ADAMTS-1 Protein Anchors at the Extracellular Matrix through the Thrombospondin Type I Motifs and Its Spacing Region*

Cellular disintegrin and metalloproteinases (ADAMs) are a family of genes with a sequence similar to those of snake venom metalloproteinases and disintegrins. The ADAMTS-1 gene encodes a new type of ADAM protein with respect to possessing the thrombospondin (TSP) type I motifs. Expression of the gene is induced in kidney and heart by in vivo administration of lipopolysaccharide, suggesting a possible role in the inflammatory reaction. In this study, we characterized the ADAMTS-1 gene product by using a transient expression system in COS-7 cells. We found that the precursor and processed forms of ADAMTS-1 were secreted from cells. Under normal growth conditions, little or none of both forms was detected in the cell culture medium, and instead the majority was found associated with the extracellular matrix (ECM). In addition, when cells were cultured in the presence of heparin, the mature form of ADAMTS-1 protein was detected in the cell culture medium, suggesting that binding of ADAMTS-1 to the ECM is mediated through sulfated glycosaminoglycans such as heparan sulfate. Analyses of deletion mutants of the ADAMTS-1 protein revealed that the spacer region as well as three TSP type I motifs in the carboxyl-terminal region of the ADAMTS-1 protein are important for a tight interaction with the ECM. These results suggest that the ADAMTS-1 is a unique ADAM family protein that anchors at the ECM.

Disintegrin and metalloproteinases (ADAMs) 1 represent a new family of gene products that show significant sequence similarity to snake venom metalloproteinase and disintegrin (1,2). So far, 18 -20 ADAM genes, including fertilin, epididymal apical protein I, cyritestin, meltrin, metalloproteinase-like/ disintegrin-like/cysteine-rich (MDC) protein, macrophage cellsurface antigen MS2, and metargidin, have been identified (3)(4)(5)(6)(7)(8). Typical ADAMs are cell surface proteins that consist of a pro-, a metalloprotease-like, a disintegrin-like, a cysteine-rich, an epidermal growth factor-like, a transmembrane, and a cy-toplasmic domain. Fertilin, the first ADAM described, has been implicated in integrin-mediated sperm-egg binding (3,9); meltrin has been shown to be required for myotube formation (6). They are thought to function in cell-cell interaction. However, a recently cloned gene encoding tumor necrosis factor ␣ (TNF-␣)-converting enzyme (TACE) has been shown to be a membrane type metalloproteinase that also belongs to the ADAM family (10,11). In Drosophila, an ADAM family gene, Kuzbanian has been demonstrated to play a role in the lateral inhibition during neurogenesis by cleavage of the extracellular domain of the transmembrane receptor Notch (12,13). These observations indicate that some ADAMs, possessing a zinc binding motif, function by processing cell surface proteins. Interestingly, it has been found that mammalian disintegrinmetalloprotease (MADM or ADAM10) is also able to process pro-TNF-␣ (14,15), whereas other ADAM members, MS2 and decysin, were identified as monocytic and dendritic cell-specific genes, respectively (7,16), suggesting that some ADAMs might be involved in host defense mechanisms.
ADAMTS-1 has been identified as a gene highly expressed in the colon 26 cachexigenic tumor in vivo (17). The mouse AD-AMTS-1 gene is mapped to chromosome 16, region C3-C5 (18). The amino-terminal region of ADAMTS-1 shows sequence similarity to both snake venom metalloproteinases, such as hemorrhagic toxins, and other ADAM family proteins such as fertilin, meltrin, and MS2, indicating that ADAMTS-1 belongs to the ADAM family. The amino-terminal region of ADAMTS-1 consists of a proprotein, a metalloproteinase, and a disintegrinlike domain. Typical ADAM family proteins are membraneanchored proteins that have a transmembrane region in the carboxyl-terminal region. In contrast, the ADAMTS-1 protein does not contain a transmembrane domain but possesses three thrombospondin (TSP) type I motifs, which are the conserved motifs in both thrombospondin 1 and 2 (19), at its carboxylterminal region. The analyses of the genomic structure of AD-AMTS-1 showed that it is markedly different from those of MDC and MS2 (20,21), suggesting that ADAMTS-1 may be a distant member of the ADAM family. Because in vivo administration of lipopolysaccharide significantly enhanced the expression of ADAMTS-1 mRNA in kidney and heart, it is presumed that ADAMTS-1 might be involved in the inflammatory reaction (17).
In this study, to elucidate further the role of ADAMTS-1 in inflammatory processes, we first investigated how the AD-AMTS-1 protein is secreted and delivered. We describe here that the majority of the ADAMTS-1 protein binds to the extracellular matrix (ECM) after being secreted by producing cells. In addition, analyses of deletion mutants revealed that the TSP type I motifs and the spacer region in the carboxyl-terminal half are involved in binding to the ECM. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Construction of Expression Vectors-The full-length mouse AD-AMTS-1 cDNA was isolated as described previously (17). Both 5Ј-and 3Ј-untranslated regions of ADAMTS-1 cDNA in pBluescript vector (Stratagene) were deleted, and an XhoI site was added after the Ser-951 codon by means of PCR to generate pBL151-31. The coding region of the ADAMTS-1 cDNA was isolated from pBL151-31 by digestion with NotI and XhoI and ligated into the NotI and XhoI sites of the mammalian expression vector pcDNA3 (Invitrogen), giving rise to pCMV151. To add the FLAG epitope tag in the COOH terminus of the ADAMTS-1 protein, the double-stranded synthetic DNA corresponding to both FLAG epitope tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) and stop codon was inserted into XhoI-XbaI sites of the pCMV151 vector, generating the pCMV-151CF vector.
The expression vectors for the carboxyl-terminal truncation mutants (X3-X6) were constructed using PCR. In brief, the wild type cDNA fragment corresponding to the carboxyl-terminal region was replaced by partially deleted PCR products.
For construction of mutant DPM-1, the proprotein and metalloproteinase domains were deleted by mutagenesis based on PCR (22). In mutant DPM-1, a BamHI site was introduced after the signal peptide (1-32 amino acids) and was followed by the middle TSP type I motif region. Furthermore, mutants DPM-4, -5 and -6, which are truncation mutants of DPM-1, were also constructed using PCR. Nucleotide sequences of mutants were confirmed by direct sequencing. DNA sequencing analysis was performed by using a PCR employing fluorescent dideoxynucleotides and a model 373A automated sequencer (Applied Biosystems).
Transfection-COS-7 cells were transfected with the expression vectors (2 g) by means of LipofectAMINE (6 l) (Life Technologies, Inc.). The liposome-DNA complex solution was spread on cells in 3.5-cm dishes and incubated for 6 h. Thereafter, 1 ml of the DMEM containing 10% fetal calf serum was added into the medium, and 18 h after transfection the medium was removed, and the cells were washed with DMEM without serum. Transfected cells were grown further in serumfree DMEM.
In some experiments, either tunicamycin that was dissolved in dimethyl sulfoxide, or heparin was added into the cell culture medium.
Western Blotting-Aliquots of cellular lysate, culture medium, or ECM fraction were subjected to 8 -10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred for 3 h at 1 mA/cm 2 onto nitrocellulose filters (0.45 M). Filters were blocked overnight in Block ACE (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) and then incubated for 1 h at room temperature with an anti-FLAG monoclonal antibody, M2 (Eastman Kodak) in TBS buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl) followed by incubation with a 1:2,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG polyclonal antibodies (Amersham Pharmacia Biotech) for 1 h at room temperature. After washing with TBS buffer, bound horseradish peroxidase-conjugated antibodies were detected by chemiluminescense (ECL) (Amersham Pharmacia Biotech) and exposed to Kodak XAR autoradiography film.

Preparation of Glutathione S-Transferase (GST) Fusion Proteins and Fractionation by High Performance Liquid Chromatography (HPLC)-
GST fusion protein expression vector for the middle TSP type I motif (amino acids 535-615) of ADAMTS-1 was prepared as described previously (17). Similarly, cDNA corresponding to the carboxyl-terminal TSP type I submotifs (amino acids 832-951) was amplified by PCR and subcloned into pGEX4T3 vector (Amersham Pharmacia Biotech). GST fusion protein was expressed in Escherichia coli (BL21) and purified as described previously (23).
These GST fusion proteins were dialyzed against 10 mM Tris-HCl (pH 7.5) and applied to an HPLC system (Amersham Pharmacia Biotech) equipped with a TSK gel heparin-5PW column (Tosoh Co., Tokyo, Japan), which was equilibrated previously with 10 mM Tris-HCl (pH 7.5). After washing the column, bound materials were eluted with a linear gradient of NaCl concentration from 0 to 1 M in the same buffer. The fractions containing GST fusion proteins were monitored by SDSpolyacrylamide gel electrophoresis.

ADAMTS-1 Protein Is Secreted and Incorporated into ECM-
Because ADAMTS-1 has a putative signal peptide at the amino terminus but not a transmembrane region (17), it is a putative secretory protein. To examine whether the ADAMTS-1 protein is actually secreted from cells or not, ADAMTS-1 cDNA was expressed in COS-7 cells. For detection of the recombinant ADAMTS-1 protein, the FLAG epitope tag, consisting of eight amino acids, was added in the carboxyl terminus of the protein.
The expression vector for ADAMTS-1 was transiently transfected into COS-7 cells, and 2 days after transfection the culture supernatants were subjected to Western blot analysis using an anti-FLAG antibody. However, ADAMTS-1 protein was not detected in the conditioned cell culture medium (Fig. 1,  lane 6).
ADAMTS-1 has three TSP type I motifs in its carboxylterminal half. The TSP type I repeats were shown to be one of the ECM interaction sites of thrombospondins 1 and 2 (19). Therefore, we next examined the possibility that ADAMTS-1 is incorporated into the ECM. After transfection of the expression vector, cells were detached from dishes by EDTA treatment, and thereafter ECM proteins were extracted with Laemmli buffer. As shown in Fig. 1 size. The FLAG epitope tag was added at the carboxyl terminus of the protein. In addition, the difference in size between the lower and upper bands is about 20 kDa, corresponding to the size of the proprotein domain. Therefore, the lower band represents the mature form of the ADAMTS-1 protein after processing of the proprotein domain. These data demonstrate that both the precursor and the processed forms of the ADAMTS-1 protein are secreted from cells and incorporated into the ECM.
Analysis of ECM Binding Domain of ADAMTS-1 Protein-To identify the domains that are responsible for the binding of the ADAMTS-1 protein to the ECM, we first constructed several carboxyl-terminal deletion mutants ( Fig. 2A). These deletion mutants were expressed as FLAG-tagged protein in COS-7 cells, and the effects of deletions on binding to the ECM were investigated. When mutant X4, deleting the spacer region and the carboxyl-terminal TSP submotifs, was expressed in COS-7 cells, the precursor form of mutant X4 was detected in both the culture medium and the ECM fraction (Fig. 3, lanes 3 and 8). In contrast, the mature form of mutant X4 was detected in the culture supernatant but not in the ECM fraction (lanes 3 and  8). Similarly, the precursor form of mutant X5, deleting from the middle TSP type I motif to the carboxyl terminus, was detected in both the ECM fraction and the culture medium, whereas its mature form was detected only in the culture medium (lanes 4 and 9). Furthermore, the precursor form of mutant X6, also lacking a disintegrin-like domains, was found associated with the ECM (data not shown). These data demonstrate that the precursor form consisting of a proprotein and a proteinase domain has ECM binding capacity, whereas its mature form, lacking a proprotein domain, does not.
On the other hand, the mature form of the wild type AD-AMTS-1 protein was found associated with the ECM (Fig. 1,  lane 4, and Fig. 3, lane 10), whereas no mature form of the carboxyl-terminal deletion mutants X4 and X5 was detected in the ECM fraction (Fig. 3, lanes 8 and 9). These data suggest that the carboxyl-terminal half containing TSP type I motifs is important for the interaction of the mature protein with the ECM.
ECM Binding Domain in the Carboxyl-terminal Region-The  2 and 7), mutant X4 (lanes 3 and 8), mutant X5 (lanes 4 and 9), and control vector (lanes 1 and 6). Two days after transfection, the culture medium (lanes 1-5) and the ECM fractions (lanes 6 -10) were analyzed by Western blotting with an anti-FLAG M2 antibody. The positions of the wild type ADAMTS-1 and its deletion mutants are shown by arrows; p, precursor form; m, mature form. above results suggest a major contribution of the carboxylterminal half to the ECM binding for the mature form of ADAMTS-1 protein. To confirm the ECM binding activity of the carboxyl-terminal region, we next constructed a vector expressing the mutant (DPM-1) that lacks both a proprotein and a metalloproteinase domains (Fig. 2B). In mutant DPM-1, the signal peptide is followed by the middle TSP type I motif of the ADAMTS-1 protein. When mutant DPM-1 was transiently expressed in COS-7 cells, it was detected in the ECM fraction but not in the cell culture medium (Fig. 4, lanes 2 and 6), confirming the capacity of the carboxyl-terminal half for ECM binding. Similarly, mutant DPM-3, deleting the carboxyl-terminal TSP submotifs, was detected predominantly in the ECM fraction (lanes 3 and 7). In contrast, DPM-6, which is a truncation mutant of the spacer region, was found in both the culture medium and the ECM fraction (lanes 4 and 8). This indicates that truncation of the spacer region results in reduction of ECM binding activity and that the three TSP type I motifs alone have ECM binding activity. Moreover, the carboxyl-terminal spacer region alone (DPM-5) was found associated with the ECM (lanes 10 and 13). These results demonstrate that the spacer region in the carboxyl-terminal region contributes its tight binding to the ECM in addition to that of the three TSP type I motifs.
ADAMTS-1 Protein Is N-Glycosylated-As described above, the ADAMTS-1 proteins were expressed in COS-7 cells with a slightly larger molecular size than expected. Because AD-AMTS-1 possesses the putative N-linked glycosylation motifs, it is possible that the larger molecular size of ADAMTS-1 is caused by post-translational modification by glycosylation. Therefore, we next examined whether tunicamycin, an inhibitor of N-glycosylation, affects the molecular size of the protein.
As shown in Fig. 5A, tunicamycin treatment of cells clearly resulted in reduction of the molecular size of the precursor form of ADAMTS-1 protein (lanes 3 and 4), suggesting that the majority of ADAMTS-1 is glycosylated when expressed in COS-7 cells.
ADAMTS-1 has four putative N-linked glycosylation motifs. One is located between the disintegrin-like and the first TSP type I motif, and the others are present within the carboxylterminal spacer region of the protein (Fig. 2). To analyze the contribution of these motifs to glycosylation of the ADAMTS-1 protein in vivo, we next compared molecular size of the carboxyl-terminal deletion mutants with or without tunicamycin treatment. As shown in Fig. 5B, the precursor forms of mutant X3, deleting the carboxyl-terminal TSP submotifs, were detected as triplet bands without tunicamycin (lane 1), whereas the upper two bands were shifted to the faster migrating one by treatment with tunicamycin (lane 2), suggesting that mutant X3 is highly glycosylated. In contrast, the precursor forms of mutant X4, deleting the spacer region that includes three Nlinked glycosylation motifs, were detected as doublet bands in the absence of tunicamycin (lane 3), and the upper band disappeared by tunicamycin treatment (lane 4), suggesting that mutant X4 is partially glycosylated. However, tunicamycin treatment did not change the molecular size of the precursor form of mutant X6 (lanes 5 and 6), deleting all four N-linked glycosylation motifs in the protein. These data demonstrate that ADAMTS-1 is N-glycosylated at multiple sites within the carboxyl-terminal region.
ADAMTS-1 Is Found in the Culture Medium in the Presence of Soluble Heparin-The above results suggest that TSP type I motifs contribute to the ECM binding of the ADAMTS-1 protein. Because TSP type I motifs of thrombospondins were shown to associate with sulfated glycosaminoglycans such as heparin and heparan sulfate (19), we next examined the possibility that sulfated glycosaminoglycan is a target for the ECM binding of the ADAMTS-1 protein. To address this possibility, soluble heparin was added to the cell culture medium, and its effect on the distribution of the ADAMTS-1 protein was analyzed. As mentioned above, under normal cell culture conditions, little or no ADAMTS-1 protein was found in the cell culture medium (Fig. 6, lane 9). In contrast, the processed form of the ADAMTS-1 protein clearly appeared in the cell culture supernatant when the cells were grown in the presence of 50 g/ml soluble heparin (lane 10). Similarly, mutant DPM-1, corresponding to the carboxyl-terminal half, was not detected in the normal condition medium (lane 11) but was present in the culture medium containing soluble heparin (lane 12). These results raise the possibility that the ADAMTS-1 protein was released into the medium by competing with soluble heparin.
HPLC column using a linear gradient (Fig. 7). The middle TSP type I motif protein was eluted at 0.46 -0.66 M NaCl, whereas the carboxyl-terminal TSP submotifs protein were eluted at slightly lower salt concentration (0.4 -0.53 M NaCl). GST alone was recovered in the flow-through fraction (data not shown). These data indicate that both the middle and the carboxylterminal TSP type I motifs can bind to heparin independently at a physiological salt concentration. Collectively, our data demonstrate that the ECM association of the mature form the ADAMTS-1 protein occurs through binding to sulfated glycosaminoglycans such as heparan sulfate. DISCUSSION Typical ADAMs are classified as not only membrane-anchored proteases, but also cell surface adhesion molecules (1,2). Previously, we have identified ADAMTS-1 as an atypical ADAM family protein that does not possess a transmembrane domain (17). In this study, we have shown that COS-7 cells transfected with the expression vector for the ADAMTS-1 gene express and secrete ADAMTS-1 protein as both precursor and mature forms. In normal growth condition, little or none of the ADAMTS-1 protein was detected in the cell culture supernatant, whereas the majority of the protein was found in the ECM fraction on the culture dishes after the intact cells were detached. Our previous study showed that ADAMTS-1 mRNA expression is induced in kidney and heart after lipopolysaccharide administration, suggesting that ADAMTS-1 protein may be involved in an inflammatory process. Because this protein has the ECM binding property, it is unlikely that the AD-AMTS-1 protein would diffuse extensively in these organs during inflammation, although ADAMTS-1 is secretory type of ADAM family protein. Instead, ADAMTS-1 may be incorporated into the ECM region around the producing cells.
The ECM binding domains of the ADAMTS-1 protein were first addressed by analysis of its carboxyl-terminal deletion mutants. Truncation of the carboxyl-terminal region containing three TSP type I motifs completely abolished the ECM binding capacity of the mature form of the protein. Consistent with this finding, we observed that localization of the mutant corresponding to the carboxyl-terminal half was restricted to the ECM. These data demonstrate that the carboxyl-terminal half, including three TSP type I motifs, of the protein is important for the ECM binding of the mature form of the ADAMTS-1 protein.
The TSP type I motif is conserved in thrombospondins 1 and 2, which are multifunctional ECM proteins that influence cell adhesion, motility, and growth (19,24). The TSP type I motif was also found in multiple copies in properdin, which stabilizes the C3 convertase during activation of the alternate complement pathway (25). The TSP type I motifs of these factors have been shown to be involved in binding to sulfated glycoconjugates, such as heparin and heparan sulfate. It was shown that the TSP type I motifs in thrombospondins possess two conserved heparin binding segments: W(S/G)XWSXW and CS-VTCG (24,26,27). Sequences related to the latter segment (CSVTCG) are also found in a group of secreted proteins such as cyr61(CEF-10), which is induced by v-src, serum, and platelet-derived growth factor (28), a serum-inducible protein, fisp-12 (29), an immediate early gene expressed in virally induced avian nephroblastoma, nov (30), and a connective tissue growth factor (31). Among these factors, cyr61 was shown to bind to the ECM (28). In the case of ADAMTS-1, the middle TSP I motif has sequences similar to these heparin-binding segments in thrombospondins, WGPWGPW, and CS(R/K)TCG, whereas the carboxyl-terminal submotifs have only the latter sequence (17). In this study, we have shown that both the middle TSP type I motif and the carboxyl-terminal TSP submotifs of the ADAMTS-1 protein are able to bind to heparin. The carboxyl-terminal TSP submotif protein binds to heparin with slightly lesser affinity than the TSP type I motif in the middle of the molecule. This lower affinity of the carboxylterminal submotifs might be the result of incomplete conservation in the amino-terminal sequence of the motifs. On the other hand, we also showed that growth of transfected cells in the presence of soluble heparin led to the appearance of the mature ADAMTS-1 protein in the culture medium. Taken together, these data demonstrate that the interaction between its three TSP type I motifs and sulfated glycosaminoglycans in the ECM, such as heparan sulfate, plays a role in the ECM binding of the ADAMTS-1 protein.
On the other hand, we also found that truncation of the spacer region intervening between the middle and carboxylterminal TSP type I motifs significantly reduced the ECM binding of the protein. Moreover, the spacer region alone was able to associate tightly with the ECM. Therefore, the carboxylterminal spacer domain is also important for tight binding to the ECM in addition to the three TSP type I motifs. In our previous study, we called this region the "spacer region" because it does not show any homologies with known proteins. However, the present study has identified the spacer region as one of the ECM binding domains of the ADAMTS-1 protein.
Analyses of carboxyl-terminal deletion mutants also revealed that the precursor form, consisting of a proprotein domain and a metalloproteinase domain, has the ability to bind to the ECM, whereas its mature form does not. One possibility is that the proprotein domain of ADAMTS-1 possesses an ECM binding site. However, when the proprotein domain alone was expressed in COS-7 cells, it was present only in the culture medium. 2 Therefore, it seems that a conformational change in the amino-terminal region, triggered by an interaction between the proprotein and the proteinase domains, might lead to exposure of an unknown ECM binding site on the surface of the molecule.
Like other ADAMs, the amino-terminal half of the AD-AMTS-1 protein consists of a proprotein domain, a metalloproteinase domain, and disintegrin-like domains. We found that the wild type or deletion mutants of the ADAMTS-1 protein were detected as both the precursor and mature forms when transiently expressed in COS-7 cells. Because there are two typical cleavage site (RRRR, 178 -182) (RKKR, 233-236) for the furin-like protease within the proprotein domain of the mouse ADAMTS-1 protein, it is possible that the precursor of ADAMTS-1 protein is processed by a furin-like protease. Furin is a widely distributed serine endoprotease in the Golgi. It cleaves a wide range of precursor proteins at the consensus RX(K/R)R (32,33). Furin cleavage sites are found in a number of precursor proteins that are transported to the cell surface by a constitutive pathway rather than one that is involved in storage in granules (34). On the other hand, ADAM family proteins are classified into two groups based on the presence or absence of a zinc binding motif (HEXXH) in their protease domains. In addition, it is thought that the zinc binding motif is masked by the conserved cysteine in the proprotein domain. Because the ADAMTS-1 protein has a zinc binding motif in its metalloproteinase domain, the present data suggest that AD-AMTS-1 is secreted from cells as a proteolytically active form by cleavage with a furin-like enzyme.
ADAMTS-1 possesses putative N-linked glycosylation motifs. In this study, we have shown that ADAMTS-1 is actually N-glycosylated at multiple sites within the carboxyl-terminal region. Unexpectedly, when cells were cultured in the presence of tunicamycin, the processed form of the ADAMTS-1 protein was not detected in the ECM fraction. It is possible that processing of the ADAMTS-1 protein may be regulated by N-glycosylation in the Golgi. On the other hand, it is clear that glycosylation of the protein is not required for the association with the ECM because the precursor forms of the wild type and deletion mutants were found associated with the ECM after tunicamycin treatment.
Recent studies show that TACE and ADAM10 (MADM) cleave a membrane-bound TNF-␣ precursor protein and release soluble TNF-␣. In addition, in Drosophila, the Kuzbanian gene product is responsible for cleavage of the extracellular domain of Notch. From these observations, membrane-bound ADAM family proteins with a metalloproteinase consensus sequence are proposed as candidates for protein ectodomain shedding protease of cytokines such as the c-kit ligand, transforming growth factor ␣, and Fas ligand, cytokine receptors such as the interleukin-6 receptor, and adhesion molecules such as L-selectin (35)(36)(37)(38). In this study, we demonstrated that the AD-AMTS-1 protein is secreted from cells, and the majority of the protein is incorporated into the ECM. Therefore, it is presumed that the target molecule(s) of the ADAMTS-1 protein may be different from those of membrane-anchored ADAM proteinases.