Matrilysin (matrix metalloprotease-7) cleaves
membrane-bound annexin II and enhances binding of
tissue-type plasminogen activator to cancer cell surfaces
Jun Tsunezumi
1,2
, Kazuhiro Yamamoto
1
, Shouichi Higashi
1,2
and Kaoru Miyazaki
1,2
1 Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan
2 Graduate School of Integrated Sciences, Yokohama City University, Japan
Matrix metalloproteinases (MMPs) form a group of
more than 20 zinc-dependent enzymes that are
involved in the processing of several components of
the extracellular matrix (ECM). They play roles in
many physiological processes, such as bone remodeling
and organogenesis, and have additional roles in the
reorganization of tissues during pathological
conditions such as inflammation and invasion and
metastasis of cancer cells [1,2]. Many recent studies
have provided evidence that the biological activities of
various cell surface molecules are proteolytically
modulated by several MMPs, including membrane-
type MMPs, gelatinase A (MMP-2), gelatinase B
(MMP-9), stromelysin (MMP-3), and matrilysin
(MMP-7) [3–6]. These metalloproteinases are likely to
regulate cellular functions by activating, inactivating or
releasing membrane proteins. Such regulation of cell
surface proteins, as well as MMP-catalyzed degra-

the 35 kDa fragment of annexin II from the cell surface, it significantly
increased tPA binding to the cell membrane. A synthetic N-terminal non-
apeptide of annexin II bound to tPA more efficiently than intact annexin II.
This peptide formed a heterodimer with intact annexin II in test tubes and
on cancer cell surfaces. These and other results suggested that the nonapep-
tide generated by matrilysin treatment might be anchored to the cell mem-
brane, possibly by binding to intact annexin II, and interact with tPA via
its C-terminal lysine. It is supposed that the cleavage of cell surface annex-
in II by matrilysin contributes to tumor invasion and metastasis by enhanc-
ing tPA-mediated pericellular proteolysis by cancer cells.
Abbreviations
ECM, extracellular matrix; MMP, matrix metalloproteinase; PVDF, poly(vinylidene difluoride); siRNA, small interfering RNA; TAPI-1, N-(R)-[2-
(hydroxyaminocarbonyl)-methyl]-4-methylpentanoyl-
L-naphthylalanyl-L-alanine-2-aminoethyl amide; tPA, tissue-type plasminogen activator.
4810 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS
degrade or process a variety of matrix and nonmatrix
molecules [7]. Unlike most MMPs, which are expressed
by stromal cells, matrilysin is principally expressed by
epithelial cells [8]. This enzyme seems to be one of the
most important MMPs in human colon cancers,
because the expression of matrilysin is highly corre-
lated with malignancy and metastatic potential of the
cancers, especially in their liver metastasis [9]. It has
recently been reported that active matrilysin specifi-
cally binds to the surface of colon cancer cells and
induces notable cell aggregation due to processing of
the cell membrane protein(s). Furthermore, these
aggregated cells showed greatly enhanced metastatic
potential in the nude mouse model [10,11]. Therefore,
it seems important to identify cell surface proteins that

cascade on cancer cells.
Results
Cleavage of annexin II by matrilysin
It was previously found that active matrilysin specifi-
cally binds to surfaces of colon cancer cells and
induces prominent cell aggregation [10,11]. In the pres-
ent study, we first analyzed membrane proteins that
are cleaved by matrilysin. A membrane fraction of
WiDr human colon carcinoma cells was prepared by
the phase separation method with Triton X-114. When
the membrane fraction was treated with matrilysin,
several proteins, including a major protein of approxi-
mately 35 kDa, were released from the membrane
fraction (Fig. 1A). The N-terminal amino acid
sequence of the 35 kDa protein was determined to be
B
MAT
−+
116
97
200
(kDa)
A
97
66
45
31
21
N-
-C

LSLEGDHSTPPSAY by automated protein sequenc-
ing, and this sequence was identical to the amino acid
sequence from residues 10 to 23 of annexin II
(Fig. 1B).
To determine whether annexin II is directly cleaved
by matrilysin, we used both a recombinant human
annexin II and a natural annexin II purified from
CaR-1 human colon carcinoma cells. Matrilysin effec-
tively cleaved the 36 kDa recombinant annexin II and
converted it to the 35 kDa form (Fig. 2). This cleavage
was inhibited by an MMP inhibitor, N-(R)-[2-(hydrox-
yaminocarbonyl)-methyl]-4-methylpentanoyl-l-naphthyl-
alanyl-l-alanine-2-aminoethyl amide (TAPI-1), but not
by a mixture of inhibitors for serine, aspartic and
cysteine proteinases. The N-terminal amino acid
sequence of the 35 kDa, cleaved annexin II was iden-
tical to that of the membrane-derived annexin II
fragment (LSLEGDHSTPPSAY). These results indi-
cate that matrilysin cleaves the peptidyl bond between
Lys9 and Leu10 of annexin II (Fig. 1B).
When the annexin II purified from CaR-1 cells was
analyzed by immunoblotting, it showed two distinct
bands at approximately 36 and 72 kDa under non-
reducing conditions, but a single 36 kDa band under
reducing conditions (Fig. 3A). The 72 kDa protein was
thought to be a homodimer of annexin II cross-linked
with a disulfide bond. Next, the natural annexin II was
incubated with matrilysin and four other MMPs, and
then analyzed by immunoblotting under nonreducing
conditions (Fig. 3B). Matrilysin and MMP-2 almost

(TAPI) (Mix.)
Fig. 2. Cleavage of purified annexin II by matrilysin (MAT). Recom-
binant annexin II (1 lgÆmL
)1
) was incubated at 37 °C for 3 h with
(+) or without ())50n
M matrilysin in the presence or absence of
the MMP inhibitor TAPI (4 l
M) or a proteinase inhibitor mixture
[Mix.; 0.2 m
M 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.16 lM ap-
rotinin, 0.025 m
M bestatin, 7.5 lM E-64, 0.01 mM leupeptin, and
5 l
M pepstatin]. The digests were analyzed by immunoblotting with
an antibody against annexin II under reducing conditions. Other
experimental conditions are described in Experimental procedures.
Arrowheads indicate native and cleaved annexin II bands at 36 and
35 kDa, respectively.
A
B
Fig. 3. Immunoblotting of purified natural annexin II and its cleav-
age by five kinds of MMP. (A) Immunoblotting of natural annexin II
purified from CaR-1 cells under nonreducing ()) and reducing (+)
conditions. 2ME, 2-mercaptoethanol. The bands at 72 and 36 kDa
correspond to dimeric and monomeric forms of annexin II, respec-
tively. (B). The natural annexin II (2 lgÆmL
)1
protein) was incubated
in 50 lL of a reaction mixture without (None) or with 5 n

released its soluble form. When CaR-1 cells were trea-
ted with active MMP-2, however, no annexin II frag-
ment was detectable in the culture supernatant,
suggesting that the cleavage of annexin II on the cell
surface is specific for matrilysin (data not shown).
It is known that annexin II binds to glycosaminogly-
cans such as heparan sulfate proteoglycans and sialo-
glycoproteins and phospholipids on the cell surface,
and many of the interactions are mediated by calcium
ions [22,23]. Serotonin (5-hydroxytryptamine) interacts
with N-acetylneuraminic acid, which is often contained
in glycolipids and glycoproteins on the cell surface
[24]. We examined the synergistic effects of matrilysin
with serotonin, heparin and EDTA on the release of
annexin II from the cell surface. WiDr cells and CaR-1
cells were treated with serotonin, heparin or EDTA in
the presence or absence of matrilysin, and the released
annexin II was analyzed by immunoblotting (Fig. 5).
When WiDr cells were treated with each of these
reagents, the intact (or full-length) annexin II was
released into the culture supernatant at a higher level
than the 35 kDa annexin II released by the matrilysin
treatment alone. When the cells were treated with sero-
tonin or heparin in the presence of matrilysin, release
MAT
–
+
–
+
–

M matrilysin (MAT) at 37 °C for 3 h. Proteins
released into the culture medium were concentrated by trichloro-
acetic acid precipitation and analyzed by immunoblotting under
reducing conditions with the antibody against annexin II. As a con-
trol, whole lysate of WiDr cells was run on the same gel. (B) CaR-1
cells were treated with (MAT) or without (None) matrilysin as
above, and the amount of annexin II remaining on the cell surface
was measured by cell ELISA. Each value represents the
mean ± SD of three independent results. (C) CaR-1 cells were
treated with (MAT) or without (None) matrilysin as above, and ann-
exin II remaining on the cell surface was visualized by immunofluo-
rescence staining. Detailed experimental conditions are described
in Experimental procedures.
Fig. 5. Release of membrane-bound annexin II by matrilysin and
three reagents. CaR-1 cells and WiDr cells in monolayer cultures
were incubated in the serum-free medium without ()) or with (+)
50 n
M matrilysin (MAT) in the presence or absence (None) of
0.2 m
M serotonin or 1 mgÆmL
)1
heparin at 37 °C for 3 h as
described in Fig. 4. Alternatively, the same cultures were incubated
with 5 m
M EDTA at 25 °C for 5 min. Proteins released into the
culture medium were analyzed by immunoblotting with the
antibody against annexin II as described in Fig. 4. Other experi-
mental conditions are described in Experimental procedures.
J. Tsunezumi et al. Cleavage of annexin II by matrilysin
FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4813

ple buffer and subjected to immunoblotting for tPA (top panel) and enolase as an internal loading control (center panel). Annexin II released
into the culture supernatant by the matrilysin/serotonin treatment is shown in the lower panel. (B) Detection of tPA bound to the cell surface
by cell ELISA. CaR-1 cells were pretreated without (None) or with 50 n
M matrilysin (MAT) on 96-well plates for 3 h, and incubated with tPA
and TAPI-1 as above. To quantify tPA bound to the cell surface, the cultures were subjected to cell ELISA according to the method
described in Experimental procedures. Each value represents the mean ± SD of triplicate assays. (C) Enzymatic activity of tPA bound to the
cell surface. CaR-1 cells were treated with the indicated concentrations of matrilysin and then with tPA as shown above. The catalytic activ-
ity of tPA bound to the cell surface was assayed using the fluorogenic peptide 3145v as a substrate. Each value represents the mean ± SD
of triplicate assays. Annexin II released into the culture supernatant by the matrilysin treatment is shown in the lower panel. (D) Effects of
annexin II siRNA on tPA binding to matrilysin-treated cells. CaR-1 cells were inoculated onto 24-well culture plates and treated with annex-
in II siRNA or a control RNA. Two days later, these cells were treated with matrilysin and then tPA as described above. The cells were
washed, lysed in the SDS/PAGE sample buffer and subjected to immunoblotting for tPA, annexin II (ANX-II) and enolase-1 as an internal
loading control. The cleaved annexin II (Sol. ANX-II) released into the culture medium is shown in the upper panel. Other experimental condi-
tions are described in Experimental procedures.
Cleavage of annexin II by matrilysin J. Tsunezumi et al.
4814 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS
and serotonin on tPA binding to CaR-1 cells
(Fig. 6A). Unexpectedly, the single treatment with
matrilysin significantly increased the binding of exoge-
nous tPA to CaR-1 cells, while releasing the cleaved
annexin II into the culture supernatant. Furthermore,
when CaR-1 cells were treated with both matrilysin
and serotonin, tPA binding to the cell surface was
greatly enhanced by the presence of serotonin. The
enhancement of tPA binding to the cell surface was
coincident with the release of cleaved annexin II. The
enhancement of tPA binding to the cell surface by
matrilysin was confirmed when the amount of tPA on
the cell surface was assayed by cell-based ELISA
(Fig. 6B). Moreover, the assay of tPA activity on the

about twice that bound to the control plates (Fig. 7A).
Next, tPA binding was examined in the presence or
absence of Ac-STVHEILCK on the plates precoated
with the purified, native annexin II or with the
annexin II cleaved by matrilysin. The tPA binding was
slightly but significantly more efficient on the cleaved
annexin II than on the native one, and on either plate
the addition of the soluble N-terminal peptide signifi-
cantly suppressed tPA binding to the plate (Fig. 7B).
These results suggested that tPA was able to bind
Ac-STVHEILCK. The competitive effect of the syn-
thetic peptide was also examined for tPA binding to
CaR-1 cells. tPA, with or without the peptide, was
applied to the cells pretreated with or without matrilysin
(Fig. 7C). Although Ac-STVHEILCK at 0.2 mm had
no effect on the nontreated cells, it significantly inhibited
tPA binding to the CaR-1 cells pretreated with
matrilysin. These results strongly suggested that the
matrilysin-enhanced tPA binding to cell membranes
depended, at least in part, on the N-terminal peptide
fragment Ac-STVHEILCK, which was generated by the
matrilysin-catalyzed cleavage of annexin II.
To obtain further evidence that the N-terminal
peptide binds tPA on the cell surface, CaR-1 cells,
without matrilysin treatment, were incubated with
the N-terminal peptide and then with tPA. Treat-
ment of CaR-1 cells with the peptide increased the
amount of tPA bound to the cell surface (Fig. 8A).
This implies that the N-terminal peptide binds both
an unidentified cell surface molecule and tPA on the

J. Tsunezumi et al. Cleavage of annexin II by matrilysin
FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4815
presence of heparin (Fig. 8C, lane 5). In addition,
the 37 kDa annexin II heterodimer was faintly
detected even when the purified annexin II was
digested by matrilysin (Fig. 8C, lane 2), although it
did not increase in amount when the cleaved
annexin II was incubated with the uncleaved form
(lane 3). These results suggested that the 37 kDa
nonapeptide–intact annexin II complex might be
produced on the matrilysin-treated cancer cells.
However, we failed to recover the 37 kDa annexin II
complex from the matrilysin-treated cells (data
not shown). This is probably due to the low
concentration of the peptide in the matrilysin-treated
cells.
It has been reported that tPA binds to a lysine
residue via its kringle-2 domain [27]. This suggests
that tPA binds to the C-terminal lysine residue of
Ac-STVHEILCK, which is produced from annexin II
by matrilysin treatment. To test this possibility, we
performed a competition assay using e-aminocaproic
acid as a C-terminal lysine analog. e-Aminocaproic
acid at 10 mm strongly inhibited tPA binding to both
the matrilysin-treated cells and the untreated cells
(Fig. 8D). This competitive effect was much more
evident than that obtained with 0.2 mm
Ac-STVHEILCK (Fig. 7C). However, 1 mm e-amino-
caproic acid scarcely inhibited tPA binding (data not
shown). These results support the hypothesis that the

digested (Cleaved) annexin II were individually coated on 96-well
microtiter plates overnight. Using these annexin II-coated wells, the
tPA binding assay in the presence (+) or absence ()) of 200 l
M
Ac-STVHEILCK was carried out as described above. (C) Competitive
inhibitory effect of Ac-STVHEILCK on tPA binding to CaR-1 cells.
CaR-1 cells were incubated with 50 n
M matrilysin on 96-well plates
at 37 °C for 3 h. The tPA binding to the CaR-1 cells in the presence
(+) or absence ()) of 200 l
M Ac-STVHEILCK was analyzed as
shown in Fig. 5B. In (A), (B) and (C), the data represent the
mean ± SD of triplicate assays.
Cleavage of annexin II by matrilysin J. Tsunezumi et al.
4816 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS
Discussion
The present study identified annexin II as a novel mem-
brane-bound substrate for matrilysin. Matrilysin
cleaved annexin II on the surfaces of human colon
cancer cells, releasing a major C-terminal sequence of
annexin II from the cell membrane. The matrilysin
treatment of cancer cells facilitated the binding of tPA
to the cell surface. We previously showed that active
matrilysin efficiently binds to cholesterol sulfate on the
cell membranes of colon cancer cells, retaining its enzy-
matic activity [11]. This MMP, together with cholesterol
sulfate, was localized in the lipid microdomain so-called
raft of cell membrane [11]. Annexin II is also localized
in the membrane domain raft [28]. Thus, it is highly
likely that the membrane-bound active matrilysin

treated with (+) or without () ) matrilysin (MAT) on 24-well or 96-well culture plates and then incubated with 5 n
M tPA plus 5 lM TAPI-1 in
the presence (+) or absence ())of10m
M e-aminocaproic acid (eACA). The amounts of tPA on the cell surface were analyzed by immuno-
blotting (left panel) and cell ELISA (right panel). Each bar represents the mean ± SD of triplicate assays. Other experimental conditions are
described in Fig. 6 and Experimental procedures.
J. Tsunezumi et al. Cleavage of annexin II by matrilysin
FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4817
efficiently cleaves annexin II on cancer cell surfaces.
MMP-2 and MMP-9 cleaved purified annexin II, but
they appeared not to cleave annexin II on the cell sur-
face, indicating that the cleavage of the membrane-
bound annexin II is specific for matrilysin. The specific
cleavage of cell surface annexin II by matrilysin may
result from the specific binding of matrilysin to the
cancer cells. In our previous study, among three MMPs
tested (matrilysin, MMP-2 and MMP-3), only matrily-
sin was able to bind to the cancer cells [10] and choles-
terol sulfate [11].
Annexin II is expressed in epithelial cells of various
tissues, including the epidermis, pancreas and breast
[19–21], and vascular endothelial cells [25]. In these
kinds of cells, some annexin II molecules are found on
the cell surface. Annexin II is known to interact with
membrane phospholipids and glycosaminoglycans such
as heparin, heparan sulfate [22,23] and fucoidan as a
sulfated fucopolysaccharide, in a calcium-dependent or
calcium-independent manner [29,30]. Serotonin is
known to interact with glycolipids and glycoproteins
containing N-acetylneuraminic acid [24]. In this study,

decreased tPA binding (Fig. 6). Second, tPA bound to
the synthetic peptide coated on plastic plates in a dose-
dependent manner, and the tPA binding was more effi-
cient than that to the entire annexin II molecule
(Fig. 7A,B). Third, pretreatment of colon cancer cells
with the synthetic peptide significantly increased tPA
binding to the cells, whereas the peptide competitively
suppressed tPA binding to the matrilysin-treated cells
(Figs 7C and 8A). All these results support the hypoth-
esis that the N-terminal annexin II peptide produced
by matrilysin remains bound to cell membranes and
functions as a receptor for tPA. tPA is known to have
high affinity for lysine. tPA binds to lysyl–Sepharose
through its kringle-2 domain, and this interaction is
blocked by l-lysine or e-aminocaproic acid as a C-ter-
minal lysine analog [27]. The kringle-2 domain of tPA
directly interacts with e-aminocaproic acid [35]. Krin-
gle-2-mediated tPA binding to the C-terminal lysines
plays an important role in the degradation of fibrin
clots [36,37]. Partial degradation of fibrin by plasmin
generates C-terminal lysines, which function as new
binding sites for tPA, resulting in further activation of
plasminogen on the fibrin clot [38]. On the basis of
these facts, it seems very likely that tPA binds to the
C-terminal lysine of the N-terminal annexin II frag-
ment Ac-STVHEILCK remaining on cell membranes.
Although we cannot exclude the possibility that tPA
binds to the C-terminal lysines of other protein frag-
ments that are produced by the matrilysin activity, the
result of the siRNA experiment with annexin II shown

which is often regarded as the annexin II light chain,
to form a heterotetramer complex [26]. In this study,
we detected the possible annexin II dimer of approxi-
mately 72 kDa, but we failed to detect p11 in the
annexin II complex with a specific antibody (data not
Cleavage of annexin II by matrilysin J. Tsunezumi et al.
4818 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS
shown). Our data indicated that exogenous N-terminal
annexin II peptide bound to the cancer cell surface, and
the bound peptide was recovered as a 37-kDa heterodi-
mer complex with the intact annexin II molecule from
the cell surface when the cells were treated with heparin.
This heterodimer complex was linked by a disulfide
bond, and was produced efficiently when the peptide
was incubated with the intact annexin II in test tubes.
These results strongly suggest that the N-terminal ann-
exin II peptide remains as the 37 kDa complex with the
intact annexin II on the surfaces of matrilysin-treated
cells. However, this possibility was not confirmed,
because we failed to detect this nonapeptide–annexin II
complex in the matrilysin-treated cancer cells (data not
shown). Thus, it is also possible that the N-terminal
annexin II peptide binds to cell membranes through p11
or other membrane molecules.
The plasminogen activator–plasmin system is well
known to play important roles not only in fibrinolysis
but also in ECM degradation during tissue remodeling
[39,40]. Like urokinase-type plasminogen activator,
tPA binds to some membrane proteins, including ann-
exin II [41]. The binding of tPA or urokinase-type

cleavage of membrane protein(s) [13,14]. However, the
cleavage of annexin II by matrilysin appeared not to
induce cell–cell adhesion, because the suppression of
annexin II synthesis by RNA interference did not inhi-
bit cell aggregation (data not shown). Therefore, it is
likely that membrane proteins other than annexin II
are also degraded or processed by matrilysin. These
actions of matrilysin may also contribute to the malig-
nant growth of cancer cells. Understanding the patho-
logical significance of the cleavage of annexin II and
other membrane substrates by matrilysin seems to be
important in designing new targets for cancer therapies.
Experimental procedures
Antibodies and other reagents
The sources of reagents used were as follows: human
recombinant annexin II was from AmProx (Carlsbad, CA,
USA); human Glu-plasminogen and Lys-plasminogen were
from Hematologic Technologies (Essex Junction, VT,
USA); tPA and Protease Inhibitor Cocktail Set III were
from Calbiochem (San Diego, CA, USA); human recom-
binant MMP-9, human recombinant interstitial collage-
nase (MMP-1) and MMP-3 were from Chemicon
(Temecula, CA, USA); human recombinant matrilysin and
6-aminohexanoic acid (e-aminocaproic acid) were from
Wako Pure Chemical Industries (Osaka, Japan); the MMP
substrates 3145v (Pyr-Gly-Arg-MCA) and 3105v (Boc-Glu-
Lys-Lys-MCA) and the synthetic MMP inhibitor TAPI-1
were from Peptides Institute (Osaka, Japan); and serotonin
(5-hydroxytryptamine hydrochloride) was from Sigma
Aldrich (St Louis, MO, USA). Commercial antibodies

were transferred onto poly(vinylidene difluoride) (PVDF)
membranes (Millipore, Billerica, MA, USA) and visualized
by the alkaline phosphatase method or the enhanced chemi-
luminescence method (GE Healthcare, Amersham, UK)
with specific antibodies.
Suppression of annexin II expression by RNA
interference
A predesigned siRNA corresponding to the target sequence
for human annexin II (5¢-UGGAAAGCAUCAGGAAA
GAGGUUAA-3¢) and a control RNA were obtained from
iGENE (Tsukuba, Japan). Cells were inoculated on the day
before transfection at a cell density of approximately 30%
saturation in 24-well culture plates and treated with the
siRNA or the control RNA by using the HiperFect reagent
(Qiagen, Tokyo) according to the manufacturer’s protocol.
Two or three days later, the cells were used for experiments.
Phase separation of membrane-associated
molecules in Triton X-114 solution
To separate membrane-associated proteins from soluble
ones, we used phase separation of the Triton X-114 solu-
tion, as described previously [11]. WiDr cells (approxi-
mately 3 · 10
8
cells) were dissolved in 1.2 mL of 50 mm
Tris/HCl (pH 7.5) containing 150 mm NaCl and 5 mm
CaCl
2
(TBSC) supplemented with 0.1% Triton X-114 and
centrifuged at 95 g at 4 °C for 5 min. The resultant super-
natant was added to Triton X-114 to make a final concen-

TBSC and 0.01% Brij 35 at 37 °C for the indicated lengths
of time. The reaction was stopped by mixing with the SDS
sample buffer, and the reaction mixture was analyzed by
SDS/PAGE and immunoblotting. In some experiments,
membrane fractions instead of the purified annexin II were
used as the substrates. For determination of N-terminal
sequences, the digested proteins were separated by SDS/
PAGE, transferred to PVDF membranes, and stained with
Coomassie Brilliant Blue R-250. Stained protein bands were
cut from the membranes and analyzed with a Procise 49X
cLC protein sequencer (Applied Biosystems, Foster City,
CA, USA).
Cleavage of cell surface annexin II by matrilysin
Cancer cells were harvested by trypsinization, inoculated at
a density of 5 · 10
6
cells per 60 mm culture dish in the
growth medium, and incubated for 2 days. The cultures
were washed twice with the serum-free DMEM/F12 med-
ium and then incubated in 2 mL of the serum-free medium
containing 50 nm matrilysin at 37 °C for 3 h. Proteins
released into the culture medium were collected, precipi-
tated in 10% trichloroacetic acid, washed with ethanol, and
analyzed by immunoblotting.
Immunofluorescence staining of cell surface
annexin II
CaR-1 cells were inoculated onto four-well Lab-Tek chamber
slides (Nagel Nunc; Naperville, IL, USA) in the growth
medium for 2 days. The cultures were washed twice with the
serum-free medium, and incubated in the medium containing

with 50 nm matrilysin at 37 °C for 3 h. After the incubation,
each culture was washed three times with NaCl/P
i
containing
1mm CaCl
2
,1mm MgCl
2
and 6 mm glucose, incubated
with 5 n m tPA and 5 lm TAPI-1 as an MMP inhibitor at
37 °C for 1 h, and washed three times. The cells were fixed
with 10% formaldehyde for 10 min, washed three times with
NaCl/P
i
containing 0.1% Tween-20, and blocked with 1.2%
BSA in NaCl/P
i
. Finally, each culture was sequentially incu-
bated with a monoclonal antibody against tPA and with a
biotinylated second antibody (Vector Laboratories) at 37 °C
for 1 h. The intensity of immunoreactive signals for tPA was
measured by the alkaline phosphatase method with p-nitro-
phenylphosphate as a substrate. Alternatively, tPA on the
cell surface was detected by measuring its enzyme activity.
In this assay, cells carrying the exogenous tPA were incu-
bated with 200 lm fluorogenic peptide 3145v as substrate at
37 °C for 40 min. Substrate hydrolysis was determined by
measuring fluorescence at 390 nm for excitation and at
460 nm for emission, using a Plate Chameleon spectrofluo-
rometer (Hidex, Turku, Finland).

Shinomura Y (2006) Role of matrix metalloproteinase-7
(matrilysin) in human cancer invasion, apoptosis,
growth, and angiogenesis. Exp Biol Med 231, 20–27.
8 Miyazaki K, Hattori Y, Umenishi F, Yasumitsu H &
Umeda M (1990) Purification and characterization of
extracellular matrix-degrading metalloproteinase, matrin
(pump-1), secreted from human rectal carcinoma cell
line. Cancer Res 50, 7758–7764.
9 Hasegawa S, Koshikawa N, Momiyama N, Moriyama
K, Ichikawa Y, Ishikawa T, Mitsuhashi M, Shimada H
& Miyazaki K (1998) Matrilysin-specific antisense oligo-
nucleotide inhibits liver metastasis of human colon
cancer cells in a nude mouse model. Int J Cancer 76,
812–816.
10 Kioi M, Yamamoto K, Higashi S, Koshikawa N, Fujita
K & Miyazaki K (2003) Matrilysin (MMP-7) induces
homotypic adhesion of human colon cancer cells and
enhances their metastatic potential in nude mouse
model. Oncogene 22, 8662–8670.
11 Yamamoto K, Higashi S, Kioi M, Tsunezumi J, Honke
K & Miyazaki K (2006) Binding of active matrilysin to
cell surface cholesterol sulfate is essential for its mem-
brane-associated proteolytic action and induction of
homotypic cell adhesion. J Biol Chem 281, 9170–9180.
12 Massey-Harroche D, Mayran N & Maroux S (1998)
Polarized localizations of annexins I, II, VI and XIII in
epithelial cells of intestinal, hepatic and pancreatic
tissues. J Cell Sci 111, 3007–3015.
13 Siever DA & Erickson HP (1997) Extracellular ann-
exin II. Int J Biochem Cell Biol 29, 1219–1223.

worth MA & Pour PM (1993) Enhanced expression of
annexin II in human pancreatic carcinoma cells and pri-
mary pancreatic cancers. Carcinogenesis 14, 2575–2579.
21 Sharma MR, Koltowski L, Ownbey RT, Tuszynski GP
& Sharma MC (2006) Angiogenesis-associated protein
annexin II in breast cancer: selective expression in inva-
sive breast cancer and contribution to tumor invasion
and progression. Exp Mol Pathol 81, 146–156.
22 Kassam G, Manro A, Braat CE, Louie P, Fitzpatrick
SL & Waisman DM (1997) Characterization of the hep-
arin binding properties of annexin II tetramer. J Biol
Chem 272, 15093–15100.
23 Shao C, Zhang F, Kemp MM, Linhardt RJ, Waisman
DM, Head JF & Seaton BA (2006) Crystallographic
analysis of calcium-dependent heparin binding to
annexin A2. J Biol Chem 281, 31689–31695.
24 Sturgeon RJ & Sturgeon CM (1982) Affinity chroma-
tography of sialoglycoproteins, utilising the interaction
of serotonin with N-acetylneuraminic acid and its deriv-
atives. Carbohydr Res 103, 213–219.
25 Hajjar KA, Jacovina AT & Chacko J (1994) An endo-
thelial cell receptor for plasminogen/tissue plasminogen
activator. I. Identity with annexin II. J Biol Chem 269,
21191–21197.
26 Becker T, Weber K & Johnsson N (1990) Protein–pro-
tein recognition via short amphiphilic helices; a muta-
tional analysis of the binding site of annexin II for p11.
EMBO J 9, 4207–4213.
27 de Munk GA, Caspers MP, Chang GT, Pouwels PH,
Enger-Valk BE & Verheijen JH (1989) Binding of

33 Ryzhova EV, Vos RM, Albright AV, Harrist AV,
Harvey T & Gonzalez-Scarano F (2006) Annexin 2: a
novel human immunodeficiency virus type 1 Gag
binding protein involved in replication in monocyte-
derived macrophages. J Virol 80, 2694–2704.
34 Cesarman GM, Guevara CA & Hajjar KA (1994) An
endothelial cell receptor for plasminogen/tissue plasmin-
ogen activator (t-PA). II. Annexin II-mediated enhance-
ment of t-PA-dependent plasminogen activation. J Biol
Chem 269, 21198–21203.
35 van Zonneveld AJ, Veerman H & Pannekoek H
(1986) On the interaction of the finger and the krin-
gle-2 domain of tissue-type plasminogen activator with
fibrin. Inhibition of kringle-2 binding to fibrin by
epsilon-amino caproic acid. J Biol Chem 261, 14214–
14218.
36 Verheijen JH, Caspers MP, Chang GT, de Munk GA,
Pouwels PH & Enger-Valk BE (1986) Involvement of
finger domain and kringle 2 domain of tissue-type plas-
minogen activator in fibrin binding and stimulation of
activity by fibrin. EMBO J 5, 3525–3530.
37 Bakker AH, Weening-Verhoeff EJ & Verheijen JH
(1995) The role of the lysyl binding site of tissue-type
plasminogen activator in the interaction with a forming
fibrin clot. J Biol Chem 270, 12355–12360.
38 de Vries C, Veerman H & Pannekoek H (1989) Identifi-
cation of the domains of tissue-type plasminogen activa-
tor involved in the augmented binding to fibrin after
Cleavage of annexin II by matrilysin J. Tsunezumi et al.
4822 FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS

trypsin-2 enhances in vivo invasion of human tongue
carcinoma cells. J Dent Res 81, 831–835.
46 Jin X, Yagi M, Akiyama N, Hirosaki T, Higashi S, Lin
CY, Dickson RB, Kitamura H & Miyazaki K (2006)
Matriptase activates stromelysin (MMP-3) and pro-
motes tumor growth and angiogenesis. Cancer Sci 97,
1327–1334.
47 Davis GE, Pintar Allen KA, Salazar R & Maxwell SA
(2001) Matrix metalloproteinase-1 and -9 activation by
plasmin regulates a novel endothelial cell-mediated
mechanism of collagen gel contraction and capillary
tube regression in three-dimensional collagen matrices.
J Cell Sci 114, 917–930.
J. Tsunezumi et al. Cleavage of annexin II by matrilysin
FEBS Journal 275 (2008) 4810–4823 ª 2008 The Authors Journal compilation ª 2008 FEBS 4823