©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The C-terminal Region of Membrane Type Matrix Metalloproteinase Is a Functional Transmembrane Domain Required for Pro-gelatinase A Activation (*)

(Received for publication, July 21, 1994; and in revised form, October 20, 1994)

Jian Cao (1) Hiroshi Sato (1) Takahisa Takino (1) (2) Motoharu Seiki (1)(§)

From the  (1)Department of Molecular Virology and Oncology, Cancer Research Institute and the (2)Department of Oral Surgery, School of Medicine, Kanazawa University, Kanazawa, Ishikawa 920, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We identified a new matrix metalloproteinase (membrane type matrix metalloproteinase (MT-MMP)) that has a potential transmembrane (TM) domain at the C terminus and reported its expression on the surface of invasive tumor cells. The expression of MT-MMP induced specific activation of 72-kDa pro-gelatinase A (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M.(1994) Nature 370, 61-65). Thus, MT-MMP on the cell surface is thought to play an important role in various physiological and pathological processes accompanying tissue remodeling. In this study, we demonstrated that the potential TM domain deduced from the amino acid sequence functions as a membrane linker when it is fused to a secretory protein, tissue inhibitor of matrix metalloproteinases-1. The pro-gelatinase A activation function of MT-MMP was abolished by truncation of the TM domain and recovered by fusing the MT-MMP mutant with the TM domain of interleukin 2 receptor alpha-chain. The truncated MT-MMP was released from the cells into the medium and detected as processed or modified forms. In spite of the deletion of the TM domain some portions of the mutant MT-MMP were still retained on the surface of cells. Thus, MT-MMP has an additional device to keep it on the cell surface. The TM domain however, plays an essential role in the pro-gelatinase A activation function of MT-MMP, probably regulating its fine orientation or the localization that is necessary to interact with substrate.


INTRODUCTION

For tumor cell invasion, degradation of the extracellular matrix in the vicinity of the cell surface is thought to be essential(1, 2, 3, 4, 5, 6, 7) . Many investigators have reported that the plasma membrane fractions of invasive tumor cells contain MMP (^1)activators(6, 8, 9, 10, 11) . Urokinase type plasminogen activator (uPA), associating on the tumor cell surface through a specific receptor(12) , is an initiator of the MMP activation cascade, activating plasminogen to plasmin, which then activates interstitial collagenase and stromelysin 1(6, 13, 14, 15) . The 72-kDa gelatinase A is another MMP that is reportedly activated on the invasive tumor cell surface(8, 16, 17) and retained there through interaction with a receptor-like molecule (7, 18) . However, neither uPA nor plasmin can activate pro-gelatinase A. Instead, it is activated by the cells cultivated with 12-O-tetradecanoylphorbol-13-acetate(16, 17) , concanavalin A(8, 19) , or in collagen gel (9) and by invasive tumor cells(10, 20) . The plasma membrane fraction prepared from the cells can activate pro-gelatinase A, and the reaction was sensitive to chelating reagents and tissue inhibitor of metalloproteinases-2 (TIMP-2)(8, 17) . Thus, the putative pro-gelatinase A activator may be a member of the MMP family.

We isolated a cDNA clone that encodes a hitherto unknown MMP named MT-MMP(21, 22) . MT-MMP is expressed as a 63-kDa protein on the surface of transfected cells with MT-MMP cDNA. At the same time, the cells caused specific activation of 72-kDa pro-gelatinase A(22) . Since MT-MMP has a potential TM domain and was not released into the culture medium, the TM domain is thought to play a direct role in MT-MMP integration into the plasma membrane. In this study, we examined whether the potential TM domain is indeed required for anchoring MT-MMP onto the cell surface by constructing deletions and chimeric molecules. The pro-gelatinase A activation function was also examined in relation to the subcellular localization of MT-MMP mutants.


MATERIALS AND METHODS

Construction of Plasmids

The SV40 early promoter of the pSG5 plasmid (Stratagene, La Jolla, CA) was used to express MT-MMP as reported(22) . Chimeric proteins between TIMP-1 (23) and the TM domains of MT-MMP (TIMP/TM) or human interleukin 2 receptor alpha-chain (IL-2R) (24, 25) (TIMP/TM) were constructed as follows. The cDNA fragments encoding the C terminus of MT-MMP (Gly to Val) harboring the deduced TM domain (Ala to Phe) or the TM domain of IL-2R (Val to Ile) were amplified by polymerase chain reaction. Fragments were then ligated to the 3`-end of the TIMP-1 cDNA fragment to express both proteins as fusion products and subcloned into the pSG5 expression vector. A deletion mutant lacking the deduced TM domain of MT-MMP (DeltaMT-MMP) was constructed by inserting a BamHI linker and a termination codon at Gly. Five extra amino acids (Gly-Phe-Arg-Ser-Tyr) derived from the BamHI linker were added to the truncated C terminus. A chimera between DeltaMT-MMP and the heterologous TM domain of IL-2R (DeltaMT-MMP/TM) was constructed using the introduced BamHI site in the DeltaMT-MMP plasmid and the amplified TM DNA fragment described above. The predicted products were 57 kDa for DeltaMT-MMP, 60 kDa for DeltaMT-MMP/TM, 24.8 kDa for TIMP/TM, and 26.6 kDa for TIMP/TM (Fig. 1).


Figure 1: A schematic illustration of chimeric proteins. Domains of membrane type-matrix metalloproteinase (MT-MMP) (21) are shown: signal peptide (Sig), propeptide (Pro), catalytic (Catalytic), hinge (Hinge), hemopexin-like (Hemopexin), and potential transmembrane (TM, box with diagonal lines) in the hemopexin-like domain. Zn indicates the zinc-binding site in the catalytic domain. Interleukin 2 receptor alpha-chain (IL-2 R(alpha)) (24, 25) is shown: signal peptide, extracellular domain, TM domain (black box), and cytoplasmic domain. Tissue inhibitor of metalloproteinases 1 (TIMP-1) (23) is shown: signal peptide and inhibitor domain (meshed box). Chimeric proteins (TIMP/TM, TIMP/TM, and DeltaMT-MMP/TM) and a truncated mutant (DeltaMT-MMP) were constructed by cDNA manipulation as described under ``Materials and Methods'' and expressed by a eukaryotic expression vector (pGS5).



Cell Culture and Transfection

COS-1 cells were cultured in Dulbecco's modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FCS) and 2 mM glutamine under a 5% CO(2) atmosphere. Plasmids were transfected into cells using calcium phosphate as described(26) .

Immunoprecipitation

Immunoprecipitation proceeded essentially as described(22) . After transfection for 36 h, the cells were labeled with 30 µCi/ml [S] methionine (SJQ0079, Amersham Corp.) in methionine-free DMEM (10% FCS) and incubated for 4 h at 37 °C. Thereafter, the medium was collected and clarified by centrifugation. Cells were then lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 50 mM NaCl, 0.02% NaN(3), 1% Nonidet P-40, 0.25 mM dithiothreitol). Both the conditioned media and cell lysates were incubated with monoclonal antibodies (mAbs) against TIMP-1 (50-1H7) or MT-MMP (113-5B7, 114-2F2) (mAbs were provided by Fuji Chemical Industries, Ltd., Takaoka, Japan). Antigen-antibody complexes were precipitated with protein A coupled to Sepharose beads by centrifugation. After washing with lysis buffer, immunoprecipitates were suspended and boiled in sample buffer (50 mM Tris.HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol), and then electrophoresis proceeded in an SDS-polyacrylamide gel with a discontinuous buffer system. The radioactivity was detected by a Bioimage Analyzer BAS1000 (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Preparation of Cell Membrane Fraction

The cell membrane was extracted as described(17) . COS-1 cells transfected with mutant and wild type MT-MMP expression plasmids were cultured for 24 h in serum-free DMEM. Cells were collected and homogenized using a Dounce homogenizer. The whole-cell homogenate was centrifuged at 3000 times g for 10 min, and the supernatant fraction was collected. The cell organelle-enriched fraction was pelleted by centrifugation at 100,000 times g for 2 h. The pellet was resuspended in saline and layered onto a discontinuous sucrose density gradient (20, 30, 50, and 60% sucrose) and centrifuged at 100,000 times g for 3 h. The plasma membrane-enriched fraction at the 30-50% sucrose interface was collected and pelleted again. The pellets were dissolved in HEPES/KOH buffer (25 mM HEPES/KOH, pH 7.5, 0.1 mM CaCl(2)) at a final protein concentration of 3 mg/ml.

Western Blotting

Membrane fractions of the COS-1 cells transfected with various plasmids were mixed with an equal volume of 2 times SDS gel-loading buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiothreitol, 4% SDS, 0.2% bromphenol blue, 20% glycerol). The samples were resolved by 10% polyacrylamide gel electrophoresis. The separated proteins were transferred onto a nitrocellulose membrane (Amersham Corp.) using a semidry system (Biometra, Gottingen, Germany). The membrane was blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature and then probed with mAbs against TIMP-1 and MT-MMP (113-5B7) for 16 h at room temperature. The final concentration of each mAb was 1 µg/ml in 3% BSA/PBS. After extensive washing with TBS (20 mM Tris-HCl, pH 7.6, 137 mM NaCl), the membrane was reprobed with anti-mouse IgG conjugated with horseradish peroxidase (Cappel, West Chester, PA) 1:4000 in TBS for 1 h at room temperature. The blot was developed in a 20-ml buffer containing 50 mM Tris-HCl, pH 7.3, containing 5 µl of 30% H(2)O(2), 5 mg of 3,3`-diaminobenzidine tetrahydrochloride. The reaction was terminated by rinsing the blot in water.

Antibody Binding Surface Protein Assay

The procedure was performed as described(27) . COS-1 cells were co-transfected with beta-galactosidase and DeltaMT-MMP or MT-MMP expression plasmids and then metabolically labeled with [S]methionine for 6 h at 36 h after transfection. The labeled cells were washed three times with 3% BSA/PBS and then incubated with anti-MT-MMP (114-2F2, 5 µg/ml) and anti-beta-galactosidase (5 µg/ml; Promega, Madison, WI) at 37 °C for 40 min, rinsed three times with 3% BSA/PBS, and lysed in lysis buffer on ice. The cell lysates were clarified by centrifugation and immunoprecipitated with protein A-Sepharose as described above.

Activation of Pro-gelatinase A

Plasma membrane fractions (10 µg) were mixed with 0.5 µl of FCS as a source of pro-gelatinase A and incubated for 2 h at 37 °C. The reaction was terminated by the addition of sample buffer, and the samples were analyzed by gelatin zymography.

Gelatin Zymography

Zymography was performed as described(28) . Samples were mixed with SDS sample buffer in the absence of a reducing agent and incubated for 20 min to denature MMPs and dissociate MMP-TIMP complexes. Electrophoresis proceeded on 10% polyacrylamide gels containing 0.1% SDS and gelatin at a final concentration of 0.1% (w/v). Thereafter, gels were washed in 2.5% Triton X-100 for 1 h to remove the SDS. During this process, pro-enzymes are activated autocatalytically. Gels were then incubated for 24 h at 37 °C in a reaction buffer (50 mM Tris-HCl, pH 7.6, 0.15 M NaCl, 10 mM CaCl(2), 0.02% NaN(3)) and stained with 0.1% Coomassie brilliant blue R-250. The location of gelatinolytic activity is detectable as a clear band in the background of uniform staining.


RESULTS

The TM Domain of MT-MMP Functions as a Membrane Linker

To assess the role of the putative TM domain of MT-MMP as a membrane linker, we prepared a fusion protein between the TM domain and a secretory protein, TIMP-1, by manipulating the cDNAs of both proteins (Fig. 1). Thus a plasmid was constructed that expressed chimeric TIMP/ TM. The TM domain of the IL-2 receptor alpha-chain (IL-2R) was used as a positive control, and a fusion protein TIMP/TM was constructed. TIMP-1 and fusion proteins were expressed in COS-1 cells by transient transfection of the plasmids. After labeling with [S]methionine, cell lysates were prepared and examined by immunoprecipitation using anti-TIMP-1 mAb. TIMP-1 can be detected as a 28-kDa band after glycosylation of the translated 21-kDa product(29) . TIMP/TM and TIMP/TM were detected in cell lysates as 32- and 34-kDa bands, respectively, that were proportional to the calculated molecular weights ( Fig. 1and 2A).

Among these proteins, only TIMP-1 was detected in the culture medium, whereas the other chimeric proteins were not. Thus, both of the TM domains fused to TIMP-1 seem to inhibit the release of the products into the medium. However, it is possible that overexpressed chimeric proteins disturb the normal secretory machinery in the cells. To examine this possibility, the effect of chimeric protein expression upon TIMP-1 secretion was analyzed by co-transfecting the chimeric plasmids together with that expressing TIMP-1. TIMP-1 was secreted normally, whereas both chimeras were detected only in the cell lysate (Fig. 2B).


Figure 2: Expression of TIMP-1 and its chimeras in COS-1 cells. Plasmids encoding TIMP-1 and its chimeras (TIMP/TM and TIMP/TM) were transiently transfected into COS-1 cells. Cells were then labeled with [S]methionine for 4 h, and proteins in the cell lysate and in the medium were immunoprecipitated with a monoclonal antibody against TIMP-1 (50-1H7). Precipitates were separated on SDS-containing polyacrylamide gels and the radioactivity was detected by means of autoradiography. Molecular size markers are indicated in the figures. A, the transfected plasmids were pSG5 vector alone (Control), the expression plasmids for TIMP-1 (TIMP), TIMP/TM, and TIMP/TM. Molecular mass of the products were 28, 32, and 34 kDa, respectively, as indicated in the figure. Other bands were nonspecific. B, TIMP-1 was co-expressed with TIMP/TM (TIMP/TM+ TIMP) or TIMP/TM (TIMP/TM+ TIMP), and others were the same as in A.



To ensure the localization of the chimeras on the cell surface, transfected cells were grown on coverslips and examined by indirect immunofluorescence staining by incubating the cells with anti-TIMP-1 mAb without permeabilization(22) . Whereas TIMP-1-producing cells were not stained with anti-TIMP-1 antibody, TIMP/TM- and TIMP/TM-producing cells were positively stained on the cell surface (data not shown). These results indicated that the TM domain of IL-2R can trap a naturally secretory TIMP-1 on the cell surface and that the activity of the TM domain of MT-MMP is also comparable with that of IL-2R. Thus, the TM domain of MT-MMP is a membrane linker.

The Role of the TM Domain on Pro-gelatinase A Activation Function

To examine the role of the TM domain of MT-MMP in pro-gelatinase A activation function, the C terminus including the TM domain was truncated (DeltaMT-MMP) as described under ``Materials and Methods'' (Fig. 1). COS-1 cells that produce pro-gelatinase A into the culture supernatant were used as the recipient for transfection assay. Pro-gelatinase A activation was monitored by gelatin zymography. Transfection with the MT-MMP plasmid induced processing of pro-gelatinase A (66-kDa) into the activated form (62-kDa) via an intermediate form (64-kDa) (Fig. 3B)(17, 22) , whereas the truncated mutant DeltaMT-MMP failed to activate it. The defect was not because of the low levels of DeltaMT-MMP expression as demonstrated by immunoprecipitation (Fig. 3A), and pro-gelatinase A activation was not induced even when the doses of the transfected DeltaMT-MMP plasmid were increased (data not shown). Thus, the TM domain of MT-MMP is indispensable for pro-gelatinase A activation.


Figure 3: Deletion of the TM domain from MT-MMP and its effect on pro-gelatinase A activation. COS-1 cells were transfected with the expression plasmids pSG5 (Control), DeltaMT-MMP, and MT-MMP. A, transfected cells were labeled with [S]methionine for 4 h, and cell lysates were prepared and immunoprecipitated with anti-MT-MMP monoclonal antibody (113-5B7). B, COS-1 cells transfected with MT-MMP, DeltaMT-MMP, or vector plasmid were cultured in serum-free DMEM for 12 h. Conditioned medium (15 µl) was mixed with SDS electrophoresis sample buffer (without dithiothreitol) and separated in a 10% polyacrylamide gel containing 1 mg/ml gelatin. After gelatin digestion as described under ``Materials and Methods,'' the gel was stained with 0.1% Coomassie brilliant blue R-250.



The Sequence Specificity of the TM Domain of MT-MMP

It is still possible to argue that the TM domain of MT-MMP is required for pro-gelatinase A activation in a sequence-specific manner rather than as a membrane linker. To test this notion, the TM domain of IL-2 receptor alpha-chain was fused to DeltaMT-MMP (DeltaMT-MMP/TM), and the recovery of pro-gelatinase A activation function was investigated. Transient expression of DeltaMT-MMP/TM in COS-1 cells induced pro-gelatinase A activation as efficiently as MT-MMP. Then, plasma membrane fractions of the transfected cells were prepared, and their activities against the exogenous pro-gelatinase A that is in fetal calf serum (FCS) were examined (Fig. 4). Plasma membrane fractions from the transfected cells did not contain any gelatinolytic activity by themselves. After incubation with FCS, only preparations from the cells that express MT-MMP and DeltaMT-MMP/TM retained the activity to generate active forms of gelatinase A, whereas that from the DeltaMT-MMP-transfected cells did not (Fig. 4). Thus, we concluded that the heterologous TM domain was sufficient to recover the biochemical function of MT-MMP and that particular sequences in the TM domain were not essential for this function.


Figure 4: Pro-gelatinase A activation by the plasma membrane fraction. COS-1 cells transfected with plasmids were homogenized, and plasma membrane fractions were prepared by sucrose density gradient centrifugation. Membrane fractions (10 µg of protein) were incubated with (+) or without(-) fetal calf serum (Serum) for 120 min at 37 °C, and gelatinolytic activities were analyzed by gelatin zymography.



Secretion of the Mutant MT-MMP

Plasmids encoding MT-MMP and DeltaMT-MMP were transfected into COS-1 cells together with the TIMP-1 plasmid, and the products in the culture media were examined by immunoprecipitation with mAbs. Both MT-MMP and DeltaMT-MMP were detected at similar levels in the cell lysates (Fig. 5). MT-MMP was not released into the culture medium as reported, and the co-expressed secretory protein (TIMP-1) was released and accumulated in the medium (Fig. 5)(22) . However, DeltaMT-MMP was detected in the culture medium, though at lower levels as compared with the co-expressed TIMP-1 (Fig. 5). Two fragments (64- and 54-kDa) detected in the medium were specifically reacted with anti-MT-MMP mAbs recognizing different epitopes (113-5B7 and 114-2F2), and they were not the same size as those in the cell lysate. It is not clear at this moment what kind of processing or modification is associated with the molecules released into the culture medium. However, it is clear that deletion of the TM domain from MT-MMP facilitated the release of the fragments into the medium.


Figure 5: Expression of DeltaMT-MMP and its release into culture medium. COS-1 cells were co-transfected with pSG5 (Control), MT-MMP, or DeltaMT-MMP plasmid together with TIMP-1 plasmid. Cells were labeled with [S]methionine, and proteins in the media or cell lysates were immunoprecipitated with anti-MT-MMP mAb (113-5B7) and anti-TIMP-1 mAb (50-1H7). Specific bands detected by mAbs are indicated. Two bands in the media from DeltaMT-MMP+TIMP (indicated by arrows) were detected by another anti-MT-MMP mAb (114-2F2) (data not shown).



To confirm the loss of cell surface expression of DeltaMT-MMP further, antibody binding studies against the cell surface molecules (27) were performed. Metabolically labeled, transfected cells were first incubated with mAbs without permeabilization, and excess antibodies were removed. The cells were then lysed, and immune complexes that formed on the surface were precipitated with protein A-Sepharose. The negative control was beta-galactosidase, a cytoplasmic protein, and it was detected only in the cell lysate (Fig. 6A). On the contrary, both MT-MMP and DeltaMT-MMP were precipitated by this procedure as shown in Fig. 6A. The secretory protein TIMP-1 was not precipitated under these conditions (data not shown). Thus, a substantial fraction of DeltaMT-MMP remains on the cell surface, although it is thought to be gradually shed into the medium. These results suggested that MT-MMP has an additional device to keep it on the surface of cells other than its TM domain. Western blotting of the plasma membrane preparations, which were used for the study as described in the previous section (Fig. 4), demonstrated that both DeltaMT-MMP and MT-MMP co-purified with the membrane fractions (Fig. 6B). This is also another indication that DeltaMT-MMP is still associating with the cell surface.


Figure 6: Localization of DeltaMT-MMP and MT-MMP on the plasma membrane. A, the beta-galactosidase (beta-Gal) expression plasmid was co-transfected into COS-1 cells with pSG5, DeltaMT-MMP, and MT-MMP plasmids. Living cells were then incubated with mAbs against MT-MMP and beta-Gal, and then excess antibodies were washed and the cells were lysed. Immune complexes were precipitated together with protein A-Sepharose (Surface). By contrast, lysates were prepared from the cells that were not incubated with mAbs and immunoprecipitated as described in Fig. 5(Lysate). B, plasma membrane fractions of COS-1 cells transfected with pSG5, DeltaMT-MMP, and MT-MMP plasmids were analyzed by Western blotting using anti-MT-MMP mAb (113-5B7) and anti-TIMP-1 mAb.




DISCUSSION

MT-MMP is an MMP recently discovered by cDNA cloning(21, 22) . We deduced the cell surface expression of MT-MMP from its TM-like structure at the C terminus, which consists of a 24-hydrophobic amino acid stretch. Although it indeed localizes on the cell surface and induces specific activation of exogenous pro-gelatinase A, we did not have any evidence to show how the protein is linked to the plasma membrane and its importance in the biochemical function of the enzyme. The function of the putative TM domain of MT-MMP as a membrane linker was demonstrated by constructing a fusion protein with the secretory protein TIMP-1. The TM domain changed the TIMP-1 localization from culture medium onto the cell surface, as did the authentic TM domain of IL-2R. Thus, we concluded that the TM domain is sufficient to retain MT-MMP on the cell surface.

Plasma membrane integration of MT-MMP through the TM domain was essential for its pro-gelatinase A activation function, because the activity was abolished by truncation of the domain and recovered by making a fusion of the mutant with a heterologous TM domain of IL-2 receptor alpha-chain.

Truncation of the TM domain facilitated release of the product into the culture medium. However, a substantial portion of the product was still retained on the cell surface as shown by the mAb binding study. Thus, MT-MMP seems to have additional devices other than the TM domain to retain it on the cell surface. However, DeltaMT-MMP did not activate pro-gelatinase A despite its localization on the cell surface. So far, we do not know what causes this functional difference between the cell surface MT-MMP and DeltaMT-MMP. It is possible, however, that membrane integration is essential to maintain the appropriate orientation of the molecule on the cell surface, thus enabling the molecule to interact with substrate or accessory molecules at that site.

The DeltaMT-MMP fragments released into the medium have a different molecular mass (54 and 64 kDa) from that in and on the cells (57 kDa). Since sizes of the released fragments were discrete, specific processing or modification of the product seems to associate with the step at or after release from the cells. The medium containing the secreted DeltaMT-MMP fragments did not show pro-gelatinase A activation activity (data not shown).

MT-MMP (63-kDa) expressed in the transfected cells is thought be in the latent form having a propeptide domain, because the apparent molecular mass is close to that of the calculated gene product. Also, a small deletion in the propeptide domain, which should be cleaved if it is activated, resulted in a smaller product as expected from the latent protein sequence (data not shown). The activation mechanism of MT-MMP might differ from that of the other MMPs where the propeptide sequence can be cleaved by autocatalytic mechanisms(17, 30, 31, 32, 33) . Only MT-MMP and stromelysin 3 (34) have an extra sequence between the propeptide and catalytic enzyme domain(21, 22) . RXKR sequences that are reported to be the consensus for subtilisin-like processing endopeptidases (35, 36) were conserved between the two. Many secretory peptide hormones, growth factors, and viral envelope proteins have this consensus and are processed during exocytosis(37, 38, 39) . However, this process seems to be inefficient at least for some of the proteins. For example, only less than 15% of human immunodeficiency virus gp160 can be processed into gp120 in the cells(40) . Thus, MT-MMP processing may be inefficient, at least in the transfected cells, and only a small portion of the translated product can be processed.

Another question that remains to be clarified is whether MT-MMP directly cleaves the propeptide domain of pro-gelatinase A. Otherwise, activation may be indirectly mediated by a secondary molecule that is the real substrate of MT-MMP. We are currently attempting to extract MT-MMP from the membrane fraction and demonstrate direct activity against pro-gelatinase A. Goldberg's group (17) reported that an initial cleavage site in the propeptide domain occurs between Asn and Leu. Thus, it will be of interest to determine whether MT-MMP can cleave this sequence.

In this study, we demonstrated the function of the TM domain of MT-MMP as a membrane linker and as an essential structural requirement for pro-gelatinase A activation. A chimeric molecule such as DeltaMT-MMP/TM will be valuable in dissecting the specific elements of MT-MMP further.


FOOTNOTES

*
This work was supported by the Special Coordination Fund for Promoting Science and Technology from the Ministry of Science and Technology of Japan; by a grant-in-aid for cancer research from the Ministry of Education, Science, and Culture of Japan; by the Japanese Foundation for Multidisciplinary Treatment of Cancer; and by the Foundation for Promotion of Cancer Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Takara-machi 13-1, Kanazawa, Ishikawa 920, Japan. Tel.: 81-762-34-4504; Fax: 81-762-60-7840.

(^1)
The abbreviations used are: MMP, matrix metalloproteinase; MT-MMP, membrane-type matrix metalloproteinase; TIMP-1, tissue inhibitor of metalloproteinases-1; TM, transmembrane domain; IL-2R, interleukin-2 receptor alpha-chain; mAb, monoclonal antibody; BSA, bovine serum albumin; PBS, phosphate-buffered saline; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle's medium.


ACKNOWLEDGEMENTS

We thank Drs. J. Tanaka and M. Fujii for critical discussion and Dr. Iwata (Fuji Chemical Industries, Ltd., Takaoka) for preparing monoclonal antibodies.


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