©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of the 92-kDa Gelatinase by Stromelysin and 4-Aminophenylmercuric Acetate
DIFFERENTIAL PROCESSING AND STABILIZATION OF THE CARBOXYL-TERMINAL DOMAIN BY TISSUE INHIBITOR OF METALLOPROTEINASES (TIMP) (*)

(Received for publication, December 29, 1994)

Steven D. Shapiro (2) (3)(§) Catherine J. Fliszar (1) Thomas J. Broekelmann (2) (3) Robert P. Mecham (2) (3) Robert M. Senior (2) Howard G. Welgus (1)

From the  (1)From theDivisions of Dermatology and (2)Respiratory/Critical Care, Department of Medicine, Washington University School of Medicine at Jewish Hospital and the (3)Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The matrix metalloproteinase 92-kDa gelatinase is a major product of inflammatory cells. Macrophages synthesize and secrete this proteinase as a proenzyme in association with tissue inhibitor of metalloproteinases (TIMP) (92), whereas neutrophils store and release it from secondary granules as a TIMP-free proenzyme (92). Metalloproteinase proenzymes can be activated in vitro by a variety of agents, including organomercurials and proteinases, resulting in loss of an 8-10-kDa NH(2)-terminal domain which disrupts the interaction of a conserved cysteine residue with the catalytic zinc molecule. We report that the activation and processing of 92-kDa gelatinase differs depending on its association with TIMP and the nature of the activating agent. We observed that 92 undergoes classic activation to 82 kDa by stromelysin, whereas exposure to 4-aminophenylmercuric acetate (APMA) results in a final product of 83 kDa that still contains the ``prodomain'' cysteine. Association with TIMP appears to stabilize the COOH-terminal domain, whereas 92 is converted by APMA to a final product of 67 kDa lacking the COOH-terminal portion. In the continued presence of APMA, which maintains cysteine-zinc disruption, the 67-kDa species is at least as active as the classic 82 kDa. In contrast, activation of 92 by stromelysin initially generates the 82-kDa form which is followed by final conversion to a 50-kDa species that lacks the catalytic domain of the parent molecule. Therefore, although stromelysin activation of 92 is initially efficient, the active 82-kDa form is short-lived and is replaced by an inactive 50-kDa product. This complex pattern of activation of the 92-kDa gelatinase may serve to restrict its proteolytic capacity following exposure to stromelysin and may serve to regulate proteinase activity in vivo.


INTRODUCTION

The 92-kDa gelatinase (gelatinase B, MMP 9) (^1)is a member of the metalloproteinase family of structurally related, matrix-degrading enzymes. These proteinases play a major role in tissue remodeling and repair associated with development and inflammation(1, 2) . Abnormal expression of MMPs may contribute to destructive processes, including tumor invasion/metastasis(3, 4) , arthritis(5) , atherosclerosis(6) , aneurysm formation(7) , and perhaps pulmonary emphysema related to cigarette smoking(8) .

The expression of metalloproteinases is subject to transcriptional regulation by various cytokines, growth factors, and other agents (9, 10, 11, 12, 13, 35) . Regulation of MMP activity is also tightly controlled post-translationally in the extracellular space via coordinated activation of secreted proenzymes as well as through interaction of MMPs with the TIMPs(14, 15) . Proenzyme activation may be achieved by various means in vitro that disrupt the interaction between the active site zinc molecule, coordinated in the catalytic domain, and a cysteine present within the proenzyme domain(16, 17) . Activation results in loss of the 8-10-kDa NH(2)-terminal prodomain, leaving the mature, active proteinase. The mechanisms of MMP activation in vivo are currently unknown. Cleavage of the COOH-terminal domain has also been described for selected MMPs during the activation process(18, 19) . The effect of COOH-terminal processing on enzyme function is unknown.

The 92-kDa gelatinase (gelatinase B), a major product of monocyte/macrophages, and the 72-kDa gelatinase (gelatinase A), produced by many cell types, especially fibroblasts, each efficiently degrade gelatins of all genetic types, native collagen types IV and V, fibronectin, entactin, and insoluble elastin(20, 21, 22, 23) . These proteinases are unique among MMPs in that they are secreted in physical association with specific TIMPs (72-kDa gelatinase-TIMP-2 complex and 92 kDa gelatinase-TIMP-1 complex)(24, 25) . TIMPs bind to the COOH-terminal domain of the progelatinases. The 92-kDa gelatinase is also produced by neutrophil precursors and stored in secondary granules. Because TIMP is not produced by neutrophils, the 92-kDa gelatinase in these cells is released TIMP-free(26) . The functional consequences of the unusual association of proenzyme and inhibitor are unknown. Furthermore, the mechanisms of zymogen activation of the 92-kDa progelatinase-TIMP complex secreted by macrophages versus the TIMP-free 92-kDa progelatinase released by neutrophils are incompletely understood.

The present study was initiated following the unexpected finding that activation of recombinant 92-kDa gelatinase proenzyme (TIMP-free) by stromelysin resulted in an initial burst in enzyme activity, but by 24-h proteolytic activity had markedly diminished. We now report that activation and processing of the 92-kDa gelatinase results in various processed forms with differing catalytic activities. The final product is dependent upon the association of the 92-kDa gelatinase with TIMP and the particular activating agent.


MATERIALS AND METHODS

Reagents

APMA was obtained from Sigma. Truncated stromelysin containing the catalytic domain only was a generous gift of V. Baragi, Parke Davis (Ann Arbor, MI). Gelatin was prepared from Vitrogen-100 Type I collagen (Celtrix, Santa Clara, CA) by heating to 60 °C for 15 min.

Purification of 92-kDa Gelatinase

92-kDa gelatinase complexed to TIMP was obtained from U937 cells as described previously (23) . U937 cells were grown in RPMI 1640 with 10% heat-inactivated fetal calf serum (JRH Biosciences, Lenexa, KS). Prior to stimulation with 10M phorbol 12-myristate 13-acetate, medium was changed to serum-free + lactalbumin hydrolysate (0.2%) (Life Technologies, Inc.). After exposure to 10M phorbol 12-myristate 13-acetate for 48 h, the conditioned medium was collected and subjected to reactive red 120-agarose affinity chromatography (Sigma) equilibrated in 0.02 M Tris, pH 7.5, containing 0.005 M CaCl(2) and 0.15 M NaCl. 92-kDa gelatinase was eluted using a linear NaCl gradient (0-2.0 M) and the peak collected. The sample was then dialyzed overnight against 0.02 M Tris, pH 7.5, containing 0.005 M CaCl(2) and 0.5 M NaCl and the dialysate subjected to gelatin-agarose (Sigma). The gelatinase was removed from the column with a 0-10% Me(2)SO (Sigma) gradient. The fractions were collected, the Me(2)SO was dialyzed out, and the samples then applied to SDS-PAGE followed by Coomassie staining under both reduced and nonreduced conditions. Fractions free of dimerized 92-kDa gelatinase and of all other proteins but the 92-kDa gelatinase-TIMP complex were used for the studies described.

Recombinant 92-kDa gelatinase (TIMP-free) was obtained from p2AHT2a cells transfected with the adenovirus E1A gene, which inhibits TIMP and MMP transcription, and the 92-kDa gelatinase gene driven by an SV40 promoter (kind gift of Dr. Gregory Goldberg, Washington University, St. Louis, MO). Cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% heat-inactivated fetal calf serum and penicillin (10^5 units/ml) and streptomycin (10^5 µg/liter). Medium was changed to serum-free + lactalbumin hydrolysate (0.2%) for 24 h, and the conditioned media were collected and purified using the same protocol described above for TIMP-associated 92-kDa gelatinase. Small amounts of 92 complex were separated from the major 92 species by the 0-10% Me(2)SO gradient gelatin chromatography. SDS-PAGE Coomassie gels and Western blots using separate polyclonal antibodies directed against the 92-kDa gelatinase and TIMP-1 demonstrated that 92 consisted of two equimolar bands on Coomassie stain which were identified by immunoblots as the 92-kDa gelatinase and TIMP-1. Recombinant 92 consisted of pure 92-kDa gelatinase in the complete absence of TIMP-1 (not shown).

Activation of the 92-kDa Gelatinases

The 92-kDa progelatinase was activated by incubating at 37 °C for the various times indicated with freshly prepared APMA at a final concentration of 2 mM or with truncated stromelysin 1:100 (M/M). Note that indistinguishable results were obtained for all major observations using trypsin-activated full-length native stromelysin 1:15 (M/M). For most experiments, however, truncated stromelysin was used because it is spontaneously active. We have previously shown (27) that truncated stromelysin exhibits identical substrate specificity to its native full-length counterpart, but is 4-6-fold as active on a molar basis.

Purification of Processed Forms of 92-kDa Gelatinase by Gel Filtration

Gel filtration chromatography was performed to purify the various processed forms of the 92-kDa gelatinase. Samples were applied to a Waters 650 protein purification system with a Pharmacia Superdex 75 HR 10/30 column equilibrated in 0.02 M Tris, pH 7.5, containing 0.005 M NaCl. 100-µl samples (15 µg) were applied at 0.5 ml/min, and the peak fractions were collected. The products were displayed by SDS-PAGE.

Isolation and Purification of TIMP

TIMP was purified from the conditioned media of monolayer cultures of human skin fibroblasts as described previously(28) . Either this native TIMP, or the recombinant TIMP generously provided by David Carmichael, Synergen Corp. (Boulder, CO), which exhibit identical metalloproteinase inhibitory activities(29) , were used in these experiments.

NH(2)-terminal Sequencing

Amino acid sequence analysis was performed on the various activated and processed forms of the 92-kDa gelatinase. Samples were subjected to polyacrylamide gel electrophoresis and the proteins transferred to ProBlott membranes (Applied Biosystems). Bands were visualized by staining with 0.1% Coomassie Blue, and the products were excised and then sequenced by Edman degradation using an Applied Biosystems 473 sequenator as described(30) .

Hydrolysis of Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OEt (thiopeptolide)

Hydrolysis of the thiopeptolide substrate (Bachem Bioscience, King of Prussia, PA) was determined as described previously (31) to assess the general catalytic activity of the activated and processed forms of the 92-kDa gelatinase. Accordingly, 7 mg of the thiopeptolide substrate was dissolved in 50 µl of methanol and brought to 1 ml by the addition of 950 µl of assay buffer (0.05 M HEPES, pH 7.0, 0.01 M CaCl (2)). 0.1 mM 5,5`-dithiobis(2-nitrobenzoic acid), 40 mg, was dissolved in 2 ml of ethanol and brought up to 10 ml with assay buffer. Reaction mixtures contained 100 µl each of enzyme solution and 5,5`-dithiobis(2-nitrobenzoic acid) and 10 µl of the thiopeptolide substrate in a total volume of 1 ml. Reactions were performed at room temperature for 5 min. Enzymatic activity is proportional to the rate of color change quantified by spectrophotometric determination of OD at 410 nm (Gilford RESPONSE) over time.

Gelatin Zymography

Processed forms of the 92-kDa gelatinase were applied without reduction to a 10% polyacrylamide slab gel impregnated with 1 mg/ml gelatin. Gel electrophoresis was performed at 4 °C. After electrophoresis the gel was incubated in 2.5% (v/v) Triton for 30 min and then for 2 h at 37 °C in 50 mM Tris, pH 7.5, containing 5 mM CaCl(2) and 0.001 mM ZnCl(2). The gel was then stained with 0.125% Coomassie Blue.

Degradation of Gelatin

Gelatin (5 µg) was incubated for 30 min at 37 °C with four concentrations of the different HPLC-purified activated and processed forms of the 92-kDa gelatinase. Concentrations represented 4-fold increments and were chosen based on preliminary data, demonstrating the capacity of each form to degrade gelatin. Reaction mixtures were incubated in 30 µl final volume (0.05 M Tris, pH 7.5, containing 0.01 M CaCl(2), and 0.15 M NaCl). The reactions were stopped with SDS sample buffer containing dithiothreitol, boiled, and subjected to SDS-PAGE. Protein bands were stained with Coomassie Blue and densitometrically scanned in a Gilford spectrophotometer (560 nm). Gelatin degradation was calculated as the percent reduction in density compared with the control gelatin band. Linear extrapolation was used to determine the amount of each enzyme required for 50% degradation of the gelatin.

Elastin Degradation

Bovine ligament elastin (Elastin Products, Owensville, MO) was radiolabeled with [^3H]sodium borohydride (DuPont NEN) as described previously(19, 23) . Elastin degradation was quantified by measuring solubilization of insoluble [^3H]elastin at 37 °C and pH 7.5. Data are expressed as micrograms of elastin degraded ± S.D. of triplicate samples. These calculations are based on measurements of [^3H]elastin counts/min, corrected for buffer blanks. The radiolabeled elastin used for these studies had a specific activity of approximately 1900 cpm/µg.


RESULTS

Elastolytic Activity Markedly Decreases over Time following Activation of TIMP-free 92-kDa Progelatinase (92) by Stromelysin

In the course of studying the elastolytic activity of 92-kDa gelatinase, we observed that activation of 92 by stromelysin resulted in initial elastolytic activity that markedly diminished over time (not shown). To determine the basis for this observation, we correlated enzymatic activity with the appearance of processed 92-kDa gelatinase species on Coomassie-stained SDS-PAGE. As shown in Fig. 1, after 4 h of activation by stromelysin, the 92-kDa proenzyme was converted predominantly to the 82-kDa active form with some processing to a smaller 50-kDa species. This material was then applied to insoluble [^3H]elastin for 1 h and exhibited substantial elasin degrading activity, solubilizing 0.88 µg of elastin/µg enzyme/h of incubation at 37 °C. Following 72 h of activation by stromelysin, however, the 92 was completely processed to smaller species, predominantly a 50-kDa form. This material was incubated with insoluble [^3H]elastin and exhibited markedly decreased elastolytic capacity, solubilizing 0.05 µg of elastin/µg of enzyme/h. These activation products were also tested for their general catalytic activity by exposing them to a thiopeptolide substrate(31) . The 4-h 82-kDa product had five times the activity of the 72-h 50-kDa species. Thus, over time, stromelysin activation leads to continued processing of the 92 with loss of catalytic activity. Interestingly, activation of the 92 by APMA did not result in loss of elastolytic activity over time (not shown). These findings stimulated further studies to define modes of activation and processing of the 92-kDa gelatinase and their effects on the catalytic activity of the enzyme species generated.


Figure 1: Stromelysin-mediated activation and processing of 92 over time and effect on catalytic activity. Purified 92 was activated with stromelysin for 4 or 72 h at 37 °C. The reaction products were applied to SDS-PAGE and Coomassie-stained. The activated products were also incubated with insoluble [^3H]elastin and thiopeptolide. Note, that after 4 h of activation most of the proenzyme has been activated to the 82-kDa form which has substantial catalytic activity. However, by 72 h, the predominant band migrated at 50 kDa and had little catalytic activity.



Activation and Processing of 92-kDa Progelatinase, TIMP-associated (92) versus TIMP-free (92), by Organomercurials and Stromelysin

92 was purified from U937 cells. As reported previously, each molecule of secreted 92-kDa progelatinase is stoichiometrically bound to a molecule of TIMP (15, 25) . As demonstrated in Fig. 2, incubation of 92 with APMA for 18 h at 37 °C resulted in complete conversion of the 92-kDa proenzyme (lane 1) to an 83-kDa form (lane 2). Similar activation by stromelysin resulted in conversion to an 82-kDa form, with small amounts of a 50-kDa species similar to that seen in Fig. 1(lane 3). Interestingly, activation of 92 exhibited a different pattern of processing. Incubation of 92 with APMA for 18 h at 37 °C produced a 67-kDa enzyme (lane 5). Stromelysin activation of 92 generated a major 50-kDa product, with small amounts of the 82-kDa species remaining (lane 6). Of note, activation of 92 to the 82-83-kDa forms was delayed compared with 92 and often incomplete.


Figure 2: Activation and processing of 92 and 92 by stromelysin and APMA. 92 and 92 were incubated with APMA or stromelysin for 18 h at 37 °C. The reaction products were applied to SDS-PAGE and Coomassie-stained. Note, the association with TIMP (lanes 1-3) largely inhibits processing past the 82-83-kDa forms. Note also the different 92 forms generated by APMA (67 kDa, lane 5) versus stromelysin (50 kDa, lane 6).



Time Course of Activation and Processing of TIMP-associated and TIMP-free 92-kDa Gelatinase by APMA

92 purified from U937 cells, recombinant 92, and 92 + exogenously added TIMP (2 times molar excess of TIMP) were incubated with APMA for 0, 1, 4, and 18 h to define the time course of activation and processing of the 92-kDa gelatinase (Fig. 3). Most of the 92 was activated to the 83-kDa form by 1 h. Small amounts of an 86-kDa intermediate, previously described(32) , were also visualized at 1 h. No further activation occurred over the time course. 92 underwent activation to both the 83-kDa form and another 74-kDa intermediate species by 1 h. After 4 h, the majority of the gelatinase migrated at a molecular mass of 67 kDa, and the conversion to the final processed 67-kDa form was complete by 18 h. The addition of exogenous TIMP to 92, followed by APMA treatment, resulted in conversion to the 83-kDa form by 1 h without further processing. Stromelysin activation of 92 resulted in conversion to the 82-kDa species within 1 h (not shown). The appearance of the 50-kDa processed form was evident by 4 h (Fig. 1), with complete conversion to 50 kDa by 18 h (Fig. 2).


Figure 3: Time course of APMA-mediated activation and processing of 92 and 92 and effect of exogenous TIMP. 92 purified from U937 cells, recombinant 92, and 92 + exogenous TIMP (2-fold molar excess) were incubated with APMA for 0, 1, 4, and 18 h at 37 °C. The reaction products were applied to SDS-PAGE and Coomassie-stained. Note, conversion of the 92-kDa proenzyme to the 83-kDa form in the presence of endogenous (lanes 1-4) or exogenous (lanes 9-11) TIMP within 1 h. In the absence of TIMP (lanes 5-8) there is more extensive processing to a 67-kDa final form over time.



TIMP Stabilizes NH(2)-terminal-activated Gelatinase and Prevents Further Processing

To further examine the capacity of TIMP to prevent the complete processing of 92-kDa gelatinase, we added TIMP to partially APMA-activated 92 to determine whether further processing could be inhibited. Fig. 4demonstrates that after 1 h of incubation with APMA, pro-92 (lane 1) was activated to the 83-kDa form and partially to a 74-kDa intermediate form (lane 2). The reaction mixture was then divided into two parts. TIMP was added at this time to one of them, and no further processing occurred despite overnight incubation with APMA (lane 3). In the other sample, without added TIMP, 92 was nearly completely processed to 67 kDa following overnight incubation with APMA. The presence of TIMP also appeared to stabilize processing of stromelysin-activated 92 to truncated forms smaller than 82 kDa, as seen by the difference in the proportion of 82- versus 50-kDa species following stromelysin activation in Fig. 2. Similar TIMP-addback experiments were not performed, since exogenous TIMP would inhibit the activating agent, stromelysin.


Figure 4: Effect of exogenous TIMP on APMA-activated 92. 92 proenzyme (lane 1) was activated by APMA at 37 °C for 1 h (lane 2). After 1 h, APMA activation continued for an additional 17 h either in the presence (+TIMP, lane 3) or the absence (-TIMP, lane 4) of TIMP. Reaction products were applied to SDS-PAGE and Coomassie-stained. Note, conversion of the proenzyme to the 83-kDa form and a smaller intermediate by 1 h. Exogenous recombinant TIMP added (seen in lane 3 migrating at 21 kDa) inhibits further processing. In the absence of TIMP, there is complete conversion to the 67-kDa species.



Gelatinolytic Activity of the Activated and Processed Forms of the 92-kDa Gelatinase

To determine the relative catalytic potential of the different activation products, 92 was activated by APMA and stromelysin at 37 °C for various times. Equal amounts (5 µg) from each reaction mixture were applied to SDS-PAGE and Coomassie-stained to estimate the relative quantities of each product. Smaller (50 ng), equal amounts of each mixture were also subjected to gelatin zymography (2-h incubation at 37 °C) to determine catalytic activity. As shown in Fig. 5, the 92-kDa proenzyme was partially activated by zymography conditions, resulting in a small zone of lysis (note, this is a nondenaturing gel). Activation with APMA demonstrated significant gelatinolytic activity of both the 83- and 67-kDa forms. Stromelysin-activated 92 generated large amounts of the 82-kDa enzyme at 1 and 4 h. By 18 h most of the enzyme was converted to the 50-kDa species. During stromelysin activation, a transient 65-kDa species was frequently observed, and it had greater specific activity than the 82-kDa form. Activity of the 50-kDa form was barely detectable, even when it was the predominant species. It should be noted that gelatinolytic activity by zymography reflects both the ability of an enzyme form to refold and its subsequent catalytic activity under the experimental conditions.


Figure 5: Gelatinolytic activity of the activated and processed forms of the 92-kDa gelatinase. 92 was activated by APMA and truncated stromelysin (22 kDa) at 37 °C for 1, 4, and 18 h. A, equal amounts (5 µg) from each reaction mixture were applied to SDS-PAGE and Coomassie-stained. The 92-kDa proenzyme(-) was activated by APMA to the 83-kDa form by 1 h and further processed after 4 and 18 h. Stromelysin activation resulted in complete conversion to 82 kDa after 1 h. Further processing was evident at 4 h with more extensive processing after 18 h. B, 0.05 µg of each mixture was also subjected to gelatin zymography. Following Coomassie staining, white bands represent zones of lysis of the gelatin substrate. Note, activity of the 92-kDa proenzyme, activated in situ(-). Regardless of activating agent, the predominant active forms migrate at 82-83 and 67 kDa. There is minimal activity of the 50-kDa stromelysin activation product.



To more definitively quantify the gelatin-degrading capacity of the activated and processed forms of 92, without introducing the variable of partial denaturation/refolding encountered in zymography, each species was isolated by HPLC gel-filtration, and the amount of enzyme required to degrade 50% of a known amount of gelatin was determined. As shown in Table 1, the APMA-activated 83-and 67-kDa forms are nearly equivalent gelatinases. However, their activity is dependent on the continued presence of APMA. When APMA is removed from the buffer following APMA activation, there is no detectable catalytic activity.



Stromelysin-activated 82-kDa enzyme had similar gelatinolytic activity to the APMA-induced forms. However, the 50-kDa species showed only 20-25% of this activity. Nevertheless, this assay revealed greater activity of the 50-kDa form than might have been expected from the zymography data (Fig. 5) and the results of NH(2)-terminal sequencing (see below). This activity could be related to small amounts of the 65-kDa intermediate, transient species which may have co-purified with the 50-kDa form. Thus, much of the activity observed may have come from the active, larger species. Alternatively, this 50-kDa preparation might also retain a small amount of the NH(2)-terminal, catalytic site-containing portion of 92-kDa gelatinase (see below).

NH(2)-terminal Sequence Analysis of Activated and Processed Forms of the 92-kDa Gelatinase

To determine the precise structural nature of the processed forms of 92-kDa gelatinase, these products were purified by HPLC and subjected to NH(2)-terminal sequence analysis (Table 2). Activation by APMA yields the 83-kDa active form with the NH(2)-terminal sequence MRT at amino acid 75 of the proenzyme domain (excluding signal peptide), which is 3 amino acids upstream and containing the conserved cysteine that interacts with zinc. This is in agreement with a recent publication (33) and explains why the continued presence of APMA is necessary for sustained catalytic activity. The mature processed 67-kDa APMA-activated product has the same NH(2)-terminal sequence as the 83-kDa form, strongly suggesting that the 67-kDa species results from processing at the COOH terminus.



Stromelysin activation (Table 2) yields the 82-kDa species which is the classic NH(2)-terminal active form of MMPs described previously(32) . NH(2)-terminal sequence begins with FQT (amino acid 88), which is the start of the catalytic domain just downstream from the conserved cysteine. The 50-kDa form has the NH(2)-terminal sequence EPE (amino acid 429). Accordingly, this 50-kDa fragment starts past the catalytic site, within the proline-rich hinge region. We were unable to identify a fragment containing the catalytic zinc-binding domain, but we assume that a minor fragment of 50 kDa remains which has this motif based on the small but detectable activity of the HPLC-generated 50-kDa form (Table 1) and the barely detectable zymographic activity present at this M(r) (Fig. 5). Presumably, much of this NH(2)-terminal ``half'' of the 92-kDa gelatinase has been degraded to lower M(r) forms during stromelysin activation.


DISCUSSION

In this report we demonstrate that activation and processing of the 92-kDa gelatinase can result in several final products, depending upon association of the proteinase with TIMP and the activating agent applied. Native 92-kDa gelatinase produced by mononuclear phagocytes is secreted in physical association with TIMP (92). The presence of TIMP results in classic NH(2)-terminal activation and prevents further processing. TIMP-free 92-kDa gelatinase (92), as secreted from neutrophil secondary granules, undergoes further processing to a COOH-terminally truncated, active proteinase following exposure to either organomercurial agents or MMPs (stromelysin). This 67-kDa form is the final active product when activation is achieved with APMA. However, this is only a transient product when activation is mediated by stromelysin. In the latter case, over time, the enzyme continues to undergo cleavage, resulting in its diminished catalytic activity.

Organomercurial agents activate MMPs by chelating the highly conserved prodomain cysteine residue, interrupting its interaction with the active site zinc(14, 15, 16, 17) . Freeing of the zinc leads to autolytic activity whereby the enzyme first cleaves a portion of the proenzyme domain yielding an 86-kDa intermediate species which has been described previously(32) . We consistently observed this product, however, only when the reaction was stopped within 1 h of activation. This intermediate alters the structure of the protein, resulting in autolytic cleavage of nearly the entire proenzyme domain. However, the next cleavage is not COOH-terminal to the conserved cysteine, as one might expect, but just upstream or NH(2)-terminal yielding an 83-kDa product, as recently described (33) and shown here. The retention of the cysteine explains why the continued presence of APMA is required for catalytic activity. In the presence of TIMP, this is the final product of APMA activation; however, the enzyme has limited catalytic potential due to inhibition caused by the associated TIMP molecule.

In the absence of TIMP, APMA allows further autolytic processing to a 67-kDa form. Given the same NH(2)-terminal sequence of both the 83- and 67-kDa forms, this processing event must come from the COOH terminus (Fig. 6). This COOH-terminal truncated product has equal to or greater catalytic activity than the 83-kDa form. Increased catalytic activity after loss of a portion or all of the COOH-terminal domain has been observed for other MMPs(27) , with the exception of interstitial collagenase degradation of interstitial collagens, where the presence of the COOH-terminal domain is required, probably for substrate binding(34) .


Figure 6: Domain structure of activated and processed forms of the 92-kDa gelatinase. The domain structure of the 92-kDa gelatinase is shown (top), including the proenzyme, catalytic domain that coordinates the active site zinc molecule, the fibronectin-like domain (Fn), the type V collagen-like domain (coll V), and the COOH-terminal domain. The APMA- and stromelysin-activated and processed products are aligned based on NH(2)-terminal cleavage sites (Table 2).



It is unlikely that activation with APMA mimics processes that occur in vivo. Interaction of MMPs with other proteinases may more closely reflect activation in vivo. Stromelysin has been reported to activate the 92-kDa gelatinase to an 82-kDa product (NH(2)-terminal sequence FQT) with loss of the entire NH(2)-terminal proenzyme domain, including the Cys(32) . However, this 82-kDa form is the final product only in the presence of TIMP. 92, or neutrophil gelatinase, undergoes further processing. A 50-kDa protein was isolated that contained the COOH-terminal collagen-like and hemopexin-like domains. This fragment represented cleavage in the Pro-rich hinge region, past the catalytic domain. Although this fragment cannot have catalytic activity, we did observe greatly reduced, but detectable, enzymatic function in the HPLC-purified 50-kDa form and at 50-kDa by gelatin zymography. Most likely, this was caused by the NH(2)-terminal, catalytic domain-containing fragment also produced by this cleavage in the hinge region. We were unable to isolate this fragment, however, and therefore the reason it is likely to have been largely degraded to smaller nonfunctional fragments. Nevertheless, it is clear that stromelysin causes extensive processing of 92, ultimately leading to diminished catalytic activity.

Although this study was conducted in vitro, it may provide important clues to post-translational mechanisms of regulating proteinase activity in vivo. Neutrophils and macrophages recruited during an inflammatory response have the capacity to release the 92-kDa gelatinase. Within this inflammatory mileu, the enzyme may come in contact with other proteinases and inhibitors that regulate the proteolytic activity of the gelatinase. The current study demonstrates that proteinases may not only activate the 92-kDa gelatinase, but upon continued exposure may inactivate the enzyme, thereby limiting further tissue damage. Such inactivation may rather rapidly follow activation, as for stromelysin processing of 92-kDa gelatinase. Finally, these data demonstrate that the catalytic function, processing, and fate of neutrophil-released 92-kDa progelatinase free of associated TIMP may be very different from macrophage-secreted 92-kDa progelatinase-TIMP complex. Interestingly, 92-kDa gelatinase released from neutrophils appears to be more labile than the 92 produced by macrophages. Perhaps neutrophil gelatinase is ``turned over'' more quickly in vivo, consistent with a role in acute inflammatory processes which typically involve neutrophils.


FOOTNOTES

*
This work was supported by Grants RO-1 HL47328, HL2401, AR35805, PO-1 HL29594 and by the Montsanto-Searle/Washington University Biomedical Agreement. 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.

§
Recipient of a Career Investigator Award from the American Lung Association. To whom correspondence should be addressed: Respiratory and Critical Care Division, Jewish Hospital at Washington University Medical Center, St. Louis, MO 63110. Tel.: 314-454-7524; Fax: 314-454-8293.

(^1)
The abbreviations used are: MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; APMA, 4-aminophenylmercuric acetate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.


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