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INTRODUCTION |
Bone morphogenetic protein
(BMP)-11 is a
zinc-dependent metalloproteinase (1, 2) that is fundamental
to dorsal-ventral patterning and tissue morphogenesis of vertebrates
(3, 4). During early embryogenesis, BMP-1 fragments chordin, a
BMP antagonist, thereby allowing BMPs to bind to cognate receptors.
During tissue morphogenesis, BMP-1 cleaves the latent peptides of
extracellular matrix (ECM) macromolecules, including type I-III
procollagen (5, 6), probiglycan (7), prolysyl oxidase (8), and type VII
procollagen (9) as well as chains of laminin-5 (10, 11). Effort has
been focused on identifying substrates of BMP-1, but little is known
about its activation and in particular where, in relation to the cell,
it cleaves its substrates. BMP-1 is synthesized as an inactive
proenzyme (for review see Ref. 12), and the site of removal of the
prodomain is expected to determine the site of action of BMP-1. The
active proteinase can be isolated from the medium of cultured human
cells and tendon organ cultures. However, this does not exclude the
possibility that the prodomain of BMP-1 can be cleaved within the cell,
with secretion of the active molecule.
Pro-BMP-1 comprises a signal peptide, a prodomain, a catalytic domain,
three CUB domains, and an epidermal growth factor-like domain
(12). The function of the prodomain is unknown, although latency is a
likely role. Expression of recombinant BMP-1 lacking a prodomain showed
that its absence does not abrogate secretion (13), but this does not
rule out a function for the prodomain in the secretion of BMP-1 and the
regulation of its site of action. A major question is whether the
prodomain maintains BMP-1 in an inactive form until it is transported
to its site of action, whereupon removal of the prodomain leads to the
release of active BMP-1.
Evidence from expression studies in bacteria suggests that cleavage of
the prodomain is required for BMP-1 activity (5, 6). Removal of the
prodomain is predicted to occur after the dibasic motif
117RSRR120 (14, 15), which is a consensus
sequence for proprotein convertases (PCs). Furin, PACE4, PC5/6, and PC7
are the four members that recognize the general
RX(K/R)R motif (15, 16). The cellular site where this
maturation occurs is not well defined, despite extensive studies of the
localization of PCs. For example, furin has a transmembrane domain that
targets the protein to the trans-Golgi network (TGN) and is
known to cycle between the TGN and the plasma membrane (PM) via the
endosome retrieval pathway (17). There is also evidence to suggest that
furin can be cleaved at the PM and released as a secreted form into the
ECM (18). Furin is involved in the processing of a wide variety of
molecules, for example, notch (19), fibrillin (20), type V procollagen
(21), MT1-MMP (22), and MT3-MMP (23). Furin also autoactivates with cleavage of its prodomain being required for exit from the endoplasmic reticulum (ER). After cleavage, the prodomain remains associated with
the mature molecule and inhibits the furin. Upon reaching the acidic
environment of the TGN, the prodomain dissociates and furin becomes
active (24).
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MATERIALS AND METHODS |
Cell Culture and Reagents--
BMP-1 antibody (N-ter antibody)
was from Oncogene, and the anti-FLAG M2 antibody was from Sigma.
All PCR products and plasmid purifications were carried out using
Qiagen kits. Prestained protein markers were obtained from Bio-Rad.
Decanoyl-RVKR-chloromethyl ketone was obtained from Bachem. A
full-length cDNA for BMP-1 (P13497) was cloned from a human
placental cDNA library and inserted at the
KpnI/XbaI of pcDNA3 (Invitrogen). HT-1080
human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM
(Invitrogen), supplemented with 100 mM
L-glutamine and 10% fetal bovine serum (FBS) (complete
DMEM), at 37 °C with 5% CO2. The mutant BMP-1 proteins
were stably expressed in HT-1080 cells. Transfections were carried out
using Lipofectin reagent (Invitrogen) with 10 µg of plasmid/100-mm
tissue culture dish, according to the manufacturer's instructions.
Pools were selected and maintained with 250 µg/ml Geneticin. The
culture media was collected and cleared of cell debris by
centrifugation at 1600 × g for 10 min and concentrated using Centriprep-30 (Amicon, Inc.). The samples were used immediately or stored at
80 °C.
Plasmid Constructs and Transfections--
The FLAG epitope
(DYKDDDDK) was added to the C terminus of the BMP-1 construct (5' of
the stop codon) by PCR modification of the wild-type construct. A
forward primer (5'-TCGTGAGAACATCCAGCCAGGGCA-3') and a reverse
primer
(5'-CCCCCCTCGAGTCACTTGTCATCGTCGTCCTTGTAGTCCTGGGGGGTCCGGTTTCTTTTCTGCACTCGGAATTTGAGC-3') with a XhoI site (boldface) and the FLAG epitope
(underlined) were used to amplify a fragment of ~1500 bp. An internal
BamHI site and the XhoI were used to generate a
fragment that was introduced in place of the wild-type fragment. The
117RSRR120 to
117RSAA120 (pro-BMP-1AA) mutant was
generated by site-directed mutagenesis of the
KpnI/XcmI fragment of the BMP-1 FLAG
construct. KpnI is located prior to the start codon, and
XcmI is located at nucleotide 383. The mutations were made
by standard procedures using strand overlap PCR (25) using
Pwo polymerase (Roche Applied Science). A forward primer containing a KpnI site (boldface)
(5'-GCGGTACCCGGGGATCCGATAT-3', KpnII), a reverse
primer (5'-AGGGTCTCTCCACAGGCTGGGCAC-3', seq8), and oligonucleotides in
both directions containing the desired mutation (underlined)
(5'-ACGTCGCCGCTGCTGCGCTACGGGATC-3', furR; 5'-GATCCCGTAGCGCAGCAGCGGCGACGT-3', furF) were used.
Pwo DNA polymerase was used to minimize base
misincorporation during the polymerase chain reactions. Briefly, a DNA
fragment was amplified using the KpnII primer and the
antisense mutant primer (furR), and an overlapping fragment was
amplified using the sense mutant primer (furF) and the downstream seq8
primer. Both fragments were gel-purified (Qiagen), mixed, and
re-amplified with the KpnII and seq8 primers. The product was digested using appropriate restriction enzymes (KpnI and
XcmI), gel-purified, and introduced in place of the
corresponding wild-type fragment in BMP-1 FLAG. DNA sequencing (Big
Dye, ABI Biosystems) was used to verify the mutations and to ensure
that the cDNA clones were error-free.
Western Blotting--
Nonidet P-40 extracts were prepared as
follows. Cells were rinsed three times with phosphate-buffered saline
and incubated on ice for 15 min with 500 µl of Nonidet P-40 buffer
(1% Nonidet P-40, 50 mM Tris, pH 7.6) containing 10 mM EDTA and protease inhibitor mixture (Roche Applied
Science). Cells in Nonidet P-40 buffer were scraped on ice, and lysates
were subjected to a 5-min centrifugation at 14,000 × g
at 4 °C. Supernatants were retained and stored at
80 °C for
further analysis. Secreted proteins were concentrated on Centriprep-30
membranes and separated by discontinuous SDS-PAGE (4/10% or 4/7%).
Most samples were grown for 5-24 h prior to harvest in DMEM with 0%
FBS. For BFA and MON treatment, cells were grown for 4-5 h in DMEM
with 0% FBS in the presence of 3.5 µM inhibitor. FI was
used at 20 and 40 µM in 0% DMEM for 4-5 h. Pro-BMP-1
and pro-BMP-1AA were examined by Western blot analysis in
which the primary antibody was either the mouse monoclonal FLAG
antibody or the rabbit polyclonal N-ter antibody. Secondary antibodies
were either horseradish peroxidase-conjugated to anti-mouse or
anti-rabbit IgG and were detected by the enhanced chemiluminescence
method (Supersignal West Dura Extended Duration, Pierce).
Assay of Procollagen C-proteinase--
Recombinant BMP-1 was
assayed for procollagen C-proteinase activity using human
14C-labeled type I procollagen substrate and analysis of
the cleavage products on SDS gels as described (38). In brief,
14C-labeled type I procollagen was obtained from the medium
of human skin fibroblasts that had been cultured in DMEM supplemented
with ascorbic acid (25 µg/ml), L-glutamine, and a mixture
of uniformly labeled 14C-L-amino acids (1 µCi/ml). The procollagen was purified by ammonium sulfate
precipitation (176 mg/ml) and ion exchange chromatography and was then
concentrated by ultrafiltration on Centriprep-100 membranes. The assays
were carried out at 37 °C for 4 h.
In Vitro Furin Digest and Deglycosylation--
For furin
digests, 1-2 µg of protein was incubated overnight at 37 °C in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 1 mM
-mercaptoethanol,
protease inhibitor, with or without 5 units of recombinant furin
(Affinity Bioreagents). PNGase and neuraminidase (New England BioLabs)
were used to digest protein samples per the manufacturer's instructions.
Immunofluorescence--
HT-1080 cells were grown on coverslips
in complete DMEM to ~60% confluency. Cells were then rinsed three
times with PBS and fixed and permeabilized with cold methanol
(
20 °C) for 5 min. Fixed cells were washed three times with PBS
and incubated for 20 min at room temperature with either 1210 (rabbit
IgG) antibody or anti-TGN46 (sheep IgG) antibody in PBS supplemented
with 1 mg/ml bovine serum albumin. After washing, cells were incubated for 20 min with rhodamine-conjugated anti-rabbit or anti-sheep IgG
(Santa Cruz Biotechnology). Cells were washed with PBS, incubated for 1 min with DAPI (4 µg/ml), further washed with PBS, mounted in Mowiol
4-88 (Calbiochem), and observed with a Zeiss fluorescence microscope.
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RESULTS |
Pro-BMP-1 Processing in HT-1080 Cells--
The tetrabasic motif at
117RSRR120 was the presumed C-terminal end of
the prodomain, with activation being carried out by a member of the
furin-like proprotein convertase (PC) family. Previous work had
established that pro-BMP-1 is cleaved following an Arg-Arg sequence,
based on the reactivity of the antiserum raised to the N terminus of
the cleaved product (25). In this study, we wanted to know where the
prodomain was removed, in particular whether this occurred
intracellularly, at the PM or extracellularly. In preliminary
experiments we engineered a pro-BMP-1 molecule containing a FLAG
epitope at the C terminus. Previous work had established that the FLAG
epitope did not interfere with expression or proteolytic activity of
BMP-1 (3). The potential PC cleavage site in pro-BMP-1 was changing
from 117RSRR120 to
117RSAA120 by site-directed mutagenesis
(pro-BMP-1AA, Fig. 1).

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Fig. 1.
Schematic representation of pro-BMP-1
containing a C-terminal FLAG epitope. SP, signal
peptide; PD, prodomain; MET, metalloproteinase
domain; C1-3, CUB domains 1-3; E, epidermal
growth factor-like domain; S, BMP-1-specific sequence, and
FLAG, C-terminal FLAG epitope tag. The partial sequence of
the putative prodomain is shown below with the
underlined RSRR furin recognition motif. The prodomain
cleavage site is at Arg120. Amino acid substitutions of the
furin recognition motif 117RSRR120 to RSAA
(pro-BMP-1AA) were carried out. The asterisk
represents the binding site of the anti-N-ter antibody, which
recognizes the prodomain. The FLAG antibody recognized recombinant
pro-BMP-1 and BMP-1, which both contained the FLAG epitope.
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HT-1080 cells were transfected separately with cDNAs encoding
pro-BMP-1 and pro-BMP-1AA, and the proteins in the cell
lysate and culture medium were examined by Western blot analysis using
the anti-prodomain antibody (N-ter antibody) and the anti-FLAG M2
antibody (Fig. 2). The N-ter antibody
recognized a protein of ~95 kDa in Nonidet P-40 extracts of cells
transfected with pro-BMP-1 and pro-BMP-1AA. A protein of
~105 kDa was detected in the medium of cells transfected with
cDNA for pro-BMP-1AA but not pro-BMP-1. The anti-FLAG
antibody reacted with the ~95-kDa protein in the cell extract and a
protein of ~85 kDa in the medium of cells expressing pro-BMP-1 as
well as the ~105-kDa protein in the medium of cells expressing
pro-BMP-1AA. Taken together, these results showed that the
~85-kDa protein in the medium corresponded to BMP-1 (which lacked the
prodomain), the ~95-kDa protein in the Nonidet P-40 extract
corresponded to pro-BMP-1 (retaining the prodomain), and the ~105-kDa
pro-BMP-1AA (retaining the prodomain).

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Fig. 2.
Cleavage of pro-BMP-1 in HT-1080 cells.
HT-1080 cells were transfected with cDNA encoding pro-BMP-1 or
pro-BMP-1AA, and the proteins were examined in Nonidet P-40
extracts of the cell layer (C) or medium (M)
using either the N-ter-antibody (A) or the anti-FLAG
antibody (B). Samples were separated by 4/10% discontinuous
SDS-PAGE. A, pro-BMP-1 and pro-BMP-1AA were
detected in the cell layer (C), and pro-BMP-1AA
was detected in the culture medium (M).
Pro-BMP-1AA was detected in the medium but at a noticeably
higher molecular weight. The N-ter antibody did not react with BMP-1 in
the medium or with the vector control (not shown). B, BMP-1
was observed in the culture medium. Pro-BMP-1AA was
detected in the cell layer and in the medium. Anti-FLAG Western blots
had a common background band observed in the vector control and all
Nonidet P-40 extracts (asterisk).
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From these results we drew several conclusions. For example, the
processing of pro-BMP-1 occurs only at the tetrabasic motif; removal of
the P1 and P2 arginine residues blocked cleavage of the prodomain.
Furthermore, no alternate cleavage site was used when the prodomain
cleavage site was removed. The results also showed that removal of the
prodomain is not required for secretion of BMP-1. The medium of cells
expressing pro-BMP-1 contained a protein of ~15 kDa that was
recognized by the N-ter antibody (data not shown). This was most likely
to be the cleaved prodomain of pro-BMP-1. However, pro-BMP-1 was only
detected in the cell extracts and not in the culture medium.
Pro-BMP-1AA migrated slower (~105 kDa) than intracellular
pro-BMP-1 (~95 kDa). To determine the reason for this, the
glycosylation state of the protein in cell extracts and culture medium
was examined by treatment with PNGase (which removes all
N-linked glycosylation) or neuraminidase (which removes
sialic acid residues). As shown in Fig.
3, digestion of intracellular pro-BMP-1
with PNGase resulted in a marked decrease in molecular weight, which
was consistent with the removal of the five N-linked glycans
that occur on pro-BMP-1 (26). Digestion of pro-BMP-1AA with
PNGase resulted in the protein migrating to the same position as
deglycosylated pro-BMP-1. Digestion of secreted pro-BMP-1AA
with neuraminidase resulted in a decrease in molecular weight, which
was indicative of sialylation of the secreted protein. Of particular relevance to this study, the absence of the ~105-kDa form
of pro-BMP-1AA in the cell extracts and the presence of
TGN-mediated addition of sialylic acid residues of this protein showed
that the rate of transit of pro-BMP-1AA through the TGN was
rapid. The deglycosylation studies showed that the secreted and
intracellular forms of pro-BMP-1AA differed only in the
state of glycosylation. The clearly detectable difference in migration
of secreted pro-BMP-1AA before or after treatment with
neuraminidase and the identical migration of the intracellular
pro-BMP-1AA before and after neuraminidase treatment
demonstrated that: (i) the majority of protein detected on Western
blots of the Nonidet P-40 extracts had been extracted from the ER or in
transit to the Golgi and (ii) cleavage of the prodomain and sialylation
of the pro-BMP-1 molecule occurs at a late stage in the secretory pathway. The absence of pro-BMP-1 in the medium, coupled with the rapid
transit of pro-BMP-1 through the late secretory pathway, was consistent
with complete cleavage of the prodomain in the late secretory
pathway.

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Fig. 3.
Post-translational modification of pro-BMP-1
and pro-BMP-1AA in HT-1080 cells. A,
proteins were extracted from cells expressing pro-BMP-1, treated with
PNGase, and analyzed by Western blotting with the N-ter antibody. The
lower molecular weight band corresponds to deglycosylated pro-BMP-1.
B, proteins were extracted from lysates (C) or
medium (M) of cells expressing pro-BMP-1AA and
either treated or untreated with PNGase or neuraminidase
(Neur) prior to SDS-PAGE and analysis by Western blotting
with the N-ter antibody. PNGase treatment resulted in the removal of
all post-translational glycosylation. Treatment of
pro-BMP-1AA from the medium with neuraminidase resulted in
a small molecular weight change, whereas intracellular
pro-BMP-1AA showed no migration difference compared with
untreated.
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Pro-BMP-1 Does Not Exhibit Procollagen C-proteinase
Activity--
To determine if the prodomain inhibited proteolytic
activity of BMP-1, pro-BMP-1AA and BMP-1 were assayed for
procollagen C-proteinase activity using type I
L-[U-14C]procollagen as substrate
(25). The results showed that BMP-1 is an effective C-proteinase, which
is consistent with previous observations of the recombinant protein (3,
5). However, pro-BMP-1AA was inactive (Fig.
4). The C-proteinase activity of BMP-1
was inhibited by EDTA. In control experiments, medium from cells
transfected with empty vector lacked C-proteinase activity (Fig.
4).

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Fig. 4.
Pro-BMP-1 was inactive in assays of
procollagen C-proteinase. Concentrated media from overnight
cultures of stable transfectants grown in DMEM without FCS were
incubated with 1 µg of
L-[U-14C]procollagen. Proteins were separated
in SDS gels, and the radiolabeled proteins were detected by
14C-phosphorimaging. BMP-1 converted pro- 1(I) and
pro- 2(I) chains to pN 1(I) and pN 2(I), respectively, by removal
of the C-propeptides. EDTA (E) inhibited the procollagen
C-proteinase activity of BMP-1. Pro-BMP-1AA did not cleave
the procollagen chains. Far right two lanes are empty vector
and water control samples, respectively. All samples were
incubated at 37 °C for 4 h.
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Furin-like/Proprotein Convertase Activity Was
Responsible for Prodomain Cleavage of Pro-BMP-1--
Of the PC family
of proteinases, furin, PACE4, PC5/6, and PC7 cleave target molecules in
the constitutive secretory pathway (16) utilizing the tetrabasic motif
of RX(K/R)R. The arginine residues at P1 and P4 are critical
for specific cleavage, whereas additional arginine residues at
positions P6 and P8 contribute to enhance cleavage (18), with P6
controlling the pH dependence of the cleavage (17). BMP-1 has arginine
residues at the potential P1, P2, P4, P6, and P8 positions
(113RGRSRSRR120), which strongly suggests that
pro-BMP-1 is a substrate for a furin-like PC.
Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (FI) is a potent inhibitor
of furin-like proprotein convertases (27), leading to inhibition of
cleavage of PC substrates. To investigate if these specific PCs are
responsible for the maturation of pro-BMP-1, FI was added to HT-1080
cells expressing pro-BMP-1 (Fig. 5). In control samples, pro-BMP-1 occurred in the cell lysate and BMP-1 occurred in the medium (see Figs. 2, 5A, and 5B).
Pro-BMP-1 was not observed in the culture medium in the absence of FI.
However, treatment of the cells with 20 µM FI resulted in
the secretion of the ~105-kDa form of pro-BMP-1 that we had observed
in the culture medium of cells expressing pro-BMP-1AA. Of
particular interest, the levels of pro-BMP-1 did not appear to be
markedly affected by the presence of FI (Fig. 5A), which was
indicative that the secretory pathway was functional and the cells were
viable. When the experiments were carried out in the presence of 40 µM FI, the ~105-kDa pro-BMP-1 was efficiently
synthesized by the cells (as shown by the presence of the protein in
the cell lysate (see Fig. 5, A and B) but was not
secreted into the culture medium (see Fig. 5B). Presumably,
at this elevated concentration, FI inhibited the secretory
machinery.

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Fig. 5.
Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone
(FI), a specific furin inhibitor, blocks the cleavage of pro-BMP-1 in a
dose-dependent manner. A, stable
transfectants were treated with 0, 20, or 40 µM FI, and
proteins in the Nonidet P-40 extracts (C) of the cells and
concentrated media (M) were analyzed by Western blot
analysis using the N-ter antibody. Pro-BMP-1 was secreted in the
presence of FI. No differences were observed in the Nonidet P-40
extracts upon FI treatment. B, same as in A, but
the anti-FLAG antibody was used to detect the proteins. BMP-1 was
observed in the medium (M) of untreated cells. At 20 µM FI, both pro-BMP-1 and BMP-1 were observed in the
medium, but pro-BMP-1 was the predominant species. At 40 µM FI, the levels of pro-BMP-1 in the medium were much
reduced compared with 20 µM FI. The asterisk
indicates a background band in all samples (including empty vector
controls, data not shown).
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We wanted to know if furin could cleave the prodomain of pro-BMP-1. To
investigate this possibility, we treated pro-BMP-1 with recombinant
furin in vitro. To obtain pro-BMP-1, we incubated BMP-1-secreting cells with 20 µM FI and concentrated the
pro-BMP-1 by ultrafiltration to decrease the ratio of furin inhibitor
to pro-BMP-1. Proteins were incubated with recombinant furin, and the
products were examined by Western blot analysis using the anti-FLAG
antibody. The results showed that furin cleaved pro-BMP-1 in
vitro to yield a protein of the expected size of BMP-1 (Fig. 6). Recombinant furin did not cleave
pro-BMP-1AA (data not shown).

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Fig. 6.
In vitro cleavage of pro-BMP-1 by
recombinant furin. Pro-BMP-1-transfected HT1080 cells were treated
with 20 µM FI, and the medium was concentrated to remove
the majority of the inhibitor. Samples were incubated overnight
in vitro at 37 °C with or without 5 units of recombinant
furin. Western blot analysis was carried out on the samples using the
anti-FLAG antibody. The fastest migrating band (asterisk)
was the same background band seen in all anti-FLAG antibody Western
blots.
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Kridel et al. (28) carried out substrate hydrolysis on MMP-9
and identified a dibasic motif, RR, as the potential P1 and P2 amino
acids of the cleavage site. GM6001 treatment showed no inhibition of
maturation or alteration of secretion profiles for either pro-BMP-1 or
proBMP1AA (data not shown).
Cellular Localization of Prodomain Cleavage--
The data on the
secretion and post-translational modification of pro-BMP-1 and
pro-BMP-1AA, together with those on the effects of FI,
suggested that the prodomain of pro-BMP-1 could be cleaved by furin (or
a similar PC) within the cell or at the cell-ECM boundary.
Interestingly, furin contains a transmembrane domain, which localizes
the protein to the TGN, although active cycling between the TGN and the
PM occurs (17). Additional evidence suggests that the catalytic region
of furin can be cleaved to produce a secreted enzyme (18). In the next
series of experiments, we used brefeldin A (BFA) and monensin (MON) to
test the possibility that the prodomain of pro-BMP-1 is cleaved within
the ER and Golgi, respectively. BFA depolymerizes the Golgi apparatus
and causes it to fuse with the ER. This effectively blocks protein
transport from ER to Golgi (29, 30). MON is a
Na+/H+ ionophore, which interferes with
transport to the late Golgi, effectively blocking protein transport
from Golgi to TGN (31). Additionally, MON alters the pH of the TGN,
which may inhibit the activity of TGN-resident proteins, for example
furin (31).
Western blot analysis of cells treated separately with BFA and MON
showed that BMP-1 was not secreted (Fig.
7A). This confirmed that BFA
and MON had stopped protein secretion. The results also showed that
neither pro-BMP-1 nor pro-BMP-1AA was secreted (Fig.
7A). Analysis of BFA- and MON-treated cells with the
anti-FLAG antibody (Fig. 7B) showed identical results. Importantly, treatment of the pro-BMP-1- or
pro-BMP-1AA-transfected cells with either BFA or MON showed
no accumulation of BMP-1. This indicated that removal of the prodomain
occurs post-Golgi.

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Fig. 7.
Brefeldin A (BFA) and
monensin (MON) block the normal cleavage of pro-BMP-1
and the secretion of BMP-1 and pro-BMP-1AA.
A, stable transfectants of either pro-BMP-1,
pro-BMP-1AA, or empty vector control (PC) in
HT-1080 fibroblasts were grown in the presence of 3.5 µM
BFA or MON for 5 h, and the proteins in Nonidet P-40 extracts of
the cells (C) or medium (M) were examined by
Western blot analysis using the N-ter antibody. There was no observed
secretion of pro-BMP-1 or pro-BMP-1AA. Pro-BMP-1 and
pro-BMP-1AA migrated marginally slower in BFA-treated
cells. B, Western blot analysis was carried out using the
anti-FLAG antibody. No pro-BMP-1 or BMP-1 were observed in the medium
of the cells, except after extended exposure of the gels, when low
levels of secretion of BMP-1 and pro-BMP-1AA were observed
from MON-treated cells.
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Pro-BMP-1 Is Converted to BMP-1 during Sialylation of the
Molecule--
A consistent observation was that pro-BMP-1 was
never detected in the culture medium (unless the cells were treated
with FI). Furthermore, we had not detected BMP-1 in cell lysates.
Therefore, pro-BMP-1 was either cleaved in the medium by a mechanism
that was highly efficient so that pro-BMP-1 occurred transiently, or it
was cleaved inside the cell in the late secretory pathway or at the
plasma membrane. If efficient cleavage occurred in the culture medium,
then we would expect to detect small amounts of pro-BMP-1 in highly
concentrated solutions. Likewise, if efficient cleavage occurred within
cells, then we would expect to see low levels of BMP-1 in concentrated
solutions of cell extracts. In the next series of experiments, we
examined highly concentrated solutions of the culture medium and
Nonidet P-40 cell extracts of cells taken from 90-95% confluent
100-mm dishes. Care was taken to cool the extracts rapidly and prepare
them for 4/7% discontinuous SDS-PAGE in the presence of proteinase
inhibitors to avoid nonspecific cleavage of the proteins (see
"Materials and Methods"). Cells expressing pro-BMP-1AA
were used in additional control experiments. The results showed the
absence of pro-BMP-1 in the culture medium, except in medium of cells
expressing pro-BMP-1AA (see Fig.
8A). In contrast, BMP-1 was
detected in the Nonidet P-40 extract and culture medium (see Fig.
8B). Intracellular BMP-1 was detected with the anti-FLAG
antibody and not with the N-ter antibody, consistent with the protein
being BMP-1. We noted that intracellular BMP-1 migrated faster than
secreted BMP-1 (see exert panel in Fig. 8B). This
suggested to us that a proportion of the intracellular BMP-1 was
post-translationally immature. To test this hypothesis, we digested the
BMP-1 with PNGase and neuraminidase.

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Fig. 8.
Cleavage of the prodomain of pro-BMP-1 occurs
within the cell boundary. Cells expressing pro-BMP-1 and
pro-BMP-1AA were grown to 90-95% confluency prior to
Nonidet P-40 extracts being prepared, and the medium was concentrated
by ultrafiltration. Proteins were analyzed by 4/7% discontinuous
SDS-PAGE and Western blotting. A, Western blot analysis of
Nonidet P-40 extracts (C) and concentrated media
(M) from cells expressing pro-BMP-1,
pro-BMP-1AA, and EV (empty vector control) using the N-ter
antibody. B, same as in A, but the anti-FLAG
antibody was used. The expected secretion profile was observed, except
that a novel band was observed in the Nonidet P-40 extract
(C). The migration of this protein, which was slightly
slower in the medium of the cells, corresponded to BMP-1. The
asterisk represents a background band seen in all anti-FLAG
antibody Western blots.
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Treatment of intracellular pro-BMP-1 with PNGase (Fig.
9A) showed that the protein
was N-linked glycosylated. Furthermore, treatment of the
secreted form of BMP-1 showed that this protein was also
N-linked glycosylated (see right-hand panel of
Fig. 9B). Treatment of the secreted BMP-1 with neuraminidase
(see right-hand panel of Fig. 9B) resulted in a
marked sharpening and downward shift of BMP-1, which showed that this
protein was sialylated. Results from the anti-FLAG antibody
(Fig. 9, B and C) showed that intracellular
pro-BMP-1 had no sialic acid modification (as shown by its
insensitivity to treatment, see Fig. 9C), whereas both intracellular and secreted BMP-1 molecules were sialylated. Upon treatment with neuraminidase, secreted BMP-1 and intracellular mature
BMP-1 migrated to the same position in SDS gels. The presence of sialic
acid modification on intracellular BMP-1, whereas intracellular pro-BMP-1 lacks sialic acid, implies that sialic acid modification is
required prior to removal of the prodomain. Additionally, the observation that secreted BMP-1 had more sialylation compared with
intracellular mature BMP-1FLAG implies that removal of the prodomain
and sialylation occurs in the same compartment. These results provide
evidence that cleavage of the prodomain occurs in the
trans-Golgi network.

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Fig. 9.
Digests with neuraminidase and PNGase confirm
the presence of intracellular BMP-1. Samples were prepared as in
Fig. 8, treated with PNGase and neuraminidase, and separated on 4/7%
discontinuous SDS-PAGE, and the proteins were analyzed by Western blot
analyzing using the antibodies as shown. A, neuraminidase
treatment of intracellular pro-BMP-1 showed no size shift, whereas
treatment with PNGase showed a large shift downward in migration to the
level of unglycosylated pro-BMP-1. Pro-BMP-1 was not detected in the
medium of the cells. B, PNGase treatment of intracellular
pro-BMP-1 resulted in a fast migrating band, which corresponded to
deglycosylated pro-BMP-1. Likewise, PNGase treatment resulted in a
marked increase in mobility of the secreted BMP-1 (see right-hand
panel). Neuraminidase treatment resulted in a significant
sharpening of the BMP-1 band in the culture medium samples, and the
appearance of BMP-1 in the cell lysates, which migrated to the same
position as secreted de-sialylated BMP-1. C,
pro-BMP-1 and BMP-1 were detected in the cell lysates. Neuraminidase
treatment resulted in a slight increase in mobility of BMP-1.
Number sign (#), deglycosylated BMP-1; asterisk,
a background band seen in all anti-FLAG antibody Western blots.
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In further experiments we carried out temperature drop experiments in
which the pro-BMP-1-transfected cells were incubated at 20 °C to
stop vesicular transport from the TGN (32), and extracts of the cells
were examined by Western blotting. The results showed a small amount of
mature BMP-1 in the Nonidet P-40 extracts and none in the culture
medium of cells treated at 20 °C (data not shown). These results are
consistent with the proposal that cleavage of pro-BMP-1 to BMP-1 begins
in the TGN.
Immunolocalization of BMP-1 to the trans-Golgi Network and Plasma
Membrane--
To pinpoint the localization of BMP-1 inside cells, we
carried out immunofluorescence studies using a neoepitope antibody (1210) that recognizes the N terminus of mature BMP-1 but not pro-BMP-1
(25). The results showed TGN and plasma membrane localization of BMP-1
(Fig. 10A). For comparison,
Fig. 10B shows labeling of TGN46, which is a TGN marker
(33). Localization of mature BMP-1 to the PM was similar to that
obtained with Na+/K+ ATPase (data not
shown).

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Fig. 10.
Immunofluorescence using a neoepitope
antibody to BMP-1 shows localization to the TGN and plasma
membrane. HT-1080 cells expressing pro-BMP-1 were grown on
coverslips and prepared for immunofluorescence as described under
"Materials and Methods." A, cells labeled with the 1210 antibody/rhodamine (red) and DAPI (blue) showed
localization of BMP-1 to the TGN (closed arrowheads). The
1210 antibody recognized BMP-1 at the plasma membrane of the cells
(open arrowheads). B, cells labeled with
TGN46/rhodamine (red) and DAPI (blue) showed
labeling of the TGN (closed arrows). DAPI
staining was restricted to the nucleus.
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DISCUSSION |
In this study we showed that the prodomain of pro-BMP-1 can be
removed inside the cell by a furin-like (paired basic) proprotein convertase. This removal is required for procollagen C-proteinase activity of BMP-1 but is not essential for its secretion. Lee et
al. (34) examined secretion of BMP-1 in several cell types and
showed that TGF-
1 induced the secretion of BMP-1 from fibroblasts. Both pro-BMP-1 and BMP-1 were secreted, supporting the results reported
here that removal of the prodomain is not required for secretion.
Interestingly, TGF-
1 is activated by furin (15, 35), which implies
that secretion of pro-BMP-1 under TGF-
1 inducement involves a
complex feedback system between BMP-1, TGF-
1, furin, or a related
PC.
Decanoyl-RVKR-chloromethyl ketone, the intracellularly active furin
inhibitor, blocked the removal of the prodomain of pro-BMP-1. The
inhibitor functions by either binding active furin or by inhibiting autoactivation (18). To show that furin could remove the prodomain of
pro-BMP-1, we incubated pro-BMP-1 with recombinant furin in vitro. The results showed that furin was able to cleave pro-BMP-1. The scissile bond was predicted between P1 Arg120
and P1' Ala121. Site-directed mutagenesis of the P1 and P2
arginine residues resulted in accumulation of pro-BMP-1AA
in cell culture medium and failure of recombinant furin to remove the
prodomain in vitro.
The identity of the PC or PCs responsible for the removal of the
prodomain of pro-BMP-1 were not identified, and it is likely that more
than one PC is capable of carrying out this function. The fact that FI
was an effective inhibitor of the removal of the prodomain indicated
that furin is a good candidate for the PC responsible for cleavage of
pro-BMP-1. PACE4, PC5/6, and PC7 are additional candidates, because
they are resident within the constitutive secretory pathway, although
only furin and PC7 (like BMP-1 (14)), have ubiquitous tissue
distribution (16, 18).
The secretion profile of pro-BMP-1AA was identical to that
of the pro-BMP-1 in FI-treated cells. Moreover, the majority of
intracellular pro-BMP-1 (~95 kDa) detected on SDS gels was located in
the ER and early Golgi compartments. Furthermore, TGN modifications
were identified on the secreted form of pro-BMP-1 (~105 kDa) but not the intracellular form (compare Figs. 3B and 9B).
This showed that secretion rates from the ER were fast, and, that TGN-
and post-TGN-located BMP-1 occurred transiently within the cell
boundary. The rapid conversion of pro-BMP-1 inside cells presumably
maximizes the efficiency of BMP-1 in ECM assembly, which might not be
possible if pro-BMP-1 were secreted into the ECM. This explained the
practical difficulties of detecting BMP-1 within the cell boundary. The fact that no pro-BMP-1 was identified outside the cell shows that there
was efficient removal of the prodomain within the cell boundary. In
some experiments we attempted to use immunofluorescence to co-localize
BMP-1 and furin within the cell. However, the inherent difficulties
associated with detecting extremely low levels of furin in most
cultured cell lines precluded this approach (36). Single
immunofluorescence was possible using an antibody raised to a peptide
corresponding to the neoepitope of BMP-1 (25). The results from these
experiments showed that mature BMP-1 (i.e. lacking the
prodomain) occurs in the TGN and at the plasma membrane.
Numerous ECM molecules, other than BMP-1, require PCs for their
maturation. Fibrillin, a major component of microfibrils in the ECM,
requires removal of its C terminus by furin for incorporation into the
matrix (20), although the cellular site of this maturation was not
determined. Type V procollagen is a component of the heterotypic collagen fibrils in the cornea and has been implicated in regulating the size of the narrow-diameter fibrils in the cornea (37). The
1(V)
N-propeptides and
2(V) C-propeptides of type V procollagen are
removed by BMP-1, whereas the
1(I) C-propeptides are removed by
furin (21).
Collectively, therefore, the concerted actions of furin and related PCs
together with BMP-1 initiate the critical steps in the assembly of the
extracellular matrix. It is intriguing to speculate why cells evolved
to use BMP-1 as a convertase for extracellular matrix macromolecules
when furin-like PCs would appear to have all the attributes of
effective ECM convertases, including being located in the late
secretory pathway. Presumably, the use of two convertases, perhaps in
different post-TGN compartments, provides a further level of control
with which to assemble an ordered and stable ECM.