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J. Biol. Chem., Vol. 277, Issue 18, 16022-16027, May 3, 2002

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Substrate Binding of Gelatinase B Induces Its Enzymatic Activity in the Presence of Intact Propeptide^*

Gregory A. Bannikov, Tatiana V. Karelina, Ivan E. Collier, Barry L. Marmer, and Gregory I. Goldberg^§¶

From the  Department of Medicine, Division of Dermatology and ^§ Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

Received for publication, November 15, 2001, and in revised form, January 15, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of gelatinase B (matrix metalloprotease 9) in human placenta is developmentally regulated, presumably to fulfill a proteolytic function. Here we demonstrate that gelatinolytic activity in situ, in tissue sections of term placenta, is co-localized with gelatinase B. Judging by molecular mass, however, all the enzyme extracted from this tissue was found in a proform. To address this apparent incongruity, we examined the activity of gelatinase B bound to either gelatin- or type IV collagen-coated surfaces. Surprisingly, we found that upon binding, the purified proenzyme acquired activity against both the fluorogenic peptide (7-methoxycoumarin-4-yl)-acetic acid (MCA)-Pro-Leu-Gly-Leu-3-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl-Ala-Arg-NH₂ and gelatin substrates, whereas its propeptide remained intact. These results suggest that although activation of all known matrix metalloproteases in vitro is accomplished by proteolytic processing of the propeptide, other mechanisms, such as binding to a ligand or to a substrate, may lead to a disengagement of the propeptide from the active center of the enzyme, causing its activation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Morphogenesis (1, 2), tissue repair (3, 4), angiogenesis (5, 6), uterine involution, and bone resorption (7) are characterized by intensified tissue remodeling that begins with degradation of the existing extracellular matrix. Resident cells of tissues can secrete a specialized group of enzymes, matrix metalloproteases (MMPs)¹ (8), that can degrade extracellular matrix macromolecules such as collagens and proteoglycans (9). Malignant cells can exploit these same proteases to promote tumor invasion and metastasis (10, 11). MMPs are secreted in a proenzyme form. The activation of MMPs and interaction with their specific inhibitors, TIMPs, (12-23) determine the fate of these enzymes in the extracellular space. Since we initially demonstrated (24) that the propeptide of interstitial collagenase (MMP-1) is cleaved upon activation, proteolytic processing became a generally accepted pathway for activation of MMPs. In vitro, the processing of the propeptide leads to the removal of a conserved Cys residue, triggering the "cysteine switch" activation mechanism (25) and a loss of about 10 kDa in molecular mass. Processing of a propeptide can be initiated by a variety of agents and usually includes an autocatalytic step. Activation of both the monomer and the dimer of purified gelatinase B (GelB) (26) is no exception to this rule (27).

The role of proteolytic processing in the mechanism of MMP activation in vivo remains poorly understood. In most cases, the physiological role of GelB is inferred based on its correlation with enzyme expression, although neither enzyme activation nor the appearance of a proteolytically activated form has been demonstrated (28-32). Proteolytically activated species of GelB have not been found in the in vivo model of dermal-epidermal separation triggered by antibodies to the hemidesmosomal protein BP-180 (28, 33). In this model, cleavage of serpin a1 proteinase inhibitor by GelB is a critical event. The data gleaned from the literature demonstrate no evidence of proteolytically activated species of GelB in a number of normal and pathological conditions. For example, an increased level of GelB, exclusively in its proenzyme form, was found in samples of synovial fluid and tissue from patients with inflammatory arthritis (29). Mice injected with lymphoma cells expressing GelB developed thymic lymphoma more rapidly than those injected with control lymphoma cells; however, no activated enzyme form was detected (30). Expression of GelB has been found to be conducive to the formation of metastases by murine prostate tumor cells, but the presence of activated enzyme was not evident (31). GelB was implicated in the pathogenic mechanism of autoimmune encephalomyelitis (32), whereas only the proform of the enzyme was present in cerebrospinal fluid.

Expression of GelB in the placenta is developmentally regulated, and it is commonly accepted that this enzyme plays an important role in implantation, placental development, and the invasion of the trophoblast into the uterine epithelium (34-42). Again, there is no evidence of a proteolytically activated species of GelB in human placental tissue (35, 39, 43). Is expression of GelB incidental to the processes of tissue metabolism, or does a nonproteolytic mechanism of GelB activation exist?

Here we report that the mere binding of the GelB proenzyme to gelatin or type IV collagen substrates induces its enzymatic activity without cleavage of its propeptide. These results, together with the in situ zymography experiments on tissue sections of human placenta, suggest that GelB activation in vivo may occur via an alternative mechanism.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Enzyme Purification and Activation-- The proform of GelB enzyme was purified from conditioned medium of transfected p2AHT2a cells as described previously (27). Monomer and dimer forms of the enzyme were separated by elution from gelatin-Sepharose (Sigma) affinity column using a 0-10% Me₂SO gradient. Purified enzyme was activated with stromelysin (MMP-3) (MMP-3/MMP-9 ratio = 1:40 (w/w)) at 37 °C for periods of 1-3 h in 5 mM Tris-HCl (pH 7.5) buffer containing 0.005% Brij-35 and 1 mM CaCl₂. The activated stromelysin used for GelB activation was obtained by treatment of stromelysin proenzyme with L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK)-treated trypsin at 1:20 (w/w) ratio for 20 min at 20 °C. Trypsin was inhibited with an 8-fold excess of soybean trypsin inhibitor before the addition of stromelysin to the MMP-9 activation reaction.

Gelatinolytic Activity (in Situ Zymography) in Sections of Human Placenta-- Fresh tissue of human term placenta was a kind gift of Dr. Yoel Sadovsky (Department of Obstetrics and Gynecology, Washington University School of Medicine). Frozen sections of tissue were blotted to remove excess liquid and incubated with either TBS (control), TBS containing 1-10 nM TIMP-1, or inhibitory mouse monoclonal antibodies to human GelB (clone GE-213; Chemicon International) or gelatinase A (MMP-2) (clone CA-4001; Chemicon International) at a concentration of 5-60 µg/ml for 30 min at room temperature. Slides were dipped into an autoradiography emulsion (NTB2; Eastman Kodak Co.) diluted 2.5 times with Tris-HCl (pH 7.5) buffer containing 4 mM CaCl₂ and preheated to 40 °C. In a separate experiment, slides were incubated with emulsion containing 5 mM EDTA instead of CaCl₂. The coated slides were incubated in a humid chamber for 16-32 h, developed, and photographed under the light microscope. The transparent pattern on a black background reflects zones of gelatinolytic activity.

Immunohistochemistry-- Frozen sections of human term placenta were fixed with 3.5% paraformaldehyde and incubated with monoclonal antibodies to human GelB (clone GE-213; Chemicon International), human gelatinase A (clone CA-4001; Chemicon International), rabbit anti-mouse GelB (Chemicon International catalogue number AB19047), and collagen type IV (clone col-94; Sigma) as indicated. The reactions were developed with secondary antibodies: fluorescein isothiocyanate-conjugated AffinPure donkey anti-rabbit IgG (catalogue number 711-095-152) and tetramethylrhodamine isothiocyanate-conjugated AffinPure donkey anti-mouse IgG (catalogue number 715-025-150), both from Jackson Immunoresearch Laboratories.

Binding of GelB to Gelatin and to a Type IV Collagen-coated Surface-- 96-Well plates (Costar Tissue culture or Dynex Technologies Microfluor 2) were incubated overnight at room temperature with 150 µl/well Tris-HCl (pH 7.5) buffer containing either enzyme immuno assay grade gelatin (Bio-Rad; 1 mg/ml) or 150 µl of 125 µg/ml bovine type IV collagen (CC083; Chemicon International). The plates were blocked with bovine serum albumin (1 mg/ml) for 1 h and washed with the same buffer. Various forms of GelB were added at the indicated concentrations in 100 µl of TBS buffer containing 0.005% Brij-35, and the plates were incubated at 4 °C with agitation. The bound enzyme was extracted with 100 µl of 12.5 mM Tris-HCl (pH 7.5) buffer containing 40% glycerol, 5% SDS, and 0.02% bromphenol blue and analyzed using either gelatin zymography or Western blot as described previously (44). Control experiments using [³⁵S]methionine metabolically labeled pro-GelB and active GelB enzymes demonstrated the 95% effectiveness of the extraction method (data not shown). Quantitation of each sample was accomplished by scanning gels with a UMAX UC1260 scanner and analyzing the images by comparison with the known concentration of MMP-9 enzyme using Collage 2.7 software.

Assay of GelB Activity-- Measurement of GelB activity against the fluorogenic peptide substrate MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (Calbiochem) was performed in Microfluor 2 plates filled with 50 mM Tris-HCl (pH7.5) buffer (100 µl/well) containing 100 mM NaCl, 10 mM CaCl₂, 0.005% Brij-35, and 10-30 µM substrate. The reaction was monitored with a fluorometer (PerkinElmer Life Sciences LS50B) running FLDM software at _ex 328 nm and _em 393 nm at room temperature. To assay the activity of bound GelB against gelatin, a mixture of cold gelatin and denatured [³H]collagen (rat type I; 0.28 mCi/mg; PerkinElmer Life Sciences) was adjusted to a final total protein concentration of 2.8 µCi/100 µg/ml and used to coat the 96-well FlashPlates (PerkinElmer Life Sciences) as described above. Fifty µl of pro-MMP-9 or activated MMP-9 dimer at a concentration of 1.4 mg/ml in TBS containing 0.005% Brij-35 and 0.5 mM EDTA were added to the well in triplicate, and plates were incubated at 4 °C for 30 min. Unbound enzyme was removed using three washes with cold TBS buffer containing 0.005% Brij-35 and 5 mM CaCl₂. The last wash was replaced with 50 mM Tris-HCl (pH 7.5) buffer containing 100 mM NaCl and 10 mM CaCl₂. The digestion of gelatin was measured by counting the radioactivity associated with the well during the 0-70 min time interval using a Packard TopCount scintillation counter.

Inhibition of Gelatin-bound GelB with TIMP-1-- Wells containing GelB bound to gelatin were preincubated with TIMP-1 for 15 min at room temperature in 100 µl of 100 mM Tris-HCl (pH7.5) buffer containing 100 mM NaCl, 10 mM CaCl₂, 0.005% Brij-35, and 1 mg/ml bovine serum albumin. The fluorogenic peptide substrate was then added to the wells, and enzymatic activity was measured as described above. K_i values for pro-GelB and activated GelB were calculated with the assumption that the binding of TIMP-1 to the immobilized enzyme is simple noncooperative binding and that the remaining enzyme activity at any inhibitor concentration is proportional to the remaining free enzyme. Hence, the remaining enzyme activity at any inhibitor concentration is given by the following equation: a₀ × {1  i₀/(1 + K_i × i₀)}, where a₀ is the activity in the absence of inhibitor, K_i is the inhibitory binding constant, and i₀ is the concentration of free inhibitor.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Gelatinolytic Activity in Term Placenta Is Co-localized with GelB with Intact Propeptide-- GelB is secreted in a proenzyme form and can be activated in vitro by proteolytic processing of the propeptide (27, 45-47) with a corresponding loss of apparent molecular mass. In tissues where GelB is expressed and presumably active, the presence of the proteolytically activated form of the enzyme has not been demonstrated (28-32, 35, 39, 43). To address this apparent incongruity, we have investigated the gelatinolytic activity in human placenta in situ. Immunohistochemical staining of tissue sections from term placenta revealed the presence of GelB at the placental villus margin, corresponding with the location of the trophoblast bilayer, with minimal expression in the villus core (Fig. 1A). A small quantity of gelatinase A was also found (Fig. 1B). Double immunostaining with antibodies against GelB and type IV collagen showed a complete overlap of the two proteins at the trophoblast basal membrane (Fig. 1, CE). In addition, microcapillaries inside the villi devoid of type IV collagen also have a small amount of GelB (Fig. 1E).

View larger version (58K): [in this window] [in a new window]   Fig. 1.   Localization of GelB and gelatinase A in tissue of human placenta. Immunohistochemistry was performed as described under "Experimental Procedures." Sections of human placenta were stained with either (A) mouse anti-human GelB or (B) anti-human gelatinase A monoclonal antibodies. The GelB protein is associated with the outer surface of trophoblast villi. The gelatinase A protein is localized in a few scattered cells. ×75; scale bars, 100 mm. Double immunostaining (CE) was performed using rabbit anti-mouse GelB and anti-type IV collagen monoclonal antibodies. The sections were than decorated with fluorescein isothiocyanate-conjugated AffinPure donkey anti-rabbit IgG and tetramethylrhodamine isothiocyanate-conjugated AffinPure donkey anti-mouse IgG secondary antibodies. The secondary antibodies were visualized using a filter allowing visualization of only fluorescein isothiocyanate-conjugated IgG (GelB; C) or tetramethylrhodamine isothiocyanate-conjugated IgG (collagen type IV; D) and double exposure (E) for simultaneous visualization of both proteins. Yellow is developed where the two antigens (GelB and type IV collagen) are co-localized. Green indicates the presence of GelB antigen alone. ×186; scale bars, 40 mm.

Comparison of immunohistochemical staining of sections of placental villi with an in situ zymogram assay (Fig. 2) revealed that the gelatinolytic activity and GelB were co-localized. Inclusion of EDTA in emulsion completely abolished the gelatinolytic activity in this assay. Preincubation of the tissue sections with TIMP-1 (see "Experimental Procedures") inhibited gelatinolytic activity at TIMP-1 concentrations as low as 3 nM. This activity was also inhibited (Fig. 2B) in a dose-dependent fashion by preincubation of the sections with monoclonal inhibitory antibody against GelB (clone GE-213). Inhibition could be observed at an antibody concentration of 5 µg/ml (data not shown), whereas almost complete inhibition was achieved at an antibody concentration of 60 µg/ml. Monoclonal inhibitory antibody against gelatinase A (clone CA-4001) had no noticeable effect on the in situ gelatinolytic activity (Fig. 2C). These observations suggest that the gelatinolytic activity in term placenta is due to the presence of GelB. We next examined whether an active form of GelB, as judged by the presence of lower molecular mass enzyme species, could be detected in the placenta tissue. The extracts from excised tissue sections were subjected to zymogram and Western blot analysis (Fig. 3). Both monomer and dimer forms of GelB were found to have a molecular mass corresponding to that of an unprocessed proenzyme form. These data, together with the immunohistochemical staining and in situ zymography results, suggest that the GelB present in human placenta is enzymatically active, despite the fact that its proteolytically activated form cannot be detected.

View larger version (86K): [in this window] [in a new window]   Fig. 2.   Gelatinolytic activity (in situ zymography) in sections of human placenta. In situ zymography was performed as described under "Experimental Procedures." The tissue sections were preincubated for 30 min with (A) buffer, (B) 20 µg/ml inhibitory antibodies against GelB (clone GE-213; Chemicon International), and (C) 60 µg/ml inhibitory antibodies against gelatinase A (clone CA-4001; Chemicon International). The tissue sections were coated with emulsion and incubated for an additional 38 h to develop gelatinolytic activity. Gelatinolytic activity is associated with trophoblast villi and is co-localized with GelB (see Fig. 1).

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Zymogram and Western blot analysis of MMP-9 extracted from tissue sections of human placenta. Sections of human placenta were extracted with 2× electrophoresis sample buffer (12.5 mM Tris-HCl (pH 7.5), 40% glycerol, and 5% SDS), and zymography and Western blot analysis were performed as described under "Experimental Procedures." Lane 1, purified pro-GelB dimer; lane 2, purified activated GelB dimer; lane 3, purified pro-GelB monomer; lane 4, purified activated GelB monomer; lanes 5 -7, various amounts of extract of human placenta sections. Lanes 8 and 9, Western blot analysis of placenta tissue extracts with monoclonal antibodies against human GelB (clone GE-213). Proteolytically processed forms of GelB dimer or monomer cannot be detected in placenta tissue extracts by zymography or Western blot analysis.

Substrate Binding of GelB Proenzyme Is Accompanied by Partial Activation in the Absence of the Proteolytic Processing of the Propeptide-- In view of the results described above, we examined whether the interaction of GelB with physiologically relevant ligand(s) can induce enzyme activation in the absence of proteolytic processing of the propeptide. The results presented in Fig. 4 show the effect of gelatin substrate binding on the activity of GelB compared with the enzyme in solution. Average specific activities of pro-GelB and activated GelB (either free or gelatin-bound) are summarized in Table I. No significant changes in specific activity were observed upon binding to gelatin of proteolytically activated GelB. In contrast, binding of GelB proenzyme to gelatin induced an approximate 600-fold increase in specific activity compared with that of the proenzyme in solution. The specific activity of bound proenzyme, however, was about 10-fold lower than that of activated GelB. We next examined whether the increase in activity upon binding of GelB proenzyme to gelatin could be attributed to the appearance of the proteolytically activated enzyme species. To investigate this possibility, SDS extracts of gelatin-bound enzyme were subjected to Western blot analysis (Fig. 5). The control experiments with [³⁵S]methionine-labeled enzyme showed that 95% of the gelatin-bound enzyme is recovered using the SDS extraction procedure (data not shown). Because the activity of bound proenzyme was 10-fold lower than that of activated GelB, the amount of activated enzyme in the extracts is expected to reach a level of at least 10%. The quantitative comparison of the extracts with mixtures of pro-GelB and activated GelB of known ratios (Fig. 5) showed that the amount of the activated enzyme form in the extracts did not exceed 0.25-0.5%. This number is at least 20-fold lower than that expected if the activity of the bound proenzyme is to be explained by the presence of the proteolytically activated enzyme species. These results suggest that the increase in proteolytic activity of GelB upon binding to gelatin can be attributed to an enzyme with an unprocessed propeptide.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Proteolytic activity of pro-GelB and stromelysin-activated GelB. Enzymatic activity of pro-GelB and stromelysin-activated GelB in solution (A) or bound to gelatin-coated surface (B) was measured as described under "Experimental Procedures" using fluorogenic peptide as a substrate. The amount of GelB bound to gelatin was determined using quantitative zymography of the extracted enzyme as described under "Experimental Procedures." A, 1 and 2, activated dimer (4 and 2 ng, respectively); 3, activated monomer (2 ng); 4, proform of dimer (2800 ng); and 5, proform of monomer (3000 ng). B, 1, activated dimer (2.1 ng); 2 and 5, proform of dimer (17.1 and 10.2 ng, respectively); 3, activated monomer (2.3 ng); 4, proform of monomer (12.7 ng). Specific activities calculated from these data are summarized in Table I.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Western blot analysis of molecular species of GelB extracted from gelatin-coated wells. Lanes 1 and 2 contain 10 ng of purified pro-GelB and the activated form of GelB, respectively. Lanes 3-7 contain a mixture of 10 ng of purified pro-GelB with decreasing amounts of activated enzyme (lane 3, 10%; lane 4, 5%; lane 5, 2%; lane 6, 1%; and lane 7, 0.5%). Lanes 8-10 contain 5, 10, and 20 ng of GelB extracted from gelatin-coated wells.

To examine whether this activity is limited to a small fluorogenic peptide substrate, we assayed the activity of bound proenzyme against a gelatin substrate. The wells of FlashPlates were coated with [³H]gelatin, and its release was followed after binding of the enzyme. The specific activity of the bound enzyme (Table I) was calculated based on the amount of released gelatin and the amount of enzyme bound to a well, determined as described above after extraction. The results of this experiment show that both pro-GelB and activated GelB were active against [³H]gelatin with specific activities of 27.5 and 242.5 pg gelatin/min/ng, respectively. The ratio of specific activities of bound activated versus GelB proenzyme against [³H]gelatin is in good agreement with that obtained for the peptide substrate.

To further characterize the gelatin-bound GelB, we examined the inhibitory activity of TIMP-1 against both proenzyme and activated forms of the enzyme (Fig. 6). The GelB bound to gelatin-coated wells was incubated with TIMP-1 (concentration, 0-30 nM). Fitting the inhibition curves to the equation for simple noncooperative binding gave K_i values of 9.3 ± 3.4 and 2.2 ± 1.0 nM for the activated and proenzyme forms, respectively. These results show that the enzymatic activity of either enzyme form when bound to gelatin was inhibited by TIMP-1 with similar kinetics.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   TIMP-1 inhibition of pro-GelB and activated GelB bound to gelatin-coated wells. Pro-GelB (open squares) and activated GelB (closed squares) bound to gelatin-coated wells were preincubated with TIMP-1 at the indicated concentrations, and enzyme activity was measured as described under "Experimental Procedures" using the fluorogenic peptide as a substrate. Fitting of the inhibition curves to the equation for simple noncooperative binding gave a Ki of 9.3 ± 3.4 and 2.2 ± 1.0 nM for activated GelB and pro-GelB, respectively.

Although gelatin presents a most effective substrate for the study of gelatinases, GelB can bind a number of extracellular matrix components, including laminin, fibrin, and type I and type IV collagens (52, 55). Hence, we compared the effect of GelB proenzyme binding to gelatin and to type IV collagen on its enzymatic activity. The results indicate that binding to either substrate induces proteolytic activity of the proenzyme to a similar degree, whereas the GelB propeptide remained unprocessed (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Matrix metalloproteases are secreted as proenzymes and can be activated in solution by a variety of agents. Initially, we showed (24, 48, 49) that activation of purified pro-MMP1, induced by either partial proteolysis or treatment with organomercurial compounds, resulted in the removal of the propeptide with a corresponding loss in molecular mass of the active enzyme species. Since then, it has been established that activation of all known enzymes of this family is accompanied by the proteolytic processing of a propeptide, with removal of the conserved cysteine residue triggering a "cysteine switch" activation mechanism (25). The more detailed analysis of the pathway of MMP-1 activation using organomercurials (24, 48, 49) showed that the initial activation of the proenzyme occurred without a loss of molecular mass. Subsequently, the enzyme undergoes autoproteolytic conversion to a 44-kDa intermediate and finally to the 42-kDa stable active enzyme (48, 49). This result indicates that enzyme activity can develop even in the presence of the unprocessed propeptide. Mutagenesis of the conserved propeptide sequence in stromelysin (MMP-3) produced enzyme variants with an increased tendency to undergo spontaneous activation (50), also suggesting that destabilizing the propeptide may lead to the development of proteolytic activity.

Purified GelB can be converted in vitro to an active form by cleavage of the propeptide by metalloproteases MMP-1, MMP-2, MMP-3, MMP-7, tissue kallikrein, and plasminogen activator (34). The role of proteolytic processing of the propeptide in the physiological activation mechanism of GelB remains unclear (see "Introduction"). Using in situ zymography and inhibitory antibodies in combination with immunohistochemistry, we have shown that GelB expressed in human placental tissue is co-localized with type IV collagen and is enzymatically active. This occurs despite the fact that its proteolytically processed forms cannot be detected in tissue extracts. We have also shown that the mere binding of GelB to gelatin- or type IV collagen-coated surfaces was sufficient to induce proteolytic activity of the enzyme. Activation of the proform of GelB, resulting from its binding to a substrate, is not accompanied by a loss of its NH₂-terminal propeptide, indicating that its activation is due to a conformational change. Absence of a detectable amount of NH₂-terminal-truncated species in this model also suggests that the enzymatically active proform of GelB that is bound to gelatin is not capable of autocatalytic cleavage of the propeptide under these conditions.

The specific activity of bound pro-GelB against the peptide substrate is about 600-fold higher than the background specific activity of pro-GelB in solution. However, it is about 10-fold lower than that of either free or bound proteolytically activated enzyme. The simplest explanation for the relatively low specific activity of bound pro-GelB is that a conformational change induced by binding results in a lesser accessibility of its active center compared with the enzyme with a processed propeptide. However, this interpretation is not plausible because the ratios of specific activities of bound activated GelB to bound proenzyme against the small peptide substrate and high molecular mass substrate, gelatin, are essentially the same. Similar inhibition of activated and proenzyme forms of GelB bound to gelatin by TIMP-1 also argues against this possibility. Alternatively, one can speculate that only a fraction of substrate-bound pro-GelB is activated, so that interaction with only a subset of the heterogeneous binding sites can support the conformational rearrangements needed for enzyme activation. Heterogeneous, low and high affinity collagen type I binding sites for pro-MMP-9 with K_d values in the 6 × 10¹³ to 9 × 10⁸ M range have been reported (52). Denatured collagen, gelatin, may present an even more complex mixture of binding sites (53). The crystal structure of latent gelatinase A, an enzyme closely related to GelB, reveals that the propeptide of this molecule interacts with the fibronectin-like gelatin binding domain through hydrogen bonding and a salt bridge (54). Hence, binding of the fibronectin-like domain to gelatin can potentially disrupt its interaction with the propeptide, destabilizing its interaction with the active center and consequently leading to enzyme activation.

Our results suggest that binding of pro-GelB to the extracellular matrix in vivo may represent a physiological pathway of enzyme activation. GelB can potentially bind a number of extracellular matrix molecules and cell surface receptors (51). Which of these interactions can mediate GelB activation remains to be elucidated. Further investigation of the relationship between GelB binding and activation is needed for a better understanding of the mechanisms of its physiological function.

	ACKNOWLEDGEMENTS

We thank Dr. Yoel Sadovsky for the generous gift of samples of human placenta. We thank Drs. Arthur Eisen and Yoel Sadovsky of Washington University School of Medicine for critical reading of the manuscript.

	FOOTNOTES

^* This work was supported by Grants R01 AR40618 and R01 AR39472 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (National Institutes of Health).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8172; Fax: 314-362-8159; E-mail: goldberg@medicine.wustl.edu.

Published, JBC Papers in Press, February 11, 2002, DOI 10.1074/jbc.M110931200

	ABBREVIATIONS

The abbreviations used are: MMP, matrix metalloprotease; TIMP, tissue inhibitor of metalloprotease; GelB, gelatinase B; TBS, Tris-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				P. Baluk, W. W. Raymond, E. Ator, L. M. Coussens, D. M. McDonald, and G. H. Caughey Matrix metalloproteinase-2 and -9 expression increases in Mycoplasma-infected airways but is not required for microvascular remodeling Am J Physiol Lung Cell Mol Physiol, August 1, 2004; 287(2): L307 - L317. [Abstract] [Full Text] [PDF]

				M. Bjorklund, P. Heikkila, and E. Koivunen Peptide Inhibition of Catalytic and Noncatalytic Activities of Matrix Metalloproteinase-9 Blocks Tumor Cell Migration and Invasion J. Biol. Chem., July 9, 2004; 279(28): 29589 - 29597. [Abstract] [Full Text] [PDF]

C. A. Owen, Z. Hu, C. Lopez-Otin, and S. D. Shapiro Membrane-Bound Matrix Metalloproteinase-