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J. Biol. Chem., Vol. 275, Issue 48, 37638-37644, December 1, 2000
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From the
Received for publication, July 11, 2000, and in revised form, August 31, 2000
Cultured fibroblasts secrete an 88-kDa serine
protease that cleaves insulin-like growth factor binding protein-5
(IGFBP-5). Because IGFBP-5 has been shown to regulate IGF-I actions,
understanding the chemical identity and regulation of this protease is
important for understanding how IGF-I stimulates anabolic functions.
The protease was purified from human fibroblast-conditioned medium by
hydrophobic interaction, lectin affinity, and heparin Sepharose affinity chromatography followed by SDS-polyacrylamide gel
electrophoresis. An 88-kDa band was excised and digested with
lysyl-endopeptidase. Sequencing of the high pressure liquid
chromatography-purified peptides yielded the complement components C1r
and C1s. To confirm that C1r/C1s accounted for the proteolytic activity
in the medium, immunoaffinity chromatography was performed. Most of the
protease activity adhered to the column, and the eluant was fully
active in cleaving IGFBP-5. SDS-polyacrylamide gel electrophoresis with silver staining showed two bands, and IGFBP-5 zymography showed a
single 88-kDa band. Amino acid sequencing confirmed that the 88-kDa
band contained only C1r and C1s. C1r in the fibroblast medium underwent
autoactivation, and the activated form cleaved C1s. C1s purified from
the conditioned medium cleaved C4, a naturally occurring
substrate. The purified protease cleaved IGFBP-5 but had no activity
against IGFBP-1 through -4. C1 inhibitor, a protein known to inhibit
activated C1s, was shown to inhibit the cleavage of IGFBP-5 by the
protease in the conditioned medium. In summary, human fibroblasts
secrete C1r and C1s that actively cleave IGFBP-5. The findings define a
mechanism for cleaving IGFBP-5 in the culture medium, thus allowing
release of IGF-I to cell surface receptors.
Insulin-like growth factor-I
(IGF-I)1 is a potent trophic
factor for multiple cell types (1). The mitogenic potential of IGF-I is
controlled by a family of high affinity IGF binding proteins (IGFBPs)
that are ubiquitously present in interstitial fluids (2). The
concentrations of IGFBPs and their affinity constants are such that at
equilibrium most of the IGF-I is bound (3). Factors that disrupt this
binding, such as proteolysis, result in the release of IGF-I to
receptors (4, 5).
IGFBP-5 has been shown to be an important regulator of IGF-I actions in
mesenchymal cell types (6). Both cultured human fibroblasts and smooth
muscle cells secrete an IGFBP-5 protease that is specific for IGFBP-5
and cleaves it into a 22-kDa fragment, which has more than 1000-fold
reduction in its affinity for IGF-I (7, 8). Studies utilizing a
protease-resistant IGFBP-5 mutant have shown that high
concentrations of the mutant IGFBP-5 (at least a 5-fold molar excess
over IGF-I) completely inhibited IGF-I-mediated receptor activation
(9). In contrast, if a 1:1 molar ratio of native IGFBP-5 to IGF-I is
added to extracellular matrix, IGFBP-5 can act to potentiate the
mitogenic effect of IGF-I (10). Therefore, the factors that control
proteolytic cleavage of IGFBP-5 represent an important mechanism for
controlling the amount of IGF-I that is available to interact with
receptors. Although some broad spectrum proteases, such as plasmin,
thrombin, or matrix metalloproteases-2 and -9, have been shown to
cleave several forms of IGFBPs, the identity of IGFBP protease
activities that are specific for a single form of IGFBP has been
difficult to determine (9, 11-14). Recently, a partially purified
fraction of fibroblast-conditioned medium containing the
pregnancy-associated plasma protein-A protease was shown to
specifically cleave IGFBP-4 (15). However a homogenous preparation of a
protease that specifically cleaves IGFBP-5 has not been reported. For
these reasons, we purified and characterized the IGFBP-5-specific
protease that is present in fibroblast-conditioned medium.
Cell Culture--
Human dermal fibroblasts (GM 10) were
purchased from Coriell Institute (Camden, NJ). The cells were grown to
confluency in 175-cm2 tissue culture flasks (Falcon
Labware, Fairfield, NJ) in minimum essential media (Life Technologies,
Rockville, MD) supplemented with 10% bovine serum (Colorado Serum Co.,
Denver, CO). To collect conditioned medium, the monolayers were washed
three times with PBS, and 150 ml of serum-free minimum essential medium
was added per flask. The medium was collected after 48 h,
centrifuged at 16,000 × g for 20 min to remove
cellular debris, and then stored at Protein Purification--
Ammonium sulfate (Mallinckrodt, Baker,
Paris, KY) was added to 12 liters of conditioned medium to 85%
saturation, and the mixture was stored for 14 h at 4 °C, then
centrifuged at 23,500 × g for 1 h. The pellet was
extracted with 600 ml of 20 mM Tris, 4 mM
CaCl2, 10 mM NaCl, pH 7.4. The extract was
adjusted to 1.0 M ammonium sulfate, stirred for 2 h at
4 °C, and centrifuged at 23,500 × g for 45 min. The
supernatant was applied to a 4.4 × 3.5 cm butyl-Sepharose
column-4 (Fast Flow, Amersham Pharmacia Biotech). The flow-through
material was discarded, and the column was eluted with 20 mM Tris-HCl, pH 7.4, then tested for proteolytic activity.
The fractions that were eluted were tested for activity by incubating
between 1 and 10 µl with 100 ng of IGFBP-5 for 14 h at 37 °C
in 50 µl of 0.05 M Tris, 4 mM
CaCl2, pH 7.2 (7). The products of the reaction were
analyzed by SDS-PAGE with immunoblotting using a specific anti-IGFBP-5
antiserum, as described previously (16). The eluant, approximately 350 ml, was applied directly to a wheat germ-agarose column (1.6 × 5.0 cm; Sigma Chemical Co., St. Louis, MO) that had been equilibrated
with 20 mM Tris, 4 mM CaCl2, 0.4 M NaCl, pH 7.2. The fractions were eluted with the same
buffer containing 0.5 M
N-acetyl-D-glucosamine. The fractions containing
proteolytic activity were determined as described above, and then the
fractions with the greatest amount of activity were pooled
(approximately 100 ml of eluant) and diluted 1:1 with 20 mM
Tris, 4 mM CaCl2, 2 mM
MnCl2, 0.4 M NaCl, pH 7.2. This solution was
applied to a concanavalin A-agarose column (Sigma; 1 × 9 cm) that
had been equilibrated with the same buffer. The column was eluted with
the same buffer containing 0.5 M
Immunoaffinity Chromatography--
Antiserum to C1r and C1s was
prepared using synthetic peptides that contained the following
sequences: C1r (CLYPKEHEAQSNASLDVFLGHTNVEE) and C1s
(CVEGNREPTMYVGSTSVQTSRLAKS). The peptides (6.1 mg of C1r and 8.8 mg of
C1s) were each conjugated to 4 mg of maleimide-activated KLH (Pierce,
Rockford, IL). Each peptide was linked with KLH in 1.4 ml of 0.83 M Na2H2PO4, pH 7.2, containing 0.9 M NaCl, 0.35 M EDTA. Following
dialysis in the same buffer without EDTA, the mixture was lyophilized.
New Zealand White rabbits were immunized with 1 mg of linked KLH
peptide in complete Freund's adjuvant. The rabbits were injected with
0.5 mg of KLH peptide in incomplete Freund's adjuvant at monthly
intervals. To prepare an affinity column, 6 ml of C1r and C1s antisera
were each diluted to 12 ml with 20 mM
NaH2PO4, pH 7.0, and each solution was applied
to a protein G-Sepharose-4 (Fast-flow, 1.0 × 7.0 cm; Amersham
Pharmacia Biotech) affinity column. The IgG was eluted with 0.1 M glycine HCl, pH 2.5, and immediately neutralized with 1.0 M Tris to pH 7.2. The purity of the IgG was 95-99% based
on SDS-PAGE with silver staining. 15.4 mg of the anti-C1r and 15.4 mg
of anti-C1s IgG were mixed and dialyzed against 0.02 M
sodium acetate, pH 5.0, containing 0.15 M NaCl, then
oxidized for 1 h with 0.01 M NaIO4. Oxidation was terminated with glycerol, and the mixture was then dialyzed against 0.1 M sodium acetate, pH 4.5, containing
0.10 M NaCl. The solution was mixed with Affi-Prep Hz
support (Bio-Rad, Hercules, CA) for 24 h at 4 °C, then washed.
Approximately 10 ml of the pool of active fractions that had been
eluted from heparin-Sepharose was diluted to 20 mM Tris, 50 mM NaCl, 4 mM CaCl2, pH 7.2 and applied to the anti-C1r/C1s affinity column (1.5 × 5.5 cm) at 3 ml/h. The active fractions were eluted in that buffer containing 1.0 M NaCl.
Amino Acid Sequence Analysis--
The partially purified
material (4-5 ml) was concentrated ~50-fold on an Ultrafree 0.5-ml
centrifugal filter (Biomax-10K NMWL, Millipore, Bedford,
MA) prior to SDS-PAGE. Both material that had been purified through the
heparin-Sepharose step and material that had been
immunoaffinity-purified were sequenced. The proteins were separated by
SDS-PAGE, 9% gel. The gel was stained with Coomassie Blue, R-250
(Sigma) then destained for 2 h, and bands corresponding to
molecular mass estimates of 88, 180, 280, and > 300 kDa
were excised. These gel slices were washed three times in 450 µl of 50% acetonitrile/0.2 M Tris, pH 9.0. The gel pieces were
incubated in 200 µl of 0.1 M Tris, pH 9.2, containing 0.4 µg of lysyl-endopeptidase (Wako Bioproducts, Richmond, VA) for
14 h at 37 °C, and then they were sonicated for 30 min at room
temperature. An additional 0.2 µg of lysyl-endopeptidase was added,
and the incubation was continued for 5 h at 37 °C. The peptides
were extracted by adding 100 µl of 0.1 M Tris, pH 9.0, followed by 30 min of sonication. This was repeated using 400 µl of
buffer then followed by two additional extractions with 400 µl of
60% acetonitrile, 0.2% trifluoroacetic acid. All four extracts were
pooled and diluted to 6% acetonitrile with 0.04% trifluoroacetic
acid. The pH was adjusted to 3.0, and the solution was applied to a
reverse-phase HPLC C18 (2 mm × 15 cm) column (VYDAC, Hesperia,
CA). The column was eluted with 0.04% trifluoroacetic acid and a
linear acetonitrile gradient (10-60%) over 75 min. Selected peptides
were repurified using the same solvents except that the gradient was
extended to 125 min. Multiple internal peptides that were obtained from
each band were sequenced. Automated Edman degradation chemistry was
used to determine the NH2-terminal sequence for the unknown
peptides (17). The following systems were employed for these analyses:
I. Applied Biosystems model 494 protein sequencer, model 140C
microgradient system, and model 785A absorbance detector. The
PTH-derivatives were identified by reverse-phase HPLC analysis
in an on-line fashion and utilized a PerkinElmer Life Sciences/Brownlee
2.1-mm inner diameter PTH-C18 column. II. PerkinElmer Life
Sciences/Applied Biosystems model 492 cLC protein sequencer model 140D
microgradient system and model 785A absorbance detector. The online
reverse-phase HPLC analysis of the PTH-derivatives was performed on a
PerkinElmer Life Sciences/Brownlee 0.8-mm inner diameter PTH-C18 column.
Gel Electrophoresis and Immunoblotting--
Purified protein
fractions or concentrated conditioned media samples were loaded onto
9% SDS-polyacrylamide gels and electrophoresed as described previously
(18). Appropriate protein markers were run in a parallel lane to
determine molecular weight. For some analyses, the gel was fixed with
5% acetic acid, 10% ethanol, and analyzed by silver staining. For
other analyses, the proteins were transferred to polyvinylidene
difluoride membranes, as described previously (19). The membranes were
incubated with a 1:500 dilution of C1r or C1s antiserum in 2.0 ml of
PBS containing 1% BSA, pH 7.0, for 14 h. After extensive washing
in PBS, they were incubated for 3 h, diluted 1:1500 with goat
anti-rabbit IgG conjugated to alkaline-phosphatase in TBS with 0.1%
BSA, and then the immune complexes were detected as described
previously (16). Pure C1r (400 ng) (Calbiochem, La Jolla, CA) or C1s
(400 ng) (Enzyme Research, South Bend, IN) were run in parallel lanes
to confirm that the antibodies recognized proteins of the correct
molecular weight. For immunoblotting of C4, a 7.5% gel was
used. The anti-C4 antiserum (Calbiochem) was used at a
1:500 dilution. For immunoblotting for IGFBP-1, -2, -3, and -4, the
specific antisera utilized and the conditions used have been described
previously (7).
IGFBP-5 Zymography--
Seven micrograms of IGFBP-5 was mixed
with 4.0 ml of acrylamide gel solution (10% gel), and the gel was
polymerized (5). The IGFBP-5 protease-containing fractions were
concentrated 5-20× by centrifugation using Ultrafree 0.5-ml
centrifugal filters. The filter was exposed to 10 µl of 3× Laemmli
sample buffer, and this was pooled with the concentrate (20 µl) then
electrophoresed at room temperature. The gel was washed in 2.5% Triton
X-100 at 4 °C for 1 h, followed by extensive washing in
distilled water. The gel was incubated in 0.05 M Tris, 4 mM CaCl2, pH 7.4, overnight at 37 °C to
allow for proteolysis and for capillary transfer to a polyvinylidene
difluoride membrane (9). The membrane was immunoblotted using a 1:1000
dilution of IGFBP-5 antiserum as described above. The electrophoretic
mobility of the bands that were detected was compared with prestained
molecular weight standards (Life Technologies).
Analysis of Protease Activation--
To determine if the
purified C1r could undergo autoactivation, 500 ng of material was
incubated in 0.02 ml of 0.025 M MES, 125 mM
NaCl, 2 mM EGTA, pH 7.2, for 15-90 min. The products of the reaction were then analyzed by SDS-PAGE (9% gel) using reducing conditions, 0.1 M dithiothreitol, followed by
immunoblotting for C1r and C1s. To determine the ability of the
purified C1s to be cleaved by C1r, 400 ng of the highly purified
protease was exposed to 800 ng of C1r in 30 µl of the buffer listed
previously, incubated at 37 °C for 2 h, and then analyzed by
SDS-PAGE (9% gel) under reducing conditions, followed by
immunoblotting for C1s. To determine if the purified IGFBP-5 protease
had activity in cleaving C4, 180 or 540 ng of the highly
purified protease was incubated with 600 ng of C4
(Calbiochem) in 25 µl of 25 mM MES, 125 mM
NaCl, 2 mM EGTA, pH 7.2, for 18 h and the products
analyzed SDS-PAGE (7% gel) with immunoblotting for C4.
Duplicate tubes containing 200 or 500 ng of pure C1s (Enzyme Research)
were also analyzed. To determine the ability of the purified IGFBP-5
protease to cleave IGFBP-5, between 3 and 50 ng of protein was
incubated with 100 ng of IGFBP-5 in 60 µl of 0.05 M Tris,
pH 7.2, with 4 mM CaCl2 for variable time
periods up to 3.5 h at 37 °C. To determine the effects of
protease inhibitors on the ability of IGFBP-5 protease in conditioned
media to cleave IGFBP-5, 25 µl of fibroblast conditioned media was
incubated with 100 ng of IGFBP-5 for 16 h at 37 °C in the
buffer listed previously. The following inhibitors were analyzed, PB-145 (10 Silver stain analysis of the material purified through the
heparin-Sepharose step showed a prominent 88-kDa band, a high molecular weight band that did not enter the gel, and two other bands with molecular mass estimates of 180 and 280 kDa (Fig.
1A). Minor bands were detected
at 92 and 240 kDa. Amino acid sequence analysis of the 88-kDa band
showed that it contained the C1r and C1s sequences. Eleven peptides
that were sequenced had sequences corresponding to human C1r, and eight
peptides had sequences corresponding to human C1s (Fig.
2). Each of these peptides was distinct
and was not a mixture of the two sequences. Likewise, single HPLC peaks yielded pure peptides that were either pure C1r or pure C1s, but, within a single digested band, peaks that contained either C1r or C1s
were detected. When the faint band that was detected at 92 kDa was
carefully excised from the gel and separated from the lower 88-kDa
band, sequences corresponding to C1r only were obtained. The 180-kDa
band gave the sequences of the Because the heparin-Sepharose-purified material had several bands that
were detected by silver staining, the material was further purified by
immunoaffinity chromatography. Silver stain analysis of the material
that was eluted showed three detectable bands (Fig.
3A). Sequence analysis of the
88-kDa band confirmed the presence of C1r and C1s, and no other peptide
sequences were detected. Sequence analysis of the band that did not
enter the gel revealed thrombospondin-1. The 160-kDa band yielded
rabbit IgG, suggesting that this band had been cleaved and eluted
during immunoaffinity chromatography. Immunoblotting for C1s showed an intense 88-kDa band and faint bands of >200 kDa and 68 kDa. Following reduction, bands with estimated molecular masses of 92 and 70 and 30 kDa were detected (Fig. 3B). Immunoblotting for C1r showed two bands at 88 and 92 kDa and a band with an estimated molecular mass
of 180 kDa. Analysis of IGFBP-5 protease activity showed that the
material was fully active and yielded the expected 22-kDa band.
Approximately 2 ng of the material degraded 100 ng of IGFBP-5 (2 µg/ml) in 3.5 h (Fig. 3C). This result establishes
that the material purified by this procedure that cleaved IGFBP-5 into a 22-kDa fragment contained only C1r and C1s, and no other proteases could be identified in the 88-kDa band or any of the protein
contaminant bands.
The Complement Component C1s Is the Protease That Accounts
for Cleavage of Insulin-like Growth Factor-binding Protein-5 in
Fibroblast Medium*
,,,¶
Department of Medicine, University of North Carolina
School of Medicine, Chapel Hill, North Carolina 27599-7170 and the
§ Department of Protein Chemistry, Monsanto, Inc., Chesterfield,
Missouri 63198
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
-methylmannopyranoside (Sigma), and the fractions were tested
for activity as described previously. The pool of active fractions was
diluted to 30 mM NaCl, 42 mM
-D-mannopyranoside, and applied to a 2.5 × 6 cm
heparin-Sepharose column (Amersham Pharmacia Biotech) that had been
equilibrated with 20 mM Tris, 4 mM
CaCl2, 30 mM NaCl, pH 7.2. The column was eluted with the same buffer containing either 0.5 or 1.0 M
NaCl, and the fractions were tested for protease activity.
7 M), a synthetic peptide with
sequence similarity to anti-thrombin III (20), heparin co-factor II
(107 M), antithrombin III (107
M). In additional experiments, the ability of the purified
protein to cleave other forms of IGFBPs was determined by incubating 50 ng of purified protease with 150 ng of pure IGFBP-1, -2, -3, and -4 as
stated for IGFBP-5. The products of the reactions were then analyzed by
SDS-PAGE (12.5% gel) followed by immunoblotting for each specific IGFBP.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and 2 chains of collagen
type VI. The 280-kDa band contained two peptides with sequences
corresponding to tenascin-C, and the band that did not enter the gel
contained multiple peptides with sequences encoding collagen type VI or
thrombospondin-1. Both tenascin-C and thrombospondin-1 have been shown
to bind to IGFBP-5 (21, 22). Immunoblotting of the identical material
with anti-C1r antiserum showed a prominent 88-kDa band that co-migrated
with the intact C1r standard and a second band that had a molecular
mass estimate of >200 kDa (Fig. 1B). When analyzed
following reduction, the major C1r band was detected that had a
molecular mass estimate of 94 kDa, and less abundant bands were
detected at 62 and 38 kDa (Fig. 1B). These corresponded to
size estimates of pure activated C1r. Immunoblotting for C1s showed
prominent bands with molecular mass estimates of 88 and >200 kDa
(non-reduced) and 86, 68, and 28 kDa (following reduction). IGFBP-5
zymography of this same material showed a prominent band of activity
with an 88-kDa molecular mass estimate, and a less intense band was
detected that had an estimated molecular mass of 190 kDa (Fig.
2C). Four independently run gels, followed by excision of
the 88-kDa band and sequence analyses, showed only C1r and C1s, and no
other proteins were present in this band. When IGFBP-5 protease
activity was analyzed, 25 ng of this material (400 ng/ml) cleaved 100 ng of IGFBP-5 in 1.5 h (Fig. 2D). There was a
concentration-dependent increase in activity, and cleavage was detected using protease concentrations as low as 3.5 ng (60 ng/ml).
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Fig. 1.
Properties of IGFBP-5 protease purified
through the heparin-Sepharose step. Material that had been
purified through the heparin-Sepharose step, as described under
"Experimental Procedures," was analyzed by the following methods.
A, silver stain analysis. Lane 1, pure C1s and
bovine serum albumin standards; lane 2, 2 µl of the active
fraction; lane 3, 4 µl of the active fraction. The
arrows denote the positions of the four bands that were
detected. For sequencing, 250 µl of the active fraction was run in
two lanes, that were stained with Coomassie Brilliant Blue R250. The
same four bands that had been detected by silver stain analysis were
excised, digested, and sequenced. B, immunoblotting for C1r
and C1s. The fraction that was analyzed by silver staining was also
analyzed by immunoblotting for C1r (lanes 2 and
6) and C1s (lanes 4 and 8) under
non-reducing (lanes 1-4) and reducing (lanes
5-8) conditions. Pure C1r standard (lanes 1 and
5) and pure C1s standard (lanes 3 and
7) were also analyzed. The single arrow
(lanes 1-4) denotes the positions of C1r (lanes
1 and 2) or C1s (lanes 3 and 4)
(non-reduced). The arrows shown for lanes 5-8
denote the positions of intact C1r and C1s, and their two major
proteolytic fragments that are detected after reduction. C,
IGFBP-5 zymography. A pool of active heparin-Sepharose-purified
material (lane 1) and immunoaffinity-purified material
(lane 2) were analyzed. The results show IGFBP-5 proteolytic
activity with molecular mass estimates of 88 and 190 kDa (lane
1) and 88 kDa (lane 2). D, IGFBP-5 protease
activity. Increasing concentrations (3.5-50 ng) the pool of
heparin-Sepharose-purified material were incubated with 100 ng of
IGFBP-5 for 1.5 h at 37 °C then analyzed for IGFBP-5
proteolytic activity by immunoblotting. Lane 1, 50 ng;
lane 2, 25 ng; lane 3, 10 ng; lane 4,
3.5 ng; lane 5, control, 100 ng of IGFBP-5, no protease. The
arrows denote the positions of intact IGFBP-5 and its major
proteolytic fragment, 22 kDa. As shown in the figure, activity was
detected at 3.5 ng (lane 4) and was maximal at 25 ng
(lane 2).
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Fig. 2.
Sequences of peptides from C1r and C1s.
The HPLC-purified peptides that are shown were sequenced. The sequences
of contiguous peptides are underlined. The arrow
denotes the position of the cleavage site that is required for
activation.
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Fig. 3.
Purity and protein characteristics of
immunoaffinity purified proteins. The heparin-Sepharose-purified
material was further purified by immunoaffinity chromatography with a
column that had been prepared with anti-C1r and -C1s antisera. The pool
of the most active fractions was then analyzed by the methods listed.
A, Coomassie stain analysis. Lanes 1 and
2 contain 250 and 125 µl of the eluted pool of fractions
from the immunoaffinity column. These bands were subsequently excised,
digested, and sequenced. The protein staining bands are noted with
arrows. A non-reduced, pure C1s standard and BSA are shown
in lane 3. The arrows denote the positions of the
three detectable bands. B, C1r and C1s immunoblotting. The
material that was eluted from the immunoaffinity column was
immunoblotted separately for C1r and C1s. Lane 1, C1s,
non-reduced; lane 2, C1s, reduced; lane 3, C1r,
non-reduced. C, IGFBP-5 protease activity. The capacity of
the purified material to degrade IGFBP-5 was determined as described
under "Experimental Procedures." The arrows denote the
position of intact IGFBP-5 and its major proteolytic 22-kDa fragment.
Lane 1, material that was loaded onto the column; lane
2, material excluded from the column; lane 3, 4 µl of
eluant; lane 4, 1 µl of eluant; lane 5, 0.25 µl of eluant; lane 6, 0.05 µl of eluant; lane
7, conditioned medium; lane 8, IGFBP-5 standard.
C1r has been shown to cleave C1s, which is secreted as a zymogen, to
generate its active form (23, 24). To determine if pure C1r could
activate our purified C1s, activated C1r was incubated with our
purified material, and then it was analyzed by immunoblotting after
reduction for evidence of activation. Immunoblotting showed full
activation of our purified C1s that was comparable to activation of a
purified C1s standard (Fig.
4a). To exclude the
possibility that all of the IGFBP-5 protease activity was due to C1r,
activated C1r was incubated with IGFBP-5. A high concentration
(e.g. 2-5 µg of C1r) was required to detect cleavage
(Fig. 4b). Furthermore, cleavage proceeded slowly, and the
fragments that were obtained had estimated molecular masses of 24 and
26 kDa. In contrast, a much lower concentration of activated C1r
(e.g. 400-800 ng) cleaved C1s into the 67- and 30-kDa forms
(Fig. 4b). To determine if C1r in the purified samples could
further autoactivate, and if its autoactivation was associated with C1s
activation, we incubated the heparin-Sepharose-purified material for
0.5, 1, and 2 h at 37 °C, then determined the degree of C1r and
C1s activation by immunoblotting. As shown in Fig.
5, a time-dependent increase in activated C1r and C1s was detected. These findings suggest that C1r
is functioning to activate C1s, which is cleaving IGFBP-5. To confirm
that the purified protease could cleave C4, the known physiologic substrate for C1s, its capacity to cleave C4
was compared with pure C1s. The purified protease had greater activity
in cleaving C4 than did pure C1s (Fig.
6). To determine the specificity of the
protease for IGFBP-5, 50 ng of material purified through the immunoaffinity chromatography step was incubated with pure IGFBP-1, -2, -3, -4, or -5. As shown in Fig. 7, only
IGFBP-5 was degraded by the purified protease. Because the IGFBP-5
proteolytic activity in crude fibroblast-conditioned medium is specific
for IGFBP-5 (7), this finding further supports the conclusion that we
have purified the protease that accounts for the IGFBP-5 proteolytic activity in fibroblast-conditioned medium.
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To further confirm that C1r and C1s accounted for IGFBP-5 proteases in
fibroblast-conditioned medium, several serine protease inhibitors that
had been shown to inhibit IGFBP-5 proteolysis to some extent were
incubated with crude material that had only been purified through the
first step, and their ability to inhibit IGFBP-5 cleavage was
determined. As shown in Fig. 8, several
of these inhibitors at least partially inhibited IGFBP-5 proteolysis. C1 inhibitor, the physiologic inhibitor of C1s activation (25), was the
most potent inhibitor of the IGFBP-5 protease activity contained in the
fibroblast-conditioned medium. The other inhibitors that had less of an
effect on C1s activation (data not shown) also had less of an effect on
IGFBP-5 proteolysis.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We have reported previously that human fibroblasts secrete an IGFBP-5 protease that cleaves IGFBP-5 into predominantly a 22-kDa fragment, and this fragment has a low affinity for IGF-I (8). This proteolytic activity is a serine protease and is specific for IGFBP-5 (7). Because IGFBP-5 is an important modulator of IGF-I bioactivity, the factors that regulate the activity of this protease have the potential to regulate the ability of IGFBP-5 to modulate IGF-I actions. For that reason, we were interested in determining the molecular identity of IGFBP-5 protease activity in fibroblast-conditioned medium. To that end, we purified a large quantity of IGFBP-5 protease from human fibroblast-conditioned medium and subjected it to amino acid sequence analysis. Sequence analysis yielded several peptides that encoded C1r and C1s. Because these proteins could not be separated, it is not possible to definitively determine which protease is actually cleaving IGFBP-5; however, C1s is known to have a much broader range of substrates (25). Furthermore, a very low concentration (60 ng/ml) of the C1r/C1s mixture cleaved IGFBP-5 rapidly. In contrast, IGFBP-5 cleavage by C1r required a much higher concentration, proceeded slowly, and yielded fragments that had size estimates that were different from those that were detected after cleavage by the purified IGFBP-5 protease. These results do not exclude the possibility that C1r may be cleaving IGFBP-5 to some extent in the medium, but they suggest that C1s accounts for the major portion of the IGFBP-5 protease activity. Because C1s is secreted at least in part as an inactive zymogen, the primary role of C1r may be to cleave and activate C1s, which subsequently cleaves IGFBP-5. This conclusion is also supported by the known specificity of both enzymes for specific recognition sequences (25).
That these enzymes were the predominant protease activity for IGFBP-5 in fibroblast-conditioned medium is proven by several points. First, during purification, other proteases that have been shown to cleave IGFBP-5, such as ASPO-5 and MMP-2 and -9 were removed by various chromatographic steps; however, most of the proteolytic activity was retained. Furthermore, when pure MMP-2, -9, or ASPO-5 are incubated with IGFBP-5, the rate of proteolysis is slow and the fragment sizes that are generated are distinct from those generated by the IGFBP-5 protease activity (7). Finally, MMP-2 and -9 are not specific for IGFBP-5 and degrade other forms of IGF binding proteins. In contrast, the proteolytic activity in fibroblast media and the immunoaffinity-purified material were specific for IGFBP-5 and did not cleave other forms of IGF binding proteins. Second, immunoaffinity chromatography of partially purified conditioned medium showed that most of the IGFBP-5 protease activity in the medium could be accounted for by C1r and C1s that adhered to the antibody affinity column. Third, the specific C1s protease inhibitor, C1 inhibitor, inhibits IGFBP-5 cleavage by crude conditioned medium, and its ability to inhibit C1r and C1s activation correlates with its ability to inhibit IGFBP-5 proteolysis. Furthermore, extensive sequencing of all of the protein bands that were detected in the most highly purified material did not yield sequences corresponding to any other protease. Following immunoaffinity chromatography of fibroblast medium, approximately 84% of the IGFBP-5 proteolytic activity adhered to the column (data not shown). Taken together, these data strongly suggest that C1s is the predominant protease cleaving IGFBP-5 in fibroblast medium and C1r is responsible for its activation.
Recently, it was shown that a part of the IGFBP-4 protease in fibroblast medium could be ascribed to PAPP-A, which is a metalloprotease (15). However, in that study PAPP-A was not purified to homogeneity, and therefore the results did not exclude the possibility that other proteases were present in fibroblast medium that could potentially degrade IGFBP-4. Furthermore, the extent to which any of these proteases is active is determined not just by the concentration of protease, but whether it is secreted as the zymogen, the percentage activation and whether protease inhibitors are present. When the secretion of C1r and C1s was analyzed using several test conditions, partial activation of these proteases was noted. Therefore, partially activated forms exist in fibroblast medium, making it more likely that they account for IGFBP-5 protease activity.
The physiologic significance of activated C1r/C1s in fibroblast medium and their roles in modulating IGF-I actions remains to be determined. However, we have shown that a protease-resistant form of IGFBP-5 inhibits IGF-I actions (11). Therefore, the fact that these proteases are present in the medium in partially activated forms that can cleave IGFBP-5 into fragments with very low affinity suggests that they are capable of modulating IGF-I bioactivity. Furthermore, inhibition of their activity with a specific inhibitor (e.g. C1 inhibitor) results in inhibition of IGFBP-5 cleavage, suggesting that activation can be modulated in physiologic fluids that contain this inhibitor. In additional studies, we have detected C1 inhibitor in fibroblast-conditioned medium by immunoblotting (data not shown). Therefore, the variables that regulate C1r and C1s synthesis and activation, as well as the secretion of C1 inhibitor, have the potential to alter IGFBP-5 cleavage and thereby modulate IGF-I actions.
Other investigators have reported that activated C1r and C1s occur at sites outside the liver (25, 26) and that they have proteolytic functions other than complement activation (27-30). Specifically, pleural fluid and joint fluid contain activated C1r and C1s, as do cell culture supernatants from glial cells (25, 31). Several central nervous system cell types have been shown to contain C1s peptide and messenger RNA. Spleen, liver, brain, and kidney have been shown to contain C1r and C1s mRNA (25). Several studies have also shown that C1r and C1s activation may lead to cleavage of peptides other than the complement components (27-30), and several of the proteins that have been shown to be substrates for C1s are not traditional components of the complement pathway. Taken together, these findings suggest that C1r and C1s may have roles other than complement activation.
For several years, investigators have hypothesized a linkage between
inflammation (i.e. acute complement activation during injury) and subsequent cellular repair processes. Because IGF-I and
IGFBP-5 are secreted by several cells, such as macrophages or
fibroblasts, which are involved in repair that occurs in response to
injury, and both peptides are present in pericellular fluids (32), on
cell surfaces, and in extracellular matrix of several mesenchymal cell
types (33), it is possible that there is a coordinated linkage between
activation of C1r and C1s during acute injury and the subsequent
release of IGF-I to receptors that are present on the surface of cell
types that are involved in tissue repair. IGFBP-5, which is also
secreted by the connective tissue cell types that are involved in
tissue repair (34, 35), could be an important component for controlling
the amount of IGF-I that can be released to receptors after C1r and C1s
activation (35). Whether such a linkage exists deserves further
exploration. In summary, we have determined that the predominant
protease component of fibroblast-conditioned medium is ascribable to
the complement subcomponents C1r and C1s. C1s is the major protease
that cleaves IGFBP-5, but its full activation requires the presence of
activated C1r. Further studies are necessary to determine the
physiologic role of these proteases in activating a cascade of events
leading to IGF-I receptor stimulation.
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ACKNOWLEDGEMENT |
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We thank George Mosley for his help in preparing the manuscript.
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FOOTNOTES |
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* This work was supported by Grant AGO23331 from the National Institutes of Health.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.
¶ To whom correspondence should be addressed: Division of Endocrinology, CB 7170, 6111 Thurston-Bowles, University of North Carolina, Chapel Hill, NC 27599-7170. Tel.: 919-966-4735; Fax: 919-966-6025; E-mail: endo@med.unc.edu.
Published, JBC Papers in Press, September 11, 2000, DOI 10.1074/jbc.M006107200
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ABBREVIATIONS |
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The abbreviations used are: IGF-I, insulin-like growth factor-I; IGFBP-5, insulin-like growth factor binding protein-5; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; C1r, complement 1r; C1s, complement 1s; PBS, phosphate-buffered saline; KLH, keyhole limpet hemocyanin; TBS, Tris-buffered saline; BSA, bovine serum albumin; MES, 2-(N-morpholino)ethanesulfonic acid; MMP-2, matrix metalloprotease-2; PAPP-A, pregnancy-associated protein-A; PTH, phenylthiohydantoin.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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