From the Institute of Molecular Pathology, University
of Copenhagen, Denmark and the ¶ Burnham Institute, La Jolla,
California 92037
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ABSTRACT |
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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 ). In this report we provide
evidence that the metalloprotease domain of ADAM 12 is catalytically
active. We used the trapping mechanism of
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.
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INTRODUCTION |
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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-, 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 ), 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.
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EXPERIMENTAL PROCEDURES |
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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 IgSite-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 2-macroglobulin (
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
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.
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RESULTS |
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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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ADAM 12 Is an Active Metalloprotease--
To test the hypothesis
that ADAM 12 has proteolytic activity toward 2M, the 92- and 68-kDa
forms of ADAM 12-S protein were prepared in serum-free medium. They
were incubated with purified
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
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
2M (24, 31). As further
confirmation that these bands arose from the cleavage of
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
2M was blocked by this inhibitor. Activity was also
completely inhibited in the presence of EDTA (data not shown).
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
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
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·
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 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
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
S or
C179
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
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
S mutation in the cysteine switch. ADAM 12-S
zymogen with a C179
S mutation therefore contains a
latent rather than an inactive metalloprotease domain.
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DISCUSSION |
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In the present study we used the trapping mechanism of 2M as an
assay for proteolytic activity of ADAM 12. The mechanism of action of
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
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
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 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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; 2M,
2-macroglobulin; MMP, matrix metalloprotease; NEM, N-ethylmaleimide; SVMP, snake venom metalloprotease; nt,
nucleotide(s); TACE, TNF-
-converting enzyme.
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REFERENCES |
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