Metalloprotease-Disintegrin MDC9: Intracellular Maturation and
Catalytic Activity*
Monireh
Roghani
,
J. David
Becherer§,
Marcia L.
Moss§,
Ruth E.
Atherton¶
,
Hediye
Erdjument-Bromage**,
Joaquin
Arribas
,
R. Kevin
Blackburn§§,
Gisela
Weskamp
,
Paul
Tempst¶**, and
Carl P.
Blobel
¶¶¶
From the
Cellular Biochemistry and Biophysics
Program, ** Molecular Biology Program, Sloan-Kettering Institute,
Memorial Sloan-Kettering Cancer Center, ¶ Cell Biology and
Molecular Biology Program, Graduate School of the Cornell University
Medical College, New York, New York 10021, the
§ Department of Molecular Biochemistry and the
§§ Department of Analytical Chemistry, Glaxo
Wellcome Research and Development Inc., Research Triangle Park,
North Carolina 27709, and the 
Laboratori
de Recerca Oncologica, Hospital General, Psg. Vall d'Hebron, 08035 Barcelona, Spain
 |
ABSTRACT |
Metalloprotease disintegrins are a
family of membrane-anchored glycoproteins that are known to function in
fertilization, myoblast fusion, neurogenesis, and ectodomain shedding
of tumor necrosis factor (TNF)-
. Here we report the analysis of the
intracellular maturation and catalytic activity of the widely expressed
metalloprotease disintegrin MDC9. Our results suggest that the
pro-domain of MDC9 is removed by a furin-type pro-protein convertase in
the secretory pathway before the protein emerges on the cell surface.
The soluble metalloprotease domain of MDC9 cleaves the insulin B-chain,
a generic protease substrate, providing the first evidence that MDC9 is
catalytically active. Soluble MDC9 appears to have distinct specificities for cleaving candidate substrate peptides compared with
the TNF-
convertase (TACE/ADAM17). The catalytic activity of MDC9
can be inhibited by hydroxamic acid-type metalloprotease inhibitors in
the low nanomolar range, in one case with up to 50-fold selectivity for
MDC9 versus TACE. Peptides mimicking the predicted
cysteine-switch region of MDC9 or TACE inhibit both enzymes in the low
micromolar range, providing experimental evidence for regulation of
metalloprotease disintegrins via a cysteine-switch mechanism. Finally,
MDC9 is shown to become phosphorylated when cells are treated with the
phorbol ester phorbol 12-myristate 13-acetate, a known inducer of
protein ectodomain shedding. This work implies that removal of the
inhibitory pro-domain of MDC9 by a furin-type pro-protein convertase in
the secretory pathway is a prerequisite for protease activity. After
pro-domain removal, additional steps, such as protein kinase
C-dependent phosphorylation, may be involved in regulating
the catalytic activity of MDC9, which is likely to target different
substrates than the related TNF-
-convertase.
 |
INTRODUCTION |
Metalloprotease-disintegrin proteins (also known as
MDC1 proteins,
metalloprotease/disintegrin/cysteine-rich
proteins; ADAMs, a disintegrin
and metalloprotease(1)) are a family of
membrane-anchored glycoproteins that play a role in sperm-egg binding
and fusion (2-10), muscle cell fusion (11), neurogenesis, and
modulation of the Notch receptor signaling pathway in Drosophila
melanogaster and in Caenorhabditis elegans (12-16) and
processing of the pro-inflammatory cytokine TNF-
(17-19) (for
recent reviews see Refs. 1 and 20). Metalloprotease disintegrins are
usually comprised of several different protein modules as follows: an
N-terminal signal sequence is followed by a pro-domain, metalloprotease
domain, disintegrin domain, cysteine-rich domain, epidermal growth
factor repeat, transmembrane domain, and cytoplasmic tail (see Fig.
2A). About half of the currently known metalloprotease
disintegrins are predicted to be active metalloproteases due to a
catalytic site consensus sequence (HEXXH) in their
metalloprotease domain. The family members that lack a catalytic site
are not predicted to be active metalloproteases. Both catalytically
active and inactive metalloprotease disintegrins may play a role in
cell-cell and cell-matrix interactions (1, 20).
It has been postulated that catalytically active metalloprotease
disintegrins may have a role in the shedding or release of the
ectodomain of membrane-anchored precursor proteins (17, 18, 20).
Several different types of membrane proteins undergo protein ectodomain
shedding (21), including cytokines such as TNF-
(17, 18) and the kit
ligand (22), cytokine receptors like the TNF-
receptors (23, 24) and
the p75 nerve growth factor receptor (25), adhesion proteins such as
L-selectin (26, 27), and other proteins, including the
-amyloid precursor protein (28, 29), the angiotensin-converting
enzyme (30, 31), and the protein tyrosine phosphatases LAR and PTP
(32). Shedding of many of these protein ectodomains can be stimulated
by phorbol esters such as PMA and can be inhibited by hydroxamic
acid-based metalloprotease inhibitors (21).
In this study, we have evaluated the biosynthesis and in
vitro catalytic activity of the widely expressed metalloprotease disintegrin MDC9 (33). Our results demonstrate that the pro-domain of
MDC9 is removed in the secretory pathway, most likely by a furin-type
pro-protein convertase, and that MDC9 is phosphorylated when cells are
stimulated with PMA. Furthermore, this study provides the first
evidence that the soluble metalloprotease domain of MDC9 is
catalytically active, has a different peptide substrate specificity
than the related TNF-
convertase, and that the catalytic activity of
MDC9 can be inhibited by hydroxamic acid-based metalloprotease inhibitors. These results are discussed in the context of a potential role of MDC9 in protein ectodomain shedding.
 |
MATERIALS AND METHODS |
Cell Culture--
COS-7 cells were grown in Dulbecco's modified
Eagle's medium (10% fetal calf serum, 1% glutamine, 1%
penicillin/streptomycin). Transient transfections of COS-7 cells were
performed using LipofectAMINE (Life Technologies, Inc.) or via
electroporation (Bio-Rad Genepulser II).
Western Blot Analysis--
Cells were lysed in TBS containing
1% Nonidet P-40 and protease inhibitors (4) on ice for 5 min, and
centrifuged for 10 min in a Sorvall tabletop centrifuge. Cleared
lysates were mixed with sample loading buffer, heated for 5 min at
95 °C in 50 mM dithiothreitol, separated by SDS-PAGE,
and transferred to nitrocellulose. The nitrocellulose filters were
blocked with 5% reconstituted dry milk, incubated with primary and
secondary antibodies, and bound antibodies were visualized using the
ECL chemiluminescence detection kit as described (34). Where indicated,
the samples were deglycosylated with endoglycosidase H or PNGase F as
described previously (35).
Cloning and Site-directed Mutagenesis--
The cDNA encoding
the soluble MDC9 metalloprotease (consisting of nucleotides 1-1253
(33)) was synthesized as follows: a cDNA encoding for a Myc epitope
tag (EQKLISEEDL) and an XbaI restriction site was added to
the 3' end of the cDNA by polymerase chain reaction. The resulting
polymerase chain reaction product was cleaved with EcoRI and
XbaI and subcloned into a pcDNA3 vector (Invitrogen, San
Diego, CA). The transformer site-directed mutagenesis kit (CLONTECH Laboratories Inc.) was used to
introduce specific mutations, which were confirmed by DNA sequencing.
Cell-surface Biotinylation and
Immunoprecipitation--
Transfected COS-7 cells were grown to
confluency on 6-well plates, washed in PBS at 4 °C, and incubated
with the non-membrane-permeable biotinylation reagent NHS-LC-biotin
(Pierce) for 45 min on ice. After washing with 0.1 M
glycine in PBS, the cells were lysed directly on the dish with cell
lysis buffer (see above). Cleared cell lysates were subjected to
immunoprecipitation as described (33). The immunoprecipitated material
was separated by SDS-PAGE, transferred to nitrocellulose, probed with
horseradish peroxidase-coupled streptavidin, and detected using the ECL
chemiluminescence detection kit (Amersham Pharmacia Biotech).
Metabolic Labeling, Secretory Pathway Inhibitors, and Treatment
with Recombinant Furin--
Transfected COS-7 cells were incubated in
methionine/cysteine-free growth medium for 1 h and then
pulse-labeled for 20 min with 200 µCi/ml Pro-Mix Label (NEN Life
Science Products) (70% [35S]methionine and 30%
[35S]cysteine). After the cells were washed in PBS,
unlabeled growth medium was added for a chase period of up to 18 h. Where indicated, the secretory pathway inhibitors brefeldin A (5 µg/ml) and monensin (2, 10, and 25 µg/ml) were added during all
steps of the experiment prior to lysis. For in vitro furin
treatment, MDC9 precursor, immunoprecipitated from brefeldin A-treated
cells, was incubated with 3 or 6 units of recombinant furin (kindly
provided by Dr. R. Fuller) for 40 min at 37 °C in a modified furin
cleavage buffer (35, 36). All immunoprecipitated materials were
separated by SDS-PAGE. Following electrophoresis, the gels were fixed,
incubated in Enhance solution (DuPont), dried, and exposed to Kodak XAR autoradiography film.
Purification of Soluble MDC9-MP-Myc and N-terminal Sequence
Determination--
The soluble MDC9 metalloprotease was purified from
supernatants of transiently transfected COS-7 cells using the
anti-Myc-tag monoclonal antibody 9E10 coupled to protein G-Sepharose
beads (37). The beads were washed with PBS, and the bound protein was
eluted in 100 mM glycine, pH 3, neutralized immediately
with 1 M Tris/HCl, pH 8, and concentrated with a
Centricon-10 concentrator (Amicon). The N terminus of the purified
soluble MDC9 metalloprotease was determined as follows: after
separation by SDS-PAGE, the sample was transferred to a polyvinylidene
difluoride membrane (ProBlott, Applied Biosystems), stained with
Coomassie Blue R-250, destained in 50% methanol, 10% acetic acid, and
rinsed with doubly distilled H2O. The band of interest was
excised, and the N-terminal amino acid residues were analyzed by
automated Edman degradation, using an Applied Biosystems 477A
sequenator, with instrument and procedure optimized for femtomole level
analysis as described (38). The N-terminal sequence of the soluble MDC9
metalloprotease was identified as A206VLPQTR, which
corresponds to the sequence immediately following the putative
pro-protein convertase cleavage site RRRR205 in mouse MDC9
(33).
Insulin B-chain Cleavage Assay--
Purified soluble MDC9
MP-Myc, or recombinant purified TNF-
convertase (TACE) (18), or
purified recombinant MMP-1 (39) were incubated with 50 µM
oxidized insulin B-chain in 100 µl of reaction buffer (50 mM Tricine, pH 7.4, 200 mM NaCl, 10 mM CaCl2) at 37 °C for 1 or 6 h, in the
presence or absence of 10 mM 1,10-phenanthroline. The
reaction was stopped by addition of trifluoroacetic acid. The insulin
B-chain and its cleavage products were detected by matrix-assisted
laser-desorption/ionization time-of-flight (MALDI-TOF) mass
spectrometry using a Reflex III instrument (Bruker Franzen, Bremen,
Germany) as described (40).
Inhibitor Titration of MDC9 to Determine Enzyme
Concentration--
The MMP peptide substrate dinitrophenyl-proline
cyclohexylalanineGC(Me)HK(N-methylanthranilic
acid)NH2 (41), which is cleaved by MDC9 (see Table I), was
diluted from a Me2SO stock to 30 µM in 10 mM Hepes, pH 7.5, containing 0.0015% Brij-35 (Buffer A). 60 µl of substrate was then added to 30 µl of CGS27023 (a potent hydroxamic acid-based inhibitor of MDC9, see below and Table III) at
concentrations ranging from 0 to 10,000 nM. The reaction
was initiated by addition of MDC9 (10 µl), so that the final dilution was 100-3000-fold and the reactions were run for 1-4 h at 22 °C. Initial velocities were calculated by measuring the fluorescence units
of the reaction time and dividing by the fluorescence units when
complete turnover of the substrate was observed (final reaction time).
The initial velocities were plotted as a function of inhibitor concentration, and the curve was fit to Equation 1, a modified version
of the Morrison equation (42). As an approximation, the
Ki(app) was proposed to be comparable to
the Ki as the substrate concentration was at least
5-fold below the Km. Note: an absolute number for
the Km could not be determined as the substrate was
not soluble at high concentrations.
|
(Eq. 1)
|
|
(Eq. 2)
|
Incubation of Candidate Substrate Peptides with MDC9 and
Determination of Cleavage Sites and Kinetics--
Peptides
corresponding to 12 amino acid residues surrounding the membrane
proximal cleavage sites of selected proteins that are known to be
released from the plasma membrane by metalloproteases (see Table I)
were synthesized by Synpep (Dublin, CA). All peptides contained a
dinitrophenyl (Dnp) group at their N terminus to facilitate detection
at 350 nm. The peptide sequences and the reported cleavage sites are
presented in Table I. MDC9 (1 nM) was incubated with each
of the putative peptide substrates (30 µM) in 10 mM Hepes, pH 7.2, containing 0.0015% Brij (Sigma).
Reactions were timed to allow approximately 5-20% turnover of the
substrate. Reactions were quenched using 1% heptafluorobutyric acid,
and products were separated by high pressure liquid reverse phase
chromatography (C18 column, Vydac, Hisperia, CA) with absorbance
monitored at 350 nm. Turnover was quantitated by integrating peak areas
of the substrate and product(s). Liquid chromatography-mass
spectrometry was used to determine the masses of the products and
therefore the cleavage site(s) recognized by MDC9. Briefly, digestion
mixtures were passed over a hypersil C18 column, and after UV detection at 350 nm, the sample was routed into the ionspray source of a Sciex
API-III triple quadrupole mass spectrometer. Specificity constants were
calculated from initial velocities using the equation: kcat/Km = (% turnover/100)/([E]·time). Conditions of kcat/Km were verified by
running the reactions at more than one substrate concentration. The
enzyme concentration for MDC9 was determined as described above. For
the TNF and the MMP peptide substrates, two products were formed, and
individual specificity constants were determined for each of the
products since they were generated independently from one another (data
not shown).
Evaluation of the Inhibitory Potential of Cysteine Switch
Peptides--
Synpep Corp. (Dublin, CA) synthesized all of the
Cys-switch peptides (see Table IV). All solvents and buffers used were
sparged with argon to avoid oxidation of the thiol group. Peptides were dissolved in dimethyl formamide to yield a stock solution of 50 mM and were diluted as described below such that the final
concentration of dimethyl formamide in the assay was 1%. MDC9 activity
was measured using streptavidin-coated scintillation proximity assay
(SPA) beads and a biotinylated peptide corresponding to the sequence of
the cleavage site in pro-TNF-
. The digests were performed by the
addition of 25 µl of titrated inhibitor in 3% dimethyl formamide (10 mM Hepes, pH 7.5) to 25 µl of substrate
(biotin-SPLAQA*VRSSSRTP(3H) S-NH2) in Hepes
with 0.1% bovine serum albumin. Reactions were initiated by addition
of 25 µl of recombinant MDC9 or recombinant TACE (in Hepes with 0.1%
bovine serum albumin). The digestion was quenched by addition of
streptavidin SPA beads (Amersham Pharmacia Biotech) in EDTA. The final
concentration of substrate in the assay was 200 nM. Enzyme
was 1.0 nM, and cysteine-switch peptides were titrated from
100 µM with 3-fold dilution, 11 points per curve. Under
the above conditions the substrate concentrations are significantly
less than Km, and the Ki can be
determined directly by plotting percent inhibition versus
the log of the inhibitor concentration, where
Ki(app) = Ki (1 + [S]/Km). No effects on the Ki
were observed whether 0.5 mM cysteine was included or not.
Inhibition Constants Against Batimastat, Marimastat, CGS 27023, and TNF-
Protease Inhibitor--
Synthesis of batimistat, CGS
27023, and the TNF-
protease inhibitor has been described (18, 43).
The substrate Dnp-PChaGC(Me)HK(NMA)NH2 (41) was diluted
from a Me2SO stock solution to a concentration of 30 µM in buffer containing 10 mM Hepes, pH 7.5, and 0.0015% Brij-35 (Buffer A). A 60-µl aliquot was removed and
added to a black Polyfiltronics 96-well plate. Inhibitor was diluted
1/10 from 10 mM Me2SO stock solutions.
Three-fold serial dilutions in Me2SO were performed across
a Costar 96-well round bottom plate. The inhibitor was diluted 30-fold
into Buffer A. An aliquot of inhibitor, 30 µl, was then added to the
black Polyfiltronics 96-well plate. MDC9 was diluted 60-300-fold into
Buffer A, and 10 µl was added. Fluorescence was monitored at an
excitation of 343 nm and emission of 450 nm. Reactions were run from
0.5 to 4 h at 22 °C and liquid chromatography/mass spectrometry
analysis was performed to determine cleavage sites in the substrate.
In a similar manner, Ki values were determined for
19-kDa truncated collagenase (MMP-1), 20-kDa truncated collagenase 3 (MMP-13), stromelysin-1 (MMP-3), and 50-kDa truncated gelatinase B
(MMP-9) using the same fluorogenic substrate. Assays were conducted in
a total volume of 0.180 ml of assay buffer (200 mM NaCl, 50 mM Tris, 5 mM CaCl2, 10 µM ZnSO4, 0.005% Brij 35, pH 7.6) in each well of a black 96-well microtiter plate. 19-kDa collagenase-1, 20-kDa
collagenase-3, stromelysin-1, and 50-kDa gelatinase B concentrations were adjusted to 500 pM, 30 pM, 5 nM, and 100 pM, respectively. A dose response
was generated using an 11-point 3-fold serial dilution with initial
starting concentrations of 100, 10, or 1 µM. Inhibitor
and enzyme reactions were incubated for 30 min at room temperature and
then initiated with 10 µM fluorogenic substrate. The
product formation was measured after 45-180 min. Finally, Ki values for these inhibitors against TACE were
determined using the same SPA assay as described earlier for the
cysteine-switch peptides. In all cases, the conditions were such that
the substrate concentrations were significantly less than
Km, and the Ki can be determined
directly by plotting percent inhibition versus the log of
the inhibitor concentration, where
Ki(app) = Ki (1 + [S]/Km).
Phorbol Ester Treatment of CHO Cells--
A CHO cell line stably
expressing MDC9 or a control CHO cell line stably transfected with a
pcDNA3 vector alone was labeled either with 35S-Pro-Mix
Label (NEN Life Science Products) (70% [35S]methionine
and 30% [35S]cysteine) or incubated in the presence 1 mCi/ml of 32P for 3 h. Cells were then treated for 5 min in the presence or absence of 1 µM PMA.
Immunoprecipitated material was separated by SDS-PAGE and detected by
autoradiography as described above.
 |
RESULTS |
Intracellular Maturation of MDC9--
To evaluate the
biosynthesis and maturation of mouse MDC9 in the secretory pathway,
pulse-chase experiments were performed using transiently
transfected COS-7 cells expressing MDC9. Immunoprecipitations at
different time points show that MDC9 is first synthesized as a
precursor of 110 kDa (Fig. 1A)
which is later processed to an 84-kDa form. Only the processed form of
MDC9 with a molecular mass of 84 kDa is immunoprecipitated from
cell-surface biotinylated cells, demonstrating that pro-domain removal
most likely occurs intracellularly (Figs. 1C and
2B, and see Ref. 33). The subcellular localization of
pro-domain removal was further defined by assessing the effect of the
secretory pathway inhibitors brefeldin A and monensin on MDC9
processing. Brefeldin A, which blocks vesicle budding in the
endoplasmic reticulum (44), completely blocked processing of MDC9 at 5 µg/ml. Monensin, which is thought to prevent transport past the
medial-Golgi apparatus (45), partially blocked MDC9 processing at 2 and
10 µg/ml and completely blocked processing at 25 µg/ml (Fig.
1B). Western blot analysis of an endoglycosidase H-treated
sample of MDC9-expressing COS-7 cells showed that both the precursor
and mature forms of MDC9 were sensitive to endo H (Fig. 1C, lane
2). Treatment of an identical sample with PNGase, which removes
most or all N-linked carbohydrate residues, resulted in a
faster migrating mature form of MDC9 compared with the endo H-treated
sample. In contrast, the MDC9 precursor appeared to comigrate in the
endo H- and PNGase-treated samples. The cell surface-labeled processed
form of MDC9 displayed a similar change in migration on SDS-PAGE after
endo H and PNGase treatment compared with the processed form of MDC9
detected by Western blot. This result suggests that at least one, but
not all, of the N-linked carbohydrate moieties of mature
MDC9 acquires resistance to endo H treatment through the conversion of
high mannose glycans into complex carbohydrates in the medial-Golgi
network. Taken together, these results suggest that MDC9 is
proteolytically processed in the secretory pathway after passage
through the medial-Golgi apparatus.

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Fig. 1.
Pulse-chase analysis of MDC9 maturation in
COS-7 cells and in vitro cleavage by recombinant
furin. A, MDC9 was immunoprecipitated from transiently
transfected COS-7 cells with antibodies against the MDC9 cytoplasmic
tail at different time points. B, comparison of MDC9
immunoprecipitated from transfected COS-7 cells immediately after pulse
labeling (lane 1) and after a 3-h chase (lane 2),
with MDC9 immunoprecipitated after a 3-h chase in the presence of the
secretory pathway inhibitors brefeldin A (5 µg/ml, lane 3)
or monensin (2, 10, and 25 µg/ml, lanes 4-6).
C, Western blot of (upper panel) and
immunoprecipitation from cell-surface biotinylated COS-7 cells
(lower panel)-expressing MDC9. Prior to electrophoresis, the
samples were deglycosylated with endo H (lane 2), or with
PNGase F (lane 3), or mock-incubated as control (lane
1). In both cases the primary antibody was against the MDC9
cytoplasmic tail. D, MDC9 immunoprecipitated after a pulse
label and 3-h chase from untreated COS-7 cells expressing MDC9
(lane 1) was compared with the products resulting from
treatment of the MDC9 precursor with 3 units/20 µl or 6 units/20 µl
of recombinant furin (lanes 3 and 4,
respectively). The MDC9 precursor was immunoprecipitated from cells
incubated with brefeldin A during the 3-h pulse (lane
2).
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Since MDC9 contains a consensus cleavage sequence for the pro-protein
convertase furin between its pro- and metalloprotease domain, and
because furin resides predominantly in the trans-Golgi network (45),
furin or related pro-protein convertases are good candidates for
processing MDC9 in the secretory pathway. To provide direct evidence
for a potential role of furin in processing MDC9, we immunoprecipitated
the precursor of full-length MDC9 from brefeldin A-treated COS-7 cells
and incubated it with recombinant furin (36) (Fig. 1D). The
furin-treated MDC9 precursor co-migrated with the MDC9 as it is
processed in vivo in COS-7 cells. Increasing amounts of
furin also generated an additional faster migrating band, perhaps due
to an additional cleavage site that is not accessible to the
membrane-anchored form of furin or a related pro-protein convertase
in vivo. Alternatively, this site may be less efficiently cleaved and therefore only utilized when relatively high concentrations of furin are added in vitro.
To evaluate the sequence requirements for intracellular processing and
metalloprotease activity of MDC9, the following mutations were
introduced into both a soluble and a membrane-anchored form of MDC9 by
site-directed mutagenesis (see Fig.
2A): 1) removal of the furin
cleavage site (
4R) between the pro- and metalloprotease domains; 2)
conversion of the putative pro-domain cysteine-switch residue (46, 47),
which is predicted to inhibit the protease during biosynthesis by
binding to the active site, into an alanine (pro-Cys
Ala); 3)
mutation of the "catalytic" glutamic acid in the metalloprotease
active site (MP-Glu
Ala) (48).

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Fig. 2.
Processing of full-length and soluble MDC9
mutants. A, diagram of the domain organization of MDC9
and other metalloprotease disintegrins, and of the mutations generated
for this study. B, Western blot (top panel), or
immunoprecipitation of cell-surface biotinylated material (bottom
panel) from COS-7 cells transfected with vector alone (lane
1), or expressing full-length wild-type MDC9 (lane 2),
MDC9 lacking the predicted furin cleavage site ( 4R, lane
3), MDC9 with a catalytic site mutation (MP Glu Ala,
lane 4) and MDC9 with a pro-domain cysteine-switch mutation
(pro-Cys Ala, lane 5). Lane 6 shows a Western
blot of a separate sample of wild-type full-length MDC9 expressed in
COS-7 compared with the 4R mutant (lane 7), which were
separated on a 7.5% SDS-gel to better resolve subtle differences in
their relative motility. A rabbit polyclonal antibody against the MDC9
cytoplasmic domain was used as primary antibody. C, Western
blots of cell lysates (top panel) and of culture
supernatants (bottom panel) of COS-7 cells transfected with
the vector (lane 1), or expressing soluble wild-type MDC9
(lane 2), or soluble MDC9 4R (lane 3), pro-Cys
Ala (lane 4), or MP Glu Ala (lane 5). The
9E10 monoclonal antibody against the Myc tag was used as primary
antibody. Pro-MP refers to the soluble precursor protein, whereas MP
refers to the processed metalloprotease domain lacking the pro-domain.
The 51- and 80-kDa proteins that are also present in the nontransfected
control lane most likely represent proteins with which the 9E10
monoclonal antibody against the Myc tag cross-reacts. D,
material resulting from affinity purification of MDC9 was separated
under non-reducing conditions by SDS-PAGE and stained with Coomassie
Brilliant Blue. IgG indicates immunoglobulin that coelutes from an
affinity column with the 9E10 monoclonal antibody. N-terminal sequence
analysis of the soluble MDC9 metalloprotease (MP) confirmed
the identity of the purified protein and demonstrated that pro-domain
removal had occurred by cleavage next to the furin cleavage site (RRRR,
see "Materials and Methods"). DTT, dithiothreitol;
WT, wild type.
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The effects of the mutations on intracellular processing were first
tested by comparing the relative amounts of MDC9 precursor and
processed forms in Western blots and in cell-surface biotinylated samples (Fig. 2B). On Western blots, the 110-kDa precursor
of MDC9 and the processed form of 84 kDa can both be detected (Fig. 2B). However, only the processed form of 84 kDa can be
immunoprecipitated from cell surface-labeled samples (Figs.
1C and 2B, see also Ref. 33). Both removal of the
predicted furin cleavage site, and the Glu
Ala mutation in the
catalytic site did not detectably affect MDC9 processing and appearance
on the cell surface (Fig. 2B). However, removal of the
putative cysteine-switch residue in the pro-domain led to a dramatic
decrease of the processed 84-kDa form of MDC9 in the cell lysate as
well as on the cell surface. The
4R and the Pro-Cys
Ala
mutations also resulted in small amounts of additional faster migrating
bands compared with wild-type MDC9, which most likely result from
aberrant processing.
Similar results were obtained with a recombinant soluble pro- and
metalloprotease domain of MDC9 (Fig. 2C). The wild-type soluble protein and all three mutant forms were detected as proteins of
~47 kDa in Western blots of COS-7 cell lysates (Fig. 2A,
predicted molecular mass of soluble wild-type MDC9 is 44.29 kDa). The
supernatant of cells expressing the soluble wild-type and the MP Glu
Ala mutant contained mainly a ~28-kDa protein, suggesting that
the pro-domain had been removed intracellularly (Fig. 2C, lower
panel, lanes 2 and 5). N-terminal sequence analysis of
the affinity purified soluble wild-type MDC9 metalloprotease domain
(Fig. 2D, lane 1) confirmed that processing had occurred
immediately after the predicted pro-protein convertase cleavage site in
MDC9 (RRRR205-AVLPQTR, see "Materials and Methods").
Soluble MDC9
4R was secreted both as an unprocessed precursor of 47 kDa and as a processed form of ~33 kDa (Fig. 2C, lane 3).
Evidently processing of MDC9 can occur in the absence of a furin
cleavage site at an adjacent position. This finding prompted a
re-evaluation of the processing of the full-length MDC9
4R mutant by
comparing its migration to that of the wild-type MDC9 on a Western blot
of proteins that had been separated on a 7.5% SDS-polyacrylamide gel.
Under these conditions the full-length
4R mutant migrated slightly
slower than the wild-type protein (Fig. 2B, lanes 6 and
7), consistent with a cleavage at an adjacent but distinct
site. Similar to the behavior of the full-length pro-Cys
Ala
mutant, very little of the soluble pro-Cys
Ala MDC9 mutant could be
detected in the supernatant, although the protein was clearly present
in the cell lysate (Fig. 2C, lane 4).
Catalytic Activity of Soluble MDC9, TACE, and MMP-1 on the Insulin
B-chain--
To determine whether MDC9 is catalytically active, the
soluble wild-type metalloprotease was affinity purified from COS-7 cell
supernatants (Fig. 2D). The affinity purified material was incubated with the insulin B-chain peptide, a generic protease substrate consisting of 30 amino acid residues (49). The resulting insulin B-chain fragments were identified by mass spectrometry (Fig.
3, A-C). After 1-h incubation
with MDC9, cleavage between insulin B-chain residues Tyr16
and Leu17, and Tyr26 and Thr27
could be detected (Fig. 3A). After 6 h, the substrate
had been completely converted into products resulting from cleavage
between Tyr16 and Leu17, and Tyr26
and Thr27. No processing was seen after 6 h in the
presence of the metalloprotease inhibitor 1,10-phenanthroline (Fig.
3A) or after co-incubation of the insulin B-chain for 6 h with a similar amount of the MP Glu
Ala catalytic site mutant
purified under identical conditions (data not shown). When the insulin
B-chain was incubated with purified soluble TACE (Fig. 3B)
or MMP-1 (Fig. 3C) under identical conditions, in each case
only one cleavage between Tyr16 and Leu17 was
observed. As would be expected, 1,10-phenanthroline inhibited TACE- and
MMP-1-mediated cleavage of the insulin B-chain (Fig. 3, B
and C). Under the conditions used in this study, the
apparent cleavage specificity of MDC9, TACE, and MMP-1 on the insulin
B-chain is thus more restricted than what has been reported for most
other proteases, including related snake venom metalloproteases (49, 50).

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Fig. 3.
MALDI-TOF analysis of insulin B-chain
cleavage products produced by soluble MDC9. Matrix-assisted
laser-desorption/ionization time-of-flight (MALDI-TOF) mass
spectrometric traces of products generated from incubating the oxidized
insulin B-chain with affinity purified soluble MDC9 for 1, 6, or 6 h in the presence of 1,10-phenanthroline (A); recombinant
TACE (B); and MMP-1 for 6 h or 6 h in the presence
of 1,10-phenanthroline (C). The numbers
above the major peaks indicate the experimental monoisotopic
masses (m/z). D, sequence of the insulin B-chain
and the predicted protonated monoisotopic masses of the oxidized
insulin B-chain and of the corresponding cleavage products. The
predicted protonated monoisotopic masses were calculated with Procomp
software, assuming that both cysteine residues in the insulin B-chain
have the mass of the oxidized form of cysteine, cysteic acid
(C(O3H)). The cleavage sites are marked by
arrowheads.
|
|
Kinetic Analysis of Candidate Substrate Peptide Cleavage by
MDC9--
Several different types of membrane proteins are known to be
released from the plasma membrane by metalloproteases. To evaluate further the cleavage specificity of MDC9 and to test whether MDC9 might
in principle have a role in protein ectodomain shedding, the soluble
MDC9 metalloprotease was incubated with peptides corresponding to
cleavage sites of specific membrane proteins that are known to be
released from the membrane by metalloproteases (21). Table I shows that MDC9 was able to cut the
cleavage site peptide of the
-amyloid precursor protein (
-APP)
(28), tumor necrosis factor
(TNF-
) (51), the p75 TNF receptor
(p75-TNFR) (52), and the c-kit ligand-1 (KL-1) (53). To determine the
cleavage sites, the fragments of peptides that were processed by
affinity purified MDC9 were analyzed by mass spectrometry. As indicated in Table I, MDC9 did not cleave any of the peptides tested here at the
same site that is known to be used by sheddases in cells in
vivo. The c-kit ligand and TNF-
peptide were cut in more than one position by recombinant MDC9. Furthermore, we found that affinity purified MDC9 was able to process an MMP substrate peptide that is not
processed by TACE.2 Finally,
affinity purified soluble MDC9 did not process cleavage site peptides
for the interleukin-6 receptor (54), the p55 TNF receptor (p55-TNFR)
(55), pro-transforming growth factor
(TGF-
) (56), and
L-selectin (55) under the conditions used here.
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Table I
Peptides mimicking the membrane-proximal cleavage site of proteins
known to be shed by metalloproteases were synthesized and evaluated
for cleavage by MDC9
An asterisk denotes the predicted cleavage site based on published
studies (see "Results" for references). Those peptides that were
resistant to MDC9 proteolysis are denoted by minus. For those peptides
where multiple cleavages occurred, the products are listed in order of
abundance with P1 > P2 > P3.
|
|
The cleavage kinetics of the
-APP peptide, the TNF-
peptide, and
also of the MMP peptide by MDC9 were determined as described under
"Materials and Methods." The kinetic analysis revealed that the
kcat/Km of
-APP cleavage
by MDC9 is 4.3-fold higher than the
kcat/Km for cleavage of the
P1 site in TNF-
, 6.8-fold higher than the
kcat/Km for cleavage of the P2 site in TNF-
, and 21-fold higher than the
kcat/Km for cleavage of the
MMP substrate peptide by MDC9 (Table
II).
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Table II
kcat/Km values were determined, as described under
"Materials and Methods," for three of the peptides listed in Table
I
%cv indicates the coefficient of variation in percent.
|
|
Hydroxamic Acid Derivatives and Cysteine-switch Peptides as
Inhibitors of MDC9 Metalloprotease Activity--
Since TACE can be
inhibited by hydroxamic acid-based metalloprotease inhibitors, we asked
whether (a) hydroxamic acid derivatives are also inhibitors
of the MDC9 metalloprotease activity, and (b) whether there
is some specificity of four previously described hydroxamic acid-based
inhibitors toward MDC9 or TACE. Table III shows the Ki of different inhibitors toward TACE and MDC9, as well as toward the matrix-type metalloproteases MMP-1, MMP-3,
MMP-9, and MMP-13. Under the in vitro cleavage conditions used here, CGS 27023 was the most selective for MDC9 over TACE (~50-fold), whereas marimastat was the most selective for TACE over
MDC9 (~12-fold). These data further demonstrate that MDC9 has a
unique inhibitor profile which does not resemble that of TACE or any of
the MMPs tested here.
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Table III
Inhibition constants were determined for each enzyme using the MMP
fluorescent substrate assay
For TACE, which does not cleave the MMP fluorescent substrate, the TNF
SPA peptide assay was used instead (see "Materials and Methods").
|
|
Metalloprotease disintegrins are predicted to be kept inactive during
biosynthesis via a cysteine-switch mechanism (46, 47). The predicted
cysteine-switch residue is defined as an odd-numbered cysteine that is
only present in the pro-domain of metalloprotease disintegrins that
contain a catalytic site but not in those that lack a catalytic site.
To provide experimental support for this hypothesis, we examined the
inhibitory potential of peptides mimicking the predicted
cysteine-switch sequence of human or mouse MDC9, human TACE, human
ADAM10 (KUZ/MADM), and mouse ADAM12 (meltrin
) on the catalytic
activity of mouse MDC9 and human TACE. As shown in Table
IV, the TACE and mouse MDC9 cysteine-switch peptides were the best inhibitors of TACE and MDC9. The
human MDC9 cysteine-switch peptide, which differs from the mouse
peptide in one amino acid residue, inhibited both proteases somewhat
less efficiently. The cysteine-switch peptides of ADAM10 and ADAM12 did
not significantly inhibit TACE or MDC9 up to a concentration of 100 µM.
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Table IV
IC50 (µM) values for various Cys-switch peptides
were determined against TACE and MDC9 using the SPA TNF peptide assay
(see "Materials and Methods")
(>150) denotes those peptides for which an IC50 could not be
determined but where the IC50 is higher than 150 µM since in all cases inhibition of the enzyme by these
Cys peptides was less than 20% at 100 µM. h, human; m,
mouse.
|
|
Phorbol Ester-dependent Phosphorylation of
MDC9--
Metalloprotease-induced protein ectodomain shedding of a
large variety of cell-surface proteins, including cytokines, cytokine receptors, adhesion proteins, and the
-amyloid precursor protein, can be triggered by addition of the phorbol ester PMA to cells, presumably by activating protein kinase C (21, 57). A CHO cell line
stably expressing MDC9 (see Fig. 4,
lane 2) was labeled with 32P in the absence or
presence of the phorbol ester PMA. MDC9 immunoprecipitated from these
cells was only found to be labeled with 32P in extracts of
PMA-treated cells (Fig. 4, lane 5) but not in unstimulated
cells (Fig. 4, lane 4).

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Fig. 4.
Phorbol ester-dependent
phosphorylation of MDC9. Immunoprecipitation of MDC9 from
[35S]Met/Cys-labeled (lane 2) or from
32P-labeled CHO cells stably expressing MDC9, incubated in
the presence (lane 5) or absence of PMA (lane 4).
Control immunoprecipitations were performed on CHO stably transfected
with the expression vector and labeled with 35S (lane
1) or 32P (lane 3).
|
|
 |
DISCUSSION |
This study provides the first analysis of the intracellular
maturation and catalytic activity of the metalloprotease disintegrin MDC9. Several lines of evidence demonstrate that MDC9, like MDC15 (35),
is processed in a late compartment of the secretory pathway, most
likely by a pro-protein convertase such as furin. First, processing of
MDC9 can be prevented by addition of the secretory pathway inhibitors
brefeldin A (44) and monensin (45), suggesting that MDC9 is processed
after passage through the medial-Golgi apparatus. Second, incubation of
pro-MDC9 with recombinant furin in vitro results in
pro-domain removal. Third, N-terminal sequence analysis of a soluble
secreted MDC9 metalloprotease domain confirmed that processing occurs
next to the predicted furin cleavage site (RRRR) in cells. Finally,
after deletion of the furin cleavage site, both full-length
membrane-anchored MDC9 and a soluble pro- and metalloprotease-domain
construct appear to be processed at a different site than the wild-type
proteins. Nevertheless, removal of the furin site did not noticeably
affect the efficiency of processing of full-length membrane-anchored
MDC9. One explanation for this result could be that additional
cleavages in the MDC9 pro-domain normally occur after processing at the
furin site, something that has also been observed for mMDC15 (35). Such additional cleavage sites in the pro-domain may facilitate its removal
after cleavage by furin and may only become apparent after the furin
cleavage site is eliminated.
The first evidence for catalytic activity of MDC9 was obtained by
incubating affinity purified soluble MDC9 with the insulin B-chain, a
generic protease substrate. Our results demonstrate that MDC9, TACE,
and MMP-1 can all cleave the insulin B-chain, albeit with somewhat
different specificity. Cleavage of the insulin B-chain and of the
general protease inhibitor
2-macroglobulin (58) may thus
be useful initial assays to assess the catalytic activity of other
metalloprotease disintegrins.
Because metalloprotease disintegrins have been hypothesized to function
in protein ectodomain shedding, we evaluated the ability of soluble
MDC9 to cleave selected candidate substrate peptides that mimic the
cleavage sites of proteins that are shed from the plasma membrane by a
metalloprotease activity. Of the eight peptides tested here, the
-APP peptide was the most efficiently cleaved, followed by the
pro-TNF-
, p75-TNFR, and KL-1 peptides. MDC9 did not process peptides
mimicking the cleavage site of the p55-TNFR, L-selectin,
pro-TGF-
, and the interleukin-6 receptor. Mass spectrometric analysis of the products generated by MDC9 revealed that none of the
four substrate peptides were cleaved at the site that is used during
release of the corresponding protein from cells. Therefore MDC9 is most
likely not the secretase responsible for processing the proteins that
were included in the present study. Indeed, TACE can process TGF-
,
L-selectin, and p55-TNFR peptides in the correct position
in vitro.3
Furthermore, a targeted deletion of TACE results in the inability of
cells to release TNF-
, TGF-
, and L-selectin from the
cell surface, although it remains formally possible that some of these substrates are cleaved by another protease that needs TACE to become
activated (17, 59).
With respect to
-APP processing, TACE is apparently the major
protein kinase C-dependent
-secretase in cultured cells,
and it also cleaves the
-APP peptide at the major
-secretase site in vitro (28, 60). Although MDC9 clearly does not target the major
secretase site in vitro, in light of the
relatively efficient processing of the
-APP peptide observed here it
will be interesting to determine whether MDC9 may have some role in
-APP processing, perhaps restricted to certain cell types. In this
context it is worth pointing out that MDC9 cleaves
-APP within a
peptide sequence (HHQK) which apparently plays a role in inducing
neurotoxic microglia (61). Finally, we note that a standard MMP peptide
substrate is cleaved in a similar fashion by MDC9 and MMPs -1, -3, -9, and -13, but not by TACE, and that MDC9 cleaves the TNF-
peptide at
the same two sites as MMP-1 and
MMP-9.4 It will thus also be
interesting to evaluate whether MDC9 may have substrates in the
extracellular matrix in vivo.
These results provide the first evidence for differences in the
substrate specificity and cleavage site selection between two distinct
metalloprotease disintegrins, at least as far as soluble peptide
substrates and soluble proteases are concerned. In addition to any
inherent substrate specificity of metalloprotease disintegrins in
vitro, additional factors may determine which substrates are
cleaved in vivo. These determinants may include potential
targeting or recognition events between the substrate and other parts
of the metalloprotease disintegrin protein, such as the disintegrin
domain, cysteine-rich region, or epidermal growth factor repeat (20).
Furthermore, access to substrates may also depend on regulated or
constitutive co-localization in the same subcellular compartment(s)
(35).
An evaluation of the inhibitory potential of different hydroxamic
acid-based metalloprotease inhibitors, which are known to potently
inhibit TACE (17, 18, 62), revealed that these compounds are also
potent inhibitors of MDC9 and of certain matrix-type metalloproteases.
Furthermore, we observed differences in the selectivity of the
compounds tested here toward TACE or MDC9. An extension of this
approach may therefore yield even more selective inhibitors of these or
other metalloprotease disintegrins.
To address whether removal of the prodomain, for example by furin, is a
prerequisite for catalytic activity (33, 35, 63, 64), we tested whether
peptides corresponding to the cysteine-switch sequence of various
metalloprotease disintegrins inhibit the catalytic activity. In matrix
type metalloproteases (46, 47) and the snake venom metalloproteases
adamalysin II (47), the free sulfhydryl of the cysteine-switch residue
in the pro-domain is thought to bind to the Zn2+ ion in the
catalytic site. Our results suggest that MDC9 and TACE are indeed both
inhibited by their respective cysteine-switch peptides. The inhibitory
potential of the mouse MDC9 cysteine-switch peptide for mouse MDC9 is
similar to that of the adamalysin II cysteine-switch peptide for
adamalysin II (47). The relatively potent inhibition of mouse MDC9 by
its cysteine-switch peptide may explain why mutating the
cysteine-switch residue strongly decreased transport of full-length and
soluble MDC9 to the cell surface. In analogy to the protease
subtilisin, where the pro-domain is thought to function as
intramolecular chaperone (65), the interaction of the free sulfhydryl
residue with the active site may be necessary for the pro- and
metalloprotease domains of MDC9 to fold properly. If so, removing the
cysteine-switch residue might prevent productive folding, resulting in
retention of the mutant protein by endoplasmic reticulum-resident
chaperones and subsequent degradation (66). In contrast to MDC9,
mutating the cysteine-switch residue in soluble human ADAM12 (meltrin
) did not affect its secretion (58), suggesting differences in the contribution of the cysteine-switch peptide to how these two proteins fold.
Finally, we demonstrate that MDC9 is phosphorylated after addition of
PMA to cells. Since protein ectodomain shedding can be triggered with
phorbol esters (21, 57), this result raises the intriguing possibility
that the metalloprotease activity of MDC9 could be regulated by
inside-out signaling due to protein kinase C-dependent
phosphorylation of the cytoplasmic tail. However, since PMA stimulation
can result in the phosphorylation of a variety of different proteins,
the physiological significance of this result remains to be determined.
In conclusion, this study demonstrates that MDC9 is made as a precursor
which is processed by a pro-protein convertase in the secretory
pathway. Processing removes the presumably inhibitory pro-domain of
MDC9 and is thus most likely a prerequisite for the protease to become
active. After pro-domain removal, there may be additional mechanisms to
regulate protease activity, such as phosphorylation of the cytoplasmic
tail by protein kinase C. Furthermore, this study provides the first
evidence that MDC9 is catalytically active, that it has a different
substrate specificity than TACE, and that it can be inhibited by
hydroxamic acid-based inhibitors. The catalytically active
metalloprotease domain of MDC9 will be an important tool for further
biochemical and functional analysis of this widely expressed
metalloprotease disintegrin protein.
 |
ACKNOWLEDGEMENTS |
We thank Dr. R. Fuller for kindly providing
recombinant furin; Dr. J. E. Rothman for aliquots of the 9E10
anti-Myc tag monoclonal antibody; Dr. P. C. Andrews for providing
the Procomp software; Dr. R. Andrews and C. Haffner at Glaxo Wellcome
for the synthesis of the hydroxamate inhibitors used in this study; and
L. Lum and J. Schlöndorff for comments on the manuscript.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant R55GM51988, by a grant from Glaxo Wellcome (to
C. P. B.), by National Science Foundation Grant DBI-9420123 (to
P. T.), and by the Memorial Sloan-Kettering Cancer Center Support
Grant NCI-P30-CA-08748.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 in part by the Ford Foundation.
¶¶
To whom correspondence should be addressed: Cellular
Biochemistry and Biophysics Program, Sloan-Kettering Institute,
Memorial Sloan-Kettering Cancer Center, Box 368, 1275 York Ave., New
York, NY 10021. Tel.: 212-639-2915; Fax: 212-717-3047; E-mail:
c-blobel{at}ski.mskcc.org.
The abbreviations used are:
MDC, metalloprotease/disintegrin/cysteine-rich
proteins; ADAMs, a disintegrin
and metalloprotease; TNF, tumor necrosis
factor; TNFR, TNF receptor; PMA, phorbol 12-myristate 13-acetate; TACE, TNF-
convertase;
-APP,
-amyloid precursor protein; PAGE, polyacrylamide gel electrophoresis; PNGase F, peptide
N-glcosidase; PBS, phosphate-buffered saline; TGF-
, transforming growth factor
; MMP, matrix metalloproteinase; MP, metalloprotease; endo H, endoglycosidase H; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; SPA, scintillation proximity assay; Dnp, dinitrophenyl; MALDI-TOF, matrix-assisted laser-desorption/ionization time-of-flight; CHO, Chinese hamster ovary.
2
M. Moss, submitted for publication.
3
R. Black, personal communication.
4
M. Moss, unpublished observation.
 |
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