From the Department of Medicine III, Okayama
University Medical School, 2-5-1 Shikata-cho, Okayama 780-8558, Japan,
the § Department of Nephrology, The First Teaching Hospital,
Beijing Medical University, Beijing, P. R. China 100034, and the
Department of Pathology, Northwestern University Medical School,
Chicago, Illinois 60611
Received for publication, July 27, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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Collectrin, a novel homolog of
angiotensin-converting enzyme-related carboxypeptidase (ACE2),
was identified during polymerase chain reaction-based cDNA
subtraction and up-regulated in 5/6 ablated kidneys at hypertrophic
phase. Collectrin, with 222 amino acids, has an apparent signal peptide
and a transmembrane domain; the sequence is conserved in mouse, rat,
and human and shares 81.9% identity. Human collectrin has
47.8% identity with non-catalytic extracellular, transmembrane, and
cytosolic domains of ACE2; however, unlike ACE and ACE2, collectrin
lacks active dipeptidyl carboxypeptidase catalytic domains. The
collectrin mRNA transcripts are expressed exclusively in the
kidney. In situ hybridization reveals its mRNA expression in renal collecting ducts, and immunohistochemistry shows
that it is localized to the luminal surface and cytoplasm of collecting
ducts. Immunoprecipitation studies, using
[35S]methionine-labeled renal cortical and inner medullar
collecting duct cells, i.e. M-1 and mIMCD-3, indicate that
the protein size is ~32 kDa. During the development of mouse kidney,
mRNA signal is detectable at day 13 of gestation, and the protein
product is observed in the ureteric bud branches. Its expression is
progressively increased during later stages of the gestation extending
into the neonatal periods and then is decreased in adult life.
Up-regulated expression of collectrin in the hypertrophic
kidneys after renal ablation and restricted spatio-temporal expression
during development indicates a possible role(s)in the process of
progressive renal failure and renal organogenesis.
Reduction of nephron number in various immunological and
non-immunological renal diseases has been implicated in the development of subsequent glomerulosclerosis and interstitial scarring of the
kidney that is followed by a decline in renal function. Such a
reduction can be experimentally achieved by 5/6 renal ablations in rat,
a model in which glomerular hyperfiltration in renal nephrons plays a
central role in the development of glomerulosclerosis (1). In
Wistar-Kyoto and Harlan Sprague Dawley rats, partial ablation of renal
mass initiates a cycle of progressive renal injury in the remnant
kidney associated with glomerular and tubular hypertrophy,
hyperfiltration, and systemic hypertension (2). The remaining viable
renal mass undergoes the following phases: 1) hypertrophic phase that
occurs after 2-4 weeks of ablation, 2) the quiescent phase that
follows during the next 4-10 weeks with minimal histological
alterations, and 3) the development of segmental glomerular sclerosis
and tubulointerstitial fibrosis that ensue after 10 weeks (3). To
investigate the molecular mechanism(s) and the genes that may be
involved in the initiation of such a pathobiologic response, we
employed PCR1-based
subtractive hybridization method, i.e. representational difference analysis of cDNA (cDNA-RDA; Refs. 4 and 5) to screen
the differentially expressed genes in remnant kidney at hypertrophic
phase of 5/6 nephrectomized mice (6). In the process of cDNA
subtraction, the cDNA fragment of a novel gene, NX-17, was isolated, and its mRNA expression was up-regulated in 5/6 ablated remnant kidney compared with control kidney (6).
In the present study, we have reported cDNA cloning of the full
coding sequence of NX-17. The gene product of NX-17 has been designated
as collectrin, because it is a novel transmembrane glycoprotein
specifically expressed in collecting tubules of the kidney. With
homology searches, collectrin was determined to be a novel homolog of
ACE-related carboxypeptidase (ACE2; Refs. 7, 8), which has been
recently identified as the human homolog of ACE (8). ACE2, like ACE, is
a membrane-associated and secreted enzyme expressed predominantly on
endothelium, but unlike ACE it is highly restricted in humans to heart,
kidney, and testis (8). ACE2 has a single carboxypeptidase active site
and catalyzes the cleavage of angiotensin I (Ang I) to Ang1-9, whereas
ACE has two active site domains and converts Ang I to angiotensin II
(Ang II) (8). Collectrin shares 47.8% identity with C-terminal regions including non-catalytic extracellular, transmembrane, and cytosolic domains of ACE2, and this segment does not share homology with ACE.
Although collectrin lacks catalytic domains, its highly restricted expression in collecting ducts and differential expression in embryonic
and ablated kidneys suggest that it has a unique role in the
pathobiology of collecting ducts that may be related to the
organogenesis and organ failure of the kidney.
Isolation of a Full-length Collectrin cDNA and Nucleotide
Sequencing--
Using cDNA-RDA, we previously described the
isolation of differentially expressed genes in the 5/6 nephrectomized
mouse remnant kidney (3, 4). Among the various novel genes,
NX-17 (collectrin partial-length cDNA) had a ~2-fold
up-regulated mRNA expression in nephrectomized mouse with 5/6
ablated kidney (5). Using this partial-length cDNA, the full coding
sequence of collectrin cDNA from mouse, rat, and human was isolated
with the Marathon cDNA amplification kit
(CLONTECH, Palo Alto, CA). Total kidney RNAs from
8-week-old CD-1 (ICR) mice and 4-week-old Harlan Sprague Dawley rats
(Charles River Co., Yokohama, Japan) were extracted by guanidinium
isothiocyanate-CsCl ultracentrifugation (8-11), and mRNAs were
further isolated by FastTrack 2.0 Kit (Invitrogen, San Diego, CA).
Human kidney poly(A)+ RNA was purchased from
CLONTECH. Double-stranded cDNAs were
synthesized from 1.0 µg of mRNA and subjected to the rapid
amplification of 5'- and 3'-cDNA ends (RACE) reactions using
5'-GCCCGTCTGGATTATTGTATTCGGTGTG-3' (506-533 bp, S1) and
5'-CATGTCCAAGGGATCACAAGGGATGCC-3' (669-695, AS1) as primers. The 5'-
and 3'-RACE products were subcloned into the pCR2.1-TOPO vector
(Invitrogen) and sequenced by automated DNA sequencing (ABI PRISM 310 Genetic Analyzer, Perkin-Elmer, Foster City, CA). At least four
different clones were sequenced to ensure the fidelity of
Taq polymerase.
Structural and Homology Analysis--
Kyte and Doolittle
hydrophobicity/hydrophilicity plot analyses (12) were performed using
GENETYX-WIN (Software Development, Tokyo, Japan). An online homology
search was performed using BLAST (National Library of Medicine).
Subsequently, structural analyses were performed with online programs
available on the ExPASy Molecular Biology and Network Protein Sequence
Analysis servers. Multiple sequence alignment was performed using
ClustalW and DIALIGN. The presence of the transmembrane domain was
assessed by SOSUI and the cytoplasmic localization of collectrin by
PSORT II.
Preparation of Rabbit Polyclonal Anti-collectrin
Antibody--
Antibodies were raised in rabbits against the synthetic
peptide, GIRQRRRNNKGPPGV, the sequence of which was derived from the amino acid stretch between residues 165 and 179 of the intracellular domain of collectrin (Fig. 1A). This segment of collectrin
is highly conserved and identical between the mouse and rat. The cystine residue was added to the N terminus for conjugation of the
peptide to keyhole limpet hemocyanin (KURABO, Osaka, Japan). To assess
the specificity of the anti-collectrin antibody, ELISA (enzyme-linked
immunosorbent assay) and competitive inhibition ELISA assays were
performed as described previously (13).
Immunoprecipitation--
SV40 MES 13 (glomerular mesangial cells
from an SV40 transgenic mouse, CRL-1927, ATCC), M-1 (kidney cortical
collecting duct cells from an SV40 transgenic mouse, CRL-2038, ATCC)
and mIMCD-3 (kidney inner medullar collecting duct cells, CRL-2123,
ATCC) were grown in a mixture of Dulbecco's modified Eagle's medium, Ham's F12 medium, and 5% fetal bovine serum. For immunoprecipitation, the cells were grown to ~70% confluence in a cell culture flask (~5 × 106 cells) and labeled with
[35S]methionine and [35S]cysteine (0.25 mCi/ml, Amersham Pharmacia Biotech) for 12 h. The cells were
washed with culture medium and lysed in immunoprecipitation buffer (IP
buffer; 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2,
10 mM benzamidine-HCl, 10 mM
Northern Blot Analyses--
Northern blot analyses were
performed using mRNAs isolated from various CD-1 mouse and
Sprague-Dawley rat tissues including brain, heart, liver, lung, spleen,
kidney, intestine, muscle, and thymus. In addition, total RNAs were
isolated in various developmental stages of embryonic, newborn, and
adult kidneys from the CD-1 mouse. 2 µg of poly(A)+ RNA
isolated from adult tissues were subjected to 2.2 M
formaldehyde, 1% agarose-gel electrophoresis and were capillary
transferred to the Hybond N+ nylon membranes (Amersham
Pharmacia Biotech). For embryonic, newborn, and adult mouse kidneys, 20 µg of total RNA were used. Human 12-Lane MTN blot was purchased from
CLONTECH. On this membrane, 2 µg of
poly(A)+ RNA from human brain, heart, skeletal muscle,
colon, thymus, spleen, kidney, liver, small intestine, placenta, lung,
and peripheral blood leukocytes were blotted. The membranes were
hybridized with [ In Situ Hybridization Studies--
The kidneys of CD-1 mice were
harvested after intracardiac catheter perfusion with 4%
paraformaldehyde in phosphate-buffered saline, fixed at 4 °C for
3 h, and dehydrated in ethanol. The whole embryo of the CD-1 mouse
at day 13 of gestation was also fixed with 4% paraformaldehyde in PBS.
Tissues and embryos were embedded in paraffin; 4-µm-thick sections
were prepared and mounted on RNase-free ProbeOn Plus glass slides
(Fisher Scientific). Oligodeoxynucleotide spanning residues 370-398 of
mouse collectrin cDNA (5'-CTCTTCCTGCAGCTGAAGTACAGTCGGCC-3', AS2;
Fig. 1) linked to a 3'-biotinylated tail
[3'-(TAG)5-BBB-(TAG)2-BBB-5'] were
synthesized and used as antisense probe (Research Genetics). Biotinylated poly(T) and poly(A) probes were used as positive and
negative controls, respectively. In situ hybridization was performed using MicroProbeTM System (Fisher Scientific) and in situ sampler kit (Research Genetics, Huntsville, AL) following the
manufacturer's protocol. In brief, the tissue sections were rapidly
dewaxed (AutoDewaxer), cleared with alcohol (AutoAlcohol), and
rehydrated with a Tris-based buffer, pH 7.4 (Universal Buffer). The
tissue sections were digested with a stable pepsin solution (0.5 mg/dl)
for 3 min. The probes (1 µg/ml) were applied to the tissues and
incubated at 45 °C for 30 min. Finally, the biotin-labeled hybrids
were detected by horseradish peroxidase-linked streptavidin and
diaminobenzidine, and the slides were counterstained with hematoxylin
and coverslipped with Clear Mount (Research Genetics).
Immunofluorescence Study--
CD-1 mouse adult kidneys were
embedded in OCT compound (EM Science, Washington, PA) and snap frozen
in liquid nitrogen. 4-µm-thick cryostat sections were prepared and
air-dried. The sections were briefly washed with phosphate-buffered
saline and incubated with specific rabbit anti-collectrin serum for 30 min. After washing with PBS, rhodamine-conjugated donkey anti-rabbit
IgG (Chemicon) was applied and incubated for 30 min. Serial sections
were also stained with rabbit anti-aquaporin-2 serum and
rhodamine-conjugated donkey anti-rabbit IgG (Chemicon). Various
developmental stages of CD-1 mouse kidneys were also prepared and
stained with rabbit anti-collectrin serum and fluorescein
isothiocyanate-conjugated anti-rabbit IgG (Sigma-Aldrich). The
slides were rewashed with phosphate-buffered saline twice, covered with
a drop of buffered glycerol, coverslip mounted, and examined with an
immunofluorescence microscope equipped with epi-illumination.
Immunohistochemical Studies--
Immunohistochemisty was
performed utilizing the MicroProbeTM System and UltraProbe staining
kit (Biømeda, Foster City, CA). Briefly, 4% paraformaldehyde-fixed
CD-1 mouse kidney paraffin sections (4-µm) were dewaxed, cleared, and
rehydrated. The tissues were digested with stable pepsin at 0.5 mg/dl
for 2 min. Following a brief wash in 1× immuno/DNA buffer (Biømeda),
a 1:100 dilution of antibody specific for collectrin in primary
antibody diluent (Biømeda) was applied to the slides and incubated for
30 min at 50 °C. The slides were then washed in 1× immuno/DNA
buffer, and the biotinylated donkey anti-rabbit antibody (Chemicon,
Temecula, CA) was applied for 5 min at 50 °C. The biotin-labeled
hybrids were detected with alkaline phosphatase-linked streptavidin,
and the signal was developed with the streptavidin AP detection system (Biømeda). The sections were counterstained with hematoxylin and eosin stain.
Isolation and Sequence Analysis of Mouse, Rat, and Human
Collectrin--
By RACE-PCR using S1 and AS1 primers, cDNA
containing the full-coding sequence of mouse collectrin was obtained
(GenBankTM/EBI accession no. AF178085). The mouse
collectrin cDNA included 1262 bp, and it contained a 666-bp open
reading frame flanked by 5'- and 3'-untranslated regions. The potential
initiation codon was located at position 87-89, and the open reading
frame encoded 222 amino acid residues with a predicted molecular mass
of ~25 kDa (Fig. 1A). The S1
and AS1 primer sequences matched the corresponding EST clones of both
rat and human deposited in GenBankTM/EBI. Using these
primers, 1181 bp of rat collectrin (AF178086) and 1345 bp of human
collectrin (AF229179) cDNAs were also isolated by RACE-PCR (Fig.
1B). A ClustalW multiple alignment indicates that the
primary protein structure of mouse, rat, and human collectrin was
highly conserved throughout the entire sequence (Fig. 1B).
They shared 84.3 and 81.9% identity at the nucleotide and amino acid
levels, respectively (Table I).
Structural and Homology Analysis of Collectrin--
Kyte and
Doolittle hydrophobicity/hydrophilicity plot analysis (12) predicted
that the protein had two hydrophobic domains (Fig. 1C). The
N-terminal hydrophobic domain (amino acids 1-14) was predicted as a
signal sequence by von Heijne's method (16), and a predicted cleavage
site was located between amino acid residues 14 and 15. The second
hydrophobic domain stretched between residues 142 and 164, and it
seemed to have the characteristics of a transmembrane domain (SOSUI;
Ref. 17). Thus, collectrin seemed to be a type 1a membrane protein. A
mitochondrial targeting sequence, an endoplasmic reticulum retention
signal, and a peroxisomal targeting signal were not present. The
extracellular domain included 127 amino acid residues, and it contained
two N-linked glycosylation and one O-glycosylation sites
(Fig. 1, A and C). Homology searching using the
Homology searches through human genome sequences indicates that the
human collectrin cDNA matches the 159446-base BAC clone GS1-594A7
(AC003669, NT 001172) localized to chromosome Xp22. Four exons were
found on the BAC clone, and they corresponded to residues
142-1345 of the human collectrin cDNA (Fig. 2C). The exon(s) corresponding to nucleotides 1-141 of the human collectrin cDNA was not discovered in searches of human genome sequences deposited in GenBankTM/EBI. The ACE2 gene
was also located on BAC clone GS1-594A7, and the alignment between ACE2
mRNA and the BAC clone GS1-594A7 revealed that the ACE2
gene spanned 40 kilobases, comprised 18 exons, and was located ~26
kilobases from the collectrin gene.
Collectrin Is a Kidney-specific Gene and Is Localized to the Kidney
Collecting Ducts in Vivo--
Tissue distribution of collectrin
mRNA was investigated by Northern blot analyses using mouse, rat,
and human collectrin cDNAs as the hybridization probes (Fig.
3, A-D). A single transcript of ~1.8 kilobases was observed exclusively in the kidney, and it was
not detected in any other tissues. The transcript size was similar in
mouse, rat, and human, and no alternative splicing variants were
observed. To investigate the mRNA localization of collectrin,
in situ hybridization was performed using CD-1 mouse kidney
(Fig. 3, E-H). The hybridizing signals of collecrin
mRNA were observed in collecting tubules in the renal cortex (Fig. 3, E and F) and medulla (Fig. 3, G and
H), indicating the whole segment of collecting duct
expressed collectrin mRNA. By in situ hybridization of
the whole mouse embryo at 13 days of gestation, collectrin
mRNA was not expressed in any other tissues except kidney (data not
shown).
The localization of collectrin was further investigated. To test the
specificity of rabbit anti-collectrin antibody, immunoprecipitation studies using M-1 and mIMCD-3 collecting duct cell lines and SV40 MES
13 glomerular mesangial cell line were performed. A ~32-kDa single
band was observed in an anti-collectrin-immunoprecipitated protein
fraction derived from M-1 and mIMCD-3 collecting duct cells, and the
band was absent in SV40 MES 13 cells (Fig.
4). Preimmune sera also did not show a
specific band corresponding to the size of collectrin. The specificity
of anti-collectrin antibody was further verified by competitive
inhibition ELISA assay as described previously (data not shown)
(13).
Collectrin was localized in the cytoplasm of collecting duct cells in
the cortex (Fig. 5A) and
medulla (Fig. 5C). High magnification micrographs revealed
linear immunoreactivity along the luminal surface of collecting ducts
(Fig. 5, B and D). The epithelial cells in the
renal pelvis also expressed collectrin (Fig. 5C). The
immunostaining of serial sections indicated that the distribution of
collectrin (Fig. 5E) matched with aquaporin-2, which is the marker for collecting ducts (Fig. 5F). To further confirm
the localizaion of collectrin, the avidin-biotin complex method was employed. Collectrin was expressed in the cytoplasm of collecting tubule in the cortex (Fig. 5G) and medulla (Fig.
5H), and the linear staining of the luminal surface was also
observed. These data indicate that collectrin is a kidney-specific gene
and localized to the cells lining the renal collecting tubules and
ducts.
Collectrin Is Developmentally Regulated in Embryonic and Adult
Kidneys--
Because collectrin is specifically expressed on
collecting ducts, its expression was examined in various developmental
stages of the CD-1 mouse kidney. By Northern blot analysis, the
mRNA signal was detectable at 13 days of gestation, and its
expression was progressively increased during the later stages of
gestation extending into the neonatal periods. mRNA expression was
then greatly reduced in the adult life of the CD-1 mouse (Fig.
6). By immunofluorescence studies,
collectrin was observed in the ureteric bud branches, the progenitor of
collecting ducts, at day 13 of gestation of the CD-1 mouse (Fig.
7, A and B). During subsequent stages of gestation, collectrin was localized in the developing collecting ducts, and expression was increased during fetal
and neonatal life (Fig. 7, C-J). In adult kidneys, the
immunoreactivity of collectrin was rather decreased (Fig. 7,
K and L).
In this investigation, isolation and characterization of a novel
renal-specific type 1a transmembrane glycoprotein, designated as
collectrin, is described. Homology analysis indicates that collectrin
has a long-ordered homology with ACE2, which is the first human homolog
of ACE and was identified from 5' sequencing of a human heart failure
ventricle cDNA library (8). A renal and pulmonary splicing isoform
of ACE (A31759) consists of two homologous repeats of catalytic
domains, whereas the testicular isoform (S05238) consists of only one
catalytic domain (20). The ACE2 protein also has one active site domain
and shares 41.8% identity with ACE. Although ACE is ubiquitously
expressed in the vasculature, ACE2 is restricted to heart, kidney, and
testis (8). In contrast to ACE and ACE2, collectrin lacks the
metalloprotease catalytic domains, although it shares 47.8% identity
with the non-catalytic extracellular, transmembrane, and intracellular domains of ACE2. Both ACE2 and collectrin revealed
tissue-restricted expression in kidney. However, ACE2 is present
throughout the endothelium and in proximal tubular epithelial cells,
whereas collectrin is present in collecting duct epithelia only.
Homology searches of human collectrin cDNA in the human genome
indicate that it is localized to chromosome Xp22. Interestingly,
ACE2 is localized in the same BAC clone that is close to the
collectrin gene. Exon/intron organization and chromosome localization
of ACE, ACE2, and collectrin suggests that these
three genes evolved from a common ancestral gene.
The catalytic activity of ACE2 has been reported; removing the
C-terminal Leu from Ang I and generating Ang1-9. In contrast, ACE
cleaves the His-Leu dipeptide and converts Ang I to Ang II (Ang1-8;
Ref. 8). A multiple sequence alignment indicates that the catalytic
domain is apparently absent, and extensive searches for domain
structures or consensus sequences have not revealed any additional
information as yet. Clinical and experimental studies in animals have
revealed that the ACE inhibitors or Ang II receptor antagonists
decrease proteinuria and slow the progression of diabetic nephropathy
and various forms of glomerulonephritis. The finding that the systemic
renin-angiotensin system (RAS) is not activated in most types of
chronic renal disease has led to the suggestion that a local intrarenal
RAS plays important roles in the relentless progression of renal
disease. In this regard, it is interesting to note that renal tubular
cells are capable of generating Ang II and Ang II type 1 (AT1) receptor
proteins that are implicated in various kidney diseases. In
Sprague-Dawley rats with subtotal nephrectomy, renin and Ang II are
up-regulated and implicated in the pathogenesis of tubulo-interstitial
fibrosis (21), as is the case in uninephrectomized Wistar-Kyoto rats,
where ACE has been reported to be up-regulated in proximal tubules
(22). Because Ang II is involved in renal cell growth and matrix
production via the activation of AT1 receptor (23), Ang II may be
responsible for the tubulo-interstitial lesions seen in this model.
Along these lines it is conceivable that up-regulated expression of the
angiotensinogen gene, as observed in proximal tubular cell line under
high glucose conditions, may contribute to a certain degree to the
progression of tubulo-interstitial lesions in diabetic nephropathy
(24).
Studies reported in the literature suggest that intrarenal RAS,
especially in the tubular cells, plays a critical role in the
progression of interstitial tissue injury. The latter of course is
certainly linked to the deterioration of renal functions in various
kidney diseases. Conceivably, collectrin, which is homologous to ACE2
and up-regulated in the hypertrophic phase of the ablated kidney in 5/6
nephrectomized Sprague-Dawley rats, would be another key molecule that
may be relevant to the renal tissue injury. In contrast to ACE and
ACE2, collectrin does not contain dipeptidyl carboxypeptidase domains,
and thus it may play a role in hypertrophic phase kidneys via other yet
to be characterized mechanism(s) rather than via RAS activation. To
explore such a role in the progression of tubulo-interstitial renal
tissue injury, determination of the biological activity of collectrin
will be the subject of future investigations.
Because the administration of RAS antagonists causes widespread
structural and growth abnormalities in the kidney, the enhanced expression of RAS genes in fetal and early postnatal life seems to
predict critical functions related to renal growth and development (25,
26). Actually, targeted inactivation of the angiotensinogen gene
induced adulthood developmental abnormalities including vascular hypertrophy, focal tubular dropout with interstitial inflammatory infiltrates, and marked atrophy of the renal papilla (27, 28). This
phenotype is also seen in mice that completely lack ACE (29, 30).
Although mice lacking AT1A (31-34) receptor genes survive in normal numbers, and their renal morphology is reported to be normal
with the exception of some minor abnormalities of the inner medulla and
papilla, mice lacking both AT1A and AT1B genes
do not develop a renal pelvis resulting in the buildup of urine and progressive kidney damage (35). Ang II and its receptor are transiently
up-regulated at the renal outlet at birth and related to peristaltic
movements of the pelvis to transfer 50-fold-increased urine, compared
with embryonic life (35). The evidence that collectrin up-regulates the
perinatal period and is expressed in collecting ducts and epithelia of
renal pelvis suggests that collectrin may have a role in the
development of collecting ducts and renal pelvis. Because collectrin
has homology with ACE2, a novel member of RAS, collectrin may be
involved in the functions related to the RAS, e.g. the
urinary peristaltic machinery during the perinatal period.
The identification of collectrin, a novel homolog of ACE2, adds
complexity to the RAS and may facilitate the discovery of novel role(s)
of the RAS in the pathophysiology of renal collecting ducts and pelvis.
At present, the obvious questions that one can raise would be why
collectrin is lacking catalytic domains and what are its
functional activities. However, its up-regulated expression in the
hypertrophic kidneys after renal ablation and embryonic and early
postnatal kidneys points toward the possible role of collectrin in the
process of progressive renal failure and renal organogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino-n-caproic acid, 2 mM
phenylmethylsulfonyl fluoride, and 1% Triton X-100) by vigorous shaking at 4 °C for 2 h. The lysates were centrifuged at
12,000 × g for 30 min at 4 °C, and the supernatants
were incubated with preimmune rabbit serum and protein A-Sepharose
CL-4B (Amersham Pharmacia Biotech). Immunoprecipitation was performed
by adding 10 µl of rabbit anti-collectrin antibody to 0.5 ml of
supernatant and gently swirled at 4 °C for 1 h. The
antibody-antigen complexes were further incubated with 80 µl of
protein A-Sepharose CL-4B for 1 h at 4 °C on a rocking
platform. The protein A-Sepharose was centrifuged at 4 °C, and the
pellets were washed five times with 1 ml of IP buffer. The
immunoprecipitated complexes were dissolved in a gel-loading buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 100 mM dithiothreitol, and 0.1% bromphenol blue) and subjected
to 12.5% SDS-polyacrylamide gel electrophoresis under reducing
conditions. The gels were treated with a solution containing 10%
acetic acid and 20% methanol and dried using a Bio-Rad Gel Dryer
(Bio-Rad). Autoradiograms were prepared using the Bio-Imaging Analyzer
System (BAS-1800 II, FUJIFILM, Japan).
-32P]dCTP-radiolabeled mouse, rat,
and human collectrin, and GAPDH (1 × 106 cpm/ml)
cDNA at 42 °C in ExpressHybTM Hybridization Solution
(CLONTECH) for 18 h. Filters were washed under
high stringency condition, i.e. four times in 1× SSC, 0.1%
SDS at 24 °C, followed by two times at 50 °C in 0.1× SSC, 0.1% SDS.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Amino acid sequences of mouse, rat, and human
collectrin. A, nucleotide and deduced amino acid
sequence of mouse collectrin. The 1262-bp mouse collectrin cDNA
(AF178085) contains a 666-bp open reading frame flanked by 5'- and
3'-untranslated regions. The poly(A)+ signal (AATAAA) is
observed at the 3'-untranslated region and is indicated by a bold
box. The putative signal sequence is observed at the N-terminal
(underlined) with the cleavage site (arrow).
Collectrin is a membrane-bound glycoprotein, and the putative
transmembrane domain is double underlined. The potential
N-linked and O-linked glycosylation sites are
indicated by filled triangles ( ) and filled circles (
),
respectively. The sense (S1) and antisense (AS1) primers used for 5'-
and 3'-RACE are boxed. The antisense probe (AS2) used for
in situ hybridization is indicated by a dotted
line. The synthetic peptide used for antibody generation is
indicated by bold underline. B, multiple sequence
alignment of mouse, rat, and human collectrin. ClustalW multiple
alignment indicates that the primary protein structure of mouse
(AF178085), rat (AF178086), and human (AF229179) collectrin is
conserved. The conserved amino acid sequences in all species are
indicated by a dash. C, Kyte and Doolittle
hydrophobicity/hydrophilicity plot analysis of mouse collectrin. Two
hydrophobic domains, i.e. signal peptide and transmembrane
domain (TM), are noted. N-linked and
O-linked glycosylation sites are indicated by
N-Gly and O-Gly, respectively.
Sequence similarity of mouse, rat, and human collectrin at nucleotide
and amino acid levels
BLAST program through the NCBI, National Institutes of Health (18)
revealed significant and long-ordered homology with the human
angiotensin-converting enzyme homolog (ACEH)
(GenBankTM/EBI AF241254), angiotensin-converting
enzyme-related carboxypeptidase (ACE2) (GenBankTM/EBI
AF291820), and DKFZP434A014 protein (GenBankTM/EBI
AL110224, UniGene no. Hs.178098). The nucleotide sequences of
ACEH, ACE2, and DKFZp434A014 matched, and they were derived from
identical genes. The full coding sequence of ACEH and ACE2 mRNA
encoded 805 amino acids, whereas DKFZp434A014, which was originally
identified and deposited in the German Genome Project, contained a
coding sequence with 804 amino acids. Multiple sequence alignment based on segment-to-segment comparison (DIALIGN 2; Ref. 19), indicated that
human collectrin shared 47.8% identity with the C-terminal end of ACE2
(amino acids 614-805) corresponding to non-catalytic extracellular,
transmembrane, and cytosolic domains (Fig.
2, A and B). The
comparison between ACE and ACE2 suggests that the ACE2 protein contains
a single catalytic domain (amino acids 147 to 555) and shares 41.8%
identity with both catalytic domains of the human
angiotensin-converting enzyme (ACE, GenBankTM/EBI no.
A31759). Collectrin lacks carboxypeptidase active site domains and thus
it does not share the segments or domains of ACE. The multiple sequence
alignment of the three proteins ACE, ACE2, and collectrin is shown in
Fig. 2B.
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Fig. 2.
Comparison of collectrin, ACE, and ACE2
protein sequences. A and B, multiple
sequence alignment of ACE, ACE2, and collectrin. ACE (A31759), ACE2
(AF241254, AF291820), and human collectrin (AF229179) are aligned by
DIALIGN based on a segment-to-segment comparison. The C terminus of
ACE2 shares 47.8% identity with collectrin and ACE protein have
similarity and shares 41.8% identity with ACE. A schematic drawing of
the domain similarity is indicated (B). ACE consists of two
homologous repeated domains of a catalytic site (gray box).
ACE2 has one active site domain (gray box) and shows
significant homology with the catalytic domain of ACE. Collectrin shows
similarity with the C-terminal domain of ACE2; however, it lacks a
catalytic domain for the dipeptidyl carboxypeptidase. C,
exon/intron boundaries of human collentrin and ACE2. Homology searches
of the human genome sequences indicate that collectrin cDNA
(AF229179) and ACE2 (AF241254, AF291820) match to the 159446-base BAC
clone GS1-594A7 (AC003669, NT 001172), which is located on chromosome
Xp22. Exon/intron structures are schematically indicated. The
vertical bars in the BAC clone represent locations of exons
and vertical lines on collectrin and ACE2 cDNAs indicate
exon/intron boundaries. Four exons of collectrin cDNA and 18 exons
of ACE2 cDNA are found on the BAC clone GS1-594A7.
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Fig. 3.
Localization of mRNA transcripts of
collectrin. A-D, Northern blot analyses of collectrin
in mouse, rat, and human tissues. 2 µg of poly(A)+ RNAs
from mouse (panel A) and rat (panel B) were
subjected to 2.2 M formaldehyde, 1% agarose-gel
electrophoresis and capillary transferred to nylon membranes. The
membranes were hybridized with [ -32P]dCTP-radiolabeled
mouse, rat, and human collectrin cDNAs. The membrane was reprobed
with GAPDH (1 × 106 cpm/ml) and is shown in
panel C. A single transcript of ~1.8 kilobases is observed
exclusively in the kidneys and is not detected in any other tissues.
Transcript size is similar in the mouse, rat, and human, and no
alternative splicing variants are observed. E-H, in
situ hybridization of mouse collectrin. The kidneys of CD-1 mice
tissues were embedded in paraffin, and the sections were hybridized
with mouse collectrin antisense primer (AS2) with 3'-biotinylated
Brigati's tail [3'-(TAG)5-BBB-(TAG)2-BBB-5']
(Fig. 1). The biotin-labeled hybrids were visualized by horseradish
peroxidase-linked streptavidin and diaminobenzidine reaction. The
hybridizing signal of collectrin mRNA is observed in collecting
tubules in the renal cortex (panels E and F) and
medulla (panels G and H). No signal is observed
on glomeruli or other segments of the tubules. Bar = 100 µm in panels E and G; bar = 50 µm in panels F and H.
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Fig. 4.
Immunoprecipitaion study of collectrin using
collecting duct cell lines. M-1 (kidney cortical collecting duct
cell) and mIMCD-3 (kidney inner medullar collecting duct cell line)
were grown and labeled with [35S]methionine and
[35S]cysteine (0.25 mCi/ml) for 12 h. The cells were
lysed in immunoprecipitaion buffer and incubated with preimmune rabbit
serum and anti-collectrin antibody. The antibody/antigen complexes were
precipitated using protein A-Sepharose CL-4B and subjected to 12.5%
SDS-polyacrylamide gel electrophoresis under reducing conditions. Using
a specific anti-collectrin antibody, a ~32-kDa single band is
observed in M-1 and mIMCD-3 collecting duct cells (arrow).
The use of preimmune sera did not reveal any specific bands
corresponding to 32 kDa.
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Fig. 5.
Immunohistochemical localization of
collectrin in the kidney. A-F, immunofluorescence
studies of collectrin in CD-1 mouse kidney. Mouse kidney cryostat
sections were stained with rabbit anti-collectrin antibody and rabbit
anti-aquaporin-2 antibody. Rhodamine-conjugated donkey anti-rabbit IgG
was used as secondary antibody. Using specific antibody, collectrin was
localized in the cytoplasm of collecting duct cells in cortex
(A) and medulla (C). High magnification
photographs indicate a linear immunoreactivity along the luminal
surface (B and D). The epithelial cells in the
renal pelvis also express collectrin (C). Immunoreactivity
is not observed in the glomerulus (G, panel A).
The immunostaining of serial sections indicates that the distribution
of collectrin (E) matches with aquaporin-2 (F),
which is the marker for collecting ducts. Photographs in panels
E and F were obtained from the medulla of CD-1 mouse
kidneys. G and H, localization of collectrin
shown by the avidin-biotin complex (ABC) method. The ABC method was
performed utilizing the MicroProbeTM System and UltraProbe staining
kit (Biømeda). The sections were incubated with 1:100 dilution of
antibody specific for collectrin and the biotinylated donkey
anti-rabbit antibody. The biotin-labeled hybrids were detected with an
alkaline phosphatase-linked streptavidin, and the signal was developed
with the streptavidin AP detection system. The sections were
counterstained with hematoxylin and eosin stain. The immunoreactivity
of collectrin is observed as red in the cytoplasm and the
luminal surface of collecting ducts in the cortex (G) and
deep medulla (H). Bar = 100 µm in
A; bar = 50 µm in B;
bar = 250 µm in C; bar = 10 µm in
D-H).
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Fig. 6.
Northern blot analyses of mouse collectrin
mRNA expressed in various developmental stages of mouse
kidney. 20 µg of total RNAs from mouse kidneys of various
developmental stages were subjected to 2.2 M
formaldehyde,1% agarose-gel electrophoresis and capillary transferred
to nylon membranes. The membranes were hybridized with
[ -32P]dCTP-radiolabeled mouse collectrin cDNA. The
membrane was reprobed with GAPDH (1 × 106 cpm/ml).
mRNA signal of collectrin is detectable at 13-day of gestation and
its expression is progressively increased during the later stages of
gestation extending into the neonatal periods. The mRNA expression
is then much reduced in adult life of the CD-1 mouse. Lanes
13d and 17d, renal total RNA isolated from 13-day- and
17-day-old mouse fetuses. Lanes NB, 1W,
2W, and AD represent newborn, 1-week-, 2-week-,
and 8-week-old mice renal total RNAs, respectively.
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Fig. 7.
Immunohistochemical localization of
collectrin in various developmental stages of mouse kidney. By
immunofluorescence study, collectrin is observed in the ureteric bud
branches at day 13 of gestation (13d) of the CD-1 mouse
(A and B). Immunoreactivity of collectrin is
increased in day 17 of gestation (17d), and it is expressed
in the developing collecting ducts (C and D).
After birth, the enhanced expression of collectrin is maintained during
postnatal periods, i.e. newborn (NB,
E, and F), 1-week-old (1W,
G, and H), and 2-week-old (2W,
I, and J) mice. Collectrin is expressed in the
collecting ducts in the cortex, medulla, and epithelial cells lining
the pelvis. In 8-week-old adult kidneys (AD, K,
and L), immunoreactivity of collectrin is rather decreased.
Bar = 500 µm in A, C,
E, G, I, and K;
bar = 100 µm in B, D,
F, H, J, and L.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by the Uehara Memorial Foundation, The Naito Foundation, ONO Medical Foundation, Grant-in-aid for Encouragement of Young Scientists, Ministry of Education, Science and Culture, Japan (10770199) (to J. W.) and Grant-in-aid for Scientific Research (B), Ministry of Education, Science and Culture, Japan (11470218), the Uehara Memorial Foundation (to H. M.), the International Society of Nephrology/Kirin Fellowship Award (to H. Z.) and DK28492 (to Y. S. K.).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) AF178085, AF178086, and AF229179.
¶ To whom correspondence should be addressed: Department of Medicine III, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel.: 81-86-235-7235; Fax: 81-86-222-5214; E-mail: junwada@md.okayama-u.ac.jp.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M006723200
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ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; ACE, angiotensin-converting enzyme; cDNA-RDA, representational difference analysis of cDNA; RACE, rapid amplification of 5'- and 3'-cDNA ends; Ang II, angiotensin II; AT1, angiotensin II type 1 receptor; RAS, renin-angiotensin system; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; bp, base pair(s).
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