©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanism of Activation of Human Neutrophil Gelatinase B
DISCRIMINATING BETWEEN THE ROLE OF Ca IN ACTIVATION AND CATALYSIS (*)

(Received for publication, April 6, 1995)

Chun Hui Bu Tayebeh Pourmotabbed (§)

From the Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Gelatinase B is a Zn- 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(2)-terminal sequence analyses showed that E` and E" had the same NH(2) 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(2)-terminal sequence analysis indicated that amino acid residues 75-87 had been removed from the NH(2) 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(2)-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.


INTRODUCTION

Matrix metalloproteinases (MMP) (^1)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) .

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(2)-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(2)-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(2)-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(2)-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(2) occurs with a four-step truncation of the NH(2)-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 alpha(2)-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(2)-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.

MMPs require Ca 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.

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 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.


EXPERIMENTAL PROCEDURES

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 (MnCl(2)bullet4H(2)O), and anhydrous cadmium chloride were from Johnson Mattehey Materials Technology. Buffers and substrate were prepared in water purified to geq18 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 leq1 ppb as determined by inductively coupled argon plasma emission spectroscopy (ICAP 61E, Thermogarrel Ash Co.)(26) . ^14C-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.

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 ^14C-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(2), and different concentrations of either ZnCl(2), CoCl(2), MnCl(2), or CdCl(2) 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(2) 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(2) in the presence and absence of 5 mM CaCl(2) 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.


Figure 3: Treatment of E" by trypsin. Pro-gelatinase B (13 µg/ml) was activated by APMA and Ca 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(2), 1 µM ZnCl(2), 1% Triton X-100, and 0.02% NaN(3), 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(2)-terminal Amino Acid Sequencing

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).


RESULTS

Effect of Caon Activation of Recombinant Gelatinase B Initiated by APMA and Trypsin

Activation of pro-gelatinase B can be initiated by treatment with APMA in the presence of Ca, 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 ^14C-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 ^14C-peptide product, and the plot of product formation as a function of time generated a sigmoidal curve. However, the time course of digestion of ^14C-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(2)-terminal sequence analyses of pro-gelatinase B, E`, and E" were performed. The NH(2) 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(2) termini, i.e.MRTPRCGVPD. This indicated that Ca-independent processing of pro-enzyme to an inactive intermediate, E`, was accomplished by removal of NH(2)-terminal pro-peptide. Ca-dependent processing of E` to E", however, was not from NH(2)-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.


Figure 1: Effect of Ca 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(2) 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.




Figure 2: Time course of digestion of ^14C-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(2), 0.1 M NaCl, 0.66 mg/ml ^14C-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 ^14C-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".



Pro-gelatinase B could also be activated by trypsin; thus, the effect of Ca 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 Caon the Catalytic Activity of APMA and Trypsin-activated Gelatinase B

The enzymatic activities of MMPs are also thought to be dependent on Ca(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).


Figure 4: Ca 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.



The observed dichotomy of the Ca 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(2)-terminal sequence analyses of E" and trypsin-treated E" yielded a sequence of MRTPRCGVPDLGR and FQTFEGDLKWHHHNI, respectively, indicating that the NH(2)-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(2)-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(2) terminus of E" is accompanied by the loss of Ca dependence of the enzyme.

Removal of NH(2)-terminal Fragment from E" by Trypsin also Abolishes the Metal Specificity of Gelatinase B

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 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 ^14C-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.



On the other hand, all metal ions tested were able to restore the activity of trypsin-treated E" in the absence of Ca. 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(2) terminus of E" by trypsin may result in exchange of catalytic zinc with other metal ions.


DISCUSSION

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(2)-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 alpha(2)-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(2)- 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.

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, 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(2)-terminal sequence analyses demonstrated that E` and E" have the same NH(2) 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(2)- 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(2) 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(2)-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.

Pro-gelatinase B can also be activated in vitro by proteases such as trypsin in the presence of Ca(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(2)-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(2) terminus of E" by trypsin, generating a new NH(2) 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.

On the basis of these data, two possible mechanisms can be envisioned for Ca-dependent activity of gelatinase B. First, the NH(2)-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(2)-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(2)-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(2)-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(2)-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(2) 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(2)-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.

In conclusion, our data demonstrate that gelatinase B has a unique mechanism of activation and Ca 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(2)-terminal autoinhibitory fragment (step3A1) or stabilizes the active site through interaction with NH(2)-terminal residues (step3A2). Removal of the NH(2)-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.


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(2)-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(2)-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(2)-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(2)-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(2)-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(2)- and COOH-terminal residues (dottedline).




FOOTNOTES

*
This work was supported by National Institutes of Health Grant AR-41843 and the Arthritis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, University of Tennessee, 858 Madison Ave., Memphis, TN 38163. Tel.: 901-448-4361; Fax: 901-448-7360.

^1
The abbreviations used are: MMP, matrix metalloproteinase; APMA, 4-aminophenylmercuric acetate; TPCK, N-tosyl-L-phenylalanine chloromethylketone.


ACKNOWLEDGEMENTS

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(2)-terminal sequence analyses.


REFERENCES

  1. Woessner, J. F. (1991)FASEB J.5,2145-2154 [Abstract/Free Full Text]
  2. Birkedal-Hansen, H., Moore, W. G. I., Bodden, M. K., Windsor, L. J., Birkedal-Hansen, B., DeCarlo, A., and Engler, J. A.(1993)Crit. Rev. Oral Biol. Med.4,197-250 [Abstract]
  3. Matrisian, L. M. (1992)Bioessays14,455-463 [Medline] [Order article via Infotrieve]
  4. Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seili, M. (1994)Nature370,61-65 [CrossRef][Medline] [Order article via Infotrieve]
  5. Alvarez, O. A., Carmichael, D. F., and De Clerck, Y. A.(1990)J. Natl. Cancer Inst.82,589-595 [Abstract]
  6. Albini, A., Melchiori, A., Santi, L., Liotta, L. A., Brown, P. D., and Stetler-Stevenson, W. G.(1991)J. Natl. Cancer Inst.83,775-779 [Abstract]
  7. Schultz, R. M. (1991)Cancer Treat. Res.54,119-33 [Medline] [Order article via Infotrieve]
  8. DeClerck, Y. A., Yean, T. D., Chen, D., Shimada, H., and Langley, K. E.(1991) Cancer Res.51,2151-2157 [Abstract]
  9. DeClerck, Y. A., Perez, N., Shimada, H., Boone, T. C., Langley, K. E., and Taylor, S. M. (1992)Cancer Res.52,701-707 [Abstract]
  10. Bernhard, E. J., Gruber, S. B., and Muschell, R. J.(1994)Proc. Natl. Acad. Sci. U. S. A.91,4293-4297 [Abstract]
  11. Grant, G. A., Eisen, A. Z., Marmer, B. L., Roswit, W. T., and Goldberg, G. I.(1987) J. Biol. Chem.262,5886-5889 [Abstract/Free Full Text]
  12. Stetler-Stevenson, W. G., Krutzsch, H. C., Wacher, M. P., Margulies, I. M. K., and Liotta, L. A.(1989)J. Biol. Chem.264,1353-1356 [Abstract/Free Full Text]
  13. Nagase, H., Enghild, J. J., Suzuki, K., and Salvesen, G.(1990)Biochemistry 29,5783-5789 [Medline] [Order article via Infotrieve]
  14. Springman, E. B., Angleton, E. L., Birkedal-Hansen, H., and Van Wart, H. W.(1990) Proc. Natl. Acad. Sci. U. S. A.87,364-368 [Abstract]
  15. Van Wart, H. E., and Brikedale-Hansen, H.(1990)Proc. Natl. Acad. Sci. U. S. A.87,5578-5582 [Abstract]
  16. Chen, L., Noelken, M. E., and Nagase, H.(1993)Biochemistry32,10289-10295 [Medline] [Order article via Infotrieve]
  17. Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., Grant, G. A., and Goldberg, G. I. (1989)J. Biol. Chem.264,17213-17221 [Abstract/Free Full Text]
  18. Triebel, S., Blaser, J., Reinke, H., Knauper, V., and Tschesche, H.(1992)FEBS Lett.298,280-284 [CrossRef][Medline] [Order article via Infotrieve]
  19. Okada, Y., Gonoji, Y., Naka, K., Tomita, K., Nakanishi, I., Iwata, K., Yamashita, K., and Hayakawa, T.(1992)J. Biol. Chem.267,21712-21719 [Abstract/Free Full Text]
  20. Mordomi, T., Ogata, Y., Sasaguri, Y., Morimatsu, M., and Nagase, H.(1992) Biochem. J.285,603-611 [Medline] [Order article via Infotrieve]
  21. O'Connell, J. P., Willenbrock, F., Docherty, A. J. P., Eaton, D., and Murphy, G. (1994)J. Biol. Chem.269,14967-14973 [Abstract/Free Full Text]
  22. Okada, Y., Nagase, H., and Harris, E. D., Jr.(1986)J. Biol. Chem. 261,14245-14255 [Abstract/Free Full Text]
  23. Housley, T. J., Baumann, A. P., Braun, I. D., Davis, G., Seperack, P. K., and Whilhelm, S. M. (1993)J. Biol. Chem.268,4481-4487 [Abstract/Free Full Text]
  24. Seltzer, J. L., Welgus, H. G., Jeffrey, J. J., and Eisen, A. Z.(1976)Arch. Biochem. Biophys.173,355-361 [Medline] [Order article via Infotrieve]
  25. Lowry, C. L., McGeehan, G., and Levine, H. I.(1992)Protein: Struct. Funct. Genet.12,42-48
  26. Winefordner, J. D., Fitzgerald, J. J., and Omenetto, N.(1975)Appl. Spectrosc.25,369-383
  27. Nagai, Y., and Hori, H. (1972)Biochim. Biophys. Acta263,564-573 [Medline] [Order article via Infotrieve]
  28. Pourmotabbed, T., Solomon, T. L., Hasty, K. A., and Mainardi, C. L.(1994) Biochim. Biophys. Acta1204,97-107 [Medline] [Order article via Infotrieve]
  29. Hibbs, M. S., Hasty, K. A., Seyer, J. M., Kang, A. H., and Mainardi, C. L.(1985) J. Biol. Chem.260,2493-2500 [Abstract]
  30. Freimark, B. D., Feeser, W. S., and Rosenfeld, S. A.(1994)J. Biol. Chem. 269,26982-26987 [Abstract/Free Full Text]
  31. Park, A. J., Matrisian, L. M., Kells, A. F., Pearson, R., Yuan, Z., and Navre, M.(1991) J. Biol. Chem.266,1584-1590 [Abstract/Free Full Text]
  32. Stetler-Stevenson, W. G., Talano, J. A., Gallagher, M. E., Krutzsch, H. C., and Liotta, L. A. (1991)J. Am. Med. Sci.302,163-170
  33. Reinemer, P., Grams, F., Huber, R., Kleine, T., Schnierer, S., Piper, M., Tschesche, H., and Bode, W.(1994)FEBS Lett.338,227-233 [CrossRef][Medline] [Order article via Infotrieve]
  34. Bode, W., Reinemer, P., Huber, R., Kleine, T., Schniere, S., and Tschesche, H.(1994) EMBO J.13,1263-1269 [Abstract]
  35. Pourmotabbed, T., Aelion, J., Tyrrell, D., Hasty, K., Bu, C. H., and Mainardi, C. (1995) J. Protein Chem., in press
  36. Lovejoy, B., Hassell, A. M., Luther, M. A., Weigl, D., and Jordan, S. R.(1994) Biochemistry33,8207-8217 [Medline] [Order article via Infotrieve]

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