(Received for publication, June 8, 1995; and in revised form, July 10, 1995)
From the
Membrane-type matrix metalloproteinase (MT-MMP), which we have identified recently, is unique in its transmembrane (TM) domain at the C terminus and mediates activation of pro-gelatinase A on the cell surface (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M. (1994) Nature 370, 61-65; [Medline] Takino, T., Sato, H., Yamamoto, E., and Seiki, M.(1995) Gene (Amst.) 115, 293-298). In addition to MT-MMP, a novel MMP-related cDNA of 2.1 kilobases was isolated from a human placenta cDNA library. The cDNA contains an open reading frame for a new MMP. The deduced protein composed of 604 amino acids was closely related to MT-MMP in the amino acid sequence (66% homology at the catalytic domains) and has a potential TM domain at the C terminus. Monoclonal antibodies raised against the synthetic peptide recognized a 64-kDa protein as the major product in the transfected cells. TIMP-1 fused with the potential TM domain was localized on the cell surface while native TIMP-1 is in the culture medium. Thus, we called the second membrane-type MMP, MT-MMP-2 and renamed MT-MMP, MT-MMP-1. MT-MMP-1 and -2 are thought to form a distinct membrane-type subclass in the MMP family since all the others are secreted as soluble forms. Like MT-MMP-1, expression of MT-MMP-2 induced processing of pro-gelatinase A (68-kDa in gelatin zymography) into the activated form of 62-kDa fragments through a 64-kDa intermediate form. Expression of MT-MMP-2 mRNA was at the highest levels in the brain where MT-MMP-1 was at the lowest level compared to other tissues. MT-MMP-1 and -2 are thought to be utilized for extracellular matrix turnover on the surface of cells under different genetic controls.
Matrix metalloproteinases (MMPs) ()are a family of
enzymes that share a common domain structure composed of propeptide,
catalytic, hinge, and hemopexin-like domains (exception is
MMP-7
matrilysin, which lacks a hemopexin-like
domain)(3, 4) . These enzymes are responsible for the
turnover of extracellular matrix by degrading native macromolecules,
including a variety of collagens and glycoproteins such as fibronectin
and laminin, and play crucial roles in tissue remodeling during
morphogenesis, wound healing, angiogenesis, and also in many
pathological conditions such as tumor invasion and rheumatoid
arthritis(5, 6, 7, 8, 9, 10) .
11 MMPs encoded by different genes are known as the MMP family members,
and they have different substrate specificity against extracellular
matrix macromolecules. 10 of them are produced by cells as a soluble
zymogen form, but the last one, which we recently discovered, has a
transmembrane domain at the C terminus and is expressed as a membrane
protein(1, 2, 11) . Thus, we called this
ectoenzyme membrane-type MMP (MT-MMP).
All of the MMPs are expressed
as inactive zymogens and need proteolytic activation for them to
function. Although serine proteases such as plasmin, neutrophil
elastase, and trypsin can activate several MMP zymogens in a test
tube(12, 13) , little is known about the activators in
tissue. MMP-2gelatinase A cannot be activated by these serine
proteinases (14, 15) but is activated on the surfaces
of fibroblasts (16) and tumor cell lines treated with
12-O-tetradecanoylphorbol-13-acetate or concanavalin A (17, 18, 19) . Thus, a unique activator on
the cell surface was expected to be responsible for gelatinase A
activation, and it turned out to be MT-MMP. Expression of MT-MMP in the
transfected cells induced specific activation of gelatinase A in a
cell-mediated manner(1) . Appearance of the activated form of
gelatinase A in the tissue is also a characteristic feature of invasive
carcinomas(20, 21) . Expression of MT-MMP was detected
there, and the product was immunolocalized in and on the carcinoma
cells(1) . Since gelatinase A is an important enzyme for
basement membrane invasion by degrading type IV collagen, MT-MMP on the
tumor cell surface is thought to play a critical role in the invasive
phenotype of tumor cells.
MT-MMP cDNA was isolated from a human
placenta cDNA library using a MMP-related gene fragment as a
probe(2) . The probe with a possible new MMP gene sequence was
obtained from the amplified cDNA fragments by polymerase chain reaction
(PCR) using degenerate oligonucleotide primers corresponding to the
conserved sequences within the MMP family. The structural
characteristic of MT-MMP is the transmembrane domain at the C terminus
and two more additional insertions. One is the insertion of 8 amino
acids in the catalytic domain, and the other is the insertion of 11
amino acids between the propeptide and the catalytic domain.
MMP-11stromelysin-3 (22) has a similar insertion of 10
amino acids between propeptide and catalytic domain, and RXKR
sequences, potential recognition sites for subtilisin-like processing
enzymes(23) , are conserved between them. No insertion
corresponding to the 8 amino acids of MT-MMP was found in the other
members of the MMP family.
Both MT-MMP and stromelysin-3 were identified by cDNA cloning instead of the conventional biochemical purification of the enzymes. Thus, new MMP members may be obtained further by survey of the MMP-related genes. To identify yet unknown MMP members, we extended our previous study to survey the MMP-related cDNAs amplified from various human tissues by reverse transcription-PCR. In this study, we identified a fragment of another new MMP-related gene from a human melanoma tissue. This fragment was used as a probe to screen a human placenta cDNA library, and a cDNA fragment that encodes a new MMP having a TM domain at the C terminus was obtained. Thus, MT-MMPs form a distinct subgroup in the MMP family.
A human placenta cDNA library (Clontech), which was constructed using oligo(dT)- and random-primed cDNA was screened with the MMP-X2 cDNA fragment as a probe.
By checking cDNA libraries from various sources by PCR, a human placenta library was found to contain MMP-X2 cDNA fragments and screened to isolate a longer size of the cDNA fragment. We isolated a 2.1-kb cDNA having a long open reading frame that potentially encodes a 604-amino acid protein (Fig. 1).
Figure 1: Nucleotide and the deduced amino acid sequence encoded by MMP-X2 cDNA. The entire nucleotide sequence of the 2.1-kilobase pair MMP-X2 cDNA fragment is presented, and the longest open reading frame that initiates from ATG and terminates at the TGA codon was translated. PRCGVPD, which is the most conserved sequence among the MMP family, is boxed. Zinc binding site is doubleunderlined. The predicted transmembrane domain at the C terminus is underlined, and asterisks indicate hydrophobic amino acids in this domain. The nucleotide sequence data are in the GSDB/DDBJ/EMBL/NCBI nucleotide sequence data base with the accession number D50477.
Figure 2: Domain structure of MT-MMP-2. A, alignment of amino acid sequences of the MMP family members. The typical domains are indicated in the figure (signal, propeptide, catalytic, hinge hemopexin-like domains from the N terminus). Specific insertions characteristic to MT-MMPs are indicated by the upwardarrows (IS-1 to IS-3). B, homology between the extra sequences characteristic to MT-MMPs are indicated. Asterisks are the conserved amino acids between the two.
MT-MMP has three characteristic insertions compared to other MMP family members. They are the 11-amino acid insertion (IS-1) between propeptide and catalytic domain, the 8 amino acids (IS-2) in the catalytic domain, and the 75 amino acids (IS-3) at the C terminus, having a TM composed of the 24 hydrophobic amino acids. All such insertions exist in MMP-X2 at the same positions with significant homology (Fig. 2B). In the first insertion, the RXKR sequence, a potential processing site for subtilisin-like enzymes, was conserved(23) . The extra 75 amino acids at the C terminus contain a potential TM domain composed of 24 amino acids with higher hydrophobicity. Thus, MMP-X2 is also expected to be a membrane-type MMP as well. Based on this structural homology to MT-MMP and the localization of the product on the cell surface as demonstrated in the following section, we will call the MMP-X2 gene product MT-MMP-2 and rename the previously identified MT-MMP as MT-MMP-1.
Figure 3:
Identification of MT-MMP-2 gene product.
Immunoprecipitation of MT-MMP-2 is shown. COS-1 cells transfected with
MT-MMP-2 plasmid (MT-MMP-2), TIMP-1 plasmid (TIMP-1) or vector plasmid
(control) were labeled with [S]methionine for 5
h at 37 °C, and then cell lysates and culture medium were prepared
and immunoprecipitated with mAbs against MT-MMP-2 (117-4E1 and
117-13B6) or TIMP-1 (50-1H7). Immunoprecipitates were
separated by SDS-polyacrylamide gel electrophoresis, and the
radioactivity was detected by autoradiography. MT-MMP-2 protein (64
kDa) and TIMP-1 protein (28 kDa) are indicated by arrows.
Figure 4:
Function of the potential transmembrane
domain. A, expression of TIMP-1 and its chimeras with the TM
domains of MT-MMP-1 or -2 in COS-1 cells. COS-1 cells were transfected
with TIMP-1 plasmid (TIMP-1), its chimeras (TIMP-1/MT-1 and
TIMP-1/MT-2), or vector plasmid (control). After labeling the cells
with [S]methionine for 5 h at 37 °C, the
lysates and cultured medium were immunoprecipitated with the mAb
against TIMP-1 (50-1H7). TIMP-1 protein and its chimeric proteins were
detected as 28- and 32-kDa proteins, respectively. B,
immunofluorescence staining. COS-1 cells were transfected with TIMP-1
plasmid (TIMP-1) or TIMP-1/MT-2 plasmid (TIMP-1/MT-2) and incubated
with anti-TIMP-1 mAb (50-1H7) prior to fixation. After washing
excess mAb, cells were fixed with acetone and stained with fluorescein
isothiocyanate-conjugated anti-mouse IgG.
TIMP-1, TIMP/MT-1, and TIMP/MT-2 were expressed as 28-, 32-, and 32-kDa proteins in the cell lysate, respectively. The sizes of the chimeras were as expected from the construct of the fusion genes. TIMP-1 was predominantly detected in the medium, and a small amount was in the cell lysate. However, TIMP/MT-1 was found exclusively in the cell lysate but not in the medium as reported(11) . The localization of TIMP/MT-2 was exactly the same as that of TIMP/MT-1. Thus, the TM domain of MT-MMP-2 has a function similar to that of the MT-MMP-1 in preventing secretion of the fusion protein to the media.
Next, we examined whether the TIMP-1 portion of the chimera is expressed on the cell surface. To show this, transfected cells were incubated with anti-TIMP-1 mAb without fixation, and then indirect immunofluorescence staining was carried out using fluorescein isothiocyanate-conjugated anti-mouse IgG (Fig. 4B). TIMP/MT-2-producing cells were stained as positively as that of TIMP/MT-1, but TIMP-1 producer cells were negative. Thus, we concluded that MT-MMP-2 is the second member of the membrane-type MMP.
Figure 5: Activation of pro-gelatinase A by MT-MMP-2 expression. A, activation of pro-gelatinase A in COS-1 cells. COS-1 cells cotransfected with gelatinase A plasmid and MT-MMP-2 plasmid (MT-MMP-2), MT-MMP-1 plasmid (MT-MMP-1), or vector plasmid (control) were cultured in serum-free DMEM for 24 h. Conditioned media were collected and analyzed by gelatin zymography. B, activation of pro-gelatinase A by MT-MMP-2 and effect of TIMPs in HT 1080 cells. MT-MMP-2 plasmid was cotransfected into HT-1080 cells together with TIMP-1 plasmid (lane3), TIMP-2 plasmid (lane4), or vector plasmid (lane2), or vector plasmid alone was transfected (lane1). After transfection, the cells were cultured in serum-free DMEM for 24 h. Aliquots were analyzed as described under ``Materials and Methods.'' The conditioned medium of the cells treated with concanavalin A was also analyzed (lane5).
Figure 6:
Tissue distribution of MT-MMP-2 mRNA. A, RNA blot analysis of MT-MMP-2 mRNA in human tissues.
Northern blot (Multiple Tissue Northern, Clontech) was probed with P-labeled cDNA for MT-MMP-2 or MT-MMP-1, and the
radioactivity was analyzed by a Bioimage Analyzer BAS 1000. The same
membrane was rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe to confirm the amounts of RNA loaded. B, RNA blot analysis of MT-MMP-2 mRNA in cultured human cell
lines. Total RNA (10 µg) extracted from the cells was separated by
electrophoresis and transferred to a nylon membrane. This membrane was
hybridized and rehybridized as described above. The size of mRNA is 12
kb for MT-MMP-2, 4.5 kb for MT-MMP-1, and 1.8 kb for
glyceraldehyde-3-phosphate dehydrogenase.
Expression of MT-MMP-1 and -2 mRNA in various cell lines was also examined (Fig. 6B). MT-MMP-2 mRNA was detected in bladder carcinoma T24 and larynx carcinoma Hep2 cells, where MT-MMP-1 transcripts were detected at low levels. By contrast, MT-MMP-1 transcripts were predominant in squamous cell carcinoma OSC-19 and human embryonal lung fibroblasts where MT-MMP-2 mRNA levels were low. Thus, expression of the genes for MT-MMP-1 and -2 is regulated differently, although they share similar protein structures and functions in activating pro-gelatinase A.
In addition to MT-MMP-1 (MT-MMP in the previous paper), which we previously reported(1, 2) , we identified a new MMP gene that was expressed in a human oral malignant melanoma and a human placenta. The isolated 2.1-kilobase pair cDNA contained a sufficient coding frame for MT-MMP-2. However, the transcript in tissues or cell lines was at 12 kb in size. Since the cDNA lacks the typical polyadenylation signal (AATAAA), the 3`-non-coding region of the gene is thought to be missing in the cloned fragment. MT-MMP-1 and -2 are approximately the same in their molecular weights, but they are encoded by 4.5- and 12-kb transcripts, respectively. The most plausible explanation for the difference of the mRNA sizes is the different length of non-coding regions at their 3`-ends.
MT-MMP-2 is the most closely related to MT-MMP-1 in the amino acid sequence (66% homology at the catalytic domain) and in the characteristic insertions compared to other MMPs. These insertions may be important for their function and regulation. For example, the first insertions (IS-1) between the propeptide and catalytic domain contain the conserved RXKR sequences. A similar insertion also exists in stromelysin-3 but not in others (Fig. 2B). These sequences may be the recognition site for processing, since immediately downstream of the RXKR sequences is the reported N terminus of the processed forms of stromelysin 3 and MT-MMP-1(34, 35) . Since RXKR is the consensus sequence for subtilisin-like enzymes, autocatalytic activation mechanism, which is common for other MMPs, may not be applicable for these three MMPs. Indeed, 4-aminophenylmercuric acetate or SDS that induces autocatalytic activation of pro-MMPs cannot activate stromelysin-3 and MT-MMPs.
There exist 8 amino acid insertions (IS-2) in the catalytic domains of MT-MMP-1 and -2 at the same position. Three of the eight amino acids at both ends were conserved. Although the significance of this insertion is not clear, it may modulate substrate specificity of the enzymes from its position in the catalytic domain like the gelatin-binding domain of two gelatinases(36) .
Additional sequences (IS-3) containing the TM domains are found downstream of the hemopexin-like domains of MT-MMPs. Both MT-MMPs are expressed as membrane proteins embedded into the plasma membrane through the TM domains at the C terminus. Thus, these two MMPs form a unique membrane-type subclass in the MMP family, while the others are expressed as a soluble form. We previously aligned the most C-terminal portion of the cysteine residue of MT-MMP-1 to that of the hemopexin-like domain of other MMPs and thought that the TM domain is an insertion in the hemopexin-like domain(1, 2) . However, it is more appropriate that the IS-3 locate downstream of the hemopexin-like domains of MT-MMP-1 and -2 rather than splitting the domain as shown in the alignment in Fig. 2. With this alignment, it becomes possible for MT-MMPs to form a cysteine bridge in the hemopexin-like domain, the conserved structure among the MMPs, outside of the cells.
Expression of MT-MMP-2 in cells, like that of MT-MMP-1, induced activation of the pro-gelatinase A to the fully activated form (62 kDa) through the intermediate form (64 kDa). Thus, both MT-MMPs have similar biochemical activities at least in part, though it remains to be elucidated whether these MT-MMPs have different substrate specificity or not. If both MT-MMPs are similar in function, why are two separate genes required for the organism? The organism may have to utilize these MT-MMPs in different tissue environment or situations. Consistent with this idea, the tissue distribution of the MT-MMP-1 and MT-MMP-2 mRNAs was different in the human tissues. In contrast to MT-MMP-1, which is expressed widely in various tissues, MT-MMP-2 is expressed in only restricted tissues such as brain, heart, and placenta. In particular, brain tissue expresses MT-MMP-2 at the highest level but expresses MT-MMP-1 at the lowest level. The high level expression of MT-MMP-2 in brain may suggest a specific role of the product in the central nervous system. In cell lines, squamous cell carcinoma OSC-19 and human embryonal lung fibroblasts express MT-MMP-1 mRNA at higher levels but MT-MMP-2 mRNA at lower levels. The reverse was also the case; the MT-MMP-1 mRNA level was low in T24 cells where MT-MMP-2 mRNA was expressed predominantly.
Since MT-MMP-2 was originally detected in
oral malignant melanoma, whether MT-MMP-2 is also involved in
activation of gelatinase A in tumor tissues like MT-MMP-1 is of
interest. Our preliminary findings with lung carcinomas where MT-MMP-1
was overexpressed indicated that MT-MMP-2 was not expressed frequently
there. ()
In addition to the activation of pro-gelatinase
A, some of the proteins, such as -amyloid precursor protein, tumor
necrosis factor-
, and V-2 vasopressin receptor, are reported to be
processed on the cell membrane by MMP-like
activities(37, 38, 39, 40) . MT-MMPs
may be responsible for the processing of many biologically important
proteins on the cell surface as a regulator. These possibilities remain
to be examined.
In this study, we reported MT-MMP-2 as a new member of the MMP family. Since both MT-MMP-1 and -2 have TM domains at the C terminus, we proposed a new subclass for MT-MMPs in the MMP family. Studies of MT-MMPs should provide clues to understand the cell surface events controlling extracellular matrix turnover and diverse biological responses.
Note added in Proof-Two new MT-MMP-2s were reported at the Gordon Research Conference on Matrix Metalloproteinases (Andover, NH July 16-21, 1995). The one reported by Will et al. (Will, B., and Hinzmann, B.(1995) Eur. J. Biochem. 231, 602-608) was published just prior to this article. Thus, we agreed to rename MT-MMP-2 in this paper as MT-MMP-3.