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Institute for Biochemistry II (B.K., T.B.), University of Göttingen, Gosslerstrasse 12d, D-37073 Göttingen, Germany; Strangeways Research Laboratory (S.C.), Worts Causeway, Cambridge CB1 4RN, United Kingdom; and Metabolic Unit (J.Z.), Department of Medicine, University Hospital, CH-8091 Zürich, Switzerland
Address all correspondence and requests for reprints to: Thomas Braulke, Ph.D., Institute for Biochemistry II, University of Göttingen, Gosslerstrasse 12 D, D 37073 Göttingen, Germany. E-mail: braulke{at}ukb2-00.uni-bc.gwdg.de
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Abstract |
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These studies provide the first direct biochemical evidence that human pregnancy serum contains a 50-kDa IGFBP protease with properties of a soluble disintegrin metalloproteinase that appears to be potentially involved in regulating IGF bioavailability for placental and fetal growth.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In serum of patients with GH receptor deficiency, with noninsulin-dependent diabetes, after surgery or during gestation a disappearance of intact IGFBP-3 has been observed due to an increase in IGFBP-3 protease activity (4, 5, 6, 7, 8). However, proteolytic IGFBP-3 forms have been found still in 150-kDa ternary complexes (9, 10) but with altered functional properties (11). The formation of proteolytic IGFBP-3 fragments with lower affinity to IGFs (7, 11), accompanied by faster elimination half-life as demonstrated in rat pregnancy serum (12), is speculated to be an important mechanism making IGFs more available to the cells (13) and affecting placental and fetal growth. Additionally, IGFBP-2, -4, and -5 protease activities have been reported in pregnancy serum (8, 14). Whereas both proteolysis of IGFBP-3 and -5 are cation-dependent and are blocked by serine protease inhibitors (7, 12, 14), the identity of the serum proteases are unknown. Recent antibody blocking experiments have suggested that matrix metalloproteinases may represent the IGFBP-3 proteases in rat serum during late pregnancy (15).
In the present study, we analyzed IGFBP protease activities, present in two protein size classes in human pregnancy serum. Following purification, a major 50-kDa protease was identified with properties of a disintegrin metalloproteinase that cleaves gelatin and IGFBP-3, -4, and -5.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Antibodies
Polyclonal antibodies raised against human matrix
metalloproteinase (MMP)-1 (20), human MMP-2 (21), human MMP-3 (also
recognizes MMP-10) (22), pig MMP-9 (also recognizes human MMP-9) (23),
and human MMP-13 (24) were gifts from Dr. R. Hembry (Strangeways
Research Laboratory, Cambridge, UK) and have been described elsewhere.
A preadsorbed rabbit antiserum (R70) directed against a disintegrin
domain peptide sequence conserved between human and rodents (25) was
kindly provided by Dr. P. Glynn (University of Leicester, UK).
Peroxidase-conjugated donkey antisheep IgG and goat antirabbit IgG came
from Jackson Immunoresearch (West Grove, PA) and Dianova (Hamburg,
Germany), respectively.
IGFBP protease assay
One microliter of serum or 5–50 µl of column fractions
dialyzed against 20 mM Tris/HCl, pH 7.4, containing 150
mM NaCl were incubated with [125I] IGFBPs
(10–15,000 cpm) for 2–18 h at 37 C. When indicated, the
metalloproteinase inhibitors EDTA (4 mM), 1,10
phenanthroline (2 mM), TIMP-1, TIMP-2 (10 µg/ml), TAPI
(0.1 mM), or the serine protease inhibitors
phenylmethanesulfonyl fluoride (PMSF, 10 mM) or 3,4
dichloroisocoumarin (0.5 mM) were included. After
solubilization, the samples were subjected to SDS-PAGE (12.5%
acrylamide) under nonreducing conditions and visualized by
autoradiography. Prestained molecular mass marker proteins (myosin, 200
kDa; phosphorylase b, 97.4 kDa; BSA, 66 kDa; ovalbumin, 46 kDa;
carbonic anhydrase, 30 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.3
kDa) were purchased from Amersham (Dreieich, Germany).
Gelatin zymography
Zymography was performed with 10% SDS-polyacrylamide gel
containing gelatin (1 mg/ml; Sigma) as described previously (26). Five
to 50 µl of fractions (lyophilized and resuspended in 10 µl 50
mM Tris buffer pH 7.5 containing 5 mM
CaCl2, 150 mM NaCl; buffer A) were mixed with
1/2 volume of buffer Z (0.25 M Tris/HCl, pH 6.8, containing
10% SDS and 4% sucrose) and subjected to SDS-PAGE using a Mighty
Small II MiniGel Unit (Pharmacia Biotech, Uppsala, Sweden). After
electrophoresis, the gel was washed two times for 20 min in buffer A
containing 2.5% Triton X-100, rinsed for 20 min in water followed by
incubation in buffer A for 24 h (or as indicated) at 37 C.
Gelatinase activity was visualized by negative staining with Coomassie
Brillant Blue G-250.
[125I] IGFBP zymography
Radiolabeled IGFBP zymography was performed by a modified method
described previously (27, 28, 29). In brief, [125I] IGFBP-3,
-4, or -5 (400,000 cpm/ml) were added to 5.5 ml polyacrylamide gel
solution and the gel polymerized. Protease-containing samples were
mixed with 1/2 volume buffer Z and electrophoresed without heating
through the 10% polyacrylamide substrate gel. Gels were washed and
incubated as described for gelatin zymography except for the incubation
time of 40 h. Fragments produced from IGFBP cleavage were
continuously eluted from the [125I] IGFBP substrate gel
by capillary transfer onto nitrocellulose membranes (BioBlot-NC, Costar
Scientific Corp., Cambridge, MA). The membranes and the fixed and dried
gel were analyzed by autoradiography.
Protease purification
Eighty milliliters of pregnancy serum were sequentially
precipitated by 30 and 45% ammonium sulfate. The latter precipitate
was dissolved in 4 ml 20 mM Tris buffer, pH 7.4, containing
150 mM NaCl (buffer B), dialyzed overnight against the same
buffer, and applied to a Sephacryl S-400 column (Pharmacia Biotech;
2.6 x 82 cm) that had been equilibrated with buffer B.
Four-milliliter fractions were collected at a flow rate of 2 ml/min,
and 50 µl aliquots were assayed for IGFBP-4 protease activity. Two
peaks of activity were detected, and the fractions were pooled (pool I
and II) separately and concentrated. The sample was then dialyzed
against 50 mM Tris buffer, pH 7.4, and loaded to a
hydroxyapatite column (Pharmacia Biotech; 2.0 x 22 cm)
equilibrated with 1 mM NaCl. The column was washed with 1
mM NaCl (1 ml/min) until absorbance (280 nm) had returned
to baseline. The proteins were eluted using a linear gradient of
10–300 mM K+-phosphate buffer pH 7.4.
Four-milliliter fractions were collected, dialyzed against 10
mM PBS, pH 7.4, and 40 µl aliquots were tested for
IGFBP-4 protease activity. The protease-containing fractions were
pooled and concentrated to 5 ml for chromatofocusing. A column
(1.6 x 14 cm) was packed with Polybuffer 94 exchanger and
equilibrated with 25 mM Tris buffer, pH 8.3, according to
the manufacturer’s Instructions (Pharmacia Biotech). The sample was
loaded at 1 ml/min, and the proteins eluted with Polybuffer 74.
Western Immunoblotting
Between 1 and 5 µl of pooled column fraction were separated by
SDS-PAGE or gelatin zymography and transferred to nitrocellulose
membranes. The membranes were probed with the anti-MMP or
antidisintegrin polyclonal antisera using the ECL detection system
(Pierce Chemical, Rockford, IL) as described previously (24, 25).
[14C] Gelatinase assay
Gelatinase activity was determined using heat-denaturated
[14C] collagen (type I from rat skin) as substrate
prepared by acetylation with [14C]-labeled acetic
anhydride (Amersham, Buckinghamshire, UK) as described (30).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows that at least radiolabeled
IGFBP-4 and -5 are degraded by a pregnancy specific serum protease
in vitro into fragments of 17 and 10 kDa and 22 and 15 kDa,
respectively. Whereas recombinant, nonglycosylated [125I]
IGFBP-3 was also cleaved in the presence of pregnancy serum into
fragments of 22 and 17 kDa, a weak fragmentation of IGFBP-3 was also
observed when sera from eight nonpregnant women were examined. From
preliminary experiments, it appears that the pregnancy serum mediated
proteolysis of recombinant nonglycosylated IGFBP-3 is faster than the
recombinant glycosylated IGFBP-3.
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A). A second
doublet fragment of 10–12 kDa was generated by incubation with all
protease-containing fractions except the hydroxyapatite II fractions,
which preferentially formed the 12-kDa band.
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). Pool I displayed a major band
of activity with an estimated size of 50 kDa and after longer
incubation a second active band at 87 kDa. In gel filtration, pool II
and HA fractions I and II the 50-kDa form remained the dominant
species. These data indicate that parts of the 50-kDa gelatinase
activity in pregnancy serum either aggregate to higher molecular mass
forms or are complexed with other proteins eluting in the pool I
fractions. The HA pool II fraction was further analyzed by chromatofocusing (CF). IGFBP-4 protease activity and the 50-kDa gelatinase activity were coeluted between pH 8.1 and 6.3 coinciding in fractions with highest activity at pH 7.3 (not shown). To characterize the 50-kDa gelatinase activity, the concentrated protease-containing CF fraction was examined for reactivity with antibodies against different MMPs. Whereas in the CF fraction MMP-2 (66-kDa active form) and weak MMP-9 immunoreactivity (84-kDa active form) was detected in Western blots, no specific immunoreaction with a 50-kDa protein was detected with anti-MMP-1, -2, -3, -9, or -13 antibodies (not shown).
Two different approaches were used to measure the capability of the
50-kDa gelatinase to cleave IGFBP-4. In the first approach two aliquots
of the CF fraction were separated in gelatin-containing polyacrylamide
gels followed by removal of SDS and incubation. One part of the gel was
stained with Coomassie Blue to detect the proteolytic area, whereas
from the other nonstained part of the gel, bands were excised
corresponding to the 50-kDa gelatinase-active and to unspecific zones.
The gel pieces were then incubated with [125I] IGFBP-4
for 18 and 24 h at 37 C. Following centrifugation, the
supernatants were solubilized and analyzed by SDS-PAGE and
autoradiography. Gel pieces containing 50-kDa gelatinase activity
cleaved IGFBP-4 forming 17- to 18-kDa and 10-kDa fragments, but
unspecific gel pieces did not mediate IGFBP-4 proteolysis (Fig. 3). When concentrated CF fractions were
incubated with [125I] IGFBP-4, proteolytic fragments of
17 and 12 kDa were formed.
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).
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). Serine
protease inhibitors like PMSF (10 mM) in combination with
soybean trypsin inhibitor (15 µg/ml) did not affect the gelatinase or
the 50-kDa IGFBP-5 protease activity but completely inhibited the
30-kDa IGFBP protease. These data indicate that more than one IGFBP-3
or -5 protease is present in pregnancy serum. [125I]
IGFBP-4 zymography showed a predominant 50-kDa band in pool II and CF
fractions, suggesting that the 50-kDa gelatinase was similar or
identical to the IGFBP-4 protease (Fig. 4), which also contributes to
IGFBP-3 and-5 degradation.
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). IGFBP protease activities were weakly
(12–23%) or not at all inhibited by tissue inhibitor of matrix
metalloproteinase-1 or -2 (TIMP-1, -2; 10 µg/ml), respectively, but
were completely blocked by the metalloprotease inhibitor TAPI at 0.1
mM (Fig. 7). The 50%
inhibitory concentration (IC50) in the IGFBP-4 protease
solution assay was about 3.4 µM. Furthermore, when the CF
fraction was examined for [14C] gelatinase activity, in
the presence of 1,10-phenanthroline less than 2% of the uninhibited
activity was measured. These results suggest that the CF purified
activity contains only a cation-dependent metalloproteinase that shows
biochemical properties of metalloproteases of the disintegrin
family.
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), and
analysis of the corresponding immunoblot revealed
disintegrin-immunoreactivity at the identical position (Fig. 8B)
confirming the 50-kDa bands as disintegrin-containing protease. When
aliquots of the pool II and CF fraction were solubilized under
denaturating conditions and processed by SDS-PAGE and antidisintegrin
immunoblotting, two immunoreactive bands of 50 kDa and 28 to 30 kDa
were detected in pool II and a single immunoreactive band of 28 to 30
kDa was found in CF fractions (Fig. 8C). As positive control, the
purified 62-kDa MADM (25) was analyzed on the same gel. The data
indicate that the 50-kDa disintegrin-protease appears not to be a
monomeric enzyme that dissociates under denaturating conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our previous filtration experiments, IGFBP protease activity in pregnancy serum was completely retained by a 100-kDa cut-off membrane filter (14). When the serum was fractionated by ammonium sulfate and subjected to S 400-gel filtration, IGFBP protease activity was found in two fractions, one with an approximate size of 600 kDa and a second with estimates of approximately 50–100 kDa. Because both IGFBP protease pools contain the 50-kDa gelatinase activity, it appears that in unfractionated serum the majority of the 50-kDa gelatinase may aggregate or become associated with other proteins forming the higher molecular mass protease complex. This complex persisted intact under conditions of [125I] IGFBP zymography and did not enter the gel. In hydroxyapatite chromatography and chromatofocusing fractions derived from the 50- to 100-kDa serum pool II fraction that contained IGFBP-4 protease activity, the predominant IGFBP-3, -4, and -5 protease activity migrated at 50 kDa. On the other hand, the analysis of unfractionated pregnancy serum and of fractions derived from serum pool II by IGFBP zymography, demonstrated the presence of additional IGFBP proteases with Mr estimates of 79, 30, and 22 kDa. Although our inhibitor studies demonstrated the susceptibility of the 30-kDa IGFBP protease to serine protease inhibitors, the identity of the three proteases is unknown. However, it appears that they preferentially cleave IGFBP-5 and -3 but not IGFBP-4.
Results obtained with the 50-kDa metalloprotease excised from the
gelatin gel indicated that this enzyme might be closely related or
identical to the 50-kDa IGFBP protease. Western immunoblot analysis of
the chromatofocusing fraction, which is enriched in the 50-kDa
gelatinase activity but free of 79, 30, and 22 kDa IGFBP proteases
showed no specific reactivity with different MMP antibodies. On the
other hand, whereas TIMP-1 and -2 showed either weak or no inhibition
of IGFBP proteolytic activity, a synthetic hydroxamic acid-based
inhibitor of metalloproteases (TAPI) completely prevented the
proteolysis of IGFBP-4 and -5. TAPI has been reported to be a potent
inhibitor of tumor necrosis factor-
converting enzyme (TACE),
whereas TIMPs had no effects (19, 31, 32). Recently TACE has been
purified, cloned and identified as a new member of disintegrin
metalloproteases (33, 34), which are proposed to be involved in
cell-cell interaction (35). Although the majority of these disintegrin
metalloproteases are integral membrane proteins, also soluble members
of this protease family have been described (36). It has also been
established that disintegrin metalloproteases degrade collagen and
gelatin (37). Similarities in biochemical properties and the reactivity
with disintegrin domain-specific antibodies support the concept that
the 50-kDa IGFBP metalloprotease described in our present study
represents a new soluble member of this metalloprotease family.
It has been reported that MMPs with molecular mass estimates of 52- to 72-kDa function as IGFBP-3 and -5 proteases both in vitro and in vivo (27, 29). Furthermore, studies with TIMP-1 and with neutralizing antibodies have suggested that MMP-1 and -3 contribute to IGFBP-3 proteolysis by rat pregnancy serum (15). Media from osteoblasts and fibroblasts contain additional 97- and 92-kDa IGFBP-5 protease activities with characteristics of serine proteases, which constitute the predominant IGFBP-5 protease in media from fibroblasts (27, 38). Whereas the serine type IGFBP protease(s) contributing to the circulating IGFBP protease activity during pregnancy remain to be identified, the present study provides evidence that a 30-kDa protease is involved. In seminal plasma and in media of MG63 osteoblasts, the serine proteases prostate-specific antigen (PSA) and plasmin, respectively, have been reported to function as IGFBP proteases (39, 40, 41). However, results of a recent study have shown that plasminogen detected in a 70- to 90-kDa pregnancy serum fraction seems not to be related to the pregnancy-associated increases in IGFBP-3 protease activity (42) but may be involved in IGFBP-3 proteolysis in nonpregnancy serum observed in this study.
The origin of IGFBP proteases in pregnancy serum has not been determined. Analyzing human fetal serum from 19- to 24-week of gestation, Bang et al. (43) failed to detect increased IGFBP protease activity suggesting that the pregnancy-induced protease is produced by maternal tissues. Indeed, in situ hybridization and immunohistochemistry have revealed a close spatial association of different MMPs in cells of the amnion, decidua, and chorionic villi at all stages of pregnancy (44). The contribution of these cells of human placenta to the 50-kDa disintegrin-metalloprotease and other protease activities responsible for IGFBP proteolysis during pregnancy remains to be determined.
The substrate specificity of the 50-kDa IGFBP metalloprotease in pregnancy serum for connective tissue matrix molecules is unknown. Because matrix turnover and tissue growth occurs during pregnancy, the 50-kDa metalloprotease activity may be involved in processes during trophoblast invasion, growth, and remodeling of the placenta. The metalloprotease-mediated proteolysis of IGFBPs, which reduces their affinity for IGFs, may provide an additional mechanism to increase IGF availability required for placental growth.
In conclusion, the present study demonstrates that a novel 50-kDa disintegrin metalloprotease functions as an IGFBP-3, -4, and -5 protease that may contribute to the increase in free IGF I concentrations in pregnancy serum (11). The functional interaction of the 50-kDa metalloprotease with serine IGFBP proteases and its role in the greater than or equal to 600-kDa IGFBP protease complex during pregnancy as well as their significance in pathological processes still remains elusive.
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Acknowledgments |
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Footnotes |
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2 Supported by the Association for International Cancer
Research.
Received August 4, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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precursor by metalloproteinases. Nature 370:555–557[CrossRef][Medline]
(TNF-)
production suggest the major TNF- converting enzyme is not an MMP.
Biochem Biophys Res Commun 225:400–405[CrossRef][Medline]
from cells. Nature 385:729–733[CrossRef][Medline]
. Nature 385:733–736[CrossRef][Medline]
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