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INTRODUCTION |
Matrix metalloproteinases
(MMPs)1 are
zinc-dependent endopeptidases that degrade components of
extracellular matrix and play an essential role in tissue remodeling
under physiological and pathological conditions such as morphogenesis,
angiogenesis, tissue repair, and tumor invasion (1-3). Most MMPs are
secreted as a zymogen and are activated by serine proteases or some
activated MMPs. The activities of activated MMPs are regulated by a
family of specific inhibitors known as tissue inhibitor of
metalloproteinases (TIMPs). Among the MMP family, gelatinase A (MMP-2)
and gelatinase B (MMP-9) are critical in the invasion of tumor cells
across basement membranes because of their strong activity against type
IV collagen, a major component of basement membranes (4-6). Unlike
other zymogen of MMPs, progelatinase A is not activated by serine
proteases or soluble MMPs and had been reported to be activated by a
MMP-like activity on the surface of cancer and fibroblastic cells
(7-10). Sato et al. (11) recently identified a novel
membrane-type MMP, named MT-MMP as an activator of progelatinase A on
the cell surface. The cell-mediated activation of progelatinase A
includes two steps of processing: MT-MMP-catalyzed cleavage of
progelatinase A at a peptide bond between Asn37 and
Leu38, first converts the zymogen into an intermediate
form, and then autocatalytic cleavage of a
Asn80-Tyr81 bond converts the intermediate form
into a mature one (12). Several studies suggest that both steps are
greatly accelerated by binding of (pro)gelatinase A onto the cell
surface, and therefore, the receptor of (pro)gelatinase A on the cell
surface is important for the activation. Carboxyl-terminal
hemopexin-like domain of gelatinase A is reported to be essential for
the interaction with the cell surface receptor (12, 13). The
NH2-terminal reactive site of TIMP-2 binds to the active
site of MT-MMP to form a protease-inhibitor complex, whereas the
COOH-terminal region of TIMP-2 has an affinity to the hemopexin-like
domain of gelatinase A. Therefore, it is hypothesized that a complex
formed between MT-MMP and TIMP-2 acts as a receptor of progelatinase A. This hypothesis appears to be supported by a finding that
overexpression of MT-MMP results in an accumulation of gelatinase A on
the cell surface (11). Another candidate for the gelatinase A receptor
is integrin
v
3, which forms a sodium dodecyl sulfate stable
complex with gelatinase A also by binding to the hemopexin-like domain
(14, 15). TIMP-2 is a bifunctional regulator of the cell-mediated
activation of progelatinase A. Strongin et al. (13)
demonstrated that a small amount of TIMP-2 facilitates the activation
of progelatinase A by the MT-MMP-containing cell membrane, whereas
excess TIMP-2 strongly inhibits the activation. This could be explained
that the binding of TIMP-2 to MT-MMP provides a receptor for
progelatinase A and also leads to an inhibition of catalytic activity
of MT-MMP. However, the detailed mechanism remains to be clarified.
Recently, we examined expression levels of gelatinase A, TIMP-2, and
three MT-MMPs in human cancer cell lines and found that activation of progelatinase A has a strong inverse correlation only with the level of
TIMP-2 secreted into culture medium (16), suggesting that TIMP-2 is a
key regulator of the activation of progelatinase A. In this study, we
prepared a chemically modified TIMP-2 of which the reactive site is
destroyed, and the modified inhibitor was examined for its effect on
the cell-mediated activation of progelatinase A. Mechanisms related to
the TIMP-2 regulation of the activation of progelatinase A are discussed.
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EXPERIMENTAL PROCEDURES |
Materials--
The sources of materials used were as follows:
3167v
(7-methoxycoumarin-4-yl)-acetyl-Arg-Pro-Lys-Pro-Tyr-Ala-norvalyl-Trp-Met-N
-(2,4-dinitrophenyl)-lysine amide) was from Peptide Institute, Inc. (Osaka, Japan); potassium cyanate was from Wako Pure Chemical Industries (Osaka);
p-aminophenyl mercuric acetate (APMA) from Tokyo Kasei
(Tokyo, Japan); CNBr-activated Sepharose 4B from Pharmacia Fine
Chemicals (Uppsala, Sweden); Ultrasphere ODS 5U (2.0 × 150 mm)
from Beckman (Fullerton, CA). Bovine pancreatic trypsin treated with
N-tosyl-L-phenylalanine chloromethyl ketone was
purchased from Worthington (Freehold, NJ); the plant lectin
concanavalin A (type IV, substantially free of carbohydrates) was from
Sigma; gelatin from Difco (Detroit, MI). Recombinant human matrilysin
was a generous gift from Dr. Y. Matsuo, Oriental Yeast (Shiga, Japan).
All other chemicals were of analytical grade or the highest quality
commercially available.
Proteins--
TIMP-2-free and TIMP-2-bound forms of
progelatinase A were purified, separately, from the conditioned medium
(CM) of the T98G human glioblastoma cell line, as described previously
(17). TIMP-2 was purified from the TIMP-2-bound progelatinase A using a
SynChropak RP-4 reverse-phase column (SynChrom; Lafayette, IN) according to the method of Collier et al. (5). Rabbit
antiserum against progelatinase A was prepared in our laboratory.
Chemical Modification of TIMP-2 with KNCO--
Fifty µl of 1.0 M KNCO was added to 200 µl of protein solution, which
contained 500 pmol of TIMP-2 in 50 mM Tris-HCl (pH 7.5) containing 0.1 M NaCl and 0.01% NaN3
(Tris-buffered saline; TBS). The mixture was incubated at 37 °C for
0, 30, 60, 120, and 240 min. After incubation, 50 µl of each sample
taken from the reaction mixture was mixed with 20 µl of 1.0 M hydroxylamine hydrochloride (pH 8.0) and incubated at
25 °C for 1 h to terminate the modification reaction. The
resultant reaction mixtures were dialyzed against TBS at 4 °C.
Assay of Inhibitory Activity of TIMP-2 after Chemical
Modification--
After modification of TIMP-2 under various
conditions, various concentrations of the modified TIMP-2 were
incubated with matrilysin (33 nM) in 90 µl of TBS
containing 10 mM CaCl2 and 0.01% Brij 35 at
37 °C for 15 min. The mixtures were added with 10 µl of 1 mM 3167v, and further incubated for 40 min. The reaction
was terminated by adding 100 µl of 0.1 M EDTA (pH 7.5).
The amounts of 3167v hydrolyzed by matrilysin were measured
fluorometrically with excitation at 360 nm and emission at 460 nm. The
amount of 3167v hydrolyzed without enzyme was subtracted from the total amount of the hydrolyzed substrate.
Separation of Active and Inactive TIMP-2 after Partial
Carbamylation--
TIMP-2 (150 µg) was incubated with 0.2 M KNCO in 500 µl of TBS at 37 °C for 25 min. This
treatment resulted in a 50% reduction of inhibitory activity of
TIMP-2. The TIMP-2 sample was further incubated with 0.2 M
hydroxylamine hydrochloride at 25 °C for 1 h, and then dialyzed
extensively against TBS containing 10 mM CaCl2
at 4 °C. To separate inactive TIMP-2 from active TIMP-2, the
reaction mixture was applied to an matrilysin-Sepharose 4B column in
which 100 µg of matrilysin had been coupled to 500 µl of
CNBr-activated Sepharose 4B, and the flow-through fraction containing
inactive TIMP-2 was collected. After washing the column with TBS
containing 10 mM CaCl2, the adsorbed sample
(active TIMP-2) was eluted with TBS containing 4 M
guanidine hydrochloride and 20 mM EDTA. After the elution,
the column was washed sequentially with TBS containing 10 mM CaCl2 plus 50 µM
ZnCl2 and with TBS containing 10 mM
CaCl2 to renature the immobilized matrilysin. The TIMP-2
samples in the flow-through and eluted fractions were separately
dialyzed against phosphate-buffered saline.
Inhibition Assay of Gelatinase A Activity by Modified Derivatives
of TIMP-2--
TIMP-2-free form of progelatinase A was activated by
incubating with 1 mM APMA at 37 °C for 1 h as
described previously (17). The activated gelatinase A (89 nM) was incubated with various concentrations of the
KNCO-treated derivatives of TIMP-2 in 90 µl of TBS containing 10 mM CaCl2 and 0.01% Brij 35 at 37 °C for 15 min. The mixtures were added with 10 µl of 1 mM 3167v,
and further incubated for 40 min. The reaction was terminated by adding 100 µl of 0.1 M EDTA (pH 7.5). The hydrolyzed 3167v was
measured as described above.
Reduction and S-Carboxyamidomethylation of KNCO-treated
TIMP-2 Forms in Matrilysin-bound and Matrilysin-unbound
Fractions--
Each of the KNCO-treated TIMP-2 forms in
matrilysin-bound and matrilysin-unbound fractions (10 µM)
was incubated with 100 mM dithiothreitol in TBS containing
4 M guanidine hydrochloride and 20 mM EDTA at
50 °C for 30 min. After incubation, the samples were transferred to
a container of ice water and further incubated with 240 mM
iodoacetamide. After 2 h, the samples were dialyzed against
TBS.
Cell Culture and Preparation of CM and Cell Lysate--
HT1080
fibrosarcoma cell line was grown to semi-confluency in a 1:1 mixture of
Dulbecco's modified Eagles's medium and Ham's F-12 medium (Life
Technologies, Inc., Grand Island, NY), Dulbecco's modified
Eagle's/Ham's F-12 medium, supplemented with 10% fetal calf serum.
The cells were rinsed three times with serum-free Dulbecco's modified
Eagle's/Ham's F-12 medium, and the culture was further continued in
the presence of various concentrations of TIMP-2 or modified TIMP-2 and
a fixed concentration of concanavalin A (100 µg/ml) in serum-free
Dulbecco's modified Eagle's/Ham's F-12 medium. After 24 h, the
resultant CM was collected, clarified by centrifugation, and dialyzed
against distilled water at 4 °C. The sample was then lyophilized and
dissolved in a small volume of a sodium dodecyl sulfate-sampling buffer
consisting of 50 mM Tris-HCl (pH 6.8), 2% sodium dodecyl
sulfate, and 10% glycerol. By these procedures, the initial CM was
concentrated 20-fold. To prepare cell lysates, the cells were rinsed
three times with phosphate-buffered saline, and then dissolved in a
small volume of the sodium dodecyl sulfate-sampling buffer.
Ligand Blotting Analysis--
TIMP-2 or modified TIMP-2 was
subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
under nonreducing conditions. After electrophoresis, the proteins on
the gel were transferred onto nitrocellulose membrane, using a Bio-Rad
Mini Trans-Blot apparatus. The membrane was blocked with TBS-containing
5% skim milk at room temperature for 12 h, washed with TBS
containing 0.05% Tween 20, 10 mM CaCl2, and
0.1% bovine serum albumin (TBS-Tween), and then incubated at room
temperature with progelatinase A (5 µg/ml) in TBS-Tween. After 3 h, the membrane was washed with TBS-Tween and incubated for 3 h
with an anti-progelatinase A antiserum, which had been diluted
1000-fold with TBS-Tween. After washing with TBS-Tween, the membrane
was incubated 1000-fold diluted biotinylated anti-rabbit IgG antibody
(Vector Laboratories, Burlingame, CA), washed with TBS-Tween, and then
incubated with avidin-alkaline phosphatase (Vector) at room temperature
for 1 h. The membrane was washed extensively and then incubated in
a reaction mixture containing 5-bromo-4-chloro-3-indolylphosphate and
nitro blue tetrazolium to develop colored product on the membrane.
Gelatin Zymography--
Zymography was carried out on 10%
polyacrylamide gels containing 1 mg/ml gelatin, as described previously
(18).
Amino-terminal Sequence Analysis--
Samples were analyzed on
an Applied Biosystems 477A gas-phase sequencer. Phenylthiohydantoin
derivatives were detected using an Applied Biosystems 120A PTH analyzer
with an on-line system.
Mass Spectrometric Analysis--
Tryptic peptides of TIMP-2 (10 pmol/µl) were mixed together with an equal volume of
-cyano-4-hydroxycinnamic acid solution (10 mg of
-cyano-4-hydroxycinnamic acid was dissolved in 1 ml of 50%
acetonitrile containing 0.1% trifluoroacetic acid). The sample/matrix
solution was dropped onto a sample plate for matrix-assisted laser
desorption ionization time of flight mass spectrometry, then dried
under ambient conditions. A mass spectrum was obtained on a
Voyager-DETM STR system (PerSeptive Biosystems, Inc.,
Framingham, MA).
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RESULTS |
Effect of KNCO Treatment of TIMP-2 on the Inhibitory
Activity--
The recently determined crystal structure of the complex
formed between TIMP-1 and stromelysin suggests that the
-amino group of the NH2-terminal Cys1 of TIMP-1 binds to the
catalytic zinc atom at the active site of stromelysin, thus playing an
essential role in the inhibitory action of TIMP-1 (19). As the
structure of the NH2-terminal region of TIMP-2 is
homologous to that of TIMP-1, the
-amino group of Cys1
of TIMP-2, corresponding to that of TIMP-1 may be critical for the
inhibitory activity of TIMP-2. To examine this possibility, we
attempted to carbamylate the
-amino group of Cys1 by
treating TIMP-2 with KNCO under various conditions, and the chemically
modified derivatives of TIMP-2 were examined for their abilities to
inhibit the matrilysin-catalyzed hydrolysis of 3167v. As shown in Fig.
1A, the incubation of TIMP-2
with KNCO led to increase in the IC50 value of the
inhibition, where IC50 represents a concentration of the
modified derivatives of TIMP-2 giving a 50% inhibition of the activity
of matrilysin. When the inverse values of the IC50
versus incubation time with KNCO were plotted, the
1/IC50 value diminished with increasing time of incubation with KNCO, and 50% reduction of the 1/IC50 value was
observed when the incubation time was 25 min (Fig. 1B). The
inhibitory activity of TIMP-2 was abolished after 4 h incubation
with KNCO.

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Fig. 1.
Effect of KNCO on the inhibitory activity of
TIMP-2. TIMP-2 (2 µM) was incubated with 0.2 M KNCO in TBS at 37 °C for 0 ( ), 30 ( ), 60 ( ),
120 ( ), and 240 (×) min. After incubation, each of the samples were
treated with hydroxylamine hydrochloride, and dialyzed against TBS as
described under "Experimental Procedures." In panel A,
matrilysin (30 nM) was incubated with 0.1 mM
3167v at 37 °C for 40 min in the presence of various concentrations
of the KNCO-treated derivatives of TIMP-2. All the reaction mixtures
contained TBS, 10 mM CaCl2, and 0.01% Brij 35. The amount of 3167v hydrolyzed by matrilysin was taken as 100%, and
the relative amount of 3167v hydrolyzed by matrilysin in the presence
of each concentration of the KNCO-treated derivatives of TIMP-2 is
shown on the ordinate. In panel B, inverse values
of IC50 obtained in panel A versus the
incubation time with KNCO are plotted. IC50 represents a
concentration of the KNCO-treated derivatives of TIMP-2 that gives a
50% inhibition of the activity of matrilysin.
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Separation of Active and Inactive Fractions after Partial
Modification of TIMP-2--
As described under "Experimental
Procedures," TIMP-2 was treated with 0.2 M KNCO at
37 °C for 25 min. This modification led to loss of 50% inhibitory
activity of TIMP-2 (Fig. 1). The partially modified TIMP-2 was then
separated on an matrilysin-Sepharose 4B column. After separation,
matrilysin-bound and matrilysin-unbound fractions contained almost the
same amount of protein (data not shown), suggesting that about 50% of
the modified TIMP-2 before separation had essentially no affinity for
matrilysin. The matrilysin-bound fraction and native TIMP-2 showed
comparable abilities to inhibit the matrilysin-catalyzed hydrolysis of
3167v (Fig. 2A). In contrast, the matrilysin-unbound fraction had no inhibitory activity, as expected. The matrilysin-unbound fraction was also inactive against APMA-activated gelatinase A (Fig. 2B). These data are
consistent with the view that treatment of TIMP-2 with KNCO leads to
modification of the reactive site of TIMP-2, thus preventing formation
of the protease-inhibitor complex.

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Fig. 2.
Inhibitory activity of KNCO-treated TIMP-2
forms in matrilysin-bound and matrilysin-unbound fractions. After
treatment with KNCO, the partially modified TIMP-2 was separated, using
a matrilysin-Sepharose 4B column as described under "Experimental
Procedures." Matrilysin (30 nM, panel A) and
APMA-activated gelatinase A (80 nM, panel B)
were incubated, respectively, with 0.1 mM 3167v at 37 °C
for 40 min in the presence of various concentrations of the
KNCO-treated TIMP-2 forms in the matrilysin-bound ( ) and
matrilysin-unbound ( ) fractions. All the reaction mixtures contained
TBS, 10 mM CaCl2, and 0.01% Brij 35. The
amount of 3167v hydrolyzed by enzyme was taken as 100%, and the
relative amount of 3167v hydrolyzed by enzyme in the presence of each
concentration of the KNCO-treated TIMP-2 forms is shown on the
ordinate.
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Determination of the Site of Modification Responsible for the Loss
of Inhibitory Activity of TIMP-2--
To determine the site of
modification responsible for the loss of inhibitory activity, the
samples in matrilysin-bound and matrilysin-unbound fractions were
reduced and S-carboxyamidomethylated and then subjected to
tryptic digestion, after which the digests were separated by
reversed-phase high performance liquid chromatography. The differences
observed between the two elution profiles were only peaks B-20 and U-21
from the matrilysin-bound and matrilysin-unbound fractions,
respectively (Fig. 3, A and
B). The mass spectrometric analyses of the peptides (Fig.
4, A and B) showed
that molecular masses of B-20 and U-21 were 2345.22 and 2388.26, respectively. Based on the determined molecular mass, B-20 is assigned
as the peptide corresponding to residues 1-20 of human TIMP-2. On the other hand, difference of molecular masses between B-20 and U-21 corresponds to the mass of a carbamyl adduct, suggesting that U-21
is a peptide corresponding to residues 1-20 of TIMP-2
bearing a single carbamylated amino group. Furthermore, the
ZSZSPVHPQQAFZNADVVI sequence corresponding to residues 1-19 of TIMP-2
was determined in the NH2-terminal sequence analysis on
B-20, where Z was detected as a phenylthiohydantoin-derivative of
S-carboxyamidomethylcysteine. As expected, no
phenylthiohydantoin-derivative of amino acid was detected in the
NH2-terminal sequence analyses of U-21. These results
indicate that B-20 and U-21 are peptides derived from the
NH2-terminal region of TIMP-2 corresponding to residues
1-20, and that the
-amino group of Cys1 of U-21 is
carbamylated. The results also suggest that the carbamylation of the
-amino group of NH2-terminal Cys1 of TIMP-2
leads to the inactivation of TIMP-2.

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Fig. 3.
High performance liquid chromatography
separation of tryptic peptides of KNCO-treated TIMP-2 forms in
matrilysin-bound and matrilysin-unbound fractions. Each of the
KNCO-treated TIMP-2 forms in the matrilysin-bound (panel A)
and matrilysin-unbound (panel B) fractions was reduced and
S-carboxyamidomethylated as described under "Experimental
Procedures," and then digested with trypsin in an enzyme to substrate
ratio of 1:100 (w/w) at 37 °C for 24 h. The digest was applied
to an Ultrasphere ODS 5U column (2.0 × 150 mm) and eluted at a
flow rate of 0.5 ml/min with a linear gradient of acetonitrile
containing 0.05% trifluoroacetic acid. The column eluate was monitored
at 206 nm (solid lines), and the broken line
shows the percentage of acetonitrile in the elution medium.
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Fig. 4.
Mass spectrum of B-20 and U-21. Peaks
B-20 (panel A) and U-21 (panel B) obtained from
the ODS column (Fig. 3) were subjected to matrix-assisted laser
desorption ionization time of flight mass spectrometry, using 10 mg/ml
-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 0.1%
trifluoroacetic acid as the matrix solution.
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Effect of KNCO Treatment of TIMP-2 on the Progelatinase A Binding
Ability--
In addition to the MMPs inhibitory activity, TIMP-2 also
has an ability to interact with the hemopexin-like domain of
progelatinase A. To examine whether the carbamylation of TIMP-2 affects
the progelatinase A binding ability, the matrilysin-bound and
matrilysin-unbound fractions of KNCO-treated TIMP-2 and native TIMP-2
were tested for their progelatinase A binding abilities, using the
ligand blotting analysis as described under "Experimental
Procedures." As shown in Fig. 5, native
TIMP-2 and the KNCO-treated TIMP-2 in the matrilysin-unbound fraction
and that in matrilysin-bound one had comparable abilities to bind with
progelatinase A, suggesting that the carbamylation of TIMP-2 has
essentially no effect on the interaction with progelatinase A.

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Fig. 5.
Progelatinase A binding ability of
KNCO-treated TIMP-2 forms in matrilysin-bound and matrilysin-unbound
fractions. Indicated amounts of the KNCO-treated TIMP-2 forms in
the matrilysin-unbound fraction (NH2-terminal modified
TIMP-2), matrilysin-bound fraction (unmodified TIMP-2), and native
TIMP-2 were subjected to ligand blotting analysis as described under
"Experimental Procedures." Ordinate, molecular size in
kDa.
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Effect of Reactive Site-modified TIMP-2 and Native TIMP-2 on the
Cell-mediated Activation of Progelatinase A--
It has been
hypothesized that a complex formed between MT-MMP and TIMP-2 acts as a
receptor of progelatinase A and the formation of the ternary complex is
essential for the cell-mediated activation of progelatinase A (12, 13,
20). Since the matrilysin-unbound fraction of carbamylated TIMP-2 loses
the reactive site to interact with the active site of MMPs while
retaining the progelatinase A-binding site, the reactive site-modified
TIMP-2 may be able to prevent the formation of the ternary complex by
competing for the limited number of the TIMP-2-binding sites of
progelatinase A. To examine this possibility, various concentrations of
the reactive site-modified and -unmodified TIMP-2 forms and native TIMP-2 were added to the culture medium of concanavalin A-stimulated HT1080 cells and various species of endogenous gelatinase A in the cell
lysate and those in the CM were analyzed by gelatin zymography. As
shown in Fig. 6A, the
cell-associated mature form of gelatinase A was gradually diminished
with increasing concentrations of the reactive site-modified inactive
TIMP-2 in the matrilysin-unbound fraction. Progelatinase A and
progelatinase B in the cell lysate were not affected by the reactive
site-modified TIMP-2. These detected zymogens may be pre-secreted
proteins. In the CM, the mature form but not the intermediate form of
gelatinase A almost disappeared as the concentration of the modified
TIMP-2 was increased to 36 nM or higher, whereas the amount
of progelatinase A increased with increasing concentrations of the
TIMP-2 (Fig. 6B), suggesting that the conversion of endogenous
progelatinase A to the intermediate form was partially inhibited,
whereas the conversion of the intermediate form to the mature form was
strongly inhibited in the presence of high concentrations of the
reactive site-modified TIMP-2. The disappearance of the mature form of
gelatinase A in the CM was in parallel with the diminution of the
cell-associated mature form. Therefore, the conversion of the
intermediate form to the mature one may depend on the cell-associated
active gelatinase A. On the other hand, when increasing concentrations
of the active TIMP-2 in the matrilysin-bound fraction were added into
the culture of HT1080 cells, the cell-associated mature form of
gelatinase A increased slightly at 4.5 nM active TIMP-2,
and then sharply diminished at higher concentrations (Fig.
6A). Both the mature and intermediate forms of gelatinase A
in the CM disappeared, whereas progelatinase A increased with
increasing concentrations of the active TIMP-2, suggesting that
proteolytic processing of progelatinase A was inhibited in the presence
of active TIMP-2. The disappearance of the mature and intermediate
forms of gelatinase A in the CM was also in parallel with the
diminution of the cell-associated mature form. As the inhibition of
processing of progelatinase A by the active TIMP-2 did not lead to
increasing the amount of cell-associated progelatinase A, the
cell-associated zymogen may be released at high concentrations of
TIMP-2. The effects of native TIMP-2 on the cell-associated gelatinase
A and on the cell-mediated activation of progelatinase A were almost
the same as those of the active TIMP-2 in the matrilysin-bound fraction
(data not shown).

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Fig. 6.
Effects of NH2-terminal modified
and unmodified TIMP-2 on processing of progelatinase A in lysate and CM
of concanavalin A-stimulated HT1080 cells. HT1080 cells were
incubated for 24 h in serum-free medium with the indicated
concentrations of the KNCO-treated TIMP-2 forms in the
matrilysin-unbound fraction (NH2-terminal modified TIMP-2)
and matrilysin-bound fraction (unmodified TIMP-2) and a fixed
concentration (100 µg/ml) of concanavalin A. Cell lysates
(panel A) and CMs (panel B) were prepared from
the incubated cells and subjected to gelatin zymography as described
under "Experimental Procedures." Arrowheads indicate the
gelatinolytic bands of progelatinase A at 66 kDa (upper),
the intermediate form at 59 kDa (center), and the mature
form at 57 kDa (lower). An arrow at 90 kDa
indicates a gelatinolytic band of progelatinase B. Ordinate,
molecular size in kDa.
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DISCUSSION |
To explore the reactive site of TIMP-2 involved in the interaction
with the active site of MMPs, we treated TIMP-2 with cyanate ions under
controlled conditions, and identified an amino group essential for the
inhibitory activity of TIMP-2. We also examined effects of the reactive
site-modified TIMP-2 on the cell-mediated activation of progelatinase
A. We found that carbamylation of the
-amino group of the
NH2-terminal Cys1 of TIMP-2 led to complete
losses of its inhibitory activity and binding ability to matrilysin.
The crystal structure of the complex formed between TIMP-1 and
stromelysin suggests that the unprotonated
-amino group and carbonyl
oxygen of the NH2-terminal Cys1 of TIMP-1
coordinate the catalytic zinc atom of stromelysin, thus being involved
in the inhibitory action (19). Quite recently, the crystal structure of
the complex formed between TIMP-2 and catalytic domain of MT1-MMP was
also determined (21). According to their data, the
-amino group and
carbonyl oxygen of the NH2-terminal Cys1 of
TIMP-2 similarly interact with the catalytic zinc of the protease, suggesting that chelation of the catalytic zinc atom by the
NH2-terminal Cys1 of TIMPs is a common
mechanism for the inhibition of MMPs activity. Carbamylation
of Cys1 of TIMP-2 must lead to a reduction of
basicity of the N
nitrogen of the
-amino group,
which probably makes it unable for the N
nitrogen to coordinate the catalytic zinc atom of MMPs,
thereby abolishing the inhibitory activity of TIMP-2. There is an
alternative explanation that the carbamylated
-amino group of
Cys1 may not be able to interact with the catalytic zinc
atom due to steric hindrance. The crystal structures of the two
MMP·TIMP complexes also indicate that TIMPs have wide range contacts
with the corresponding MMPs. However, the present study showed that the
modified TIMP-2 bearing a single carbamylated
-amino group had
essentially no affinity with matrilysin. This discrepancy might be
explained by sequential interactions: the primary interaction between
the Cys1 of TIMPs and the catalytic zinc atom of MMPs may
trigger a rearrangement of residues to make secondary interactions.
Further study will be required to clarify this mechanism. Previously,
it has been reported that chemical modification of TIMP-1 with diethyl
pyrocarbonate abolishes the inhibitory activity. The modified residues
are His95, His144, and His164 of
TIMP-1, and the modification of His95 is proposed to be
responsible for the loss of activity (22). However, mutational study
has revealed that replacement of His95 to glutamine does
not affect the inhibitory activity of TIMP-1 (22). Furthermore, the
H95Q mutant is still sensitive to diethyl pyrocarbonate treatment. So
far, there is no explanation for the effect of diethyl pyrocarbonate on
the TIMP-1 activity. It is possible, however, to speculate that the
-amino group of Cys1 of TIMP-1 had been modified during
treatment with diethyl pyrocarbonate, because the
-amino group, as
well as the imidazole group, are reactive with diethyl pyrocarbonate.
As the carbamylated TIMP-2 in the matrilysin-unbound fraction had an
ability to bind to progelatinase A, it is likely that a site of TIMP-2
essential for the interaction with the hemopexin-like domain of
(pro)gelatinase A is not affected by the carbamylation. We found that
the reactive site-modified TIMP-2 could prevent an accumulation of the
active form of gelatinase A on the surface of concanavalin A-stimulated
HT1080 cells. It is hypothesized that a complex formed between MT-MMP
and TIMP-2 acts as a cell surface receptor of (pro)gelatinase A (12,
13). Accordingly, the disappearance of the cell-associated gelatinase A
could be explained by speculation that the competitive binding of the
reactive site-modified TIMP-2 to the hemopexin-like domain of
gelatinase A makes it unable for gelatinase A to be retained on the
cell surface, because TIMP-2 cannot interact with MT-MMP. We also found that the reactive site-modified TIMP-2 partially inhibited the conversion of progelatinase A to the intermediate form and strongly inhibited the conversion of the intermediate form to the mature one. As
the conversion of progelatinase A to the intermediate form is thought
to be facilitated by cell association of progelatinase A (20), the
partial inhibition of the processing of progelatinase A is likely to be
caused by the prevention of cell association of the zymogen by the
reactive site-modified TIMP-2 (Fig.
7A). We also speculate that
the conversion of the intermediate form of gelatinase A to the mature
one depends upon the cell associated activity of gelatinase A, and
therefore, deprivation of the cell-associated active form of gelatinase
A by the reactive site-modified TIMP-2 causes an inhibition of
production of the mature form. In the presence of high concentrations
of reactive site-modified TIMP-2, the disappearance of the mature form
of gelatinase A in the CM was indeed in parallel with the diminution of
the cell-associated active gelatinase A (Fig. 6). Recent studies (15,
23-26) suggest that transmembrane domainless variants of MT-MMP
convert progelatinase A to the intermediate form but hardly to the
mature one. It is also reported that cell-mediated processing of mutant
progelatinase A of which the active site residue is replaced does not
produce the mature form of the mutant (27, 28). These studies suggest the importance of cell associated activity of gelatinase A for the
conversion of the intermediate form of gelatinase A to its mature form.
Considering the importance of formation of the ternary complex
consisting of MT-MMP, TIMP-2, and (pro)gelatinase A, the inhibition of
the cell-mediated activation of progelatinase A by TIMP-2 could be
explained in two alternative ways. One explanation is that excess
TIMP-2 occupies both the active site of MT-MMP and the TIMP-2-binding
site in hemopexin-like domain of (pro)gelatinase A, thus preventing the
formation of the ternary complex (Fig. 7B). The other
explanation is that TIMP-2 inhibits the catalytic activity of MT-MMP,
thus inhibiting the proteolytic processing of progelatinase A. We found
that native TIMP-2, as well as reactive site-modified TIMP-2, could
prevent accumulation of active gelatinase A on the cell surface,
without increasing the cell-associated progelatinase A. These data
suggest that prevention of the formation of ternary complex contributes
to the TIMP-2 inhibition of the cell-mediated activation of
progelatinase A. Native TIMP-2, but not the reactive site-modified
TIMP-2, inhibited production of the intermediate form of gelatinase A. Therefore, it is also likely that inhibition of the catalytic activity
of MT-MMP by TIMP-2 contributes to inhibition of the processing of
progelatinase A. As disappearance of the mature and the intermediate
forms of gelatinase A in the CM and diminution of the cell-associated
active gelatinase A were observed at similar concentrations of
unmodified TIMP-2 (Fig. 6), prevention of formation of the ternary
complex and inhibition of MT-MMP activity may occur simultaneously, at
a critical concentration of TIMP-2 (Fig. 7B). It is likely
that both the mechanisms make TIMP-2 a potent regulator of the
cell-mediated activation of progelatinase A. As described here,
reactive site-modified TIMP-2 could inhibit the activation of
progelatinase A without inhibiting the catalytic activity of MT-MMP.
The reactive site-modified TIMP-2 might be a useful tool to distinguish
the functions of MT-MMP and cell-associated gelatinase A. We are now
using this modified TIMP-2 to explore the role of MT-MMP and/or
cell-associated gelatinase A in the processing of cell-surface
proteins.

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Fig. 7.
Hypothetical model for inhibitory effects of
reactive site-modified TIMP-2 and native TIMP-2 on formation of the
ternary complex consisting of MT-MMP, TIMP-2, and (pro)gelatinase
A. In panel A, the reactive site-modified TIMP-2
inhibits the formation of the ternary complex consisting of MT-MMP,
TIMP-2, and (pro)gelatinase A by competing for the hemopexin-like
domain of (pro)gelatinase A. The reactive site-modified TIMP-2
cannot interact with the active site of MT-MMP. In panel B,
an excess amount of native TIMP-2 inhibits the formation of
the ternary complex by occupying both the active site of MT-MMP and the
hemopexin-like domain of (pro)gelatinase A. H2N,
the -amino group of NH2-terminal Cys1 of
TIMP-2; H2NCONH, the carbamylated -amino
group of NH2-terminal Cys1 of TIMP-2;
Zn2+, catalytic zinc atom of
metalloproteinases.
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