 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 Na
EDTA, 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.
, 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.
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.).
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 K
HPO
, 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) .
/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.
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.
RT53;
2817 bp in length) encoded a 130-kDa protein that was recognized by
Western blot analysis with C575-5I (Fig. 3).
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).
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.
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.
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.
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.
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) .
; 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.
-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.
-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.
/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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.