(Received for publication, April 6, 1995)
From the
Gelatinase B is a Zn
Matrix metalloproteinases (MMP) ( These proteinases are highly homologous enzymes that share several
unique features, including a little understood mechanism of activation.
MMPs can be activated from a latent to an active form by autocatalytic
cleavage of the NH MMPs require Ca In this study, we first
present evidence that the removal of the COOH-terminal domain of
pro-gelatinase B is essential for activation of the enzyme and
Ca
Gelatinolytic activity of
the metal-free enzyme was determined by incubating the APMA or
trypsin-treated enzyme with 100 µg of metal-free heat-denatured
Figure 3:
Treatment of E" by trypsin.
Pro-gelatinase B (13 µg/ml) was activated by APMA and
Ca
Figure 1:
Effect of Ca
Figure 2:
Time
course of digestion of
Pro-gelatinase B could also be activated by trypsin;
thus, the effect of Ca
Figure 4:
Ca
The observed dichotomy of the Ca
On
the other hand, all metal ions tested were able to restore the activity
of trypsin-treated E" in the absence of Ca The activation of latent MMPs by different reagents has been
explained by the cysteine switch model(14, 15) . In
this model, activation is postulated to occur when the cysteine residue
in the NH It is well documented that APMA activates
MMP(17, 18, 20) . We have found that
APMA-initiated autoproteolytic processing followed by the activation of
gelatinase B consists of at least two consecutive steps. The first step
requires no extrinsic Ca Pro-gelatinase B can also be activated in
vitro by proteases such as trypsin in the presence of
Ca On the basis of these data, two possible
mechanisms can be envisioned for Ca In conclusion, our data demonstrate that gelatinase B
has a unique mechanism of activation and Ca
Figure 5:
A proposed mechanism for gelatinase B
activation. 1A, disruption of zinc-cysteine coordination by
APMA in pro-gelatinase B leads to autolytic cleavage of the
NH
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
- and
Ca
-dependent endopeptidase that is secreted from
cells as an inactive pro-enzyme. The enzyme can be activated in
vitro by organomercurial compounds and by trypsin. The role of
Ca
in autoproteolytic processing initiated by
4-aminophenylmercuric acetate and trypsin and in catalytic activity of
the activated enzyme was investigated by zymography and by kinetic
analysis. Treatment of unglycosylated 57.5-kDa pro-gelatinase B with
4-aminophenylmercuric acetate (1 mM) in the absence of
Ca
generated a 49-kDa inactive intermediate (E`), whereas a 41.5-kDa active species (E") was
generated in the presence of Ca
(5 mM). Upon
addition of Ca
to the reaction mixture of
Ca
-depleted E` or E" at 37 °C, E` showed a lag period in generation of the product as a
function of time, but E" presented an immediate activity. The
appearance of enzymatic activity of E` correlated with the
generation of the E" species. NH
-terminal sequence
analyses showed that E` and E" had the same NH
termini, i.e. Met-75, suggesting that
Ca
-dependent removal of COOH terminus of E` is required for activation of the enzyme. Treatment of
pro-gelatinase B with trypsin in the absence of Ca
,
led to degradation of the enzyme. In the presence of
Ca
, trypsin processed the pro-enzyme to a 40-kDa
active species. In contrast to E", this active species did not
require Ca
for activity. The Ca
dependence of E" activity was also abolished by
treatment of the enzyme with trypsin. NH
-terminal sequence
analysis indicated that amino acid residues 75-87 had been
removed from the NH
terminus of E" by trypsin,
suggesting that these residues are responsible for the
Ca
-dependent activity of the enzyme. Removal of
Ca
and catalytic Zn
inhibited the
activities of both E" and trypsin-treated E". In the
absence of Ca
, either Zn
,
Co
, Mn
, or Cd
was
able to restore the activity of trypsin-treated E". None of
the divalent cations tested however, was able to stimulate the activity
of E" in the absence of Ca
. These
experiments further suggest that binding of Ca
to E" or removal of the NH
-terminal residues of the
enzyme by trypsin induces a conformational change in the protein and
makes the active site of the enzyme accessible to various metal ions
rendering the enzyme active.
)are a family of
zinc- and calcium-dependent endopeptidases that play an important role
in the degradation of extracellular matrix in higher eukaryotic
organisms. This gene family includes collagenases (MMP-1, MMP-8),
stromelysins (MMP-3, MMP-10, and MMP-11), two gelatinases (gelatinase
A, MMP-2; gelatinase B, MMP-9), PUMP-1 (MMP-7) (1, 2, 3) , and newly discovered
membrane-type MMP (MT-MMP)(4) . These enzymes are synthesized
and secreted from connective tissue cells as latent enzymes that upon
activation initiate the degradation of extracellular macromolecules,
such as collagen and proteoglycans. It is widely assumed that normal
physiological processes are dependent upon careful spatial and temporal
regulation of the activity of these enzymes. Malignant cells have been
shown to exploit these same proteases to promote invasion and
metastasis(1, 2, 3) . It has recently been
shown that cells bearing the gelatinase B cDNA are able to metastasize
in nude mice, whereas inhibition of the gelatinase activity prevents
metastasis(5, 6, 7, 8, 9, 10) .
-terminal domain. This cleavage can occur
either by a conformational change induced by limited proteolysis of one
or more sites within pro-domain, or by reaction with organomercurial
compounds(1, 2) . Studies using
pro-MMP-1(11) , MMP-2 (12) , and MMP-3 (13) reported that fully activated enzymes obtained by
4-aminophenylmercuric acetate (APMA) treatment all lack the
NH
-terminal pro-peptides including a highly conserved
PRCGVPD sequence. A mechanism described as the ``cysteine
switch'' was suggested to explain the in vitro activation
of MMPs (14, 15) . Activation is believed to be
initiated by disruption of the interaction of the conserved cysteine in
the PRCGVPD sequence with the catalytic zinc, followed by
autoproteolytic processing of the NH
-terminal pro-domain
generating an active enzyme species. Chen et al.(16) have demonstrated that the disruption of
cysteine-zinc linkage, however, is not sufficient to activate
pro-MMP-3. In contrast to these MMPs, the data emerging from several
laboratories suggest a distinct mechanism of activation for gelatinase
B(17, 18, 19, 20, 21) .
Based on the NH
-terminal sequence analyses, it has been
demonstrated that the APMA-activated gelatinase B retains the conserved
PRCGVPD sequence in the molecule (17, 18, 19) . Using enzyme purified from
neutrophils, Tribel et al.(18) reported that
autoprocessing of gelatinase B in the presence of HgCl
occurs with a four-step truncation of the NH
-terminal
pro-peptide to Met-75 and a three-step loss of COOH-terminal fragments
to Ala-398, generating a 63-kDa species. The precise relationship of
catalytic activity to the different forms was not clear. APMA treatment
of pro-gelatinase B from U937 cells was found to give similar results
and the smallest protein product (68 kDa) was shown to be the active
form, in that it uniquely complexed with
-macroglobulin(20) . Recently, Murphy and
co-workers (21) have demonstrated that exposure of gelatinase
B to APMA causes rapid self-processing of both the
NH
-terminal pro-peptide and the COOH-terminal domain. They
concluded, however, that this does not imply that the COOH-terminal
domain is involved in activation. Together, these data suggest that
disruption of the cysteine-zinc interaction is not sufficient to
activate pro-gelatinase B and other factors may play a role in
gelatinase B activation.
as well
as Zn
for their action. It is thought that the role
of Ca
in MMPs is to stabilize the conformation of the
activated enzymes. Okada et al.(22) reported that
MMP-3 requires Ca
for its activity; recently, Housley et al.(23) showed that Ca
is
actually required for stabilization of APMA-activated MMP-3.
Ca
has also been reported to stabilize the 19-kDa
catalytic domain of MMP-1(24, 25) . However, the
precise role of Ca
in gelatinase B and the mechanism
of its action have not been elucidated.
plays a significant role in this process. Second,
we demonstrate that Ca
is absolutely required for
activity of the APMA-activated enzyme. Ca
on the
other hand, is not an essential divalent cation for the activity of the
trypsin-activated gelatinase. Based upon these data a
Ca
-dependent activation mechanism for the gelatinase
B is proposed.
Materials
Bacterial culture reagents were
obtained from Difco Laboratories (Detroit, MI). The Escherichia
coli expression system (pET-12C) and bacterial strain
BL21(DE3)pLyS were purchased from Novagen (Madison, WI). Tris, HEPES,
APMA, 1,10-phenanthroline, diisopropyl phosphorofluoride, gelatin
attached to 4% beaded agarose, TPCK-treated trypsin, soybean trypsin
inhibitor insolubilized on 4% beaded agarose, and iminodiacetic acid
chelating resin were from Sigma. Zinc chloride (ultradry, 99.99%) was
from Alfa (Ward Hill, MA). Calcium chloride hydrate (99.99+%) was
from Aldrich. High purity (99.999%) cobalt(II) chloride, manganese(II)
chloride (MnCl4H
O), and anhydrous cadmium
chloride were from Johnson Mattehey Materials Technology. Buffers and
substrate were prepared in water purified to
18 ohms by a Milli-Q
reagent water system (Millipore) and further rendered metal-free by
passage through an iminodiacetic acid chelating column (15
3
cm). This treatment reduced the Ca
and Zn
contents of the buffer to
1 ppb as determined by inductively
coupled argon plasma emission spectroscopy (ICAP 61E, Thermogarrel Ash
Co.)(26) .
C-Labeled type I collagen was prepared
from calf skin (27) and rendered metal-free by initially
dialyzing the sample (2 mg/ml) against 25 volumes of 50 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl or 10 mM HEPES-NaOH, pH 7.5, containing 0.1 M NaCl with three
changes and then passing it through a chelating column (5
1.5
cm) equilibrated with either Tris buffer or HEPES buffer.
Enzyme Purification and Assay
Recombinant
pro-gelatinase B was purified from E. coli as
described(28) . The protein concentration was determined by the
Bradford dye binding technique (a standard Bio-Rad assay) using bovine
serum albumin as a standard. To prepare metal-depleted gelatinase B,
pro-gelatinase B or activated enzymes (about 20 µg/ml) were first
dialyzed at 4 °C against 200 volumes of metal-free Tris or HEPES
buffer with four changes over 36 h, and further dialyzed against the
same buffer containing 5% (v/v) chelating resin for 2-3 h. Atomic
emission analysis indicated that the enzyme prepared by this procedure
contained 0.25 mol of Ca and 0.92 mol of
Zn
/mol of enzyme. Metal-free solutions and enzymes
were stored in polyethylene containers.
C-labeled type I collagen in a reaction mixture containing
10 mM HEPES-NaOH, pH 7.5, 0.1 M NaCl, 0 or 1
mM CaCl
, and different concentrations of either
ZnCl
, CoCl
, MnCl
, or CdCl
in a final volume of 150 µl at 37 °C for 2 h, as
described previously(29) . Duplicates were performed for each
metal ion concentration tested.
Activation of Pro-gelatinase B by
APMA
Ca-free pro-gelatinase B was incubated
with 1.0 mM APMA in 50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 0.5 µM ZnCl
in the absence or
presence of 5 mM Ca
at 37 °C for various
times (0-16 h). Metal ions were removed from the activated enzyme
solutions as described before.
Activation of Pro-gelatinase B by
Trypsin
Ca-free pro-gelatinase B was treated
with TPCK-treated trypsin (1-10 µg/ml) in 50 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 0.5 µM ZnCl
in the presence and absence of 5 mM CaCl
for various times (0-4 h) at 37 °C.
Trypsin was inactivated with 2 mM diisopropyl fluorophosphate
or removed by passage through a soybean trypsin inhibitor column (2
ml). In several experiments, pro-gelatinase was first activated by 1
mM APMA in the presence of 5 mM Ca
and further treated with TPCK-treated trypsin (10 µg/ml) at
37 °C for 0.5 h. Trypsin was removed as described before. Zymogram
gel showed that the flow-through was essentially trypsin-free (Fig. 3, lane3). Metal ions were removed from
activated enzyme solutions as described previously.
as described previously. The activated enzyme was
further treated with TPCK-treated trypsin (10 µg/ml) at 37 °C
for 0.5 h. Trypsin was removed by passage through a soybean trypsin
inhibitor column (2 ml). 2-µl samples were analyzed on a 10%
gelatin zymogram gel. Lane1, pro-gelatinase B; lane2, APMA-activated gelatinase (E"); lane3, trypsin-treated E"; lane4, flow-through of trypsin (10 µg/ml) passing through
trypsin inhibitor column; lane5, trypsin (10
µg/ml). The positions of molecular mass standards are indicated on
the left.
Gelatin Zymography
Zymography was a modification
of the procedure described by Hibbs et al.(29) .
SDS-polyacrylamide gel electrophoresis was performed in 10% acrylamide
gels containing 1.0 mg/ml gelatin. The gels were washed twice in 50
mM Tris-HCl, 5 mM CaCl, 1 µM ZnCl
, 1% Triton X-100, and 0.02% NaN
, pH
7.5, at 25 °C and incubated in the same buffer containing 0.6%
Triton X-100 for 16 h at 37 °C. The different forms of gelatinase
produced clear zones of lysis in the gels.
NH
Pro-gelatinase B and activated enzymes were
separated on a 10% polyacrylamide gel under reducing conditions and
transferred to a polyvinylidene difluoride membrane (Milipore). After
staining with Coomassie Brilliant Blue R-250, the bands of interest
were excised and sequenced on a model 475A protein sequencer (Applied
Biosystems).-terminal Amino Acid
Sequencing
Effect of Ca
Activation of pro-gelatinase B can be initiated by
treatment with APMA in the presence of Caon Activation of
Recombinant Gelatinase B Initiated by APMA and
Trypsin
, followed
by autoproteolytic processing of the enzyme to the stable
form(19, 20) . The role of Ca
in
autoproteolytic processing of pro-gelatinase B was examined using
Ca
-free unglycosylated recombinant enzyme. The
Ca
-free pro-enzyme was prepared as described under
``Experimental Procedures'' and was shown to contain 1 ppb
Ca
(0.25 mol of Ca
/mol of enzyme)
as determined by atomic emission spectroscopy. The effect of
Ca
on autoproteolytic processing of the enzyme was
then assessed by incubating the Ca
-free pro-enzyme
with APMA in the presence and absence of Ca
. A time
course of the autoproteolytic processing of the enzyme was followed by
zymography. As shown in Fig. 1A, treatment of the
57.5-kDa unglycosylated recombinant pro-gelatinase B with APMA (1
mM) in the absence of Ca
generated a 49-kDa
species (E`, lanes 2-6) over 8 h, while
treatment of the pro-enzyme with both APMA (1 mM) and
Ca
(5 mM) led to a 41.5-kDa species (E", lanes 7-11). The enzyme did not process
without activator whether Ca
was present or not (Fig. 1B, lanes 7 and 13). To
identify the active species and to assess the role of Ca
in the conversion of E` to E", the time course
of digestion of
C-gelatin by E` and E" was determined (Fig. 2A). In the absence of
Ca
, the rate of gelatin hydrolysis by both E` and E" was very slow. Upon addition of 5 mM Ca
, E` showed a distinct lag period in
the formation of acid-soluble
C-peptide product, and the
plot of product formation as a function of time generated a sigmoidal
curve. However, the time course of digestion of
C-gelatin
by E" after addition of Ca
at 37 °C was
essentially linear. Zymographic analysis at each time point indicated
that the E` was gradually converted to E" upon
addition of Ca
(Fig. 2B). The
generation of E" correlated well with the appearance of the
enzymatic activity obtained in the 3-h time course assay. The
conversion of E` to E" was apparently
Ca
-dependent. E` was stable at 37 °C in
the absence of Ca
for as long as 16 h (Fig. 2B, lane12). In the presence
of Ca
it was completely converted to E" (Fig. 2B, lane13). These
experiments suggested that E` is an intermediate and E" is the activated form of the enzyme. To identify the fragments
that were removed by Ca
-independent and -dependent
processes, the NH
-terminal sequence analyses of
pro-gelatinase B, E`, and E" were performed. The
NH
terminus of recombinant pro-gelatinase revealed a single
sequence of
APRQRQSTLVLFP, identical to that reported for
native gelatinase B(17, 19) . E` and E" had the same NH
termini, i.e.
MRTPRCGVPD. This indicated that
Ca
-independent processing of pro-enzyme to an
inactive intermediate, E`, was accomplished by removal of
NH
-terminal pro-peptide. Ca
-dependent
processing of E` to E", however, was not from
NH
-terminal domain of the enzyme. These data suggest that
pro-gelatinase requires Ca
for autoproteolytic
processing of the COOH-terminal domain of the enzyme to generate the
active species.
on
activation of recombinant pro-gelatinase B initiated by APMA (A) and trypsin (B). Recombinant pro-gelatinase B (13
µg/ml) was made Ca
-free as described under
``Experimental Procedures.'' The enzyme was incubated in 50
mM Tris-HCl, pH 7.5, 0.1 M NaCl, 0.5 µM
ZnCl
containing 1 mM APMA (A) or 1
µg/ml TPCK-treated trypsin (B) in the presence or absence
of 5 mM Ca
at 37 °C for various times.
Aliquots (2 µl) were separated in a 10% polyacrylamide gel
containing 1 mg/ml gelatin under nonreducing conditions. After gelatin
digestion, the gel was stained with Coomassie Brilliant Blue R-250. A, samples of pro-gelatinase B were treated with APMA in the
absence (lanes 2-6) or the presence (lanes
7-11) of 5 mM Ca
at 37 °C for
up to 8 h. The processing was terminated by keeping the samples on ice. Lane1, pro-gelatinase B; lanes2 and 7, 3 and 8, 4 and 9, 5 and 10, and 6 and 11,
the samples incubated for 0, 2, 4, 6, and 8 h, respectively. B, samples of
pro-gelatinase B were treated with TPCK-treated trypsin in the absence (lanes 2-6) or the presence (lanes 8-12)
of 5 mM Ca
at 37 °C for up to 4 h.
Trypsin was inactivated with 2 mM diisopropyl fluorophosphate. Lane1, pro-gelatinase B; lanes2 and 8, 3 and 9, 4 and 10, 5 and 11, and 6 and 12, the samples incubated for 0, 1, 2, 3, and 4 h,
respectively; lane7, pro-gelatinase B incubated
without activators and Ca
for 8 h; lane13, pro-gelatinase B incubated without activators but
with 5 mM Ca
for 8 h. The positions of
molecular mass standards are indicated on the
left.
C-gelatin by E` and E". E` and E" were prepared by treatment of
recombinant pro-gelatinase B with 1 mM APMA in the absence or
presence of 5 mM Ca
for 16 h and then
dialyzed to remove APMA and Ca
as described under
``Experimental Procedures.'' A, the reaction mixture
containing 50 mM Tris-HCl, pH 7.5, 0.5 µM ZnCl
, 0.1 M NaCl, 0.66 mg/ml
C-gelatin, and 9.3 µg/ml Ca
-free E` or E" was incubated with (solid) or
without (open) 5 mM Ca
at 37
°C. At the indicated time, 150-µl reaction mixtures were mixed
with 20 µl of 50 mM EDTA and digested
C-peptide was determined as described under
``Experimental Procedures.'' B, the reaction
mixtures of the E` (2 µl) at indicated time were
electrophoresed in gelatin-containing gels (12% acrylamide). Lane1, E`; lanes 2-11, the enzyme
incubated with Ca
for 0, 0.25, 0.5, 0.75, 1.0, 1.25,
1.5, 2.0, 2.5, and 3.0 h, respectively. Lane12, 16 h
in the absence of Ca
; lane13, 16 h
in the presence of Ca
; lane14, 20
ng of pro-enzyme. Upperarrow, latent 57.5-kDa
pro-enzyme; middlearrow, 49-kDa E`; lowerarrow, 41.5-kDa E".
on activation of recombinant
gelatinase B initiated by trypsin was examined as described for APMA
activation. The incubation of 57.5-kDa pro-gelatinase B with trypsin in
the absence of Ca
at 37 °C resulted in a
time-dependent degradation of the protein without generating an active
species (Fig. 1B, lanes 2-6). The
pro-enzyme, however, could be processed to a 40-kDa active species by
trypsin in the presence of 5 mM Ca
(Fig. 1B, lanes 8-12). This
suggested that Ca
stabilized the activated enzyme and
protected it from degradation by trypsin. This was supported by the
observations that in the absence of Ca
, 41.5-kDa
APMP-activated species (E") lost its catalytic activity upon
incubation at 37 °C and was rapidly inactivated by trypsin (data
not shown). It could, however, be converted to a 40-kDa active species
by trypsin in the presence of 5 mM Ca
(Fig. 3, lane3).
Effect of Ca
The
enzymatic activities of MMPs are also thought to be dependent on
Caon the Catalytic
Activity of APMA and Trypsin-activated Gelatinase B
(22, 23, 24, 25) . The effect
of Ca
on the catalytic activities of both
APMA-activated (E"), and trypsin-activated gelatinase B were
examined. As demonstrated in Fig. 4, E" had negligible
activity in the absence of Ca
and its activity was
absolutely dependent on Ca
. Trypsin-activated
gelatinase, on the other hand, exhibited significant activity in the
absence of Ca
(80% of maximal activity). Addition of
1 mM Ca
increased the activity to 100%. This
is probably due to the stabilizing effect of Ca
described above. To rule out the possibility that residual
trypsin in the gelatinase solution might be responsible for the
observed Ca
-independent activity of the enzyme, the
activated enzyme was assayed in the presence of 1,10-phenanthroline
(0.5 mM), a MMP inhibitor. This reagent was able to inhibit
the Ca
-independent activity of the trypsin-activated
enzyme (Fig. 4). This activity could also be deprived by passing
the enzyme through a gelatin-Sepharose column (data not shown). In
addition, zymography of the trypsin-activated enzyme solution passed
through a soybean trypsin inhibitor column showed no trypsin
contamination (Fig. 3, lane3). These data
indicated that the Ca
-independent activity of the
enzyme is specific and is not due to any contaminating trypsin. This
notion was further supported by the observation that the
trypsin-activated enzyme was able to degrade native type V collagen in
the absence of Ca
. The pattern of cleavage products
was identical to that obtained by APMA-activated gelatinase (data not
shown).
dependence of
APMA-activated (E") and trypsin-activated gelatinase.
Ca
-free E", trypsin-activated gelatinase and
trypsin-treated E" (13 µg/ml) were prepared as described
under ``Experimental Procedures.'' The gelatinolytic
activities were assayed in HEPES buffer containing 0.5 µM
Zn
in the presence or absence of 1 mM Ca
or presence of 0.5 mM 1,10-phenanthroline (OP). Values determined in the
presence of 1 mM Ca
are taken as 100% of
activity. The results shown are the mean of triplicate
determinations.
effect on
the activity of APMA-activated and trypsin-activated enzyme raised the
possibility that the peptide fragment(s) responsible for the
Ca
-dependent activity of E" may have been
eliminated in trypsin-activated gelatinase. This was supported by the
observation that further treatment of E" with trypsin resulted
in the conversion of 41.5-kDa E" into a 40-kDa species (Fig. 3), and abolished the Ca
requirement for
the activity of the enzyme. The activity of E" in the absence
of Ca
was about 5% of expected activity. This
activity was increased to 80% upon treatment with trypsin (Fig. 4). The NH
-terminal sequence analyses of E" and trypsin-treated E" yielded a sequence of
MRTPRCGVPDLGR
and
FQTFEGDLKWHHHNI, respectively, indicating that the
NH
-terminal fragment
MRTPRCGVPDLGR
, containing the conserved
cysteine residue, had been removed from E" by trypsin. Triebel
and co-workers (18) have demonstrated that Ala-398 is the COOH
terminus of HgCl
-activated human gelatinase B. Since no
trypsin cleavage site has been identified at the COOH terminus of this
truncated enzyme, we infer that both E" and trypsin-treated E" have the same COOH-terminal sequences. These data suggest
that removal of amino acid residues 75-87 from the NH
terminus of E" is accompanied by the loss of
Ca
dependence of the enzyme.
Removal of NH
The above observation focused our attention on the role
of amino acid residues 75-87 in the metal specificity of
gelatinase B. The metal-depleted E" and trypsin-treated E" were prepared as described under ``Experimental
Procedures'' and were shown to contain 0.25 mol of Ca-terminal Fragment from E"
by Trypsin also Abolishes the Metal Specificity of Gelatinase
B
and 0.92 mol of Zn
/mol of enzyme as determined
by atomic emission spectroscopy. The effects of different divalent
cations on the activities of the activated gelatinases were then
assessed by assaying the enzymes in the presence and absence of
Ca
using
C-gelatin substrate. In the
absence of any exogenous metal ions, the basal gelatinolytic activities
of E" and trypsin-treated E" were less than 5% of
their activities determined in the presence of Ca
(1.0 mM) and Zn
(0.5 µM) (Table 1), suggesting that the concentrations of endogenous metal
ions were negligible. Under these conditions, Zn
,
Co
, Mn
, and Cd
were tested for their abilities to restore the gelatinolytic
activity of the enzymes. As shown in Table 1, none of the metal
ions tested was able to stimulate E" activity in the absence
of Ca
. Only in the presence of Ca
(1.0 mM), was E" able to catalyze the
degradation of gelatin substrate. Addition of optimal concentrations of
either Zn
(0.5 µM), Co
(50 µM), Mn
(0.5 mM), or
Cd
(0.5 µM) increased
Ca
-supported activity of E". Higher
concentrations of each of the divalent cations were inhibitory. On the
basis of these results, it is reasonable to conclude that E" is highly specific for Ca
and its catalytic
activity is absolutely dependent on the presence of this cation.
.
The restoration of the enzymatic activity by these metal ions was not
due to the contaminated Ca
, because the
concentrations of Zn
, Co
, and
Cd
needed for half-stimulation were about 3 orders of
magnitude lower than that of Ca
(data not shown).
Co
seemed to be the most effective cation in
stimulating the activity of the enzyme. The rest of the metal ions
stimulated the activity to an extent similar to that for
Zn
. Addition of 1 mM Ca
further increased the activities of the metal-substituted
enzymes. This is probably due to stabilizing effect of Ca
on enzyme conformation. These data demonstrate that the activity
of trypsin-treated E" is directly dependent on the interaction
of the protein with a divalent cation, which could act as an
electrostatic catalyst. These observations also suggest that removal of
amino acid residues 75-87 from the NH
terminus of E" by trypsin may result in exchange of catalytic zinc with
other metal ions.
-terminal domain of the enzyme is transiently
dissociated from the zinc atom. The disruption of this interaction is
thought to be sufficient to lead to pro-MMP activation and subsequent
processing to a lower molecular weight active species. Recent study
suggested that the disruption of cysteine-zinc interaction, however, is
not sufficient to activate pro-MMP-3 (stromelysin)(16) , and
site-directed mutagenesis of the enzyme showed that other amino acid
residues in addition to cysteine are also involved in latency of
pro-MMP-3(30) . The data emerging from several laboratories
regarding the mechanism of activation of gelatinase B also deviate from
this model. Morodomi et al.(20) showed that treatment
of pro-MMP-9 (pro-gelatinase B) with APMA produces an inactive
intermediate species. Only the low molecular mass species (68 kDa) was
shown to be active by its ability to bind to
-macroglobulin. This experiment suggests that the
autoproteolytic processing steps following the disruption of
cysteine-zinc coordination are also essential for the gelatinase B
activation. It has also been demonstrated that the APMA-activated
gelatinase B retains the conserved cysteine in the molecule after
stepwise removal of both NH
- and COOH-terminal domain (17, 18, 19) . In this report, we demonstrate
that Ca
-dependent autocatalytic removal of the
COOH-terminal domain of pro-gelatinase B is a crucial step for
activation of the enzyme. The continued presence of the pro-peptide
containing the conserved cysteine in gelatinase B renders the activity
of the enzyme to be Ca
-dependent. Removal of this
fragment by trypsin abolishes the Ca
dependence of
the enzyme activity.
, whereas the second step is
Ca
-dependent. Treatment of latent recombinant
gelatinase B with APMA in the absence of Ca
generates
a 49-kDa intermediate (E`), which upon addition of
Ca
is processed to a 41.5-kDa protein. This 41.5-kDa
protein is the active species (E"). NH
-terminal
sequence analyses demonstrated that E` and E" have
the same NH
termini (Met-75). Thus, the
Ca
-dependent processing of E` to E" must come from COOH terminus and this cleavage is prerequisite for
appearance of the catalytic activity of gelatinase B. These data are
consistent with the previous observation that upon exposure to APMA and
Ca
, 92-kDa gelatinase from HT 1080 human fibrosarcoma
cells was activated by sequential processing of both NH
-
and COOH-terminal domain as determined by immunoblot
analysis(19) . The 67-kDa COOH-terminal truncated species was
apparently the active form. In this study, however, Ca
was included in the activation reaction mixtures, and its
specific role in the activation process was not addressed. Triebel et al.(18) have demonstrated that the intermediate
species that is generated upon treatment of gelatinase B with
HgCl
contains free sulfhydryl groups. This intermediate was
shown to be processed to a lower molecular mass species by three-step
COOH-terminal cleavages(18) . This indicated that the
Ca
-dependent conversion of E` to E" is not due to removal of an internal fragment(s) between the
disulfide linkages. These data suggest that the hemopexin-like
COOH-terminal domain as well as NH
-terminal pro-domain of
pro-gelatinase B are involved in the latency of the enzyme. The precise
role of the COOH-terminal domain in latency of gelatinase B and the
involvement of Ca
in this phenomenon are currently
being investigated.
(19, 20) . We were, however,
unable to assess the role of Ca
in processing of
pro-gelatinase by trypsin. The Ca
-free enzyme was
degraded by trypsin, and no apparent intermediate or active species was
formed upon treatment of Ca
-free pro-gelatinase B
with trypsin unless Ca
was present in the reaction
mixture. The role of Ca
in this reaction was
apparently to stabilize the conformation of the protein and protect the
enzyme from degradation by trypsin. To our surprise, we found that in
contrast to APMA-activated gelatinase (E"), the
trypsin-activated enzyme does not require Ca
for
activity. The observed gelatinolytic activity of the enzyme in the
absence of Ca
is not due to trypsin contamination
since the activity of the enzyme can be inhibited by the zinc chelating
agent, 1,10-phenanthroline, and by passage of the enzyme through a
gelatin-Sepharose affinity column. This observation prompted us to
further evaluate the role of Ca
in APMA- and
trypsin-activated gelatinase. We found that trypsinization of E" produces a lower molecular mass species that displays activity in
the absence of Ca
. The activity of this species could
be stimulated by either Zn
, Co
,
Mn
, or Cd
in the absence of
Ca
. Ca
-independent activity is
apparently specific for trypsin-activated enzyme, since E" is
highly specific for Ca
as a metal activator. No other
divalent cations tested (Zn
, Co
,
Mn
, or Cd
) were capable of
restoring the activity of metal-depleted E" in the absence of
Ca
. Based on the homology of gelatinase B to other
MMPs(34, 36) , it may also contain a tightly bound
structural and a loosely bound catalytic Zn
.
Considering that the enzymes used in this study contain about 1 mol of
Zn
/mol of protein, the catalytic Zn
can apparently be removed from the activated enzyme by dialysis
against chelating resin-containing buffer and replaced by other
divalent metal ions. Comparison of NH
-terminal sequence of E" and trypsin-treated E" indicated that the amino
acid residues 75-87 corresponding to MRTPRCGVPDLGR sequence had
been removed from the NH
terminus of E" by
trypsin, generating a new NH
terminus, Phe-88. These
observations strongly suggest that the catalytic activity of E" is dependent upon either removal of the peptide fragment (amino
acids 75-87) containing the conserved cysteine residue by
proteolysis or a Ca
-dependent conformational change
involving this fragment.
-dependent
activity of gelatinase B. First, the NH
-terminal peptide
fragment of E", MRTPRCGVPDLGR, acts as an autoinhibitory
fragment and masks the active site of enzyme. Binding of Ca
to the enzyme induces a local conformational change in the
protein moving the fragment away from the active site, resulting in
activation. Removal of the putative inhibitory fragment by trypsin also
abrogates the inhibition and renders the enzyme active in the absence
of Ca
. It is worth noting that this fragment contains
the highly conserved peptide sequence PRCGVPD, which has been proposed
to be involved in latency of the enzyme(14, 15) .
Although the conserved cysteine residue apparently cannot maintain the
latency of E" by coordinating with the catalytic
Zn
, this sequence is likely to inhibit the catalytic
activity of E" in the absence of Ca
. This is
supported by the observations that a synthetic peptide containing this
sequence inhibited MMP-3 (31) and MMP-2(32) . Second,
the active site conformation of E" is not stable in the
absence of Ca
. Binding of Ca
to E" induces a conformational change and stabilizes the active
site by interaction of Ca
with the
NH
-terminal residues. On the basis of the x-ray crystal
structure of collagenase, Reinemer and co-workers (33) have
concluded that in stromelysin-activated collagenase,
NH
-terminal ammonium group of Phe-79 forms a salt linkage
with the side chain carboxylate moiety of the conserved Asp-232, giving
an order to the NH
-terminal segment of the protein.
Although the geometry of the active site is not affected by the salt
bridge, in the absence of this interaction the NH
-terminal
segment of the activated collagenase is disordered, leading to a less
active enzyme (34) . Considering the homology between members
of MMPs, it is reasonable to assume that the amino acid sequence
75-87 destabilizes the active site of the APMA-activated enzyme
in the absence of Ca
leading to inactivation. We have
found that trypsinization of E" abrogates the Ca
dependence of the enzyme and generates a new NH
terminus, Phe-88. Similar to Phe-79 in collagenase, this residue
may form a salt linkage with the conserved Asp-432 to stabilize the
active site of the gelatinase. The role of Phe-88 in this process can
be mimicked by binding Ca
to the
NH
-terminal region of the APMA-activated enzyme. The
gelatinolytic activity associated with the conformational change could
be expressed by either Ca
binding to or trypsin
treatment of E". Our previous observation that Asp-432
(corresponding to Asp-232 in collagenase) is necessary for general
catalytic activity of the enzyme (35) is in accord with these
new findings.
plays a
key role in this process. As is illustrated in Fig. 5,
Ca
is a key player in several steps leading to
activation of the enzyme by APMA (steps 1A-3A). It is
involved in autoproteolytic processing of the COOH-terminal domain of
the enzyme (step2A). Ca
also
effects catalysis by inducing a conformational change in the protein (step3A) similar to the one generated by treatment
of the enzyme with trypsin (step2T). Binding of
Ca
to the enzyme dislodges the
NH
-terminal autoinhibitory fragment (step3A1) or stabilizes the active site through interaction
with NH
-terminal residues (step3A2).
Removal of the NH
-terminal fragment by trypsin (step2T), on the other hand, leads to permanent exposure of
the active site (step3T1) or stabilization of the
active site via formation of a salt linkage (step3T2), generating a fully active enzyme that does not
require Ca
. Whether blockage of the active site by
the PRCGVPD sequence or lack of proper active site conformation is
responsible for the lack of enzymatic activity of APMA-activated enzyme
in the absence of Ca
remains to be determined.
-terminal pro-domain in the absence of
Ca
, generating an inactive intermediate (E`); 2A, binding of Ca
to E` results in autoproteolytic processing of COOH-terminal domain,
generating an active enzyme species (E"); 3A1,
Ca
binding to E" induces a conformational
change in the NH
-terminal segment of E", relieving
its autoinhibition; 3A2, Ca
binding to E" induces a conformational change and stabilizes the active
site by interaction with the NH
-terminal residues; 1T, trypsin activates pro-gelatinase B in the presence of
Ca
, generating an active enzyme that does not require
Ca
; 2T, removal of the
NH
-terminal segment of E" by trypsin generates the
same molecule as described in step1T; 3T1,
trypsin-activated enzyme does not require Ca
for
activity due to relieving the autoinhibition of the
NH
-terminal segment of E"; 3T2,
trypsin-activated enzyme does not require Ca
for
activity due to stabilization of the active site by forming a salt
linkage between NH
- and COOH-terminal residues (dottedline).
We are grateful to Dr. Jacob Aelion and Thomas
O'Farrell for helpful discussion of the manuscript. We thank
Martha B. Holt (Buckman Lab, Memphis, TN) for kindly performing atomic
emission analyses. We also acknowledge the Center for Biotechnology
(St. Jude Children's Research Hospital, Memphis, TN) for
providing the service of NH-terminal sequence analyses.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.