FIGURE 4.

Blade IV of the PEX domain of MMP-9 is required for homodimerization and cell migration. A, ribbon diagram of MMP-9 PEX domain (PDB 1ITV). Each outermost β-strand from the four blades was swapped with the corresponding MMP-2 PEX domain sequences (upper panel). Lower panel, schematic diagram of substitution mutations at the outermost β-strands of four blades of MMP-9 by corresponding MMP-2 sequences. B, requirement of blade IV for MMP-9 homodimerization: COS-1 cells transfected with MMP-9 chimeric cDNAs were examined by co-immunoprecipitation (IP) assay. Mutation at blade IV of the PEX domain of MMP-9 fails to co-precipitate with wild type MMP-9 (upper panel). The aliquot of condition medium (CM) was examined by Western blotting (WB) using anti-HA and anti-Myc antibody (middle panel) and by gelatin zymography (lower panel) for control of protein expression. C, mutations of the PEX domain or the outermost IS4 and IVS4 motifs of MMP-9 fail to enhance cell migration. COS-1 cells transfected with wild type and mutant MMP-9 cDNAs were examined by a transwell cell migration assay. Three triplicate repeats were performed for each transfection and the experiment was repeated three times. *, p < 0.05. D, design of an inhibitory peptide interfering with MMP-9 homodimer formation. A peptide mimicking MMP-9/IVS4 was chemically synthesized and incubated (100 μm) with COS-1 cell-transfected cDNAs as indicated. Scrambled peptide was used as a control. Both the cell lysates (CL) and conditioned medium were examined by a co-immunoprecipitation assay (upper and middle panel). An aliquot of the conditioned medium and cell lysate were examined by Western blotting using anti-HA and anti-α/β-tubulin as a control. E, dose-dependent inhibition (from 1 μm to 1 mm) of MMP-9-mediated cell migration by IVS4 peptides. COS-1 cells transfected with an empty vector or MMP-9 cDNAs were preincubated with 1% DMSO, the IVS4 peptide (NQVDQVGY), and IVS4 scrambled peptide (VQYDNGQV) for 30 min followed by a transwell chamber migration assay in the presence of different concentrations of peptides for 6 h. Each data point was performed in triplicate and the experiments were repeated three times (*, p < 0.05).