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Originally published In Press as doi:10.1074/jbc.M301509200 on July 14, 2003

J. Biol. Chem., Vol. 278, Issue 38, 36350-36357, September 19, 2003
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Clusterin, an Abundant Serum Factor, Is a Possible Negative Regulator of MT6-MMP/MMP-25 Produced by Neutrophils*

Akira Matsuda {ddagger} §, Yoshifumi Itoh {ddagger} , Naohiko Koshikawa {ddagger}, Toshifumi Akizawa §, Ikuo Yana {ddagger} and Motoharu Seiki {ddagger} ||

From the {ddagger}Division of Cancer Cell Research, Institute of Medical Science, the University of Tokyo, Minato-ku, Tokyo 108-8639 and the §Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan

Received for publication, February 12, 2003 , and in revised form, July 10, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
MT6-MMP/MMP-25 is the latest member of the membrane-type matrix metalloproteinase (MT-MMP) subgroup in the MMP family and is expressed in neutrophils and some brain tumors. The proteolytic activity of MT6-MMP has been studied using recombinant catalytic fragments and shown to degrade several components of the extracellular matrix. However, the activity is possibly modulated further by the C-terminal hemopexin-like domain, because some MMPs are known to interact with other proteins through this domain. To explore the possible function of this domain, we purified a recombinant MT6-MMP with the hemopexin-like domain as a soluble form using a Madin-Darby canine kidney cell line as a producer. Mature and soluble MT6-MMP processed at the furin motif was purified as a 45-kDa protein together with a 46-kDa protein having a single cleavage in the hemopexin-like domain. Interestingly, 73- and 70-kDa proteins were co-purified with the soluble MT6-MMP by forming stable complexes. They were identified as clusterin, a major component of serum, by N-terminal amino acid sequencing. MT1-MMP that also has a hemopexin-like domain did not form a complex with clusterin. MT6-MMP forming a complex with clusterin was detected in human neutrophils as well. The enzyme activity of the soluble MT6-MMP was inactive in the clusterin complex. Purified clusterin was inhibitory against the activity of soluble MT6-MMP. On the other hand, it had no effect on the activities of MMP-2 and soluble MT1-MMP. Because clusterin is an abundant protein in the body fluid in tissues, it may act as a negative regulator of MT6-MMP in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells are continuously communicating with the extracellular environment through the cell surface where molecules mediating signals exist. Proteolysis is an important part of the regulation of these transmembrane signals by activation, inactivation, or functional conversion of the molecules (1–3). However, our knowledge about the proteases involved in such processes remains limited compared with that of the intracellular signaling events and responsive molecules. Recently, a number of cell-associated proteases have been identified by cDNA cloning techniques and from the whole genome sequencing projects, and this has gradually opened the way to addressing the proteolytic events in the pericellular space.

One such example is MT6-MMP, the latest member of the MT-MMP1 subgroup in the MMP family (matrixins) (4, 5). Among the six members, MT6-MMP tethers to the cells through a covalent link to the glycosylphosphatidylinositol (GPI) in the plasma membrane (6, 7) like MT4-MMP (8), whereas other MT-MMPs are integrated into the plasma membrane through a C-terminal transmembrane sequence (MT1, MT2, MT3, and MT5-MMP) (3, 9). MT6-MMP is expressed almost specifically in neutrophils (polymorphonuclear leukocytes, PMN) (4, 7), although some brain tumors also express it (5). Neutrophils express other MMPs such as MMP-8 (neutrophil collagenase) and MMP-9 (gelatinase B) as well (10), but these are also expressed in other tissues and cell types (2). Given this specificity in its expression, MT6-MMP seems to play a pivotal role in the neutrophil function. It is also interesting that stimulation of neutrophils with phorbol myristate acetate or interleukin-8 caused MT6-MMP to be released as a soluble enzyme from the cell surface or secretory vesicles (7). Because MMPs are responsible for the degradation of most of the components of the extracellular matrix, the MMPs produced by neutrophils are presumably important for invasion and migration of the cells to inflammatory sites and/or destruction of the host tissue. On the other hand, the substrates of MMPs are not restricted to the matrix components (11). Recent studies in the field have revealed their targets also include non-matrix type molecules, such as cell adhesion molecules, cytokines, growth factors, and receptors (2).

The proteolytic activity of MT6-MMP has been studied using recombinant catalytic fragments and was found to degrade type IV collagen, gelatin, fibrin, fibronectin, chondroitin sulfide proteoglycan, and dermatin sulfide proteoglycan but not types I–III collagens (7, 12). Although the substrate specificity of MMP is basically attributable to the structure of the catalytic cleft, the hemopexin-like domain is also known to modulate the activity by binding the substrates, interacting with other proteins that may function as an adaptor for possible substrates, or affecting the enzyme structure (13–18). Thus, it is important to examine MT6-MMP with a hemopexin-like domain.

In this study, we established a Madin-Darby canine kidney (MDCK) cell line that stably expresses a soluble form of MT6-MMP with a hemopexin-like domain to study the properties of the intact MT6-MMP focusing on the function of the C-terminal domain. Mature MT6-MMP processed at the furin motif was purified as a 45-kDa protein and a 46-kDa protein having a single cleavage in the hemopexin-like domain. During the purification, 73- and 70-kDa proteins were found to associate with MT6-MMP by forming stable complexes. These proteins were identified as clusterin, a major component in serum, by N-terminal amino acid sequencing. This study examined the specificity and roles of the complex formation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, FLAG peptide, monoclonal mouse anti-FLAG M2 antibody, and anti-FLAG M2-agarose were purchased from Sigma. Monoclonal mouse anti-human clusterin antibody was from Research Diagnostics, Inc. (Flanders, NJ). G418 (geneticin) and hygromycin B were from Invitrogen. Protease inhibitor mixture and FuGENETM 6 were from Roche Applied Science. A quenched fluorescent peptide substrate, Mca-PLGL-Dpa-AR-NH2, was obtained from Peptide Institute, Inc. (Osaka, Japan). Soluble form of MT1-MMP was kindly provided by Dr. Y. Okada at Keio University (Tokyo, Japan). MMP-2 was purified as described previously (16). TIMP-1 and -2 were a kind gift from Dr. K. Iwata (Dai-ichi Pure Chemical, Takaoka, Japan).

Cell Culture and Transfection—Madin-Darby canine kidney (MDCK) cells and human breast carcinoma MCF-7 cells were purchased from the American Type Culture Collection (Manassas, VA). MDCK cells were maintained in DMEM (Sigma) supplemented with 10% fetal bovine serum and 0.1 mg/ml of kanamycin (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2. MCF-7 cells were maintained in RPMI 1640 medium (Sigma) supplemented with 10% fetal bovine serum and 0.1 mg/ml kanamycin. For transfection, cells were seeded in 6-well plates at 1 x 105 cells/well, and transfection was performed after 16 h. Expression plasmids for proteins were transfected using FuGENETM 6 (Roche Applied Science) according to the manufacturer's instructions.

Construction of Expression Plasmids—cDNA of MT6-MMP was used as a template to generate DNA fragments for sMT6-F (Met1–Gly514) by PCR employing two primers: a forward primer (5'-AGT GGA TCC CCA CCA TGC GGC TGC GGC TCC G-3') and a reverse FLAG insertion primer (5'-ACT CTC GAG TCA TCA CTT GTC ATC GTC GTC CTT GTA GTC ACC AGA GCT CGG GGC GG-3'). The fragment was sub-cloned into the pcDNA3.1(+) expression vector (Invitrogen).

FLAG epitope (DYKDDDDK)-tagged MT1-MMP (MT1F) and MT6-MMP (MT6F) were constructed as described previously (6) and sub-cloned into the pCEP4 expression vector (Invitrogen). The MT6F plasmid was used as a template to generate DNA fragments for MT6FdCAT and MT6FdPEX. MT6FdCAT and MT6FdPEX were catalytic domain (Tyr108–Gly280)-deleted and hemopexin-like domain (Cys317–Cys518)-deleted mutants of MT6F, respectively. All the mutant constructs were generated by PCR using the overlap extension method as described previously. All PCR products were confirmed by DNA sequencing.

Establishment of Stable Cell Lines of sMT6-F—The expression vector for sMT6-F was transfected into MDCK cells using FuGENETM 6 (Roche Applied Science). At 2 days after transfection, the cells were selected in DMEM supplemented with 10% fetal bovine serum, 0.1 mg/ml kanamycin (Invitrogen), and 800 µg/ml G418. After culture for 10 days, each single clone was selected by limiting dilution. Positive clones were then used for protein purification.

Purification of sMT6-F—The stable sMT6-F transfectant was cultured in a cell factory (Nunc, Rochester, NY) until confluent. The cells were washed three times with PBS and replenished with serum-free DMEM. After incubation for 72 h, the conditioned medium (CM) was collected, clarified by centrifugation, and then concentrated using ammonium sulfate to a final saturation of 80%. The precipitated protein was collected by centrifugation and then dissolved in and dialyzed against Tris-buffered saline (TBS: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl) containing 0.05% Brij 35. This fraction was then applied to an anti-FLAG M2-agarose column, and the column was washed with the same buffer. Specific elution was carried out using FLAG peptide (100 µg/ml).

Western Blot Analysis—Samples were subjected to SDS-PAGE under reducing or non-reducing conditions (16), and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, MA). After blocking with 10% fat-free dry milk in PBS for 1 h, the membrane was probed with primary antibody specific to each antigen. The membrane was further probed with horseradish peroxidase-conjugated anti-mouse IgG (Amersham Biosciences) and detected using ECL Plus (Amersham Biosciences).

Gelatin Zymography—Gelatin zymography was performed as described previously (19). The samples were mixed with SDS sample buffer without a reducing agent and separated on 7.5% acrylamide gels containing gelatin (0.8 mg/ml). The gelatin-containing gel was renatured by washing with 2.5% Triton X-100-containing buffer for 1 h and incubated for 12 h at 37 °C. The gelatin remaining in the gel was stained with Coomassie Brilliant Blue R-250, and gelatinolytic activity was detected as clear bands against a blue background.

N-terminal Amino Acid Sequencing of Purified sMT6-F and Its Binding Proteins—The sample was subjected to SDS-PAGE under reducing conditions and transferred to PVDF membrane (Millipore). After the staining of the membrane with Coomassie Brilliant Blue R-250, the stained bands were excised, and the Beckman Coulter LF3000 amino acid sequencer was used.

Gel Permeation Chromatography—The sMT6-F-clusterin complex was subjected to gel permeation chromatography on HiLoad 16/60 Superdex 200 pg (Amersham Biosciences) equilibrated with TBS buffer containing 0.05% Brij 35 at a flow rate of 0.5 ml/min, and a 1-ml fraction was collected. The calibration was performed using thyroglobulin (669 kDa), ferritin (440 kDa), adolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa) as protein standards.

Dissociation of sMT6-F from sMT6-F-Clusterin Complex—The sMT6-F-clusterin complex was separated by gel permeation chromatography. The complex-containing fractions were pooled and then dialyzed against 50 mM Tris-HCl (pH 7.5) buffer. After 12 h dialysis, the pooled fraction was denatured using 6 M urea. The denatured complex was applied to a Q-anion exchange column (Bio-Rad). The dissociated clusterin was eluted with the same buffer containing 0.1 M NaCl, whereas clusterin-free sMT6-F was eluted with buffer containing 0.2 M NaCl. Subsequently, the separated sMT6-F and clusterin were individually dialyzed against 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 5 mM CaCl2, 50 µM ZnCl2, and 0.01% Brij 35 at 4 °C for 2 and 12 h, respectively. The final preparation was frozen at –80 °C before use.

Assay of Enzyme Activities—Purified enzyme was assayed using a fluorescence-quenched peptide substrate (Mca-PLGL-Dpa-AR-NH2) (20). Purified enzyme (0.7 nM) was incubated with substrate (2 µM)in50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 5 mM CaCl2, and 0.05% Brij 35 at 37 °C for 1 h. The concentration of enzyme was determined by active site titration with TIMP-2.

Reaction mixtures of enzyme and inhibitors were preincubated at 25 °C for 15 min with TIMP-1, TIMP-2, and clusterin. The apparent inhibition constant Ki(app) was calculated by using Equation 1,

(Eq. 1)
where I is the final inhibitor concentration, Vo the rate of substrate hydrolysis without inhibitor, and Vi the rate of hydrolysis with inhibitor.

Immunoprecipitation—Expression plasmids were transfected into MCF-7 cells using FuGENETM 6. Stable transfectants were selected using hygromycin B (300 µg/ml) and lysed with RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1% deoxycholic acid, and 0.1% SDS) in the presence of a protease inhibitor mixture (Roche Applied Science). The cell lysate was clarified by centrifugation at 15,000 rpm for 15 min. The supernatant was incubated with anti-FLAG M2-agarose (Sigma) at 4 °C for 6 h. The agarose was washed three times with RIPA buffer and precipitated. The bound proteins were eluted with FLAG peptide and analyzed by Western blotting using anti-FLAG M2 antibody or anti-human clusterin antibody. Other experimental conditions were described in our previous report (18).

Preparation of Anti-MT6-MMP Polyclonal Antibody—A polyclonal antibody against MT6-MMP was raised in rabbits immunized with a purified sMT6-F. Because the sMT6-F preparation may contain residual clusterin, the antibody was extensively absorbed using the clusterin preparation from the MDCK cells. The antibody was confirmed to be specific to MT6-MMP without showing cross-reactivity against other MT-MMPs (Fig. 10A) and not to react with clusterin using MDCK and MCF-7 cells (data not shown).



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  		FIG. 10.
Detection of a clusterin-MT6-MMP complex in human PMNs. A, immunoreactivity of anti-MT6-MMP polyclonal antibody for MT-MMPs. COS-1 cells were transiently transfected with plasmids for MT-MMPs tagged with a FLAG epitope downstream of the furin motif. The cell lysate was analyzed by Western blotting (WB) with anti-FLAG M2 antibody (upper panel) or anti-MT6-MMP polyclonal antibody (lower panel). B, immunoprecipitation of MT6-MMP expressed in PMNs. PMNs (1 x 107 cells/lane) were lysed in PBS containing 1% Triton X-100 and subjected to immunoprecipitation (IP) with either rabbit IgG or anti-MT6-MMP polyclonal antibody. Precipitates were solubilized and analyzed by Western blotting using anti-clusterin antibody (left panel) or biotinylated anti-MT6-MMP polyclonal antibody (right panel). The biotinylated antibody was visualized using avidin-conjugated horseradish peroxidase.

 

Isolation of PMNs—Human PMNs with >98% purity were isolated from fresh heparinized blood of healthy volunteers by dextran sedimentation followed by separation on a density gradient (21). The viability was confirmed by the trypan blue exclusion assay.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Purification of MT6-MMP—The translated pro-MT6-MMP has a hydrophobic amino acid stretch at the C terminus that acts as a signal for GPI anchoring. To prepare a soluble form of MT6-MMP, the hydrophobic stretch was deleted and substituted with the FLAG tag sequence (sMT6-F) (Fig. 1A). MDCK cells that stably express sMT6-F were prepared, and the conditioned medium containing the secreted sMT6-F was collected. The sMT6-F in the culture medium was detected as a band with an expected molecular size of 47 kDa under reducing conditions as demonstrated by Western blotting using anti-FLAG M2 monoclonal antibody (anti-FLAG mAb). sMT6-F was accumulated in the medium according to the time in culture without significant degradation (Fig. 1B). A weak band of 21 kDa presumably representing a degradation product was also detected depending on the culture conditions.



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  		FIG. 1.
Stable expression of soluble MT6-MMP. A, schematic representation of wild-type (MT6-MMP) and soluble MT6-MMP (sMT6-F). sMT6-F was a C-terminal membrane-spanning domain deletion mutant and tagged with a FLAG epitope for detection and purification. SP, signal peptide; Pro, propeptide; Furin Motif, furin cleavage site; Cat, catalytic domain; PEX, hemopexin-like domain; GPI signal, glycosylphosphatidylinositol anchor signal; FLAG, FLAG epitope (DYKDDDDK). B, a plasmid for sMT6-F and an empty vector (mock) were stably transfected into MDCK cells. Confluent cells were washed three times with PBS and cultured further in serum-free DMEM. After 24, 48, and 72 h, each conditioned medium was collected and analyzed by Western blotting using anti-FLAG M2 antibody.

 

After concentration of the culture medium with ammonium sulfate, sMT6-F was purified using an agarose beads column conjugated with the anti-FLAG mAb. Most of the proteins did not bind to the column as shown in Fig. 2 (lanes 1 and 2). The proteins bound to the column were eluted with FLAG peptide and analyzed by SDS-PAGE under reducing conditions. Five major bands corresponding to 47, 45, 35, 27, and 21 kDa were detected in the elution fraction (lane 3). Among them, the 47- and 21-kDa bands had the FLAG sequence from the reactivity to the antibody, but the others did not (lane 5). SDS-PAGE under non-reducing conditions revealed four major bands of 73-, 70-, 46-, and 45-kDa proteins (lane 4). Both the 46- and 45-kDa proteins were detected by anti-FLAG mAb (lane 6) and showed gelatinolytic activity, whereas the 73- and 70-kDa proteins did not (lane 7, and also refer to Fig. 5 for better resolution). As the 73- and 70-kDa proteins were eluted from the column by FLAG peptide and were not detected in the preparations from the non-transfected MDCK cells (data not shown), they are presumably associating with sMT6-F.



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  		FIG. 2.
Purification of soluble MT6-MMP by affinity chromatography. Purified proteins on an anti-FLAG monoclonal affinity column were subjected to SDS-PAGE (left panel, lanes 3 and 4) and Western blotting (center panel, lanes 5 and 6) using anti-FLAG M2 antibody under reducing or non-reducing conditions. Purified fraction (lane 4) was also analyzed by gelatin zymography (lane 7). Lane 1, conditioned medium; lane 2, flow-through.

 


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  		FIG. 5.
Separation of sMT6-F-clusterin complex by gel permeation chromatography. A, separation of clusterin-bound and -free sMT6-F by gel permeation chromatography. Purified proteins (clusterin-bound and -free sMT6-F) were subjected to chromatography on a gel permeation column of Superdex 200 at a flow rate of 0.5 ml/min. Fractions of 1 ml were collected. Protein standards (MW Markers indicated by arrowheads) were used to calibrate the column: thyroglobulin (669 kDa), ferritin (440 kDa), adolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa). B, starting material (starting) and purified samples (FN. 47, 50, 53, 57, 60, 64, 70, 75, 78, 80, 83, and 87) were analyzed by SDS-PAGE under non-reducing conditions. Each protein was detected by silver staining. Bands corresponding to clusterin and sMT6-F are indicated by arrows. Asterisks indicate the fractions used for the following assay. C, comparison of proteolytic activity between clusterin-bound sMT6-F (FN 57 and 60) and -free sMT6-F (FN 80 and 83). Each fraction was assayed using fluorescence-quenched peptide substrate. D, the same fractions were analyzed by gelatin zymography.

 

Identification of Canine Clusterin as the Protein Binding to sMT6-F—To analyze the polypeptide components of the bands detected under non-reducing conditions (Fig. 3A), they were extracted from the gel and examined further under reducing conditions (Fig. 3B). The 73-kDa protein in Fig. 3A was composed of two polypeptide chains of 45 and 35 kDa possibly linked with disulfide bonds (Fig. 3B, 2nd lane), and the 70-kDa protein also contained two polypeptides of 45 and 27 kDa (Fig. 3B, 3rd lane). The 46-kDa protein in Fig. 3A was separated into 27- and 21-kDa polypeptides (Fig. 3B, 4th lane). The smaller polypeptide has the FLAG tag from the reactivity to the antibody (Fig. 2, lane 5) and is thought to contain the C-terminal fragment of sMT6-F. The 27-kDa polypeptide presumably corresponds to the fragment containing the catalytic domain of sMT6-F, because the 46-kDa protein, from which this fragment is derived, retained the gelatinolytic activity (Fig. 2, lane 7, and also refer to Fig. 5 for better resolution). The 45-kDa protein in Fig. 3A was composed of a single polypeptide of sMT6-F (Fig. 3B, 5th lane). A shift of molecular mass from 45 kDa under non-reducing conditions to 47 kDa under reducing conditions was observed, and this reflects the disruption of the intramolecular disulfide bond in the hemopexin-like domain.



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  		FIG. 3.
Identification of purified proteins that shifted their molecular weight on SDS-PAGE under non-reducing conditions. A, purified sMT6-F and associated proteins were separated by SDS-PAGE under non-reducing conditions and stained with Coomassie Brilliant Blue R-250. B, the 73-, 70-, 46-, and 45-kDa proteins (1st 4 bands) were excised from the gel and subjected to SDS-PAGE under reducing conditions. Each protein was detected by silver staining. Total, purified sMT6-F and associated proteins.

 

Then each polypeptide was extracted from the gel and subjected to N-terminal amino acid sequencing (Fig. 4). The N terminus of the 47-kDa polypeptide (108 amino acids from the translation start site) exactly matched the downstream sequence of the site of processing by furin-like enzymes that generate the mature form of MT6-MMP (4, 5), thus indicating that this is the intact sMT6-F. The 27-kDa polypeptide had the same sequence indicating that this is the N-terminal fragment of sMT6-F containing the catalytic domain. On the other hand, the N-terminal sequence of the 21-kDa polypeptide coincided with the sequence in the hemopexin-like domain that starts from the 348-amino acid position. Thus, the 46-kDa sMT6-F has a cleavage within the hemopexin-like domain, and the two polypeptide chains are connected to each other by a disulfide bond within the hemopexin-like domain ("nicked" sMT6-F).



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  		FIG. 4.
N-terminal sequence analysis of sMT6-F and its associated proteins. Purified sMT6-F and associated proteins (47, 45, 35, 27, and 21 kDa) were separated by SDS-PAGE under reducing conditions and electrotransferred onto a PVDF membrane. After the proteins were stained by Coomassie Brilliant Blue R-250, each protein band was excised and subjected to N-terminal amino acid sequencing analysis. Other experimental conditions were described under "Experimental Procedures."

 

The N-terminal amino acid sequence of the 45-kDa polypeptide derived from the 73- and 70-kDa proteins exactly coincided with the {beta} subunit of the canine clusterin, whereas the sequences of the 35- and 27-kDa polypeptides corresponded to the {alpha} subunit of clusterin. Clusterin is known to be composed of {alpha} and {beta} subunits connected by five disulfide bonds (22, 23). The difference in the molecular size of the two forms of the {alpha} subunit sharing the same N terminus may reflect the difference in modification or heterogeneity downstream.

sMT6-F Forming a Complex with Clusterin Has No Proteolytic Activity—To analyze the nature of the clusterin-sMT6-F complex, the eluate from the affinity column was applied to a Superdex 200 gel permeation column. The applied sample was eluted forming three major peaks corresponding to molecular sizes larger than the 669-kDa marker proteins, 540 and 62 kDa, respectively (Fig. 5A). Collected fractions were analyzed further by SDS-PAGE under non-reducing conditions (Fig. 5B). The fraction with the highest molecular weight peak (FN. 53) contained an unknown protein of several hundred kilodaltons and a small amount of sMT6-F. The second peak (FN. 57–70) with a broad range of 560–215 kDa contained two forms of clusterin and two forms of sMT6-F. However, the intensity of the intact sMT6-F band (45 kDa) was more abundant than the nicked molecule (46 kDa) in this fraction. The third peak contained only sMT6-F alone presumably as a monomer from the estimated molecular size, and the nicked molecule was predominant in this fraction compared with the intact form.

To examine the difference in proteolytic activity of sMT6-F between the clusterin complex and the free form, the activity of the clusterin-containing (FN. 57 and 60) and -free fractions (FN. 80 and 83) was analyzed using the fluorescence-quenched peptide substrate. The fractions free of clusterin showed significant proteolytic activity (Fig. 5C), but the activity of the clusterin-containing fraction was negligible. On the other hand, all fractions showed almost equal gelatinolytic activity in zymography (Fig. 5D), suggesting that the activity of sMT6-F is inhibited as a result of clusterin binding.

Dissociation of the Clusterin Complex—The cluterin-sMT6-F complex was stable in the buffer containing high concentrations of NaCl (0.15 to 1 M) or 1% detergents (Triton X-100, n-octyl-{beta}-D-glucoside, n-dodecyl-{beta}-D-maltoside, CHAPS, and deoxycholic acid) (data not shown). The complex in the pooled fraction (FN. 55–73) was then treated with 6 M urea for dissociation and subjected to anion exchange column chromatography. Elution was carried out with a urea-containing buffer with increasing ionic strength. Clusterin was eluted with the buffer containing 0.1 M NaCl, whereas sMT6-F was eluted with 0.2 M NaCl (Fig. 6A). The 0.2 M NaCl fraction contained intact sMT6-F with minor contamination by the nicked molecule. To refold the denatured enzyme, the concentration of urea was gradually decreased by dialysis at 4 °C. The recovery of the enzyme activity during dialysis was monitored using the peptide substrate. The activity recovered rapidly according to the time of dialysis and reached a plateau after 1 h (Fig. 6C). The integrity of the enzyme was also checked by SDS-PAGE (Fig. 6B). Prolonged dialysis for more than 3 h caused auto-degradation of the enzyme. Thus, we used the 2-h dialyzed fractions as the refolded enzyme for subsequent experiments.



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  		FIG. 6.
Dissociation of sMT6-F and clusterin from sMT6-F-clusterin complex. A, sMT6-F-clusterin complex was denatured in buffer containing 6 M urea and 50 mM Tris-HCl (pH 7.5), and applied to an anion exchange column. The column was washed with 50 mM Tris-HCl (pH 7.5) containing 6 M urea, and eluted with 0.1 M NaCl (lane 2) and 0.2 M NaCl (lane 3). The sMT6-F-clusterin complex and eluted fraction were analyzed by SDS-PAGE under non-reducing conditions. Bands were visualized by silver staining. B and C, denatured sMT6-F was refolded by dialysis against 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 5 mM CaCl2, 50 µM ZnCl2 and 0.01% Brij 35 at 4 °C for 0–3, 6, and 18 h. Each sample was analyzed by SDS-PAGE under non-reducing conditions and detected by silver staining (B). Proteolytic activity was also tested by assay using fluorescence-quenched peptide substrate (C).

 

Clusterin Is a Negative Regulator Specific to MT6-MMP— The refolded sMT6-F showed dose-dependent proteolytic activity against the peptide substrate (data not shown), and the activity was inhibited by TIMP-1 and TIMP-2 in a stoichiometric fashion as reported previously (Fig. 7A) (12). We then used the clusterin preparation isolated from the anion exchange column to examine the effect on the proteolytic activity after refolding (Fig. 7B). Increasing doses of clusterin inhibited the activity of sMT6-F in a dose-dependent manner, although the inhibition was considerably weaker than that by TIMPs. The Ki(app) value was 120 nM. On the other hand, it did not affect the activities of either MMP-2 or the soluble form of MT1-MMP. Thus, the inhibitory effect of clusterin is specific to sMT6-F and not a result of its nonspecific interaction with MMPs.



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  		FIG. 7.
Inhibition of sMT6-F activity by clusterin. A, sMT6-F (0.7 nM) was preincubated with the indicated concentrations of TIMP-1 or TIMP-2 at room temperature for 15 min, and the enzyme activity was measured using a fluorescence-quenched peptide substrate. B, purified sMT6-F (0.7 nM), soluble MT1-MMP (0.6 nM), or p-aminophenylmercuric acetate (APMA)-activated MMP-2 (0.7 nM) was preincubated with different concentrations of purified clusterin at room temperature for 15 min, and the enzyme activities were analyzed using a fluorescence-quenched peptide substrate.

 

Complex Formation of Human Clusterin with GPI-anchored MT6-MMP—To confirm that human clusterin can form a complex with the GPI-anchored form of MT6-MMP, we expressed MT6-MMP having a FLAG tag downstream of the furin site (MT6F) (Fig. 8A) in human breast carcinoma MCF-7 cells that express clusterin constitutively. The FLAG tag MT1-MMP (MT1F) that was not inhibited by the canine clusterin was used as a negative control. Stable transfectants expressing either MT6F or MT1F were obtained (Fig. 8B). The cells were lysed, and then immunoprecipitation was carried out using anti-FLAG mAb. Clusterin in the precipitates was analyzed by Western blotting using anti-human clusterin antibody. Clusterin was specifically co-precipitated with MT6F but not with MT1F (Fig. 8C). Thus, the formation of a complex between MT6-MMP and human clusterin is not a nonspecific interaction.



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  		FIG. 8.
Specific binding of human clusterin to MT6-MMP but not MT1-MMP. A, schematic presentation of MT6-MMP and MT1-MMP constructs used in this experiment. MT6F and MT1F have a FLAG epitope downstream of the furin motif. SP, signal peptide; Pro, propeptide; Furin Motif, furin cleavage site; Cat, catalytic domain; PEX, hemopexin-like domain; GPI signal, glycosylphosphatidylinositol anchor signal; TM, transmembrane domain; FLAG, FLAG epitope (DYKDDDDK). B, detection of MT6-F and MT1F expressed in MCF-7 cells. Plasmids for MT6F and MT1F, and an empty vector (mock) were stably transfected into MCF-7 cells. The hygromycin-resistant cell populations were subjected to Western blotting analysis using anti-FLAG M2 antibody. The asterisks indicate the nonspecific band. The upper arrow indicates MT1F, and the lower arrow indicates MT6F. C, immunoprecipitation (IP) of human clusterin with MT6-MMP and MT1-MMP. The cells expressing either MT6-MMP or MT1-MMP were lysed and subjected to immunoprecipitation experiments using anti-FLAG antibody. The proteins bound to the antibody was eluted with FLAG peptide and analyzed by Western blotting (WB) using antibodies for clusterin (upper panel) and anti-FLAG (middle panel). Conditioned medium was also analyzed for clusterin by Western blotting (lower panel).

 

To examine the importance of the ectodomains of MT6-MMP in forming a complex with human clusterin, deletion mutants lacking either the catalytic domain (MT6FdCAT) or the hemopexin-like domain (MT6FdPEX) were prepared (Fig. 9A) and expressed in the MCF-7 cells (Fig. 9B). As demonstrated in Fig. 9C, MT6FdCAT co-precipitated with clusterin but MT6FdPEX did not, indicating that the hemopexin-like domain is responsible for clusterin binding.



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  		FIG. 9.
Identification of the domain of MT6-MMP binding to human clusterin. A, schematic presentation of MT6-MMP and its mutant used in this study. SP, signal peptide; Pro, propeptide; Furin Motif, furin cleavage site; Cat, catalytic domain; PEX, hemopexin-like domain; GPI signal, glycosylphosphatidylinositol anchor signal; FLAG, FLAG epitope (DYKDDDDK). B, Western blotting of MT6-MMP and its mutants detected by anti-FLAG M2 antibody. An expression plasmid for MT6-MMP and that for mutants and an empty vector (mock) were stably transfected into MCF-7 cells. The hygromycin-resistant cell population was selected and used for analysis by Western blotting using anti-FLAG M2 antibody. The upper arrow indicates MT6F; the middle arrow indicates MT6FdPEX, and the lower arrow indicates MT6FdCAT. C, immunoprecipitation (IP) of human clusterin with MT6-MMP and its mutants. The transfectants were lysed and subjected to immunoprecipitation using anti-FLAG antibody. The proteins eluted from the precipitates with a FLAG peptide were analyzed by Western blotting (WB) using anti-clusterin or anti-FLAG antibodies (upper and middle panels). The conditioned medium was also analyzed by Western blotting using anti-clusterin antibody (lower panel).

 

MT6-MMP Expressed in Neutrophils Forms a Complex with Clusterin—To confirm the complex formation with natural products, we purified human neutrophils and examined the endogenous MT6-MMP. For this study, a rabbit polyclonal antibody against recombinant sMT6-F was prepared, and its specificity was confirmed by Western blotting as in Fig. 10A. No cross-reactivity was detected with other MT-MMPs. The antibody also did not react with clusterin itself in MDCK and MCF-7 cells at all (data not shown).

The neutrophils collected were lysed and subjected to immunoprecipitation using the antibody. As demonstrated in Fig. 10B, a mature form of MT6-MMP (54 kDa) was precipitated as detected by Western blotting. An additional band of 42 kDa was also detected in the precipitate and presumably represents a degradation product. Thus, MT6-MMP is expressed in neutrophils as reported (7). Clusterin was detected in the precipitate indicating that a significant amount of endogenous MT6-MMP also forms a complex with clusterin. Although clusterin is reported to interact with immunoglobulin (24), it did not precipitate with the control IgG under our assay conditions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, clusterin was identified as a binding protein for human MT6-MMP. The interaction seemed to be specific to MT6-MMP because MT1-MMP did not bind clusterin. The MDCK cell line used to produce sMT6-F in this study is known to express endogenous clusterin constitutively (25) and has also been used to produce other recombinant MMPs by us and others (7, 26, 27). We have expressed soluble forms of MT1-MMP and MT4-MMP and MMP-2 in the cells and purified the proteins. Neither MMP-2 nor the soluble form of MT1-MMP associated with the clusterin, but the soluble form of MT4-MMP, which is similar to MT6-MMP in amino acid sequence (45%) and GPI anchoring, also bound clusterin (data not shown). A soluble form of MT5-MMP and MMP13 was also expressed in MDCK cells and purified by other groups, but the association of clusterin with these enzymes has not been reported (26, 27). Thus, clusterin seems to bind only a class of MMPs related to MT6-MMP in a specific manner.

The deletion mutant either lacking the catalytic domain (MT6FdCAT) or the hemopexin-like domain (MT6FdPEX) was expressed in MCF-7 cells as a GPI-anchored form. MT6FdCAT co-precipitated with clusterin but MT6FdPEX did not. Thus, the hemopexin-like domain is responsible for the clusterin binding. In a previous study, the catalytic fragment of MT6-MMP was also expressed in MDCK cells and purified without an association with clusterin (7). This is consistent with our finding that the hemopexin-like domain is responsible for clusterin binding. In addition to the intact sMT6-F, our preparation contained a molecule having a nick in the hemopexin-like domain. The nicked molecule was found less in the clusterin complex and more in the clusterin-free fractions, whereas the reverse was the case for the intact molecule. This phenomenon also indicates that the integrity or specific sequence of the hemopexin-like domain is important for the clusterin binding. Thus, it is not the result of nonspecific binding of clusterin to denatured proteins. However, it is still possible that partial denaturation in the region induces interaction with clusterin. Even in this case, this is a specific feature of MT6-MMP and MT4-MMP not found in other MMPs examined. In addition, clusterin forming a complex with MT6-MMP was detected in human neutrophils indicating that the two are interacting even in natural producer cells.

The separation of the complex by gel permeation column chromatography indicated that the main peak of the clusterin complex (540 kDa) is larger than the expected size of the stoichiometric complex (120 kDa). Thus, the complex is thought to contain multiple clusterin and sMT6-F molecules, presumably due to the nature of clusterin to interact (22). The interaction between clusterin and sMT6-F seems stable and irreversible under physiological conditions, because no dissociation occurred in the buffer containing high concentrations of NaCl or detergents. However, it is still possible for dissociation to occur when a conformational change to clusterin or the hemopexin-like domain is induced by proteolytic cleavage or association with other proteins under certain conditions.

The clusterin-sMT6-F complex did not show proteolytic activity against the synthetic peptide substrate, although the clusterin-free fraction did. However, gelatinolytic activities in the both fractions were detected by zymography, in which sMT6-F was dissociated from clusterin by detergent, suggesting that clusterin inhibits sMT6-F by forming a complex. The complex dissociated in the presence of 6 M urea and the components were separated by anion exchange column chromatography. Refolding of the denatured protein restored the proteolytic activity which was inhibited again on the addition of the purified and refolded clusterin. It is notable that the inhibitory activity of the purified clusterin is considerably weak compared with that in the complex. The refolding of clusterin may be too incomplete to restore full activity. It is not clear how clusterin bound to the hemopexin-like domain suppresses the catalytic activity. The formation of a high molecular weight aggregate may prevent the substrate from accessing the catalytic site or clusterin binding to the hemopexin-like domain may facilitate interaction with the catalytic domain and disturb the catalytic function. The mechanisms remain to be investigated.

Clusterin is a heterodimeric glycoprotein composed of {alpha} and {beta} subunits connected by five disulfide bonds and abundant in every tissue fluid in the body such as plasma, milk, urine, cerebrospinal fluid, and semen (22, 23). The expression of clusterin is reported to be induced in various disease stages and by stress (28). The gene expression is regulated by a transcription factor mediating heat-shock stress, called heat shock factor 1 (HSF1) (23, 29), and thus the product is thought to play a role in protecting tissue from damage by stress. Although clusterin was identified almost 20 years ago (30), its specific function has not yet been clarified (22, 23, 28). However, one of the characteristic features of this protein is the ability to interact with a wide array of components in serum and on the cell surface including complements (31), immunoglobulins (24), lipids (32), {beta}-amyloid peptide (33), and prion peptide (34) etc. It also binds to the Staphylococcus aureus cell surface (35). Because clusterin is abundant in serum and binds immunoglobulins, complements, and bacteria, it is reasonable to speculate that clusterin plays some role at the inflammatory site where neutrophils accumulate. It should be also noted that the level of clusterin in serum is about 10 times the Ki(app) value for MT6-MMP (36). Thus, it is reasonable to suppose that clusterin regulates neutrophil function by inhibiting MT6-MMP and prevents excessive destruction of the host tissue. Indeed, we observed that a substantial amount of MT6-MMP expressed in human neutrophils forms a complex with clusterin. Clusterin-deficient mice have been generated, and it was reported that excessive tissue damage compared with wild-type mice was observed at inflammatory sites caused by an autoimmune reaction (37). Together with the results of this study, the excessive tissue damage in the clusterin null mice might be a reflection of the lack of the tissue-protective function of clusterin caused by inhibiting a certain class of MMPs at the site of inflammation. The expression of clusterin is also induced in brains with tumors (38). Because some brain tumors are reported to express MT6-MMP (5), the clusterin expressed there may also act to prevent tissue damage possibly caused by MT6-MMP. Thus, we propose that clusterin is a possible negative regulator of MT6-MMP produced by neutrophils and brain tumors.


   FOOTNOTES
 
* This work was supported by the Special Coordination Fund for promoting science and technology from the Ministry of Science and Technology of Japan and by a grant-in-aid for cancer research from the Ministry of Education, Science and Culture of Japan. 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

Present address: Dept. of Matrix Biology, Kennedy Institute of Rheumatology, Imperial College, Faculty of Medicine, Hammersmith, London W6 8LH, UK. Back

|| To whom correspondence should be addressed: Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan. Tel.: 81-3-5449-5255; Fax: 81-3-5449-5414; E-mail: mseiki{at}ims.u-tokyo.ac.jp.

1 The abbreviations used are: MT-MMP, membrane-type matrix metalloproteinase; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; Mca, (7-methoxycoumarin-4-yl)acetyl; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PVDF, polyvinylidene difluoride; FN, fraction; PMN, polymorphonuclear leukocytes; GPI, glycosylphosphatidylinositol; MDCK, Madin-Darby canine kidney; mAb, monoclonal antibody. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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