FIGURE 1.

Hpx domain is dispensable for proMMP-2 activation but important for collagenolytic activity. A, schematic representation of mutant constructs used in the experiments. S, signal peptide; Pro, propeptide; FLAG, FLAG tag (DYKDDDDK); Cat, catalytic domain; L1, linker 1 (hinge); HPX, hemopexin domain; L2, linker 2; TM, transmembrane domain; Zn, catalytic zinc ion. B, COS7 cells were transfected with empty vector (Mock), MT1F, MT1F-ΔHpx, and MT1F-ΔHpxΔL2 as indicated. Cells were then incubated with purified proMMP-2 in serum-free culture medium for 18 h. ProMMP-2 activation in the media was analyzed by zymography (upper panel), and cell lysates were analyzed for expression of the proteins by Western blotting using anti-FLAG M2 antibody (lower panel). The arrows indicate MT1-MMP mutants expressed. P, proMMP-2; A, active MMP-2. C, transfected cells were subjected to surface biotinylation as described under “Materials and Methods” and analyzed by Western blotting using anti-FLAG M2 antibody. The upper panel is biotinylated samples, and the bottom panel is whole cell lysates. D, in situ gelatin degradation assay was carried out as described under “Materials and Methods.” Transfected COS7 cells were seeded on Alexa Fluor 488-labeled gelatin-coated 4-well chamber slides and cultured for 18 h. The cell surface-localized active form of FLAG-tagged MT1-MMP mutants was visualized by staining with anti-FLAG M1 antibody in the presence of 1 mm CaCl₂ without permeabilization. Green channel (F-gelatin) and red channel (FLAG M1) fluorescences were captured in each field using ×10 objective lens. Merged images are also shown in the bottom. The bar indicates 200 μm. E, in situ solid-phase collagen degradation assays were carried out as described under “Materials and Methods.” The bar indicates 100 μm.