From the Departments of Cardiovascular Research and Biophysics, Berlex Biosciences, Richmond, California 94804
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ABSTRACT |
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A novel cDNA has been identified from human
heart that encodes an unusual mosaic serine protease, designated corin.
Corin has a predicted structure of a type II transmembrane protein and contains two frizzled-like cysteine-rich motifs, seven low density lipoprotein receptor repeats, a macrophage scavenger receptor-like domain, and a trypsin-like protease domain in the extracellular region.
Northern analysis showed that corin mRNA was highly expressed in
the human heart. In mice, corin mRNA was detected by in
situ hybridization in the cardiac myocytes of the embryonic heart
as early as embryonic day (E) 9.5. By E11.5-13.5, corin mRNA was most abundant in the primary atrial septum and the trabecular ventricular compartment. Expression in the heart was maintained through
the adult. In addition, mouse corin mRNA was also detected in the
prehypertrophic chrondrocytes in developing bones. By fluorescent in situ hybridization analysis, the human corin gene was
mapped to 4p12-13 where a congenital heart disease locus, total
anomalous pulmonary venous return, had been previously localized. The
unique domain structure and specific embryonic expression pattern
suggest that corin may have a function in cell differentiation during development. The chromosomal localization of the human corin gene makes
it an attractive candidate gene for total anomalous pulmonary venous return.
Serine proteases are essential for a variety of biological
processes including food digestion, complement activation, and blood coagulation (1-3). In Drosophila, serine proteases
are also involved in developmental pathways. For example, serine
proteases encoded by the nudel, gastrulation
defective, easter, and snake genes are key components
of a proteolytic cascade that is critical for the establishment of the
dorsal-ventral pattern in developing embryos (4-6). Genetic defects in
these genes often lead to the disruption of the dorsal-ventral axis,
resulting in embryonic lethality (7).
Most serine proteases of the trypsin family are secreted proteins.
Several members from this family have been identified that contain an
integral transmembrane domain. Hepsin, for example, is a serine
protease expressed on the surface of hepatocytes. Structurally, hepsin
is a type II transmembrane protein with the transmembrane domain at its
amino terminus and the protease domain at the carboxyl terminus exposed
to the outside of the cell (8). In tissue culture studies, hepsin was
shown to contribute to hepatocyte growth (9). However, the
physiological significance of the growth stimulating activity of hepsin
remains unknown (10). In Drosophila, Stubble-stubbloid
protein, another transmembrane serine protease, shares structural
similarities with hepsin (11). Genetic studies demonstrated that
Stubble-stubbloid is essential for epithelial morphogenesis and
development of the fruit fly. Defects in the
Stubble-stubbloid gene cause malformation of legs, wings,
and bristles. Most recently, other transmembrane serine proteases were
isolated and cloned from human trachea and small intestine (12,
13). The biological function of these newly discovered membrane-bound
serine proteases has not yet been determined.
In this study, we report the cloning of a cDNA from the human heart
that encodes a novel transmembrane serine protease, designated corin.
Corin has a predicted structure of a type II transmembrane protein
containing two frizzled-like cysteine-rich motifs, seven LDL1 receptor repeats, a
macrophage scavenger receptor-like domain, and a trypsin-like protease
domain in the extracellular region. In situ hybridization
revealed that corin mRNA was expressed in the embryonic heart as
early as E9.5, and the expression in the heart was maintained through
the adult stage. In addition, corin mRNA was detected in
prehypertrophic chrondrocytes of the developing bones. The unusual
domain structures and specific expression pattern suggested that corin
may have a function in cell differentiation during embryonic development.
Materials--
Human cancer cell lines, HEC-1-A (endometrium
adenocarcinoma), U2-OS (osteosarcoma), SK-LMS-1 (vulva sarcoma), RL95-2
(endometrium carcinoma), and AN3-CA (endometrium adenocarcinoma) were
obtained from the American Type Culture Collection (ATCC). Human heart cDNA libraries and human and mouse multiple tissue Northern blots were purchased from CLONTECH (Palo Alto, CA). Mouse
tissue sections used for in situ hybridization were
purchased from Novagen (Madison, WI). Tissue culture media and
supplements were from Life Technologies Inc. All other chemicals were
obtained from Sigma.
Isolation of Human Corin cDNA Clones--
An expressed
sequence tag (EST) clone was found in a human heart cDNA library
from the Incyte EST data base that shared significant sequence homology
with trypsin, indicating that the EST may encode a novel serine
protease gene. A 2.1-kb EcoRI-XhoI insert from this EST clone was used to screen a human heart cDNA library
(CLONTECH). Approximately, 5 × 106 lambda phage clones were screened, and two positive
clones were isolated that contained inserts of 3.5 and 3.1 kb,
respectively. The DNA sequences of these two clones were determined.
Oligonucleotide primers were designed to clone further 5' end cDNA
sequences by 5' rapid amplification of cDNA ends (RACE) using
Marathon-ready human heart cDNA templates
(CLONTECH). The PCR products from 5' RACE were
cloned into pCRII vector (Invitrogen, San Diego, CA) and sequenced.
Oligonucleotide primers used in the 5' RACE experiments were
5'-CAGTTGGTTTGAACAAGTGCAGGG-3', 5'-TGCAAGGAGGGATACGCTCGCCTG-3', 5'-AATCCCAAGAACAGACTCACAGCG-3', 5'-CGGGTCACAGAGAGAGCTACCACC-3', 5'-GGTCTCCTTCTTGACATGAATCTG-3', 5'-CGGAGCCCCATGAAGTTAAACCA-3', and
5'-AACAAAAGGATCCTTGGAGGTCGGACGAGT-3'. The final 5' end sequence of
human corin cDNA was derived from at least three independent clones. The full-length cDNA sequence was compiled using the
Genetics Computer Group (GCG) software (version 9.1, Madison, WI).
Northern Analysis--
Northern blots containing
poly(A)+ RNA samples (2 µg/lane) from multiple human and
mouse tissues were purchased from CLONTECH. Human
and mouse corin cDNA probes were labeled with
[32P]dCTP using a random primed DNA labeling kit (Roche
Molecular Biochemicals). Northern hybridization was performed at
42 °C overnight in a solution containing 40% formamide, 5×
Denhardt's solution, 6× SSC, 100 µg/ml salmon sperm DNA, and 0.1%
SDS. Blots were washed with 0.2× SSC, 0.1% SDS at 60 °C and then
exposed to Fuji imaging plates. As a control, the blots were reprobed
with a human actin cDNA probe provided by
CLONTECH.
RT-PCR--
mRNA samples were isolated from Hec-1-A, U2-OS,
SK-LMS-1, and AN3-CA cells using a commercial RNA preparation kit
(Oligotex Direct mRNA Mini Kits, Qiagen). First strand cDNAs
were synthesized using SuperScript II RNase In Situ Hybridization--
Mouse adult heart and embryonic
tissue sections were deparaffinized in xylene, rehydrated, and fixed in
4% paraformaldehyde. The tissues were digested with proteinase K (20 µg/ml), then treated with triethanolamine/acetic anhydride, and
dehydrated. An 800-bp mouse corin cDNA fragment from the coding
region was cloned into pCRII (Invitrogen) in two orientations to yield
plasmids pM11 and pM41. The plasmids were linearized by
HindIII digestion. Sense and antisense probes were
synthesized using T7 RNA polymerase (T7/SP6 transcription kit, Roche
Molecular Biochemicals) and labeled with [33P]UTP
(Amersham Pharmacia Biotech). The hybridization was carried out as
described (14). The slides were dehydrated and dipped in Kodak NTB-2
emulsion and exposed for 4 weeks in light-tight boxes at 4 °C.
Photographic development was carried out in a Kodak D-19 developer. The
slides were stained with hematoxylin/eosin and analyzed using both
light- and dark-field optics of a Zeiss microscope.
Fluorescent in Situ Hybridization (FISH) Analysis--
P1 phage
clones containing the human corin gene were isolated by filter
hybridization using a human corin cDNA as the probe. One clone was
confirmed by DNA sequencing using a primer from human corin cDNA.
The DNA fragment from this P1 phage was labeled with digoxigenin-dUTP.
The labeled probe was combined with sheared human DNA and hybridized to
metaphase chromosomes derived from PHA-stimulated peripheral blood
lymphocytes in a solution containing 50% formamide, 10% dextran
sulfate, and 2× SSC. Hybridization signals were detected by
fluorescent-labeled antidigoxigenin antibodies and counter-staining
with 4,6-diaminoidino-2-phenylindole. A total of 80 metaphase cells
were analyzed of which 74 cells exhibited specific labeling.
Homology Model of the Protease Domain of Corin--
A model of
the corin protease domain (amino acids 802-1042) was built based on
the structure of bovine chymotrypsinogen A at 1.8-Å resolution (15,
16), using the homology program (Insight II, 1995, MSI, San Diego, CA).
Rotamers were used for non-identical side chain replacements (16).
Coordinates for the loop insertions were extracted from the Brookhaven
protein data bank (17). The model was refined by energy minimization
using the AMBER force field (Discover 95.0), with a
distance-dependent dielectric constant. The minimization
used the steepest descents and conjugate gradient methods as follows:
first for the loops only where insertions and deletions occurred, then
side chains, and a final round of minimization keeping the C Cloning of the Full-Length Human Corin cDNA--
A computer
search using the BLAST program identified an EST clone from a human
heart library that shared significant homology with serine protease
family members, such as trypsin. The EST clone was used to isolate the
full-length cDNA of a novel gene, designated corin for its abundant
expression in the heart. The sequence of the full-length corin
cDNA, 4933 bp in length, is shown in Fig.
1. The size of the cDNA is consistent
with the length of corin mRNA (~5 kb) detected by Northern
analysis (Fig. 4A). An ATG codon is located at position 95 that may represent the translation initiation site. The open reading
frame (ORF) spans 3126 bp with a 5'-untranslated region of 94 nucleotides before the initiation codon. At the 3' end, there is a
1.7-kb 3'-untranslated region after the stop codon at position 3221. A
polyadenylylation signal of AATAAA is present 12 nucleotides before the
poly(A)+ tail.
The Domain Structure of Human Corin--
The ORF of the human
corin cDNA encodes a polypeptide of 1042 amino acids with a
calculated mass of 116 kDa. At the amino terminus of the predicted
corin protein, there is no discernible signal peptide sequence.
Hydropathy plots using the GCG program identified a highly hydrophobic
region between amino acids 46 and 66 (Fig.
2B). This hydrophobic sequence
could serve as a potential transmembrane domain. There are positively
charged amino acid residues immediately preceding the putative
transmembrane segment, suggesting that corin is a type II transmembrane
protein with the amino terminus present in the cytosol (18). Consistent
with this hypothesis, there are 19 predicted N-linked
glycosylation sites present in the extracellular domains of corin (Fig.
1).
Analysis of the corin protein sequence showed that in the extracellular
region there are two frizzled-like cysteine-rich domains, seven LDL
receptor repeats, one macrophage scavenger receptor-like domain, and
one trypsin-like serine protease domain (Fig. 2A). As shown
in Fig. 2A, two frizzled-like cysteine-rich domains are located at amino acids 134-259 and 450-573, respectively. Amino acid
sequences of these two domains share significant similarities with the
extracellular cysteine-rich domain of the Drosophila Frizzled protein, a seven-transmembrane receptor essential for polarity
determination during the development of the fruit fly (19). The
frizzled-like cysteine-rich domains have also been found in other
proteins, such as Dfz2 in Drosophila (20), Lin-17 in
Caenorhabditis elegans (21), and FZ-1 in human (22). The sequences of the two frizzled-like cysteine-rich domains in corin are
closest to those in Lin-17 and FZ-1. As shown in Fig. 2C, all the 10 conserved cysteine residues are present in the frizzled-like cysteine-rich domains of corin.
Between amino acids 268-415 and 579-690 (Fig. 2, A and
D), there are seven cysteine-rich repeats homologous to the
LDL receptor class A repeats (23). Each repeat is about 36 amino acids
long and contains six cysteine residues as well as a highly conserved cluster of negatively charged amino acids. In the LDL receptor, these
cysteine-rich repeats bind calcium ions and play an essential role in
endocytosis of the extracellular ligands (23). Similar motifs have been
found in the extracellular domain of other membrane receptors, such as
LDL receptor-related protein (LRP1) (24), megalin (also known as LRP2
or gp330) (25), complement proteins (26), enterokinase (27), and
Drosophila proteins yolkless and nudel (28, 29).
In addition to the frizzled-like cysteine-rich domains and LDL
receptor-like repeats, there is another cysteine-rich region between
amino acids 713 and 801 in corin (Fig. 2, A and
E). This region contains 88 amino acids and is homologous to
the cysteine-rich motif found in the macrophage scavenger receptor
(30). This motif is also present in the sea urchin spermatozoa speract
receptor (31, 32) and the vertebrate serine protease, enterokinase (27).
At the carboxyl terminus of corin protein between amino acid residues
802 and 1042, there is a trypsin-like serine protease domain (Fig.
2A). This protease domain is highly homologous to the
catalytic domain of members of the trypsin superfamily. For example,
amino acid sequence identities between corin and prekallikrein (33),
factor XI (34), and hepsin (35) are 40, 40, and 38%, respectively. All
essential features of serine protease sequences are well conserved in
corin (Figs. 1 and 2F). The active site residues of the
catalytic triad are located at His843, Asp892,
and Ser985. The amino acid residues forming the substrate
specificity pocket are located at Asp979,
Gly1007, and Gly1018. These residues are
predicted to bind the substrate P1 residues, suggesting that corin
would cleave its substrate after basic residues, such as lysine or
arginine. In addition, a putative activation cleavage site was found at
Arg801, suggesting that corin would be synthesized as an
inactive zymogen and that another trypsin-like enzyme was required for
its activation.
In the protease domain, there are 12 cysteine residues. Potential
pairing of these cysteine residues can be predicted by comparing with
other well studied serine proteases, such as trypsin and chymotrypsin.
First three pairs of cysteine residues present in essentially all
members of the trypsin superfamily are located at
Cys828-Cys844,
Cys955-Cys970, and
Cys981-Cys1010. Two more pairs of cysteine
residues are present at the positions Cys790-Cys912 and
Cys926-Cys991. These two pairs of cysteine
residues are commonly found in a subfamily of two-chain serine
proteases, such as chymotrypsin and prekallikrein (33). The presence of
Cys790 and Cys912 indicated that, after the
activation cleavage at Arg801, the catalytic domain of
corin would remain attached to the rest of molecule by a disulfide
bond. Interestingly, there is one additional pair of cysteine residues,
Cys817 and Cys830, present in corin. Cysteine
residues at these two positions were not found in any other serine
proteases in vertebrates. A search of data bases showed that a
chymotrypsinogen-like serine protease from the lugworm, Arenicola
marina, had two cysteine residues at the corresponding
positions.2 A model of the
corin protease domain was built based on the structure of bovine
chymotrypsinogen A (Fig. 3). Based on
this corin model, where the C- Northern Analysis of Corin mRNA Expression--
To determine
expression of the corin gene in human tissues, Northern hybridization
was performed using human corin cDNA probes. As shown in Fig.
4A, an ~5-kb transcript was
detected only in the heart but not in other tissues including brain,
placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen,
thymus, prostate, testis, ovary, colon, and leukocytes. Since the heart is mainly composed of cardiac muscles, Northern analysis was performed to examine the presence of corin mRNA in other human muscle-rich tissues. Again, corin mRNA was detected in the heart but not in uterus, small intestine, bladder, stomach, and prostate (Fig. 4B).
To examine corin mRNA expression in mice, the full-length mouse
corin cDNA was cloned by a PCR-based strategy. Mouse corin cDNA
shared 89% sequence identities with human corin cDNA (data not
shown). Northern analysis was performed with RNA samples from mouse
tissues. As shown in Fig. 4C, a prominent transcript of ~5
kb was detected in samples derived from the heart. In contrast to
Northern analysis with human samples, low levels of corin mRNA were
also detected in samples derived from the testes and kidneys.
Mouse Corin mRNA Expression in Adult and Embryonic
Hearts--
In situ hybridization was performed to
determine the temporal and special expression of corin mRNA. In
adult mice (Fig. 5), corin mRNA was
detected in cardiac myocytes of both atrium and ventricle. The level of
expression appeared to be higher in the atrium than the ventricle.
During embryonic development, corin mRNA was first detected at E9.5
in both atrium and ventricle of the developing heart (Fig.
6B). Between E11.5 and E13.5,
corin mRNA was highly expressed in the thickened atrial wall and in the regions that underwent trabeculation in the ventricle (Fig. 6,
D and F). By E15.5, corin mRNA in the heart
was more abundant, especially in primary atrial septa (Fig.
6H). Weak signals appeared to be present in developing aorta
and vena cava but not in the esophagus and lungs (Fig. 6H).
The expression of corin mRNA in the heart was maintained in the
subsequent embryonic stages (not shown).
Corin mRNA Expression in Other Tissues--
In addition to the
heart, corin mRNA was also detected in other mouse tissues by
in situ hybridization. For example, corin mRNA was
present in the uterus of pregnant mice and in the developing kidneys.
In the uterus (Fig. 7L), corin
mRNA expression was most abundant in the decidual cells close to
the implantation site of the embryo. In the developing kidneys at
E15.5, corin mRNA was highly expressed in the stromal cells in the
medulla but not in the cortex of the kidney (Fig. 7J). This
finding was consistent with the results of Northern analysis in which a
corin transcript was found in RNA samples from mouse kidneys (Fig.
3C).
Interestingly, in situ hybridization also identified corin
mRNA in several cartilage-derived structures, such as the vertebra in the tail, the turbinate in the head, and the long bones in the limbs
(Fig. 7, B, D, F, and H). Fig. 7B
showed the expression of corin mRNA in cartilage primordia of
vertebral bodies in the posterior of an E13.5 embryo. By E15.5, the
level of corin mRNA expression in the vertebra was much lower as
the vertebra became more matured (data not shown), indicating that
corin may play a role in the differentiation of chondrocytes. This
notion was supported by the expression of corin mRNA in developing
limbs. Fig. 7, E and F, showed an early
developing digital bone that consisted of three types of cells as
follows: hypertrophic chondrocytes at the center, prehypertrophic
chondrocytes next to the hypertrophic zone, and proliferating
chondrocytes at the both ends. Corin mRNA was found mostly in the
prehypertrophic chondrocytes (Fig. 7F). Hybridization
signals were also present in perichondrium (Fig. 7F). Fig.
7, G and H, showed a long bone in a hind limb
that was at a more advanced developmental stage. The central
hypertrophic zone was replaced by vascularized tissues containing bone
marrow cells and osteroblasts. Nevertheless, similar expression pattern of corin mRNA was found in the narrow zone of the prehypertrophic chondrocytes and in the perichondrium. These results indicated that
corin expression was associated with a specific stage of chondrocyte differentiation.
Corin mRNA Expression in Human Tumor Cell Lines--
A number
of human cancer cell lines were screened by Northern and RT-PCR
analyses for the presence of corin mRNA. In most cell lines, such
as HL60, HeLa, K562, MOLT-4, RAJI, SW480, A549, and G36, corin mRNA
was undetectable (data not shown). However, corin mRNA was found in
several cell lines derived from uterus tumors or osteosarcoma. As shown
in Fig. 8, corin mRNA was detected by
RT-PCR in endometrium carcinoma cell lines HEC-1-A, AN3 CA, and RL95-2,
leiomyosarcoma cell line SK-LMS-1, as well as in osteosarcoma cell line
U2-OS. The result is consistent with the finding by in situ
hybridization in which corin mRNA was highly expressed in the
developing bones in embryos as well as in the maternal uterus.
Chromosomal Localization of the Human Corin Gene--
FISH
analysis was performed to determine the chromosomal locus of the human
corin gene. Specific fluorescent spots were found at 4p12-13, a region
adjacent to the centromere on the short arm of chromosome 4 (Fig.
9). The result was confirmed in a
subsequent experiment in which a genomic probe previously mapped to
4p15.3 was co-localized with the corin gene probe (data not shown). A search of the OMNI human genetic data base indicated that a congenital heart disease locus, total anomalous pulmonary venous return (TAPVR), was previously mapped to this region at 4p13-q12 (37).
In this study, we describe the cloning and initial
characterization of a novel cDNA from the human heart that encodes
a putative transmembrane serine protease, which we have designated as
corin. The presence of a hydrophobic transmembrane domain at its amino terminus and the absence of a signal peptide suggest that corin is a
type II transmembrane protein. In the extracellular region of corin,
there is a trypsin-like catalytic domain that contains all conserved
structural features of serine proteases, such as the catalytic triad,
the activation cleavage site, the substrate specificity pocket, and the
essential cysteine residues. Interestingly, the protease domain of
corin contains two unique cysteine residues, Cys817 and
Cys830, that are not present in other trypsin-like serine
proteases in vertebrates. Molecular modeling showed that these two
cysteine residues are likely to form a disulfide bond connecting two
Although members of the trypsin superfamily are known to contain a
variety of domain structures such as kringle and epidermal growth
factor-like domains that are important for protein-protein interactions, this is the first report of the presence of a
frizzled-like cysteine-rich domain in this extended family. Originally,
the frizzled gene was identified in Drosophila (38). The
gene encodes a seven-transmembrane receptor that is required for proper
development of hairs, bristles, and ommatidia of the fruit fly (19,
39). Later, other Frizzled proteins have been identified in many other species. They all contain a well conserved extracellular cysteine-rich domain and a seven-transmembrane domain and act as receptors for secreted Wnt glycoproteins (for review see Refs. 40 and 41). The
cysteine-rich domain, which is about 120 amino acids in length and
contains a motif of 10 invariantly spaced cysteine residues, has been
shown to be necessary and sufficient for the binding of the Wnt ligands
(20, 42). Recent studies demonstrated that Frzb, a secreted
frizzled-like protein without the seven-transmembrane domain, is
expressed in the Spemann organizer of frog embryos and can bind and
inhibit Wnt-8 (43, 44). In addition, similar frizzled-like
cysteine-rich domains have also been found in several other proteins,
including mouse collagen (XVIII) The temporal and special pattern of corin gene expression further
supported a potential developmental function of corin. In mice, corin
mRNA was detected in the cardiac myocytes of the embryonic heart as
early as E9.5 (Fig. 6B). The expression was most prominent in the primary atrial septum and the trabecular ventricular compartment by E11.5-13.5 (Fig. 6, D and F). During this
period, an active process of looping and remodeling takes place in the
embryonic heart. As a result, outflow tracts are formed, and the
original single tube-like heart is reorganized into a four-chambered
structure. Growth factors, such as bone morphogenic proteins and the
transforming growth factor- In addition to the heart, corin mRNA was identified in other
tissues, such as the pregnant uterus and developing kidneys and bones.
The expression of corin mRNA in these tissues appeared to be cell
type-specific. For example, in developing long bones corin mRNA was
specifically expressed in the prehypertrophic chrondrocytes. It is
known that skeletal bones are derived from two different processes,
intramembranous and endochondral ossification. In the former case,
mesenchymal tissues are directly converted into bones, whereas in the
latter case the mesenchymal cell is converted to bone via cartilage as
an intermediate step. The vertebrae, long bones, and certain fragments
of skull are formed by endochondral ossification (53). In these bones,
mesenchymal cells first become chondrocytes that in turn differentiate
from proliferating chondrocytes to prehypertrophic chondrocytes and
finally to hypertrophic chondrocytes. The hypertrophic chondrocytes
eventually undergo apoptosis followed by vascularization and
ossification. This process of chondrocyte differentiation has been
shown to be tightly regulated by hedgehog proteins, bone morphogenic
proteins, and parathyroid hormone-related protein (54-57). The
specific expression of corin mRNA in a subset of chondrocytes
indicated that corin may also be involved in this cell differentiation process.
Finally, by FISH analysis the human corin gene was located on the short
arm of chromosome 4 (4p12-13) (Fig. 9). A search of the OMNI human
genetic data base showed that a disease locus, total anomalous
pulmonary venous return (TAPVR), had been previously mapped to this
region. TAPVR is a rare cyanotic form of congenital heart defects in
which the pulmonary vein connected abnormally to the right atrium or
one of the venous tributaries instead of the left atrium. The molecular
mechanism responsible for this developmental defect in the heart is
unknown. A linkage study of a large Utah-Idaho family that included 14 affected individuals localized the TAPVR locus to a 30-centimorgan
interval on 4p13-q12 (37). The findings that the corin gene and the
TAPVR locus are co-localized on chromosome 4 and that corin mRNA is
highly expressed in the embryonic heart, particularly in the region
where outflow tracts were formed, suggest that corin is an attractive
candidate for the TAPVR gene. The isolation of the corin cDNA
provided a useful tool to study further this intriguing possibility.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
reverse
transcriptase (Life Technologies Inc.). Human corin-specific oligonucleotide primers (sense primer,
5'-AACAAAAGGATCCTTGGAGGTCGGACGAGT-3', and antisense primer,
5'-CGGAGCCCCATGA AGTTAATCCA-3') were used to amplify a 630-bp fragment
of corin cDNA between nucleotides 2475 and 3105. Oligonucleotide
primers TFR1 (5'-GTCAATGTCCCAAACGTCACCAGA-3') and TFR2
(5'-ATTTCGGGAATGCTGAGAAAACAGACAGA-3'), derived from the human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, were used as an
internal quantification control. PCR reactions were performed with a
thermal cycler (Perkin-Elmer, model 480). PCR products were separated
on 1% agarose gels and visualized by ethidium bromide staining.
atoms
fixed. The residues of corin (His843, Asp892,
and Ser985) corresponding to the catalytic triad of the
template structure were also held fixed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequence of human corin cDNA
and its deduced amino acid sequence. The potential codon for the
initial methionine, the translation stop codon, and the
polyadenylylation signal were in bold-face type and
underlined. The putative transmembrane domain was
double underlined. The 19 potential N-linked
glycosylation sites are in boldface type and double
underlined. An arrow indicates the putative cleavage
site for the activation of the serine protease. The active site
residues of the catalytic triad (His843,
Asp892, and Ser985) are in boldface
type and underlined.
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Fig. 2.
A, a schematic presentation of the
domain structure of corin protein. The transmembrane domain
(TM), frizzled-like cysteine-rich domains (CRD),
LDL receptor repeats (LDLR), scavenger receptor
cysteine-rich domain (SRCR), and serine protease catalytic
domain (Catalytic) are indicated. Numbers
correspond to the amino acid residues of the ORF shown in Fig. 1.
B, hydropathy plots of the deduced amino acid sequence of
corin by Goldman and Kyte-Doolittle methods, respectively (36).
Hphobic, hydrophobic; Hphilic, hydrophilic.
C, alignment of amino acid sequences of the frizzled-like
cysteine-rich domains from corin and other members of the frizzled
family, including Frizzled in Drosophila, lin-17 in C. elegans, and FZ-1 in human. D, alignment of amino acid
sequences of the seven LDL receptor repeats of corin with the consensus
sequence derived from the human LDL receptor. E, alignment
of amino acid sequences of the scavenger receptor-like cysteine-rich
domains from corin and human enterokinase (Entk), sea urchin
speract receptor (q17064) and human scavenger receptor I
(o15393). Asterisks indicate conserved residues.
F, alignment of amino acid sequences of protease domains
from human corin, prekallikrein (KAL), enterokinase
(ENTK), trypsin (TRP1), and bovine
chymotrypsinogen A (CTRA).
atoms of these two cysteine residues
were held fixed during energy minimization, the distance between the
sulfur atoms of their side chains is about 2.5 Å after rotamer
searching. The model indicates that these two cysteines are likely to
form a disulfide bond connecting two
-sheets in the core of the
protease domain (Fig. 3).
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Fig. 3.
Molecular model of the protease domain of
corin between amino acids 802 and 1042. A corin model was built
based on the structure of bovine chymotrypsinogen A, as described under
"Experimental Procedures." The active site residues of the
catalytic triad (His843, Asp892, and
Ser985) are shown in purple. Four disulfide
bonds in the corin model (Cys828-Cys844,
Cys955-Cys970,
Cys926-Cys991, and
Cys981-Cys1010) that correspond to the
disulfide bonds in the catalytic domain of chymotrypsinogen
(Cys42-Cys58,
Cys168-Cys182,
Cys136-Cys201, and
Cys191-Cys220) are shown in blue.
The side chains of Cys817 and Cys830 of the
corin model are in an acceptable proximity to form a disulfide bond
(pink). The distance between the C- atoms from the
chymotrypsinogen template (Val31 and Gly44)
corresponding to these two cysteine residues is 5.08 Å, and the
distance between the sulfur atoms after rotamer searching of the
cysteine side chains is about 2.5 Å. The potential disulfide bond
between Cys790 and Cys912 of corin
corresponding to the disulfide bond between Cys1 and
Cys122 of chymotrypsinogen is not included in the
model.
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Fig. 4.
Northern analysis of corin mRNA
expression. Human and mouse multiple tissue Northern blots were
hybridized with human and mouse corin cDNA probes, respectively. In
human tissues (A and B), corin mRNA was
detected only in samples from heart. In mouse tissues (C),
abundant expression of corin mRNA was detected in samples from
heart. Weak signals were also detected in samples from testis and
kidney.
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Fig. 5.
Analysis of corin mRNA expression by
in situ hybridization in an adult mouse heart.
Tissue sections from atrium (B) and ventricle (A)
were stained with hematoxylin/eosin. Corin mRNA was detected by
in situ hybridization using a mouse corin cDNA probe.
Expression of corin mRNA was found in the cardiac myocytes of both
the atrium (D) and the ventricle (C) as shown by
white spots.
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Fig. 6.
Expression of corin mRNA in the
developing heart. Tissue sections were prepared from mouse embryos
at day E9.5 (A and B), E11.5 (C and
D), E12.5 (E and F), and E15.5
(G-J) and stained with hematoxylin/eosin
(A, C, E, G, and I). Corin mRNA expression
was detected by in situ hybridization in developing heart by
E9.5 (B) and E11.5 (D) as indicated by
arrows. The expression was prominent in the primary atrial
septum and the trabecular ventricular compartment by E12.5
(F). By E15.5, corin mRNA was detected in most cardiac
myocytes in both atrium (H) and ventricle (J).
Abbreviations used in E, G, and I are as follows:
Atr, atrium; V, ventricle; Ar, aorta;
Vc, vena cava; E, esophagus; Lu,
lung.
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Fig. 7.
Expression of corin mRNA in other tissues
during embryonic development. Tissue sections were stained with
hematoxylin/eosin. In situ hybridization was performed uning
a mouse corin cDNA probe, as described under "Experimental
Procedures." A and B, expression of corin
mRNA in cartilage primordia of vertebral bodies of an E13.5 embryo.
C and D, expression of corin mRNA in the
turbinate primordium around the nasal and eye cavities of an E15.5
embryo. E and F, expression of corin mRNA in
a developing digital bone in a front paw at E15.5. Corin mRNA was
detected in the region adjacent to the hypertrophic chondrocytes and in
the perichondrocytes. G and H, in a more matured
digital bone in a hind limb of an E15.5 embryo, a similar pattern of
corin mRNA expression was found in the region adjacent to the
hypertrophic chrondrocytes and in the perichondrocytes. I
and J, expression of corin mRNA in the medulla of a
developing kidney at E15.5. K and L, expression
of corin mRNA in the decidual cells of a pregnant uterus.
Abbreviations used are: V, vertebral bodies; N,
nasal cavity; E, eye cavities; Hy, hypertrophic
chondrocytes; P, perichondrocytes.
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Fig. 8.
Analysis of corin mRNA expression in
tumor cell lines by RT-PCR. RNA samples were isolated from human
tumor cell lines. RT-PCR experiments were performed using
oligonucleotide primers derived from human corin cDNA. Corin
mRNA was detected in samples from Hec-1-A, U2-OS, SK-LMS-1, RL95-2,
and AN3-CA cells (upper panel, lanes 2-6) but not in
samples from HeLa cells (upper panel, lane 1). In a control
experiment, PCR reactions were performed with specific oligonucleotide
primers for the human GAPDH gene. GAPDH mRNA was detected in
samples from all cell lines (lower panel, lanes 1-6).
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Fig. 9.
Chromosomal localization of the human corin
gene by FISH. A fluorescent-labeled genomic DNA probe containing
the human corin gene was hybridized to metaphase chromosomes derived
from PHA-stimulated peripheral blood lymphocytes. Hybridization signals
are shown as bright blue spots and indicated by white
arrows (left panel). The position of the corin locus on
human chromosome 4 is illustrated in a diagram (right
panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets in the core of the protease domain (Fig. 3). A search of
genomic data bases showed that a chymotrypsin-like protease found in
the lugworm, A. marina, also has two cysteine residues at
the corresponding positions. It is not clear whether these two cysteine
residues are maintained through a convergent or divergent evolution.
Nevertheless, the presence of such an unusual pair of cysteine residues
in both corin and the lugworm protease suggests an important biological function of the disulfide bond. One potential possibility is that the
disulfide bond may contribute to stability of the proteases.
1 chain (45), human
carboxypeptidase Z (46), and several receptor tyrosine kinases
(47-49). The function of the cysteine-rich domain in these proteins
has not been determined. Corin is unique in that it contains the
frizzled-like cysteine-rich domains and a serine protease domain. The
presence of frizzled-like domains in corin implies that corin may play
an important role in development by directly interacting with Wnt proteins.
family members, are known to play a
critical role during the embryonic heart development (50). Recent
studies in Drosophila showed that the wingless
(wg) gene, a homologue of the wnt oncogene in
mammals, is directly involved in heart formation (51). It has been
suggested that similar signaling pathways also contributed to the heart
development in vertebrate (52). It is possible that corin could
participate in such developmental pathways by interacting directly with
Wnt proteins or other growth factors.
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ACKNOWLEDGEMENTS |
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We thank Drs. W. Dole and G. Rubanyi for their encouragement and helpful discussions.
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FOOTNOTES |
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* 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) AF133845.
To whom correspondence should be addressed: Berlex Biosciences,
15049 San Pablo Ave., Richmond, CA 94804. Tel.: 510-669-4737; Fax:
510-669-4246; E-mail: qingyu_wu{at}berlex.com.
2 J. Eberhardt, GenBankTM accession number G1160388.
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ABBREVIATIONS |
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The abbreviations used are: LDL, low density lipoprotein; EST, expressed sequence tag; FISH, fluorescent in situ hybridization; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ORF, open reading frame; RT, reverse transcriptase; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; TAPVR, total anomalous pulmonary venous return; kb, kilobase pair; bp, base pair; E, embryonic day.
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REFERENCES |
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