Regulation of Human ADAM 12 Protease by the Prodomain
EVIDENCE FOR A FUNCTIONAL CYSTEINE SWITCH*
Frosty
Loechel
§,
Michael T.
Overgaard¶,
Claus
Oxvig¶,
Reidar
Albrechtsen
, and
Ulla M.
Wewer
From the
Institute of Molecular Pathology, University
of Copenhagen, Copenhagen DK-2100, and the ¶ Department of
Molecular and Structural Biology, University of Aarhus, Aarhus, Denmark
DK-8000
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ABSTRACT |
The ADAMs (a disintegrin
and metalloprotease) are a family of
multidomain proteins that are believed to play key roles in cell-cell
and cell-matrix interactions. We have shown recently that human ADAM
12-S (meltrin
) is an active metalloprotease. It is synthesized as a
zymogen, with the prodomain maintaining the protease in a latent form.
We now provide evidence that the latency mechanism of ADAM 12 can be
explained by the cysteine switch model, in which coordination of
Zn2+ in the active site of the catalytic domain by a
cysteine residue in the prodomain is critical for inhibition of the
protease. Replacing Cys179 with other amino acids results
in an ADAM 12 proform that is proteolytically active, but latency can
be restored by placing cysteine at other positions in the propeptide.
None of the amino acids adjacent to the crucial cysteine residue is
essential for blocking activity of the protease domain. In addition to
its latency function, the prodomain is required for exit of ADAM 12 protease from the endoplasmic reticulum. Tissue inhibitor of
metalloprotease-1, -2, and -3 were not found to block proteolytic
activity of ADAM 12, hence a physiological inhibitor of ADAM 12 protease in the extracellular environment remains to be identified.
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INTRODUCTION |
The ADAMs1are a family
of integral membrane or secreted glycoproteins comprised of several
distinct domains. Together with snake venom metalloproteases, they make
up the reprolysin family of zinc metalloproteases (1-5). The
archetypical ADAM protein has a prodomain, metalloprotease
domain, disintegrin-like domain, cysteine-rich region,
and in the case of membrane-anchored ADAMs, a transmembrane and
cytoplasmic domain. We recently made use of an
2-macroglobulin (
2M) trapping assay to
demonstrate that human ADAM 12-S, the secreted form of ADAM 12, is an
active metalloprotease (6).
Members of the metzincin superfamily of metalloproteases, including the
reprolysins and matrix metalloproteases (MMPs), are synthesized as
inactive precursors, in which an NH2-terminal prodomain is
responsible for maintaining latency of the protease (7, 8). MMPs are
generally secreted as proenzymes; the latent proform is subsequently
converted to the active form by proteolytic cleavage of the prodomain.
In contrast, it appears that ADAM proteases are converted from a latent
proform to an active enzyme before secretion as a result of cleavage of
the prodomain by furin or related proteases in the
trans-Golgi (6, 9).
The prodomain of all MMPs contains a highly conserved cysteine residue
that is part of the mechanism for the blocking activity of the
proenzyme (3). This cysteine residue coordinates the zinc ion located
at the active site of the catalytic domain and is the basis for the
proposed "cysteine switch" model of repression/activation for MMPs
(10, 11). The key cysteine residue of the prodomain is also highly
conserved in snake venom metalloproteases as well as in those members
of the ADAMs family which have been demonstrated to be active proteases
(1, 3, 6, 12, 13). It is therefore likely that a similar cysteine
switch is part of the mechanism by which the activity of these
proteases is regulated.
It is not clear to what extent the regulatory activity of an ADAM or
MMP prodomain should be attributed to cysteine coordination of the
active site zinc and how much is the result of interaction of other
regions of the prodomain with sites in the metalloprotease domain.
Treatment of pro-MMPs with thiol-modifying agents results in activation
with ensuing autocleavage of the propeptide (10, 14). This supports the
view that the cysteine-zinc interaction is the key one. On the other
hand, Chen et al. (15) showed that disruption of the
zinc-cysteine interaction in MMP-3 by chemical modification of the
cysteine was not sufficient to activate the proenzyme, whereas
subsequent treatment with 4-aminophenylmercuric acetate did result in
activation (15). This suggests that, at least in the case of MMP-3, the
prodomain is capable of performing its role of maintaining latency even
in the absence of a cysteine switch. Single amino acid substitutions in
the cysteine switch region of MMP proenzymes, whether of the cysteine
itself or of adjacent amino acids, have been shown to result in
spontaneous activation of the proenzyme (16-18). The details of how
the cysteine switch functions in MMP proenzymes have therefore not been
fully resolved, and the relative importance of the cysteine switch to the latency mechanism of ADAM proteases remains an open question.
We decided to explore whether the cysteine switch model can explain
latency of the ADAM 12-S proenzyme. Our approach was to substitute
amino acids in the prodomain, based on what has been learned from
previous studies on MMPs, and to determine the effect this had on
protease activity of the ADAM 12 proenzyme, using
2M as a substrate.
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EXPERIMENTAL PROCEDURES |
Materials--
N-Ethylmaleimide (NEM),
phenylmethylsulfonyl fluoride, and Nonidet P-40 were from Sigma. BB-94
and BB-3103 were from British Biotech Pharmaceuticals Ltd. (Oxford,
United Kingdom). Restriction endonucleases were from New England
Biolabs or Roche Molecular Biochemicals. Recombinant human tissue
inhibitors of metalloprotease (TIMP)-1, -2, and -3 were provided by
Gillian Murphy (University of East Anglia, Norwich, U. K.); they were
expressed in NS0 mouse myeloma cells and purified as described
previously (19, 20).
Plasmid Constructs--
Plasmids for expression of full-length
ADAM 12-S (p1151), for expression of the same protein lacking a furin
cleavage site at the junction between the prodomain and the
metalloprotease domain (p1197), and for expression of ADAM 12-S lacking
both the prodomain and the metalloprotease domain (p1095) have been
described previously (6, 21). Nucleotide and amino acid numbering is according to the ADAM 12-S sequence deposited in the GenBank data base
(accession number AF023477).
Plasmids coding for ADAM 12 proteins with amino acid substitutions in
the cysteine switch region were constructed as follows. First, plasmid
1197 was modified to give two unique restriction sites in place of the
codons for amino acids 177-183. This was done using the method
described previously by performing strand overlap polymerase chain
reaction on plasmid 1197 to generate a PmlI DNA fragment
(nucleotides 716-2163) containing the desired mutation and using this
to replace the wild-type PmlI DNA fragment in plasmid 1151 (6). The resulting plasmid (p1265) contains, in addition to the mutated
furin cleavage site, the sequence GTC CGC GGA GTT AAC, which represents
the codon for Val175 followed by a KspI site and
an HpaI site. Ligation of an appropriate double-stranded
oligonucleotide to p1265 digested with KspI and HpaI allowed insertion of the desired codons.
A modified form of plasmid 1265 was prepared containing a
Glu351
Gln mutation to eliminate catalytic activity.
Insertion of the appropriate double-stranded oligonucleotide at the
KspI/HpaI sites yielded plasmid 1376, coding for
full-length ADAM 12-S with a mutated furin cleavage site and the
Glu351
Gln mutation, and plasmid 1377, containing these
mutations plus a Cys179
His mutation in the cysteine switch.
A plasmid for the expression of an ADAM 12-S polypeptide lacking the
prodomain (p1229) was constructed using the pSecTagB vector
(Invitrogen). A DNA fragment containing nucleotides 925-2523 of human
ADAM 12-S, with a BamHI site at the 5'-end and an
XbaI site at the 3'-end, was prepared by polymerase chain
reaction amplification using Pfu DNA polymerase
(Stratagene). It was inserted at the BamHI/XbaI
sites of pSecTagB. The resulting polypeptide contains an Ig
-chain
signal peptide at the NH2 terminus followed by amino acids
207-738 of ADAM 12-S (metalloprotease, disintegrin, and cysteine-rich domains).
Table I provides an overview of these
ADAM 12-S expression plasmids. Standard recombinant DNA techniques were
used throughout (22). 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 Pharmacia Biotech).
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Table I
ADAM 12-S expression plasmids
Additional plasmids are listed in Tables II and III and correspond to
plasmid 1197 with various amino acid substitutions in the cysteine
switch of the prodomain.
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Transfection Assays and Immunoblotting--
COS-7 cells were
transfected with ADAM 12-S expression plasmids by electroporation as
described previously (6). Unless stated otherwise, cells were grown in
medium containing 10% fetal bovine serum (Life Technologies, Inc.).
48-72 h post-transfection, the medium was harvested, concentrated
10-fold using an Amicon Centricon-10 filter, and processed for
SDS-PAGE. To evaluate ADAM 12 polypeptides located intracellularly, a
cell extract was prepared by washing the cell layer with
phosphate-buffered saline and lysing by incubation in 150 mM NaCl, 20 mM Tris (pH 7.4), 1% Nonidet P-40
on ice for 10 min. After centrifugation at 7,000 × g
for 5 min at 4 °C, the supernatant was prepared for SDS-PAGE.
Samples were denatured and reduced by boiling in SDS sample buffer
containing dithiothreitol, subjected to SDS-PAGE on Tris-glycine gels
(Novex) and transferred to a polyvinylidene difluoride membrane (22). The membranes were incubated either with the 14E3 monoclonal antibody specific for the ADAM 12 cysteine-rich region or with rabbit antiserum 104 specific for the same region (21). After incubation with the
appropriate peroxidase-conjugated second antibody, detection was
performed using the chemiluminescence SuperSignal kit from Pierce or
the ECL-Plus kit from Amersham.
Deglycosylation was performed on denatured protein that had been boiled
in SDS sample buffer. Protein was precipitated with 8 volumes of
acetone to remove excess SDS. The protein pellet was resuspended by
boiling in 100 mM 2-mercaptoethanol, 0.1% SDS for 2 min.
One aliquot was diluted with an equal volume of 150 mM
NaCl, 20 mM Tris (pH 7.4), 1% Nonidet P-40. EDTA was added to 10 mM, and digestion with N-glycosidase F
(Roche Molecular Biochemicals) was performed for 16 h at 30 °C.
One aliquot was diluted with an equal volume of 100 mM
sodium citrate (pH 5.5), phenylmethylsulfonyl fluoride was added to 5 mM, and digestion with endoglycosidase H (Roche Molecular
Biochemicals) was performed for 16 h at 30 °C.
Protease Assays--
The
2M complex formation assay was used
(23-25). ADAM 12 protein from transfected COS-7 cells was prepared in
serum-free medium (UltraDOMA medium from BioWhittaker) and concentrated
10-fold using an Amicon Centricon-10 filter. Assays were carried out in 100 mM NaCl, 50 mM Tris (pH 7.4), 10 mM CaCl2, and 0.02% sodium azide. The
2M
substrate was added either in the form of fetal bovine serum at a final
concentration of 25% or purified
2M at a concentration of 1 µg/µl. ADAM 12 proforms were alkylated by the addition of 1 mM NEM and incubation at 20 °C for 15 min before the
addition of
2M. Reactions were terminated after incubation at
37 °C for 16 h by boiling in SDS sample buffer as described above.
The ability of ADAM 12-S protease to react with
2M purified from
three different sources was tested. Adult human and bovine
2M was
purified as described previously (26). Fetal bovine
2M was purified
by a slightly modified procedure. Briefly, fetal bovine serum (Life
Technologies, Inc.) was precipitated with PEG-6000, and the 4-20%
precipitate was redissolved in 10 mM sodium phosphate, 10 mM NaCl, 2 mM EDTA (pH 7.4) and loaded onto a
DEAE-Sephacel column. The column was eluted with a linear gradient from
10 to 300 mM sodium chloride.
2M-containing fractions,
as determined by a protease protection assay, were pooled and loaded on
a Zn2+-iminodiacetic acid-Sepharose 4B column. The column
was washed with 2 volumes of 20 mM sodium phosphate, 150 mM sodium chloride (pH 7.0) and eluted with 100 mM EDTA (pH 7.0). As a final step, the eluate was
fractionated on a Superose 6 column in 100 mM sodium phosphate, pH 8.0, resulting in 95% pure
2M as judged by SDS-PAGE. All column materials were from Amersham Pharmacia Biotech. All preparations contained 3.7-3.9 thiol esters/
2M tetramer, as
measured by the appearance of thiols upon treatment with methylamine.
Because side-by-side assays showed that human
2M was cleaved more
efficiently than either fetal or adult bovine
2M in purified form,
all assays described in this study were performed either with purified
human
2M or fetal bovine serum. We reported previously that an ADAM
12-S proenzyme with a mutated cysteine switch did not react with
purified bovine
2M (6). This was apparently the result of the
preparation of bovine
2M used in the previous study because bovine
2M, when purified and used as above, demonstrated unequivocal
activity both with wild-type ADAM 12-S and mutant ADAM 12-S proenzymes.
The relative efficiency of cleavage, however, was often higher for the former.
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RESULTS |
Evaluating Proteolytic Activity of ADAM 12-S Proforms by the
2M
Trapping Assay--
We employed the
2M trapping assay that we used
previously to demonstrate that human ADAM 12 is an active
metalloprotease (6). Fig. 1A
shows the results of a typical assay, in which ADAM 12/
2M reaction
products were detected by immunoblotting with an antibody specific for
ADAM 12. When wild-type ADAM 12-S encoded by plasmid 1151 was secreted
by cells growing in serum-containing medium, both a 92- and a 68-kDa
form were seen, representing the latent proform and the catalytically
active protease, respectively. The majority of the protein was found in
the furin-cleaved 68-kDa form. A cluster of high
Mr bands is evident, which results from proteolytic cleavage of
2M followed by covalent cross-linking of
ADAM 12 to
2M via its thiol ester (Fig. 1A, lane 1). To
be able to assess the proteolytic activity of the ADAM 12-S proform, two amino acid residues at the junction between the ADAM 12 prodomain and metalloprotease domain were mutated (plasmid 1197), thereby eliminating the furin cleavage site. As we have shown previously, cells
transfected with plasmid 1197 secreted the 92-kDa proform, consisting
of the prodomain, metalloprotease domain, and disintegrin-like and
cysteine-rich domains (6). This proform is a latent protease and
therefore does not react with
2M, as evidenced by the absence of the
characteristic high Mr bands (Fig. 1A,
lane 2).

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Fig. 1.
2M trapping assays on ADAM 12-S
proteins expressed in COS cells. Medium from COS cells transfected
with various plasmids was concentrated, subjected to SDS-PAGE, and
immunoblotted with monoclonal antibody 14E3, specific for ADAM 12. Panel A, analysis of reaction products of ADAM 12 polypeptides with 2M from fetal bovine serum. In lanes
1-3, cells were grown in the presence of 10% fetal bovine serum,
such that reaction occurred as ADAM 12 proteins were secreted to the
medium. Plasmid 1151 codes for wild-type ADAM 12-S, plasmid 1197 codes
for ADAM 12-S with a mutated furin cleavage site, and plasmid 1214 contains both a mutated furin cleavage site and a Cys179
Ala mutation. In lanes 4-9, cells were grown in
serum-free medium. The 2M trapping assay was performed subsequently
by the addition of fetal bovine serum to concentrated medium, either in
the presence or absence of NEM. Samples were analyzed on a 6%
polyacrylamide gel. Panel B, analysis of reaction
products of ADAM 12 polypeptides with purified human 2M. Protease
assays were carried out on concentrated serum-free medium, and the
samples were then subjected to electrophoresis on a 6% polyacrylamide
gel.
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The prodomain of ADAM 12 contains a conserved cysteine residue at
position 179 which is predicted to participate in the cysteine switch
for maintaining latency of the protease domain. When an ADAM 12-S
proform containing a Cys179
Ala mutation (plasmid 1214)
was secreted to serum-containing medium, the presence of
2M reaction
products showed that this proform is a constitutively active rather
than a latent protease (Fig. 1A, lane 3). Consistent results
were obtained when assays were performed by first preparing ADAM 12-S
protein in serum-free medium and then adding
2M in the form of fetal
bovine serum (Fig. 1A, lanes 4-9). In addition, these
assays showed that the latent ADAM 12-S proform encoded by plasmid 1197 could be activated by treatment with NEM, which presumably alkylates
the free cysteine in the prodomain, thereby destroying the cysteine
switch (Fig. 1A, lane 7). NEM treatment had no significant
effect on activity of the 68-kDa form nor on activity of the proform
containing the Cys179
Ala mutation. Purified human
2M reacted with ADAM 12-S in a similar fashion. It is a more
efficient substrate than the preparation of purified bovine
2M which
we used in a previous study; the intensity of the
2M reaction
products is comparable to that obtained with fetal bovine serum and
clearly shows the proteolytic activity of the proform containing the
Cys179
Ala mutation (Fig. 1B, lane 3). The
reaction products of
2M with the proenzyme encoded by plasmid 1214 are larger than those generated by the wild-type ADAM-12 protease, as
would be expected for complexes containing 92-kDa rather than 68-kDa
ADAM 12 polypeptides.
Transfection with ADAM 12-S proform plasmids often yielded an
additional band that migrated at 68 kDa in addition to the strong band
at 92 kDa (Fig. 1, panel A, lanes 7-9 and
panel B, lane 3). It is conceivable that this
band represents active ADAM 12-S protease, generated either by
inefficient cleavage by furin at an alternate site or by autocleavage
of the ADAM 12 proenzyme (see next "Results" section). Some of the
2M reaction products generated by activated ADAM 12 proforms
comigrated with the products of wild-type ADAM 12 polypeptide (Fig.
1A, lanes 7-9) and possibly derive from reaction of 68-kDa
rather than 92-kDa polypeptides. Therefore, for the purpose of
evaluating whether a given ADAM 12-S proform was proteolytically active, we judged it to be active only in the event that it produced
2M reaction products larger than those produced by the wild-type 68-kDa polypeptide.
Which Amino Acids Are Required for a Functional Cysteine
Switch?--
We proceeded to generate a series of proforms with
various amino acid substitutions in the cysteine switch region,
centered around Cys179. A proform that did not react with
2M was judged to be a latent protease on the condition that it
reacted after treatment with NEM. A proform that reacted with
2M
when secreted into serum-containing medium or when prepared in
serum-free medium that was subsequently incubated with
2M in the
absence of NEM was judged to be a constitutively active protease, in
which the cysteine switch of the prodomain had been inactivated by the
amino acid substitution.
Mutants of the ADAM 12-S proform were expressed in COS cells and
assayed for proteolytic activity. The results are summarized in Table
II. The polypeptide encoded by plasmid
1197 has the wild-type ADAM 12 cysteine switch, and it has no activity
unless assayed in the presence of NEM. Replacing Cys179
with alanine or histidine yields a constitutively active protease (mutants 1214 and 1336). Cysteine is therefore essential for a functional cysteine switch and cannot be replaced even by an amino acid
such as histidine, which is capable of coordinating zinc. The two
histidine residues in the vicinity of Cys179 could
conceivably be involved in coordination of the active site Zn2+, similar to the way in which three histidines of the
protease domain bind to Zn2+. However, mutant 1313 shows
that changing these two amino acids does not affect the ability of the
prodomain to maintain latency. Gly180 is highly conserved
among members of the metzincin superfamily, but mutant 1358 demonstrates that it is not essential for function of the ADAM 12 cysteine switch. Finally, replacement of the ADAM 12 cysteine switch
with the cysteine switch of ADAM 17 or with a consensus snake venom
metalloprotease or MMP cysteine switch, yields a fully functional
prodomain. We conclude that cysteine is the crucial amino acid of the
ADAM 12 cysteine switch and that none of the adjacent amino acid
residues is essential.
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Table II
Protease activity of ADAM 12-S proforms with amino acid substitutions
in the cysteine switch of the prodomain
These ADAM 12 polypeptides all contain the KR207 NG
mutation to prevent cleavage of the prodomain by furin. Note that for
mutants 1312 and 1311, Gly177 is not part of the consensus
sequence.
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Replacing Cys179 with Ala results in an inactivated
cysteine switch (mutant 1214). We next asked whether an active cysteine
switch could be re-created in the prodomain by placing a cysteine
residue in the vicinity of position 179. A series of ADAM 12 proforms was generated in which this inactive cysteine switch was modified by
substituting cysteine for the adjacent amino acid residues one at a
time. The results are presented in Table
III. When cysteine was present at
positions
1, +1, or +2 relative to the wild-type position, the result
was a constitutively active protease, i.e. the cysteine
switch was nonfunctional. A prodomain containing cysteine at position
+3 or +4 was functionally indistinguishable from the wild-type
prodomain (mutants 1335 and 1343 compared with 1197). Therefore, not
only is cysteine the key amino acid residue, but there is a certain
flexibility in its position in the prodomain. Together, these data
demonstrate that the latency mechanism of the ADAM 12 prodomain can be
explained by the cysteine switch model as proposed in 1990 for MMPs
(10, 11).
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Table III
Effect of moving the cysteine residue of the cysteine switch to a
different position
These ADAM 12 polypeptides all contain the KR207 NG
mutation to prevent cleavage of the prodomain by furin.
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In the Absence of a Functional Cysteine Switch, ADAM 12 Proenzyme
Is Capable of Autocleavage--
As mentioned above, ADAM 12 proforms
in which the cysteine switch was altered either chemically (by
alkylation with NEM) or genetically (by substitution of another amino
acid for Cys179) often yielded a 68-kDa band in addition to
the major 92-kDa product. To test for autocatalysis of the ADAM 12-S
proenzyme, the 92-kDa proform polypeptide with a wild-type cysteine
switch was prepared in serum-free medium by transfecting cells with
plasmid 1197. This protein is stable when incubated at 37 °C (Fig.
2, lane 1). Treatment with
NEM, in the absence of
2M, resulted in the appearance of a 68-kDa
band (Fig. 2, lane 2). The same polypeptide with a
Glu351
Gln mutation in the catalytic domain to abolish
proteolytic activity did not generate a 68-kDa band (Fig. 2,
lanes 3 and 4). We therefore concluded that
conversion of the 92-kDa polypeptide to a 68-kDa form was caused by
autocatalysis. A similar result was obtained for an ADAM 12-S proform
where the cysteine switch was inactivated genetically. Cells
transfected with plasmid 1336, encoding the proenzyme with a mutated
furin cleavage site and with a Cys179
His mutation in
the cysteine switch, secreted predominantly the 92-kDa form, with a
lesser amount of 68-kDa polypeptide (Fig. 2, lane 5). The
processing was a result of autocleavage rather than cleavage by furin
because the same proform with the Glu351
Gln mutation
yielded a 92-kDa band only (Fig. 2, lane 6).

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Fig. 2.
Immunoblot to assay for autocleavage of ADAM
12-S proenzyme. ADAM 12-S protein was prepared in serum-free
medium, concentrated, and incubated for 16 h at 37 °C in 100 mM NaCl, 50 mM Tris (pH 7.4), 10 mM
CaCl2, 1 mM ZnCl2, and 0.02%
sodium azide. NEM at a concentration of 1 mM was included
for the samples in lanes 2 and 4. Samples were
analyzed on a 6% polyacrylamide gel and detected with monoclonal
antibody 14E3. Plasmid 1197 codes for full-length ADAM 12-S with a
mutated furin cleavage site. Plasmid 1376 codes for the corresponding
polypeptide where proteolytic activity has been ablated by a
Glu351 Gln mutation. Plasmid 1336 codes for ADAM 12-S
with a mutated furin cleavage site and a Cys179 His
mutation in the cysteine switch. Plasmid 1377 codes for the
corresponding polypeptide where proteolytic activity has been ablated
by a Glu351 Gln mutation.
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The Prodomain Is Required for Secretion of ADAM 12 Protease--
When wild-type ADAM 12-S is expressed in COS-7 cells, a
steady-state level of the 92-kDa proenzyme can be detected in cell extracts by Western blotting; these are presumably polypeptides that
have not yet reached the trans-Golgi, where a furin-like protease removes the prodomain (Fig.
3A, lane 1). If an ADAM 12-S protein lacking the prodomain is synthesized (encoded by plasmid 1229),
it accumulates in the cell and is not secreted (Fig. 3A, lanes
2 and 5). Retention of this polypeptide is related to
the metalloprotease domain because an ADAM 12-S polypeptide lacking both the prodomain and the metalloprotease domain is secreted efficiently (Fig. 3A, lanes 3 and 6). To
determine in which subcellular compartment the polypeptide encoded by
construct 1229 was located, we analyzed the glycosylation pattern of
intracellular and secreted ADAM 12 proteins. ADAM 12-S has five
predicted N-glycosylation sites, two of which are in the
prodomain (21). Processed and secreted 68-kDa ADAM 12-S protease is
glycosylated, as evidenced by increased mobility on SDS-PAGE after
treatment with N-glycosidase F (Fig. 3B, lanes 1 and 2). These carbohydrates are resistant to removal by
endoglycosidase H, as expected for a protein that has traversed the
Golgi (Fig. 3B, lane 3). ADAM 12-S protein synthesized without the prodomain (plasmid 1229) is endoglycosidase H-sensitive and
therefore has not been processed by enzymes in the medial Golgi (Fig.
3B, lanes 4-6). We conclude that the prodomain is required
for translocation from the ER to the Golgi apparatus, possibly by
assisting the metalloprotease domain in folding to a
secretion-competent conformation.

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Fig. 3.
Immunoblots of intracellular and secreted
ADAM 12-S polypeptides. COS cells were transfected with plasmids
coding for full-length wild-type (wt) ADAM 12-S (1151), ADAM
12-S lacking the prodomain (1229), or ADAM 12-S lacking both the
prodomain and the metalloprotease domain (1095). Panel
A, ADAM 12 polypeptides located intracellularly (lanes
1-3) and in the medium (lanes 4-6) were analyzed on
an 8% polyacrylamide gel followed by Western blotting with monoclonal
antibody 14E3. Panel B, lane 1, shows ADAM 12-S
protein secreted by COS cells after transfection with plasmid 1151. Lane 4 shows ADAM 12-S protein located intracellularly after
transfection with plasmid 1229. In lanes 2 and 5 the samples were digested with N-glycosidase F
(N-GlycoF) before electrophoresis. In lanes 3 and
6, the samples were treated with endoglycosidase H
(Endo H). Proteins were analyzed on a 6% polyacrylamide
gel.
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Testing Potential Inhibitors of ADAM 12 Protease--
Regulation
of ADAM 12 protease activity intracellularly appears to be carried out
by the cysteine switch of the prodomain, but cleavage of the prodomain
by furin means that ADAM 12 is secreted as a constitutively active
protease. Are there specific inhibitors in the extracellular
environment which take over the function of regulating ADAM 12 after
removal of the prodomain? It is well established that MMPs are
regulated by a group of physiological inhibitors, the TIMPs (7, 27). It
had long been assumed that the specificity of TIMPs was restricted to
inhibition of MMPs, but recently TIMP-3 has been shown to inhibit ADAM
17 protease (28). We tested the ability of recombinant TIMPs -1, -2, and -3 to inhibit ADAM 12-S protease. 68-kDa active protease was
prepared by transfecting cells with plasmid 1151 and growing them in
serum-free medium. After incubation with inhibitor for 15 min, purified
human
2M was added, and the reaction was allowed to proceed for
16 h in the presence of the inhibitor. There was no inhibition by any of the TIMPs at concentrations up to 500 nM (Table
IV). Longer preincubation with TIMP-3, up
to 2 h before the addition of the substrate, had no effect.
Because MMPs are inhibited by picomolar concentrations of TIMPs, and
TIMP-3 inhibits the ADAM 17 protease with an apparent
Ki of 182 pM, we conclude that ADAM 12 is not specifically inhibited by TIMP-1, -2, or -3. It should be noted,
however, that these assays were carried out using
2M as the
substrate; it is conceivable that inhibition of ADAM proteases by TIMPs
is substrate-dependent. It will therefore be of interest to
test for inhibition by TIMPs once the physiological substrates of ADAM
12 protease are identified.
We tested two synthetic MMP inhibitors for their effect on ADAM 12 protease activity. Hydroxamate inhibitors such as BB-94 and BB-3103
inhibit MMPs in in vitro enzyme assays when present at low
nanomolar concentrations, and BB-94 inhibits ADAM 17 protease with an
apparent Ki of 0.54 nm (28). However, no effect on
ADAM 12 protease activity was observed with these hydroxamate inhibitors unless they were added at markedly higher concentrations (Table IV). This may reflect a difference in substrate specificity between ADAM 12 and ADAM 17.
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DISCUSSION |
The study of the ADAM family of metalloproteases is a relatively
young field, but the extensive research that has been carried out on
MMPs can serve as a basis for functional analyses of ADAMs. The most
intensively studied proenzyme in the metzincin superfamily is proMMP-3
(stromelysin-1) (7, 8). The three-dimensional structure has been
determined by x-ray diffraction analysis, and it supports the cysteine
switch model of activation in that the active site of the proenzyme is
filled by the propeptide, and Cys75 interacts directly with
the catalytic zinc ion (29). However, elimination of the cysteine
switch by alkylation of Cys75 does not activate the
proenzyme (15). There have been conflicting reports on the effect of
substituting another amino acid residue for Cys75. In some
expression systems this leads to activation of MMP-3, presumably by
autocatalysis (16, 30), but it has also been reported that the mutant
proenzyme remains latent (17). Alteration of amino acid residues in the
vicinity of Cys75 can lead to activation of proMMP-3, and
there is evidence that the amino-terminal region of the prodomain is
involved in maintenance of latency (16, 17, 30).
The experiments we describe in this paper demonstrate that
Cys179 is a key part of the latency mechanism of the ADAM
12 zymogen. Chemical modification of the thiol group or substitution of
another amino acid for Cys179 results in proteolytic
activity despite the presence of the prodomain. These results are fully
consistent with the cysteine switch model proposed in 1990, which
states that the protease is "off" when the prodomain cysteine
ligates the catalytic Zn2+, and the protease is "on"
when the cysteine is dissociated (10, 11). In view of the fact that
none of the amino acids in the vicinity of Cys179 is
essential for maintenance of latency, we suggest that a functional cysteine switch can be regarded as a prodomain scaffolding that positions the sulfur atom of a cysteine side chain in the immediate vicinity of the zinc ion. This model is supported by the observation that a mutant ADAM 12 prodomain that fails to repress protease activity
can be rescued by substituting a cysteine residue for another
amino acid in the propeptide (Table III).
The cysteine switch mechanism appears to be utilized only within the
metzincin protease family. Latency in other protease families generally
results from the propeptide rendering the active site sterically
inaccessible to substrates (8, 31). The latency mechanism of ADAM 12, as well as of most other metzincin proteases, can be viewed as having
two components: steric obstruction of the active site and coordination
of the catalytic Zn2+ by a prodomain cysteine residue. The
relative contribution of the two components to latency may vary from
protease to protease and might for example explain why proMMP-3 can
remain latent even after dissociation of cysteine from
Zn2+. In the case of ADAM 12, steric obstruction by the
prodomain is apparently not sufficient to inhibit the protease
completely in the absence of a cysteine switch. It should be stressed,
however, that the assays used in this study were carried out using
2M as the substrate, and thus there may be a partial inhibitory
effect that would be revealed by repeating these assays once the
natural substrate has been identified.
Chemical or genetic inactivation of the ADAM 12 cysteine switch yields
a proteolytically active proform that is not only capable of cleaving
an exogenous substrate, in this case
2M, but becomes autocatalytic,
in which case the prodomain itself becomes a substrate. We do not know
whether autocleavage plays any role in the mechanism for activation of
wild-type ADAM 12 proenzyme. Removal of the prodomain does not require
autocatalysis because as we have reported previously, the 92-kDa
proenzyme is processed to the 68-kDa form, even in a mutant ADAM 12 protein whose catalytic activity has been eliminated by replacing
Glu351 with Gln (6). However, it remains to be seen whether
ADAM 12 and other ADAM proteases carry out subsequent trimming of the NH2 terminus, either by autocatalysis or by intermolecular
reaction. It is known that the NH2-terminal amino acid
influences both activity and specificity of some members of the MMP
family of proteases, so the precise cleavage position of the propeptide
can be a critical determinant of protease function (7).
The cysteine switch may have evolved as a way of supplementing a
latency mechanism that relied solely on tight binding of the prodomain
to the protease domain. For proteases intended to be secreted in an
active form, reducing the affinity of the propeptide/catalytic domain
interaction would have a potential advantage in that it could allow for
more efficient dissociation of the propeptide after cleavage by furin.
By including a cysteine switch component in the latency mechanism, this
could be achieved without sacrificing efficiency of inhibition. In this
model, after cleavage of the polypeptide chain at the
propeptide/catalytic domain junction, the propeptide would not have
sufficient affinity for the catalytic domain to act as a competitive
inhibitor of the active enzyme. But when tethered to the protease
domain, before cleavage by furin, it would have sufficient affinity to
position the crucial cysteine residue for coordination of the active
site Zn2+. This cysteine switch would ensure that protease
activity was fully suppressed in the endoplasmic reticulum and Golgi
apparatus before the appropriate activation by furin in the
trans-Golgi immediately prior to secretion.
In addition to its function of repressing activity of the
metalloprotease domain, the ADAM 12 prodomain is required for secretion of the protease. Retention of ADAM 12-S protein lacking the prodomain in the lumen of the endoplasmic reticulum is not related directly to
its protease activity because when COS cells synthesize the same
protein with a Glu351
Gln mutation to eliminate
activity, this polypeptide is also retained.2 The most likely
explanation for retention is that the prodomain is required for proper
folding of newly synthesized ADAM 12 into an active protease. In the
absence of the prodomain, the misfolded ADAM 12 polypeptide would be
retained in the endoplasmic reticulum and ultimately degraded. We
performed
2M trapping assays on intracellular ADAM 12 lacking the
prodomain (coded for by plasmid 1229) and were unable to detect
protease activity, even though the assay is sensitive enough to detect
activity of intracellular ADAM 12-S proforms with a mutated cysteine
switch.2 This result is not unexpected because studies on
proteases from diverse families have consistently shown that the
prodomain is required for folding of a polypeptide into an active
protease (32-35).
An area that remains to be investigated is regulation of ADAM protease
activity in the extracellular environment. The presence of specific
physiological inhibitors would seem particularly important for
regulation of proteases that are secreted in an active form rather than
as zymogens. Based on structural homology, ADAM proteases fall into two
groups. One is comprised of ADAMs 10 and 17, and the other is comprised
of ADAM 12 along with nine other ADAMs whose activity has not yet been
demonstrated experimentally (5, 6, 36). TIMP-3 is a candidate for
regulation of ADAMs 10 and 17 in vivo because it inhibits
ADAM 17 efficiently in in vitro assays (28). The failure of
TIMPs-1, -2, and -3 to inhibit ADAM 12 protease in vitro
raises the possibility that there is an unidentified proteinaceous
inhibitor whose role is to regulate activity of the second group of
ADAM proteases, analogous to the role of TIMPs in regulating MMPs. An
alternative hypothesis is that cleavage of physiological substrates is
initiated by their binding to recognition sites in the ADAM 12 disintegrin or cysteine-rich domains and that inhibition by TIMPs
occurs by blocking this substrate-selection step rather than by direct
inhibition of the catalytic domain. Of relevance here is the fate of
ADAM prodomains after cleavage by furin. Is the role of the prodomain
restricted to folding of the protease and maintenance of latency prior
to cleavage by furin in the trans-Golgi? Or does the
propeptide acquire a new function after secretion into the
extracellular space, where it could potentially act as a competitive
inhibitor of one or more ADAM proteases?
ADAM prodomains are substantially larger than MMP prodomains (179 amino
acids for ADAM 12 versus 82 amino acids for MMP-3). This
could be an indication that the prodomain of an ADAM protein has
functions in addition to its role in folding and inhibition of the
metalloprotease domain. One could envision, for example, that an ADAM
prodomain is responsible for regulating the activity of the
disintegrin-like and cysteine-rich domains by blocking their binding
sites for potential ligands until the protein is secreted. Further
studies will be needed to test this possibility.
In conclusion, we have shown that the prodomain of human ADAM 12 modulates the activity of the metalloprotease domain by means of a
cysteine switch. The results of this study will be of use in future
investigations of latency in both ADAMs and MMPs, ultimately leading to
a better understanding of the biological relevance of such
molecular mechanisms.
 |
ACKNOWLEDGEMENTS |
We thank Gillian Murphy for the kind gift of
TIMPs-1, -2, and -3; Bent Børgesen for photographic assistance; and
Brit Valentin and Aase Valsted for technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Danish
Cancer Society, the Danish Medical Research Council, and the VELUX, Novo-Nordisk, Munksholm, Haensch, Thaysen, Wærum, Bojesen, Beckett, Hartmann, and Meyer Foundations (to U. M. W., and R. A.).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.
2
F. Loechel and U. M. Wewer, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
ADAM, a
disintegrin and metalloprotease;
2M,
2-macroglobulin;
MMP, matrix metalloprotease;
NEM, N-ethylmaleimide;
TIMP, tissue inhibitor of
metalloprotease;
PAGE, polyacrylamide gel electrophoresis.
 |
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