BIOCHEMICAL-CHARACTERIZATION OF HUMAN COLLAGENASE-3

The cDNA of a novel matrix metalloproteinase, collagenase 3 (MMP-13) h as been isolated from a breast tumor library (Freije, J. M. P., Diez-I tza, I., Balbin, M., Sanchez, L. M,, Blasco, R., Tolivia, J., and Lope z-Otin, C. (1994) J. Biol. Chem. 269, 16766-16773), and a potential ro le in tumor progression has been proposed for this enzyme. In order to establish the possible role of collagenase-3 in connective tissue tur nover, we have expressed and purified recombinant human procollagenase -3 and characterized the enzyme biochemically. The purified procollage nase-3 was shown to be glycosylated and displayed a M(r) of 60,000, th e N-terminal sequence being LPLPSGGD, which is consistent with the cDN A-predicted sequence. The proenzyme was activated by p-aminophenylmerc uric acetate or stromelysin, yielding an intermediate form of M(r) 50, 000, which displayed the N-terminal sequence L(58)EVTGK. Further proce ssing resulted in cleavage of the Glu(84)-Tyr(85) peptide bond to the final active enzyme (M(r) 48,000). Trypsin activation of procollagenas e-3 also generated a Tyr(85) N terminus, but it was evident that the C -terminal domain was rapidly lost, and hence the collagenolytic activi ty diminished. Analysis of the substrate specificity of collagenase-3 revealed that soluble type II collagen was preferentially hydrolyzed, while the enzyme was 5 or 6 times less efficient at cleaving type I or III collagen. Fibrillar type I collagen was cleaved with comparable e fficiency to the fibroblast and neutrophil collagenases (MMP-1 and MMP -8), respectively. Unlike these collagenases, gelatin and the peptide substrates Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 and Mca-Pro-Cha-Gly-Nva -His-Ala-Dpa-NH2 were efficiently hydrolyzed as well, as would be pred icted from the similarities between the active site sequence of collag enase-3 (MMP-13) and the gelatinases A and B. Active collagenase-3 was inhibited in a 1:1 stoichiometric fashion by the tissue inhibitors of metalloproteinases, TIMP-1, TIMP-2, and TIMP-3. These results suggest that in vivo collagenase-3 could play a significant role in the turno ver of connective tissue matrix constituents.