From the
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706,
Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin 53706,
FibroGen Inc., South San Francisco, California 94080
Received for publication, January 23, 2003
, and in revised form, March 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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There are indications that ADAMTS proteases play a broad range of roles in development, reproduction, disease, and homeostasis. Aside from the role of ADAMTS-2 in biosynthetic processing of procollagens I and II, the phenotype of Adamts2-null mice suggests a role in male fertility as well (31). ADAMTS-1, -4, and -5/11 appear to constitute a subset of highly homologous ADAMTS family members with aggrecanase activity, important to homeostasis of cartilage and etiology of the arthritides (32, 33, 34). ADAMTS-1 was originally identified as a gene product induced by inflammation and associated with cachexia (26). The phenotype of Adamts1-null mice also suggests roles for ADAMTS-1 in growth, organogenesis, and female fertility (35), whereas both ADAMTS-1 and ADAMTS-8 have been shown to have anti-angiogenic activity (36). Roles for ADAMTS-like proteins in fertility and organogenesis in a broad spectrum of species are suggested by the finding that the gon-1 gene, which encodes an ADAMTS-like product, is essential for gonadal morphogenesis in Caenorhabditis elegans (37). Mutations in the gene for ADAMTS-13 have been shown to be causal for thrombotic thrombocytopenic purpura, perhaps due to its demonstrated ability to process von Willebrand factor, showing ADAMTS-13 to have an important role in human vascular homeostasis (38).
Despite the seeming importance of ADAMTS proteases in many biological processes, there has been little characterization of the regulation of their expression and activity. In a previous study of the regulation of procollagen C-proteinase activity and expression of BMP-1 and mTLD, by transforming growth factor-1 (TGF-
1), we noted elevation of both pCP and pNPI activities in MG-63 osteosarcoma cells treated with TGF-
1 (39). In the present study, we show that TGF-
1 induces expression of ADAMTS-2 mRNA, apparently at the transcriptional level, resulting in expression of fully processed and active 132-kDa ADAMTS-2 protein from MG-63 cells. Processing of the ADAMTS-2 pro-domain is shown to be via a two-step process involving sequential cleavage by furin-like proprotein convertases at two consensus sites. Surprisingly, purified ADAMTS-2 protein is shown to have pNP activity not only against procollagens I and II but against procollagen III as well, whereas processing of the procollagen III N-propeptide is shown to be decreased in EDSVIIC fibroblast cultures. These latter two findings indicate that the distinction that has been made between pNPI and pNPIII is a false one. Implications of the various data are discussed.
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EXPERIMENTAL PROCEDURES |
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Poly(A+) RNA was prepared from cultured MG-63 human osteosarcoma cells as described previously (39). Unless otherwise noted in the text, TGF-1 treatment of cells harvested for RNA was at 2 ng/ml for 24 h. To test stability of ADAMTS-2 mRNA, just confluent MG-63 cells, untreated or treated with TGF-
1 as described above, were then treated with 10 µg/ml actinomycin D for varying times in Dulbecco's modified Eagle's medium (DMEM) containing 0.1% fetal bovine serum (FBS) containing or lacking 2 ng/ml TGF-
. Stability was determined by RNase protection assays. RNase protection and Northern blot assays were performed as described by Lee et al. (39).
Production of Recombinant ProteinFor expression of recombinant ADAMTS-2 in 293-EBNA cells, a human ADAMTS-2 cDNA insert extending from nts 112 to 3633 of the published sequence (21), corresponding to the full-length protein minus signal peptide sequences and with PCR addition of sequences encoding DYKDDDDK-Stop to the 3'-end of the insert, was ligated between the NheI and BamHI sites of episomal expression vector pCEP-Pu/BM40s (40). Coding was thus for full-length human ADAMTS-2 differing from the native protein only in replacement of the native signal peptide by BM40 signal peptide sequences, to enhance secretion, and by addition of a C-terminal FLAG tag. Experiments in Fig. 7 were performed with recombinant ADAMTS-2 from a clonal line of transfected 293 cells constitutively expressing ADAMTS-2 and prepared toward the end of the study. To prepare this clonal line, in-frame BM40s/ADAMTS-2/FLAG coding sequences were excised from pCEP-Pu with AflII and BamHI and inserted between corresponding sites of expression vector pcDNA3.1 (Invitrogen), which was transfected into 293 cells. 293 cells were grown in growth medium consisting of DMEM supplemented with 1 mM L-glutamine and 10% FBS, whereas 293-EBNA cells were grown in the same growth medium supplemented with 250 µg/ml G418 (Invitrogen). Transfection of both types of cells was at 90% confluence with 10 µg of expression vector per 100-mm culture dish, using LipofectAMINE (Invitrogen). After 36 h, transfected 293-EBNA cells were selected, and surviving cells were allowed to grow to confluent mass cultures in growth medium containing 5 µg/ml puromycin (Sigma) and 250 µg/ml G418. After 72 h, transfected 293 cells were selected in growth medium containing 500 µg/ml G418 (Invitrogen), and surviving colonies of cells were ring cloned. Culture media of 36 clones were analyzed by immunoblot, and the clone expressing the highest levels of ADAMTS-2 was used for the studies of Fig. 7.
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To prepare purified recombinant ADAMTS-2, mass cultures of transfected 293-EBNA cells were grown to confluence, washed twice with phosphate-buffered saline (PBS), and switched to serum-free DMEM containing 40 µg/ml soybean trypsin inhibitor (Sigma) with or without 5 µg/ml soluble heparin (Sigma). Conditioned media were harvested 24 h later, and protease inhibitors were added to final concentrations of 0.4 mM phenylmethylsulfonyl fluoride and 10 µg/ml leupeptin. Harvested media were centrifuged to remove debris, and supernatants were stored at -70 °C. FLAG-tagged ADAMTS-2 in media was bound to 1 ml of FLAG M2 matrix, and the matrix was washed with 10 ml of binding buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4), using the supplier's protocols (Sigma). ADAMTS-2 was eluted with 1 mg/ml FLAG peptide (Sigma) in binding buffer, and fractions were collected and analyzed by Western blot. Purified full-length recombinant human ADAMTS-1, ADAMTS-4, and ADAMTS-5 were provided by Wyeth Research, Cambridge, MA.
Preparation of Cell Lysate and ECMFor the experiments of Fig. 7, confluent cells were rinsed twice with PBS, detached in PBS containing 5 mM EDTA, pelleted by centrifugation, and lysed in 1x SDS-PAGE loading buffer. ECM remaining on dishes was rinsed twice with PBS and then scraped into 1x SDS-PAGE loading buffer. Treatment of cells in serum-free growth media with 20 µM of furin inhibitor decanoyl-RVKR-chloromethyl ketone (Bachem) was as described previously (41).
Induction of Secreted ADAMTS-2 by TGF-MG-63 cells at 70% confluence were trypsinized and replated in low salt, low cysteine, low sulfate DMEM containing 10% dialyzed FBS (Hyclone), 4 mM L-glutamine, and 30 mM sodium chlorate. After 48 h, cells were trypsinized again and replated in the same type of chlorate-containing media. The next day, cells were switched to serum-free, chlorate-containing DMEM with 2 ng/ml TGF-
1 (R & D Systems) and 40 µg/ml soybean trypsin inhibitor, and conditioned media were harvested 48 h later. Cell layers were then treated 2 h with 0.006 IU/ml heparitinase (Seikagaku America) in serum-free DMEM at 37 °C. These media were removed and pooled with conditioned media from the previous step, and pooled media were incubated 20 h with heparin-Sepharose beads (Amersham Biosciences) at 4 °C. Beads were centrifuged 1 min at 1,000 x g at 4 °C and washed three times with PBS, and protein was eluted by boiling beads for 5 min in 30 µl of 4x SDS-PAGE loading buffer.
ImmunoblotsSamples subjected to SDS-PAGE were transferred to Immobilon-P membranes (Millipore) as described (39). Blots were blocked 1 h with 2% bovine serum albumin in T-PBS (PBS, 0.05% Tween 20) and incubated overnight with primary antibody diluted 1:5000 in the same solution. Blots were washed with T-PBS and blocked again by 2% bovine serum albumin in T-PBS, followed by incubation with 1:5000 diluted secondary antibody. After washing 6 times in T-PBS, blots were incubated 4 min in SuperSignal West Pico substrate (Pierce) and exposed to film. The blot in the right panel of Fig. 9B was washed overnight in T-PBS after transfer, blocked 1 h with 3% bovine serum albumin in T-PBS, incubated overnight with primary antibody diluted 1:2000 in the same solution and, the next day, washed as above. Antibodies 1429, 1435, 1374, and 1442, raised against peptides whose sequences are presented in Fig. 3, were prepared and affinity-purified using techniques described previously (39). Peptide antibodies directed against sequences in the pro-1(I) C-telopeptide (LF-67), pro-
1(III) C-propeptide (LF-69), and N-telopeptide (LF-71) have been described previously (42) and were the kind gifts of Dr. Larry Fisher (NIDCR, National Institutes of Health, Bethesda). Although antibody LF-71 was raised against pro-
1(III) N-telopeptide sequences, it appears to recognize only forms that retain the N-propeptide (i.e. pro-
1(III) and pN
(III) chains) and does not recognize forms from which the N-propeptide has been removed (i.e. pC
1(III) and mature
1(III) chains), perhaps due to conformational changes affecting availability of epitopes3 (42).
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Proteinase AssaysADAMTS-2, prepared and purified as above, and/or mTLL-1, prepared and purified as described previously (7), were incubated for 20 h with 210 ng of types IIII procollagen in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2 at 37 °C. Reactions were stopped by adding 10x SDS-PAGE loading buffer and boiling 5 min. Procollagen substrates, radiolabeled with [2,3-3H]proline, were prepared and purified essentially as described previously (7). Samples were subjected to SDS-PAGE on 5% acrylamide gels, which were treated with EN3HANCE (DuPont) and exposed to film.
Amino Acid Sequence AnalysisProteins on 5% acrylamide SDS-PAGE gels were transferred, as above, to Sequi-Blot polyvinylidene difluoride membranes (Bio-Rad). Bands were excised, and N-terminal amino acid sequences were determined by automated Edman degradation at the Harvard University Microchemistry Facility.
Enzymatic Deglycosylation AssaysEnzymatic deglycosylation was performed using the Glycopro deglycosylation kit (ProZyme). Briefly, purified protein in 50 mM sodium phosphate buffer, pH 7.0, was denatured in 0.08% SDS, 40 mM 2-mercaptoethanol for 5 min at 100 °C. Triton X-100 was added to the cooled sample to chelate SDS, which was then incubated 3 h at 37 °C with various combinations of glycosidases, as described in the text, and final concentrations of 0.1 unit/µl PNGase F, 0.1 unit/µl sialidase A, 0.025 milliunit/µl endo-O-glycosidase, 0.06 milliunit/µl -14-galactosidase, and 0.8 milliunit/µl glucosaminidase. Reactions were stopped with 10x SDS-PAGE loading buffer and boiling for 5 min.
Culturing of EDSVIIC and Normal Human Dermal Fibroblasts Human dermal fibroblasts (kind gifts of Dr. Peter H. Byers, University of Washington, Seattle) were grown in the presence of dextran sulfate to augment efficiency of enzymatic conversion of procollagens to collagens, essentially as indicated in Smith et al. (25). Cells were grown to 80% confluence and treated overnight in growth medium containing 50 µg/ml ascorbate. The next day, cells were switched to serum-free medium containing 50 µg/ml ascorbate, 40 µg/ml soybean trypsin inhibitor, and 0.01% dextran sulfate (Amersham Biosciences). After 20 h, cell layers were rinsed twice with PBS and scraped into hot SDS-PAGE loading buffer.
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RESULTS |
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In contrast to MG-63 cells, we had previously been unable to detect pNPI activity in cultured human keratinocytes, even upon treatment with TGF-1 (39). We were thus interested in determining whether this lack of activity was due to a lack of expression of ADAMTS-2 in keratinocytes or, as is the case for BMP-1 and mTLD of pCP (39), whether pNPI might be secreted by keratinocytes in an inactive form in response to TGF-
1. To address this issue, an RNase protection assay was performed on RNA from human keratinocytes cultured in the presence or absence of TGF-
1. As can be seen (Fig. 1B), no ADAMTS-2 mRNA is detected by the highly sensitive RNase protection assay in cultures of nonfibrogenic keratinocytes, even after treatment with TGF-
1.
Incubations of MG-63 cell cultures with TGF-1 concentrations, ranging from 0.01 to 10 ng/ml, showed induction of ADAMTS-2 mRNA to be dose-dependent (Fig. 2A), with maximal induction at
1.0 ng/ml. Induction at higher concentrations was somewhat less, possibly due to toxic effects of TGF-
1 at higher concentrations (43). Kinetics of induction of ADMTS-2 mRNA were examined by incubating MG-63 cultures in 2 ng/ml TGF-
1 for varying times (Fig. 2B). As can be seen (Fig. 2B), noticeable induction first occurs at
6 h, with maximal induction at
24 h post-treatment. Thus, kinetics for induction of ADAMTS-2 mRNA overlap those for induction of procollagen C-proteinases BMP-1 and mTLD, in which initial and maximal increases were at 12 and 24 h post-induction, respectively (39), and overlap the kinetics of induction of RNAs for various extracellular matrix proteins, as such induction generally occurs within 35 h of TGF-
induction of a variety of cell types (44). Treatment of confluent MG-63 cultures with cycloheximide prior to TGF-
1 treatment inhibited induction of ADAMTS-2 (Fig. 2C). Thus, as with BMP-1 and mTLD mRNAs (39), induction of ADAMTS-2 mRNA by TGF-
1 is indirect and requires prior protein synthesis. To obtain insights into roles that transcriptional and post-transcriptional processes might have on increases in steady state levels of ADAMTS-2 mRNA in TGF-
1-treated cells, just-confluent MG-63 cells, untreated with TGF-
or pretreated with 2 ng/ml TGF-
1 for 24 h, were incubated with 10 µg/ml of the transcriptional inhibitor actinomycin D for varying times (in media containing or lacking 2 ng/ml TGF-
1), followed by determination of ADAMTS-2 mRNA levels by RNase protection assays (Fig. 2D). Although levels of ADAMTS-2 RNA were higher at all time points in TGF-
1-treated cells, the rate of decrease in ADAMTS-2 RNA levels over time was approximately the same for treated and untreated cells. This similarity in stabilities of ADAMTS-2 mRNA in TGF-
1-treated and -untreated cells is consistent with the probability that increased steady state levels of ADAMTS-2 mRNA induced by TGF-
1 in MG-63 cells are due to transcriptional effects, rather than to post-transcriptional enhancement of RNA stability.
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Levels of Secreted ADAMTS-2 Protein Are Up-regulated by TGF-1 Treatment of MG-63 CellsTo determine whether TGF-
1 induction of higher steady state levels of ADAMTS-2 mRNA is paralleled by increased secretion of ADAMTS-2 protein, we attempted to detect endogenous ADAMTS-2 protein in media of TGF-
1-treated or -untreated MG-63 cells. Toward this end, we produced four polyclonal antibodies directed against four different peptides corresponding to different regions of ADAMTS-2 (Fig. 3). Only one of the four antibodies, antibody 1435, directed against sequences within the protease domain, was able to detect endogenous ADAMTS-2 produced by MG-63 cell cultures. Moreover, as seen in Fig. 4A, 1st and 2nd lanes, antibody 1435 detected endogenous ADAMTS-2 protein only in media of cells treated with TGF-
1. The size of the induced band was
132-kDa. This is larger than the molecular weight of 107 previously estimated for active ADAMTS-2 isolated from fetal calf skin, based on mobility on SDS-PAGE gels (20), but is similar to the 131,697 molecular weight predicted by the human cDNA sequence (21) for full-length ADAMTS-2 in which only the signal peptide has been removed. Thus, this result left open the possibility that the major form of ADAMTS-2 secreted by MG-63 cells was the inactive precursor form. It should be noted that the
132-kDa band recognized by antibody 1435 is unlikely to represent ADAMTS-3 or ADAMTS-14, as these proteins, although closely related to ADAMTS-2, are considerably diverged in sequence from ADAMTS-2 in the region corresponding to the peptide against which antibody 1435 was raised (Fig. 3).
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The Nature of Secreted ADAMTS-2To obtain further insights into the nature of secreted ADAMTS-2, recombinant ADAMTS-2 with a C-terminal FLAG tag was produced in 293-EBNA human embryonic kidney cells that had been transfected with human ADAMTS-2 cDNA in an expression vector. As can be seen (Fig. 4B), anti-FLAG antibodies recognize a doublet of 132- and
118-kDa bands in media of transfected, but not untransfected, cells. As reported for other ADAMTS proteins (30), these bands were observed only in media of transfected cells cultured in the presence of heparin, which is thought to compete for ADAMTS proteins bound to sulfated glycosaminoglycans in the cell layer. As can be seen in Fig. 4C, antibodies 1435 and 1374, directed against sequences in the ADAMTS-2 protease domain, and antibody 1442, directed against the C-terminal domain (see also Fig. 3), all recognize the 132-kDa band. In contrast, antibody 1429, directed against putative proregion sequences, does not, suggesting the 132-kDa form to represent ADAMTS-2 in which at least a portion of the proregion has been removed. Interestingly, antibodies 1374 and 1442 also recognize the 118-kDa band (Fig. 4C), whereas antibody 1435 does not. Because antibody 1435 recognizes sequences in the more N-terminal portion of the protease domain than does antibody 1374, this suggests that the 118-kDa band represents ADAMTS-2 in which some sequences from the N-terminal portion of the protease domain have been removed. Antibody 1435 recognizes an
95-kDa band not recognized by anti-FLAG antibodies (e.g. compare Fig. 4, B and C) and which, therefore, is missing ADAMTS-2 C-terminal sequences. It is not clear whether this band is not recognized by antibodies 1374 and 1442 or whether it is not apparent on Western blots using these antibodies because they produce a lower signal than antibody 1435 and because the 95-kDa form is a relatively minor species. The minor 95-kDa form was not characterized further.
Importantly, when recombinant ADAMTS-2 and samples from media of MG-63 cells treated with TGF-1 are run in adjacent SDS-PAGE lanes and analyzed by Western blot with antibody 1435, the 132-kDa forms from the two samples comigrate exactly (Fig. 4A). Thus, the same 132-kDa form likely represents the predominant form of secreted ADAMTS-2 in both the recombinant system and in TGF-
1-treated MG-63 cells.
To determine whether the ADAMTS-2 produced in the recombinant system was an active procollagen N-proteinase, purified recombinant ADAMTS-2 was incubated with human type II procollagen. As can be seen (Fig. 5), the recombinant ADAMTS-2 cleaves procollagen II to produce pC1(II) chains and together with the procollagen C-proteinase mTLL-1 cleaves procollagen II to produce mature
1(II) chains, with apparent absence of nonspecific cleavages. Thus, the ADAMTS-2 produced in the recombinant system is an active procollagen N-proteinase, although it was not clear from these data whether this activity might be furnished by the 132-, 118-, or 95-kDa form of the protein.
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Human ADAMTS-2 has eight potential sites for Asn-linked glycosylation (Ref. 21 and Fig. 3). Thus, a significant proportion of the apparent molecular masses estimated for various ADAMTS-2 forms by SDS-PAGE may be due to carbohydrate side chains. To determine the degree and nature of glycosylation of ADAMTS-2, the recombinant protein was treated with the enzymes peptide N-glycosidase F (PNGase F), endo-O-glycosidase, sialidase A, -14-galactosidase, and glucosaminidase. Treatment with a mixture of all five enzymes increased the mobilities of the
132- and
118-kDa forms to
110 and
90-kDa, respectively, upon SDS-PAGE (Fig. 6, lane 5). The 110-kDa form is thus similar in size to the 107,450 molecular weight predicted by the cDNA coding sequence for ADAMTS-2 proteolytically processed at the more C-terminal of two potential recognition sequences for cleavage by furin-like proprotein convertases (see Fig. 3).
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To better elucidate the nature of ADAMTS-2 glycosylation, the recombinant protein was separately incubated with each of the various enzymes from the mixture. PNGase F, which removes virtually all N-linked oligosaccharides from glycoproteins, produced a marked shift in mobility for both the 132- and 118-kDa forms, consistent with glycosylation of at least some of the 8 potential sites (Fig. 6, lane 2). Sialidase A (sialidase from Arthrobacter ureafaciens) removes mono-, di-, and trisialyl residues, the most commonly occurring modifications, from Gal(13)GalNAc O-linked cores. Treatment of ADAMTS-2 with both PNGase F and sialidase A produced a further increase in electrophoretic mobility, compared with samples treated with PNGase F alone (Fig. 6, lanes 2 and 3), thus indicating the presence of sialylated O-linked oligosaccharides. Endo-O-glycosidase (endo-
-N-acetylgalactosaminidase) removes Gal
(1, 2, 3)GalNAc cores from serine and threonine residues, but only after modifying monosaccharides have first been removed by exoglycosidases. Treatment of ADAMTS-2 with PNGase F, sialidase A, and endo-O-glycosidase did not clearly produce an additional increase in electrophoretic mobility, compared with treatment with PNGase F and sialidase A alone (Fig. 6, lanes 3 and 4), suggesting the absence of appreciable quantities of simple sialylated O-linked Gal
(13)GalNAc O- linked cores. In contrast,
-14-galactosidase and glucosaminidase, which remove less common modifying
-1,4-linked galactose and
-16-linked N-acetylglucosamine residues, respectively, from Gal
(13)GalNAc O-linked cores, were found to enable endo-O-glycosidase to induce a detectable mobility shift in ADAMTS-2 (Fig. 6, lane 5). Thus, ADAMTS-2 appears to be decorated with O-linked Gal
(13)GalNAc cores, which are in turn decorated with modifying
-1,4-linked galactose and
-16-linked N-acetylglucosamine residues.
As noted above, the 110-kDa form of recombinant ADAMTS-2 obtained from deglycosylation using all five glycosidases is similar in size to the
107 molecular weight predicted by the sequence for ADAMTS-2 processed at the more C-terminal of two potential furin cleavage sites. To determine whether the 110-kDa form might indeed result from cleavage at such a site, we incubated transfected 293 cells in the presence of the highly specific furin inhibitor decanoyl-RVKR-chloromethyl ketone. As can be seen (Fig. 7A), in the presence of furin inhibitor, cells secrete into medium a
150-kDa form as the predominant ADAMTS-2 species, in addition to the 132- and 118-kDa forms.
To characterize further ADAMTS-2 biosynthesis, we compared ADAMTS-2 forms detected in medium with those detected in cell layer and cell layer-associated ECM, using both anti-FLAG antibody and antibody 1435. Both antibodies detected the 132- and 118-kDa forms in medium, cell layers, and ECM (Fig. 7B). Interestingly, although neither antibody detected the 150-kDa form in medium, cell layer, or ECM, in the absence of furin inhibitor, both antibodies detected a cell layer-associated 141-kDa form of ADAMTS-2, not found in either ECM or medium (Fig. 7B). Upon treatment with decanoyl-RVKR-chloromethyl ketone, medium, cell layer, and ECM also contained the 150-kDa form (Fig. 7C). Antibody 1429, which is directed against putative proregion sequences (Fig. 3) and which does not recognize the 132- and 118-kDa secreted forms (Figs. 4C and 7C), recognizes the 150-kDa form in medium, cell layer, and ECM of cultures incubated with the furin inhibitor (Fig. 7C). This is consistent with the identity of the 150-kDa species as a precursor form of ADAMTS-2. In addition, antibody 1429 recognizes the 141-kDa form of ADAMTS-2 found in cell layers of cultures either treated or untreated with furin inhibitor (Fig. 7C). As antibody 1429 is directed against the prodomain sequences located between the two potential furin cleavage sites (Fig. 3), these results suggest the 141-kDa form to represent a processing intermediate in which cleavage has occurred at a site N-terminal to the more C-terminal furin site. The presence of the 141-kDa form in cell layers in the absence of furin inhibitor and absence of the 150-kDa form suggest that cleavage at the more N-terminal site is more rapid than that occurring at the more C-terminal site, thus allowing accumulation of the 141-kDa processing intermediate. Persistence of the 141- and 132-kDa forms in the presence of furin inhibitor probably reflects incomplete inhibition of cleavage at the two furin consensus sites, whereas persistence of the 118-kDa form probably reflects cleavage by non-furin-like proteases (see below). The presence of the 141-kDa form in cell layers, but not in ECM or media, indicates that it is likely an exclusively intracellular and/or cell-associated form.
Sufficient 132- and 118-kDa ADAMTS-2 was isolated to enable automated Edman degradation and determination of the N-terminal amino acid sequences of each. As expected from the similarity in size between the 110-kDa deglycosylated version of the 132-kDa form and the molecular weight of 107 predicted by the sequence for ADAMTS-2 processed at the more C-terminal furin site, the N-terminal sequence of the 132-kDa form was HAADDDYNIE, showing cleavage occurred between residues Arg259 and His260 of the published sequence, at the more C-terminal consensus furin recognition sequence 256RARR259 (Fig. 3). N-terminal sequences for the 118-kDa band showed it to be heterogeneous and to represent two protein species, one of which had been processed between residues GFSS402 and 403AFVV and the other of which had been processed between residues SYDC465 and 466LLDD. Both of these cleavages occurred within the protease domain (Fig. 3), one immediately N-terminal to and the other C-terminal to the Zn2+-binding active site. Both of these cleavages occur C-terminal to the sequences recognized by antibody 1435 (Fig. 3), explaining the inability of this antibody to detect the 118-kDa form.
ADAMTS-2 Has Both pNPI and pNPIII ActivitiesIn the course of characterizing recombinant ADAMTS-2, it was tested for pNP activity against procollagens IIII. Surprisingly, ADAMTS-2 was found to have pNP activity not only against procollagens I and II (pNPI activity) but against procollagen III (pNPIII) as well (Fig. 8). Moreover, incubating varying amounts of ADAMTS-2 with fixed amounts of the three procollagen types showed ADAMTS-2 to have similar levels of pNPI and pNPIII activity.
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If ADAMTS-2 is truly a pNPIII, then TGF- induction of ADAMTS-2 in MG-63 cells should be accompanied by induction of pNPIII activity. This appears to be the case, as evidenced by appearance of pC
1(III) chains in media of MG-63 cells treated, but not in media of those untreated, with TGF-
1 (Fig. 9A). As a final test of the identity of ADAMTS-2 as a pNPIII, processing of procollagen III was compared in control dermal fibroblasts and those derived from an individual with EDSVIIC. It has been shown that the effects of dermatosparaxis/EDSVIIC on type I procollagen processing are best observed in cell layers of fibroblasts cultured in the presence of neutral polymers (25, 45). Addition of the polymers, which appear to act via a volume exclusion mechanism, potentiates normal processing of procollagen to collagen and association of processed forms with cell layers, such that defects in processing are accentuated when mutant and control fibroblast cultures are compared (25, 45). As can be seen (Fig. 9B, left panel), whereas pro-
1(I) chains are almost entirely converted to mature
1(I) chains in the control fibroblast cell layer, conversion to
1(I) chains is impaired in the EDSVIIC culture, with accumulation of large amounts of incompletely processed pN
1(I) chains. Such results reflect loss of pNPI activity normally provided by ADAMTS-2, whereas residual pNPI activity is thought to be provided by another ADAMTS family member (46). When the same samples were analyzed on a separate Western blot using antibodies that recognize pro-
1(III) and pN
1(III), but not pC
1(III) or mature
1(III) chains, processing of procollagen III was seen to be impaired in the EDSVIIC culture, with accumulation of pN
1(III) chains in the EDSVIIC cell layer, and no evidence of such chains in the cell layer of the normal control. This result strongly supports an in vivo role for ADAMTS-2 as a pNPIII.
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DISCUSSION |
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In addition to demonstrating induction of ADAMTS-2 mRNA by TGF-1, data presented herein also indicate that TGF-
1 induces secretion of ADAMTS-2 protein. Moreover, this protein is identical in size to an
132-kDa form of recombinant ADAMTS-2 that appears to represent the mature active form of the protease, as the prodomain has been removed via cleavage by a furin-like protease at a consensus recognition site. Thus, in addition to increasing formation of ECM by inducing synthesis of matrix components, matrix-metalloproteinase inhibitors (44), lysyl oxidase (47), and pCPs such as BMP-1 and mTLD (39), TGF-
1 also appears to affect ECM formation by inducing production and secretion of the active form of ADAMTS-2.
ADAMTS-3 and -14 have greater homology to ADAMTS-2 than do any other ADAMTS family members, and the three proteases have been suggested to constitute a subfamily (46, 49). In addition, both ADAMTS-3 and -14 have been shown recently (46, 49) to have pNPI activity in vitro and have been suggested as possible sources for the residual pNPI activity observable in bone, tendon, cartilage, skin, and other tissues of EDSVIIC patients, dermatosparaxic cattle, and Adamts2-/- mice. Nevertheless, correlation in the present study between the level of induction of the 132-kDa band recognized by polyclonal antibody 1435 and the level of induction of ADAMTS-2 mRNA, the fact that the 132-kDa band is identical in size to mature ADAMTS-2, and the fact that ADAMTS-3 and -14 are considerably diverged in sequence from ADAMTS-2 in the region recognized by antibody 1435, all make it highly likely that the 132-kDa band induced by TGF-
1 corresponds to ADAMTS-2.
Previously, Colige et al. (46) have reported that levels of ADAMTS-14 RNA in human dermal fibroblasts are unaffected by TGF-, and they cite unpublished data as showing the same to be true for ADAMTS-2 and -3 RNA. It is not stated which type of TGF-
was employed in that study, although if TGF-
1 was used, then reasons for the different results obtained for ADAMTS-2 RNA in that study and in the present study are unclear. However, as dermal fibroblasts were used in the previous study and MG-63 cells are used here, these differences may suggest that TGF-
1 is capable of affecting ADAMTS-2 expression in some cell types but not in others.
A surprise in the present study was the finding that ADAMTS-2 has pNPIII activity, and that levels of pNPIII and pNPI activity appear similar in this single protease. This finding is contrary to accepted dogma based on various biochemical studies that have reported previously (14, 15, 16, 17, 18, 19) that the protease responsible for pNPI activity is unable to process procollagen III, whereas the protease responsible for pNPIII activity is unable to process procollagen I. In addition, processing of type III procollagen N-propeptides appears to occur more slowly than processing of type I procollagen N-propeptides in tissues and tissue culture systems (50), which has also suggested the operation of two different N-proteinases. Reasons for the difference between previous biochemical findings and those of the current study are unclear but may be related to use here of relatively large amounts of highly purified ADAMTS-2. In contrast, most earlier studies (16, 17, 18, 19) have employed tissue or cell culture isolates of relatively low purity, and such preparations may have contained contaminants (e.g. endogenous inhibitors and/or other proteases) that affected proteolytic activity. Other studies (14, 15) that have employed more highly purified preparations of pNPI from tissue or organ culture have nevertheless found this activity to be associated with large 500-kDa complexes containing several polypeptides, some of which might similarly have affected activity. Additional polypeptides, and/or differences in the accessibility of ADAMTS-2 to procollagens I and III, may also result in the different kinetics with which N-propeptides are processed from the two procollagen types in tissues and tissue culture systems.
Importantly, it should be noted that one of the earlier studies (19) cited above described a marked decrease in processing of the procollagen III N-propeptide in skin of dermatosparactic cattle. Thus, as dermatosparaxis is the bovine equivalent of EDSVIIC, this previous report provides genetic evidence that supports the evidence presented here that ADAMTS-2 is both a pNPI and a pNPIII, and that ADAMTS-2 plays an in vivo role as a pNPIII, such that defects in the ADAMTS2 gene lead to a defect in processing of procollagen III in EDSVIIC skin fibroblasts. These results suggest that the phenotypes of dermatosparaxis and EDSVIIC may arise from defects in the processing of both types I and III procollagen N-propeptides. As ADAMTS-3 and ADAMTS-14 have been postulated to provide residual pNPI activity found in dermatosparactic tissues (46, 49), one or both of these two proteinases may be capable of pNPIII activity, perhaps explaining the residual low level processing of procollagen III N-propeptides previously noted in dermatosparactic calf skin (19). It should be noted that pNPIII activity is not intrinsic to all ADAMTS family members, as ADAMTS-1, -4, and -5 were negative for pNPIII activity in our in vitro assays (data not shown).
Interestingly, Apte and colleagues (49) have recently shown ADAMTS-2 mRNA to be at dramatically higher levels in 7 days post-conception murine gastrulas than at later gestational times. This is despite the fact that mRNAs of the major fibrillar collagens are at relatively low levels at 7 days post-conception and are found at abundant levels only markedly later in development4 (51). This difference in temporal patterns of expression suggests that, in addition to its expanded role as both pNPI and pNPIII, ADAMTS-2 may also be involved in proteolytic processing of substrates other than the major fibrillar collagens. In this regard, it is of interest that the procollagen C-proteinases, each of which processes the C-propeptides of procollagens IIII, have been found to process a wide range of additional substrates involved in formation of the ECM and in other morphogenetic processes, such as BMP signaling (7, 44, 48, 52, 53, 54). The possibility of such multiple roles for ADAMTS-2, and perhaps for other ADAMTS family members as well, presents an interesting avenue for future research.
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FOOTNOTES |
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¶ Present address: College of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea.
|| Supported by National Institutes of Health Predoctoral Training Grant T32 GM07215 in Molecular Biosciences.
** Present address: AstraZeneca R & D Charnwood, Loughborough, Leics LE11 5RH, UK.
Present address: Graduate School of Biostudies, Kyoto University, Kyoto 60-8502, Japan.
¶¶ To whom correspondence should be addressed. Tel.: 608-262-4676; Fax: 608-262-6691; E-mail: dsgreens{at}facstaff.wisc.edu.
1 The abbreviations used are: ECM, extracellular matrix; N-propeptide, N-terminal propeptide; C-propeptide, C-terminal propeptide; pCP, procollagen C-proteinase; pNP, procollagen N-proteinase; EDSVIIC, Ehlers-Danlos Syndrome type VIIC; ADAMTS, a disintegrin and metalloproteinase with thrombospondin type I motifs; TGF-1, transforming growth factor-
1; BMP-1, bone morphogenetic protein-1; mTLD, mammalian Tolloid; mTLL-1, mammalian Tolloid-like 1; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PNGase F, peptide N-glycosidase F.
2 I. C. Scott, W. N. Pappano, and D. S. Greenspan, unpublished observations.
3 L. W. Fisher and P. H. Byers, personal communication.
4 B. M. Steiglitz and D. S. Greenspan, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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