Identification of a Region of beta -Amyloid Precursor Protein Essential for Its Gelatinase A Inhibitory Activity*

Shouichi HigashiDagger 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Because beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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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 beta -amyloid precursor protein (APP).2

APP is a type I integral membrane protein that was initially identified as a precursor of beta -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 beta -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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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-[Nbeta -(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-Nepsilon -(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 DH5alpha . 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-beta -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 alpha -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 DH5alpha . 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-beta -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).


[<UP>I</UP>]<SUB><UP>50</UP></SUB><UP> = </UP>K<SUB>d</SUB><UP> · </UP>(<UP>1 + </UP>[<UP>S</UP>]<UP>/</UP>K<SUB>m</SUB>) (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.
<UP>&Dgr;&Dgr;</UP>G=RT<UP> · 1n</UP>(K<SUB>da</SUB>/K<SUB>db</SUB>) (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.
<UP>&Dgr;&Dgr;</UP>G=RT<UP> · 1n</UP>(<UP>IC<SUB>50 a</SUB>/IC<SUB>50 b</SUB></UP>) (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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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 (open circle ). 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.

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.

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. alpha , beta , and gamma  represent the sites of cleavage by alpha -, beta -, and gamma -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 Delta Delta 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 (Delta Delta G = 16 kJ/mol), Asp591 (Delta Delta G = 24 kJ/mol), and Leu593 (Delta Delta G = 20 kJ/mol) of APP mainly stabilize the interaction between gelatinase A and the inhibitor. We also found that Ile586 (Delta Delta G = 6.6 kJ/mol), Met594 (Delta Delta G = 8.0 kJ/mol), and Pro595 (Delta Delta 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 (open circle ), APP-IP (black-triangle), APP-IP·Delta C1 (triangle ), and APP-IP·Delta C2 (×); in B, APP-IP () and its NH2-terminal residues-deleted variants APP-IP·Delta N1 (open circle ), APP-IP·Delta N2 (black-triangle) and APP-IP·Delta N3 (triangle ); in C, APP-IP () and its internal residues-replaced variants APP-IP·N/A (open circle ), APP-IP·D/A (black-triangle) and APP-IP·L/A (triangle ). 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.


                              
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Table I
IC50 values for gelatinase A inhibition by APP-derived peptides


                              
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Table II
Contribution of residues of APP in gelatinase A inhibitory activity

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, open circle ), matrilysin (3.6 nM, black-triangle), and the catalytic domain of MT1-MMP (3.0 nM, ×) were incubated with 50 µM 3163v, and stromelysin (5.9 nM, triangle ) 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 (open circle ), APP-IP·L/A (black-triangle), or APP-IP·D/A (triangle ) 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 (open circle ), the endoproteinase Asp-N-treated APP-IP (black-triangle), 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Delta Delta G = 6.6 kJ/mol), Tyr588 (Delta Delta G = 16 kJ/mol), Asp591 (Delta Delta G = 24 kJ/mol), Leu593 (Delta Delta G = 20 kJ/mol), Met594 (Delta Delta G = 8.0 kJ/mol), and Pro595 (Delta Delta 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  (Delta Delta G > 10 kJ/mol) and open circle  (10 kJ/mol > Delta Delta 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 alpha -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 alpha -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 alpha -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.

Dagger 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, beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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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