EMMPRIN (CD147), an Inducer of Matrix Metalloproteinase Synthesis, Also Binds Interstitial Collagenase to the Tumor Cell Surface

Phage Display Library.

mRNA was prepared from human fibroblasts with the Oligotex mRNA kit (Qiagen, Valencia, CA) and used for cDNA synthesis with the Directional RH primer cDNA synthesis kit (Novagen, Madison, WI). After second-strand synthesis, the cDNA ends were flushed with T4 DNA polymerase and ligated to EcoRI/HindIII directional linkers. The cDNA was then digested with EcoRI and HindIII and ligated to T7Select1-1b vector arms (Novagen). The ligated DNA was packaged into bacteriophage T7 using the T7Select1-1 Packaging Extract (Novagen). The host strain of bacteria, BLT 5403 (Novagen), was then grown to A_{600 nm} = 0.8–1.0 and mixed with the packaged cDNA (at a ratio of 10⁶ phage/10 ml cells) in LB media containing 50 μg/ml carbenicillin (Novagen). Molten top agarose at 45°C–50°C was added to the phage/host mixture (10:1) and immediately poured onto a 150-mm plate containing LB/carbenicillin medium. The plate was incubated at room temperature overnight until the plaques were nearly confluent. The phage was then eluted by covering the plate with phage extraction buffer [100 mm NaCl, 20 mm Tris, and 6 mm MgSO₄ (pH 8.0)] at 4°C overnight. The phage lysate was clarified with chloroform and subjected to screening by biopanning.

Screening of Phage Display Library.

Twenty four-well cell culture plates were prepared for biopanning as suggested by the manufacturer (Novagen). The wells were coated with immunopurified EMMPRIN protein (Ref. 12 ; 1 μg/ml in Tris-buffered saline) at 4°C overnight and washed with Tris-buffered saline five times. Unreacted sites were blocked with 5% blocking reagent overnight at 4°C and washed. In the first round of screening, the phage lysate was applied to the EMMPRIN-coated plate (0.5 ml lysate/well) for 30 min at room temperature. The plate was then washed five times with Tris-buffered saline. The bound phages were eluted by adding 0.5 ml of elution buffer (1% SDS) at room temperature for 20 min. The eluted phages were then added to a culture of the host cells (BLT 5403) in LB media and incubated at 37°C with shaking for 3 h, at which time lysis was observed. The lysed culture was centrifuged, and the supernatant was collected for the next round of biopanning. A total of five rounds of screening was carried out. DNA from the phages isolated during the final round of screening was purified and sequenced using the T7 SelectUp primer (GGAGCTGTCGTATTCCAGTC) and the T7 SelectDown primer (AACCCTCAAGACCCGTTTA; Novagen).

Immunoaffinity and Ligand Affinity Chromatography.

EMMPRIN was isolated from extracts of membranes from LX-1 human lung carcinoma cells by immunoaffinity chromatography using E11F4 monoclonal antibody against EMMPRIN immobilized on Sepharose beads, as described previously (12) .

For manufacture of the ligand affinity medium, EMMPRIN protein (0.5 mg) was first dissolved in coupling buffer [0.1 m NaHCO₃ and 0.5 m NaCl (pH 8.3) containing 0.5% NP40]. The coupling solution was then mixed with CNBr-activated Sepharose 4B gel (Pierce; 0.25 g of dried powder swelled and washed in 1 mm HCl for 30 min) at 4°C. After overnight incubation, the gel was washed three times with 5 ml of coupling buffer, followed by incubation in 0.1 m Tris-HCl (pH 8) for 2 h to block any remaining active groups. Then the gel was washed using three cycles of 0.1 m acetate buffer, 0.5 m NaCl (pH 4), and 0.1 m Tris and 0.5 m NaCl (pH 8). After washing, the gel was resuspended in 5 ml of 10 mm Tris buffer (pH 8.3).

Extracts of human fibroblasts [10⁸ cells in 5 ml of 10 mm Tris, 0.15 m NaCl, and 0.5% NP40 (pH 8.3)] were added to the EMMPRIN-coupled gel and incubated at 4°C overnight with rotation. The gel was then washed with 10 mm Tris and 0.15 m NaCl containing 30 mm octyl glucoside until the A_{280 nm} was less than 0.05. Binding proteins were eluted with 0.1 m glycine buffer (pH 2.5) containing 30 mm octyl glucoside. The eluate was neutralized to pH 7 by the addition of 1 m Tris (pH 9.5) and concentrated for further analysis.

ELISA of MMP-1.

MMP-1 protein was measured in the eluates from EMMPRIN-Sepharose and in immunopurified EMMPRIN preparations using a commercial ELISA system (Amersham, Piscataway, NJ) according to the manufacturer’s instructions. Briefly, 5 or 10 μl of eluate were added to microtiter plates coated with antibody to MMP-1 and incubated for 2 h at 25°C. The plates were washed with phosphate buffer and incubated with anti-MMP-1 antiserum for 2 h. After washing, the plates were incubated with peroxidase-conjugated secondary antibody for 1 h, and processed for color development and measurement at A_{450 nm} in a microplate spectrophotometer. The concentration of MMP-1 in the eluate was estimated from a standard curve.

SDS-PAGE, Silver Staining, and Western Blotting.

Proteins were dissolved in SDS sample buffer containing 0.1 m DTT and heated at 95°C for 5 min. The samples were then subjected to electrophoresis on 10% SDS polyacrylamide gels. The gels were either stained using the Sterling silver staining system (National Diagnostics, Atlanta, CA) or electroblotted onto nitrocellulose membranes and incubated with antibody against EMMPRIN (E11F4; Ref. 12 ) or against MMP-1 (Calbiochem, La Jolla, CA) for 1 h at room temperature. The immunoreactive protein bands were detected with horseradish peroxidase-conjugated antimouse IgG and chemiluminescence reagent (New England Nuclear Life Science, Boston, MA).

Immunocytochemistry.

LX-1 human lung carcinoma cells were seeded into chamber culture slides and cultured for 48 h at 37°C in 5% CO₂ air. The cells were then washed with PBS, fixed in 1% paraformaldehyde in PBS for 45 min at room temperature, quenched with 0.1 m Tris (pH 7.4), and blocked with 1% BSA, 1% goat serum, and 2% nonfat milk in PBS at room temperature for 1 h. The LX-1 cells were then incubated with monoclonal antibody against MMP-1 (Calbiochem) for 1 h at room temperature, followed by Cy3-conjugated Texas red goat antimouse IgG. The cells were washed with PBS, mounted with coverslips, and then observed and photographed using a Zeiss Axioskop-20 microscope.

Phage Display Reveals MMP-1 as an EMMPRIN-binding Protein.

We used the T7Select Phage Display System (Novagen) to identify EMMPRIN-binding protein(s) encoded by a cDNA library prepared from human fibroblasts, as described in “Materials and Methods.” In this method, each phage becomes coated with a fusion protein comprised of the phage coat protein and a protein generated from the cDNA library used. Phages coated with putative EMMPRIN-binding protein were selected by repeated panning over 24-well plates coated with EMMPRIN. Five rounds of biopanning were carried out, and the final lysate was used for plaque assay, PCR amplification, and sequencing.

Eight clones were obtained from the procedure described above. All eight of the inserts were of identical size, i.e., 0.8 kb, and were found to have identical sequences corresponding exactly to a portion of the human MMP-1 sequence (Fig. 1)⇓ .

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Fig. 1.

Comparison of partial sequences of cDNA isolated by phage display with that of human MMP-1 cDNA. The 5′ and 3′ sequences of one of the partial cDNAs obtained are given. Eight cDNA clones were isolated after biopanning of phages on EMMPRIN; all eight clones had identical sequences.

MMP-1 Binds to EMMPRIN-Sepharose.

To confirm the binding of EMMPRIN to fibroblast-produced MMP-1, we performed ligand chromatography over Sepharose conjugated with immunopurified EMMPRIN. Fibroblast extracts were mixed with the EMMPRIN-Sepharose, which was then washed and eluted as described in“ Materials and Methods.” The eluates were subjected to SDS-PAGE, followed by silver staining. On silver staining, a prominent protein band at ∼M_r 55,000 was observed, as well as a weaker band at ∼M_r 67,000 (Fig. 2A)⇓ ; in some cases a ∼M_r 45,000 band could also be seen.

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Fig. 2.

EMMPRIN affinity chromatography of proteins extracted from human fibroblasts. A, proteins recovered from chromatography of fibroblast extracts on EMMPRIN-Sepharose were run on SDS-PAGE and silver-stained; two bands (at ∼M_r 55,000 and∼ M_r 67,000) were detected. Arrows indicate positions of the M_r 43,000 and M_r 67,000 markers. B, parallel gels to those in A were transblotted and reacted with antibody to MMP-1 or secondary antibody only (IgG); the∼ M_r 55,000 band reacted with anti-MMP-1.

Western blots were also performed on the eluates from EMMPRIN-Sepharose using antibody against human MMP-1. The protein band at∼ M_r 55,000 (the approximate size of pro-MMP-1, which is M_r 52,000) reacted with anti-MMP-1 antibody (Fig. 2B)⇓ , confirming our results from the phage display. ELISA measurements also revealed MMP-1 in the eluates from EMMPRIN-Sepharose (data not shown). The identities of the∼ M_r 67,000 and ∼M_r 45,000 proteins are not yet known.

EMMPRIN Forms a Complex with MMP-1 on the Surface of Tumor Cells.

Some tumor cells themselves produce small amounts of MMP-1. Thus, we also determined whether, in addition to binding isolated EMMPRIN protein, MMP-1 forms a complex with EMMPRIN present on the surface of LX-1 human lung carcinoma cells. We immunopurified EMMPRIN from extracts of LX-1 cell membranes using monoclonal antibody E11F4 covalently bound to Sepharose beads and tested whether MMP-1 was present in the eluted EMMPRIN preparation. Fig. 3⇓ shows a Western blot of such an EMMPRIN preparation with antibody against MMP-1. A strong band at ∼M_r 55,000, corresponding approximately in size to pro-MMP-1, reacted with the antibody, indicating the presence of MMP-1 in the EMMPRIN preparation. A weaker band at ∼M_r 45,000, which is not seen consistently, is most likely activated MMP-1 (M_r 42,000).

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Fig. 3.

Western blot of immunopurified EMMPRIN with antibody to MMP-1.

Quantitation of the MMP-1 content by ELISA gave 2.1 μg of MMP-1 per 5μ g of total protein in the EMMPRIN preparation. Because EMMPRIN and pro-MMP-1 have molecular weights of ∼58,000 and 52,000, respectively, this result suggests that EMMPRIN and MMP-1 are complexed in an equimolar ratio.

The presence of MMP-1 at the surface of LX-1 human lung carcinoma cells was confirmed by immunocytochemistry using antibody against MMP-1 (Fig. 4)⇓ .

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Fig. 4.

Immunoreactivity of LX-1 human carcinoma cells with antibody to MMP-1. A, cells stained with antibody against MMP-1. B, cells stained with secondary antibody only.