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Originally published In Press as doi:10.1074/jbc.M414379200 on May 18, 2005

J. Biol. Chem., Vol. 280, Issue 28, 26160-26168, July 15, 2005
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Mutational and Structural Analyses of the Hinge Region of Membrane Type 1-Matrix Metalloproteinase and Enzyme Processing*

Pamela Osenkowski{ddagger}§, Samy O. Meroueh¶, Dumitru Pavel¶, Shahriar Mobashery¶, and Rafael Fridman{ddagger}||

From the {ddagger}Department of Pathology, School of Medicine, Wayne State University, Detroit, Michigan 48201 and the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556

Received for publication, December 21, 2004 , and in revised form, May 4, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Membrane type 1 (MT1)-matrix metalloproteinase (MMP) is a major mediator of collagen degradation in the pericellular space in both physiological and pathological conditions. Previous evidence has shown that on the cell surface, active MT1-MMP undergoes autocatalytic processing to a major membrane-tethered 44-kDa product lacking the catalytic domain and displaying Gly285 at its N terminus, which is at the beginning of the hinge domain. However, the importance of this site and the hinge region in MT1-MMP processing is unknown. In the current study, we generated mutations and deletions in the hinge of MT1-MMP and followed their effect on processing. These studies established Gly284–Gly285 as the main cleavage site involved in the formation of the 44-kDa species. However, alterations at this site did not prevent processing. Instead, they forced downstream cleavages within the stretch of residues flanked by Gln296 and Ser304 in the hinge region, as determined by the processing profile of various hinge deletion mutants. Also, replacement of the hinge of MT1-MMP with the longer MT3-MMP hinge did not prevent processing of MT1-MMP. Molecular dynamic studies using a computational model of MT1-MMP revealed that the hinge region is a highly motile element that undergoes significant motion in the highly exposed loop formed by Pro295–Arg302 consistent with being a prime target for proteolysis, in agreement with the mutational data. These studies suggest that the hinge of MT1-MMP evolved to facilitate processing, a promiscuous but compulsory event in the destiny of MT1-MMP, which may play a key role in the control of pericellular proteolysis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinases (MMP(s))1 are zinc-dependent endopeptidases that play key roles in normal and pathological conditions because of their ability to degrade extracellular matrix components and a variety of bioactive molecules (1–3). The MMP family comprises secreted and membrane-anchored proteases that are major mediators of pericellular proteolysis. Among the membrane-anchored MMPs, MT1-MMP (MMP-14) is a type I transmembrane protease whose broad range of functions includes pericellular collagenolysis (4–6), activation of other MMPs, including MMP-2 (7, 8) and MMP-13 (9, 10), and cleavage of growth factors, cytokines, chemokines, cell adhesion receptors, protease inhibitors, and an apoptosis death receptor (11–15). MT1-MMP activity is critical for normal growth and development (16, 17), and it is involved in various pathological conditions including cancer metastasis and arthritis (18–20). Like all MMPs, MT1-MMP is inhibited by the tissue inhibitors of metalloproteinases (TIMPs), with TIMP-2, TIMP-3 and TIMP-4 being high affinity inhibitors and TIMP-1 being a weak inhibitor (21). The TIMP-2·MT1-MMP complex, which serves to inhibit catalytic activity, can also recruit pro-MMP-2 to the cell surface, facilitating its activation by a neighboring TIMP-2-free MT1-MMP (7, 8). Active MMP-2 can then activate pro-MMP-9 (progelatinase B) (22) and can also participate in the activation of pro-MMP-13 (10). Thus, MT1-MMP initiates a cascade of zymogen activation at the cell surface which promotes pericellular proteolysis.

As a membrane-anchored protein, MT1-MMP is subjected to a unique set of regulatory processes that collectively control the level of active enzyme on the cell surface, including endocytosis and recycling (23–26), membrane trafficking (27, 28), processing (29–31), and ectodomain shedding (30, 32–35). The processing of MT1-MMP is a cell surface event in which the active enzyme is autocatalytically cleaved in trans to a major 44-kDa membrane-tethered degradation form (also referred to as the 43- or 45-kDa species) lacking the catalytic domain. A series of minor degradation products of 43–40 kDa can also be detected under conditions of enzyme overexpression (31, 36–38) or exposure of cells to concanavalin A, which inhibits MT1-MMP endocytosis (39). Concomitantly, a fragment of the catalytic domain is released into the extracellular space as an inactive 18–20-kDa soluble form (30, 33). MT1-MMP processing is regulated by the level of TIMPs (TIMP-2 and TIMP-4) or by synthetic MMP inhibitors, which by binding to the active site inhibit processing, and consequently, the mature 57-kDa enzyme accumulates on the cell surface. Conversely, reduced levels of TIMPs promote processing (31, 40–42). Processed MT1-MMP is detected on the surface (25) and isolated plasma membranes (43) of human HT1080 fibrosarcoma cells under basal conditions. Culture of HT1080 cells on fibronectin (42) or within a three-dimensional collagen I gel (44) enhances processing. Clustering of {beta}1 integrins also induces MT1-MMP processing in DOV13 ovarian carcinoma cells (45). Transforming growth factor-{beta}1 induces processing of MT1-MMP in human SCC25 oral squamous cell carcinoma cells (46). Overexpression of angiopoietin-2 in U87MG glioma cells induces expression and processing of MT1-MMP to the 44-kDa species (47). Primary human hepatic myofibroblasts cultured on collagen I also exhibit enhanced MT1-MMP processing (48). Processing of MT1-MMP to the 44-kDa species was detected in the human microvascular endothelial cell-1 line under unstimulated conditions, but it was enhanced by exposure of the cells to elastin-derived peptides (49). Active and processed MT1-MMP forms were detected in human rheumatoid synovial tissue extracts (50). Extracts of primary human glioma invasive tissues contained active and processed MT1-MMP (47). In cultured cells, the extent of MT1-MMP processing is influenced by factors that regulate enzyme expression, clustering, endocytosis, and/or lysosomal degradation such as phorbol ester (29, 51–53), concanavalin A (33, 39, 51, 54, 55), bafilomycin A1 (55, 56), calmodulin inhibitors (57), and calcium ionophores (29, 58). Thus, processing of active MT1-MMP to the 44-kDa species is a physiological mechanism of enzyme turnover.

N-terminal sequencing of the 44-kDa degradation product isolated from mammalian cells expressing recombinant MT1-MMP showed that this species displays Gly285 at the N terminus, suggesting that Gly284–Gly285 is a putative processing site (31). However, direct evidence of the importance of this site in MT1-MMP processing was not established. The Gly284–Gly285 site is located at the beginning of the hinge domain (59), a stretch of residues that links the catalytic domain to the hemopexin-like domain. Studies with soluble MT1-MMP expressed in bacteria or yeast showed that the enzyme also undergoes autolysis to several degradation fragments displaying N termini located within the hinge domain (60, 61), suggesting that, in addition to the putative Gly284–Gly285 site, processing involves multiple cleavages within the hinge region. However, at present, there is no information on the importance of the hinge in MT1-MMP processing.

In the present study, we carried out structure-function relationship studies to understand the role of the hinge domain of MT1-MMP in processing. Specifically, we generated MT1-MMP mutants containing various single and double amino acid substitutions at the putative Gly284–Gly285 cleavage site and specific truncations of the hinge domain. The results of this study support a model in which the nonrestrictive structure and high mobility of the hinge allow this domain to function as a prime target for autocatalytic processing of MT1-MMP.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Nonmalignant monkey kidney epithelial BS-C-1 (CCL-26) cells were obtained from the American Type Culture Collection (ATCC). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and antibiotics.

Recombinant Vaccinia Virus—The production of the recombinant vaccinia virus (vTF7-3) expressing bacteriophage T7 RNA polymerase has been described by Fuerst et al. (62).

Recombinant Proteins and Antibodies—Human recombinant pro-MMP-2 and TIMP-2 were expressed and purified to homogeneity, as described previously (63, 64). The rabbit polyclonal antibody (pAb) 437, raised against residues 437–454 of the hemopexin-like domain of human MT1-MMP (31), the rabbit pAb 160 (a generous gift from Dr. A. Sang, Florida State University, Tallahassee), raised against residues 160–174 of the catalytic domain of human MT1-MMP (35), and the LEM2/15 monoclonal antibody (mAb) (a generous gift from Dr. A. Arroyo, Hospital de la Princesa, Madrid, Spain), raised against residues 218–233 of the catalytic domain of human MT1-MMP (65), have been described previously. The rabbit pAb to the hinge of MT1-MMP (RDI-MMP-14Habr) was purchased from Research Diagnostics, Inc. (Flanders, NJ). The RP1MMP16 pAb, raised against residues 301–343 of the MT3-MMP hinge region, was purchased from Triple Point Biologics, Forest Grove, OR.

Mutagenesis of MT1-MMP—The pTF7-EMCV-1 expression vector containing full-length wild-type human pro-MT1-MMP (pTF7-MT1-MMP) has been described previously (31). The full-length wild-type human pro-MT3-MMP cDNA was a gift from Dr. D. Pei, University of Minnesota, Minneapolis. Single and double amino acid substitutions and sequential hinge deletion mutants in pro-MT1-MMP were generated by the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using specific primers and wild-type pro-MT1-MMP cDNA in the pTF7-EMCV-1 vector as the template. An MT1-MMP/MT3-MMP chimera, in which the hinge of MT1-MMP was replaced with the hinge of MT3-MMP, was constructed by amplifying the MT3-MMP hinge region cDNA fragment, designed to contain flanking SpeI restriction sites. The pTF7-MT1-MMP {Delta}284–315 construct, which lacks the hinge region of MT1-MMP, was modified by site-directed mutagenesis to contain a SpeI restriction site in the hinge region. Then, the MT3-MMP hinge cDNA fragment was cloned into the SpeI site, which was then removed by site-directed mutagenesis. The sequences of all mutant constructs were verified by DNA sequencing.

Expression of MT1-MMP Mutants—Expression of the MT1-MMP mutants was carried out in BS-C-1 cells by the infection/transfection procedure. Briefly, BS-C-1 cells were grown in 6-well plates to 80% confluence and infected with 10 plaque-forming units (pfu)/cell of the vTF7-3 virus in infection medium (DMEM + 2.5% fetal bovine serum and antibiotics) for 30 min, as described previously (31). For dose-dependent MT1-MMP studies, increasing amounts (0–0.4 µg/well) of pTF7-MT1-MMP were used. Control cells were infected with vTF7-3 virus but received no plasmid DNA. For inhibitor studies, increasing amounts of human recombinant TIMP-2 (0–100 nM) in serum-free DMEM were added to the cells 4 h postinfection/transfection. 16–18 h postinfection, the cells were lysed with 0.2 ml/well lysis buffer (25 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, and 100 mM NaCl with protease inhibitors) on ice for 1 h and centrifuged at 13,000 rpm for 30 min. For time course studies, the cells were lysed at 5, 10, or 24 h postinfection/transfection. The cell lysates were mixed with reducing Laemmli SDS sample buffer, boiled, and resolved by 10 or 12% SDS-PAGE, followed by transfer to a nitrocellulose membrane and immunoblot analysis with various anti-MT1-MMP antibodies.

Pro-MMP-2 Activation—18 h postinfection/transfection, the medium was aspirated, and the cells were washed with phosphate-buffered saline (PBS). Then, serum-free DMEM (0.6 ml/well) supplemented with 10 nM TIMP-2 was added to the cells for 10 min. The TIMP-2-containing medium was aspirated, and the cells were washed with PBS and incubated (37 °C) with 10 nM pro-MMP-2 for 30 min. The cells were washed with PBS, lysed in 0.2 ml/well cold lysis buffer on ice for 1 h, and centrifuged (13,000 rpm) for 30 min at 4 °C. The lysate samples were mixed with Laemmli sample buffer (66) without reducing agents and without heating and subjected to gelatin zymography, as described previously (67).

Cell Surface Biotinylation—BS-C-1 cells in 6-well plates were infected with 10 pfu/cell vTF7-3 virus for 30 min in infection media followed by transfection with 0.2 µg/well of either pTF7-MT1-MMP wild-type or pTF7-MT1-MMP mutant cDNA. Control cells were infected but received no plasmid DNA. The cells were incubated (18 h, 37 °C) in serum-free DMEM and then rinsed with cold PBS containing 0.1 mM CaCl2 and 1 mM MgCl2 and then biotinylated with 0.5 mg/ml EZ-link-sulfo-NHS-biotin (Pierce) for 30 min, as described previously (67). As a control, a parallel plate of cells received PBS without biotin. The cells were lysed with 0.2 ml/well lysis buffer, centrifuged at 13,000 rpm for 30 min, and the supernatant was incubated with 50 µl of streptavidin beads (Pierce) overnight at 4 °C. The beads were washed four times with harvest buffer (0.5% SDS, 60 mM Tris-HCl, pH 7.5, 2 mM EDTA) supplemented with 2.5% Triton X-100 (final concentration). The bound biotinylated proteins were eluted with reducing Laemmli SDS sample buffer, boiled, and resolved by 10% or 12% SDS-PAGE followed by transfer to a nitrocellulose membrane. The biotinylated MT1-MMP forms were detected with anti-MT1-MMP antibodies.

Immunoprecipitation of MT1-MMP Soluble Forms—BS-C-1 cells were grown in 100-mm culture dishes to 80% confluence and infected with 10 pfu/cell of the vTF7-3 virus as described above. Cells were transfected with 2 µg/plate of either pTF7-MT1-MMP wild-type or pTF7-MT1-MMP mutant cDNA. 4 h postinfection/transfection, the cells were metabolically labeled (12 h, 37 °C) with 100 µCi/ml [35S]methionine (PerkinElmer Life Sciences). Serum-free media from 35S-labeled cells expressing recombinant MT1-MMP were concentrated 10-fold and immunoprecipitated with pAb 160 under denaturing conditions as described previously (33). The immunoprecipitates were resolved by reducing 15% SDS-PAGE followed by autoradiography.

TIMP-2 Enzyme-linked Immunosorbent Assay—BS-C-1 cells in 6-well plates were infected/transfected to express wild-type or mutant MT1-MMP as described above. As a control, some cells were infected with the vaccinia virus but received no plasmid DNA. 4 h postinfection/transfection, the media were changed and replaced with serum-free media containing 10 nM recombinant human TIMP-2. 16 h later, the cells were rinsed twice with PBS to remove unbound inhibitor, solubilized with cold lysis buffer, and centrifuged. Lysate samples were analyzed for TIMP-2 levels by enzyme-linked immunosorbent assay (QIA40, Oncogene Research Products, San Diego) as described by the manufacturer's protocol.



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  		FIG. 1.
Hinge region of MT-MMPs. A, sequence alignment of the hinge region of the six members of the MT-MMP family obtained from the T-Coffee multiple sequence alignment server. Numbers correspond to amino acid residues of the MT1-MMP hinge domain. The Gly284–Gly285 autocatalytic cleavage site at the beginning of the hinge region of MT1-MMP is underlined. B, table of the homology modeling results showing identical or similar primary sequences of the hinge regions of the MT-MMPs, using the BLASTP2 algorithm, with the BLOSUM62 matrix and default settings. NS, no significant similarity. C, table of the total hinge length and proline composition of the MT-MMPs.

 
Computational Modeling of MT1-MMP and Molecular Dynamics Studies—An initial model of the 32-residue hinge domain was constructed using Sybyl 6.91 (Tripos, Inc., St. Louis, MO). The hinge region was protonated using the Protonate program, which is part of the AMBER 7 suite of programs (68). The resulting structure was then subjected to a 1.5-ns molecular dynamics simulation at 2,000 K. From these simulations, 150 structures were selected at 10-ps intervals. These structures were each subjected to simulated annealing molecular dynamics simulations, which consisted of first heating the molecule to 2,000 K over 10 ps of simulation, followed by 15 ps of molecular dynamics simulations at 2,000 K, and finally cooled back to 0 K for 15 ps. This cycle was repeated three times, except that the last cooling step (from 2,000 to 0 K) was carried out over 375 ps of molecular dynamics simulation. This three-cycle simulated annealing protocol, repeated 150 times, resulted in a total of 72 ns of molecular dynamics simulations. The AMBER forcefield parameters were assigned to all atoms using the parm99 set of parameters, which is part of the AMBER 7 package. For the simulated annealing runs, the solvent was treated implicitly as a dielectric continuum using the Generalized-Born (68–70) (GB) approach. A 1-fs time step was used, and the Sander module was used to carry out all molecular dynamics simulations.

The resulting 150 conformers were then clustered using the NMR-CLUST program (71), which resulted in 18 clusters, and a representative conformer from each cluster was selected by the program. These conformers were then docked individually on the three-dimensional structure of pro-MT1-MMP, which was constructed previously (21). The structure consisted of the propeptide domain, the catalytic domain, and the hemopexin-like domain. The rigid body docking procedure consisted of manually translating the structure of the hinge region in space until the N and C termini of the peptide were within bonding distance of the C and N termini of the catalytic and hemopexin-like domains, respectively. 15 conformers did not meet this criterion and were discarded. Among the remaining three structures, two showed unfavorable steric interactions among atoms on the peptide and the MT1-MMP structure and were discarded. The remaining structure was attached covalently to the main protein, protonated with the Protonate program, and immersed in a box of TIP3P (72) water molecules; the fully solvated system consisted of 91,256 atoms. This system was then subjected to 5 ns of molecular dynamics simulations using the Sander module of the AMBER 7 package, and all bonds involving hydrogen atoms were constrained with the SHAKE algorithm (73), which enabled the use of a 2-fs time step.

Densitometry Analysis—Densitometry analysis of the relative levels of the MT1-MMP processing products in relation to the active (57 kDa) species was performed using the Scion Image Program (www.scioncorp.com). The value obtained for the 57-kDa species was used as a reference. The ratio of the levels of the degradation products relative to the levels of the 57-kDa active species was determined and was set to 1 for the wild-type enzyme.

Sequence Analyses of the Hinge Domain of Human MT-MMPs— Amino acid sequences of the six human MT-MMPs were obtained from SwissProt: P50281 [GenBank] , P51511 [GenBank] , P51512 [GenBank] , Q9ULZ9, Q9Y5R2, and Q9NPA2, for MT1-MMP to MT6-MMP, respectively. Sequence alignment of the hinge domains was performed using the T-Coffee multiple sequence alignment software (www.ch.embnet.org/software/TCoffee.html). Homology modeling between the hinge regions was conducted using the BLASTP2 algorithm, which is used to search for primary sequence similarities (www.ncbi.nlm.nih.gov/BLAST/). The BLOSUM62 matrix was used to determine identical and similar matches of the MT-MMP hinge regions, using the default settings.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence Alignment of the Hinge Domain of MT-MMPs— Shown in Fig. 1A is the sequence alignment of the hinge regions of the six members of the MT-MMP family. The Gly284–Gly285 peptide bond at the beginning of the hinge of MT1-MMP (Fig. 1A) is unique to this enzyme. The hinge regions of the MT-MMPs are highly variable in terms of length and composition. The hinge of MT1-MMP is the shortest, comprising 32 residues, whereas the hinge of MT2-MMP is the longest, containing 63 residues. BLASTP2 homology modeling showed no significant similarity between the hinge sequence of MT1-MMP with the other MT-MMPs (Fig. 1B). However, significant identical and similar matches were found among the hinge regions of MT2-MMP, MT3-MMP, and MT5-MMP. All of the MT-MMP hinge regions are relatively rich in proline residues because proline comprises 25–39% of the residues of the MT-MMP hinge regions (Fig. 1C). The high percentage of prolines in the hinge region may impart flexibility to this domain because prolines introduce kinks in the secondary structure of a protein. To determine whether this is the case for the MT1-MMP hinge, dynamic simulations were performed to assess the overall flexibility and mobility of the hinge region. As will be described below in more detail, the hinge domain of MT1-MMP is a highly flexible structure.



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  		FIG. 2.
Processing of MT1-MMP. A, time course of MT1-MMP expression. BS-C-1 cells in 6-well plates were infected with 10 pfu/cell of vTF7-3 virus and then transfected with 0.2 µg/well pTF7-MT1-MMP expression plasmid. At the indicated times, the cells were lysed with lysis buffer. The lysates were resolved by reducing 10% SDS-PAGE followed by immunoblot analysis using the 437 pAb to the hemopexin-like domain. B, dose-dependent expression of MT1-MMP. BS-C-1 cells in 6-well plates were infected with 10 pfu/cell of vTF7-3 virus and then transfected with 0.1 (lane 1), 0.2 (lane 2), 0.3 (lane 3), or 0.4 (lane 4) µg/well pTF7-MT1-MMP expression plasmid. After 16 h, the cells were lysed, and the lysates were resolved by reducing 10% SDS-PAGE followed by immunoblot analysis using the pAb to the hinge of MT1-MMP (RDI-MMP-14Habr). The 60- and 57-kDa species represent the pro-form and active species of MT1-MMP, respectively, as reported previously (31). The asterisk indicates the 63-kDa prepro-form of MT1-MMP. Nonspecific bands are represented as #.

 
Processing of MT1-MMP as a Function of Time and Level of Expression—As shown in Fig. 2, processing of MT1-MMP is a time- and dose-dependent event. As early as 10 h after transfection, active MT1-MMP (57 kDa) was processed to a major 44-kDa degradation product (Fig. 2A, 10 h). 24 h later, an additional product of ~40 kDa, which appeared as a smear in the gel, was also detected. This product(s) was recognized by the antibody to the hemopexin-like domain (Fig. 2A) but not by an antibody to the catalytic domain (data not shown), suggesting that its formation involves cleavage within the hinge region or the beginning of the hemopexin-like domain. The extent of MT1-MMP processing was also dependent on the level of protein expression (Fig. 2B), and at low levels the main product detected was the 44-kDa fragment (Fig. 2B, lane 2). Higher levels of protease expression correlated with higher levels of the 44-kDa species and the appearance of the ~40-kDa product ~41–40 kDa).

Site-directed Mutagenesis at the Gly284–Gly285 Site in the Hinge Region of MT1-MMP—Previously, N-terminal sequencing of the 44-kDa species indicated an N terminus starting at Gly285 (31). To establish the importance of the Gly284–Gly285 site in the processing of MT1-MMP to the 44-kDa species, we carried out a series of single amino acid substitutions at both Gly284 and Gly285. These included six single point mutations: Gly284 -> Phe, Gly284 -> Arg, Gly284 -> Trp, Gly285 -> Ala, Gly285 -> Phe, and Gly285 -> Arg (Table I). The residues for substitution were chosen to introduce either a bulkier or charged side chain in the putative Gly284–Gly285 cleavage site. The mutant MT1-MMP enzymes were then expressed in BS-C-1 cells by infection/transfection using low MT1-MMP cDNA levels to avoid forcing promiscuous hydrolysis of the enzyme caused by overexpression.


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  		TABLE I
MT1-MMP hinge mutations

 
Immunoblot analyses of the cell lysates using an antibody to the catalytic domain showed that all single point mutants were activated to the 57-kDa mature species (Fig. 3A, lanes 2–7), with levels of active form comparable with the wild-type enzyme (Fig. 3A, lane 1). Also, surface biotinylation experiments showed that all of the mutants were detected on the cell surface at similar levels (data not shown). Furthermore, all of these mutants activated pro-MMP-2 in the presence of TIMP-2 (7, 8) (Fig. 3C), indicating that TIMP-2 binding was not significantly compromised.

Five of the six single point mutants examined, Gly284 -> Phe, Gly284 -> Arg, Gly284 -> Trp, Gly285 -> Ala, and Gly285 -> Phe (Fig. 3B, lanes 2–6, respectively), showed no alteration in the pattern of processing, and like the wild-type enzyme (Fig. 3B, lane 1), they all generated a major 44-kDa degradation product, as determined with the antibody to the hemopexin-like domain. Also, all of these mutants yielded the 40-kDa product when expressed at high levels (data not shown). However, the Gly285 -> Ala (Fig. 3B, lane 5) and Gly285 -> Phe (Fig. 3B, lane 6) mutants showed greater amounts of the 44-kDa product, whereas the Gly284 -> Arg (Fig. 3B, lane 3) and Gly284 -> Trp (Fig. 3B, lane 4) exhibited reduced levels of the 44-kDa fragment, under the same conditions. Densitometry analyses showed that the Gly284 -> Arg mutant demonstrated ~50% less of the 44-kDa form than the wild-type enzyme (Fig. 3D, lanes 3 and 7, respectively). In contrast, the Gly285 -> Ala and the Gly285 -> Phe mutants exhibited ~4-fold more of the 44-kDa species (Fig. 3D, lanes 5 and 6, respectively). In contrast to the previous mutations, substitution of Gly285 with Arg (Fig. 3B, lane 7) resulted in the appearance of a series of degradation products ranging from 44 to 41 kDa, with very low levels of the 44-kDa form compared with the wild-type enzyme (Fig. 3B, lane 1). These results show that cleavage at the Gly284–Gly285 site regulates MT1-MMP processing, in particular to the 44-kDa species. Of note, despite the observed effects on the level of the 44-kDa product as seen with the Gly285 -> Ala (Fig. 3B, lane 5) and Gly285 -> Phe (Fig. 3B, lane 6) mutants, the relative levels of active enzyme (57 kDa) were not altered. Thus, fluctuations in the level of the 44-kDa degradation product caused by these mutations were not sufficient to impact the relative levels of the active form or their ability to activate pro-MMP-2.

Because substitution of either Gly284 or Gly285 with the bulky, charged Arg residue changed the pattern of MT1-MMP processing (Fig. 3B, lanes 3 and 7, respectively), we generated a double Arg mutant to increase stringency at the Gly284–Gly285 site. In addition, we generated two additional double mutants in which both Gly residues were replaced with either Ala (Gly284 -> Ala/Gly285 -> Ala), which like Gly is aliphatic and contains a small side chain, or Pro (Gly284 -> Pro/Gly285 -> Pro), which, because of its unusual constrained structural properties compared with Gly, was expected to mask the cleavage site and thus deter cleavage (Table I). As shown above with the single mutants, the double mutants also exhibited amounts of the 57-kDa species (Fig. 4A, top panel, lanes 2–4) comparable with the wild-type enzyme (Fig. 4A, top panel, lane 1), indicating that zymogen activation was not compromised by these substitutions. The Gly284 -> Ala/Gly285 -> Ala mutant (Fig. 4A, bottom panel, lane 2) displayed a processing profile similar to that of the wild-type enzyme (Fig. 4A, bottom panel, lane 1) whereas the Gly284 -> Pro/Gly285 -> Pro showed a degradation product with a slight shift in molecular mass (Fig. 4A, bottom panel, lane 3), probably because of the increased molecular mass of the Pro residue or because of a shift in the cleavage site to another nearby amide bond. The Gly284 -> Arg/Gly285 -> Arg double mutant, on the other hand, showed little or no 44-kDa species. However, this mutant was processed mostly to a ~41-kDa species (Fig. 4A, bottom panel, lane 4), which was also detected on the cell surface by biotinylation (Fig. 4B, lane 2). The molecular mass of the 41-kDa degradation product generated in the Gly284 -> Arg/Gly285 -> Arg mutant is consistent with a cleavage site downstream of Gly285, likely within the hinge region. Formation of the 41-kDa product was sensitive to TIMP-2 inhibition (Fig. 4C), and 100 nM TIMP-2 caused a 47% reduction in the levels of the 41-kDa species compared with the untreated cells, as determined by densitometry analyses, indicating that formation of the 41-kDa degradation product in the double mutant is a metalloproteinase-dependent event. At present, whether the cleavage at the alternate site that leads to the formation of the 41-kDa product is autocatalytic, as reported for the generation of the 44-kDa species under the same expression conditions (31), is unknown. Cells expressing the Gly284-> Arg/Gly285 -> Arg double mutant accumulated the mature form of MT1-MMP (57 kDa) in the presence of TIMP-2 (Fig. 4C, lanes 2–4). All of the double mutants also maintained the ability to accomplish pro-MMP-2 activation in a TIMP-2-dependent manner (Fig. 4D). Taken together, these results further show that although the Gly284–Gly285 site is preferential for processing of MT1-MMP to the 44-kDa species, if restricted, processing shifts to alternate nearby sites.



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  		FIG. 3.
Effect of single point mutations at the Gly284–Gly285 cleavage site. A and B, BS-C-1 cells in 6-well plates were infected with 10 pfu/cell vTF7-3 virus and then transfected with 0.2 µg/well pTF7-MT1-MMP expressing wild-type (WT, lane 1), Gly284 -> Phe (lane 2), Gly284 -> Arg (lane 3), Gly284 -> Trp (lane 4), Gly285 -> Ala (lane 5), Gly285 -> Phe (lane 6), or Gly285 -> Arg (lane 7) MT1-MMP. After 16 h, the cells were lysed, and the lysates were resolved by reducing 10% SDS-PAGE followed by immunoblot analysis using the mAb LEM2/15 to the catalytic domain (A) or the 437 pAb to the hemopexin-like domain (B). C, BS-C-1 cells were infected/transfected to express wild-type (lane 1), Gly284 -> Phe (lane 2), Gly284 -> Arg (lane 3), Gly284 -> Trp (lane 4), Gly285 -> Ala (lane 5), Gly285 -> Phe (lane 6), or Gly285 -> Arg (lane 7) MT1-MMP, as described in A. 18 h postinfection/transfection, the cells were incubated (10 min, 37 °C) with 10 nM TIMP-2 followed by an incubation (30 min, 37 °C) with 10 nM pro-MMP-2. Lysate samples were harvested and analyzed by gelatin zymography. L and A refer to the latent and active forms of MMP-2, respectively. Lane 8 represents the pro-MMP-2 control. D, the levels of the 57- and 44-kDa species of wild-type MT1-MMP and each of the mutants were quantified by densitometry with the Scion Image Program, using the immunoblots from A and B, respectively. These data were used to determine the ratio of the levels of the 44-kDa degradation products relative to the levels of the 57-kDa active species for each of the MT1-MMP mutants. This ratio was set to 1 for the wild-type enzyme.

 



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  		FIG. 4.
Effect of double point mutations at the Gly284–Gly285 cleavage site. A, BS-C-1 cells were infected/transfected to express wild-type (lane 1), Gly284 -> Ala/Gly285 -> Ala (lane 2), Gly284 -> Pro/Gly285 -> Pro (lane 3), or Gly284 -> Arg/Gly285 -> Arg (lane 4) MT1-MMP as described in the legend of Fig. 3A. Samples of the cell lysates were resolved by reducing 10% SDS-PAGE followed by immunoblot analysis using the LEM2/15 mAb to the catalytic domain (top panel) or the 437 pAb to the hemopexin-like domain (bottom panel). B, BS-C-1 cells infected/transfected to express wild-type (lane 1) or Gly284 -> Arg/Gly285 -> Arg (lane 2) MT1-MMP were surface biotinylated and lysed as described under "Experimental Procedures." The biotinylated proteins were captured with streptavidin beads, and the bound fractions were resolved by reducing 10% SDS-PAGE followed by immunoblot analysis using the 437 pAb. C, BS-C-1 cells were infected/transfected to express the Gly284 -> Arg/Gly285 -> Arg MT1-MMP mutant as described in Fig. 3A. 4 h post infection/transfection, the cells were incubated without (lane 1) or with increasing concentrations of TIMP-2: 25 nM (lane 2), 50 nM (lane 3), or 100 nM (lane 4). 16 h later, cells were lysed, and samples of the lysates were resolved by reducing 10% SDS-PAGE followed by immunoblot analysis using the 437 pAb to the hemopexin-like domain. The dashed arrow in A (lane 4, bottom panel), B (lane 2), and C (lanes 1–4) indicates the ~41-kDa degradation product of the Gly284 -> Arg/Gly285 -> Arg mutant. D, BS-C-1 cells were infected/transfected to express wild-type (lane 1), Gly284 -> Ala/Gly285 -> Ala (lane 2), Gly284 -> Pro/Gly285 -> Pro (lane 3), or Gly284 -> Arg/Gly285 -> Arg (lane 4) MT1-MMP. 18 h postinfection/transfection, the cells were incubated (10 min, 37 °C) with 10 nM TIMP-2 followed by an incubation (30 min, 37 °C) with 10 nM pro-MMP-2. Lysate samples were harvested and analyzed by gelatin zymography. L and A refer to the latent and active forms of MMP-2, respectively. Lane 5 shows pro-MMP-2 as a control. E, wild-type (lane 1) or Gly284 -> Arg/Gly285 -> Arg (lane 2) MT1-MMP was expressed in BS-C-1 cells by infection/transfection followed by metabolic labeling (12 h, 37 °C) with [35S]methionine, as described under "Experimental Procedures." The 35S-labeled media were subjected to immunoprecipitation with pAb 160/protein A-agarose beads, and the immunoprecipitates were resolved by reducing 15% SDS-PAGE followed by autoradiography.

 
We next asked whether the different pattern of processing observed with the Gly284 -> Arg/Gly285 -> Arg double mutant would also alter ectodomain shedding, in particular the release of the inactive 18-kDa soluble fragment, which occurs concomitantly with processing to the 44-kDa species, as described previously (30, 33). These studies showed that the conditioned media of cells expressing the Gly284 -> Arg/Gly285 -> Arg mutant (Fig. 4E, lane 2) also contained the 50- and 18-kDa soluble forms of MT1-MMP similar to the media of cells expressing the wild-type enzyme (Fig. 4E, lane 1). This result indicates that processing of MT1-MMP downstream from the Gly284–Gly285 site has no effect on the shedding of the 18-kDa soluble form, which involves an autocatalytic cleavage at the Ala255–Ile256 peptide bond within the active site (33).



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  		FIG. 5.
Deletions of the hinge region do not inhibit MT1-MMP processing. Inset, the diagram shows the series of deletions generated in the hinge (Gly284–Gly315) of MT1-MMP. The arrow indicates the Gly284–Gly285 processing site, and the dashed line shows another region involved in MT1-MMP processing. A, BS-C-1 cells were infected/transfected to express wild-type (lane 1), {Delta}312–315 (lane 2), {Delta}308–315 (lane 3), {Delta}304–315 (lane 4), or {Delta}296–315 (lane 5). The cells were then surface biotinylated, lysed, and the biotinylated proteins were captured with streptavidin beads described under "Experimental Procedures." The bound biotinylated proteins were resolved by reducing 12% SDS-PAGE followed by immunoblot analysis using the 437 pAb to the hemopexin-like domain. The dashed arrow indicates the active form (~52 kDa) and degradation product (~36.5 kDa) of the {Delta}296–315-MT1 mutant in lane 5. B, BS-C-1 cells were infected/transfected to express wild-type MT1-MMP (lane 1), {Delta}312–315-MT1 (lane 2), {Delta}308–315-MT1 (lane 3), {Delta}304–315-MT1 (lane 4), or {Delta}296–315-MT1 (lane 5). 18 h postinfection/transfection, the cells were analyzed for activation of pro-MMP-2 by gelatin zymography, as described in the legend of Fig. 3C. L and A refer to the latent and active forms of MMP-2, respectively.

 
Processing of Hinge-truncated/deleted MT1-MMP—To understand better the role of the hinge region in MT1-MMP processing, we generated a series of MT1-MMP hinge deletion mutants. The truncations of the hinge region were initiated from the C-terminal end (Gly315) of the hinge to preserve the Gly284–Gly285 site, as shown in the inset of Fig. 5. Expression of the truncated hinge enzymes in BS-C-1 cells showed that the mutants were activated and trafficked to the cell surface, as determined by surface biotinylation (Fig. 5A). However, {Delta}296–315-MT1 (Fig. 5A, lane 5) displayed much less active form compared with the wild-type enzyme (Fig. 5A, lane 1) and the other mutants (Fig. 5A, lanes 2–4). Three of the hinge deletion mutants, {Delta}312–315-MT1, {Delta}308–315-MT1, and {Delta}304–315-MT1, maintained the ability to accomplish pro-MMP-2 activation in a TIMP-2-dependent manner (Fig. 5B, lanes 2–4, respectively) at levels similar to the wild-type enzyme (Fig. 5B, lane 1). However, cells expressing {Delta}296–315-MT1 (Fig. 5B, lane 5) showed significantly reduced pro-MMP-2 activation, possibly because of the lower levels of active enzyme and a 65% reduction in TIMP-2 binding as determined by enzyme-linked immunosorbent assay (data not shown).

With regard to processing, the three hinge deletion mutants, {Delta}312–315-MT1 (Fig. 5A, lane 2), {Delta}308–315-MT1 (Fig. 5A, lane 3), and {Delta}304–315-MT1 (Fig. 5A, lane 4), exhibited a pattern of processing that was similar to that of the wild-type enzyme (Fig. 5A, lane 1), with two degradation fragments analogous to the 44- and 40-kDa species of wild-type MT1-MMP, when examined using the antibody to the hemopexin-like domain. In contrast, {Delta}296–315-MT1 (Fig. 5A, lane 5) produced only a single degradation product of ~36.5 kDa, under the same conditions. Of note, {Delta}304–315-MT1 (Fig. 5A, lane 4), which exhibits two degradation fragments, contains 8 more residues than {Delta}296–315-MT1 (Fig. 5A, lane 5 and inset). Thus, the presence of a single degradation fragment in {Delta}296–315-MT1, which preserves the Gly284–Gly285 site, suggests that this mutant lacks the downstream putative cleavage site(s) responsible for generating the smaller products (41–40 kDa) of MT1-MMP, which are likely to be located within these 8 residues of the hinge domain (indicated by the dashed bracket in the inset of Fig. 5). The change in molecular mass in the active species and degradation products of the hinge deletion mutants is likely the result of a combination of loss of amino acid residues as well as loss of glycosylation sites within the hinge region (41).



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  		FIG. 6.
Processing of an MT1/MT3-MMP hinge chimera. BS-C-1 cells were infected/transfected to express wild-type MT1-MMP (WT) or the MT1/MT3-MMP hinge chimera (MT1/3). After 16 h, the cells were lysed, and the lysates were resolved by reducing 10% SDS-PAGE followed by immunoblot (IB) analysis using the 437 pAb to the hemopexin-like domain of MT1-MMP (A) or the MT3-MMP hinge pAb (B). The dashed arrow represents the pro-form, active form, and degradation product of the MT1/MT3-MMP chimera, and their relative molecular mass is shown in italics.

 
Processing of an MT1/MT3-MMP Hinge Chimera—To understand further the structural features of the hinge involved in processing, we asked whether replacement of the entire MT1-MMP hinge with that of MT3-MMP would impact processing. The hinge of MT3-MMP contains 49 amino acids, which makes it 17 residues longer than that of MT1-MMP (shown in Fig. 1). Additionally, the MT3-MMP hinge shows no significant sequence similarity to the MT1-MMP hinge and also lacks the Gly284–Gly285 peptide bond at the beginning of the hinge. As shown in Fig. 6B, the MT3-MMP hinge antibody only recognized the MT1/MT3-MMP chimera and not the wild-type enzyme, as expected. The molecular mass of the latent (63 kDa) and active (59 kDa) species of the chimera is consistent with the presence of the MT3-MMP hinge. Cells expressing the MT1/MT3-MMP hinge chimera accomplished pro-MMP-2 activation in a TIMP-2-dependent manner, albeit at a slightly reduced level compared with wild-type MT1-MMP (data not shown), indicating that the chimera was activated, trafficked to the cell surface, and bound TIMP-2. As shown in Fig. 6A, the MT1/MT3-MMP chimera underwent processing to a major fragment of ~37 kDa and a series of minor degradation products ranging from 46 to 51 kDa (Fig. 6) which were recognized by both the antibodies to the hemopexin-like domain of MT1-MMP (residues 437–454) and the hinge of MT3-MMP (residues 301–343 in MT3-MMP). Based on antibody recognition, these data suggest that the cleavage that generates the major 37-kDa species in the MT1/MT3-MMP hinge chimera may take place within the MT3-MMP hinge domain. As with the other hinge mutants, the conditioned media of cells expressing the MT1/MT3-MMP hinge chimera contained the 18-kDa soluble form of MT1-MMP (data not shown), indicating that autocatalytic shedding (33) is not altered. Taken together, these results show that processing of MT1-MMP is not restricted by the primary sequence and length of the hinge domain.



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  		FIG. 7.
Dynamic simulation of pro-MT1-MMP. A, stereo view of the superimposition of snapshots of the full-length computational structure of MT1-MMP collected from a 5-ns molecular dynamics simulation. The protein backbone is rendered in tube representation. The hinge domain is shown in orange; the catalytic, the propeptide, and the hemopexin-like domains are shown in magenta, green, and red, respectively. The catalytic zinc ion and a structural calcium ion are shown in space-filled representation and colored in orange and white, respectively. The white and yellow arrows point to portion of the N terminus and hinge domain that experience significant fluctuations during the molecular dynamics simulation. B, plot of the fluctuation that each residue experiences over the 5-ns molecular dynamics simulation. The arrow indicates the hinge region.

 
Molecular Dynamics of Pro-MT1-MMP Reveal a Highly Mobile Hinge Domain—To gain insight into the structural features of the hinge region of MT1-MMP, which may influence its sensitivity to proteolysis, we carried out computational dynamics simulations of a full model of pro-MT1-MMP. A previously constructed model of the hemopexin-like and propeptide domains (21) and the x-ray structure of the catalytic domain of MT1-MMP together with extensive simulated annealing and fully solvated molecular dynamics afforded construction of a full model of the structure of pro-MT1-MMP, including the hinge domain. The three-dimensional structure of pro-MT1-MMP is shown in Fig. 7A, which consists of a superimposition of 41 structures collected over a 5-ns molecular dynamics simulation. The hinge domain extends from an {alpha}-helix of the catalytic domain, forming a loop starting at Pro295, and ending at Arg302. This loop then leads into a 25-residue {beta}-strand-like structure that culminates in the N terminus of the hemopexin-like domain. The overall structure of pro-MT1-MMP reveals that the hinge domain protrudes from the main protein and is distal from the active site (the closest residue on the hinge to the catalytic zinc is ~15 Å away), resulting in little interaction with the catalytic, hemopexin-like, or propeptide domains. To investigate the flexibility of the hinge domain, we resorted to a 5-ns molecular dynamics simulation of pro-MT1-MMP. Of note, as the carbohydrate composition of the sugar moieties of the MT1-MMP hinge (41) has yet to be determined, the impact of this modification could not be taken into account during the dynamics simulations. The fluctuations experienced by each residue were collected over the course of the trajectory and plotted in Fig. 7B. The most significant fluctuations were found to occur at two locations in the protein. The first large peak (fluctuations greater than 10 Å2) occurred at the N terminus. This is not unexpected because the few residues at the N and C termini are known to experience more significant motion compared with the rest of the protein, in light of their greater exposure to solvent and the fact that they are connected to the rest of the protein only on one side. The absence of a peak at the C terminus is not surprising: unlike the residues at the N terminus, the C terminus in this MT1-MMP structure is not fully solvent-exposed, and favorable electrostatic interactions anchor this terminus well. Interestingly, the other large peak that revealed significant fluctuations occurred between Gly284 and Gly315, which corresponds to the hinge domain. The most significant motion occurs toward the highly exposed loop (residues Pro295 to Arg302) that is formed within the hinge domain, as shown in Fig. 7A (yellow arrows). The primary cleavage site (Gly284–Gly285) is upstream from this hinge region at the border with the catalytic domain. We also carried out dynamics simulations of active MT1-MMP (without the pro-domain) and obtained similar data (data not shown). The molecular dynamics simulation thus reveals that the hinge domain is the most unstable structural element in MT1-MMP and undergoes significant motion relative to other domains in the protein.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We showed previously that autocatalytic processing of MT1-MMP generates a major membrane-anchored inactive fragment of 44 kDa displaying Gly285 at the N terminus (33), which is located at the beginning of the hinge region, and thus lacks the catalytic domain. Here, mutational analyses established that the Gly284–Gly285 peptide bond is the primary site for the generation of the 44-kDa form of MT1-MMP. We found that although arginine substitutions at the Gly284–Gly285 site inhibited formation of the 44-kDa species, they did not prevent processing. Instead, the MT1-MMP mutants underwent alternate cleavages at downstream sites, within the hinge region, to generate smaller degradation products (~41-kDa), similar to those detected under conditions of enzyme overexpression (31, 36–38) or inhibition of endocytosis (39). The pattern of processing exhibited by a series of MT1-MMP C-terminal hinge deletion mutants, which preserve the Gly284–Gly285 site, points to the region located between Gln296 and Ser304 of the hinge region as the main target for the additional cleavage site(s) during processing. Consistently, data from the dynamic simulation showed that the most significant motion of the MT1-MMP hinge occurs within the highly exposed loop flanked by residues Pro295–Arg302, suggesting that this area is particularly sensitive to proteolytic cleavage. Also consistent with the current results, previous studies using soluble active MT1-MMP expressed in yeast or bacteria have shown that Arg298–Thr299 (61) and Thr299–Thr300 (60) are two putative sites involved in autocatalytic processing, both of which are located within the stretch of residues limited by Gln296 and Ser304. Interestingly, this stretch of residues also contains three of the four glycosylation sites within the MT1-MMP hinge region, Thr299, Thr300, and Ser301 (41). Alanine substitutions of these residues did not prevent processing, which led to the proposition that glycosylation of the hinge does not regulate processing (41), in agreement with our data with {Delta}296–315-MT1, which lacks these residues but preserves the Gly284–Gly285 site and undergoes processing.

Autocatalytic processing of MT1-MMP also leads to the release of a soluble fragment (~18 kDa) extending from Tyr112 to Ala255 (33) and thus represents a truncated catalytic domain. Because the putative cleavage site (Ala255–Ile256) leading to the release of the 18-kDa fragment is sheltered, we proposed that cleavage at the Gly284–Gly285 peptide bond, which is on the surface, is likely to precede cleavage at the Ala255–Ile256 site (33). The results presented here indicate that regardless of where cleavage occurs within the hinge region, at the Gly284–Gly285 site or alternate downstream locations, the shedding of the 18-kDa soluble fragment is not compromised, indicating that once cleavage occurs within the hinge, the Ala255–Ile256 site can be exposed, and autocatalytic shedding of the truncated catalytic domain ensues.

Why is MT1-MMP processing such an obligatory event that could not be deterred by many structural alterations? Insight into the structure of the hinge of MMPs was first revealed when the crystal structure of full-length porcine MMP-1 was solved. Data from this structure suggested that the hinge region is an exposed area, most likely flexible, and lacks secondary structure (74). Although there is little homology among the hinge regions of MMPs, one common tie among all MMP hinge domains is the high proline content, with the MT1-MMP hinge containing 25% proline residues (Fig. 1). This is most unusual because proline is the second least common amino acid in proteins, after cysteine. It seems that nature purposefully designed the hinge to be rich in proline to prevent the formation of a secondary structure for the domain in its entirety. However, because the structure of the hinge region was not known, we carried out state-of-the-art annealing dynamics to arrive at a structure. Our computer dynamics simulations demonstrated that the hinge region of MT1-MMP is a highly mobile structure compared with the catalytic and hemopexin-like domains. Such a mobile element would be expected to accommodate multiple amino acid substitutions and deletions and still maintain proteolytic sensitivity, as shown here. These data suggest the notion that structurally, the hinge region of MT1-MMP evolved to be highly susceptible to proteolysis. This characteristic of the MT1-MMP hinge also appears to be shared with longer hinge regions such as the hinge of MT3-MMP as shown here with the MT1/MT3-MMP hinge chimera, which was also processed. In this regard, we showed previously that MT3-MMP also undergoes autocatalytic processing (75), but the nature of the processed form has yet to be determined. In collagenolytic MMPs, including MT1-MMP, the lack of a rigid secondary structure of the hinge may be critical for collagen degradation, which has been proposed to act as an intermediary between the catalytic and hemopexin-like domains to facilitate collagen binding (76, 77). At the same time, this flexibility may also make the hinge region a preferred target for proteolytic cleavage. In fact, both MMP-1 (78) and MMP-8 (79), like MT1-MMP, undergo autocatalytic processing in the hinge region. Thus, although the nature of the hinge predicted its susceptibility to proteolysis, there was little experimental evidence to support the claim that it is the main target for MT1-MMP processing. The studies carried out here are the first to provide experimental support to this prediction.

We found that mutations and deletions within the hinge region, which did not prevent processing, had no impact on the level of active MT1-MMP at the cell surface, as expected. On the other hand, when processing was reduced, as in the case of the Gly284 -> Arg mutant, the level of active MT1-MMP at the cell surface and its ability to promote pro-MMP-2 activation were not affected. This suggests that under conditions of reduced processing, compensatory mechanisms may control the pool of active enzyme at the cell surface (80). This is consistent with previous studies showing that reduction of MT1-MMP internalization by a dynamin mutant (24), cytoplasmic tail mutations (23), or by culture on extracellular matrix components (81) did not alter the level of MT1-MMP at the cell surface and/or its net proteolytic activity. Thus, MT1-MMP displayed at the cell surface is subjected to multiple modes of regulation.

The fact that MT1-MMP processing is an obligatory event also raises interesting issues about its functional consequences. Considering that the products of processing are devoid of enzymatic activity, the processing of MT1-MMP can be viewed as a self-destructive mechanism designed to remove active enzyme and terminate MT1-MMP-dependent pericellular proteolysis independently of endogenous inhibitors and/or cellular processes such as endocytosis. Consequently, inhibition of processing by synthetic MMP inhibitors promotes MT1-MMP-dependent activity in the presence of TIMP-2, possibly by inducing accumulation of active MT1-MMP at the cell surface (82). Conversely, compulsory processing may elicit functional consequences that go beyond termination of proteolysis. For instance, accumulating evidence suggests that the main product of MT1-MMP processing, the 44-kDa fragment, which maintains most of the enzyme domains with the exception of the catalytic domain, can influence MT1-MMP functions including collagenolytic activity (77), homodimerization (83), pro-MMP-2 activation and migration on collagen (84). Because MT1-MMP endows invasive cancer cells with the ability to move through interstitial collagenous matrices (85), understanding the biochemical and cellular mechanisms regulating MT1-MMP activity will help the designing of better approaches aimed at controlling this important protease.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grant NCI-CA61986 (to R. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Supported by Cancer Biology Training Grant T32-CA09531 from the NCI, National Institutes of Health. Back

|| To whom correspondence should be addressed: Dept. of Pathology, Wayne State University School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1218; Fax: 313-577-8180; E-mail: rfridman{at}med.wayne.edu.

1 The abbreviations used are: MMP, matrix metalloproteinase; DMEM, Dulbecco's modified Eagle's medium; mAb, monoclonal antibody; MT, membrane type; pAb, polyclonal antibody; PBS, phosphate-buffered saline; pfu, plaque-forming units; TIMP, tissue inhibitor of metalloproteinase. Back



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 ABSTRACT
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 DISCUSSION
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