PREPARATION AND CHARACTERIZATION OF RECOMBINANT TISSUE INHIBITOR OF METALLOPROTEINASE-4 (TIMP-4)

TIMP-4, a novel human tissue inhibitor of metalloproteinase, was ident ified and cloned (Greene, J., Wang, M., Raymond, L. A., Liu, Y. E., Ro sen, C., and Shi, Y. E. (1996) J. Biol. Chem. 271, 30375-30380). In th is report, the production and characterization of recombinant TIMP-4 ( rTIMP4p) are described. rTIMP4p, expressed in baculovirus-infected ins ect cells, was purified to homogeneity by a combination of cation exch ange, hydrophobic, and size-exclusion chromatographies. The purified p rotein migrated as a single 23-kDa band in SDS-polyacrylamide gel elec trophoresis and in Western blot using a specific anti-TIMP-4 antibody. Inhibition of matrix metalloproteinase (MMP) activities by rTIMP4p wa s demonstrated in five MMPs. Enzymatic kinetic studies revealed IC50 v alues (concentration at 50% inhibition) of 19, 3, 45, 8, and 83 nM for MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, respectively. Purified rTIMP4p demonstrated a strong inhibitory effect on the invasion of human brea st cancer cells across reconstituted basement membranes. Thus, TIMP-4 is a new enzymatic inhibitor in MMP-mediated extracellular matrix degr adation and may have therapeutic potential in treating cancer malignan t progression.