Furin-mediated Processing of Pro-C-type Natriuretic Peptide*
Chengliang Wu
,
Faye Wu
,
Junliang Pan
,
John Morser
and
Qingyu Wu
¶
From the
Department of Cardiovascular Research,
Berlex Biosciences, Richmond, California 94804 and
Jiangsu Institute of Hematology, Suzhou, Jiangsu
215006, China
Received for publication, February 4, 2003
, and in revised form, April 18, 2003.
 |
ABSTRACT
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C-type natriuretic peptide (CNP) is a member of the natriuretic peptide
family that is involved in a variety of homeostatic processes. Here we
characterize the processing essential for the conversion of the precursor,
human pro-CNP, to the biologically active hormone. In human embryonic kidney
293 and chondrosarcoma SW 1353 cells, recombinant pro-CNP was converted into a
mature peptide intracellularly as detected by Western analysis. Expression of
recombinant human corin, a proatrial natriuretic peptide convertase, did not
enhance the processing of pro-CNP in these cells. The processing of pro-CNP
was inhibited in the presence of an inhibitor of the endoprotease furin but
was not affected by inhibitors of matrix metalloproteinases and tumor necrosis
factor-
convertase. In furin-deficient human colon adenocarcinoma LoVo
cells, no conversion of recombinant pro-CNP to CNP was detected. Expression of
recombinant human furin in LoVo cells restored the ability of these cells to
process pro-CNP. Furthermore, incubation of purified recombinant human furin
with LoVo cell lysate containing pro-CNP led to the conversion of the
precursor to a mature peptide. The furin-processed CNP was shown to be
biologically active in a cell-based cGMP assay. These results demonstrate that
furin is a critical enzyme for the processing of human pro-CNP.
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INTRODUCTION
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The natriuretic peptide family consists of three structurally related
peptides: atrial natriuretic peptide
(ANP),1 brain or
B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP)
(17).
ANP and BNP are produced mainly in cardiomyocytes in the heart and are
important in maintaining normal body fluid and sodium homeostasis. CNP is
expressed in many tissues and cell types, including the brain, vascular
endothelial cells, and chondrocytes
(813).
The dominant receptor for CNP is natriuretic peptide receptor-B, whereas the
receptor for ANP and BNP is natriuretic peptide receptor-A. The biological
functions of CNP are apparently different from those of ANP and BNP. Studies
have shown that CNP inhibits the proliferation of vascular smooth muscle cells
in culture (14,
15) and prevents balloon
injury-induced coronary artery restenosis in animal models
(1618).
Recent studies of CNP-deficient mice indicate that CNP plays an important role
in chondrocyte differentiation and bone formation
(11).
The natriuretic peptides are synthesized as prepropeptides. The signal
peptide is removed to form propeptides, but a further proteolytic cleavage of
the propeptide is required to convert the precursor to a biologically active
peptide. This activation mechanism is critical in regulation of the activity
of the natriuretic peptides, but the enzyme(s) responsible for the conversion
remained uncharacterized for many years. Recently, we identified a cardiac
serine protease, corin (19),
that is a member of the type II transmembrane serine protease family
(20,
21). In cell-based functional
studies, we showed that corin converted pro-ANP to biologically active ANP in
a highly sequence-specific manner
(22,
23). Recombinant corin also
processed pro-BNP to BNP in cell-based assays
(22), indicating that corin is
a convertase for pro-ANP and pro-BNP. To date, however, the enzyme responsible
for pro-CNP processing has not been fully characterized.
In addition to its abundant expression in cardiomyocytes, the
corin gene is also expressed in several other tissues, such as the
pregnant uterus and developing kidneys and bones
(19). In developing bones,
corin mRNA was specifically expressed in the prehypertrophic chondrocyte, a
subset of chondrocytes important in bone growth and maturation. Interestingly,
pro-CNP mRNA is also expressed in the prehypertrophic chondrocyte in
developing bones (11). The
co-localization of corin and pro-CNP mRNA expression suggested the hypothesis
that corin was responsible for the processing of pro-CNP.
In this study, we test this hypothesis by examining the processing of human
pro-CNP. Our results showed that in human kidney epithelial 293 cells and
chondrosarcoma SW 1353 cells, pro-CNP was processed intracellularly. The
processing of pro-CNP was not enhanced in the presence of recombinant corin
but was inhibited by an inhibitor of furin, a widely expressed
precursor-processing enzyme. We also showed that the processing of pro-CNP did
not occur in furin-deficient LoVo cells and that transfection of a plasmid
expressing human furin in LoVo cells enabled the cells to process pro-CNP to
CNP. Our data indicate that, unlike pro-ANP, pro-CNP is processed
intracellularly by the endoprotease, furin, but not by the transmembrane
serine protease, corin.
 |
EXPERIMENTAL PROCEDURES
|
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MaterialsPenicillin, streptomycin, L-glutamine,
fetal bovine serum, and cell culture medium were purchased from Invitrogen.
Human embryonic kidney 293 cells, chrondrosarcoma SW 1353 cells, and
furin-deficient LoVo human colon adenocarcinoma cells were obtained from the
American Type Culture Collection and maintained at the Core Facility at Berlex
Biosciences. Anti-V5 antibody was purchased from Invitrogen. Recombinant furin
was obtained from New England Biolabs Inc. (Beverly, MA). Furin inhibitor
(decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone) and synthetic human CNP were
purchased from Bachem Bioscience Inc. (King of Prussia, PA). The matrix
metalloproteinase (MMP) inhibitor GM6001 was purchased from BIOMOL Research
Laboratories, Inc. (Plymouth Meeting, PA). Tumor necrosis factor-
convertase (TACE) inhibitor and tumor necrosis factor-
protease
inhibitor-1 (TAPI), was purchased from Calbiochem. Transfection reagent
LipofectAMINE 2000 was purchased from Invitrogen. All other chemical reagents
were obtained from Sigma.
Cell CultureHuman 293 cells were cultured in
-minimum essential medium (
-MEM) (Invitrogen) supplemented with
10% fetal bovine serum and 1% L-glutamine. Chrondrosarcoma SW 1353
cells were grown in Leibovitz's L-15 medium (Invitrogen) supplemented with 10%
fetal bovine serum and 1% L-glutamine. LoVo cells were cultured in
Kaighn's modification of Ham's F-12 medium (Invitrogen) supplemented with 10%
fetal bovine serum and 1% L-glutamine. Rat aortic smooth muscle
cells (Cambrex Bioscience Walkersville, Inc.) were grown in a smooth muscle
cell growth medium (Cambrex Bioscience). All cells were cultured at 37 °C
in humidified incubators with 5% CO2/95% air.
Expression VectorsPlasmid vectors expressing human pro-ANP
(pcDNAproANP) and corin (pcDNACorin) were described previously
(22). Recombinant pro-ANP and
corin expressed by these vectors contain a viral V5 and a His6 tag
at their carboxyl termini, which facilitates the detection of the proteins. A
plasmid expressing human furin was provided by E. Tuley and J. E. Sadler
(Washington University, St. Louis, MO)
(24). The full-length human
pro-CNP cDNA was cloned by an overlap PCR method
(25) using the following four
oligonucleotide primers: CNP1S, 5'-TGCCGCCCGTGTGCGCCCCTCGAC-3';
CNP2A, 5'-ACATCCCAGGCCGCTCATGGAGCC-3'; CNP3A,
5'-GGGTTCGCGGGACCTTCGGCGGCGCCCCGGGCTTG-3'; and CNP4S,
5'-GTCCCGCGAACCCCGCCGGCAGAGGAG-3'
(26). Briefly, two separate
PCR products were amplified from human genomic DNA using primers CNP1S and
CNP3A or CNP2A and CNP4S. The two PCR products were gel-purified and mixed. A
second PCR was performed using primers CNP1A and CNP4S. The final PCR product
was cloned into the expression plasmid pcDNA3.1/V5-His-TOPO (Invitrogen). The
sequence of the insert and its orientation were verified by automated DNA
sequencing. The presence of the viral V5 tag at the carboxyl terminus of
pro-CNP allows the detection of the recombinant protein by Western blotting
using an anti-V5 antibody (Invitrogen).
Transfection and Western AnalysisTransient transfection was
performed in 293, chrondrosarcoma SW 1353, or LoVo cells using LipofectAMINE
2000 (Invitrogen) according to the manufacturer's instructions. Conditioned
medium was collected 13 to 24 h after transfection and subjected to
centrifugation at 15,000 rpm to remove cell debris. Cells were lysed in a
buffer containing 100 mM Tris-HCl, pH 7.5, and 0.6% Triton X-100.
To analyze pro-ANP and pro-CNP processing, recombinant pro-ANP and pro-CNP and
their derivatives in the conditioned media were immunoprecipitated by an
anti-V5 antibody (Invitrogen). Protein samples from the conditioned media or
cell lysates were separated by SDS/PAGE and analyzed by Western blotting using
a horseradish peroxidase-conjugated anti-V5 antibody (Invitrogen).
Effect of Small Molecule InhibitorsHuman 293 cells were
transfected with the pro-CNP expressing plasmid and grown in Opti-MEM medium
for 3 h. Small molecule inhibitors, including a furin inhibitor
(decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone)
(27), an MMP inhibitor
(GM6001) (28), and a TACE
inhibitor (TAPI-1) (29) were
added separately to the medium and incubated for 24 h. The conditioned medium
containing recombinant pro-CNP was collected. Cell lysate was prepared as
described above. Pro-CNP and its derivatives were analyzed by SDS-PAGE and
Western blotting using an anti-V5 antibody.
Cleavage of Pro-CNP by Purified Recombinant FurinLoVo cells
were transfected with a pro-CNP-expressing plasmid or the control vector pcDNA
and grown in Opti-MEM medium for 24 h. The cells (
106) were
washed once with phosphate-buffered saline and lysed in 1 ml of 100
mM HEPES, 1% Triton X-100, 1 mM 2-mercaptoethanol, and 1
mM calcium chloride
(30). Purified recombinant
human furin was added to the cell lysate, and the mixture was incubated at 30
°C for 2 h. Cleavage of pro-CNP was analyzed by Western blotting using an
anti-V5 antibody.
cGMP AssayTo examine the activity of recombinant CNP, a
cGMP assay was performed using a Biotrak enzyme immunoassay kit (Amersham
Biosciences), as described previously
(23). Briefly, rat aortic
smooth muscle cells were grown in 96-well plates in a smooth-muscle cell
growth medium (Cambrex Bioscience). Confluent cells were washed once with
phosphate-buffered saline. The LoVo cell lysate (180 µl), which contained
recombinant pro-CNP and was treated with purified furin, was added to each
well and incubated at 37 °C for 10 min. In this experiment, synthetic
human CNP was used a positive control. The cells were lysed by addition of a
lysis buffer (20 µl/well) containing 2% dodecyltrimethylammonium and 50
mM sodium acetate, pH 5.8. The intracellular cGMP concentration in
CNP-stimulated aortic smooth muscle cells was then determined with the Biotrak
enzyme immunoassay kit. Each experimental condition was assayed in
triplicate.
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RESULTS
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Processing of Pro-ANP and Pro-CNP in 293 CellsTo examine
whether corin is involved in the processing of pro-CNP, transfection
experiments were performed in 293 cells. As reported in our previous studies
and shown here as a control, corin was required for the processing of human
pro-ANP to ANP. Western analysis of the conditioned medium showed the
conversion of recombinant pro-ANP to ANP when the cells were co-transfected
with plasmids expressing human corin and pro-ANP
(Fig. 1A). Expression
of recombinant human corin in the transfected cells was confirmed by Western
analysis of the cell lysates (Fig.
1B). Western blotting also showed that only pro-ANP, but
not ANP, was present in the cell lysates, indicating that pro-ANP was
processed extracellularly by corin. In contrast, processed CNP was detected in
the conditioned medium from cells transfected with the pro-CNP expressing
plasmid alone or together with the corin expressing plasmid
(Fig. 1C). Western
analysis of the cell lysates showed that pro-CNP was processed similarly in
the absence or presence of recombinant corin
(Fig. 1D), indicating
that pro-CNP was processed intracellularly by a corin-independent
mechanism.

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FIG. 1. Processing of pro-ANP and pro-CNP in transfected 293 cells.
Transfection experiments were performed in human 293 cells using plasmids
expressing human pro-ANP (pProANP) (A and B) or
pro-CNP (pProCNP) (C and D) with or without a
plasmid expressing human corin (pCorin), as described under
"Experimental Procedures." Recombinant pro-ANP, pro-CNP and their
derivatives in the conditioned media (A and C) and cell
lysates (B and D) were analyzed by SDS-PAGE and Western
blotting using an anti-V5 antibody. The Western analysis also detected in the
cell lysate the expression of recombinant human corin as two bands possibly
caused by differences in glycosylation.
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Processing of Pro-ANP and Pro-CNP in Chondrosarcoma SW 1353
CellsIt is possible that the processing of pro-CNP observed in
kidney-derived 293 cells might not reflect conditions in chondrocytes because
two cell types will have different proteomes. Both corin and pro-CNP are
expressed in chondrocytes; in these cells, corin might be the pro-CNP
convertase. To test this, we next performed experiments in SW 1353 cells that
were derived from a human chondrosarcoma. As shown in
Fig. 2A and
B, co-transfection of plasmids expressing human pro-ANP
and corin led to the conversion of pro-ANP to ANP. Corin, however, was not
required to process pro-CNP. Western analysis detected both pro-CNP and CNP in
the conditioned medium and cell lysate from the cells transfected with the
pro-CNP-expressing plasmid alone or together with the corin-expressing plasmid
(Fig. 2, C and
D). Expression of corin did not enhance the processing of
pro-CNP. The results showed that the processing of pro-CNP in SW 1353 cells
again occurred intracellularly but was less efficient than that in 293 cells.
This data is consistent with the observation that corin is not required for
the processing of pro-CNP.

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FIG. 2. Processing of pro-ANP and pro-CNP in transfected SW 1353 cells.
Transfection experiments were performed in human chondrosarcoma SW 1353 cells
using plasmids expressing human pro-ANP (pProANP) (A and
B) or pro-CNP (pProCNP) (C and D) with or
without a plasmid expressing human corin (pCorin), as described under
"Experimental Procedures." Recombinant pro-ANP, pro-CNP, and their
derivatives in the conditioned media (A and C) and cell
lysates (B and D) were analyzed by SDS-PAGE and Western
blotting using an anti-V5 antibody. The Western analysis also detected in the
cell lysate the expression of recombinant human corin, as indicated.
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Effects of Small Molecule Inhibitors on Processing of
Pro-CNPThe observation that pro-CNP was processed intracellularly
suggested that the propeptide may be processed by one of the subtilisin-like
proteases such as furin, which is known for its role in the processing of many
precursor proteins
(3135).
To test this hypothesis, we examined the effect of a furin inhibitor,
decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone
(27), on the processing of
pro-CNP. Transfection experiments were performed in 293 cells using the
pro-CNP expressing plasmid, after which the cells were incubated with the
furin inhibitor. Western analysis showed an increase of pro-CNP and a decrease
of mature CNP in the conditioned medium from the cells treated with increasing
concentrations of the furin inhibitor (Fig.
3, top). Consistent with this result, mature CNP in the
cell lysate was decreased in the presence of increasing concentrations of the
furin inhibitor (Fig. 3,
bottom). In contrast, the processing of pro-CNP was not inhibited
when the transfected cells were cultured in the presence of either a
broad-spectrum MMP inhibitor (GM6001) or a TACE inhibitor (TAPI)
(Fig. 3). These results suggest
that furin, but not MMPs or TACE, is involved in the processing of
pro-CNP.

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FIG. 3. Inhibition of pro-CNP processing by small molecule inhibitors.
Transfection experiments were performed in human 293 cells using a plasmid
expressing human pro-CNP (pProCNP). The transfected cells were grown
in the presence of increasing concentrations of a furin inhibitor
(Dec-RVKR-CMK), or a high concentration (20 µM) of an
MMP inhibitor (GM6001) or a TACE inhibitor (TAPI).
Recombinant pro-CNP and its derivatives in the conditioned media
(top) and cell lysates (bottom) were analyzed by an anti-V5
antibody.
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Processing of Pro-CNP in LoVo CellsIt is possible that the
effect of the inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone on
pro-CNP processing was not mediated through its inhibition of furin because
the compound is also known to inhibit other processing enzymes. To examine the
importance of furin in the processing of pro-CNP, we performed experiments
using furin-deficient LoVo cells that were derived from a lymph node
metastasis of a human colon adenocarcinoma and contain compound mutations in
the furin gene (36).
As shown in Fig. 4, C and
D, pro-CNP, but not CNP, was detected in the conditioned
medium and cell lysates from cells transfected with the pro-CNP expressing
plasmid alone or together with the corin-expressing plasmid. In controls, the
conversion of pro-ANP to ANP was detected in LoVo cells co-transfected with
pro-ANP- and corin-expressing plasmids
(Fig. 4, A and
B), indicating that furin is not required for the
corin-mediated processing of pro-ANP. The results demonstrate that furin
deficiency prevented the processing of pro-CNP in LoVo cells.

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FIG. 4. Processing of pro-ANP and pro-CNP in transfected LoVo cells.
Transfection experiments were performed in human colon adenocarcinoma LoVo
cells using plasmids expressing human pro-ANP (pProANP) (A
and B) or pro-CNP (pProCNP) (C and D) with
or without a plasmid expressing human corin (pCorin), as described
under "Experimental Procedures." Recombinant pro-ANP, pro-CNP, and
their derivatives in the conditioned media (A and C) and
cell lysates (B and D) were analyzed by SDS-PAGE and Western
blotting using an anti-V5 antibody. The Western analysis also detected in the
cell lysate the expression of recombinant human corin, as indicated.
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Co-transfection of Plasmids Expressing Furin and Pro-CNP in LoVo
CellsWe next tested whether expression of recombinant furin could
restore the processing of pro-CNP in LoVo cells. Transfection experiments were
performed in LoVo cells using plasmids expressing pro-CNP and human furin. As
shown in Fig. 5, Western
analysis detected both pro-CNP and CNP in cell lysates from LoVo cells
co-transfected with plasmids expressing pro-CNP and furin. Consistently,
increasing levels of processed CNP were found in the conditioned medium from
LoVo cells transfected with the pro-CNP expressing plasmid and increasing
amounts of the furin expressing plasmid
(Fig. 5).

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FIG. 5. Co-transfection of plasmids expressing furin and pro-CNP in LoVo
cells. LoVo cells were transfected with a pro-CNP expressing plasmid
together with increasing amounts of a plasmid expressing human furin, as
described under "Experimental Procedures." Recombinant human
pro-CNP and its derivatives in the conditioned media (top) and cell
lysates (bottom) were analyzed by SDS-PAGE and Western blotting using
an anti-V5 antibody. In addition to specific bands representing pro-CNP and
CNP (indicated by arrows), several weak nonspecific bands are also
present.
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Cleavage of Pro-CNP by Recombinant FurinWe also examined
the effect of purified recombinant human furin on pro-CNP processing.
Recombinant pro-CNP was expressed in LoVo cells and cell lysate was prepared
and incubated with increasing concentrations of purified recombinant human
furin. The processing of pro-CNP was analyzed by Western blotting. As shown in
Fig. 6, in the presence of
increasing concentrations of recombinant furin, increasing amounts of pro-CNP
were converted to CNP, demonstrating that purified furin is a pro-CNP
convertase.

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FIG. 6. Cleavage of pro-CNP by purified recombinant furin. Cell lysates from
LoVo cells transfected with a plasmid expressing human pro-CNP (pro-CNP) were
prepared and incubated with purified recombinant human furin (10 or 20 units)
at 30 °C for 2 h. Recombinant pro-CNP and its derivatives were analyzed by
SDS-PAGE and Western analysis using an anti-V5 antibody.
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The Activity of Recombinant CNPThe biological function of
CNP is mediated through its receptor that has guanylyl cyclase activity.
Binding of CNP to its receptor stimulates the guanylyl cyclase activity,
leading to generation of intracellular cGMP. To determine whether the
furin-processed recombinant CNP is biologically active, an aortic smooth
muscle cell-based cGMP assay was performed
(37). As shown in
Fig. 7, low levels of
cGMP-stimulating activity were detected in the cell lysate from LoVo cells
transfected with a control plasmid or the pro-CNP expressing plasmid alone.
The cGMP-stimulating activity increased 2- and 3.7-fold, respectively, when 10
and 20 units of purified recombinant furin were added to the cell lysate
containing pro-CNP (Fig. 7).
Calculated amounts of recombinant CNP processed by 10 and 20 units of purified
furin were 22.5 and 46 ng/106 cells, respectively. These results
are consistent with the data showing that purified recombinant furin cleaves
pro-CNP and demonstrate that furin-cleaved recombinant CNP is biologically
active.

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FIG. 7. Stimulation of intracellular cGMP production. Cell lysates from LoVo
cells transfected with a control vector (control) or a plasmid
expressing human pro-CNP (pro-CNP) were incubated with purified
recombinant human furin (10 or 20 units) at 30 °C for 2 h and added to
wells in a 96-well plate that had rat aortic smooth muscle cells cultured in
it. The cell culture plates were incubated at 37 °C for 10 min. The cells
were lysed by addition of a buffer containing 2% dodecyl trimethylammonium and
50 mM sodium acetate, pH 5.8. The intracellular concentration of
cGMP in rat aortic smooth muscle cells was determined using the Biotrak enzyme
immunoassay kit, as described under "Experimental Procedures." The
data are presented as mean ± S.D. from three independent experiments.
**, p < 0.01 versus samples containing pro-CNP but no
furin by Student's t test.
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DISCUSSION
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In this study, we characterized the processing of human pro-CNP in
cell-based experiments, and we present evidence that pro-CNP is processed by
furin but not corin. In transfected epithelial 293 and chondrosarcoma SW 1353
cells, we found that recombinant human pro-CNP was processed intracellularly,
in contrast to pro-ANP, which was processed extracellularly. Expression of
recombinant human corin did not enhance the processing of pro-CNP in these
cells, whereas, in control experiments, recombinant corin cleaved pro-ANP to
ANP under similar experimental conditions. Thus, corin is unlikely to play a
role in processing pro-CNP despite the fact that both corin and pro-CNP mRNA
expression was detected in prehypertrophic chondrocytes in developing bones
(11,
19). The functional
significance of corin expression in chondrocytes remains to be determined.
The observation that pro-CNP was processed intracellularly suggests that
the propeptide may be processed by furin, a prohormone convertase that is
predominantly localized in the trans-Golgi network and involved in
processing a variety of constitutively secreted precursor proteins
(3135).
Consistent with this hypothesis, addition of a small molecule compound that
blocks furin activity to the culture medium inhibited the processing of
recombinant pro-CNP in transfected 293 cells. In contrast, potent inhibitors
of MMPs (GM6001) and TACE (TAPI) had little effect on pro-CNP processing in
the transfected cells, indicating that MMPs and TACE are unlikely to play an
important role in pro-CNP processing. The importance of furin in pro-CNP
processing was supported by additional studies using LoVo cells, which are
deficient in furin (36).
Western analysis showed that there was no detectable conversion of recombinant
pro-CNP to CNP in these cells. Transfection of a plasmid expressing human
furin in LoVo cells restored the ability of these cells to process pro-CNP.
Finally, incubation of purified recombinant human furin with LoVo cell lysate
containing recombinant pro-CNP led to conversion of the precursor to the
mature peptide. We also showed that the furin-processed CNP was biologically
active in a cell-based cGMP assay. Together, these data provide strong
evidence that furin is critical in the processing of pro-CNP. Thus, the
molecular mechanisms responsible for the processing of pro-CNP and pro-ANP are
different despite the fact that these peptides have high sequence
homology.
The identification of furin as an intracellular processing enzyme for
pro-CNP provides an insight into the post-translational modification of the
peptide. CNP was first isolated as a 22-amino acid peptide (CNP-22) from
porcine brain (38). Subsequent
studies have shown that a larger 53-amino acid peptide (CNP-53) exists as the
major form of CNP in porcine, human, and ovine brain tissues
(3941)
and in cultured human endothelial cells
(13). Human CNP-53 and CNP-22
are carboxyl-terminal fragments generated by proteolytic cleavages at pro-CNP
sequences
Arg45-Ser46-Arg47-Leu48-Leu49-Arg50
-Asp51
and
Lys76-Gly77-Ala78-Asn79-Lys80-Lys80
-Gly81,
respectively (26). The
RSRLLR
D cleavage site matches the consensus furin recognition sequence,
RXXR
(3135),
indicating that CNP-53 is probably the product produced by the furin-mediated
processing. In our SDS-PAGE and Western analyses, the processed recombinant
CNP form appeared as a
9-kDa band, consistent with the calculated
molecular mass of 5.8 kDa for human CNP-53 plus a carboxyl-terminal V5 and His
tag (
4 kDa). On Western blots, however, we did not detect the CNP-22 form
that, without the tag, has a calculated molecular mass of 2.2 kDa, indicating
that the enzyme responsible for generating the CNP-22 form is not present in
the cells we tested. The data indicate that CNP-53 is the major secreted form
of CNP generated by furin processing and that CNP-22 may be generated
subsequently from CNP-53 by another extracellular enzyme whose identity and
tissue distribution are unknown at this time
(Fig. 8). This conclusion is
also supported by a recent study in which the animo-terminal pro-CNP peptide
150, a product from the proteolytic cleavage at the RSRLLR
D site,
was detected as the only major cleaved propeptide from pro-CNP in circulating
human plasma (42). Apparently,
both pro-CNP peptide 150 and mature CNP-53 are secreted into the
circulation after pro-CNP is processed by furin
(Fig. 8). Functionally, CNP-53
and CNP-22 seem to be similar. In anesthetized rats, intravenous injection of
CNP-53 and CNP-22 elicits similar natriuretic responses, although their
activities are
100 times less potent than that of ANP
(26,
38). Thus, the furin-mediated
processing of pro-CNP is a critical step in converting the precursor to a
biologically active hormone. The functional importance of further conversion
of CNP-53 to CNP-22 remains to be determined.

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FIG. 8. A proposed model for the processing of human pro-CNP. Human pro-CNP
is synthesized as a prepropeptide of 126 amino acids
(26). After the signal peptide
(sp) is removed by the signal peptidase, pro-CNP is cleaved by furin
at the peptide bond R50-D51 to produce the propeptide 150 and CNP-53,
both of which are secreted outside the cell. CNP-53 is subsequently cleaved at
K80-G81 by an unknown extracellular enzyme to generate CNP-22. Nucleus and
plasma membrane (pm) are indicated.
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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. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
¶
To whom correspondence should be addressed: 2600 Hilltop Dr., Richmond, CA
94804. Tel.: 510-669-4737; Fax: 510-669-4246; E-mail:
qingyu_wu{at}berlex.com.
1 The abbreviations used are: ANP, atrial natriuretic peptide; BNP, brain
natriuretic peptide; CNP, C-type natriuretic peptide; MMP, matrix
metalloproteinase; TACE, tumor necrosis factor-
convertase; TAPI, tumor
necrosis factor-
protease inhibitor-1. 
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ACKNOWLEDGMENTS
|
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We thank E. Tuley and J. E. Sadler (Howard Hughes Medical Institute,
Washington University School of Medicine, St. Louis) for providing the human
furin expressing plasmid, and Bill Dole for encouragement, support, and
critical reading of the manuscript.
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