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J. Biol. Chem., Vol. 278, Issue 36, 34674-34684, September 5, 2003

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Identification of a Negatively Charged Peptide Motif within the Catalytic Domain of Progelatinases That Mediates Binding to Leukocyte ₂ Integrins^*

Michael Stefanidakis, Mikael Björklund , Eveliina Ihanus, Carl G. Gahmberg and Erkki Koivunen  

From the Department of Biosciences, Division of Biochemistry, Viikinkaari 5, University of Helsinki, FIN-00014 Helsinki, Finland

Received for publication, March 5, 2003 , and in revised form, June 19, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The _M2 integrin of leukocytes can bind a variety of ligands. We screened phage display libraries to isolate peptides that bind to the _M I domain, the principal ligand binding site of the integrin. Only one peptide motif, (D/E)(D/E)(G/L)W, was obtained with this approach despite the known ligand binding promiscuity of the I domain. Interestingly, such negatively charged sequences are present in many known ₂ integrin ligands and also in the catalytic domain of matrix metalloproteinases (MMPs). We show that purified ₂ integrins bind to pro-MMP-2 and pro-MMP-9 gelatinases and that that the negatively charged sequence of the MMP catalytic domain is an active ₂ integrin-binding site. Furthermore, a synthetic DDGW-containing phage display peptide inhibited the ability of ₂ integrin to bind progelatinases but did not inhibit the binding of cell adhesion-mediating substrates such as intercellular adhesion molecule-1, fibrinogen, or an LLG-containing peptide. Immunoprecipitation and cell surface labeling demonstrated complexes of pro-MMP-9 with both the _M2 and _L2 integrins in leukocytes, and pro-MMP-9 colocalized with _M2 in cell surface protrusions. The DDGW peptide and the gelatinase-specific inhibitor peptide CTTHWGFTLC blocked ₂ integrin-dependent leukocyte migration in a transwell assay. These results suggest that leukocytes may move in a progelatinase-₂ integrin complex-dependent manner.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The leukocyte integrin family consists of four heterodimeric glycoproteins with specific -chains (_L, _M, _X, or _D) and a common ₂-chain (CD18). They play an essential role in mediating adhesion of cells in the immune system (1). The major ligand-binding site locates to a 200-amino acid-long sequence within the -chain called I or inserted domain, which is homologous to the A domains of von Willebrand factor, repeats of cartilage matrix protein and collagen (2).

Among the ₂ integrins, _M2 is the most promiscuous binder being able to interact with a multitude of unrelated ligands. These include ICAM¹ 1–5, complement fragment iC3b, fibrinogen, urokinase-type plasminogen activator receptor, E-selectin, and various extracellular matrix proteins (3). The integrin has also been shown to have a capacity to bind certain enzymes, but whether this is important for leukocyte adhesion or immune reactions is unclear. Such enzymes showing integrin-binding activity are catalase (4), myeloperoxidase (5), and the proteinases elastase (6) and urokinase (7).

Extensive work has been done to identify the ligand-binding sites in ₂ integrin I domains, but less is known about the interacting ligand regions (8, 9). Recently, the structure of an _L I domain-ICAM-1 complex was reported (10). Low molecular weight peptides binding to the ₂ integrins are useful reagents to study integrin function, and such peptides have been derived from ICAM-2 (11), fibrinogen (12), and Cyr61 (13). We have used phage display libraries to study the peptide binding specificity of integrins and to develop potential drug leads. In our previous study (14), we isolated the bicyclic peptide CPCFLLGCC (LLG-C4) as the most active binder to the purified _M2 integrin. Leukocytes can efficiently adhere to the immobilized LLG-C4 peptide via the _M2 and _X2 integrins.

We have now extended phage display screenings to the purified _M I domain. This has resulted in the identification of a novel I domain-binding tetrapeptide motif (D/E)(D/E)(G/L)W, which is found on some of the known ₂ integrin ligands and interestingly also on the catalytic domain of MMPs. We show that the (D/E)(D/E)(G/L)W motif mediates binding between an MMP and ₂ integrin, and pro-MMP-9 gelatinase, the major MMP of leukocytes (15–18), occurs in complex with the _M2 and _L2 integrins in leukemic cell lines following cellular activation. The peptide inhibitors of the integrin-MMP complex prevent leukemia cell migration, suggesting a role for the complex in cell motility.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Antibodies and Reagents—The antibodies MEM170 and LM2/1 were against the _M and the MEM83 and TS2/4 antibodies against the _L integrin subunit (11, 19). The monoclonal antibody 7E4 (21) reacted with the common ₂-chain of the leukocyte integrins. The _M antibody OKM10 was obtained from the ATCC (Manassas, VA) (22). A monoclonal antibody against ICAM-5 (TL3) (23) was used as an antibody control. The monoclonal anti-MMP-9 antibody (GE-213) and anti-MMP-2 antibody (Ab-3) were obtained from Lab Vision Corp. (Fremont CA) and from Oncogene^TM research products, respectively. Affinity-purified rabbit anti-MMP-9 polyclonal antibodies were from the Borregaard Laboratory (24). The rabbit anti-mouse horseradish peroxidase-conjugated secondary antibody was from Dakopatts A/S (Copenhagen, Denmark). Inh1 (2R-2-(4-biphenylylsulfonyl)amino-N-hydroxypropionamide) was purchased from Calbiochem. Human recombinant ICAM-1 was obtained commercially by R&D Systems (Minneapolis, MN). ICAM-1-Fc fusion protein was expressed in Chinese hamster ovary cells and purified as described (14). The synthetic peptides CTT, STT, LLG-C4, and RGD-4C were obtained as described previously (14, 25). W A CTT was ordered from Neosystem, Strasbourg, France. Pro-MMP-2 and pro-MMP-9 were obtained commercially (Roche Applied Science). In zymography, the commercial pro-MMP-9 showed the 92-kDa monomer, 200-kDa homodimer, and 120-kDa NGAL complex bands. The integrins ₁₁ and ₃₁ were purchased from Chemicon International (Temecula, CA). Human plasma fibrinogen and lovastatin were from Calbiochem.

Phage Display—Phage display selections were made using a pool of random peptides CX_7–10C and X_9–10, where C is a cysteine and X is any amino acid (14, 25). Briefly, the _M I domain-GST or the GST fusion protein was immobilized on microtiter wells at 20 µg/ml concentrations, and the wells were blocked with BSA. The phage library pool was first subtracted on wells coated with GST, and then unbound phage was transferred to _M I domain-GST-coated wells in 50 mM Hepes, 5 mM CaCl₂, 1 µM ZnCl₂, 150 mM NaCl, 2% BSA, pH 7.5. After three rounds of subtraction and selection, individual phage clones were tested for binding specificity, and the sequences of the phage that specifically bound to the I domain were determined (14).

Peptide Biosynthesis and Chemical Synthesis—The phage peptides were initially prepared biosynthetically as intein fusions. The DNA sequences encoding the peptides were PCR-cloned from 1-µl aliquots of the phage-containing bacterial colonies that were stored at –20 °C. The forward primer was 5'-CCTTTCTGCTCTTCCAACGCCGACGGGGCT-3', and the reverse primer was 5'-ACTTTCAACCTGCAGTTACCCAGCGGCCCC-3'. The PCR conditions included initial denaturation at 94 °C for 2 min followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The PCR products were purified using the Qiagen nucleotide removal kit. They were then digested with SapI and PstI restriction enzymes and ligated to a similarly digested and phosphatase-treated pTWIN vector (New England Biolabs). Correct insertions were verified by DNA sequencing. Intein fusion proteins were produced in Escherichia coli strain ER2566 and affinity-purified on a chitin column essentially as described (26). The peptide was cleaved on the column, eluted, and finally purified by high pressure liquid chromatography. Chemical peptide synthesis was done using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as described, and the sequences were verified by mass spectroscopy (26).

Phage Binding Assay—Phage (10⁸ infective particles/well) in 50 mM Hepes, 5 mM CaCl₂, 1 µM ZnCl₂, 0.5% BSA, pH 7.5, were added to microtiter wells coated with I domain-GST fusion or GST (20 ng/well). The phages were allowed to bind in the absence or presence of a competitor peptide (15 µM) for 1 h followed by washings with PBS containing 0.05% Tween 20. The bound phage was detected using 1:3000 dilution of a peroxidase-labeled monoclonal anti-phage antibody (Amersham Biosciences) and o-phenylenediamine dihydrochloride as a substrate. The reactions were stopped by addition of 10% H₂SO₄, and the absorbance was read at 492 nm using a microplate reader.

Pepspot—The peptides were synthesized on cellulose membranes as described (27). The membrane was blocked with 3% BSA in TBS containing 0.05% Tween 20, and incubated with 0.5–5 µg/ml _M I domain for 2 h at room temperature. The DDGW peptide was used as a competitor at a 50 µM concentration. Bound _M I domain was detected using the monoclonal antibody LM2/1 (1 µg/ml) or MEM170 (5 µg/ml) and peroxidase-conjugated rabbit anti-mouse antibody (1:5000 dilution) followed by chemiluminescence detection.

Cell Culture—The human HT1080 fibrosarcoma and THP-1 and Jurkat leukemic lines were obtained from ATCC and maintained as described previously (11, 25, 28). OCI/AML-3, derived from the primary blasts of an AML patient (29), was maintained in 10% fetal bovine serum/RPMI supplemented with L-glutamine, penicillin, and streptomycin. Cell viability was assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay according to the instructions of the manufacturer (Roche Applied Science).

Purification of Integrins—_L2 (CD11a/CD18, LFA-1), _M2 (CD11b/CD18, Mac-1), and _X2 (CD11c/CD18) integrins were purified from human blood buffy coat cell lysates by adsorption to the anti-CD11a (TS 2/4), anti-CD11b (MEM170), or anti-CD11c (3.9) antibodies linked to protein A-Sepharose CL 4B. The integrins were eluted at pH 11.5 in the presence of 2 mM MgCl₂ and 1% n-octyl glucoside as described previously (28).

Expression and Purification of GST Fusion Proteins—The _L, _M, and _X I domains were produced as GST fusion proteins in E. coli strains BL 21 or JM109 and purified by affinity chromatography on glutathione-coupled beads (30, 31). GST containing CTT in the C terminus was constructed using the protocols described for LLG-C4-GST (14), and glutathione-coupled beads were employed for purification. The purity of the GST fusion proteins was confirmed by SDS-PAGE with Coomassie Blue staining and Western blot analysis. For pepspot analysis, GST was cleaved from the _M I domain with thrombin.

Binding of MMPs to Purified Integrins—The purified I domains (GST-_M, GST-_L, GST-_X) or integrins (_M2, _L2, _X2, ₁₁) (1 µg/well) were immobilized in 20 mM Tris, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂,and1mM MnCl₂, pH 7.4. The wells were washed with PBST (10 mM phosphate, 140 mM NaCl, pH 7.4, containing 0.05% Tween 20) and blocked with 3% BSA in PBST. Pro-MMP-2, pro-MMP-9, or the p-aminophenylmercuric acetate (APMA) or trypsin-activated forms (32) were incubated for 2 h at room temperature. In the inhibition experiments, CTT and Inh1 were first preincubated with the pro-MMPs for 30 min at room temperature. The wells were washed three times and incubated with anti-MMP-9 (GE-213) or anti-MMP-2 (Ab-3) antibody at a 2 µg/ml concentration in PBST for 1 h. Bound antibodies were detected using peroxidase-conjugated rabbit anti-mouse IgG (Dako, Glostrup, Denmark) and o-phenylenediamine dihydrochloride as a substrate.

Coprecipitation of ₂ Integrin and Progelatinases—Serum-free conditioned medium containing pro-MMP-2 and pro-MMP-9 was collected from human HT-1080 fibrosarcoma cells grown in the presence of 100 nM phorbol ester 4-phorbol 12,13-dibutyrate (PDBu) (Sigma) overnight at +37 °C. A 500-µl volume of the supernatant was incubated with 100 ng of GST-_M, GST-_L, or GST-_X I domain or _M2 integrin for 3 h at 25 °C. GST and GST-LLG-C4 were used to determine non-specific binding. CTT, STT, LLG-C4, and ICAM-1 were used as competitors at a 200 µg/ml concentration and the antibodies LM2/1 and TL3 at 40 µg/ml. After a 1-h incubation at +4 °C, complexes of I domain and gelatinases were pelleted with glutathione-Sepharose. Integrin complexes were captured by incubating first with the OKM10 antibody for 3 h at +4°C and then with protein G-Sepharose for 1 h. After centrifugation and washing, samples were analyzed by gelatin zymography on 8% SDS-polyacrylamide gels containing 0.2% gelatin (32).

Effect of Peptides on Pro-MMP-9 Release from Cells—THP-1 cells (40,000/100 µl) were incubated in serum-free RPMI medium for 48 h in the absence or presence of 200 µM peptide as described in the text. Aliquots of the conditioned media were analyzed by gelatin zymography.

Interaction between CTT and Pro-MMP-9 —CTT-GST and GST control (5 µg/well) were coated overnight on 96-well microtiter plates in 50 µl of TBS followed by blocking of the wells by BSA. Pro-MMP-9 or APMA-activated form (80 ng/well) was incubated in the absence or presence of competitors for 2 h in 50 µM Hepes buffer containing 1% BSA, 5 mM CaCl₂, and 1 µM ZnCl₂, pH 7.5. After washing, bound MMP-9 was determined with anti-MMP-9 and horseradish peroxidase-conjugated anti-mouse IgG as described above. To examine complexing of CTT with pro-MMP-9 in cell culture, THP-1 cells were activated with PDBu for 30 min and then incubated with CTT, W A CTT, or Inh1 (each 200 µM) at +37°C in serum-free medium. Samples were taken from the media at 0–5-h time points and analyzed by zymography and Western blotting with polyclonal anti-MMP-9 antibodies. Experiments with HT-1080 cells were performed similarly except that the medium samples were collected after 6 h.

Cell Surface Labeling, Immunoprecipitation, and Immunoblotting— Non-activated or PDBu-activated THP-1 cells (1 x 10⁷) were subjected to surface labeling using periodate tritiated sodium borohydride (33). The ³H-labeled cells were lysed with 1% (v/v) Triton X-100 in PBS, clarified by centrifugation, and precleared with protein G-Sepharose. The lysate was immunoprecipitated with polyclonal anti-MMP-9, _M (OKM-10), or ₂ (7E4) antibodies. After a 1-h incubation at +4 °C together with protein G-Sepharose, immunocomplexes were pelleted, washed, and resolved on 8–16% SDS-PAGE gels (Bio-Rad). The gels were treated with an enhancer (Amplify, Amersham Biosciences), dried, and exposed. Non-labeled THP-1 cells (1 x 10⁷) were similarly lysed and immunoprecipitated as above. The samples were resolved on 4–15% SDS-PAGE gels and transferred to nitrocellulose membranes. Immunodetection was performed with _M (MEM170) antibody (10 µg/ml) followed by peroxidase-conjugated anti-mouse IgG and chemiluminescence detection (Amersham Biosciences). The membranes were stripped of bound antibodies and reprobed with monoclonal _L chain (TS2/4) or polyclonal anti-MMP-9 antibodies.

Immunofluorescence—Immunofluorescence was performed on resting cells, or the cells were activated with PDBu for 30 min. A portion of the cells was treated with ICAM-1 or CTT to block ₂ integrins or gelatinases, respectively. Cells were bound to poly-L-lysine-coated coverslips, fixed with methanol for 10 min at –20 °C or with 4% paraform-aldehyde for 15 min at +4 °C, and permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min followed by several washings. The coverslips were incubated with rabbit anti-MMP-9 polyclonal and mouse anti-_M (OKM-10) antibodies diluted 1:500. After washing with PBS, the secondary antibodies, rhodamine (TRITC)-conjugated porcine anti-rabbit or fluorescein isothiocyanate-conjugated goat anti-mouse (Fab')₂ (Dakopatts A/S, Copenhagen, Denmark) were incubated at a 1:1000 dilution for 30 min at room temperature. The samples were mounted with Mowiol, incubated in the dark for 2 days, and examined by a confocal microscope (Leica multiband confocal imagine spectrophotometer) at a x400 magnification or a fluorescence microscope (Olympus Provis 70) at a x60 magnification.

Cell Adhesion and Migration—Fibrinogen and ICAM-1-Fc were coated at 40 µg/ml in TBS at +4 °C. Peptides (2 µg/well) were coated in TBS containing 0.25% glutaraldehyde at +37 °C. The wells were blocked with 1% BSA in PBS. THP-1 cells (50,000/well) with or without PDBu activation were added in 0.1% BSA/RPMI medium in the presence or absence of 200 µM peptides or monoclonal antibodies at 50 µg/ml. After 30–35 min the wells were washed with PBS to remove non-adherent cells, and the adhesive cells were quantitated by a phosphatase assay. The cell migration assay was conducted using transwell migration chambers (8 µm pore size, Costar) in serum-containing medium as described (14). Briefly, the membranes were coated on the upper and lower surface with 40 µg/ml GST, LLG-C4-GST, or left uncoated. The wells were blocked with 10% serum-containing medium for 2 h. THP-1 cells (50,000/100 µl) or HT1080 (20,000/100 µl) were preincubated with the peptides for 1 h before transfer to the upper chamber. The lower chamber contained 500 µl of the medium without the peptides. The cells were allowed to migrate to the lower surface of the membrane for 16 h and then stained with crystal violet and counted.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Identification of the _M I Domain-binding Peptide Motif D/E/D/E-G/L-W—By using phage peptide display libraries, we selected peptides that interact with the _M I domain. GST-binding phage were first eliminated on GST-coated wells, and the unbound phage preparations were incubated on _M I domain GST fusion protein-coated wells. The _M I domain-binding phage were enriched by three rounds of panning, and the peptide sequences were determined. With the exception of one linear peptide, the peptides were derived from the cyclic CX₇C and CX₈C libraries. The I domain-binding sequences showed only one conserved motif, a somewhat unexpected finding in terms of the known ligand binding promiscuity of the I domain. The bound peptides contained two consecutive negatively charged amino acids, i.e. glutamic and/or aspartic acids, followed by glycine and tryptophan residues (Fig. 1A). The consensus (D/E)(D/E)(G/L)W determined by this approach was clearly different from LLG-C4 and other ₂ integrin-binding peptides reported so far.

View larger version (41K): [in this window] [in a new window]   FIG. 1. Identification of an I domain-binding site in progelatinase. A, phage display peptide sequences specifically bound to the M I domain. The consensus motif is shown in boldface. Peptides with the strongest binding (CILWMDDGW) and the highest similarity (CPEELWWLC) are aligned with human MMPs (Swiss-Prot accession numbers shown in parentheses). B, phages bearing the CILWMDDGWC peptide or a control peptide were allowed to bind to immobilized M I domain-GST fusion protein (20 ng/well) in the absence or presence of 15 µM DDGW peptide or LLG-C4 peptide. Bound phages were detected using a monoclonal anti-M13 phage antibody. Mean absorbance of triplicate samples ± S.D. is shown. C, L, M, or X I domain-GST fusions were coated on microtiter wells as in B, and the binding of CILWMDDGWC peptide bearing phage or a control phage was measured. D, peptides covering the complete sequence of pro-MMP-9 were synthesized as overlapping peptides on a pepspot membrane. The M I domain (0.5 µg/ml) was allowed to bind to the peptides followed by immunodetection using anti-M I domain antibody LM2/1. The M I domain-binding peptide 13 (arrow) is shown in boldface, and the zinc binding catalytic sequence is underlined. The prodomain (Pro), catalytic domain containing the fibronectin type II repeats (Cat), and hemopexin domain (Pex) are marked to illustrate the domain structure ofpro-MMP-9. E, alanine-mutated and truncated peptides were synthesized on a pepspot filter and probed with the recombinant M I domain (5 µg/ml). Bound I domain was measured using monoclonal antibody MEM170 (5 µg/ml) followed by horseradish peroxidase-conjugated anti-mouse secondary antibody and ECL detection. The binding was quantified by densitometric scanning. The bars show M I domain binding to single peptide spots as arbitrary optical density units/mm2. Similar results were obtained in three independent experiments.

We first prepared the phage display peptides as intein fusion proteins, from which the peptides were cleaved. This allowed us to test rapidly the peptide solubility and the binding specificity before large scale chemical peptide synthesis. The peptides were cloned using oligonucleotide primers that amplify the peptide library insert from the phage vector. Consequently, all the peptides prepared contain the vector-derived sequences ADGA and GAAG in the N and C termini, respectively. Phage binding experiments using soluble peptides as competitors indicated that the peptides bearing the two adjacent negative charges bound to a common site (not shown). We chose the peptide ADGA-CILWMDDGWC-GAAG (DDGW) for further experiments as this peptide showed strong binding and was highly soluble in aqueous buffers (soluble in 50 mM NaOH at >10 mM concentrations). The peptide was also prepared by chemical synthesis. The phage bearing the DDGW sequence avidly bound to the _M I domain, and this was readily inhibited by low concentrations of the DDGW peptide, but only marginally affected by the LLG-C4 peptide, indicating different binding sites for DDGW and LLG-C4 (Fig. 1B). Control phage bearing other peptide sequences did not bind. The DDGW-bearing phage also showed also specific binding to the _L I domain that was inhibitable by DDGW, but the interaction was weaker than with the _M I domain (Fig. 1C and data not shown). No binding was observed with the _X I domain or GST used as a control (Fig. 1C).

Characterization of DELW Sequence on the Catalytic Domain of Gelatinases That Mediates Interaction with the ₂ Integrin I Domains—We searched protein databases for matches to the novel (D/E)(D/E)(G/L)W motif. One of the phage library-derived peptides, CPEELWWLC, was highly similar to the DELW(S/T)LG sequence present on the catalytic domain of MMP-2 and MMP-9 gelatinases (Fig. 1A). DELW-like sequences with double negative charges are also present in other secreted MMPs but not in the membrane-type MMPs such as MMP-14.

No MMP has been reported to bind to the leukocyte ₂ integrins. We therefore set out to study whether MMP-9 in particular could be a ligand of the ₂ integrins as MMP-9 gelatinase is the major leukocyte MMP and is induced during ₂ integrin activation. As a first step, we synthesized the whole pro-MMP-9 sequence as overlapping 20-mer peptides on a pepspot membrane. Binding assays with the _M I domain revealed a single active peptide that located to the MMP-9 catalytic domain (Fig. 1D). No binding was observed when the I domain was omitted, and the membrane was probed with antibodies only. The sequence of the I domain-binding peptide was QGDAHFDDDELWSLGKGVVV, and it contained the binding motif identified by phage display (Fig. 1D).

The active MMP-9 peptide contained four consecutive amino acids with negative charges, DDDE. To study the importance of these residues, the aspartic and glutamic acid residues that were closest to the tryptophan were replaced by alanines. At the same time the peptide length was shortened to 15-mer. The alanine mutagenesis significantly abrogated I domain binding on the pepspot filter; the OD value dropped from 2010 to 476 (Table I). To study whether the negatively charged peptide from other MMPs is also active, we synthesized the corresponding 15-mers and the double alanine mutations. Sequences from MMP-1–3, –7–9, and -13, but not the membrane-anchored MMP-14 (MT1-MMP), could bind the _M I domain. Alanine mutations always decreased the binding.

View this table: [in this window] [in a new window]   TABLE I Pepspot analysis of peptides derived from MMPs and M2 integrin ligands The ranges used are as follows: -, 0-1499 OD/mm2; +, 1500-2999; ++, 3000-4900; and +++, > 4500.

We did similar pepspot analysis for some of the known I domain ligands, which contain (D/E)(D/E)(G/L)W-like sequences. Peptides derived from myeloperoxidase, catalase, thrombospondin-1, and complement protein iC3b strongly bound the I domain in this assay, and the double alanine mutation caused a loss of binding (see Table I). Of the three iC3b peptide permutations tested, ARSNLDEDIIAEENI was the active one. The acidic residues were followed by a hydrophobic isoleucine cluster in this peptide. The soluble DDGW peptide efficiently inhibited the binding of this peptide to the I domain. Weaker I domain binding was observed with one complement factor H-derived peptide and one fibronectin-derived peptide. Peptides derived from ICAMs-1–3, neutrophil inhibitory factor, Cyr61, fibrinogen, GP1b, factor X, or E-selectin lacked activity.

Alanine-scanning mutagenesis of the DDGW peptide with the pepspot system similarly indicated the importance of the aspartic acid residues for I domain binding (Fig. 1E). Alanine mutations of the glycine or either one of the tryptophan residues also inactivated the peptide. Mutations of the isoleucine, leucine, or methionine residues were tolerated. Deletion of the ADGA sequence from the N terminus had no effect on I domain binding, but removal of the C-terminal GAAG sequence abolished the binding. As the peptides were immobilized via the C terminus on the filter, a sufficient linker sequence such as GAAG seemed important. We also tested a series of truncated cyclic peptides to identify the shortest active sequence. This analysis showed that ADGA-CEDGWC-GAAG but not ADGA-CDDGWC-GAAG was the minimal peptide that supported _M I domain binding. The longer side chain of glutamate compared with aspartate is probably required to bring the negatively charged carboxyl group in the correct position for I domain binding.

Progelatinases Bind to Purified _M2 and _L2 Integrins and Their I Domains—We next used a microtiter well-based sandwich assay to study gelatinase binding to purified integrins. Progelatinases bound in a concentration-dependent manner to the coated _M2 integrin (Fig. 2A). Curiously, MMP-2 and MMP-9 lost the integrin binding ability after activation by trypsin or APMA. The binding of pro-MMP-2 and pro-MMP-9 was observed with both _M2 and _L2 integrins and their corresponding I domains (Fig. 2, B and C). No binding was detected on the _X I domain or the ₁₁ and ₃₁ integrins.

View larger version (26K): [in this window] [in a new window]   FIG. 2. Binding of progelatinases to purified integrins and I domains. A, pro-MMP-9, pro-MMP-2, or their trypsin-activated forms, at the concentrations indicated, were allowed to bind to M2 integrin-coated wells. Appropriate MMP antibodies were used to determine binding. The results in this and other figures are represented as the means ± S.D. from triplicate wells. B, binding of pro-MMP-9 (80 ng/well) was examined on microtiter wells coated with an integrin (L2, M2, 11, and 31) or an I domain (L, M, X). The binding was determined using anti-MMP-9 antibody. C, pro-MMP-2 (80 ng/well) was allowed to bind to L2, M2, 11, or the I domains L, M, or X. The binding was determined using an anti-MMP-2 antibody.

The DDGW peptide was an efficient inhibitor, and it inhibited pro-MMP-9 binding to the _M I domain with an IC₅₀ value of 20 µM (Fig. 3, A and B). To demonstrate that the negative charges of aspartic acids are essential for the peptide activity, the peptide ADGACILWMKKGWCGAAG (KKGW) containing lysines in place of aspartic acids was prepared. As expected, the KKWG peptide was inactive and did not compete with pro-MMP-9 binding. We were also interested in testing lovastatin, as its binding site in the _L I domain is known (34, 35). Lovastatin was not able to compete with pro-MMP-9 even at a high concentration.

View larger version (39K): [in this window] [in a new window]   FIG. 3. Inhibitors of the pro-MMP-9/2 integrin complex. A, M and L I domain-GST fusions were immobilized on microtiter wells. Pro-MMP-9 (100 ng/well) was added in the presence or absence of various peptides (200 µM) or lovastatin (100 µM) in a buffer containing 0.5% BSA. Pro-MMP-9 binding was detected using a monoclonal antibody against MMP-9. The results are shown as percent binding compared with binding in the absence of inhibitors (100%), and no pro-MMP-9 was added (0%). B, DDGW peptide blocks pro-MMP-9 binding to M in a dose-dependent manner. The assay was done similarly as in A, except various concentrations of peptides were added to compete for binding. All samples were assayed as triplicates, and results shown are means ± S.D. from a representative experiment. C, pro-MMP-9 binding to M2 and L2 was examined in the absence and presence of EDTA (5 mM), MMP inhibitor-1 (100 µM), CTT (200 µM), STT (200 µM), MEM170 (40 µg/ml), or control TL3 antibody (40 µg/ml). The background of primary and secondary antibodies was measured by omitting pro-MMP-9 from the wells or by coating with ICAM-1. D, pro-MMP-9 binding to purified M and L I domain GST fusion proteins or wild type GST was studied in the absence or presence of competitors as indicated. Control shows the background when pro-MMP-9 was omitted.

Pro-MMP-9 bound like a true integrin ligand, as the cation chelator EDTA (5 mM) nearly completely prevented the binding (Fig. 3, C and D). For background measurement in the sandwich assay, we used antibodies alone (control) or coating with ICAM-1 or wild type GST. The gelatinase-binding peptide CTT (200 µM) inhibited pro-MMP-9-integrin interaction with the same efficiency as EDTA did. The control peptides STTHWGFTLS (STT) and CTTHAGFTLC (W A CTT), which lack gelatinase inhibitory activity (26), were without effect. A non-peptide chemical MMP inhibitor (Inh1) also prevented pro-MMP-9 binding. As EDTA inhibits both the gelatinase and the integrin, we used integrin-blocking antibodies and ligand peptides to demonstrate the specific binding activity of ₂ integrin. The known ligand-binding blocking antibodies MEM170, MEM83, and LM2/1 inhibited pro-MMP-9 binding. A control antibody TL3 had no effect. The I domain binding peptide LLG-C4 showed a partial inhibitory effect. RGD-4C, a ligand of _V integrins, served as control peptide and had no effect on pro-MMP-9 binding. The purity of the integrins was typically more than 90% and that of I domains 95%, making it unlikely that progelatinases would bind to impurities in the preparations.

Progelatinase-integrin complexes were also obtained by coprecipitation experiments using HT1080 conditioned medium as a source of pro-MMP-9 and pro-MMP-2, which were analyzed by zymography. The progelatinases coprecipitated with _M2 integrin or _M I domain GST fusion protein when these were used as a bait. The integrin added to the medium was immunoprecipitated with the _M antibody OKM10 (Fig. 4A). The _M I domain GST protein was pulled down with glutathione beads (Fig. 4B). CTT but not STT had an inhibitory effect. Inhibition of the I domain by LM2/1, ICAM-1, or LLG-C4 also affected the pull-down of progelatinases. GST control did not coprecipitate the gelatinases. No active forms of gelatinases were found to coprecipitate with _M2 or the I domain, when APMA-treated HT-1080 medium or APMA-activated MMP-9 was used (not shown).

View larger version (32K): [in this window] [in a new window]   FIG. 4. Coprecipitation of progelatinase with 2 integrin. A, M2 integrin (3 µg) was incubated with a 500-µl sample of HT1080 medium containing pro-MMP-9 and pro-MMP-2 in the absence or presence of CTT or STT (200 µM) for 2 h. The integrin was immunoprecipitated with the OKM10 antibody, and the immunoprecipitates were analyzed by gelatin zymography. In control experiments, integrin was omitted from the medium, and ICAM-1 was added instead. B, a 500-µl sample of HT1080 medium containing pro-MMP-9 and pro-MMP-2 was incubated with the M I domain GST (3 µg) or LLG-C4-GST control. ICAM-1, LM2/1, CTT, STT, or LLG-C4 were used as competitors. GST was pulled down with glutathione beads, and bound proteins were analyzed by zymography. Inset: lane 1, the pro-MMP-2 and pro-MMP-9 zymogens present in non-treated HT1080 medium; lane 2, lack of gelatinases pulled down with control LLG-C4-GST; and lane 3, pro-MMP-9 and pro-MMP-2 coprecipitated by M I domain GST fusion protein.

As the gelatinase inhibitors CTT and Inh1 prevented the binding of pro-MMP-9 to the integrin, it can be anticipated that CTT and Inh1 avidly bind to pro-MMP-9. To gain more insight into this, we examined binding of pro-MMP-9 to immobilized CTT peptide. Pro-MMP-9 specifically bound to the CTT-GST fusion protein (Fig. 5A) but not to LLG-C4-GST. CTT and Inh1 at 100 µM concentrations effectively competed in binding, but W A CTT did not. The pro-MMP-9 preparation did not contain detectable amounts of active MMP-9 on zymography analysis, and after pro-MMP-9 activation with APMA, the CTT-GST binding increased. CTT and Inh1 could also bind to pro-MMP-9 secreted into the medium of PDBu-activated THP-1 leukemic cells (Fig. 5B) or HT1080 fibrosarcoma cells (not shown). A time-dependent reduction in the gelatinolytic activity of pro-MMP-9 was observed with CTT (1st panel) and Inh1 (3rd panel) but not with the W A CTT peptide (4th panel). Western blot analysis indicated that CTT does not decrease the secretion of pro-MMP-9 by the cells (2nd panel). Furthermore, the CTT complex was reversible and disappeared after repeated freezing and thawing of the samples.

View larger version (52K): [in this window] [in a new window]   FIG. 5. CTT peptide binds to both latent and active MMP-9. A, binding of pro-MMP-9 or APMA-activated MMP-9 to CTT-GST was examined in the absence or presence of competitors CTT (100 µM), W A mutant CTT (100 µM), and Inh1 (100 µM). GST control was LLG-C4-GST. Binding was determined as in Figs. 2 and 3. The background in the absence of pro-MMP-9 is shown. B, THP-1 cells were incubated in serum-free medium containing CTT, Inh1, or W A CTT at 200 µM concentration. Samples from the media were collected at the time points indicated and analyzed by zymography (1st, 3rd, and 4th panels) or Western blotting (2nd panel).

Demonstration of a Cell-surface Complex between Progelatinases and ₂ Integrins—To study whether the progelatinases occur in a complex with the ₂ integrins on the leukocyte surface, we performed immunoprecipitation and colocalization studies. First, we examined THP-1 monocytic leukemia cells in the resting state and after induction by PDBu, which mimics leukocyte activation in vivo. THP-1 cell stimulation with PDBu led to up-regulation of MMP-9 (data not shown). The cell surface glycoproteins of THP-1 cells were labeled with tritium [³H] followed by immunoprecipitation with ₂ integrin and MMP-9 antibodies. In the PDBu-activated cells, the _M chain antibody OKM10 and ₂ chain antibody 7E4 immunoprecipitated two ³H-labeled proteins corresponding to the integrin _M chain (165 kDa) and ₂ chain (95 kDa) (Fig. 6A, lanes 9 and 10). Importantly, polyclonal MMP-9 antibodies immunoprecipitated the same two integrin chains (lane 7). In non-activated cells, essentially no coprecipitation of _M and ₂ was observed with MMP-9 antibodies, although the _M and ₂ chains were present. The coprecipitation of the integrin chains by MMP-9 antibodies was prevented by the CTT peptide (lane 8). The control antibody (TL3) did not precipitate any proteins.

View larger version (30K): [in this window] [in a new window]   FIG. 6. Progelatinases occur in complex with M2 and L2 in PDBu-activated THP-1 and Jurkat cells. A, THP-1 cell surface proteins were 3H-labeled using periodate-tritiated borohydride and analyzed by immunoprecipitation. CTT was used as a competitor (200 µM). The immunoprecipitated samples were resolved on an 8–16% polyacrylamide gel, and the film was exposed for 3 days. Lanes 1–4 are from non-activated cells and lanes 6–10 from PDBu-activated cells. Lane 5 shows molecular weight markers. B, lysates from PDBu-activated THP-1 cells were immunoprecipitated with integrin or MMP antibodies followed by Western blotting with M (OKM10), L (TS2/4)n or MMP-9 antibodies. Preclearings of the cell lysates were done using M (lane 6) and L (lane 7) antibodies. C, lysates from PDBu-activated Jurkat cells were subjected to immunoprecipitation followed by blotting with the L (MEM83) and MMP-9 antibodies.

With the ³H-labeled cells, we did not observe any band corresponding to pro-MMP-9, perhaps because the carbohydrates of pro-MMP-9 are poorly labeled. We therefore analyzed the PDBu-activated THP-1 cells by Western blotting (Fig. 6B). Pro-MMP-9 was readily immunoprecipitated with antibodies against MMP-9, _M, or _L but not by the control antibody. MMP-9 antibodies in turn were able to immunoprecipitate the _M but not the _L chain. MMP-2 antibodies similarly coprecipitated _M but not _L. When the cell lysate was precleared with the _M antibody OKM-10, the amount of immunoprecipitated _M and pro-MMP-9 clearly decreased (lane 6). Preclearing with the _L antibody TS2/4 did not significantly remove _M or pro-MMP-9 but abolished the _L precipitation (lane 7).

As THP-1 cells do not express high amounts of the _L chain (11), we examined the Jurkat T cell line, which expresses more _L than _M (28). We observed a significant immunoprecipitation of _L by MMP-9 and MMP-2 antibodies after PDBu activation (Fig. 6C). Furthermore, the _L antibody coprecipitated more pro-MMP-9 in comparison to the _M antibody. No pro-MMP-9 coprecipitated with MMP-2 antibodies in Jurkat or THP-1 cells.

Pro-MMP-9 and _M2 were found to colocalize on the cell surface following PDBu activation of THP-1 cells as studied by fluorescence and confocal microscopy (Fig. 7, A and B, respectively). By using a higher magnification, colocalization was primarily seen in cell surface clusters (Fig. 7B) and to a lesser extent on areas where cells contacted each other (not shown). We believe that the MMP-9 colocalizing with _M2 is the pro-MMP-9, as the activated MMP-9 did not bind to _M2. Without PDBu activation, there was hardly any colocalization of pro-MMP-9 and _M2. The secondary antibodies did not stain the cells when the primary antibodies were omitted (data not shown). When the cells were preincubated with the CTT peptide or recombinant soluble ICAM-1 to block pro-MMP-9 or _M2, the cell surface clusters did not form, and the pro-MMP-9-_M2 colocalization was not observed (not shown). ProMMP-9-_M2 colocalization was also observed on Jurkat cells follow-ing phorbol ester stimulation (not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 7. PDBu-induced colocalization of M2 and pro-MMP-9 in THP-1 cells. Cells were preincubated for 30 min at +37 °C with 50 nM PDBu. A, cells were treated with anti-M OKM10 and anti-MMP-9 antibodies followed by fluorescein isothiocyanate-labeled (green fluorescence) and TRITC-labeled (red fluorescence) secondary antibodies. Yellow indicates colocalization of M2 and pro-MMP-9. Bars, 8.5 µm. B, immunofluorescence staining shows intense colocalization of MMP-9 (polyclonal antibody) and M2 integrin (OKM-10) on the surface of PDBu-activated THP-1 cells at higher magnification as visualized by confocal microscopy (bars, 2.5 µm).

Blocking the Progelatinase-₂ Integrin Complex with DDGW Releases Cell-bound Pro-MMP-9 and Inhibits Cell Migration but Not Adhesion—As the DDGW peptide is an integrin ligand, one of the questions was whether it can support adhesion of leukocytes. We studied adhesion of human myelomonocytic THP-1 cells on immobilized glutaraldehyde-polymerized peptide. Phorbol ester-activated cells efficiently bound to the DDGW peptide, whereas there was no binding in the absence of cell activation (Fig. 8A). As a positive control, the recombinant intein-produced ADGA-CPCFLLGCC-GAAG peptide supported adhesion, but unlike the DDGW peptide, it also supported adhesion in the absence of integrin activation. The acute myeloid leukemic cell line OCI/AML-3 also avidly adhered to DDGW, whereas human fibrosarcoma HT1080 cells which lack ₂ integrins did not (not shown). As THP-1 cells were able to adhere on DDGW, we next studied the effect of the peptide on ₂ integrin-dependent adhesion to fibrinogen and ICAM-1. Interestingly, DDGW did not block cell adhesion to fibrinogen, whereas the LLG-C4 peptide blocked the adhesion as reported previously (Fig. 8B). Similarly, DDGW did not block the binding of recombinant _M I domain to immobilized fibrinogen (not shown). DDGW did not either block cell adhesion on ICAM-1-Fc fusion protein. As a control, the blocking antibody 7E4 against ₂ integrins prevented the ICAM-1 binding, indicating that the THP-1 cells bound in a ₂ integrin-dependent manner (Fig. 8C). We also found no blocking effect of DDGW on THP-1 adhesion to LLG-C4-GST fusion protein (not shown).

View larger version (28K): [in this window] [in a new window]   FIG. 8. The DDGW peptide supports THP-1 cell adhesion and induces pro-MMP-9 release but does not block adhesion to the major 2 integrin ligands fibrinogen and ICAM-1. A, THP-1 cells were allowed to bind to immobilized, glutaraldehyde-polymerized peptides with or without phorbol ester activation (50 nM), and the adherent cells were quantitated by phosphatase assay. THP-1 cells were allowed to bind to immobilized fibrinogen (B), or recombinant ICAM-1-Fc (C), in the presence or absence of 200 µM soluble peptides. All samples were assayed as triplicates, and results show means ± S.D. Identical results were obtained in two other independent experiments. D, THP-1 cells were incubated in the presence or absence of peptides at 200 µM concentration for 48 h. Aliquots of conditioned medium were analyzed by gelatin zymography. Arrows show the 92-kDa pro-MMP-9 and 220-kDa pro-MMP-9 dimer.

The second question raised by these studies was whether the DDGW peptide can release cell-bound pro-MMP-9. When THP-1 cells were cultured for 48 h in the presence of DDGW, an increase of pro-MMP-9 level was observed in the conditioned medium as studied by gelatin zymography (Fig. 8D). The peptide increased both monomeric and dimeric pro-MMP-9 in the culture medium. In contrast, CTT slightly decreased or inhibited active pro-MMP-9. KKGW and W A CTT had no effect.

Finally, we studied the role of the progelatinase-₂ integrin complex in leukocyte migration using a transwell assay in which leukocyte migration can be adjusted by the choice of coated matrix or ligand protein. We tested that the CTT, LLG, and DDGW peptides are not toxic to the THP-1 cells in a 48-h time frame at >200 µM concentrations using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. By using transwells coated with 10% serum in cell culture medium, we first studied the effect of peptides on the basal migration of THP-1 cells in the absence of any stimulus by phorbol ester or an adhesive matrix. Under such conditions, CTT, LLG-C4, or the gelatinase inhibitor Inh1 at a 200 µM concentration had no effect on THP-1 migration indicating no active involvement of gelatinases or ₂ integrins (Fig. 9A). We have shown previously that when the transwells are coated with LLG-C4-GST fusion protein, THP-1 cells adhere and migrate in a ₂ integrin-dependent manner (14). Thus, transwells were coated with LLG-C4-GST fusion protein or GST alone. Both the DDGW and CTT peptide, but not KKGW, inhibited the migration of THP-1 cells on the LLG-C4-GST substratum (Fig. 9B). The soluble LLG-C4 peptide also blocked the migration. In the presence of GST coating, cell migration was negligible. To verify that the effect of DDGW peptide was ₂ integrin-dependent, HT1080 fibrosarcoma cells lacking these integrins were allowed to migrate in the presence of CTT, DDGW, KKGW, or LLG-C4. Of these peptides, only CTT was capable of inhibiting cell migration (Fig. 9C).

View larger version (23K): [in this window] [in a new window]   FIG. 9. Peptide inhibition of THP-1 cell migration. THP-1 cells were preincubated with the peptide as indicated at a 200 µM concentration for 1 h at room temperature and applied to transwells in the absence (A) or presence (B) of LLG-C4-GST coating. Cells were allowed to migrate for 16 h at +37 °C. Cells migrated to the lower surface of the filter were stained and counted microscopically. C, HT1080 fibrosarcoma cell migration was similarly assayed in the absence of LLG-C4 coating. The bars show means ± S.D. from triplicate wells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Analysis of _M I domain-binding peptides led to the finding that MMPs, particularly the MMP-9 and MMP-2 progelatinases, are potent ₂ integrin ligands. Our studies show that pro-MMP-9, the major MMP of activated leukocytes, is colocalized with the ₂ integrin on the cell surface. Cell surface labeling and coimmunoprecipitation further demonstrate the occurrence of the complex in leukemic cell lines. Finally, we have found evidence that this proteinase-integrin complex plays a role in migration of the leukemic cells.

Although phage display has been extensively used with whole integrins (14, 36–44), to the best of our knowledge, this is the first successful phage display selection on an isolated integrin I domain. We could enrich only one binding motif even though the _M I domain can bind a variety of ligands (1–3). The peptide motif we isolated could not compete with the ICAM-1, fibrinogen, or LLG-C4 ligands. The success of phage display depends on the libraries used and the biopanning conditions. Our method favored "high affinity" interactions with the cyclic peptides yielding the (D/E)(D/E)(G/L)W motif. Interestingly, this motif shows a high degree of similarity to the CWDD(G/L)WLC peptide isolated by phage display as an RGD sequence-binding peptide (40). By recognizing the RGD ligand sequence, CWDDGWLC structurally and functionally behaves like a minimal integrin. Here we have identified the DDGW peptide in a reverse situation, as a ligand to integrin. However, the RGD sequence does not compete with the _M I domain as the GRGDSP peptide ata1mM concentration was unable to inhibit pro-MMP-9 binding to the I domain.² It will be interesting to see whether the DDGW peptide recognizes a positively and negatively charged sequence in the I domain.

Some hints for the binding site of DDGW come from the interaction of iC3b with _M2 integrin. Our pepspot analysis showed that the iC3b peptide ARSNLDEDIIAEENI, but not the control peptide ARSNLDAAIIAEENI, bound the _M I domain, and the DDGW peptide blocked this binding. The DEDIIAEENI sequence with multiple adjacent negative charges is required for efficient binding of iC3b to _M2 integrin (45). The binding site of complement protein iC3b in the I domain has been mapped indicating a role for the positively charged amino acid residue Lys²⁴⁵ for iC3b binding (46). Mutation of this residue does not affect the binding of the fibrinogen recognition peptide (47). This may account for the inability of the DDGW peptide to inhibit ICAM-1 and fibrinogen-mediated cell adhesion. These findings suggest that the Lys²⁴⁵ residue is the positively charged contact site for the DDGW peptide and the (D/E)(D/E)(G/L)W motif as well.

The pepspot analysis indicates that a class of ₂ integrin ligands contains an active (D/E)(D/E)(G/L)W motif. These include the previously identified _M2 ligands iC3b, thrombospondin-1, and the enzymes myeloperoxidase and catalase (3–5, 48). In our experiments, the peptides derived from several secreted MMPs, but not membrane-bound MT1-MMP, were also active. It is notable that the (D/E)(D/E)(G/L)W motif is relatively conserved in the secreted members of the MMP family. Whether other MMPs in addition to the progelatinases can make ₂ integrin complexes remains to be determined.

Finding of a dominant integrin-binding site in the catalytic domain of pro-MMP-9 was unexpected, because previous studies suggested an essential role for another MMP domain, the hemopexin domain, in integrin binding. The hemopexin domains mediated MMP-2 binding to the _V3 integrin (49–51) and MMP-1 binding to the ₂₁ integrin (52, 53). The cleaved hemopexin domain of MMP-2 has also been shown to occur in vivo and to inhibit angiogenesis (50). Understandably, phage peptide display and pepspot techniques have limitations, and only linear peptide sequences can be analyzed, not protein conformations. Thus, in the present study we cannot make conclusions of the function of separate MMP-9 domains in integrin binding, and it remains to be seen whether cleavage products of MMP-9, if present in vivo, can act as ₂ integrin ligands. Our studies suggest that the peptide sequence from the catalytic domain is essential for the binding of full-length pro-MMP-9 to the ₂ integrin, as the synthetic DDGW peptide could completely inhibit the integrin binding. It was important to use natural pro-MMP-9 because _M2 is known to bind to denatured proteins, and this may sometimes be the case for bacterially expressed proteins (4). Furthermore, we did not observe binding of an active MMP-2 or MMP-9 to the integrin, although the DELW(T/S)LG sequence should remain unchanged in the active enzyme. We rather found that AMPA and trypsin, activators of pro-MMP-9, released MMP-9 from THP-1 cells, apparently affecting the integrin complex.³ These results suggest that the proenzyme presents the integrin-binding site more efficiently than the active enzyme, and the ₂ integrin may even control the activation of the proenzyme.

In the three-dimensional structures of pro-MMP-2 and -9 (54, 55), the I domain-binding site is located in the vicinity of the zinc-binding catalytic sequence HEFGHALGLDH between the catalytic domain and the fibronectin type II repeats. This location suggests a mechanism for evading pro-MMP-9 inhibition by tissue inhibitors of MMPs or _2-macroglobulin. In the absence of inhibitors, the cell surface-localized pro-MMP-9 would be readily susceptible for activation and substrate hydrolysis, which may also occur in the presence of intact propeptide (20). On the other hand, because the binding site of the I domain is located in the vicinity of the catalytic groove, it also suggests an explanation for the blocking of MMP-9/₂ integrin interaction by the small molecule MMP inhibitors such as CTT and Inh1.

The activity of the DDGW peptide in the THP-1 cell migration assay suggests an important function for the integrin-progelatinase complex in leukocyte migration. Obviously, we cannot exclude the possibility that the DDGW peptide blocks binding of other ligands than gelatinases and in this way inhibits the leukocyte migration. However, as the specific gelatinase inhibitor CTT also blocks the THP-1 cell migration, these results strongly suggest that the pro-MMP-9-₂ integrin complex is the main target for DDGW. Interestingly, the DDGW peptide blocked THP-1 cell migration although it increased the level of pro-MMP-9 in the medium, which suggests that cell surface-bound rather than total MMP-9 level is a critical factor in cell migration.

	FOOTNOTES

 
^* This work was supported by the Academy of Finland, The Sigrid Jusélius Foundation, The Magnus Ehrnrooth Foundation, and the Finnish Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Two of the founding members and shareholders of the CTT Ltd. Co.

To whom correspondence should be addressed. Tel.: 358-9-19159023; Fax: 358-9-19159068; E-mail: erkki.koivunen{at}helsinki.fi.

¹ The abbreviations used are: ICAM, intercellular adhesion molecule; APMA, aminophenylmercuric acetate; _M2, CD11b/CD18, Mac-1 integrin; CTT, CTTHWGFTLC peptide; DDGW, ADGACILWMDDGWCGAAG peptide; GST, glutathione S-transferase; Inh1, matrix metalloproteinase inhibitor 1; KKGW, ADGACILWMKKGWCGAAG peptide; LLG-C4, CPCFLLGCC peptide; MMP, matrix metalloproteinase; STT, STTHWGFTLC peptide; W A CTT, CTTHAGFTLC peptide; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; TRITC, tetra-methylrhodamine isothiocyanate; BSA, bovine serum albumin; PDBu, 4-phorbol 12,13-dibutyrate; AML, acute myeloid leukemia.

² M. Björklund and E. Koivunen, unpublished observations.

³ M. Stefanidakis and E. Koivunen, unpublished observations.

	ACKNOWLEDGMENTS

 
We thank Esa Kuismanen, Anne Toivanen, Erik Mandelin, Tiina Hilden, Heli Valtanen, and Jussi Hepojoki for their generous help, discussions, and valuable comments, and Leena Kuoppasalmi and Jaana Kekkonen for excellent technical assistance.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Gahmberg, C. G. (1997) Curr. Opin. Cell Biol. 9, 643–650[CrossRef][Medline] [Order article via Infotrieve]
2. Colombatti, A., and Bonaldo, P. (1991) Blood 77, 2305–2315