Identification of a Region of
-Amyloid Precursor Protein
Essential for Its Gelatinase A Inhibitory Activity*
Shouichi
Higashi
and
Kaoru
Miyazaki
From the Division of Cell Biology, Kihara Institute for Biological
Research, Yokohama City University, Maioka-cho 641-12, Totsuka-ku, Yokohama 244-0813, Japan
Received for publication, December 3, 2002, and in revised form, February 11, 2003
 |
ABSTRACT |
Because
-amyloid precursor protein (APP) has the
abilities both to interact with extracellular matrix and to inhibit
gelatinase A activity, this molecule is assumed to play a regulatory
role in the gelatinase A-catalyzed degradation of extracellular matrix. To determine a region of APP essential for the inhibitory activity, we
prepared various derivatives of APP. Functional analyses of proteolytic
fragments of soluble APP (sAPP) and glutathione
S-transferase fusion proteins, which contain various
COOH-terminal parts of sAPP, showed that a site containing residues
579-601 of APP770 is essential for the inhibitory
activity. Moreover, a synthetic decapeptide containing the ISYGNDALMP
sequence corresponding to residues 586-595 of APP770 had a
gelatinase A inhibitory activity slightly higher than that of sAPP.
Studies of deletion of the NH2- and COOH-terminal residues
and alanine replacement of internal residues of the decapeptide further
revealed that Tyr588, Asp591, and
Leu593 of APP mainly stabilize the interaction between
gelatinase A and the inhibitor. We also found that the residues of
Ile586, Met594, and Pro595 modestly
contribute to the inhibitory activity. The APP-derived decapeptide
efficiently inhibited the activity of gelatinase A (IC50 = 30 nM), whereas its inhibitory activity toward membrane type 1 matrix metalloproteinase was much weaker (IC50 = 2 µM). The decapeptide had poor inhibitory activity toward
gelatinase B, matrilysin, and stromelysin (IC50 > 10 µM). The APP-derived inhibitor formed a complex with
active gelatinase A but not with progelatinase A, and the complex
formation was prevented completely by a hydroxamate-based synthetic
inhibitor. Therefore, the decapeptide region of APP is likely an active
site-directed inhibitor that has high selectivity toward gelatinase A.
 |
INTRODUCTION |
Matrix metalloproteinases
(MMPs)1 are a family of
zinc-dependent endopeptidases that degrade components of
extracellular matrix (ECM) and play an essential role in tissue
remodeling under physiological and pathological conditions such as
morphogenesis, angiogenesis, tissue repair, and tumor invasion (1-3).
Most MMPs are secreted as an inactive zymogen and are activated by
serine proteases or some activated MMPs. The activities of activated
MMPs are regulated by a family of inhibitors known as tissue inhibitors
of metalloproteinases (TIMPs). The members of TIMP family have broad
specificity against MMPs; the activities of almost all MMPs are
susceptible to TIMP (TIMP-1-TIMP-4) inhibition, and some members of a
disintegrin and metalloproteinase family are also inhibited by TIMP-3
(4-6). Recently, many hydroxamate-based inhibitors or other synthetic MMP inhibitors have been designed to develop drugs for the treatment of
diseases in which MMPs are involved (7-10). However, none of them is a
specific inhibitor of individual MMPs. A common architecture of
catalytic sites of MMPs probably relates to the broad specificity of
the inhibitors. Among the MMP family, gelatinase A (MMP-2) and
gelatinase B (MMP-9) are critical in the invasion of tumor cells across
basement membranes because of their strong activity against type IV
collagen, a major component of basement membranes (11-13). Unlike
other zymogen of MMPs, activation of progelatinase A is catalyzed
specifically by a novel type of MMP that has a transmembrane domain and
is thus localized on the cell surface (14). So far, six members of
membrane type MMP have been identified, and membrane type 1-MMP
(MT1-MMP)-catalyzed progelatinase A activation has been well
characterized (15-17). In the activation of progelatinase A, the
protease zymogen is firstly recruited on cell surface via MT1-MMP
complexed with TIMP-2, and then a TIMP-2-free, noninhibited form of
MT1-MMP also present on cell surface cleaves the propeptide of
progelatinase A to initiate the activation (14, 18, 19). After the
activation, gelatinase A retained on cell surface plays a role in the
local degradation of extracellular matrix. It is thought that both the
cell-associated active gelatinase A and MT1-MMP are recruited into
invadopodia, thus limiting proteolysis to the site of cell invasion
(20). Therefore, exertion of gelatinase A activity is strictly
regulated on its zymogen activation level. Recently, we further
proposed that the ECM degrading activity of gelatinase A can be
regulated through an MT1-MMP-catalyzed processing of the
-amyloid
precursor protein (APP).2
APP is a type I integral membrane protein that was initially identified
as a precursor of
-amyloid peptide, the principal component of
extracellular deposits in senile plaques observed in Alzheimer's
disease brain (21). In cultured cells, APP synthesized and maturated
through the constitutive secretory pathway is proteolytically cleaved
at the cell surface within the
-amyloid sequence, and the
extracellular domain of APP is released as a soluble APP (sAPP) into
the culture medium (22, 23). Because the extracellular domain of APP
has the abilities both to interact with components of ECM (24-28) and
to inhibit gelatinase A activity (29), this molecule is assumed to
protect ECM from the gelatinase A-catalyzed degradation. On the other
hand, we recently found that MT1-MMP-catalyzed processing of APP
releases a soluble fragment of APP that lacks the inhibitor domain
against gelatinase A.2 This inhibitor domain-less fragment
is still able to interact with ECM, and it displaces ECM-associated
sAPP or APP, thereby removing the gelatinase A inhibitor from ECM.
Together with the MT1-MMP-catalyzed activation of progelatinase A, the
removal of gelatinase A inhibitor from ECM facilitates the ECM
degradation only in the vicinity of MT1-MMP. Because the
MT1-MMP-catalyzed cleavage of APP at a peptide bond between
Asn579 and Met580 leads to release of the
inhibitor-less fragment,2 the COOH-terminal part of this
cleavage site is thought to be essential for the inhibitory activity of
sAPP. However, the exact location of the inhibitory domain in APP
remains to be determined. In this study, we identified a minimal
structural unit of the gelatinase A inhibitor domain. Unlike other
natural or synthetic MMP inhibitors, the APP-derived inhibitor was
found to have a high selectivity toward gelatinase A. Therefore, the
determination of the inhibitor structure may provide not only a
detailed understanding of the APP-mediated regulation of gelatinase A
activity but also the potential to design novel anti-cancer drugs that
block specific processes of tumor invasion.
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EXPERIMENTAL PROCEDURES |
Materials--
The sources of materials used are as follows:
human thrombin, phenylmethylsulfonyl fluoride, and glutathione are from
Sigma; gelatin was from Difco (Detroit, MI); goat anti-glutathione
S-transferase (GST) polyclonal antibody was from Rockland
(Gilbertsville, PA); p-aminophenyl mercuric acetate (APMA)
was from Tokyo Kasei (Tokyo, Japan); purified human proMMP-9
(progelatinase B) and human recombinant catalytic domain of MT1-MMP
were from Chemicon International Inc. (Temecula, CA); human recombinant
matrilysin, p-amidinophenyl methanesulfonyl fluoride
hydrochloride (APMSF), and potassium cyanate were from Wako Pure
Chemical Industries (Osaka, Japan); Affi-Gel 10 was from Bio-Rad;
endoproteinase Asp-N was from Roche Molecular Biochemicals; the
Cosmosyl 5C18 column (4.6 × 150 mm) was from Nacalai
Tesque, Inc. (Kyoto, Japan); and synthetic substrates for MMPs, 3163v
(7-methoxycoumarin-4-yl)-acetyl-Pro-Leu-Gly-Leu-[N
-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-Ala-Arg
amide) and 3168v
(7-methoxycoumarin-4-yl)-acetyl-Arg-Pro-Lys-Pro-Val-Glu-norvalyl-Trp-Arg-N
-(2,4-dinitrophenyl)-lysine
amide) were from Peptide Institute, Inc. (Osaka, Japan). All of the
custom oligo-DNA primers were provided by Rikaken Co., Ltd. (Tokyo,
Japan). All of the custom peptides were provided by Bex Co., Ltd.
(Tokyo, Japan). A synthetic hydroxamate-based inhibitor for MMPs,
KB-8301
(4-(N-hydroxyamino)-2R-isobutyl-3S-methylsuccinyl]-L-3-(5,6,7,8-tetrahydro-1-naphthyl)alanine-N-methylamide), was a generous gift from Dr. K. Yoshino (Kanebo Institute for Cancer
Research, Osaka, Japan). All other chemicals were of analytical grade
or the highest quality commercially available.
Proteins--
sAPP was purified from the conditioned medium of
the human bladder carcinoma cell line EJ-1, as described previously
(29). TIMP-2-free and TIMP-2-bound forms of progelatinase A were
separately purified from the conditioned medium of the human
glioblastoma cell line T98G, as described previously (30). TIMP-2 was
purified from the TIMP-2-bound progelatinase A using a SynChropak RP-4 reverse-phase column (SynChrom, Lafayette, IN) according to the method
of Collier et al. (12). Prostromelysin (proMMP-3) was purified from the conditioned medium of the Rous sarcoma
virus-transformed rat liver cell line RSV (Invitrogen) as described
previously (31).
Gene Construction of GST Fusion Proteins That Contain
COOH-terminal Parts of sAPP--
cDNA of human APP770
was a gift from Dr. K. Maruyama (Saitama Medical School). Various parts
of the COOH-terminal region of sAPP were expressed independently as GST
fusion proteins. cDNA encoding amino acid residues 439-678 or
579-678 of APP was amplified by polymerase chain reaction, using the
APP770 cDNA as a template and was ligated into
SmaI-EcoRI sites of pGEX-2TK vector. Mutagenesis by polymerase chain reaction-based methods was performed using the
ligated vector as a template under standard conditions as described
(32). Nucleotide sequences of amplified cDNA were confirmed by
sequence analysis. Sequences of primers for cloning the parts of APP
cDNA were as follows: 5'-CCCGGGGGTGGAATCTTTGGAACAGG-3' (sense
primer containing the SmaI site); 5'-AGTCATGTCGGAATTCTGC-3' (antisense primer containing the EcoRI site) for
APP439-678; 5'-CCCGGGGATGATTAGTGAACCAAGGATC-3' (sense primer
containing the SmaI site); and 5'-AGTCATGTCGGAATTCTGC-3' (antisense primer containing the EcoRI site) for
APP579-678. Sequences of primers for mutagenetic polymerase chain
reaction were as follows: 5'-TAAAGTTACGGAAACGATGCTCTC-3' (sense) and
5'-CCTTGGTTCACTAATCATGTTG-3' (antisense) for introduction of a stop
codon at position 586; 5'-TAACCATCTTTGACCGAAACG-3' (sense) and
5'-GAGAGCATCGTTTCCGTAAC-3' (antisense) for introduction of a stop codon
at position 594; and 5'-TAAACCGTGGAGCTCCTTCCC-3' (sense) and
5'-TTTCGTTTCGGTCAAAGATGG-3' (antisense) for introduction of a stop
codon at position 602.
Expression and Purification of GST-APP439-678--
The
expression vector of GST-APP439-678 was transfected into the
Escherichia coli strain DH5
. The transformant was
cultured in 2× YT medium (0.08% (w/v) tryptone, 0.5% (w/v) yeast
extract, and 0.25% (w/v) NaCl) at 37 °C, and the GST fusion protein
was induced by the addition of 0.5 mM
isopropyl-
-D-thiogalactopyranoside. After a 4-h
induction, the E. coli cells were broken in 50 mM Tris-HCl (pH 8.0) containing 50 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride,
and 5 mM dithiothreitol with a sonicator, and the resultant lysate was clarified by centrifugation. The supernatant was applied to
a glutathione-Sepharose 4B column that had been equilibrated with the
lysis buffer. After washing the column with the equilibration buffer,
the adsorbed protein was eluted with the equilibration buffer
supplemented with 10 mM glutathione. The eluted GST fusion protein was dialyzed against 50 mM Tris-HCl (pH 7.5)
containing 0.1 M NaCl, and its concentration was determined
by the Bradford dye method with a Bio-Rad protein assay kit, using
bovine serum albumin (BSA) as a standard.
Digestion of sAPP with Thrombin--
20 µl of 4.0 µM sAPP was incubated with various concentrations of
human thrombin in 50 mM Tris-HCl (pH 7.5) containing 0.1 M NaCl at 37 °C for 4 h. After incubation, each of
the samples was mixed with 5 µl of 50 mM APMSF, and the
mixture was incubated at 25 °C for 1 h to inactivate thrombin.
The resultant digests were subjected to SDS-PAGE or gelatinase A
reverse zymography analysis.
Digestion of GST-APP439-678 with Lysyl Endopeptidase--
10
µl of 4.0 µM GST-APP439-678 was incubated with various
concentrations of lysyl endopeptidase in 50 mM Tris-HCl (pH
7.5) containing 0.1 M NaCl at 37 °C for 1 h. After
incubation, each of the samples was mixed with 10 µl of 100 mM Tris-HCl (pH 6.8), 4% SDS, and 20% glycerol, and the
mixture was boiled for 5 min to terminate the enzyme reaction. The
resultant digests were subjected to SDS-PAGE and gelatinase A reverse
zymography analyses.
Determination of the Thrombin Cleavage Site in sAPP--
sAPP
(120 µg) was incubated with 0.2 M KNCO in 200 µl of 50 mM Tris-HCl (pH 7.5) containing 0.1 M NaCl at
37 °C for 6 h. The sAPP sample was further incubated with 0.2 M hydroxylamine hydrochloride at 25 °C for 1 h and
then dialyzed extensively against 50 mM Tris-HCl (pH 7.5)
containing 0.1 M NaCl. This treatment appeared to result in
complete carbamylation of the
-amino group of
NH2-terminal Leu18 because no
phenylthiohydantoin derivative was detected in the NH2-terminal sequence analysis on the modified sAPP. The
modified sAPP (2 µM) was then incubated with human
thrombin (25 nM) in 50 mM Tris-HCl (pH 7.5)
containing 0.1 M NaCl at 37 °C. After 4 h, the
sample was further incubated at 25 °C for 1 h with 10 mM APMSF. The resultant digest was blotted onto a
polyvinylidene difluoride membrane, using an Atto Immunodot apparatus
(Tokyo, Japan). The membrane was washed extensively with pure water and then stained with Coomassie Brilliant Blue R-250. The stained spot was
subjected to NH2-terminal sequence analysis.
Assay of Inhibitory Activity of APP-derived
Fragments--
TIMP-2-free form of progelatinase A, progelatinase B,
or prostromelysin was activated by incubation with 1 mM
APMA at 37 °C for 1 h as described previously (30, 33).
Activated gelatinase A (0.61 nM), gelatinase B (4.8 nM), stromelysin (6.2 nM), matrilysin (3.8 nM), and the catalytic domain of MT1-MMP (3.2 nM) were each incubated with various concentrations of
derivatives of APP (sAPP, GST fusion proteins, or peptides) in 190 µl of 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 10 mM CaCl2, 0.01% Brij
35, and 0.01% BSA at 37 °C for 15 min. Then 10 µl of 1 mM 3163v (1 mM 3168v in case of assay of
stromelysin activity) was added to the mixture, and the incubation was
further continued for 40 min. The reaction was terminated by adding 20 µl of 0.5 M EDTA (pH 7.5). The amounts of the synthetic
substrate hydrolyzed by MMPs were measured fluorometrically with
excitation at 360 nm and emission at 460 nm. The amount of the
substrate hydrolyzed without enzyme was subtracted from the total
amount of the hydrolyzed substrate.
Gelatin Zymography and Trypsin Reverse
Zymography--
Zymography (30) and gelatinase A reverse zymography
(29) were carried out on 10 or 12.5% polyacrylamide gels containing 1 mg/ml of gelatin, as described previously.
Western Blotting Analysis of GST Fusion Proteins--
The
expression vectors of GST fusion proteins were transfected into the
E. coli strain DH5
. Each of the transformants was cultured in 2× YT medium at 37 °C, and expression of the GST fusion proteins was induced by the addition of 0.5 mM
isopropyl-
-D-thiogalactopyranoside. After a 4-h
induction, E. coli cells were dissolved in a SDS sampling buffer consisting of 50 mM Tris-HCl (pH 6.8), 2% SDS, and
10% glycerol. The lysates were subjected to SDS-PAGE. After
electrophoresis, the proteins on the gel were transferred onto a
nitrocellulose membrane using a Bio-Rad Mini Trans-Blot apparatus. The
membrane was blocked with phosphate-buffered saline (PBS) containing
5% skim milk at 37 °C for 2 h, washed with PBS containing
0.05% Tween 20 and 0.1% BSA (PBS-Tween), and then incubated at room
temperature with anti-GST polyclonal antibody (0.2 µg/ml) in
PBS-Tween. After a 12-h incubation, the membrane was washed with
PBS-Tween and incubated for 1 h with a biotinylated anti-goat IgG
antibody (Vector Laboratories, Burlingame, CA) that had been diluted
1000-fold with PBS-Tween. After washing with PBS-Tween, the membrane
was incubated with 1000-fold diluted avidin-alkaline phosphatase
(Vector) at room temperature for 1 h. The membrane was washed
extensively and then incubated in a reaction mixture containing
5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium to
develop a colored product on the membrane.
Estimation of Energetic Contribution of Amino Acid Residues of
APP in Gelatinase A Inhibition--
Gelatinase A-catalyzed hydrolysis
of synthetic substrate was measured in the presence of various
concentrations of APP-derived inhibitors as described above. The
IC50 values were determined from the data, where
IC50 represents a concentration of the inhibitors giving a
50% inhibition of the activity of gelatinase A. In the condition that
the concentration of APP-derived inhibitor is excessive to that of
gelatinase A, IC50 is close to [I]50, where
[I]50 represents a concentration of the free inhibitor
giving a 50% inhibition. On the other hand, considering that
APP-derived inhibitor inhibits the gelatinase A activity in a
competitive manner (29), Equation 1 can be derived from the
Michaelis-Menten equation (34).
|
(Eq. 1)
|
where Kd represents a dissociation constant
between enzyme and the inhibitor, [S] is a concentration of free
substrate, and Km is the Michaelis constant for the
gelatinase A-catalyzed hydrolysis of synthetic substrate. On the other
hand, the change in binding energy between gelatinase A and various
APP-derived inhibitors before and after deletion/replacement of a
single amino acid residue is given by Equation 2.
|
(Eq. 2)
|
where Kdb and
Kda are the dissociation constants
(Kd values) for the binding of gelatinase A to various peptide inhibitors before and after the modification, respectively. Considering that a fixed concentration of the
synthetic substrate was used for the assay of gelatinase A activity,
the value of (1 + [S]/Km) can be assumed to be
constant. Therefore, in the condition that the concentration of
peptide inhibitor is excessive to that of gelatinase A, Equation 3 can be derived from Equation 2, using Equation 1.
|
(Eq. 3)
|
where IC50 b and IC50 a are the
IC50 values for inhibition of gelatinase A by various
inhibitors before and after the modification, respectively. The
energetic contributions of individual amino acid residues of the
APP-derived inhibitor in its interaction with gelatinase A were
calculated, using Equation 3.
Amino-terminal Sequence Analysis--
The samples were analyzed
on an Applied Biosystems 477A gas phase sequencer. Phenylthiohydantoin
derivatives were detected using an Applied Biosystems 120A PTH analyzer
with an on-line system.
Assay of Binding of APP-derived Inhibitory Peptide with Various
Forms of Gelatinase A--
30 µl of the TIMP-2-free form of
progelatinase A (930 nM) was incubated with 1 mM APMA at 37 °C for various lengths of time in 50 mM Tris-HCl (pH 7.5) containing 10 mM
CaCl2 and 0.01% Brij 35. After incubation, the sample was
transferred to an ice water container and was added with 270 µl of
ice-cold buffer consisting of 50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 150 mM NaCl, 0.01% Brij 35, and 0.1% Nonidet P-40 to quench the activation reaction. 100 µl
of the preparation was mixed with 20 µl of APP586-601-Affi-Gel 10 beads (200 nmol of hexadecapeptide, which has a sequence corresponding to residues 586-601 of APP770, was coupled to 1 ml of
Affi-Gel 10) and incubated in the presence or absence of 1.0 µM KB-8301 at 4 °C for 2 h with rotation. After
washing the beads with the incubation buffer, the adsorbed sample was
extracted with the SDS sampling buffer consisting of 50 mM
Tris-HCl (pH 6.8), 2% SDS, and 10% glycerol. Various gelatinase A
forms in the extract were analyzed by gelatin zymography. The
gelatinase A samples prepared as described above but incubated without
APP586-601-Affi-Gel 10 beads were also subjected to the zymographic analysis.
 |
RESULTS |
Thrombin Digestion of sAPP--
It has been reported that
recombinant APP produced in E. coli can be cleaved by
thrombin at a peptide bond between Arg585 and
Ile586 (35). This thrombin-susceptible site is near the
Asn578-Met579 bond in sAPP, which is
susceptible to MT1-MMP cleavage.2 To examine whether
thrombin cleavage of sAPP affects its gelatinase A inhibitory activity,
we digested purified sAPP with thrombin and tested the gelatinase A
inhibitory activities of the resultant fragments by reverse zymography.
We found that the digestion of sAPP (105 kDa) mainly yielded a 90-kDa
fragment (Fig. 1B), but this
fragment did not show inhibitory activity toward gelatinase A (Fig.
1A). The LEVPTDGNAG sequence corresponding to residues 18-27 of APP770 was determined in the
NH2-terminal sequence analysis of the 90-kDa fragment,
indicating that this fragment was derived from an
NH2-terminal region of sAPP. To determine the site of cleavage, we tried to isolate the fragment derived from a COOH-terminal region of sAPP by SDS-PAGE. However, isolation of the fragment was not
feasible because it was hardly stained with Coomassie Brilliant Blue
R-250. Then we treated sAPP with KNCO to mask its NH2
terminus by carbamylation and then digested the modified sAPP with
thrombin. The newly exposed NH2 terminus by the thrombin cleavage was analyzed as described under "Experimental Procedures." We found that the treatment of sAPP with KNCO did not affect the inhibitory activity against gelatinase A and that thrombin also cleaved
the modified sAPP to yield the noninhibitory 90-kDa fragment (data not
shown). The ISYGNDALMP sequence corresponding to residues 586-595 of
APP770 was determined in the NH2-terminal
sequence analysis on the digest of the modified sAPP, indicating that
thrombin cleavage occurs at the Arg585-Ile586
bond of sAPP as shown in the previous study (35). These results also
suggest that the large NH2-terminal part of sAPP (residues 18-585) lacks the structural elements essential for the inhibitory activity.

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Fig. 1.
Effect of thrombin digestion on gelatinase A
inhibitory activity of sAPP. 4 µM sAPP was digested
with the indicated concentrations of thrombin at 37 °C for 4 h
as described under "Experimental Procedures," and the resultant
digests were subjected to gelatinase A reverse zymography
(A) and SDS-PAGE (B) under nonreduced conditions.
The samples loaded were the digests equivalent to 0.2 and 5 µg of
sAPP for the reverse zymography and SDS-PAGE, respectively. The bands
of gelatinase A inhibitor (A) or protein bands of sAPP
fragments (B) were visualized by Coomassie Brilliant Blue
R-250 staining. An arrow at 105 kDa indicates a band of
sAPP. Ordinate, molecular mass in kDa.
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Gelatinase A Inhibitory Activity of GST-APP439-678--
In the
previous study (29), lysyl endopeptidase digestion of sAPP yields a
30-kDa fragment that have a gelatinase A inhibitory activity. Because
the inhibitory fragment has an NH2 terminus corresponding
to Val439 of APP770, the gelatinase A inhibitor
domain is suggested to be located in the COOH-terminal
region of sAPP. To examine whether the COOH-terminal part of sAPP has
the entire inhibitory activity, we constructed a GST fusion protein,
which contains the residues of 439-678 of APP770 and
compared the gelatinase A inhibitory activity of the fusion protein
with that of sAPP. The purity of the GST fusion protein (named
GST-APP439-678) expressed and purified as described under
"Experimental Procedures" is shown in Fig. 2A. We found that sAPP and
GST-APP439-678 inhibited the gelatinase A-catalyzed hydrolysis of
synthetic substrate with almost the same IC50 values (Fig.
2B). These results suggest that the GST fusion protein
contains the entire inhibitor domain of APP and that the
NH2-terminal part of sAPP corresponding to residues 18-438 is not required for the inhibitory activity.

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Fig. 2.
Comparison of gelatinase A inhibitory
activities of GST-APP439-678 and sAPP. The GST fusion protein
that contains the residues of 439-678 of APP770 was
purified as described under "Experimental Procedures." In
A, SDS-PAGE of the purified GST fusion protein (3 µg) was
performed under nonreduced condition followed by Coomassie Brilliant
Blue R-250 staining. Lane 1, standard proteins (Bio-Rad low
range); lane 2, the purified GST-APP439-678. In
B, APMA-activated gelatinase A (0.58 nM) was
incubated with 50 µM 3163v at 37 °C for 40 min in the
presence of indicated concentrations of sAPP ( ) or the purified
GST-APP439-678 ( ). All of the reaction mixtures contained 50 mM Tris-HCl, 150 mM NaCl, 10 mM
CaCl2, 0.01% Brij 35, and 0.01% BSA. The amount of 3163v
hydrolyzed in the absence of the derivatives of APP was taken as 100%.
The enzyme activity is shown as the relative amount of 3163v hydrolyzed
by the enzyme on the ordinate.
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Lysyl Endopeptidase Digestion of GST-APP439-678--
To further
explore the gelatinase A inhibitor domain, we digested GST-APP439-678
(65 kDa) with lysyl endopeptidase and tested the gelatinase A
inhibitory activities of the resultant fragments. We found that the
digestion of the GST fusion protein mainly yielded a 52-kDa fragment
(Fig. 3B). Unlike the case of the
90-kDa fragment derived from the thrombin digestion of sAPP, the 52-kDa
fragment showed inhibitory activity toward gelatinase A (Fig.
3A). Deduced from the amino acid sequence of APP
corresponding to residues 439-678 and the molecular size of the
inhibitory fragment, it was expected that lysyl endopeptidase had
cleaved the peptide bond between Lys601 and
Thr602 of the GST-APP439-678 to yield the inhibitory
52-kDa fragment.

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Fig. 3.
Effect of lysyl endopeptidase digestion on
inhibitory activity of GST-APP439-678 toward gelatinase A. 4 µM GST-APP439-678 was digested with the indicated
concentrations of lysyl endopeptidase at 37 °C for 1 h as
described under "Experimental Procedures," and the resultant
digests were subjected to gelatinase A reverse zymography
(A) and SDS-PAGE (B) under nonreduced conditions.
The samples loaded were the digests equivalent to 0.2 and 3 µg of the
GST fusion protein for the reverse zymography and SDS-PAGE,
respectively. The bands of gelatinase A inhibitor (A) or
protein bands of fragments derived from the GST fusion protein
(B) were visualized by Coomassie Brilliant Blue R-250
staining. The arrow at 65 kDa indicates a band of
GST-APP439-678. Ordinate, molecular mass in kDa.
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Gelatinase A Inhibitory Activities of GST Fusion Proteins That
Contain Various COOH-terminal Parts of sAPP--
To verify that the
lysyl endopeptidase cleavage occurs at the
Lys601-Thr602 bond to yield the inhibitory
fragment, we constructed a GST-APP439-601 and examined its gelatinase
A inhibitory activity. As shown in Fig.
4C, GST-APP439-601 expressed in
E. coli gave a 52-kDa band on Western blotting. This GST
fusion protein also showed the inhibitory activity comparable with that
of GST-APP439-678 (Fig. 4B), suggesting that the
COOH-terminal part of sAPP corresponding to residues 602-678 is not
essential for the inhibitory activity. In contrast to GST-APP439-601,
its COOH-terminal 8-residue truncated form GST-APP439-593 did not have
the inhibitory activity in the reverse zymographic analysis (Fig.
4B), although this fusion protein gave a 51-kDa band on
Western blotting (Fig. 4C). We also found GST-APP439-585, which has the same COOH terminus as the noninhibitory 90-kDa fragment derived from the thrombin digestion of sAPP, did not have the inhibitory activity, as expected. We further constructed a
GST-APP579-678 that lacked the NH2-terminal part of the
APP moiety of GST-APP439-678 (Fig. 4A). As shown in Fig.
4B, this fusion protein had inhibitory activity comparable
with that of GST-APP439-678.

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Fig. 4.
Gelatinase A inhibitory activities of GST
fusion proteins that contain various COOH-terminal parts of sAPP.
A shows a schematic diagram of APP comprising 770 amino
acids and its cleavage sites by various proteases. The regions of APP
expressed as the GST fusion proteins in this study are also shown in
this panel. KPI, Kuniz protease inhibitor domain;
CHO, potential Asn-linked glycosylation site.
, , and represent the sites
of cleavage by -, -, and -secretases, respectively.
MT1 and Th represent the sites of cleavage by
MT1-MMP and thrombin, respectively. K-endo represents the
deduced site of lysyl endopeptidase cleavage in GST-APP439-678
observed in Fig. 3. In B and C, each of the
lysates of E. coli cells expressing GST-APP439-678
(lane 1), GST-APP439-601 (lane 2),
GST-APP439-593 (lane 3), GST-APP439-585 (lane
4), and GST-APP579-678 (lane 5) was subjected to
gelatinase A reverse zymography (B) and Western blotting
analysis using the anti-GST polyclonal antibody (C) as
described under "Experimental Procedures." The lane
numbers in B and C also correspond to the
numbers represented on the left of the GST fusion
proteins in A.
|
|
Gelatinase A Inhibitory Activities of Peptides Derived from
APP--
The results in Fig. 4 suggest that the gelatinase A inhibitor
domain is located within a region of APP containing residues 579-601,
and some of the COOH-terminal 8 residues of GST-APP439-601 are
essential for the inhibitory activity. To examine the requirement of
other regions of APP in the inhibitory activity, we tested the
inhibitory activity of a synthetic hexadecapeptide APP586-601, which
encompasses the APP sequence between the thrombin and lysyl endopeptidase cleavage sites. As shown in Fig.
5A, the hexadecapeptide inhibited
the gelatinase A-catalyzed hydrolysis of synthetic substrate (IC50 = 320 nM), and the inhibitory activity
was comparable with that of sAPP (IC50 = 400 nM; Fig. 2B). Because the peptide had sufficient
inhibitory activity, a conformation of the inhibitor domain supported
by other regions of APP seemed unnecessary for the activity. To
determine which amino acid residues are essential for the inhibitory
activity, we synthesized various COOH-terminally deleted variants of
the hexadecapeptide and tested their inhibitory activity (Fig.
5A). We found that deletion of the COOH-terminal 4 or 6 residues of the hexadecapeptide slightly enhanced the inhibitory activity, respectively, and a decapeptide that has an ISYGNDALMP sequence corresponding to residues 586-595 of APP was the most effective inhibitor of gelatinase A (IC50 = 30 nM). Because further COOH- (Fig. 5A) or
NH2-terminal (Fig. 5B) deletion or alanine replacement of internal residues (Fig. 5C) of the
decapeptide led to a significant loss of the inhibitory activity, we
conclude that this decapeptide, named APP-derived inhibitory peptide
(APP-IP), is a minimal structural unit of the gelatinase A inhibitor
domain. The names and structures of the variants of APP-IP and their
IC50 values for inhibition of gelatinase A activity are
listed in Table I. To estimate the energetic
contributions of individual residues of APP-IP, we calculated

G values from the IC50 values before and
after deletion or alanine replacement of individual residues as
described under "Experimental Procedures." The results are summarized in Table II. We found that
Tyr588 (
G = 16 kJ/mol),
Asp591 (
G = 24 kJ/mol), and
Leu593 (
G = 20 kJ/mol) of APP mainly
stabilize the interaction between gelatinase A and the inhibitor. We
also found that Ile586 (
G = 6.6 kJ/mol), Met594 (
G = 8.0 kJ/mol), and
Pro595 (
G = 9.0 kJ/mol) modestly
contribute to the inhibitory activity.

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Fig. 5.
Gelatinase A inhibitory activities of
peptides derived from APP. APMA-activated gelatinase A (0.58 nM) was incubated with 50 µM 3163v at
37 °C for 40 min in the presence of indicated concentrations of
various APP-derived peptides. All of the reaction mixtures contained 50 mM Tris-HCl, 150 mM NaCl, 10 mM
CaCl2, 0.01% Brij 35, and 0.01% BSA. The amount of 3163v
hydrolyzed in the absence of the APP-derived peptides was taken as
100%. The enzyme activity is shown as the relative amount of 3163v
hydrolyzed by the enzyme on the ordinate. The APP-derived
peptides used were as follows: in A, APP586-601 ( ) and
its COOH-terminal residues-deleted variants APP586-597 ( ), APP-IP
( ), APP-IP· C1 ( ), and APP-IP· C2 (×); in
B, APP-IP ( ) and its NH2-terminal
residues-deleted variants APP-IP· N1 ( ), APP-IP· N2 ( )
and APP-IP· N3 ( ); in C, APP-IP ( ) and its
internal residues-replaced variants APP-IP·N/A ( ), APP-IP·D/A
( ) and APP-IP·L/A ( ). The names and structures of the
APP-derived peptides are listed in Table I. The arrows
represent the changes in IC50 values for the inhibition
upon the deletions or replacements of the indicated amino acid residues
in APP-IP.
|
|
Specificity of APP-derived Inhibitor--
To examine enzyme
specificity of the APP-derived inhibitor, we tested the inhibitory
activities of APP-IP toward various MMPs. As shown in Fig.
6A, APP-IP efficiently inhibited
the activity of gelatinase A (IC50 = 30 nM),
whereas its inhibitory activity toward the catalytic domain of MT1-MMP
(IC50 = 2 µM) was much weaker. We found that
APP-IP had poor inhibitory activities (IC50 > 10 µM) toward gelatinase B (MMP-9), matrilysin (MMP-7), and stromelysin (MMP-3), suggesting that APP-IP is a selective inhibitor of
gelatinase A. We also examined the specificity of APP586-601 and that
of sAPP. As shown in Fig. 6B, APP586-601 inhibited each MMP
activity with an IC50 value 5-10-fold higher than that of the APP-IP inhibition, but the hexadecapeptide had essentially the same
MMPs preference as that of APP-IP (Fig. 6B). The inhibitory activities of sAPP toward respective MMPs were almost the same as those
of APP586-601 (Fig. 6C). Therefore, the enzyme specificity was conserved from sAPP to the decapeptide inhibitor.

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Fig. 6.
Enzyme specificity in inhibitory activity of
APP-derived peptides. Gelatinase A (0.58 nM, ),
gelatinase B (4.6 nM, ), matrilysin (3.6 nM,
), and the catalytic domain of MT1-MMP (3.0 nM,
×) were incubated with 50 µM 3163v, and
stromelysin (5.9 nM, ) was incubated with 50 µM 3168v at 37 °C for 40 min in the presence of
indicated concentrations of APP-IP (A), APP586-601
(B), or sAPP (C). All of the reaction mixtures
contained 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, 0.01% Brij 35, and 0.01% BSA. The
amount of substrate (3163v or 3168v) hydrolyzed in the absence of the
APP-derived peptides was taken as 100%. The enzyme activity is shown
as the relative amount of substrate hydrolyzed by the enzyme on the
ordinate.
|
|
Ability of APP-derived Inhibitor to Bind to Various Forms of
Gelatinase A--
To examine whether the active site of gelatinase A
is required for its interaction with the APP-derived inhibitor,
progelatinase A was incubated with APMA for various time lengths, and
the resultant active forms or progelatinase A were tested for their
abilities to bind to immobilized APP586-601 as described under
"Experimental Procedures." As shown in Fig.
7A, the 57-kDa active form of
gelatinase A appeared during a 30-min incubation with APMA, and almost
all progelatinase A (66 kDa) was converted to the 57-kDa active form after a 1-h incubation. The 41-kDa active form, which is reported to be
a hemopexin-like domain-less form of active gelatinase A (36), was also
produced after a 2-h or longer incubation with APMA. We found that both
the active forms of gelatinase A (the 57- and 41-kDa forms) but not
progelatinase A had an ability to bind to the immobilized inhibitor
(Fig. 7B). When a hydroxamate-based inhibitor KB-8301 was
added to the reaction mixture, the binding of the active forms with the
immobilized inhibitor was completely inhibited (Fig. 7B),
although the activities of the active forms analyzed by zymography were
not affected by the KB-8301 treatment (Fig. 7A). These
results suggest that the unoccupied active site of gelatinase A is
required for the interaction between gelatinase A and the APP-derived
inhibitor, but the hemopexin-like domain of gelatinase A is not
necessary for the interaction.

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Fig. 7.
Ability of APP586-601 to bind to various
forms of gelatinase A. Progelatinase A was incubated without ( )
or with 1 mM APMA at 37 °C for indicated lengths of time
in 50 mM Tris-HCl (pH 7.5) containing 10 mM
CaCl2 and 0.01% Brij 35. After treatment with APMA, each
of the samples was incubated with APP586-601 coupled to Affi-Gel 10 beads in the absence ( ) or presence (+) of 1.0 µM
KB-8301 at 4 °C for 2 h with rotation. The gelatinase A forms
adsorbed to the beads were analyzed by gelatin zymography
(B) as described under "Experimental Procedures." The
gelatinase A samples prepared as described above but incubated without
APP586-601-Affi-Gel 10 beads were also subjected to the zymographic
analysis (A). An arrow at 66 kDa indicates the
gelatinolytic band of progelatinase A. The arrowheads
indicate the gelatinolytic bands of the 57-kDa form (upper
arrowhead) and the 41-kDa form (lower arrowhead) of
active gelatinase A, respectively. Ordinate, molecular mass
in kDa.
|
|
Comparison of Gelatinase A Binding Abilities of Various APP-derived
Peptides--
We compared time courses of binding of active gelatinase
A with various APP-derived peptides coupled to Affi-Gel 10 beads. As
shown in Fig. 8A, active
gelatinase A was rapidly adsorbed into APP-IP-immobilized beads, and
almost no gelatinase A activity remaining in the solution was detected
after a 120-min incubation. The adsorption of gelatinase A into
APP586-601-immobilized beads was much slower than that into
APP-IP-immobilized beads, probably reflecting a lower affinity
interaction of gelatinase A with the hexadecapeptide than that with the
decapeptide. Active gelatinase A was only slightly adsorbed into
APP-IP·L/A-immobilized beads (Fig. 8B). Neither adsorption
of the enzyme into APP-IP·D/A-immobilized beads nor that into
unconjugated beads was observed, suggesting that the loss of the
inhibitory activity of APP-IP upon alanine replacement of
Asp591 or Leu593 is due to the loss of the
affinity of the decapeptide for gelatinase A.

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Fig. 8.
Time courses of binding of active gelatinase
A with various immobilized APP-derived peptides. Progelatinase A
was activated by incubation with 1 mM APMA at 37 °C for
1 h. 100 µl of the activated gelatinase A (93 nM)
was mixed with 20 µl of Affi-Gel 10 beads coupled to APP586-601,
APP-IP, APP-IP·L/A, or APP-IP·D/A (200 nmol of each peptide was
coupled to 1 ml of Affi-Gel 10) or unconjugated Affi-Gel 10 beads and
incubated at 4 °C for 0, 15, 30, 60, and 120 min with rotation. In
A, supernatant was taken from the incubated mixture that
contained the beads coupled to APP586-601 ( ), APP-IP ( ),
APP-IP·L/A ( ), or APP-IP·D/A ( ) or unconjugated beads
(×), and gelatinase A activity in the supernatant was
measured, using 3163v as a substrate. The activity of gelatinase A
before incubation with the beads was taken as 100%. The enzyme
activity remained in the supernatant is shown as the relative activity
on the ordinate. In B, gelatinase A adsorbed to
the beads coupled to APP586-601 (lane 1), APP-IP
(lane 2), APP-IP·L/A (lane 3), or APP-IP·D/A
(lane 4) or unconjugated beads (lane 5) after 120 min incubation was analyzed by gelatin zymography. An arrow
and an arrowhead indicate the gelatinolytic bands of the
57-kDa form and the 41-kDa form of active gelatinase A, respectively.
Ordinate, molecular mass in kDa.
|
|
Susceptibility of APP-IP to Gelatinase A Cleavage--
To examine
whether the inhibitory sequence of APP is susceptible to gelatinase A
cleavage, APP-IP was incubated with active gelatinase A, and the
protease-treated peptide was tested for its inhibitory activity toward
gelatinase A. As shown in Fig. 9A,
the gelatinase A-treated APP-IP showed almost the same inhibitory activity as that of untreated APP-IP. In contrast to the case of
gelatinase A treatment, endoproteinase Asp-N treatment of APP-IP led to
a complete loss of the inhibitory activity (Fig. 9A). When the gelatinase A-treated APP-IP was analyzed by reverse-phase high
performance liquid chromatography, a single peptide having the intact
ISYGNDALMP sequence was eluted from the column (Fig. 9B). On
the other hand, two peptides that have ISYGN and DALMP sequences,
respectively, but not the original decapeptide, were detected in the
analysis of the endoproteinase Asp-N-treated APP-IP. Therefore, it
seems likely that APP-IP lost the inhibitory activity because of the
cleavage of a peptide bond between Asn590 and
Asp591. These results also suggest that APP-IP is resistant
to gelatinase A cleavage.

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Fig. 9.
Susceptibility of APP-IP to gelatinase A or
endoproteinase Asp-N cleavage. APP-IP (100 µM) was
incubated with 100 nM APMA-activated gelatinase A or 1.0 nM endoproteinase Asp-N or without enzyme in 50 mM Tris-HCl (pH 7.5) containing 10 mM
CaCl2 and 0.01% Brij 35 at 37 °C for 18 h. After
incubation, the mixture was boiled for 10 min to terminate the enzyme
reaction. In A, gelatinase A (0.58 nM) was
incubated with 50 µM 3163v at 37 °C for 40 min in the
presence of indicated concentrations of the gelatinase A-treated APP-IP
( ), the endoproteinase Asp-N-treated APP-IP ( ), or the
decapeptide incubated without enzyme ( ). The amount of 3163v
hydrolyzed in the absence of the peptides was taken as 100%. The
enzyme activity is shown as the relative amount of 3163v hydrolyzed by
gelatinase A on the ordinate. In B, APP-IP
incubated without enzyme (top panel, Enzyme),
the gelatinase A-treated APP-IP (middle panel), or the
endoproteinase Asp-N-treated APP-IP (bottom panel,
Asp-N) was applied to a Cosmosyl 5C18 column and
eluted at a flow rate of 0.5 ml/min with a linear gradient of
acetonitrile containing 0.05% trifluoroacetic acid. The column eluate
was monitored at 206 nm (solid lines), and the broken
lines show the percentages of acetonitrile in the elution medium.
The eluted peptides were identified by NH2-terminal
sequence analysis.
|
|
 |
DISCUSSION |
We explored an internal sequence of APP required for its
gelatinase A inhibitory activity and found that the inhibitor was localized within the ISYGNDALMP sequence corresponding to residues 586-595 of APP770. The inhibitor region was located just
in the COOH-terminal parts of the MT1-MMP and thrombin cleavage sites in APP, suggesting that the region next to the inhibitor domain has a
flexible and/or exposed structure. We recently found that the
MT1-MMP-catalyzed cleavage of APP releases a soluble fragment of APP
that lacks the inhibitor domain against gelatinase A.2 The
release of the inhibitor domain-less fragment is thought to be
important to facilitate the ECM degradation because the noninhibitory
fragment still has an ability to interact with ECM, and it displaces
ECM-associated sAPP, thereby removing the gelatinase A inhibitor from
ECM. Therefore, the location of the inhibitor domain in APP may be of
benefit in switching the APP function, because protease cleavage
separates the inhibitor domain from the NH2-terminal
ECM-binding regions of APP. The flexible structure near the inhibitor
domain also may be required for the inhibitory function of APP. We
found that APP-IP, a decapeptide containing the APP586-595
sequence, had an enhanced gelatinase A inhibitory activity
(IC50 = 30 nM) as compared with that of the
hexadecapeptide containing APP586-601 sequence (IC50 = 320 nM) or that of sAPP (IC50 = 400 nM), suggesting that the structures around the inhibitor domain slightly hamper and do not support the inhibitory function of
APP. Increased freedom may make it feasible for the isolated inhibitor
domain to adapt to interact with gelatinase A. Binding of gelatinase A
with immobilized APP-IP was indeed much faster than that with
immobilized APP586-601 (Fig. 8). In contrast to the region around the
inhibitor domain, 7 of the 8 amino acid residues within the inhibitor
domain tested were found to contribute the inhibitory activity. Among
them, Ile586 (
G = 6.6 kJ/mol),
Tyr588 (
G = 16 kJ/mol),
Asp591 (
G = 24 kJ/mol),
Leu593 (
G = 20 kJ/mol),
Met594 (
G = 8.0 kJ/mol), and
Pro595 (
G = 9.0 kJ/mol) showed
significant contributions. We found that truncation of the
COOH-terminal 8 residues from GST-APP439-601 led to a loss of the
inhibitory activity (Fig. 4B). This could be explained by
the fact that the removal of both Met594 and
Pro595 from the GST fusion protein leads to loss of the
binding energy corresponding to 17 kJ/mol, thereby leading to 730-fold
increase in IC50 value for the inhibition. As compared with
the regions around the inhibitor domain, the amino acid residues within
the inhibitor domain are highly conserved among APPs from different species of vertebrate (Fig. 10), suggesting
an evolutional importance of this domain. Because amyloid
precursor-like proteins or other proteins so far reported do not
contain the sequence of APP-IP, the decapeptide region seems to be an
APP-specific functional domain. So far, the physiological importance of
APP-derived gelatinase A inhibitor has not been supported by the data
from the APP-deficient mice; these mice do not show overt abnormalities
(37). Considering that gelatinase A-deficient mice also develop
normally (38), abnormal phenotypes caused by a defect of gelatinase A
or those caused by a lack of APP-mediated regulation of gelatinase A
activity may appear under certain conditions. In contrast to the
cryptic physiological function of gelatinase A, a strong correlation
with tumor invasion (39, 40) and tumor angiogenesis (41, 42) has been
reported. Because the APP-IP was found to be a selective inhibitor of
gelatinase A (Fig. 6) and the inhibitory activity of APP in ECM is
regulated by MT1-MMP, APP can be assumed to be a specific regulator for
the MT1-MMP-gelatinase A pathway. In this pathway, TIMP-2 is also known
as an important regulator, which can regulate the MT1-MMP-catalyzed
activation of progelatinase A in both positive and negative manners
(19). After activation of progelatinase A, the activity of gelatinase A
also can be inhibited by TIMP-2. However, it is reported that active
gelatinase A lacking the hemopexin-like domain is resistant to TIMP-2
inhibition (43). The hemopexin-like domain-less form of gelatinase A,
produced by autolysis (36) or by other protease (44), is thought to be
more potent ECM-degrading enzyme in vivo. We showed that
both the 57-kDa and the 41-kDa hemopexin-like domain-less forms of active gelatinase A bound similarly to the APP-derived inhibitor (Fig.
7), suggesting that the inhibitor indistinguishably inhibits both forms
of gelatinase A. Therefore, instead of TIMP-2, ECM-associated sAPP
could be a potent protector of ECM after production of the hemopexin-like domain-less form of gelatinase A.

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Fig. 10.
Sequences around the gelatinase A inhibitor
domain of APP from several species. The boxed area
shows the gelatinase A inhibitor domain of human APP determined in this
study and its corresponding sequence of APP from several species. The
symbols ( G > 10 kJ/mol) and (10 kJ/mol >  G > 5 kJ/mol) represent the
residues that showed significant contributions in the inhibitory
activity. MT1 and Th represent the sites of human
APP susceptible to MT1-MMP and thrombin cleavages, respectively.
K-endo represents the deduced site of lysyl endopeptidase
cleavage in GST-APP439-678 observed in Fig. 3. The numbers
at the bottom represent human APP770
numbering.
|
|
The members of TIMP family have broad specificity against MMPs, and
none of the hydroxamate-, barbiturate-, sulfodiimine-, and
thiadiazole-based inhibitors recently designed (7-10) is a specific
inhibitor of individual MMPs. The common architecture of catalytic
sites of MMPs probably relates to the broad specificity of the
inhibitors. The crystal structures of TIMP-1-stromelysin (45) and
TIMP-2-MT1-MMP (46) complexes have revealed that chelation of the
catalytic zinc by the
-amino group and carbonyl oxygen of the
NH2-terminal Cys1 of TIMPs is a common
mechanism for the inhibition of MMPs activity. Carbamylation of the
-amino group of the Cys1 of TIMP-2 indeed abrogates its
MMP inhibitory activity (47). Synthetic MMP inhibitors are also
designed to consist of zinc-chelating group and the remaining parts
accommodated into the substrate-binding sites of MMPs. Considering that
the interaction between the APP-derived inhibitor and active gelatinase
A was inhibited by KB-8301 (Fig. 7) and that sAPP inhibits gelatinase
A-catalyzed hydrolysis of peptide substrate in a competitive manner
(29), the inhibitor domain of APP probably interacts with the
substrate-binding sites of gelatinase A. Because APP-IP was resistant
to the gelatinase A cleavage (Fig. 9), the decapeptide inhibitor must
interact with the active site of gelatinase A not to provide the
cleaved products. Unlike other natural or synthetic MMP inhibitors, the
gelatinase A inhibitor domain of APP contains neither the
-amino
group available for zinc chelation nor the artificial zinc-chelating
group like hydroxamate. Therefore, in addition to the interaction with
the substrate-binding sites, other novel interaction(s) with gelatinase A must support the inhibitory function of APP-derived inhibitor. Such
novel interactions may also relate to the gelatinase A selectivity of
the inhibitor. However, clarification of detailed mechanism of the
inhibition must await analysis of the crystal structure of the
gelatinase A-APP-IP complex. Clarification of the mechanism of the
gelatinase A-selective inhibition also provides the potential to design
novel anti-cancer drugs that block specific processes of tumor invasion
or angiogenesis.
 |
ACKNOWLEDGEMENTS |
We thank Dr. K. Maruyama (Saitama Medical
School) for providing cDNA of human APP770 and Dr. Y. Takaki (RIKEN Brain Science Institute) for helpful comments. We are
also grateful to Dr. K. Yoshino (Kanebo Institute for Cancer Research,
Japan) for providing the synthetic metalloproteinase inhibitor
KB-8301.
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Cell Biology,
Kihara Institute for Biological Research, Yokohama City University,
Maioka-cho 641-12, Totsuka-ku, Yokohama 244-0813, Japan.
Tel.: 81-45-820-1905; Fax: 81-45-820-1901; E-mail:
shigashi@yokohama-cu.ac.jp.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M212264200
2
S. Higashi and K. Miyazaki, submitted for publication.
 |
ABBREVIATIONS |
The abbreviations used are:
MMP, matrix
metalloproteinase;
ECM, extracellular matrix;
TIMP, tissue inhibitor of
metalloproteinases;
MT1-MMP, membrane type 1 MMP;
APP,
-amyloid
precursor protein;
sAPP, soluble APP;
GST, glutathione
S-transferase;
APMA, p-aminophenyl mercuric
acetate;
APMSF, p-amidinophenyl methanesulfonyl fluoride
hydrochloride;
BSA, bovine serum albumin;
PBS, phosphate-buffered
saline;
APP-IP, APP-derived inhibitory peptide.
 |
REFERENCES |
1.
|
Docherty, A. J. P.,
O'Connell, J.,
Crabbe, T.,
Angal, S.,
and Murphy, G.
(1992)
Trends Biotechnol.
10,
200-207[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Matrisian, L. M.
(1992)
Bioessays
14,
455-463[Medline]
[Order article via Infotrieve]
|
3.
|
Stetler-Stevenson, W. G.,
Aznavoorian, S.,
and Liotta, L. A.
(1993)
Annu. Rev. Cell Biol.
9,
541-573[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Amour, A.,
Slocombe, P. M.,
Webster, A.,
Butler, M.,
Knight, C. G.,
Smith, B. J.,
Stephens, P. E.,
Shelley, C.,
Hutton, M.,
Knauper, V.,
Docherty, A. J.,
and Murphy, G.
(1998)
FEBS Lett.
435,
39-44[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Amour, A.,
Knight, C. G.,
Webster, A.,
Slocombe, P. M.,
Stephens, P. E.,
Knauper, V.,
Docherty, A. J.,
and Murphy, G.
(2000)
FEBS Lett.
473,
275-279[CrossRef][Medline]
[Order article via Infotrieve]
|
6.
|
Kashiwagi, M.,
Tortorella, M.,
Nagase, H.,
and Brew, K.
(2001)
J. Biol. Chem.
276,
12501-12504[Abstract/Free Full Text]
|
7.
|
Betz, M.,
Huxley, P.,
Davies, S. J.,
Mushtaq, Y.,
Pieper, M.,
Tschesche, H.,
Bode, W.,
and Gomis-Rüth, F. X.
(1997)
Eur. J. Biochem.
247,
356-363[Abstract]
|
8.
|
Brandstetter, H.,
Grams, F.,
Glitz, D.,
Lang, A.,
Huber, R.,
Bode, W.,
Krell, H. W.,
and Engh, R. A.
(2001)
J. Biol. Chem.
276,
17405-17412[Abstract/Free Full Text]
|
9.
|
Jia, M. C.,
Schwartz, M. A.,
and Sang, Q. A.
(2000)
Adv. Exp. Med. Biol.
476,
181-194[Medline]
[Order article via Infotrieve]
|
10.
|
Finzel, B. C.,
Baldwin, E. T.,
Bryant, G. L., Jr.,
Hess, G. F.,
Wilks, J. W.,
Trepod, C. M.,
Mott, J. E.,
Marshall, V. P.,
Petzold, G. L.,
Poorman, R. A.,
O'Sullivan, T. J.,
Schostarez, H. J.,
and Mitchell, M. A.
(1998)
Protein Sci.
7,
2118-2126[Abstract/Free Full Text]
|
11.
|
Liotta, L. A.
(1986)
Cancer Res.
46,
1-7[Medline]
[Order article via Infotrieve]
|
12.
|
Collier, I. E.,
Wilhelm, S. M.,
Eisen, A. Z.,
Marmer, B. L.,
Grant, G. A.,
Seltzer, J. L.,
Kronberger, A.,
He, C.,
Bauer, E. A.,
and Goldberg, G. I.
(1988)
J. Biol. Chem.
263,
6579-6587[Abstract/Free Full Text]
|
13.
|
Wilhelm, S. M.,
Collier, I. E.,
Marmer, B. L.,
Eisen, A. Z.,
Grant, G. A.,
and Goldberg, G. I.
(1989)
J. Biol. Chem.
264,
17213-17221[Abstract/Free Full Text]
|
14.
|
Sato, H.,
Takino, T.,
Okada, Y.,
Cao, J.,
Shinagawa, A.,
Yamamoto, E.,
and Seiki, M.
(1994)
Nature
370,
61-65[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Pei, D.
(1999)
J. Biol. Chem.
274,
8925-8932[Abstract/Free Full Text]
|
16.
|
Pei, D.
(1999)
Cell Res.
9,
291-303[Medline]
[Order article via Infotrieve]
|
17.
|
Yana, I.,
and Seiki, M.
(2002)
Clin. Exp. Metastasis
19,
209-215[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Strongin, A. Y.,
Marmer, B. L.,
Grant, G. A.,
and Goldberg, G. I.
(1993)
J. Biol. Chem.
268,
14033-14039[Abstract/Free Full Text]
|
19.
|
Strongin, A. Y.,
Collier, I.,
Bannikov, G.,
Marmer, B. L.,
Grant, G. A.,
and Goldberg, G. I.
(1995)
J. Biol. Chem.
270,
5331-5338[Abstract/Free Full Text]
|
20.
|
Nakahara, H.,
Howard, L.,
Thompson, E. W.,
Sato, H.,
Seiki, M.,
Yeh, Y.,
and Chen, W. T.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7959-7964[Abstract/Free Full Text]
|
21.
|
Kang, J.,
Lemaire, H. G.,
Unterbeck, A.,
Salbaum, J. M.,
Masters, C. L.,
Grzeschik, K. H.,
Multhaup, G.,
Beyreuther, K.,
and Muller-Hill, B.
(1987)
Nature
325,
733-736[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Sisodia, S. S.,
Koo, E. H.,
Beyreuther, K.,
Unterbeck, A.,
and Price, D. L.
(1990)
Science
248,
492-495[Medline]
[Order article via Infotrieve]
|
23.
|
Esch, F. S.,
Keim, P. S.,
Beattie, E. C.,
Blacher, R. W.,
Culwell, A. R.,
Oltersdorf, T.,
McClure, D.,
and Ward, P. J.
(1990)
Science
248,
1122-1124[Medline]
[Order article via Infotrieve]
|
24.
|
Small, D. H.,
Nurcombe, V.,
Moir, R.,
Michaelson, S.,
Monard, D.,
Beyreuther, K.,
and Masters, C. L.
(1992)
J. Neurosci.
12,
4143-4150[Abstract]
|
25.
|
Caceres, J.,
and Brandan, E.
(1997)
J. Cell. Biochem.
65,
145-158[CrossRef][Medline]
[Order article via Infotrieve]
|
26.
|
Kibbey, M. C.,
Jucker, M.,
Weeks, B. S.,
Neve, R. L.,
Van Nostrand, W. E.,
and Kleinman, H. K.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
10150-10153[Abstract]
|
27.
|
Beher, D.,
Hesse, L.,
Masters, C. L.,
and Multhaup, G.
(1996)
J. Biol. Chem.
271,
1613-1620[Abstract/Free Full Text]
|
28.
|
Ohsawa, I.,
Takamura, C.,
and Kohsaka, S.
(2001)
J. Neurochem.
76,
1411-1420[CrossRef][Medline]
[Order article via Infotrieve]
|
29.
|
Miyazaki, K.,
Hasegawa, M.,
Funahashi, K.,
and Umeda, M.
(1993)
Nature
362,
839-841[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Miyazaki, K.,
Hattori, Y.,
Umenishi, F.,
Yasumitsu, H.,
and Umeda, M.
(1990)
Cancer Res.
50,
7758-7764[Abstract]
|
31.
|
Umenishi, F.,
Yasumitsu, H.,
Ashida, Y.,
Yamauti, J.,
Umeda, M.,
and Miyazaki, K.
(1990)
J. Biochem. (Tokyo)
108,
537-543[Abstract]
|
32.
|
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual
, 2nd Ed.
, pp. 14.1-15.113, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
|
33.
|
Miyazaki, K.,
Funahashi, K.,
Numata, Y.,
Koshikawa, N.,
Akaogi, K.,
Kikkawa, Y.,
Yasumitsu, H.,
and Umeda, M.
(1993)
J. Biol. Chem.
268,
14387-14393[Abstract/Free Full Text]
|
34.
|
Segel, I. H.
(1975)
Enzyme Kinetics
, pp. 320-329, Wiley-Interscience, New York
|
35.
|
Igarashi, K.,
Murai, H.,
and Asaka, J.
(1992)
Biochem. Biophys. Res. Commun.
185,
1000-1004[Medline]
[Order article via Infotrieve]
|
36.
|
Howard, E. W.,
Bullen, E. C.,
and Banda, M. J.
(1991)
J. Biol. Chem.
266,
13064-13069[Abstract/Free Full Text]
|
37.
|
Zheng, H.,
Jiang, M.,
Trumbauer, M. E.,
Hopkins, R.,
Sirinathsinghji, D. J.,
Stevens, K. A.,
Conner, M. W.,
Slunt, H. H.,
Sisodia, S. S.,
Chen, H. Y.,
and Van der Ploeg, L. H.
(1996)
Ann. N. Y. Acad. Sci.
777,
421-426[Abstract]
|
38.
|
Itoh, T.,
Ikeda, T.,
Gomi, H.,
Nakao, S.,
Suzuki, T.,
and Itohara, S.
(1997)
J. Biol. Chem.
272,
22389-22392[Abstract/Free Full Text]
|
39.
|
Brown, P. D.,
Bloxidge, R. E.,
Stuart, N. S.,
Gatter, K. C.,
and Carmichael, J.
(1993)
J. Natl. Cancer Inst.
85,
574-578[Abstract]
|
40.
|
Azzam, H. S.,
Arand, G.,
Lippman, M. E.,
and Thompson, E. W.
(1993)
J. Natl. Cancer Inst.
85,
1758-1764[Abstract]
|
41.
|
Itoh, T.,
Tanioka, M.,
Yoshida, H.,
Yoshioka, T.,
Nishimoto, H.,
and Itohara, S.
(1998)
Cancer Res.
58,
1048-1051[Abstract]
|
42.
|
Brooks, P. C.,
Silletti, S.,
von Schalscha, T. L.,
Friedlander, M.,
and Cheresh, D. A.
(1998)
Cell
92,
391-400[Medline]
[Order article via Infotrieve]
|
43.
|
Olson, M. W.,
Gervasi, D. C.,
Mobashery, S.,
and Fridman, R.
(1997)
J. Biol. Chem.
272,
29975-29983[Abstract/Free Full Text]
|
44.
|
Rice, A.,
and Banda, M. J.
(1995)
Biochemistry
34,
9249-9256[Medline]
[Order article via Infotrieve]
|
45.
|
Gomis-Rüth, F. X.,
Maskos, K.,
Betz, M.,
Bergner, A.,
Huber, R.,
Suzuki, K.,
Yoshida, N.,
Nagase, H.,
Brew, K.,
Bourenkov, G. P.,
Bartunik, H.,
and Bode, W.
(1997)
Nature
389,
77-81[CrossRef][Medline]
[Order article via Infotrieve]
|
46.
|
Fernandez-Catalan, C.,
Bode, W.,
Huber, R.,
Turk, D.,
Calvete, J. J.,
Lichte, A.,
Tschesche, H.,
and Maskos, K.
(1998)
EMBO J.
17,
5238-5248[Abstract/Free Full Text]
|
47.
|
Higashi, S.,
and Miyazaki, K.
(1999)
J. Biol. Chem.
274,
10497-10504[Abstract/Free Full Text]
|
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