Review
This Review is part of a thematic series on Matrix Metalloproteinases, which includes the following articles:
Matrix Metalloproteinase Inhibition After Myocardial Infarction: A New Approach to Prevent Heart Failure?
Matrix Metalloproteinases in Vascular Remodeling and Atherogenesis: The Good, the Bad, and the Ugly
Matrix Metalloproteinases: Regulation and Dysregulation in the Failing Heart
Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases: Structure, Function, and Biochemistry
David Kass, Marlene Rabinovitch, Editors
Matrix Metalloproteinases and Tissue Inhibitors
of Metalloproteinases
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Structure, Function, and Biochemistry
Robert Visse, Hideaki Nagase
Abstract—Matrix metalloproteinases (MMPs), also designated matrixins, hydrolyze components of the extracellular
matrix. These proteinases play a central role in many biological processes, such as embryogenesis, normal tissue
remodeling, wound healing, and angiogenesis, and in diseases such as atheroma, arthritis, cancer, and tissue ulceration.
Currently 23 MMP genes have been identified in humans, and most are multidomain proteins. This review describes the
members of the matrixin family and discusses substrate specificity, domain structure and function, the activation of
proMMPs, the regulation of matrixin activity by tissue inhibitors of metalloproteinases, and their pathophysiological
implication. (Circ Res. 2003;92:827-839.)
Key Words: extracellular matrix 䡲 protease 䡲 protease inhibitors
E
by other reviews in this series.6 – 8 In the present review, we
give an overview of structure, function, and biochemistry of
MMPs and TIMPs.
xtracellular matrix (ECM) macromolecules are important
for creating the cellular environments required during
development and morphogenesis. Matrix metalloproteinases
(MMPs), collectively called matrixins, are proteinases that
participate in ECM degradation.1,2 Under normal physiological conditions, the activities of MMPs are precisely regulated
at the level of transcription, activation of the precursor
zymogens, interaction with specific ECM components, and
inhibition by endogenous inhibitors.1,2 A loss of activity
control may result in diseases such as arthritis, cancer,
atherosclerosis, aneurysms, nephritis, tissue ulcers, and fibrosis.3 Tissue inhibitors of metalloproteinases (TIMPs) are
specific inhibitors of matrixins that participate in controlling
the local activities of MMPs in tissues.4,5 The pathological
effects of MMPs and TIMPs in cardiovascular disease processes that involve vascular remodeling, atherosclerotic
plaque instability, and left ventricular remodeling after myocardial infarction are of considerable interest and are covered
Members of the Matrixin Family
The first MMP activity discovered was a collagenase in the
tail of a tadpole undergoing metamorphosis. To date, 24
different vertebrate MMPs have been identified, of which 23
are found in humans. Matrixins are also found in Hydra,9 sea
urchin,10 and Arabidopsis.11 The sequence homology with
collagenase 1 (MMP-1), the cysteine switch motif
PRCGXPD in the propeptide that maintains MMPs in their
zymogen form (proMMP), and the zinc-binding motif
HEXGHXXGXXH in the catalytic domain are the signatures
used to assign proteinases to this family. The exception is
MMP-23, which lacks the cysteine switch motif, but its
Original received December 28, 2001; resubmission received March 25, 2003; accepted March 25, 2003.
From the Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, London, UK.
Correspondence to Dr Hideaki Nagase, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, 1 Aspenlea Rd,
London W6 8LH, UK. E-mail [email protected]
© 2003 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000070112.80711.3D
827
828
Circulation Research
May 2, 2003
Figure 1. Domain structure of MMPs. The
domain organization of MMPs is as indicated: S,
signal peptide; Pro, propeptide; Cat, catalytic
domain; Zn, active-site zinc; Hpx, hemopexin
domain; Fn, fibronectin domain; V, vitronectin
insert; I, type I transmembrane domain; II, type II
transmembrane domain; G, GPI anchor; Cp,
cytoplasmic domain; Ca, cysteine array region;
and Ig, IgG-like domain. A furin cleavage site is
depicted as a black band between propeptide
and catalytic domain.
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amino acid sequence of the catalytic domain is related to
MMP-1. MMPs generally consist of a prodomain, a catalytic
domain, a hinge region, and a hemopexin domain (see Figure
1). They are either secreted from the cell or anchored to the
plasma membrane. On the basis of substrate specificity,
sequence similarity, and domain organization, vertebrate
MMPs can be divided into six groups (see Figure 1 and Table
1), as described below. An extended version of Table 1,
including MMP substrates, is available in the online data
supplement (available at http://www.circresaha.org).
grouped with “other MMPs” because the sequence and
substrate specificity diverge from those of MMP-3.
Matrilysins
The matrilysins are characterized by the lack of a hemopexin
domain. Matrilysin 1 (MMP-7) and matrilysin 2 (MMP-26),18
also called endometase,19 are in this group. Besides ECM
components, MMP-7 processes cell surface molecules such
as pro–␣-defensin, Fas-ligand, pro–tumor necrosis factor
(TNF)-␣, and E-cadherin. Matrilysin 2 (MMP-26) also digests a number of ECM components.
Collagenases
MMP-1, MMP-8, MMP-13, and MMP-18 (Xenopus) are in
this group. The key feature of these enzymes is their ability to
cleave interstitial collagens I, II, and III at a specific site
three-fourths from the N-terminus. Collagenases can also
digest a number of other ECM and non-ECM molecules.
Gelatinases
Gelatinase A (MMP-2) and gelatinase B (MMP-9) belong to
this group. They readily digest the denatured collagens,
gelatins. These enzymes have three repeats of a type II
fibronectin domain inserted in the catalytic domain, which
bind to gelatin, collagens, and laminin.12 MMP-2, but not
MMP-9, digests type I, II, and III collagens.13,14 Although
MMP-2 null mice develop without any apparent abnormality,15 mutations in human MMP-2 resulting in the absence of
active enzyme are linked with an autosomal recessive form of
multicentric osteolysis, a rare genetic disorder that causes
destruction and resorption of the affected bones.16 This
suggests that MMP-2 in humans is important for
osteogenesis.16
Stromelysins
Stromelysin 1 (MMP-3) and stromelysin 2 (MMP-10) both
have similar substrate specificities, but MMP-3 has a proteolytic efficiency higher than that of MMP-10 in general.
Besides digesting ECM components, MMP-3 activates a
number of proMMPs, and its action on a partially processed
proMMP-1 is critical for the generation of fully active
MMP-1.17 MMP-11 is called stromelysin 3, but it is usually
Membrane-Type MMPs
There are six membrane-type MMPs (MT-MMPs): four are
type I transmembrane proteins (MMP-14, MMP-15, MMP16, and MMP-24), and two are glycosylphosphatidylinositol
(GPI) anchored proteins (MMP-17 and MMP-25). With the
exception of MT4-MMP, they are all capable of activating
proMMP-2. These enzymes can also digest a number of ECM
molecules, and MT1-MMP has collagenolytic activity on
type I, II, and III collagens.20 MT1-MMP null mice exhibit
skeletal abnormalities during postnatal development that are
most likely due to lack of collagenolytic activity.21 MT1MMP also plays an important role in angiogenesis.22 MT5MMP is brain specific and is mainly expressed in the
cerebellum.23 MT6-MMP (MMP-25) is expressed almost
exclusively in peripheral blood leukocytes and in anaplastic
astrocytomas and glioblastomas but not in meningiomas.24,25
Other MMPs
Seven MMPs are not classified in the above categories.
Metalloelastase (MMP-12) is mainly expressed in macrophages26 and is essential for macrophage migration.27 Besides
elastin, it digests a number of other proteins.
MMP-19 was identified by cDNA cloning from liver28 and
as a T-cell– derived autoantigen from patients with rheumatoid arthritis (RASI).29
Enamelysin (MMP-20), which digests amelogenin, is primarily located within newly formed tooth enamel. Amelogenin imperfecta, a genetic disorder caused by defective enamel
formation, is due to mutations at MMP-20 cleavage sites.30
Visse and Nagase
TABLE 1.
Structure and Function of MMPs and TIMPs
829
Matrix Metalloproteinases
Enzyme
MMP
Human Chromosome
3D Structure (PDB Code)
Interstitial collagenase; collagenase 1
MMP-1
11q22-q23
Mature protein; 1FBL cat domain; 1CGF, 2TCL, 1AYK, 2AYK, 1HFC,
1CGL, 1CGE, 966C, 3AYK, 4AYK
Neutrophil collagenase; collagenase 2
MMP-8
11q21-q22
Cat domain; 1MNC, 1I76, 1JAO, 1MMB, 1JAN, 1JAP, 1JAQ, 1I73,
1KBC, 1A85, 1A86, 1BZS, 1JJ9, 1JH1
Collagenase 3
MMP-13
11q22.3
Cat domain; 1CXV, 1FM1, 1FLS, 456c, 830c, 1EUB Hpx domain; 1PEX
Collagenase 4 (Xenopus)
MMP-18
NA
Gelatinase A
MMP-2
16q13
proMMP-2; 1CK7; proMMP-2–TIMP-2 complex; 1GXD; cat domain;
1QIB, 1HOV, 1EAK; Hpx domain; 1GEN, 1RTG; Fn; 1CXW, 1KS0
Gelatinase B
MMP-9
20q11.2-q13.1
Pro–cat domain; 1L6J; cat domain; 1GKC, 1GKD; Hpx domain; 1ITV
Stromelysin 1
MMP-3
11q23
Pro–cat domain; 1SLM; cat domain; D8M, 1CIZ, 1CAQ, 1B8Y, 2SRT,
1HFS, 1SLN, 2USN, 1USN, 1D5J, 1BQO, 1D7X, 1D8F, 1BIW, 1UMS,
3USN, 1UMT, 1BM6, 1B3D, 1CQR, 1G4K, 1G49, 1HY7, 1G05; complex
with N-TIMP-1; 1UEA
Stromelysin 2
MMP-10
11q22.3-q23
Stromelysin 3
MMP-11
22q11.2
1HV5
Matrilysin 1; Pump-1
MMP-7
11q21-q22
Cat domain; 1MMP, 1MMQ, 1MMR
Matrilysin 2
MMP-26
11p15
MT1-MMP
MMP-14
14q11-q12
MT2-MMP
MMP-15
15q13-q21
MT3-MMP
MMP-16
8q21
MT5-MMP
MMP-24
20q11.2
MT4-MMP
MMP-17
12q24.3
MT6-MMP
MMP-25
16p13.3
Macrophage elastase
MMP-12
11q22.2-q22.3
No trivial name
MMP-19
12q14
Enamelysin
MMP-20
11q22.3
XMMP (Xenopus)
MMP-21
ND
CA-MMP
MMP-23
1p36.3
CMMP (Gallus)
MMP-27
11q24
Epilysin
MMP-28
17q21.1
Collagenases
Gelatinases
Stromelysins
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Matrilysins
Membrane-type MMPs
Transmembrane
Cat domain in complex with TIMP-2; 1BQQ, 1BUV
GPI anchor
Others
Cat domain 1JK3, 1JIZ
Groups of MMPs are listed with their trivial names and chromosomal location. The names of the PDB files of structures determined by x-ray crystallography and
NMR are listed. These files with their references can be downloaded from the protein databank (www.rcsb.org). An extended version of this table including the
substrates cleaved by the individual MMPs is available in online Table 1 (see online data supplement available at http://www.circresaha.org).
MMP-22 was first cloned from chicken fibroblasts,31 and a
human homologue has been identified on the basis of EST
sequences. The function of this enzyme is not known.
MMP-23, also called cysteine array MMP, is mainly expressed in reproductive tissues.32 The enzyme lacks the cysteine
switch motif in the prodomain. It also lacks the hemopexin
domain; instead, it has a cysteine-rich domain followed by an
immunoglobulin-like domain. It is proposed to be a type II
membrane protein harboring the transmembrane domain in the
N-terminal part of the propeptide. Because it has a furin
recognition motif in the propeptide, it is cleaved in the Golgi and
released as an active enzyme into the extracellular space.33
The latest addition to the MMP family is epilysin, or
MMP-28, mainly expressed in keratinocytes.34,35 Expression
patterns in intact and damaged skin suggest that MMP-28
might function in tissue hemostasis and wound repair.34 –36
Three-Dimensional (3D) Structures of MMPs
X-ray crystallography and nuclear magnetic resonance
(NMR) have determined the 3D structures of a number of
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Figure 2. 3D structure of MMPs: ribbon
diagram of MMP structures. A, ProMMP2–TIMP-2 complex (1GXD) is shown.45
Orange indicates propeptide; green, catalytic domain; pink, fibronectin domains;
red, hemopexin domain; and blue,
TIMP-2. Zinc atoms are pink, and calcium atoms are gray. B, In the MMP-2
propeptide,40 the cysteine of the cysteine
switch motif is shown. The arrow indicates the position of the initial cleavage
resulting in partial activation. C, The catalytic domain of MMP-1 is shown.128 The
␤-strands are numbered I through V; the
␣-helices are labeled A through C. The
N-terminal (N) to C-terminal (C) order of
the ␤-stands and ␣-helices is I-A-II-III-IVV-B-C. The histidines coordinating the
active-site zinc and the active-site glutamic acid are shown. D, The 3 fibronectin domains of MMP-240 are shown with
their 2 disulfide bonds each. E, The
hemopexin domain of MMP-1128 with 4
␤-propeller blades is shown. A disulfide
bond is seen between blades I and IV.
This figure was prepared with a Swiss
PDB Viewer129 and rendered with
POV-Ray.
MMPs (Table 1). The structure of the prodomain is known
for MMP-2, MMP-3, and MMP-9. It consists of three
␣-helices and connecting loops (see Figure 2B). The first
loop between helix 1 and 2 is a protease-sensitive “bait
region.” An extended peptide region after helix 3 lies in the
substrate-binding cleft of the catalytic domain. This region
contains the conserved cysteine switch, which forms a
fourth ligand of the active-site zinc, keeping the zymogen
inactive. It is notable that the orientation of the propeptide
backbone as it interacts with the active-site cleft is
opposite that of a peptide substrate. However, the hydrogen bonds that it makes with the active site are identical to
those of a substrate backbone.37
The polypeptide chain folds of the catalytic domains are
essentially superimposable. The chain consists of a
5-stranded ␤-pleated sheet, three ␣-helices, and connective
loops (see Figure 2C). This proteinase domain contains one
catalytic zinc, one structural zinc, and, generally, three
calcium ions. The substrate-binding cleft is formed by strand
IV, helix B, and the extended loop region after helix B. Three
histidines coordinate the active-site zinc. The loop region
contains the conserved “Met-turn,” a base to support the
structure around the catalytic zinc.38 The fourth ligand of the
catalytic zinc is a water molecule. The glutamic acid adjacent
to the first histidine is essential for catalysis.
In the orientation shown in Figure 2C, a substrate binds
into the catalytic site cleft from left to right with respect to its
N- and C-termini, and the carbonyl group of the peptide bond
coordinates with the active-site zinc. This displaces the water
molecule from the zinc atom. The peptide hydrolysis is
assisted by the carboxyl group of the glutamate, which serves
as a general base to draw a proton from the displaced water
molecule, thereby facilitating the nucleophilic attack of the
water molecule on the carbonyl carbon of the peptide scissile
bond. A pocket to the right of the active-site zinc, called the
specificity pocket or S1⬘ pocket, accommodates the side
chain of the substrate residue, which becomes the new
N-terminus after cleavage. The sizes of the S1⬘ pocket vary
among the MMPs, and this is one of the major determining
factors of substrate specificity.39
Three repeats of fibronectin type II domains found in
MMP-2 and MMP-9 are inserted between the fifth ␤-strand
Visse and Nagase
Structure and Function of MMPs and TIMPs
831
Figure 3. Stepwise activation of proMMPs.
ProMMPs secreted as inactive zymogens
can be activated by proteinases (top pathway) or by nonproteolytic agents (bottom
pathway). The catalytic domain is represented as a gray circle, with the active-site cleft
shown in white (not to scale), containing the
catalytic-site zinc (Zn). The propeptide is
schematically shown as a black line containing the bait region (black rectangle) and the
cysteine switch (C). SH indicates the sulfhydryl of the cysteine. Activation by proteinases is mediated by cleavage of the bait
region; this partly activates the MMP. Full
activation is achieved by completed removal
of the propeptide by intermolecular processing. Chemical activation relies on modification of the cysteine switch sulfhydryl (SX),
resulting in partial activation of the MMP and
intramolecular cleavage of the propeptide.
Full activity results from the removal of the
remainder of the propeptide by intermolecular processing.
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and the catalytic site helix40 (Figures 2A and 2D). The
structure of each fibronectin domain consists of two
antiparallel ␤-sheets, connected with a short ␣-helix and
stabilized by two disulfide bonds. NMR studies have
indicated that domains 2 and 3 are quite flexible, possibly
interacting simultaneously with multiple sites in the
ECM.41
The hemopexin domains have a 4-bladed ␤-propeller fold,
with a single stabilizing disulfide bond between blades I and
IV (Figure 2E). The hemopexin domains of MMP-9 form an
asymmetric homodimer through blade IV.42 The asymmetry
is the result of shifts in blade III and IV structure on
dimerization, which alters its physicochemical properties.42
The hemopexin domain of MMP-9 binds the C-terminal
domain of TIMP-1.43 However, the formations of this complex and the MMP-9 dimer are mutually exclusive, probably
because of an overlap in the TIMP-1– binding site and the
dimer interface.42 The recombinant MMP-9 hemopexin domain binds to gelatin and is able to inhibit the invasion of
melanoma cells.44 TIMP-2 binds to the hemopexin domain of
proMMP-2. The crystal structure of this complex45 (Figure
2A) shows that this interaction is through the C-terminal
domain of TIMP-2 and blades III and IV of the hemopexin
domain; the N-terminal inhibitory domain of TIMP-2 is free
to interact with other MMPs.
␤-Propeller domains with a larger number of blades are
found in other proteins, such as heterotrimeric G proteins,
clathrin, and the ␣-subunit of integrins. These domains often
mediate protein-protein interactions.46 Depending on the
specific MMP, the hemopexin-like domain is important for
substrate specificity and is required for proMMP-2 activation
and the dimerization of MT1-MMP and MMP-9.
Activation of ProMMPs
Stepwise Activation Mechanism
MMPs can be activated by proteinases or in vitro by chemical
agents,47 such as thiol-modifying agents (4-aminophenylmercuric
acetate, HgCl2, and N-ethylmaleimide), oxidized glutathione, SDS,
chaotropic agents, and reactive oxygens (see Figure 3). Low pH and
heat treatment can also lead to activation. These agents most likely
work through the disturbance of the cysteine-zinc interaction of the
cysteine switch.48,49 Studies of proMMP-3 activation with a mercurial compound have indicated that the initial cleavage occurs
within the propeptide and that this reaction is intramolecular rather
than intermolecular.50 The subsequent removal of the rest of the
propeptide is due to intermolecular reaction of the generated
intermediates.50,51 Recently, studies by Gu et al52 have shown that
NO activates proMMP-9 during cerebral ischemia by reacting with
the thiol group of the cysteine switch and forming an S-nitrosylated
derivative,52 a demonstration of the chemical activation of a
proMMP in vivo.
Proteolytic activation of MMPs is stepwise in many cases47
(see Figure 3). The initial proteolytic attack occurs at an
exposed loop region between the first and the second helices
of the propeptide. The cleavage specificity of the bait region
is dictated by the sequence found in each MMP. Once a part
of the propeptide is removed, this probably destabilizes the
rest of the propeptide, including the cysteine switch–zinc
interaction, which allows the intermolecular processing by
partially activated MMP intermediates or other active
MMPs.17,51 Thus, the final step in the activation is conducted
by an MMP.
Activation of proMMPs by plasmin is a relevant pathway
in vivo. Plasmin is generated from plasminogen by tissue
plasminogen activator bound to fibrin and urokinase plasminogen activator bound to a specific cell surface receptor. Both
plasminogen and urokinase plasminogen activator are
membrane-associated, thereby creating localized proMMP
activation and subsequent ECM turnover. Plasmin has been
reported to activate proMMP-1, proMMP-3, proMMP-7,
proMMP-9, proMMP-10, and proMMP-13.53 Activated
MMPs can participate in processing other MMPs. The stepwise activation system may have evolved to accommodate
finer regulatory mechanisms to control destructive enzymes,
inasmuch as TIMPs may interfere with activation by interacting with the intermediate MMP before it is fully
activated.54
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Figure 4. Model of proMMP-2 activation by MT1-MMP and TIMP-2. Active MT1-MMP (MT-1) on the membrane binds a molecule of TIMP-2 (T-2),
inhibiting its activity. MT1-MMP can form dimers or multimers on the cell surface through interaction of the hemopexin domains. ProMMP-2 (pM-2)
subsequently binds to the C-terminal domain of TIMP-2 through its hemopexin domain. The second, active, MT1-MMP then cleaves the bait region
of proMMP-2, thereby partly activating it. The MMP-2 (M-2) dissociates from the membrane and is fully activated by intermolecular processing.
Intracellular Activation
Most proMMPs are secreted from cells and activated extracellularly. Pei and Weiss 55 first demonstrated that
proMMP-11 (stromelysin 3) is activated intracellularly by
furin. ProMMP-11 possesses a furin recognition sequence,
KX(R/K)R, at the C-terminal end of the propeptide. Several
other MMPs, including the six MT-MMPs,2,56 MMP-23, and
epilysin (MMP-28),34,35 have a similar basic motif in the
propeptide. Because these proteins are most likely secreted as
active enzymes, their gene expression and inhibition by
endogenous inhibitors would be critical for the regulation of
activity.
Cell Surface Activation of ProMMP-2
ProMMP-2 is not readily activated by general proteinases.
The main activation of proMMP-2 takes place on the cell
surface and is mediated by MT-MMPs. This includes MT1MMP, MT2-MMP,57 MT3-MMP,58 MT5-MMP,59,60 and
MT6-MMP.24 MT4-MMP does not activate proMMP-2.61
MT1-MMP–mediated activation of proMMP-2 has been
studied extensively. The unique aspect is that it requires the
assistance of TIMP-2.62– 64 ProMMP-2 forms a tight complex
with TIMP-2 through their C-terminal domains, therefore
permitting the N-terminal inhibitory domain of TIMP-2 in the
complex to bind to MT1-MMP on the cell surface. The cell
surface– bound proMMP-2 is then activated by an MT1MMP that is free of TIMP-2. Alternatively, MT1-MMP
inhibited by TIMP-2 can act as a “receptor” of proMMP-2.
This MT1-MMP–TIMP-2–proMMP-2 complex is then presented to an adjacent free MT1-MMP for activation. Clustering of MT1-MMP on the cell surface through interactions of
the hemopexin domain facilitates the activation process65 (see
Figure 4). Jo et al66 reported that the maximum enhancement
of proMMP-2 activation is observed at a TIMP-2/MT1-MMP
ratio of 0.05, suggesting that a large number of free MT1MMP may surround the ternary complex of proMMP-2–
TIMP-2–MT1-MMP for effective proMMP-2 activation.
ProMMP-2 activation by MT2-MMP is direct and independent of TIMP-2.67 Interestingly, TIMP-4 binds to the
proMMP-2 hemopexin domain, and it inhibits MT1-MMP,
but it does not result in proMMP-2 activation by MT1MMP.68 The reason for this is not clear, but it may be due to
an incorrect molecular assembly with TIMP-4.
MT1-MMP also activates proMMP-13 on the cell surface;
this activation is more efficient in the presence of active
MMP-2.69 The activation of proMMP-13 by MT1-MMP is
independent of TIMP-2 but requires the C-terminal hemopexin domain of proMMP-13.70
Substrate Specificity of MMPs
Substrate specificities of MMPs have been studied either by
identifying the cleavage sites of protein substrates or by a
series of synthetic peptide substrates.71 In general, MMPs
cleave a peptide bond before a residue with a hydrophobic
side chain, such as Leu, Ile, Met, Phe, or Tyr. A peptide bond
with a charged residue at this position is rarely cleaved, with
the cleavage of the X-Lys bond by MMP-12 being an
exception.72 The hydrophobic residues fit into the S1⬘ specificity pocket, whose size and shape differ considerably
among MMPs.39 In addition to the S1⬘ pocket, other substrate
contact sites (subsites) also participate in the substrate specificity of the enzyme.73
In some cases, noncatalytic domains influence the enzyme
activity, particularly against large extended macromolecules
of the ECM. For example, the fibronectin domains of MMP-2
and MMP-9 are important for its activity on type IV collagen,
gelatin, and elastin.74,75 In collagenase 1 (MMP-1), the loop
region just before the catalytic site helix (183RWTNNFREY)
is essential for collagenolytic activity.76 Furthermore, the
hemopexin domain and the hinge between the catalytic and
the hemopexin domains also play key roles in collagenolysis
(review by Overall77).
Biological Activities Generated by MMPMediated Cleavage
A major function of MMPs is thought to be the removal of
ECM in tissue resorption. However, the ECM is not simply
an extracellular scaffold; it also acts as a reservoir of
Visse and Nagase
TABLE 2.
Structure and Function of MMPs and TIMPs
833
Biological Activities Generated by MMP-Mediated Cleavage
Responsible MMPs
Substrate Cleaved
Reference
Keratinocyte migration and reepithelialization
Biological Effect
MMP-1
Type I collagen
79
Osteoclast activation
MMP-13
Type I collagen
78
Neurite outgrowth
MMP-2
Chondroitin sulphate proteoglycan
84
Adipocyte differentiation
MMP-7
Fibronectin
130
Cell migration
MMP-1,- 2, -3
Fibronectin
Cell migration
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MT1-MMP
CD44
83
Mammary epithelial cell apoptosis
MMP-3
Basement membrane
131
Mammary epithelial alveolar formation
MMP-3
Basement membrane
132, 133
Epithelial-mesenchymal conversion
(mammary epithelial cells)
MMP-3
E-cadherin
134, 135
Mesenchymal cell differentiation with
inflammatory phenotype
MMP-2
Not identified
136
Platelet aggregation
MMP-1
Not identified
137
Generation of angiostatin-like fragment
MMP-3
Plasminogen
138
MMP-7
Plasminogen
139
Generation of endostatin-like fragment
Enhanced collagen affinity
Kidney tubulogenesis
MMP-9
Plasminogen
139
MMP-12
Plasminogen
140
MMPs
Type XVIII collagen
141
MMP-2, -3, -7, -9, -13 (but not MMP-1)
BM-40 (SPARC/osteonectin)
142
143
MT1-MMP
Type I collagen
MMP-3, -13
Perlecan
144
MMP-1, -2, -3
IGFBP-3
145
MMPs
IGFBP-5
146
MMP-11
IGFBP-1
147
MMPs
CTGF
148
MMP-2, MT1-MMP
Laminin 5␥2 chain
81, 82
Collagenase
Type I collagen
80
MMP-1, -3, -9
Processing IL-1␤ from the precursor
149
MMP-9
ICAM-1
150
Antiinflammatory
MMP-1, -2, -9
IL-1␤ degradation
151
Antiinflammatory
MMP-1, -2, -3, -13, -14
Monocyte chemoattractant protein-3
152, 153
Release of bFGF
Increased bioavailability of IGF1 and cell
proliferation
Activation of VEGF
Epithelial cell migration
Apoptosis (amnion epithelial cells)
Proinflammatory
Tumor cell resistance
Increased bioavailability of TGF-␤
MMP-2, -3, -7
Decorin
154
Disrupted cell aggregation and increased
cell invasion
MMP-3, MMP-7
E-cadherin
155
156
Reduced cell adhesion and spreading
MT1-MMP, MT2-MMP, MT3-MMP
Cell surface tissue transglutaminase
Fas receptor–mediated apoptosis
MMP-7
Fas ligand
157
Reduced IL-2 response
MMP-9
IL-2R␣
158
biologically active molecules, such as growth factors.2 Some
ECM components can express cryptic biological functions on
proteolysis. Hence, degradation of ECM components by
MMPs can alter cellular behavior and phenotypes (Table 2).
For example, the degradation of type I collagen by collagenase is associated with osteoclast activation,78 keratinocyte
migration during reepitheliazation,79 and apoptosis of amnion
epithelial cells before the onset of labor.80 MMP-2– and
MT1-MMP–mediated cleavage of the ␥2 chain of laminin 5
exposes a cryptic promigratory site and promotes the migration of normal breast epithelial cells.81,82 Cleavage of CD44
by MT1-MMP is associated with cell migration.83 MMP-2
expressed in the Schwann cells of peripheral nerves degrades
chondroitin sulfate proteoglycans and promotes neurite
growth.84 Therefore, the function of MMPs is much more
complex and subtle than straightforward demolition. Add to
this the ever expanding number of non-ECM proteins that are
MMP substrates and exert biological activities (for review,
see McCawley and Matrisian85 and Sternlicht and Werb2),
and the complexity of the role of MMPs in health and disease
is evident.
Endogenous MMP Inhibitors
TIMPs are specific inhibitors that bind MMPs in a 1:1
stoichiometry. Four TIMPs (TIMP-1, TIMP-2, TIMP-3, and
TIMP-4) have been identified in vertebrates,5 and their
expression is regulated during development and tissue remodeling. Under pathological conditions associated with unbal-
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May 2, 2003
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Figure 5. Inhibition of MMP by TIMP: complex of TIMP-2 with catalytic domain of MT1-MMP (1BQQ).90 On the left, TIMP-2 (top) and
the catalytic domain of MT1-MMP (bottom) are shown separately. TIMP-2 is shown as a ribbon diagram. The disulfide bonds stabilizing
the protein are shown. The ␤-stands are labeled A through J; the ␣-helices are numbered 1 through 4. The catalytic domain of MT1MMP is shown as a ribbon structure with a transparent surface. The location of the catalytic site cleft is indicated by a dashed rectangle. Within the active-site cleft, the active-site zinc is visible as a pink sphere, and the entrance to the S1⬘ specificity pocket is labeled.
In the complex on the right, the catalytic domain is rotated around the x-axis. The N-terminal cysteine that chelates the active-site zinc
is clearly visible. The figure was prepared with a Swiss PDB Viewer129 and rendered with POV-Ray.
anced MMP activities, changes of TIMP levels are considered
to be important because they directly affect the level of MMP
activity.
TIMPs (21 to 29 kDa) have an N- and C-terminal domain
of ⬇125 and 65 amino acids, respectively, with each containing three conserved disulfide bonds.86,87 The N-terminal
domain folds as a separate unit and is capable of inhibiting
MMPs.86 NMR first solved the structure of the N-terminal
domain of TIMP-2 in 1994.88 The complete structure of
TIMP-1 and of the inhibition mechanism was determined by
X-ray crystallographic studies of the TIMP-1–MMP-3 complex,89 and soon after, that of the TIMP-2–MT1-MMP complex was determined.90 The overall shape of the TIMP
molecule is like a wedge, which slots into the active-site cleft
of an MMP in a manner similar to that of the substrate. Figure
5 shows the structures of the MT1-MMP catalytic domain,
TIMP-2, and their interaction.90 The main sites of interaction
of TIMP-2 with the catalytic domain are the N-terminal four
residues and the CD-loop region adjacent to them. The
N-terminal four residues bind in the catalytic site cleft,
making backbone contacts similar to those of a substrate.
Residues at 1 and 3 are strictly conserved cysteines that form
disulfide bonds in the main body of the protein. Cys1 is
instrumental in chelating the active-site zinc with its
N-terminal ␣-amino group and carbonyl group, thereby
expelling the water molecule bound to the catalytic zinc.
TIMPs inhibit all MMPs tested so far, except that
TIMP-1 fails to inhibit MT1-MMP.91 However, the inhibitory property of TIMP-3 is different from the rest,
inasmuch as it inhibits ADAM-17 (TACE),92 ADAM-10,93
ADAM-12,94 and the aggrecanases (ADAMTS-4 and
ADAMTS-5). 95 Kinetic studies have indicated that
TIMP-3 is a better inhibitor for ADAM-17 and aggrecanases than for MMPs. Another unique feature of TIMP-3
is that it binds tightly to sulfated glycosaminoglycans.96 A
possible role for TIMP-3 heart failure was observed with a
reduction in the levels of TIMP-3, corresponding with
adverse matrix remodeling in a cardiomyopathic hamster
model and in the failing human heart.97
Application of TIMPs as a therapeutic tool for cardiovascular disease and cancer through gene therapy or direct
Visse and Nagase
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protein application is still in an early phase of development
(review by Baker et al98). However, there is a clear potential
for the application of TIMPs as endogenous inhibitors,
especially because the results of clinical trials with small
molecule inhibitors have been disappointing.99 For example,
adenoviral overexpression of TIMP-1 in a mouse model of
atherosclerosis showed a reduction in the lesion.100 Local
expression of TIMP-1 in a rat model of aneurysm prevented
aneurism degradation and rupture.101 However, expressing
wild-type TIMPs could have drawbacks because multiple
MMPs may be inhibited, and in the case of TIMP-3, ADAMs
and ADAMTSs may be inhibited as well. Probably the best
route to success will be the development of engineered
TIMPs with altered specificity, to allow targeting of specific
proteinases.
Proteins such as plasma ␣-macroglobulins are general
endopeptidase inhibitors that inhibit most proteinases by
trapping them within the macroglobulin after proteolysis of
the bait region of the inhibitor.102 MMP-1 reacts with ␣2macroglobulin more readily than with TIMP-1 in solution.103
Several other proteins have been reported to inhibit MMPs.
Tissue factor pathway inhibitor-2 is a serine protease inhibitor that inhibits MMPs.104 A C-terminal fragment of the
procollagen C-terminal proteinase enhancer protein has been
shown to inhibit MMP-2.105 The secreted form, membranebound ␤-amyloid precursor protein, has also been reported to
inhibit MMP-2.106 RECK, a GPI-anchored glycoprotein that
downregulates the levels of MMP-9 and active MMP-2 and
suppresses angiogenic sprouting, leading to tumor celldeath,107 inhibits the proteolytic activity of MMP-2, MMP-9,
and MT1-MMP.107,108 MMP-2, but not MMP-1, MMP-3, and
MMP-9, is inhibited by chlorotoxin, a scorpion toxin that has
anti-invasive effects on glioma cells.109 However, the mechanisms of MMP inhibition by these proteins are not known.
Biological Functions of TIMPs
In addition to metalloproteinase-inhibiting activities, TIMPs
have other biological functions. TIMP-1 and TIMP-2 have
erythroid-potentiating activity110,111 and cell growth–promoting activities.112,113 Zhao et al114 found TIMP-1 in the nucleus
of fibroblasts, and Ritter et al115 reported that TIMP-1 binds
to the cell surface of MCF-7 breast carcinoma cells and
subsequently translocates to the nucleus. During nephron
morphogenesis, TIMP-2 participates in metanephric mesenchymal growth and in the morphogenesis of the ureteric bud,
and the former activity is not due to MMP inhibition.116
Overexpression of TIMP-1, TIMP-2, and TIMP-3 reduces
tumor growth (see Gomez et al4 for review). TIMP-2, but not
TIMP-1, inhibits endothelial cell growth induced by basic
fibroblast growth factor.117 These activities are also distinct
from MMP inhibition, and their mechanism largely remains
to be discovered.
TIMP-3 has proapoptotic activity, possibly through the
stabilization of TNF-␣ cell receptor 1, Fas, and TNF-related
apoptosis, inducing ligand receptor-1, as shown for some
tumor cells.118,119 On the other hand, TIMP-1 and TIMP-2
have antiapoptotic activity.120,121 TIMP-3 is associated with
Sorsby’s fundus dystrophy, an autosomal-dominant disease
that causes blindness due to macular degeneration.122 Muta-
Structure and Function of MMPs and TIMPs
835
tions are all found in the C-terminal domain and include the
substitution of a residue for a cysteine,123 a nonsense mutation,124 or a splice mutation,125 resulting in the deposition of
the mutant TIMP-3 in Bruch’s membrane. Qi et al126 reported
that the S156C mutant exhibited some reduction in MMP
inhibitory activity, which was considered to promote angiogenesis. How this affects macular degeneration is not clear,
but the S156C and S181C mutants form multiple complexes
due to aberrant protein interaction and increased cellular
adhesiveness, which may impinge on the turnover of Bruch’s
membrane.127
Conclusion and Future Prospects
MMPs are important components in many biological and
pathological processes because of their ability to degrade
ECM components. It has become clear that the ECM is not a
mere scaffold for cells but that it also harbors cryptic
biological functions that can be revealed on proteolysis. This
puts a new light on the interplay between cells, the ECM, and
its catabolism.
Considerable advancements have been made in the understanding of biochemical and structural aspects of MMPs,
including their activation and catalytic mechanisms, substrate
specificity, and the mechanism of inhibition by TIMPs.
Nonetheless, there are important questions that remain outstanding. The structure of the proMMP-2–TIMP2 complex is
a big step toward understanding how proMMP-2 assembles
with TIMP-2 and MT1-MMP on the cell surface, but the
precise molecular assembly in time and space during cell
migration is yet to be investigated. In addition, although
collagenase was the first member of the family to be
discovered, the mechanism by which collagenases cleave
triple-helical collagens is not understood. An explanation as
to how TIMP-3, but not other structurally related TIMPs,
inhibits metalloproteinases of the ADAM family awaits
future structural studies.
Structural analyses have also led to the design of potent
synthetic matrixin inhibitors, some of which have exhibited
efficacy in animal models of cancer and arthritis, but unfortunately, clinical trials have shown no significant benefit.
Such discrepancies may be due to the fact that the trials were
conducted on patients with advanced stages of disease. Other
possibilities are that the inhibitor concentration reached in
vivo was insufficient to inhibit target enzymes in the tissue or
that nontarget enzymes were inhibited. Currently, 23 MMPs
and ⬎30 ADAM metalloproteinases are known in humans,
but their biological functions are not clearly understood. The
design of specific inhibitors for these metalloproteinases is an
important future challenge. Such inhibitors are useful not
only for gaining insights into the biological roles of MMPs
but also for the development of therapeutic interventions for
diseases associated with unbalanced ECM degradation.
Acknowledgments
This study was supported by The Wellcome Trust (grant 057508).
We would like to thank Dr L. Troeberg for critical reading of the
manuscript.
836
Circulation Research
May 2, 2003
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Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases: Structure,
Function, and Biochemistry
Robert Visse and Hideaki Nagase
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Circ Res. 2003;92:827-839
doi: 10.1161/01.RES.0000070112.80711.3D
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Supplementary data
Table 1.
3. MMPs, substrates and structures
Online Table
Groups of MMPs are listed with their trivial names and substrates cleaved are subdivided into
“ECM” and “other substrates”. It should be noted that many of the substrates listed have been
tested in vitro only. This does not mean that they are also degraded in vivo, but it indicates
that the potential for cleavage exists. This list is an expansion of that provided by Woessner
and Nagase1, references are provided for substrates not listed therein. The names of the PDB
files of structures determined by X-ray crystallography and NMR are listed. These files with
their references can be downloaded from the protein databank (www.rcsb.org). cat; catalytic
domain only, Hpx; hemopexin domain only.
MMP
ECM substrates
Other substrates
Human
Chromo
some
3D structure (PDB code)
MMP-1
Collagens (type I, II, III, VII, VIII, X, and XI),
gelatin, fibronectin, vitronectin, laminin, entactin,
tenascin, aggrecan, link protein, myelin basic
protein, versican
Autolytic, C1q, α2-Macroglobulin, ovostatin, α1-PI, α1antichymotrypsin, IL1-β, proTNFα, IGFBP-2, IGFBP3, casein, serum amyloid A, proMMP-1, proMMP-2,
proMMP-9
11q22q23
Neutrophil
collagenase;
Collagenase 2
MMP-8
Collagens (type I, II, and III), aggrecan,
Autolytic, C1q, α2-Macroglobulin, ovostatin, α1-PI,
substance P, fibrinogen2, angiotensin I, angiotensin II,
bradykinin, plasmin C1-inhibitor
11q21q22
Collagenase 3
MMP-13
Autolytic, C1q, α2-Macroglobulin, casein, fibrinogen2,
factor XII2, α1-antichymotrypsin, proMMP-9
11q22.3
Collagenase 4
(Xenopus)
Gelatinases
Gelatinase A
MMP-18
Collagens (type I, II, III, IV3, VI, IX, X, and
XIV), collagen telopeptides, gelatin, fibronectin,
SPARC, aggrecan, perlecan , large tenascin-C3
Collagen type I (rat)4
mature protein; 1FBL
cat domain; 1CGF, 2TCL,
1AYK, 2AYK, 1HFC,
1CGL, 1CGE, 966C,
3AYK, 4AYK
cat domain; 1MNC, 1I76,
1JAO, 1MMB, 1JAN,
1JAP, 1JAQ, 1I73, 1KBC,
1A85, 1A86, 1BZS, 1JJ9,
1JH1
cat domain; 1CXV, 1FM1,
1FLS, 456c, 830c, 1EUB
Hpx domain; 1PEX
MMP-2
Collagens (type I, II5, III, IV, V, VII, X, and XI),
gelatin, elastin, fibronectin, vitronectin, laminin,
entactin, tenascin, SPARC, aggrecan, link
protein, galectin-36, versican, decorin, myelin
basic protein
16q13
Gelatinase B
MMP-9
Collagens (type IV, V, XI, and XIV), gelatin,
elastin, vitronectin, laminin, SPARC, aggrecan,
link protein, galectin-36, versican, decorin,
myelin basic protein,
Autolytic, α1-PI, α2-Macroglobulin7, α1antichymotrypsin, IL1-β, proTNFα, IGFBP-3, IGFBP5, substance P, serum amyloid A8, proMMP-1,
proMMP-2, proMMP-9, proMMP-13, latent TGFβ9,
MCP-3 (monocyte chemoattractant protein-3)10,
FGFR1 (fibroblast growth factor receptor 1), big
endothelin-111, plasminogen12
Autolytic, α2-Macroglobulin, ovostatin, α1-PI, IL1-β,
proTNFα, substance P, casein, carboxymethylatedtransferrin, angiotensin I, angiotensin II, plasminogen,
proTGFβ29, IL-2Rα13, release of VEGF (substrate not
known)14
Stromelysins
Stromelysin 1
MMP-3
Collagens (type III, IV, V, VII, IX, X, and XI),
collagen telopeptides, gelatin, elastin, fibronectin,
vitronectin, laminin, entactin, tenascin, SPARC,
Autolytic, α2-Macroglobulin, ovostatin, , α1PI, α2−antiplasmin16, α1-antichymotrypsin, IL1-β,
proTNFα, IGFBP-3, substance P, T-kininogen, casein,
11q23
Enzyme
Collagenases
Insterstitial
collagenase;
Collagenase 1
NA
2
20q11.2q13.1
proMMP-2; 1CK7
proMMP-2–TIMP-2
complex; 1GXD
cat domain; 1QIB, 1HOV,
1EAK
Hpx domain; 1GEN, 1RTG
Fn; 1CXW, 1KS0
pro-cat domain; 1L6J
cat domain; 1GKC, 1GKD
Hpx domain; 1ITV
pro-cat domain; 1SLM
cat domain; D8M, 1CIZ,
1CAQ, 1B8Y, 2SRT,
aggrecan, link protein, decorin, myelin basic
protein, perlecan, versican15, fibulin
carboxymethylated transferrin, antithrombin-III, serum
amyloid A, fibrinogen17, plasminogen, osteopontin18,
proMMP-1, proMMP-3, proMMP-7, proMMP-8,
proMMP-9, proMMP-13, IGFBP-3, E-cadherin19,20,
pro-HB-EGF, u-PA21, fibrin, PAI-122
Collagens (type III, IV, and V), gelatin, elastin,
fibronectin, aggrecan, link protein,
gelatin24, fibronectin24, collagen type IV24,
laminin24
Autolytic, casein, proMMP-1, proMMP-723, proMMP8, proMMP-923
α1-PI, α2-Macroglobulin, ovostatin, IGBFP-1, casein,
α2−antiplasmin25, plasminogen activator inhibitor-225,
casein24, carboxymethylated-transferrin24
11q22.3q23
22q11.2
1HV526
Autolytic, α1-PI, α2-Macroglobulin, proTNFα, casein,
carboxymethylated transferrin, osteopontin18,
proMMP-1, proMMP-2, pro-MMP-7 proMMP-9,
plasminogen,
pro-α-defensin27, Fas-L 28,29, β4 integrin, E-cadherin20
α1-PI31,32, α2-Macroglobulin31, fibrinogen30,31,
proMMP-930
11q21q22
cat domain; 1MMP,
1MMQ, 1MMR
α2-Macroglobulin, ovostatin, α1-PI,
proTNFα, fibrinogen2, factor XII2, fibrin17, CD4433,
tissue transglutaminase34, proMMP-2, proMMP-13
proTNFα35 , tissue transglutaminase34, proMMP-2
14q11q12
Stromelysin 2
MMP-10
Stromelysin 3
MMP-11
Matrilysins
Matrilysin 1
Pump-1
MMP-7
Collagens (type I, and IV), gelatin, elastin,
fibronectin, vitronectin, laminin, entactin,
tenascin, SPARC, aggrecan, link protein, decorin,
myelin basic protein, fibulin, versican,
Matrilysin 2
MMP-26
Collagen type IV yes30; no31, gelatin30,31,
fibronectin30,31, vitronectin31
MT1-MMP
MMP-14
MT2-MMP
MMP-15
MT3-MMP
MMP-16
MT5-MMP
MMP-24
Collagens (type I, II, and III), gelatin, fibronectin,
tenascin, vitronectin, laminin, entactin, aggrecan,
perlecan
fibronectin35, tenascin35, entactin35, laminin35,
aggrecan, perlecan,
Collagen type III36, gelatin36, fibronectin36,
vitronectin36, laminin36,
Fibronectin 37,
Gelatin37, chondroitin sulphate proteoglycan37,
dermatan sulphate proteoglycan 37
1HFS, 1SLN, 2USN,
1USN, 1D5J, 1BQO,
1D7X, 1D8F, 1BIW,
1UMS, 3USN, 1UMT,
1BM6, 1B3D, 1CQR,
1G4K, 1G49, 1HY7, 1G05
complex with N-TIMP-1;
1UEA
11p15
Membranetype MMPs
A)Transmembrane
α1-PI36, α2-Macroglobulin36, casein36, proMMP-2,
tissue transglutaminase34
ProMMP-238,39 37, tissue transglutaminase34
3
15q13q21
8q21
20q11.2
cat domain in complex with
TIMP-2; 1BQQ, 1BUV
B) GPI –
anchor
MT4-MMP
MMP-17
gelatin40
MT6-MMP
MMP-25
Collagen type IV43, gelatin43,44, fibronectin43,44,
chondroitin sulphate proteoglycan44, dermatan
sulphate proteoglycan44
Others
Macrophage
elastase
MMP-12
No trivial name
MMP-19
Enamelysin
XMMP
(Xenopus)
CA-MMP
MMP-20
MMP-21
Collagens (type I, V, and IV), gelatin, elastin,
fibronectin, vitronectin, laminin, entactin,
osteonectin46, aggrecan, myelin basic protein
Collagen type IV51, gelatin51, laminin51, entactin51,
large tenascin-C51, fibronectin51, aggrecan52,
COMP52
Amelogenin, aggrecan52, COMP52
gelatin54
MMP-23
gelatin
CMMP (Gallus)
MMP-27
gelatin54
Epilysin
MMP-28
Fibrinogen41, fibrin41, proTNFα41, proMMP-2 yes40;
no41,42
Fibrinogen43, fibrin43, α1-PI44, proMMP-245
12q24.3
α2-Macroglobulin, α1-PI, proTNFα, fibrinogen2,
factor XII2, casein, plasminogen47,48
11q22.2q22.3
autolysis51, fibrinogen51, fibrin51
12q14
autolysis53
casein54
11q22.3
ND
55
16p13.3
1p36.3
autolysis54, casein54
56
casein
11q24
17q21.1
4
cat domain 1JK3 49, 1JIZ50
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9