From the Department of Biochemistry and Molecular
Biology, University of Miami School of Medicine, Miami, Florida 33101 and the § Kennedy Institute of Rheumatology, Imperial
College School of Medicine, 1 Aspenlea Road, Hammersmith, London W6 8LH
United Kingdom
Received for publication, November 30, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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The proteoglycan aggrecan is an important
major component of cartilage matrix that gives articular cartilage the
ability to withstand compression. Increased breakdown of aggrecan is
associated with the development of arthritis and is considered to be
catalyzed by aggrecanases, members of the ADAM-TS family of
metalloproteinases. Four endogenous tissue inhibitors of
metalloproteinases (TIMPs) regulate the activities of functional matrix
metalloproteinases (MMPs), enzymes that degrade most components of
connective tissue, but no endogenous factors responsible for the
regulation of aggrecanases have been found. We show here that the
N-terminal inhibitory domain of TIMP-3, a member of the TIMP family
that has functional properties distinct from other TIMPs, is a strong
inhibitor of human aggrecanases 1 and 2, with Ki
values in the subnanomolar range. This truncated inhibitor, which lacks
the C-terminal domain that is responsible for interactions with
molecules other than active metalloproteinases, is produced at high
yield by bacterial expression and folding from inclusion bodies.
This provides a starting point for developing a biologically
available aggrecanase inhibitor suitable for the treatment of arthritis.
Tissue inhibitors of matrix metalloproteinases
(TIMPs)1 are important
regulators of matrix metalloproteinases (MMPs) that participate in the
degradation of the extracellular matrix (1). To date, four isoforms of
TIMP have been identified in humans that are designated TIMP-1, -2, -3, and -4 (2); these are homologous in sequence and have similar secondary
and tertiary structures including six well conserved disulfide bonds.
Structural and functional studies of TIMP-1 and TIMP-2 (3-6) have
shown that the full inhibitory activity of TIMPs resides in the
N-terminal domain that is stabilized by three disulfide bonds.
Inhibition studies with recombinant TIMPs have shown that each TIMP
binds to MMPs with varying degrees of affinity, implicating that they
have distinct functions in vivo (2, 7).
TIMP-3 was originally discovered as a transformation-induced
protein in chicken fibroblasts (8), which was later shown to have
inhibitory activity against MMPs (9). In addition to its function as an
inhibitor of MMPs, TIMP-3 has been reported to inhibit the shedding of
cell surface-anchored molecules such as tumor necrosis factor- Recently a subgroup of eight ADAMs have recently been identified,
designated ADAM-TS proteinases. The ADAM-TS group, unlike typical
membrane-anchored ADAMs, lacks a transmembrane domain and a cytoplasmic
domain at the C terminus. Instead, these metalloproteinases contain a
varying number of thrombospondin type-1 domains (17). Among them,
ADAM-TS4 and ADAM-TS5, purified from interleukin-1-treated bovine
articular cartilage, cleave cartilage aggrecan efficiently and thus are
referred to as aggrecanase 1 and aggrecanase 2 (18, 19). These enzymes
are considered to play an important role in the breakdown of aggrecan
in cartilage under physiological and pathological conditions (18-22).
Until this study, however, no endogenous inhibitor of the aggrecanases
has been identified. Here we show that the N-terminal domain of TIMP-3
(N-TIMP-3), expressed at high yield in Escherichia coli and
folded from inclusion bodies to give a functional metalloproteinase
inhibitor, is a highly potent inhibitor of both aggrecanases. This
truncated protein, or variants engineered to enhance the specificity
for aggrecanases, may represent a route for the treatment of arthritis.
Materials--
Restriction enzymes were obtained from New
England BioLabs. ABC Western blot analysis kits were purchased from
Seikagaku Kogyo, Japan. The cDNA encoding human TIMP-3 was obtained
from a human placenta cDNA library. Human recombinant TIMP-1 was
expressed in CHO K-1 cells and purified from the conditioned medium
(23), and human TIMP-2 was purified from human uterine cervical
fibroblasts as described previously (24). Recombinant human TIMP-4, was expressed in E. coli.2 Recombinant human
MMP-1 lacking the C-terminal domain (MMP-1 Construction of the N-TIMP-3 Bacterial Expression
System--
Human TIMP-3 cDNA encoding the N-terminal region of
mature human TIMP-3, residues Cys1 to Asn121,
was amplified by PCR using a Vent PCR kit (New England BioLabs). The
reactions were carried out for 25 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min. An additional initiator methionine (bold) together with a NdeI
restriction site at the 5'-end and a NotI site
(underlined) at the 3'-end were introduced by specific
primers, 5'-GTCATATGTGCACATGCTCG-3' (forward) and 5'-CGGCCGCGTTACAACCCAGGTG-3' (reverse). The PCR
products were cloned into the pET42 vector (Novagen) using the
NdeI and NotI sites to produce the coding
sequence for truncated TIMP-3 (N-TIMP-3) with a His tag attached to the
C terminus.
Expression in E. coli and Folding of N-TIMP-3--
The plasmids
containing wild-type N-TIMP-3 were transformed into BL21 (DE3) cells
(Novagen), cultured in 3 liters of Luria-Bertani medium containing 50 µg/ml kanamycin and induced with 1 mM
isopropyl- Inhibition Kinetic Studies--
N-TIMP-3 was characterized as an
inhibitor of MMP-1 Aggrecanase Assay--
Activities of ADAM-TS4 and ADAM-TS5 were
measured by incubating enzyme with purified bovine aggrecan (500 nM) in 100 µl of 50 mM Tris-HCl buffer, pH
7.5, containing 0.1 M NaCl and 10 mM CaCl2, for 2 h at 37 °C and terminating the
reaction with 10 mM EDTA. The digestion products were then
deglycosylated with chondroitinase ABC (0.1 units/10 µg aggrecan) and
then with keratinase (0.1 units/10 µg aggrecan) and keratinase II
(0.002 units/10 µg aggrecan) for 2 h at 37 °C in 0.1 M Tris-HCl, pH 6.5, containing 50 mM sodium acetate. The enzymatically treated products were analyzed by Western blotting using BC-3 antibody (27) or an antibody against the GELE1480 neoepitope (28). To determine apparent inhibition
constant Ki(app) values of inhibitors against
the aggrecanases, ADAM-TS4 or ADAM-TS5 (at a final concentration of 50 pM) was incubated with various concentrations of the
inhibitor in 44 µl of the above buffer at room temperature for 30 min
and then a solution of bovine aggrecan (5.5 µl) was added. The
reaction products were detected with anti-GELE1480
antibody. The concentrations of ADAM-TS4 and ADAM-TS5 were confirmed by
titration with recombinant N-TIMP-3.
Expression and Folding of N-TIMP--
Approximately 100 mg of
unfolded N-TIMP-3 was purified by Ni2+-NTA affinity
chromatography of the inclusion bodies from 3 liters of bacterial
culture. Separation of the soluble protein obtained after in
vitro folding by ion exchange chromatography with CM-52 cellulose
gave a single protein peak. SDS-PAGE analysis under reducing conditions
showed a single band with an apparent molecular weight of 16 kDa
following staining with Coomassie Blue R250, in agreement with a
molecular mass calculated for N-TIMP-3 (including the His tag), of
15,230. A slightly faster electrophoretic mobility under nonreducing
conditions indicates that the intrachain disulfide bonds are present
(Fig. 1, inset). Assays for
MMP-1 inhibition indicate that the specific activity of the later
fractions in the protein peak was greater than that of the earlier
fractions. Titration of MMP-1 with the later fractions showed full
enzyme inhibition with a 1:1 stoichiometry, whereas the earlier
fractions exhibited only partial inhibition (Fig. 1). Approximately 300 µg of fully active N-TIMP-3 was obtained from 6 mg of the unfolded protein, corresponding to an overall yield of over 1.5 mg/liter of
bacterial culture. N-TIMP-3 can be concentrated up to 0.2 mg/ml without
precipitation. Only the fractions that gave total inhibition of MMP-1
with a 1:1 molecular stoichiometry were used for characterization of
the recombinant N-TIMP-3.
Inhibition of MMPs by N-TIMP-3--
N-TIMP-3 inhibited recombinant
MMP-1 Inhibition of ADAM-TS4 and ADAM-TS5--
The ability of N-TIMP-3
to inhibit ADAM-TS4 and ADAM-TS5 was examined first by detecting
aggrecan cleavage at the classical aggrecanase site, the
Glu373-Ala374 bond, using BC-3 antibody. As
shown in Fig. 2, A and
B, the actions of ADAM-TS4 and ADAM-TS5 were completely
inhibited by N-TIMP-3 at the concentration of 250 and 25 nM, respectively. By contrast, TIMP-1 or TIMP-2 even at a
concentration of 1 µM did not inhibit these enzymes (data
not shown). However, the assay for aggrecanase activity, which detects
the cleavage at the Glu373-Ala374 bond, was
not suitable for determining inhibition constants of N-TIMP-3 because
it required at least 1 nM aggrecanase. Therefore, a
quantitative and sufficiently sensitive assay system was necessary. This was accomplished by using anti-GELE antibody that recognizes the
fragment cleaved at the Glu1480-Gly1481 bond,
a site recently shown to be cleaved readily by aggrecanases (28). With
50 pM ADAM-TS4, the release of the GELE-fragment was linear
for up to 2 h at 37 °C (data not shown) and with 50 pM ADAM-TS5 for up to 4 h (Fig.
3). Thus, 50 pM aggrecanases
was used to quantify the inhibitory activity of inhibitors. As shown in
Fig. 2, C and D, N-TIMP-3 and a synthetic
hydroxamate inhibitor BB-16 at a concentration of 250 nM
completely blocked cleavage of aggrecan by ADAM-TS4 and ADAM-TS5,
whereas TIMP-1 and -2 showed little or no inhibition. TIMP-4, however,
at this relatively high concentration partially (about 35%) inhibited
ADAM-TS4, but not ADAM-TS5. The inhibitory activity of varying
concentrations of N-TIMP-3 was further measured against ADAM-TS4 and
ADAM-TS5, and the apparent Ki values
(Ki(app)) were calculated to be 3.3 and 0.66 nM, respectively (Fig. 4,
A and B). N-TIMP-3 formed a 1:1 molar
stoichiometric complex with both aggrecanases (data not shown).
TIMPs expressed in connective tissues play important roles in the
control of extracellular matrix metabolism, and the ability of TIMPs to
inhibit the activities of MMPs in vitro has been well documented (2). TIMP-3, however, has several properties distinct from
those of other TIMPs, which include its ability to bind tightly to the
extracellular matrix (9, 31), apoptotic effects on a number of cells
(10, 32), and inhibition of TACE (15) and ADAM 10 (16). This work
provides additional important information, specifically that TIMP-3 is
a potent endogenous inhibitor of aggrecanases, metalloproteinases that
play a key role in the degradation of articular cartilage aggrecan.
Inhibition of aggrecanases was not observed with TIMP-1 or TIMP-2.
TIMP-4 is however a weak inhibitor of ADAM-TS4 but not ADAM-TS5.
The property of TIMP-3 binding to matrix components is considered to be
important in the localized regulation of MMP activity, and its
interaction with the polyanionic extracellular matrix is mainly due to
the N-terminal domain of TIMP-3 (31). The inhibitor may be extracted
from the tissue with sulfated compounds such as suramin and pentosan or
enzymatic treatment with heparinase III or chondroitinase ABC (31), but
its strong binding to the extracellular matrix has made it difficult to
purify sufficient quantities of TIMP-3 for detailed characterization
from tissues or even by expression of the recombinant protein in
mammalian cells (33). In this study we have established a high yield
bacterial expression and in vitro folding procedure for the
N-terminal inhibitory domain of TIMP-3, which allows us to obtain about
5 mg of fully active TIMP-3 from a 3-liter bacterial culture.
Inhibition kinetic studies with N-TIMP-3 indicate that TIMP-3 is a
relatively weak inhibitor of MMP-3 (stromelysin 1), whereas TIMP-3
inhibits MMP-1 (collagenase 1) and MMP-2 (gelatinase A) to a similar
extent as TIMP-1. The most significant unique property of TIMP-3 that
we have demonstrated here is that it is a potent endogenous inhibitor
of aggrecanases 1 and 2 (ADAM-TS4 and ADAM-TS5). The
Ki(app) values determined in the presence of 0.5 M aggrecan substrate were 3.30 nM for
ADAM-TS4 and 0.66 nM for ADAM-TS5. The
Km of ADAM-TS4 and ADAM-TS5 for the cleavage of the
Glu1480-Gly1481 bond is less than 0.1 M.3 Because the
true Ki value is equal to
Ki(app)/(1 + [S]/Km), the
Ki values of TIMP-3 for ADAM-TS4 and ADAM-TS5 will
be at least 6-fold lower than the Ki(app)
values. These data suggest that a primary physiological function for
TIMP-3 may be the inhibition of aggrecanases because its affinity
toward ADAM-TS4 and ADAM-TS5 is much stronger than those for the three
MMPs tested.
Aggrecanases are believed to play a crucial role both in the normal
turnover of aggrecan in cartilage and in diseases such as
osteoarthritis and rheumatoid arthritis (20-22). On the other hand,
TIMP-3 mRNA is expressed in cartilage and skeletal tissue during
development of mouse embryo (34), suggesting that this inhibitor may
play a role during skeletal tissue development. TIMP-3 is also
expressed in normal bovine and human articular chondrocytes and in
synoviocytes, and elevated TIMP-3 expression was found in human
osteoarthritic synoviocytes (35). The expression of TIMP-3 in
chondrocytes in culture is up-regulated by transforming growth
factor- Whereas the general mechanism of inhibition of MMPs by TIMPs has been
well characterized by crystallographic studies (6), it is not known
which structural features confer on TIMP-3 its unique ability to
inhibit metalloproteinases from families other than the MMPs. Both
aggrecanases have little sequence similarity with MMPs, although the
overall polypeptide folds may be predicted to be similar. The primary
structures of the proteinase domains of ADAM-TS4 and ADAM-TS5 are about
48% identical to each other. They are members of the reprolysin
family, but their sequence identities with adamalysins are only
20-25% and with TACE and ADAM 10, only 10-15%. Thus, it is not
apparent how TIMP-3, but not TIMP-1, -2, or -4, can inhibit those
additional groups of metalloproteinases that are only distantly related
to MMPs. We are currently investigating key structural elements in
TIMP-3, by mutagenesis and molecular modeling, that confer its ability to inhibit aggrecanases and other members of the ADAM family of metalloproteinases. The identification of such elements may provide us
with strategies to engineer TIMPs that are more soluble than TIMP-3 and
specifically inhibit aggrecanases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor (10), L-selectin (11), interleukin 6 receptor (12), and
syndecans-1 and -4 (13). The release of these molecules is thought to
be catalyzed by membrane-bound ADAMs (a disintegrin
and a metalloproteinase domain), multidomain proteins containing an N-terminal propeptide, a metalloproteinase, a
disintegrin-like, a transmembrane, and a cytoplasmic domain. The
primary structures of the metalloproteinase domains of the MMPs and the
ADAMs have little sequence similarity except near the catalytic
Zn2+-binding motif,
HEXXHXXGXXH (14). Direct evidence for
the apparently unique ability of TIMP-3 to inhibit a broad spectrum of
metalloproteinases is provided by the demonstration of its inhibitory
action against TACE or ADAM-17 (15) and ADAM-10 (16). These properties
suggest that TIMP-3 has an important and distinct role in regulating
ADAM activities in biological systems.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C), MMP-2, and MMP-3
lacking the C-terminal domain (MMP-3
C) were prepared and activated
as described previously (24-26). Human recombinant ADAM-TS4 and
ADAM-TS5 expressed in Drosophila S2 cells (18, 19) and the
synthetic hydroxamate metalloproteinase inhibitor, BB-16
(2S,3R-N-[3-N-hydroxycarboxyamide)-2-(2-methylpropyl)-butanoyl]-O-methyl-L-tyrosine-N-methylamide) were kindly provided by DuPont Pharmaceuticals. The BC-3 antibody that
recognizes the new N-terminal 374ARGSVILTVK of aggrecan
cleaved at the Glu373-Ala374 bond (27) was a
gift from Dr. Clare E. Hughes of Cardiff University. A neoepitope
antibody to the peptide sequence ATTAGELE that recognizes the C
terminus of aggrecan fragments cleaved at the
Glu1480-Gly1481 bond was prepared by DuPont
Pharmaceuticals as described (28). Aggrecan was isolated from bovine
cartilage nasal septum according to Hascall and Sajdera (29).
-D-thiogalactopyranoside. After 3 h, the
cells were harvested by centrifugation at 4,000 rpm for 20 min, washed
twice with ice-cold wash buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM EDTA), and broken with a
French press. Inclusion bodies were collected by centrifugation at
12,000 rpm for 20 min at 4 °C, and the recombinant protein was
extracted with 6 M guanidine hydrochloride, 50 mM Tris-HCl, pH 8.0, and 10 mM
-mercaptoethanol at room temperature with constant stirring for
2 h. The solution containing extracted protein was applied to a
5-ml Ni2+-NTA-agarose column (Qiagen) and washed with 50 mM Tris-HCl, pH 8.0 containing 20 mM imidazole
and 6 M guanidine hydrochloride. The column was
subsequently washed with a solution composed of 4 volumes of 50 mM Tris-HCl, pH 8.0 containing 6 M guanidine
hydrochloride and 6 volumes of isopropyl alcohol to remove bacterial
endotoxins, and the protein was eluted with 50 mM Tris-HCl,
pH 8.0 containing 60 mM imidazole and 6 M
guanidine hydrochloride. The product was homogeneous on SDS-PAGE. The
eluted protein was diluted to 20 µg/ml with 50 mM
Tris-HCl, pH 8.0 containing 20% glycerol and 6 M guanidine
hydrochloride, treated with 20 mM cystamine with stirring
for 16 h at 4 °C. The solution was then dialyzed twice against
15 volumes of 50 mM Tris-HCl, pH 8.0 containing 20%
glycerol, 150 mM NaCl, 10 mM CaCl2,
5 mM
-mercaptoethanol, and 1 mM
2-hydroxyethyl disulfide for 24 h at 4 °C, twice against 20 mM Tris-HCl, pH 8.0 containing 20% glycerol for 8 h
at 4 °C and then centrifuged at 12,000 rpm for 30 min at 4 °C.
The supernatant was applied to a column (2.5 × 5 cm) of
carboxymethyl cellulose (Whatman CM-52) that had been equilibrated with
20 mM Tris-HCl, pH 8.0, and the bound protein was eluted
using a linear gradient ranging from 0 to 1 M NaCl in the
same buffer; all buffers contained 20% (v/v) glycerol. The
concentration of N-TIMP-3 was determined by absorbance at 280 nm using
the extinction coefficient (
280 = 17,570 M
1 cm
1) of N-TIMP-3.
C, MMP-2, MMP-3
as described previously for
N-TIMP-1 (4, 23). Inhibition constants (Ki values)
were calculated as described by Morrison and Walsh (30). For titration
of N-TIMP-3, various concentrations of the inhibitor were incubated
with MMP-1
C (100 nM) for 1 h at 37 °C, and the
residual activity was measured using 1 M Knight substrate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Titration of MMP-1 C
with N-TIMP-3. MMP-1
C at a concentration of 100 nM
was mixed with N-TIMP-3 fractions from the CM-cellulose separation at
various concentrations ranging from 0 to 200 nM in a total
volume of 100 µl and incubated at 37 °C for 1 h. The residual
MMP-1 activity was measured using Knight substrate.
, fully active
fraction,
, partially active earlier fractions. Inset,
SDS-PAGE analysis of the fully active N-TIMP-3 (1.67 g) with
(lane 1) and without (lane 2) reduction.
C, MMP-2, and MMP-3
C with Ki values of
1.2 ± 0.5, 4.3 ± 0.5, and 66.9 ± 2.8 nM,
respectively; the corresponding Ki values of
N-TIMP-1 are 3.0 ± 0.4, 1.1 ± 0.1, and 1.9 ± 0.1 nM, respectively (23) indicating distinct specificities
that must be dependent on differences in the structures of the
N-terminal domains of these two TIMPs. It is notable that the affinity
of N-TIMP-3 for MMP-1
C was greater than that for MMP-2 and
MMP-3
C.
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Fig. 2.
Inhibition of the aggrecanases by TIMPs and
the hydroxamate inhibitor, BB-16. ADAM-TS4 (A) or
ADAM-TS5 (B) at the concentration of 1 nM was
incubated with N-TIMP-3 at the concentrations of 0 (lane 1),
25 nM (lane 2), 50 nM (lane
3), 100 nM (lane 4), and 250 nM
(lane 5) for 30 min at room temperature, and then with 500 nM aggrecan at 37 °C for 30 min. After terminating the
reaction with 10 mM EDTA, the aggrecan fragments were
detected using BC-3 antibody. ADAM-TS4 (C) or ADAM-TS5
(D) at a concentration of 50 pM were incubated
with either TIMP-1, -2, -3, -4 or BB-16 at a concentration of 250 nM for 30 min at room temperature and then reacted with
aggrecan at a final concentration of 500 nM for 2 h at
37 °C. After terminating the reactions with 10 mM EDTA,
the products were analyzed using the anti-GELE polyclonal antibody.
Lane: 1, TIMP-1; 2, TIMP-2;
3, N-TIMP-3; 4, TIMP-4; 5, BB-16; and
6, buffer control.
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Fig. 3.
Aggrecanase assay using antibody that
recognizes the GELE neoepitope. Aggrecan (500 nM) was
incubated with 50 pM ADAM-TS5 at 37 °C for the indicated
period of time. An aliquot was removed, and the reaction stopped with
10 mM EDTA. The samples were then analyzed by Western
blotting with the antibody that recognizes the GELE neoepitopes as
described under "Experimental Procedures."
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Fig. 4.
Dose-dependent inhibition of
ADAM-TS4 and ADAM-TS5 by N-TIMP-3. ADAM-TS4 (A) or
ADAM-TS5 (B) at a concentration of 50 pM was
incubated with various concentrations of N-TIMP-3 in a total volume of
44 µl for 30 min at room temperature. A solution of bovine aggrecan
(5.5 µl) was added to give a final concentration of 500 nM, and the mixture was incubated for 2 h at 37 °C.
The reactions were stopped with 10 mM EDTA and the product
was analyzed by Western blotting for GELE-containing fragments using
the anti-GELE polyclonal antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(36), oncostatin M (37). Recent studies of Takizawa et
al. (38) showed that the treatment of human rheumatoid synovial
fibroblasts with an antiarthritic agent, calcium pentosan polysulfate,
increases the synthesis of TIMP-3 protein without altering its mRNA
levels, and this effect is further enhanced in cells treated with both
calcium pentosan polysulfate and interleukin 1. The levels of other
TIMPs and MMPs were not affected by this treatment. Because the
degradation of aggrecan in cartilage occurs in the early stages of
arthritis, the level of TIMP-3 in cartilage is likely to be an
important factor in relation to the development of arthritis. Elevated
TIMP-3 production therefore may be beneficial for protecting cartilage
from degradation, because it can prevent not only the action of
aggrecanases and MMPs in the cartilage but also the release of TNF-
,
one of the key inflammatory cytokines, from the synovial living cells
by inhibiting TACE.
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ACKNOWLEDGEMENT |
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We thank Dr. Clare E. Hughes for the generous gift of BC-3 antibody.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AR 40994 and Wellcome Trust Grants Nos. 057508 and 061707.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.
¶ To whom correspondence should be addressed: Dept. of Biomedical Sciences, Florida Atlantic University, 777 Glades Rd., Boca Raton, FL 33431. Tel.: 561-297-0407; Fax: 561-297-2221; E-mail: kbrew@fau.edu.
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.C000848200
2 M. Tanaka, L. Troeberg, and H. Nagase, manuscript in preparation.
3 M. Tortorella and H. Nagase, unpublished work.
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ABBREVIATIONS |
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The abbreviations used are:
TIMP, tissue
inhibitor of metalloproteinases;
MMP, matrix metalloproteinase;
MT-MMP, membrane-type matrix metalloproteinase;
ADAM, a disintegrin and a
metalloproteinase domain;
ADAM-TS, a disintegrin and a
metalloproteinase domain with thrombospondin type-1 domains;
TACE, tumor necrosis factor- converting enzyme;
PCR, polymerase chain
reaction;
PAGE, polyacrylamide gel electrophoresis;
NTA, nitrilotriacetic acid.
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