Human ADAM 12 (Meltrin alpha ) Is an Active Metalloprotease*

Frosty LoechelDagger §, Brent J. GilpinDagger , Eva Engvall, Reidar AlbrechtsenDagger , and Ulla M. WewerDagger parallel

From the Dagger  Institute of Molecular Pathology, University of Copenhagen, Denmark and the  Burnham Institute, La Jolla, California 92037

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins with structural homology to snake venom metalloproteases. We recently described the cloning and sequencing of human ADAM 12 (meltrin alpha ). In this report we provide evidence that the metalloprotease domain of ADAM 12 is catalytically active. We used the trapping mechanism of alpha 2-macroglobulin to assay for protease activity of wild-type and mutant ADAM 12 proteins produced in a COS cell transfection system. We found that ADAM 12 is synthesized as a zymogen, with the prodomain maintaining the metalloprotease in a latent form, probably by means of a cysteine switch. The zymogen could be activated chemically by alkylation with N-ethylmaleimide. Cleavage of the prodomain at a site for a furin-like endopeptidase resulted in an ADAM 12 protein with proteolytic activity. The protease activity was sensitive to inhibition by 1,10-phenanthroline and could be eliminated by mutation of the critical glutamate residue at the active site. The demonstration that the ADAM 12 metalloprotease domain is functional may have important implications for future studies that explore the role of ADAM 12 protein in development and disease.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The ADAMs1 are a family of integral membrane or secreted glycoproteins comprised of several distinct domains. The archetypical ADAM protein has a prodomain, metalloprotease domain, disintegrin domain, cysteine-rich region, and in the case of membrane-anchored ADAMs, a transmembrane and cytoplasmic domain. At least 18 members of the ADAMs family have now been identified and sequenced (1-10), and together with snake venom metalloproteases (SVMPs), they make up the reprolysin family of zinc metalloproteases. SVMPs were the first members of this family to be identified and have been extensively studied (11-13). Full-length SVMPs are processed to generate a metalloprotease, which is able to degrade proteins of the basement membrane such as type IV collagen and laminin (14), and a disintegrin domain, which can inhibit the function of platelets by interacting with platelet integrin GPIIb-IIIa (15). Because of the domain composition and striking similarity to SVMPs, the ADAMs are thought to play an important role in cellular interactions and tissue integrity.

Members of the metzincin superfamily of proteolytic enzymes, including the reprolysin and the matrix metalloprotease (MMP) families, contain the distinctive zinc binding consensus sequence HEXXH (11, 16). However, only a subset of the ADAMs, including ADAM 12, contain this motif and are therefore considered to be potentially active metalloproteases. Protease activity has actually been demonstrated so far for only a few members of the ADAMs family, ADAM 10 and TACE, which have been shown to cleave precursor tumor necrosis factor-alpha , and the Drosophila ADAM 10 homolog KUZ, which cleaves Notch (5, 7, 17-20).

We recently reported the cloning and sequencing of human ADAM 12 (meltrin alpha ), which can be expressed either as a secreted or a membrane-bound protein (21). We have now begun to assign functions to the individual domains by assaying activity of wild-type and mutant ADAM 12 proteins expressed in a COS cell transient transfection system. We report here that the metalloprotease domain of ADAM 12 is proteolytically active. Full-length ADAM 12 is a zymogen with the prodomain maintaining the metalloprotease in a latent state most likely via a cysteine switch mechanism.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Plasmid Constructs-- A plasmid for expression of full-length ADAM 12-S (p1151) was constructed using the vector pcDNA3 (Invitrogen). Standard recombinant DNA techniques were used throughout (22). A cDNA clone in pBluescript SK(+) containing nt 1-3445 of ADAM 12-L and another cDNA clone containing nt 1854-3333 of ADAM 12-S were used as a source of ADAM 12 sequence (21). The ADAM 12-L insert was excised with EcoRV and XhoI and cloned at the EcoRV/XhoI sites of pcDNA3, giving an expression plasmid for ADAM 12-L (p1138). A PflMI/XhoI fragment containing nt 2091-3333 of ADAM 12-S was then exchanged with the corresponding PflMI/XhoI fragment of p1138, yielding plasmid p1151.

The construction of plasmid p1095, a minigene which expresses an ADAM 12-S protein where the signal peptide, prodomain, and metalloprotease domain have been replaced by an Ig kappa -chain leader sequence, has already been described (21).

Site-directed Mutagenesis-- Plasmids coding for mutant ADAM 12 proteins were produced by replacing the PmlI DNA fragment (nt 721-2168) of plasmid p1151 with PmlI fragments containing the desired mutations. These were generated by strand overlap polymerase chain reaction using Pfu DNA polymerase (Stratagene), a forward and reverse primer hybridizing upstream and downstream of the PmlI sites, and complementary oligonucleotides containing the desired mutation. Briefly, a DNA fragment was produced using the forward primer and the antisense mutant primer, and an overlapping fragment was produced using the sense mutant primer and the downstream primer. The two fragments were gel-purified, then mixed and amplified with Pfu DNA polymerase and the forward and reverse primers. Digestion of the product with PmlI yielded the desired 1447-base pair DNA fragment. The sequences of the oligonucleotide primers are available from the authors upon request. Sequencing to confirm the accuracy of the mutations was performed using the Vistra DNA Sequencer 725 (Amersham).

Transfection Assays and Immunoblotting-- COS-7 cells (ATCC, CRL 1651) were grown in Dulbecco's modified Eagle medium with Glutamax I and 4500 mg/ml glucose, 50 units/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum (Life Technologies, Inc.) at 37 °C in 5% CO2. For transient transfections, COS-7 cells were electroporated with a Bio-Rad Gene Pulser II using 250 V and 1000 µF for 4 × 106 cells and 10 µg of plasmid in 0.4 ml of phosphate-buffered saline containing 14 mM HEPES (pH 7.2), with an electrode gap of 0.4 cm. After electroporation, 6 × 105 viable cells were plated in 35-mm tissue culture dishes. 24 h post-transfection, cells were refed with medium containing 5% fetal bovine serum or with serum-free medium (UltraDOMA medium from BioWhittaker). 72 h post-transfection, the medium was harvested and concentrated 10-fold using an Amicon Centricon-10 filter. Samples were denatured and reduced by boiling in SDS sample buffer containing dithiothreitol, subjected to SDS-polyacrylamide gel electrophoresis on Tris/glycine gels (Novex) and transferred to nitrocellulose membranes (22). The membranes were incubated with medium from 14E3 hybridoma cells (21) and then with peroxidase-conjugated rabbit anti-rat immunoglobulins (DAKO). Detection was performed using the chemiluminescence SuperSignal kit from Pierce.

Protease Assays-- The alpha 2-macroglobulin (alpha 2M) complex formation assay was used (23, 24). Assays on concentrated serum-free medium from transfected cells were carried out in 100 mM NaCl, 50 mM Tris (pH 7.4), 10 mM CaCl2, and 0.02% sodium azide. The following compounds were added at the indicated final concentrations: 10 units/ml bovine alpha 2M (Boehringer Mannheim); 1 mM 1,10-phenanthroline (Sigma); 5 µM or 500 µM HgCl2 (Sigma); 1 mM N-ethylmaleimide (Sigma). Reactions were terminated after incubation at 37 °C for 16 h by boiling in SDS sample buffer as described above.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Selection of ADAM 12 Functional Motifs to Be Tested in a COS Cell Expression System-- Sequence comparison of ADAM 12 with other reprolysins and with MMPs reveals that the ADAM 12 metalloprotease domain contains the zinc binding sequence that characterizes the active site of the these proteases (Fig. 1). The highly conserved HEXXHXXGXXH motif is present, including the glutamate residue that is required for the catalytic mechanism (16). In addition, ADAM 12 retains the "Met-turn" that appears to be essential for the structural integrity of the zinc-binding site (16). In those MMPs that have an enzymatically active protease domain, the prodomain serves to maintain the enzyme in a latent state via a cysteine switch mechanism (25, 26). We predict that in human ADAM 12, the cysteine residue at position 179 in the prodomain complexes to the active site zinc atom in the metalloprotease domain. In addition, examination of the amino acids at the boundary of the prodomain and the metalloprotease domain shows that they match the consensus sequence for cleavage by furin-type endopeptidases (27-29). Removal of the prodomain by cleavage at this site would be expected to convert the full-length ADAM 12 zymogen to a mature enzymatically active metalloprotease.


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Fig. 1.   Sequence comparison of ADAM 12 with related metalloproteases. A, alignment of putative cysteine switch, furin cleavage site, zinc-binding active site, and methionine turn of ADAM 12 with selected other ADAMs, SVMPs and MMPs. The most frequent residue at each position is highlighted in reverse font, whereas amino acids similar to this are shaded. The regions selected for alignment are the predicted cysteine switch in the prodomain, the region around the pro-/metalloprotease junction, the zinc-binding active site, and methionine turn in the metalloprotease domain. Aligned sequences include human (h) ADAMs 12 (AF023476), 15 (U46005), 8 (D26579), 9 (U41766), and TACE (U69612); mouse (m) ADAMs 12 (D50411), 1 (U22056), 10 (AF011379), and ADAMTS-1 (D67076); Xenopus (x) ADAMs 13 (U66003) and 16 (U78185); SVMPs jararhagin (X68251) and adamalysin-2 (S46443); and the human MMPs stromelysin-3 (MMP-11, X57766) and matrilysin (MMP-7, Z11887). Consensus sequences for furin cleavage and zinc binding motif are indicated below the alignment. B, a schematic drawing of the human ADAM 12-S protein. The domains are labeled S (signal peptide), P (prodomain), M (metalloprotease domain), D (disintegrin domain), and C (cysteine-rich domain). Also indicated are the critical amino acid residues for the cysteine switch in the prodomain, the recognition site for a furin-type endopeptidase at the junction of the prodomain and the metalloprotease domain, and the catalytic site for the metalloprotease.

COS-7 cells were transiently transfected with plasmid p1151 containing the coding sequence of human ADAM 12-S downstream of a cytomegalovirus promoter. Soluble, secreted ADAM 12-S protein was detected by SDS-polyacrylamide gel electrophoresis and immunoblotting of culture medium 72 h post-transfection using the monoclonal antibody 14E3 against the cysteine-rich region of ADAM 12. When full-length ADAM 12-S was expressed in COS cells, two bands were seen on the blot with estimated sizes of 92 and 68 kDa (Fig. 2A, lane 3). We tentatively assigned the 92-kDa band to full-length ADAM 12-S, and the 68-kDa band to ADAM 12-S after cleavage of the prodomain. These sizes are larger than those calculated from the primary structure (78 and 58 kDa) and most likely reflect glycosylation of the ADAM 12 polypeptides (21, 30).


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Fig. 2.   Immunoblots of wild-type and mutant ADAM 12 proteins expressed in COS cells. Medium from COS cells transfected with various plasmids was concentrated, subjected to SDS-polyacrylamide gel electrophoresis electrophoresis, and immunoblotted with monoclonal antibody 14E3. A, analysis of ADAM 12-S protein produced by COS cells in the presence or absence of serum. Lanes 1 and 4 show cells transfected with a negative control plasmid (pcDNA3 with no insert). Lanes 2 and 5 are from cells transfected with an ADAM 12-S minigene (plasmid p1095). Lanes 3 and 6 are from cells transfected with a full-length ADAM 12-S expression construct (plasmid p1151). Serum-free medium was used in lanes 1-3, and medium with 5% fetal bovine serum was used in lanes 4-6. Samples were analyzed on a 4-20% polyacrylamide gel. B, detection of proteolytically active forms of ADAM 12-S by reaction with alpha 2M. Protease assays were carried out on concentrated serum-free medium, and the samples were then subjected to electrophoresis on a 6% polyacrylamide gel. The samples on lanes 1-3 are from cells transfected with plasmid p1151. Lane 1 is conditioned medium alone, in lane 2 the medium was incubated with purified alpha 2M, and lane 3 includes both alpha 2M and 1,10-phenanthroline. Lanes 4 and 5 are from cells transfected with plasmid p1151(E351 right-arrow Q), incubated in the absence and presence of alpha 2M, respectively. Lanes 6-9 are from cells transfected with plasmid p1151(KR207 right-arrow NG). Lane 6 shows protein incubated in the absence of alpha 2M, and lanes 7-9 show protein incubated in the presence of alpha 2M. HgCl2 was included in the lane 8 sample and NEM in the lane 9 sample.

ADAM 12-S protein that was secreted into medium containing fetal bovine serum generated an additional set of high Mr bands (Fig. 2A, lane 6). These bands are reminiscent of the cross-linked products seen when the protease inhibitor alpha 2M is cleaved by an active protease (24, 31). We hypothesized that the high Mr bands observed in this experiment arose by cleavage of alpha 2M by the catalytically active metalloprotease domain of ADAM 12, giving cross-linked ADAM 12/alpha 2M proteins that could be detected by monoclonal antibody 14E3. This interpretation was supported by the fact that when COS cells were transfected with an ADAM 12-S minigene, the ADAM 12-S polypeptide lacking a prodomain and metalloprotease domain did not give rise to the high Mr products (Fig. 2A, lanes 2 and 5).

ADAM 12 Is an Active Metalloprotease-- To test the hypothesis that ADAM 12 has proteolytic activity toward alpha 2M, the 92- and 68-kDa forms of ADAM 12-S protein were prepared in serum-free medium. They were incubated with purified alpha 2M and then analyzed by immunoblotting with monoclonal antibody 14E3. A portion of the ADAM 12 protein shifted to the same large complexes seen after incubation with serum (Fig. 2B, lanes 1 and 2), confirming that these bands arise from the reaction of ADAM 12 with alpha 2M and not with some other component of serum. The ladder of slower migrating bands matches the pattern of monovalent and multivalent complexes seen in previous studies of endopeptidases and alpha 2M (24, 31). As further confirmation that these bands arose from the cleavage of alpha 2M by the metalloprotease activity of ADAM 12, the reaction was carried out in the presence of the metalloprotease inhibitor 1,10-phenanthroline. As can be seen in Fig. 2B, lane 3, the reaction of ADAM 12 with alpha 2M was blocked by this inhibitor. Activity was also completely inhibited in the presence of EDTA (data not shown).

For those members of the metzincin superfamily where the mechanism of the metalloprotease has been studied in detail, the glutamate residue present at the zinc-binding site is absolutely required for catalytic activity (16). As a definitive test for the proteolytic activity of ADAM 12, we used site-directed mutagenesis to change this glutamate residue of ADAM 12 to glutamine. COS cells synthesized both the 92- and 68-kDa forms of ADAM 12 containing the E351 right-arrow Q mutation, but these proteins were no longer able to react with alpha 2M (Fig. 2B, lanes 4 and 5). Thus, the E351 right-arrow Q mutation eliminated the protease activity of ADAM 12.

The ADAM 12 Prodomain Maintains the Metalloprotease in a Latent State-- We had tentatively attributed the production of two forms of ADAM 12-S by COS cells to processing of the full-length 92-kDa form by a COS cell endopeptidase. Elimination of the furin recognition site at the boundary between the prodomain and metalloprotease domain of ADAM 12 should give a precursor protein from which the prodomain can no longer be cleaved. By site-directed mutagenesis we changed the critical amino acids at the -1 and -2 positions of the predicted furin recognition site (28). When COS cells synthesized an ADAM 12 polypeptide containing the KR207 right-arrow NG mutation, only a 92-kDa form was detected in the medium (Fig. 2B, lane 6). This confirmed that the 92-kDa form is full-length ADAM 12-S containing the prodomain and that it is processed by a furin-type endopeptidase. Additionally, this experiment showed that the 92-kDa product is a latent form of the ADAM 12 protease in that it is inactive when assayed with alpha 2M (Fig. 2B, lane 7). Therefore, the protease activity detected with wild-type ADAM 12-S must derive from the 68-kDa processed form. We next attempted to activate the ADAM 12 zymogen by treatment with sulfhydryl-reactive compounds. Hg(II) and N-ethylmaleimide (NEM) have been reported to activate latent MMPs by dissociating a cysteine residue in the prodomain from the active site zinc atom (25). ADAM 12 zymogen was converted to an active form by NEM but not by Hg(II) (Fig. 2B, lanes 8 and 9). The ADAM 12·alpha 2M complexes formed by the NEM-activated zymogen were slightly larger than those seen with wild-type ADAM 12, reflecting the reaction of a 92- and 68-kDa ADAM 12 polypeptide, respectively.

The Role of the Cysteine Switch of the Prodomain-- Based on sequence comparison to other metalloproteases, the cysteine residue at position 179 of ADAM 12 is part of the cysteine switch of the prodomain, which maintains the metalloprotease domain in a latent state (Fig. 1). There is evidence that the cysteine switch of zinc metalloproteases involves binding of the cysteine thiol to the active site zinc atom but that amino acid residues surrounding the cysteine also contribute in a manner that is not well understood (32-36). As a first step in analyzing the cysteine switch of ADAM 12, the protein with the KR207 right-arrow NG mutation at the furin cleavage site was modified by changing Cys179 to either serine or alanine. Expression in COS cells yielded a 92-kDa polypeptide that displayed no protease activity when assayed with alpha 2M (Fig. 3A, lanes 5 and 8). In contrast to the proenzyme with a wild-type Cysteine switch, these mutants could not be activated with NEM (Fig. 3A, lanes 3, 6, and 9). One possible explanation for these results is that the cysteine switch of ADAM 12 performs two sequential functions: first assisting in the folding of the metalloprotease domain into an active conformation, and subsequently by maintaining the metalloprotease in a latent state until removed by a furin-type protease. According to this hypothesis, the 92-kDa ADAM 12 protein containing the C179 right-arrow S or C179 right-arrow A mutation is not a latent protease but is in fact inactive. We tested this hypothesis by allowing COS cells to synthesize an ADAM 12 protein with an intact furin cleavage site but containing the C179 right-arrow S mutation. After the synthesis, folding, and processing of this polypeptide, the COS cells secreted the 68-kDa form, which could be assayed for protease activity. Fig. 3B shows the result of this experiment. The 68-kDa ADAM 12-S polypeptide was proteolytically active regardless of whether it was generated from a precursor with a wild-type cysteine switch or a precursor with the C179 right-arrow S mutation in the cysteine switch. ADAM 12-S zymogen with a C179 right-arrow S mutation therefore contains a latent rather than an inactive metalloprotease domain.


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Fig. 3.   Immunoblots of prodomain mutants expressed in COS cells. Assays were carried out as in Fig. 2. A, effect of the cysteine switch on the proteolytic activity of ADAM 12. The 92-kDa ADAM 12-S protein containing the KR207 right-arrow NG mutation was produced with the wild-type cysteine residue at position 179 (lanes 1-3) or with this residue changed to serine (lanes 4-6) or alanine (lanes 7-9). The medium was incubated with purified alpha 2M in lanes 2, 5, and 8. The medium was incubated with both alpha 2M and NEM in lanes 3, 6, and 9. B, effect of a modified cysteine switch on the activity of processed 68-kDa ADAM 12-S polypeptide. Wild-type (WT) ADAM 12-S protein (lanes 1 and 2) or protein containing the C179 right-arrow S mutation (lanes 3 and 4) was produced and assayed by incubation with alpha 2M (lanes 2 and 4).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In the present study we used the trapping mechanism of alpha 2M as an assay for proteolytic activity of ADAM 12. The mechanism of action of alpha 2M has been studied in detail, and it is known that the formation of covalent complexes is dependent on an enzymatically active protease cleaving the bait region of alpha 2M. It is therefore a useful reagent for identification of catalytically active forms of proteases (23, 31). By site-directed mutagenesis, we altered critical amino acid residues of ADAM 12 chosen on the basis of structural comparisons with other members of the reprolysin family, expressed the mutant proteins in COS cells, and then tested for reactivity with alpha 2M. Substitution of the essential glutamate residue (Glu351) at the predicted active site of the ADAM 12 protease completely eliminated protease activity.

We have shown that ADAM 12-S is synthesized as a latent metalloprotease. It could be activated chemically by treatment with NEM, which presumably alkylates Cys179 and destroys the cysteine switch of the prodomain. Physiologically, activation is probably achieved by proteolytic cleavage of the prodomain, resulting in unmasking of the catalytic center when the cysteine dissociates from the zinc atom present at the catalytic site of the metalloprotease domain. COS cells synthesize a 92-kDa ADAM 12-S zymogen and convert it to the 68-kDa active form by cleaving at a recognition site for furin-type endopeptidases. We have not determined which of the furin-type endopeptidases is responsible for this cleavage, but it is likely to be furin itself, based on published reports of the processing of both stromelysin-3 and meprin A by furin in COS cells (37, 38). It is most likely that the cleavage occurs prior to secretion, as seen with stromelysin-3, because extended incubation of secreted ADAM 12 did not result in further conversion. A fraction of the ADAM 12-S zymogen does not get processed by COS cells and is detectable as a 92-kDa protein in the medium, possibly because endopeptidase activity in the COS cells cannot keep pace with the rate of secretion. We cannot exclude, however, that conversion of ADAM 12 from a latent to an active protease also occurs extracellularly in physiological situations.

Since the cysteine switch model was first proposed to explain the mechanism by which the prodomain of MMPs maintains the metalloprotease domain in a latent state (25), a number of studies have supported the role of cysteine and zinc ion coordination in maintaining latency, but it has also become evident that the contribution of other amino acid residues in the prodomain cannot be ignored (32-34). We present here the first data on the cysteine switch of an ADAM protein. Our results are consistent with the notion that a cysteine switch is part of the latency mechanism of ADAM metalloproteases, as it is for MMPs and SVMPs (36). The assignment of Cys179 to the cysteine switch based on sequence comparisons is supported by the fact that sensitivity to NEM alkylation disappears when Cys179 is mutated to another amino acid. The observation that these mutant forms of the ADAM 12 prodomain are still able to maintain the metalloprotease in a latent state is probably because of contributions from amino acid residues in the vicinity of Cys179, as has been seen in studies of MMPs (32, 33). The "cysteine switch" of the ADAM 12 prodomain would thus retain sufficient binding affinity for the active site of the metalloprotease domain even after elimination of the cysteine-zinc interaction, such that protease activity is blocked under the assay conditions used here. We suggest that NEM destroys the latency mechanism of the ADAM 12 cysteine switch both by virtue of alkylating the cysteine residue and by steric hindrance of the interactions of neighboring amino acids with amino acids in the metalloprotease domain.

Many proteases are synthesized as zymogens, and it is of interest to compare the modes of activation of some of these, in particular MMPs, with ADAMs. Most MMPs are synthesized and secreted in a latent form and are subsequently activated by proteolytic cleavage of the prodomain, thus providing one means of modulating their activity (39). Our data on ADAM 12, and previously published data on ADAM 10 and TACE, suggest that ADAM proteases may be constitutively active once they are secreted or displayed on the cell surface (5, 17). Another important means of modulating protease activity is by inhibitors, either those with low specificity such as alpha 2M or those with high specificity such as tissue inhibitors of metalloproteases, which are specific for MMPs (40, 41). To date, we have no information on naturally occurring specific inhibitors of ADAM proteases, but they most likely exist.

Other types of cells that produce ADAM 12 may process it differently than do transfected COS cells. One could envisage for example that some cells produce an endopeptidase that can cleave between the metalloprotease and disintegrin domains, and thus synthesize an ADAM 12 protein lacking protease activity but with an enhanced activity of the disintegrin or cysteine-rich domains; this may be the case in C2 cells (30). Also, it is not known whether the disintegrin and cysteine-rich domains are required for the proteolytic activity of ADAM 12. It is conceivable that they are essential for maintaining the metalloprotease domain in an active conformation, or that they contribute to the specificity of ADAM 12 protease by binding to selected substrates.

The substrate specificities of several SVMPs and MMPs have been studied extensively. Many of them are capable of cleaving basement membrane components, including laminins, type IV collagen, entactin, and fibronectin. Some, but not others, can cleave gelatins, interstitial collagens, and proteoglycans (39, 42). Degradation of the extracellular matrix may lead to loss of tissue integrity and be an important component of the pathology of several diseases such as arthritis and cancer. With the recent discovery of ADAMs, the mammalian homologs of SVMPs, the substrate specificity and pathophysiological role of these proteins is now an exciting focus of investigation. ADAM 10, the Drosophila ADAM 10 homolog KUZ, and TACE have been shown to be involved in protein ectodomain shedding, but no ADAM has yet been demonstrated to degrade components of the extracellular matrix. The physiological substrate of ADAM 12 remains to be identified.

In conclusion, we have demonstrated that ADAM 12-S is synthesized as a zymogen with the prodomain maintaining the metalloprotease in a latent state. Cleavage of the prodomain by a furin-type protease results in an ADAM 12 protein with proteolytic activity.

    ACKNOWLEDGEMENTS

We thank Drs. A. M. Mercurio and U. P. Thorgeirsson for stimulating discussions and comments on the manuscript. We thank Bent Børgesen for photographic assistance and Hong Xu, Brit Valentin, and Aase Valsted for technical assistance.

    FOOTNOTES

* This work was supported by grants from the Danish Cancer Society and the Danish Medical Research Council (to U. M. W. and R. A.). Our laboratories were also supported by the VELUX, Novo-Nordisk, Munksholm, Haensch, Thaysen, Wærum, Bojesen, Beckett, Hartmann, and Meyer Foundations (to U. M. W. and R. A.) and by the National Institutes of Health (to E. E.).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.

§ Supported by a fellowship from the Danish Cancer Society.

parallel To whom correspondence should be addressed: Institute of Molecular Pathology, University of Copenhagen, Frederik V's Vej 11, DK-2100, Copenhagen, Denmark. Tel.: 45-3532-6056; Fax: 45-3532-6081; E-mail: molera{at}inet.uni-c.dk.

1 The abbreviations used are: ADAM, a disintegrin and metalloprotease; alpha 2M, alpha 2-macroglobulin; MMP, matrix metalloprotease; NEM, N-ethylmaleimide; SVMP, snake venom metalloprotease; nt, nucleotide(s); TACE, TNF-alpha -converting enzyme.

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Abstract
Introduction
Procedures
Results
Discussion
References

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