(Received for publication, July 21, 1994; and in revised form, October 20, 1994)
From the
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 -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.
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 ()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.
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 -chain (IL-2 R(
)) (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
MT-MMP/TM
) and a
truncated mutant (
MT-MMP) were constructed by cDNA manipulation as
described under ``Materials and Methods'' and expressed by a
eukaryotic expression vector (pGS5).
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.
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), MT-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,
MT-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.
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.
Figure 5:
Expression of MT-MMP and its release
into culture medium. COS-1 cells were co-transfected with pSG5 (Control), MT-MMP, or
MT-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
MT-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 MT-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
-galactosidase, a cytoplasmic protein, and it
was detected only in the cell lysate (Fig. 6A). On the
contrary, both MT-MMP and
MT-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
MT-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
MT-MMP and MT-MMP co-purified with the membrane fractions (Fig. 6B). This is also another indication that
MT-MMP is still associating with the cell surface.
Figure 6:
Localization of MT-MMP and MT-MMP on
the plasma membrane. A, the
-galactosidase (
-Gal)
expression plasmid was co-transfected into COS-1 cells with pSG5,
MT-MMP, and MT-MMP plasmids. Living cells were then incubated with
mAbs against MT-MMP and
-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,
MT-MMP, and MT-MMP plasmids were analyzed by Western
blotting using anti-MT-MMP mAb (113-5B7) and anti-TIMP-1
mAb.
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 -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,
MT-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
MT-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 MT-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
MT-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 MT-MMP/TM
will be valuable in dissecting the specific elements of MT-MMP
further.