Identification of Amino Acid Residues of Matrix Metalloproteinase-7 Essential for Binding to Cholesterol Sulfate*

Matrix metalloproteinase-7 (MMP-7; matrilysin) induces homotypic adhesion of colon cancer cells by cleaving cell surface protein(s) and enhances their metastatic potential. Our previous study (Yamamoto, K., Higashi, S., Kioi, M., Tsunezumi, J., Honke, K., and Miyazaki, K. (2006) J. Biol. Chem. 281, 9170–9180) demonstrated that binding of MMP-7 to cell surface cholesterol sulfate (CS) is essential for the cell membrane-associated proteolytic action of the protease. To determine the region of MMP-7 essential for binding to CS, we constructed chimeric proteases consisting of various parts of MMP-7 and those of the catalytic domain of MMP-2; the latter protease does not have an affinity for CS. Studies of these chimeric proteases and other mutants of MMP-7 revealed that Ile29, Arg33, Arg51, and Trp55, in the internal sequence, and the C-terminal three residues corresponding to residues 171–173 of MMP-7 are essential for binding to CS. An MMP-7 mutant, which had the internal 4 residues at positions 29, 33, 51, and 55 of MMP-7 replaced with the corresponding residues of MMP-2 and the C-terminal 3 residues deleted, had essentially no affinity for CS. This mutant and wild-type MMP-7 showed similar proteolytic activity toward fibronectin, whereas the mutant lacked the ability to induce the colon cancer cell aggregation. In the three-dimensional structure of MMP-7, the residues essential for binding to CS are located on the molecular surface in the opposite side of the catalytic cleft of the protease. Therefore, it is assumed that the active site of MMP-7 bound to cell surface is directed outside. We speculate that the direction of the cell-bound MMP-7 makes it feasible for the protease to cleave its substrates on cell surface.

The matrix metalloproteinases (MMPs) 2 comprise a family of zinc-dependent endopeptidases that degrade components of extracellular matrix (ECM) and are believed to play pivotal roles in tissue remodeling under physiological and pathological conditions such as morphogenesis, angiogenesis, tissue repair, and tumor invasion (1)(2)(3)(4). However, it has recently been suggested that several MMPs proteolytically modulate the biological functions of various cell surface proteins, including growth factor precursors, growth factor receptors, or cell adhesion molecules (4); such regulation as well as MMP-catalyzed degradation of ECM is important for tumor growth, invasion, metastasis, and progression. A typical MMP consists of an N-terminal propeptide of about 80 amino acids, a catalytic domain of about 170 amino acids, and a C-terminal hemopexin-like domain of about 200 amino acids. Membrane type MMPs further have a transmembrane domain or a glycosylphosphatidylinositol anchor on the C-terminal side of the hemopexin-like domain and are thus localized on the cell surface. It has been reported that membrane type 1 MMP cleaves several cell surface proteins, such as syndecan-1 (5), ␤-amyloid precursor protein (6), integrin ␣v subunit (7), low density lipoprotein receptor-related protein (8), CD44 (9), and CD147 (10). The anchoring of this MMP with the cell membrane is probably a benefit in its interaction with cell surface substrates. MMP-7 (matrilysin) is the smallest member of the MMP family, of which the pro-form consists of only a propeptide and a catalytic domain and lacks the C-terminal hemopexin-like domain. Although various MMPs are overexpressed both in stromal and tumor cells in cancer tissues, MMP-7 has been detected specifically in tumor cells but not in stromal cells (11). Expression of MMP-7 is correlated well with malignancy and metastasis of cancers, especially in liver metastasis of colon cancer (12). Although MMP-7 has neither transmembrane domain nor glycosylphosphatidylinositol anchor, this protease processes several cell surface proteins, such as Fas-ligand (13), protumor necrosis factor-␣ (14), syndecan-1 (15), and E-cadherin (16). Recent studies further suggest that MMP-7-catalyzed cleavage of Notch on the cell surface leads to dedifferentiation of pancreatic acinar cells by activating the Notch signaling pathway (17). We previously reported that active MMP-7 efficiently binds to the surface of human colon cancer cells and induces E-cadherin-mediated cell aggregation by processing a cell membrane protein(s). The aggregated cells showed dramatically enhanced metastatic potential in the nude mouse model (18). More recently, we identified cholesterol sulfate (CS) as a major cell surface substance to which active MMP-7 binds (19) and demonstrated that binding of MMP-7 to CS is essential for its membrane-associated proteolytic action and induction of the cell aggregation.
Although mode of interaction between MMP-7 and liposome consisting of anionic or cationic lipids has been suggested (20), detailed amino acid residues of the protease contributing to its interaction with CS have not been identified. In this study, we constructed various mutants of MMP-7 and identified residues of the protease essential for binding to CS. Clarification of the interaction between MMP-7 and CS provides the potential to develop MMP-7-targeted novel anti-cancer drugs that specifically block the membrane-associated proteolytic action of this MMP.
Construction of Expression Vector for MMP Mutants-Gene constructions carried out in this study are described in the supplemental material.
Expression and Purification of MMP Mutants-The expression vectors of various MMP mutants were transfected separately into the Escherichia coli strain DH5␣. The transformants were cultured in 2ϫ YT medium (0.08% (w/v) tryptone, 0.5% (w/v) yeast extract, and 0.25% (w/v) NaCl) at 37°C, and recombinant proteins were induced by the addition of 1.0 mM isopropyl-␤-D-thiogalactopyranoside. After a 5-h induction, E. coli cells were broken in 50 mM Tris-HCl (pH 8.0) containing 50 mM NaCl and 5 mM EDTA with a sonicator, and the resultant inclusion bodies were collected by centrifugation. The inclusion bodies were solubilized in 50 mM Tris-HCl (pH 8.0) containing 6 M guanidine HCl and 100 mM dithiothreitol with gentle stirring for 2 h at 25°C. The solubilized sample was first clarified by centrifugation and then applied to a Cosmosyl 5C4 column (4.6 ϫ 100 mm) and eluted with a linear gradient of 0 -80% acetonitrile containing 0.1% trifluoroacetic acid for 70 min at a flow rate of 0.5 ml/min. The column effluent was monitored at 280 nm. The recombinant proteins eluted at about 56 -62% acetonitrile were collected. One mg each of the collected proteins was freeze-dried and dissolved again with 200 l of 50 mM Tris-HCl (pH 8.0) containing 6 M guanidine HCl and 100 mM dithiothreitol. The dissolved samples were then refolded by the rapid dilution method using a refolding buffer containing 1.0 M arginine, as described previously (21). The refolded proteins were dialyzed extensively against 50 mM sodium HEPES (pH 7.5) containing 150 mM NaCl and 10 mM CaCl 2 , and concentrated using a Centriprep YM-10 ultrafiltration device (Millipore Corp., Bedford, MA). The concentrations of the purified proteins were determined by Bradford's dye method with a Bio-Rad protein assay kit, using bovine serum albumin as a standard.
Assay of Effect of CS on Peptidolytic Activities of MMP-7 and Its Variants-Pro-forms of the MMP mutants (1 M) were activated by incubation with 1 mM p-aminophenyl mercuric acetate at 37°C for 2 h, as described previously (22). As described in the supplemental material, we also constructed MMP-7 mutants that have a spacer sequence between the first methionine residue and N terminus of the catalytic domain instead of the propeptide sequence. This spacer sequence is designed to contain the C-terminal 9 residues of the propeptide of MMP-7 and a peptide bond susceptible to autocatalytic cleavage (Fig.  1B). The N-terminal spacer region of each mutant was removed autocatalytically during the preparation described above. Active forms of the MMP mutants were first measured for their activities toward a synthetic substrate 3163v, as described previously (22). The peptidolytic activities of the representative mutants are listed in supplemental Table S2. MMP-7 (2.0 nM) and the mutants, of which concentrations were adjusted to give the activity equivalent to that of 2.0 nM MMP-7, were each incubated with various concentrations of CS or an equimolar mixture of CS and CL (CS/CL) in 190 l of 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 10 mM CaCl 2 , and 0.01% bovine serum albumin at 25°C for 15 min. Then 10 l of 1 mM 3163v was added to the mixture, and the incubation was further continued at 37°C for 30 min. The reaction was terminated by adding 20 l of 0.5 M EDTA (pH 8.0). The amounts of the synthetic substrate hydrolyzed by proteases were measured fluorometrically with excitation at 326 nm and emission at 400 nm. The amount of the substrate hydrolyzed without enzyme was subtracted from the total amount of the hydrolyzed substrate.
Separation of Free and CS-bound Forms of MMP-7 or Its Mutant-MMP-7 and its mutant were each incubated with various concentrations of CS in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 10 mM CaCl 2 , and 0.05% polyethylene glycol 4000 at 25°C for 15 min. The sample was then centrifuged at 21,000 ϫ g for 15 min, and the supernatant was collected. The resultant precipitate containing the CS-bound form of proteases was resuspended in the assay buffer. The peptidolytic activities in the supernatant and the resuspended precipitate were measured as described above.
Cell Lines and Culture Conditions-Human colon cancer cell line Colo201 was obtained from the Japanese Cancer Resources Bank. This cell line was maintained in Dulbecco's modified Eagle's/Ham's F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum.
Assay of MMP-7 Variant Binding to Cell Surface-Assay of the binding of MMP-7 variants to the surface of Colo201 cells was carried out as described previously (19). The MMP-7 vari-ants bound to the cell surface were detected by Western blotting analysis using the anti-MMP-7 antibody 11B4G (19).
Cell Aggregation Assay-The cell aggregation assay was carried out as described previously (18). The degree of cell aggregation was quantified by the following equation: cell aggregation (%) ϭ (1 Ϫ Nt/Nc) ϫ 100, where Nt (test) and Nc (control) represent the number of single cells in the presence or absence of a test sample, respectively.
Measurement of Kinetic Parameters-MMP-7 and its mutants or these enzymes plus CS, concentrations of which are indicated in the legend of Fig. 8A, were incubated, separately, with various concentrations of DNP-RPLALWRS in 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 10 mM CaCl 2 , and 0.05% polyethylene glycol 4000 at 37°C for 30 min. The total volume was 100 l. The reaction was terminated by adding 25 l of 0.5 M EDTA (pH 8.0). Each sample was then diluted with the reaction buffer supplemented with 0.1 M EDTA, so that total concentration of the DNP-bearing peptides was adjusted to be 20 M, based on the concentration of original peptide substrate. The amounts of the substrate hydrolyzed by proteases were measured fluorometrically with excitation at 280 nm and emission at 360 nm. The amount of the substrate hydrolyzed without enzyme was subtracted from the total amount of the hydrolyzed substrate. The steady-state kinetic parameters for peptide hydrolysis were obtained by double-reciprocal plot analysis.

Effect of Deletion of C-terminal Residues of MMP-7 on Its
Affinity for CS-It has been reported that MMP-7 has an affinity for CS, whereas MMP-2 and MMP-3 have no affinity with this acidic lipid (19). To examine whether the interaction between MMP-7 and CS affects the activity of MMP-7, we measured the peptidolytic activity of MMP-7 in the presence of various concentrations of CS. As shown in Fig. 1C, the activity of MMP-7 was reduced with increasing concentrations of CS and reached constant in the presence of 6.3 M or higher concentrations of CS. The activity of MMP-7 in the presence of saturating concentrations of CS was about 20% of that in the absence of CS. The IC 50 value, which is defined in this study to be a concentration of CS giving halfmaximal reduction of the MMP-7 activity, was 0.65 M. CS did not affect the peptidolytic activity of the catalytic domain of MMP-2 ( Fig.  1C), probably reflecting the inability of this protease to bind to CS. The mechanism of the CS-mediated reduction of the peptidolytic activity of MMP-7 is discussed below. To evaluate the CS binding affinities of MMP-7 and its variants, we compared the IC 50 values for the CS-mediated reduction of their peptidolytic activities in the following studies.
We previously constructed a FLAG-tagged MMP-7 (22), which has an 8-residue FLAG epitope peptide on the C-terminal side of MMP-7. When the FLAG-tagged MMP-7 and wildtype MMP-7 were compared for their affinities for CS, the FLAG-tagged MMP-7 showed a slightly lower affinity for CS (data not shown). Therefore, the CS-binding site of MMP-7 may locate near the C terminus of the protease. To examine whether the C-terminal residues of MMP-7 contribute to the interaction with CS, we constructed MMP-7 mutants, named MMP-7⌬C3 and MMP-7⌬C8, that had the C-terminal 3 and 8 residues of MMP-7 deleted, respectively (Fig. 1A), and the effects of CS on the activities of these mutants were compared. As shown in Fig. 1C, the peptidolytic activities of MMP-7⌬C3 (IC 50 ϭ 3.1 M) and MMP-7⌬C8 (IC 50 ϭ 4.0 M) were reduced by CS with similar IC 50 values. These IC 50 values are about 5-fold higher than the value for the CS-mediated reduction of MMP-7 activity (0.65 M), suggesting that the C-terminal 3 residues of MMP-7 partially contribute to its interaction with CS. Because the artificially added FLAG tag may obstruct the interaction between MMP-7 and CS, we constructed MMP-7 and its variants without adding the FLAG tag ( Fig. 1, A and B). Since we could not use an anti-FLAG antibody to purify the variants of MMP-7, we purified them before the refolding, using reversed phase high performance liquid chromatography, as described under "Experimental Procedures." Effect of CS on Peptidolytic Activities of Chimeric MMP Mutants Consisting of Various Parts of MMP-7 and Those of  Fig. 1C suggest that some regions of MMP-7 other than its C terminus also contribute to the high affinity interaction between the protease and CS, because the C-terminally deleted mutants of MMP-7 still had affinities for CS. To explore the regions of MMP-7 contributing to the affinity for CS, we constructed chimeric MMP mutants that consist of various N-terminal parts of the catalytic domain of MMP-2 and remaining C-terminal parts of MMP-7 ( Fig. 2A), and effects of CS on their activities were compared. To facilitate understanding, we use active MMP-7 numbering for the amino acid residue numbers of the catalytic domain of MMPs (Fig. 2D) and the chimeric mutants. As shown in Fig. 2B, the activity of a mutant, named M2n-11-M7c, which consists of residues 1-10 of MMP-2 and residues 11-173 of MMP-7 was reduced by CS with a low IC 50 value (0.49 M). Because this IC 50 value is close to the value for CS-mediated reduction of the MMP-7 activity (0.65 M), the N-terminal part corresponding to residues 1-10 of MMP-7 is unlikely to be important for its binding to CS. As compared with M2n-11-M7c, M2n-37-M7c (IC 50 ϭ 3.9 M) and M2n-56-M7c (IC 50 ϭ 7.2 M) showed 8-and 15-fold lower affinity for CS, respectively, suggesting that the region corresponding to residues 11-55 of MMP-7 contributes partially to the interaction with CS. On the other hand, M2n-56-M7c, M2n-83-M7c, and M2n-156-M7c showed almost the same affinity for CS (IC 50 ϭ 7.2 M), therefore suggesting that the region corresponding to residues 56 -156 of MMP-7 is also unlikely to be important for its binding to CS. Similar to the case of MMP-7, the activity of M2n-11-M7c in the presence of saturating concentrations of CS was about 20% of that in the absence of CS, whereas the activities of M2n-37-M7c, M2n-56-M7c, M2n-83-M7c, and M2n-156-M7c reached less than 10% (Fig. 2B).

MMP-2 Catalytic Domain-The data shown in
The region corresponding to residues 11-37 of MMP-7 may correlate with the level of activity of MMP-7 upon its complex formation with CS.
To verify that the observed activities of MMP-7 and its mutants in the presence of saturating concentrations of CS are those of enzyme-CS complexes but are not caused by a partial dissociation of the complexes, MMP-7 and M2n-37-M7c were first incubated with various concentrations of CS, and then free and CS-bound forms of the enzymes were separated by centrifugation. As shown in Fig. 2C, the activity of MMP-7 in the supernatant disappeared as the concentration of CS was CL Enhances CS Binding Affinities of MMP-7 and Its Variants-We previously reported that CS is localized in rafts or caveolae on the cancer cell surface (19). Since these microdomains are rich in CL, CS may form a complex with CL on the cell surface. To examine whether CL affects the affinity between MMP-7 and CS, we tested the effect of an equimolar mixture of CS and CL on the peptidolytic activity of MMP-7. As shown Fig. 3A, the activity of MMP-7 was reduced with increasing concentrations of the CS/CL mixture and reached constant in the presence of 1.6 M or higher concentrations of the mixture. The activity of MMP-7 in the presence of saturating concentrations of the CS/CL mixture was about 10% of that in the absence of the mixture. The IC 50 value for the CS/CL-mediated reduction of MMP-7 activity was 0.12 M, indicating that MMP-7 has about 5-fold higher affinity for CS/CL as compared with its affinity for CS. Because CL alone did not affect the peptidolytic activity of MMP-7 (data not shown), it is likely that CL enhances the affinity between MMP-7 and CS. We also examined the effect of the CS/CL mixture on the peptidolytic activities of the chimeric MMP mutants, of which constructions are shown in Fig. 2A. The 1/IC 50 values for the CS-or the CS/CL-mediated reduction of the activities of these mutants are compared in Fig. 3B. We found that CL enhanced about 5-fold each the CS binding affinities of wild-type MMP-7, M2n-11-M7c, and M2n-37-M7c, whereas it did not enhance the affinities of M2n-56-M7c, M2n-83-M7c, and M2n-156-M7c, suggesting that the region corresponding to residues 37-55 of MMP-7 correlates with the CL-enhanced CS binding affinity of this protease. We also found that the regions corresponding to residues 11-36 and 37-55 of MMP-7 contribute, respectively, to its high affinity interaction with CS/CL (Fig.  3B). As compared with the CS binding affinity of MMP-7, its affinity for CS/CL was reduced more prominently upon the replacement of residue 11-56. We further analyzed the CL-enhanced CS binding affinities of other variants of MMP-7 in the following studies, because the analysis was thought to be a benefit in detecting sensitively the contributions of individual amino acid residues.
Contributions of Amino Acid Residues Located in a Region Corresponding to Residues 11-55 of MMP-7 to Its Interaction with CS-The data shown in Figs. 2 and 3 strongly suggest that the region corresponding to residues 11-55 of MMP-7 contains amino acid residues contributing to the affinity for CS. To explore the residues, we first divided the region of MMP-7 into five sections, and the residues in the each section were replaced with the corresponding residues of MMP-2 as schematically represented in Fig. 4. We found that the replacements of residues 24 -31, 32-36, and 49 -55 of MMP-7 led to 5.8-, 3.1-, and 4.6-fold reductions of the affinity for CS/CL, respectively (Fig.  4), whereas that of residues 13-23 and 37-48 did not affect the affinity significantly. When all of the residues 24 -36 and 49 -55 of MMP-7 were replaced with the corresponding residues of MMP-2, the resultant mutant, named MMP-7(24 -36,49 -55/ M2), showed 420-fold reduced affinity for CS/CL as compared with MMP-7. In the regions corresponding to the residues 24 -36 and 49 -55 of MMP-7, 13 residues are different between MMP-7 and MMP-2 (Fig. 5). To examine which residues contribute the affinity for CS/CL, we next replaced individual residues in these regions of MMP-7 with the corresponding residues of MMP-2, as shown in Fig. 5. We found that the replacements of the pair Ile 29 and Arg 33 and the pair Arg 51 and Trp 55 of MMP-7 each led to a 10-fold reduction of the affinity for CS/CL. Other replacements, including that of Arg 51 or Trp 55 , also reduced the affinity for CS/CL, whereas the replacements of Arg 24 , Pro 27 , His 28 , Leu 34 , and Val 35 of MMP-7 did not affect the affinity. When all of the Ile 29 , Arg 33 , Arg 51 , and Trp 55 of MMP-7 were replaced with the corresponding residues of MMP-2, the resultant mutant, named MMP-7(29,33,51,55/ M2), showed 420-fold reduced affinity for CS/CL as compared with MMP-7 (Fig. 5). Since MMP-7(29,33,51,55/M2) and MMP-7(24 -36,49 -55/M2) had almost the same affinities for Cholesterol Sulfate-binding Site of MMP-7 DECEMBER 19, 2008 • VOLUME 283 • NUMBER 51 CS/CL, it is likely that in the regions of MMP-7 and MMP-2, corresponding to the residues 24 -36 and 49 -55, only the 4 residues at positions 29, 33, 51, and 55 are responsible for the difference in their CS/CL binding affinities. Collectively, except for the C-terminal 3 residues, the internal Ile 29 , Arg 33 , Arg 51 , and Trp 55 of MMP-7 mainly contributed to the affinity for CS.
Effects of Modifications of MMP-7 on Its CS Binding Affinity, Cell Binding Ability, or Fibronectin Cleaving Activity-To verify that the internal 4 residues of MMP-7 contributing to the affinity for CS/CL are also important for its interaction with CS, we measured the peptidolytic activity of MMP-7(29,33,51,55/M2) in the presence of various concentration of CS. As shown in Fig. 6A, the activity of the MMP-7 mutant was reduced by CS with a high IC 50 value (50 M), suggesting that the internal 4 residues also contribute significantly to the affinity for CS. When the C-terminal 3 residues of this mutant were deleted, the resultant mutant, named MMP-7(29,33,51,55/M2)⌬C3, showed essentially no affinity with CS (Fig. 6A).
We previously demonstrated that CS located on the cell surface mainly mediates the binding of MMP-7 to colon cancer cell line Colo201 (19). To examine whether the internal 4 residues and the C-terminal 3 residues of MMP-7 also contribute to the interaction between the protease and CS on the cell surface, we tested the abilities of the MMP-7 mutants to bind to Colo201 cells. As shown in Fig. 6B, wild-type MMP-7 bound effectively to the cells, whereas MMP-7⌬C3 bound to them with significantly reduced affinity, suggesting that the C-terminal 3 residues contribute to the cell binding ability of MMP-7. We could not detect the binding of MMP-7(29,33,51,55/M2) or MMP-7(29,33,51,55/M2)⌬C3 to the cells by Western blotting analysis, although these mutants and wildtype MMP-7 were similarly immunoreactive (Fig. 6B). These data are consistent with the view that the internal 4 residues are very important for the cell binding affinity of MMP-7, and the replacement of these residues reduces the affinity into the undetectable level.
We found that neither the replacement of the internal 4 residues nor the deletion of the C-terminal 3 residues of MMP-7 affected significantly the peptidolytic activity of this protease (Table S2). However, these modifications of MMP-7 may alter the activity toward macromolecular substrates. To test this possibility, cleavages of fibronectin catalyzed by MMP-7 and its variants were compared. As shown in Fig. 6C, MMP-7 and MMP-7(29,33,51,55/M2)⌬C3 similarly cleaved fibronectin and produced almost the same fragments, suggesting that the modifications of MMP-7 does not significantly alter the activity toward macromolecular substrates.
Cancer Cell Aggregation-inducing Abilities of MMP-7 Variants Having Low CS Binding Affinity-It has been reported that binding of MMP-7 to CS on cell surface is essential for its induction of cancer cell aggregation (19). To examine whether the loss of the CS binding affinity of MMP-7 upon the deletion or replacement of its residues also leads to loss of the ability of  the protease to induce the cell aggregation, MMP-7 and its variants having low affinities for CS were incubated with Colo201 cells, and their cell aggregation-inducing abilities were com-pared. As shown in Fig. 7, wild-type MMP-7 rapidly induced the cell aggregation, and 90% of Colo201 cells were aggregated within 30 min. The rates of the cell aggregation induced by MMP-7⌬C3 and MMP-7(29,33,51,55/M2) were relatively slow, and 80 and 40% of the cells were aggregated after the 60-min incubation, respectively (Fig. 7B). In contrast, MMP-7(29,33,51,55/ M2)⌬C3 did not induce the cell aggregation even after the 60-min incubation. Collectively, the variants of MMP-7 having low CS binding affinity also had low potency to induce the cell aggregation.
Enzyme Kinetics-To investigate the mechanism of the CS-mediated reduction of MMP-7 activity, the kinetic parameters of MMP-7, MMP-7⌬C3, MMP-7(29,33,51,55/ M2), and their CS-bound forms toward a synthetic peptide substrate DNP-RPLALWRS were measured. As shown in Fig. 8A, V max value was practically equal for MMP-7 and its CS-bound form, whereas the K m value of the enzyme was about onefifth of that of the CS-bound one, suggesting that the mode of CS-mediated reduction of the MMP-7 activity is a competitive inhibition. Similar to the case of MMP-7, V max values of MMP-7⌬C3 and MMP-7(29,33,51,55/M2) were not affected by CS, whereas K m values of the mutants were enhanced about 5-fold upon the binding of CS.

DISCUSSION
We explored the amino acid residues of MMP-7 contributing to its high affinity interaction with CS and found that residues Ile 29 , Arg 33 , Arg 51 , and Trp 55 in the internal sequence and Arg 171 , Lys 172 and Lys 173 in the C terminus of MMP-7 were essential for the interaction. In the three-dimensional structure of MMP-7 (23), the internal 4 residues are located on the molecular surface in the opposite side of the catalytic cleft (Fig. 9A). On the other hand, structure of the C-terminal regions of MMP-7, including the Arg 171 , Lys 172 , and Lys 173 , had not been determined in the x-ray analysis, probably because of high flexibility of this region. Considering that the flexible C-termi- nal strand of MMP-7 is long enough to bring the C-terminal 3 residues close to the internal 4 residues (Fig. 9A), all of the 7 residues essential for binding to CS may form a CS-binding patch on the molecular surface of MMP-7 upon its binding to CS. It should be noted that the structure in Fig. 9A is the active site-inhibited form of MMP-7. As described below, the CS binding affinity of MMP-7 is reduced significantly upon its binding to inhibitors. Therefore, the inhibitor-bound structure of MMP-7 may be different from its conformational state that has high affinity for CS. Prediction of the effect of inhibitor on the CS-binding residues of MMP-7 has been difficult thus far, because no three-dimensional structure of the inhibitor-free form of MMP-7 has been determined. Comparison of the three-dimensional structures of inhibitor-free and inhibitorbound forms of the catalytic domain of MMP-1 (Protein Data Bank codes 1AYK and 4AYK, respectively) suggests, however, that the residues of the protease corresponding to Ile 29 , Arg 33 , Arg 51 , and Trp 55 of MMP-7 do not move significantly upon inhibitor binding. Subtle changes in the location or orientation of the residues of MMP-7 induced by inhibitors may lead to dramatic reduction of the CS binding affinity. Since 5 of the 7 residues of MMP-7 essential for binding to CS are basic ones, electrostatic interaction between the positive charge of these residues and negative charge of a sulfate group of CS is probably a main force stabilizing the high affinity interaction between the protease and the lipid. Considering that 5 basic residues of MMP-7 contribute to the interaction, several CS molecules probably interact with one molecule of the protease. Like other lipids, CS molecules probably form clusters in aqueous solution. We speculate that MMP-7 binds to the clustered CS molecules in the solution. CL may enhance the apparent affinity between MMP-7 and CS by facilitating the cluster formation of CS molecules. However, further studies are needed to clarify the mechanism. The remaining 2 essential residues of MMP-7 were hydrophobic ones that may interact with a hydrophobic portion of CS. A recent report suggested that two basic residues and one hydrophobic residue of factor C, a horseshoe crab serine protease, contribute to its recognition of acidic lipid lipopolysaccharide on the Gram-negative bacteria surface (24). Use of both basic residues and hydrophobic ones may be a common strategy for proteins to recognize acidic lipids. Since the residues of MMP-7 essential for binding to CS are located on the molecular surface in the opposite side of the active site, it is assumed that the active site of MMP-7 is directed outside when the protease binds to CS on the cell surface. We speculate that the direction of the cell-bound MMP-7 makes it feasible for the protease to cleave its substrates on cell surface. Interestingly, the 7 residues essential for binding to CS are not necessarily conserved among MMP-7s from different mammalian species (Fig. 9B). For instance, rat MMP-7 has Gln 33 instead of Arg 33 and lacks all of the C-terminal 3 basic residues, thereby lacking 4 of the 5 basic residues essential for binding to CS. Considering that deletion of the C-terminal 3 basic residues of human MMP-7 significantly reduced its affinity for cells (Fig. 6B), rat MMP-7 may not bind effectively to CS on the cell surface. It has been reported that rat MMP-7 binds to heparan sulfate proteoglycans on the surface of rat uterine cells (25,26). We previously reported, however, that CS mainly mediates the binding of MMP-7 to colon cancer cell lines, and cell surface proteoglycans are unlikely to be the sites for the MMP-7-binding (19). The discrepancy between these studies may be explained by the difference in the CS binding affinity between the human and rat proteases. However, whether or not the rat MMP-7 has lower affinity for CS as compared with humans will need to be determined, because other basic residues of the rat protease may contribute to its interaction with CS.
We previously found that hydroxamate-based MMP inhibitors abrogate the binding of MMP-7 to CS on the cell surface (19) and speculate upon two possibilities to explain how the small inhibitor blocks the interaction between MMP-7 and CS. One possibility is that the CS-binding site and the inhibitorbinding site of MMP-7 are overlapping or are in close proximity; thus, the binding of the inhibitor competitively blocks the interaction between MMP-7 and CS. The other possibility is that the binding of the inhibitor induces the conformational change of MMP-7, and the CS-binding site of this proteinase is allosterically altered. The data in the present study support the latter possibility, because the CSbinding site was in the opposite side of the catalytic cleft of MMP-7. An allosteric linkage between the substratebinding site and ligand-binding site is well known in some serine proteases. For instance, covalent incorporation of peptide-mimetic small inhibitors into the active site of blood coagulation factor VIIa induces a conformational change in its cofactor-binding site and enhances the affinity between the protease and its cofactor tissue factor (27). Tissue factor-binding, on the contrary, accelerates the activity of factor VIIa.
Although the kinetic data ( Fig.  8A) suggest that the mode of CS-mediated reduction of the MMP-7 activity is a competitive inhibition, it is unlikely that CS competes directly with substrate for the catalytic site of MMP-7, because MMP-7-CS complex was found to have the peptidolytic activity (Fig.  2C). Considering that active site-directed inhibitors reduce the affinity between MMP-7 and CS and, inversely, CS reduces the affinity between the substrate and the enzyme, we present in Fig. 8B a model for CS-mediated reduction of MMP-7 activity. In this model, the MMP-7-CS complex and free MMP-7 catalyze the reaction with the same catalytic rate constant (k cat ). If K m ϭ 0.26 mM and KЈ m ϭ 1.2 mM, which are the K m values of MMP-7 and MMP-7-CS complex, respectively, obtained from the result in Fig. 8A, are applied to Equation 5 (Eq. 5) in Fig. 8B, the ratio of K i /KЈ i is calculated to be 0.22, indicating that the affinity between MMP-7 and CS is reduced about 5-fold upon the occupation of the catalytic site of MMP-7 by substrate. Equation 6 (Eq. 6) in Fig. 8B indicates that this model is categorized to be the competitive inhibition. Therefore, the model is in good agreement with the nature of CS-mediated reduction of MMP-7 activity and that of interaction among MMP-7, CS, and the catalytic site-occupying molecules, such as substrates and substrate-mimetic inhibitors.
The association of MMPs with cancer cell invasion and metastasis has suggested that these proteases represent attractive targets for the development of novel anti-tumor therapies. However, to date, no MMP inhibitor has been developed successfully as anti-tumor drugs mainly because of deleterious side effects. Considering that MMPs play essential roles under both physiological and pathological conditions, inhibition of activities of MMPs other than the target MMPs probably causes the side effects. The broad specificity of the MMP inhibitors so far designed must be a stiff obstacle for developing safe and effective drugs. We have recently identified a ␤-amyloid precursor protein-derived decapeptide having the ISYGNDALMP FIGURE 9. Location of amino acid residues of MMP-7 essential for binding to CS in three-dimensional structure or primary structure of the protease. A, the crystal structure of MMP-7 cited from Protein Data Bank code 1MMQ is shown in magenta. A view of the catalytic cleft of MMP-7 bearing the catalytic zinc ion and a hydroxamate-based inhibitor (inhibitor) is shown on the left, and that of the opposite side of the catalytic cleft is on the right. The residues Ile 29 , Arg 33 , Arg 51 , and Trp 55 of the protease are colored yellow. The C-terminal strand (broken line), of which the structure is not determined, may bring the part of the C-terminal 3 residues (yellow broken line) close to the 4 residues Ile 29 , Arg 33 , Arg 51 , and Trp 55 to form a CS-binding patch (white circle) on the surface upon the binding to CS. B, amino acid sequences around the residues of MMP-7 essential for binding to CS from several species are shown. The stars represent the residues of human MMP-7 essential for binding to CS. Red and blue letters represent acidic and basic residues, respectively. The numbers at the bottom represent the amino acid residue numbers in active MMP-7 numbering. sequence as an MMP-2-selective inhibitor, which interacts with the active site of MMP-2 (28). It has also been reported that the reactive site-modified tissue inhibitor of metalloproteinase-2 specifically blocks the membrane type-1 MMP-catalyzed activation of pro-MMP-2 (29). However, no MMP-7-selective inhibitor has been identified thus far. Moreover, MMP-7 also has physiological roles besides the pathological ones; the data from the MMP-7-defficient mice suggest that this protease is responsible for the activation of prodefensins and thereby participates in innate host defense. Therefore, resistance to infection may be affected by inhibition of MMP-7 activity. On the other hand, MMP-7 induces aggregation of colon cancer cells by cleaving cell surface protein(s) and enhances their metastatic potential (18). This protease also cleaves Notch on the cell surface and promotes the dedifferentiation of pancreatic acinar cells by activating the Notch signaling pathway (17); the dedifferentiation of the pancreatic cells is associated with an increased risk for tumorigenesis. Therefore, it seems likely that MMP-7-catalyzed processing of cell surface proteins associates preferentially with pathological stages. We showed that the variants of MMP-7 having low CS binding affinity also had low potency to induce the cell aggregation (Fig. 7), suggesting that binding of MMP-7 to the cell membrane facilitates MMP-7catalyzed processing of cell surface proteins. These data also suggest that the cell membrane-associated proteolytic action of MMP-7 and its proteolytic activities toward ECM substrates can be uncoupled by blocking the CS-binding site of MMP-7. For instance, anti-MMP-7 antibodies that recognize the CSbinding site probably block the binding of MMP-7 to the cell surface, MMP-7-catalyzed processing of cell surface proteins, and MMP-7 stimulation of cancer metastasis without preventing its production of defensins. Since modifications of the residues forming the CS-binding site of MMP-7 did not affect its fibronectin-cleaving activity, this protease is unlikely to use the CS-binding site as the substrate-binding exosite to cleave this ECM protein. Therefore, blocking of the CS-binding site of MMP-7 may not affect the ECM-degrading activity of the protease. Taken together, our finding provides the potential to develop MMP-7-targeted novel anti-cancer drugs that block specifically the membrane-associated proteolytic action of this MMP, thereby having restricted side effects.