The cDNA of a novel matrix metalloproteinase, collagenase-3 (MMP-13) has been isolated from a breast tumor library (Freije, J. M. P., Diez-Itza, I., Balbin, M., Sanchez, L. M., Blasco, R., Tolivia, J., and Ló pez-Otin, C. (1994) J. Biol. Chem. 269, 16766 -16773), and a potential role in tumor progression has been proposed for this enzyme. In order to establish the possible role of collagenase-3 in connective tissue turnover, we have expressed and purified recombinant human procollagenase-3 and characterized the enzyme biochemically. The purified procollagenase-3 was shown to be glycosylated and displayed a M r of 60,000, the N-terminal sequence being LPLPSGGD, which is consistent with the cDNA-predicted sequence. The proenzyme was activated by p-aminophenylmercuric acetate or stromelysin, yielding an intermediate form of M r 50,000, which displayed the N-terminal sequence L 58 EVTGK. Further processing resulted in cleavage of the Glu 84 -Tyr 85 peptide bond to the final active enzyme (M r 48,000). Trypsin activation of procollagenase-3 also generated a Tyr 85 N terminus, but it was evident that the C-terminal domain was rapidly lost, and hence the collagenolytic activity 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 efficiency to the fibroblast and neutrophil collagenases (MMP-1 and MMP-8), respectively. Unlike these collagenases, gelatin and the peptide substrates Mca-ProLeu-Gly-Leu-Dpa-Ala-Arg-NH 2 and Mca-Pro-Cha-GlyNva-His-Ala-Dpa-NH 2 were efficiently hydrolyzed as well, as would be predicted from the similarities between the active site sequence of collagenase-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 turnover of connective tissue matrix constituents.

TNF-K K converting enzyme (TACE; ADAM-17) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-K K to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP-3 but not by TIMP-1, -2 and -4. These results suggest that TIMP-3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF-K K secretion is involved.z 1998 Federation of European Biochemical Societies.

for pro matrix metalloproteinase activation. APMIS 1999;107:3844. The activation of pro matrix metalloproteinases (MMPs) by sequential proteolysis of the propeptide blocking the active site cleft is regarded as one of the key levels of regulation of these proteinases. Potential physiological mechanisms including cell-associated plasmin generation by urokinase-like plasminogen activator, or the action of cell surface MTI-MMPs appear to be involved in the initiation of cascades of pro M M P activation. Gelatinase A, collagenase 3 and gelatinase B may be activated by MT-MMP based mechanisms, as evidenced by both biochemical and cell based studies. Hence the regulation of MT-MMPs themselves becomes critical to the determination of M M P activity. This includes activation, assembly at the cell surfaces as TIMP-2 complexes and subsequent inactivation by proteolysis or TIMP inhibition.

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described`metallosheddase' cleavage sites of tumour necrosis factor K K, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.z 2000 Federation of European Biochemical Societies.

). The kinetics of activation were not affected by removal of the hemopexin-like C-terminal domain. The specific activities of both collagenase-3 and ⌬ 249 -451 collagenase-3 were found to be similar using two quenched fluorescent substrates, but ⌬ 249 -451 collagenase-3 failed to cleave native triple helical collagens (types I and II) into characteristic one-and three-quarter fragments. It was noted, however, that the ␤1,2(I) chains of type I collagen were susceptible to ⌬ 249 -451 collagenase-3, which indicates that the catalytic domain displays telopeptidase activity, thereby generating ␣1,2(I) chains that are slightly shorter than those in native type I collagen. It can be concluded that the C-terminal domain is only essential for the triple helicase activity of collagenase-3. Binding of procollagenase-3 and active collagenase-3 to type I collagen is mediated by the C-terminal domain. Both collagenase-3 and ⌬ 249 -451 collagenase-3 hydrolyzed the large tenascin C isoform, fibronectin, recombinant fibronectin fragments, and type IV, IX, X, and XIV collagens; thus, these events were independent from C-terminal domain interactions. In contrast, the minor cartilage type XI collagen was resistant to cleavage. Kinetic analysis of the mechanism of inhibition of wild-type and ⌬ 249 -451 collagenase-3 by wild-type and mutant tissue inhibitors of metalloproteinase (TIMPs) revealed that the association rates for complex formation were influenced by both Nand C-terminal domain interactions. The C-terminal domain of wild-type collagenase-3 promoted increased association rates with the full-length inhibitors TIMP-1 and TIMP-3 and the hybrid N.TIMP-2/C.TIMP-1 by a factor of up to 33. In contrast, the association rates for complex formation with TIMP-2 and N.TIMP-1/C.TIMP-2 were only marginally affected by C-terminal domain interactions.The matrix metalloproteinases (MMPs) 1 are a family of zincdependent enzymes that have the capacity to degrade most protein components of the extracellular matrix. Their uncontrolled activity contributes to the tissue destruction that is observed during such diverse pathologies as arthritis and cancer. Four main subfamilies of MMPs have been defined according to their substrate specificity, primary structures, and cellular localization: the collagenases, stromelysins, gelatinases, and membrane-type matrix metalloproteinases.Human procollagenase-3 (MMP-13) is a new member of the collagenase subfamily of MMPs, which consists of three members showing an overall sequence homology of 55% (1-4). Human collagenase-3 expression has been demonstrated in breast tumors and in osteoarthritic cartilage (1, 5), indicating that the enzyme plays a role in degradation of collagen during disease progression. Furthermore, its expression in cartilage was strongly induced at both the message and protein levels by interleukin-1␣. Biochemical characterization of human collagenase-3 has shown that it is a powerful collagenolytic enzyme, preferentially cleaving type II collagen, while it is five or six times less ...

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Concanavalin A-stimulated fibroblasts expressing MT1-MMP and fibroblast-derived plasma membranes were able to process human procollagenase-3 via a M r 56,000 intermediate form to the final M r 48,000 active enzyme which, by analogy with progelatinase A activation, may represent a model system for in vivo activation. Inhibition experiments using tissue inhibitor of metalloproteinases, plasminogen activator inhibitor-2, or aprotinin demonstrated that activation in the cellular model system was due to MT1-MMP/gelatinase A and excluded the participation of serine proteinases such as plasmin during procollagenase-3 activation. We have established that progelatinase A can considerably potentiate the activation rate of procollagenase-3 by crude plasma membrane preparations from concanavalin Astimulated fibroblasts, thus confirming our results using purified progelatinase A and MT1-MMP. This new activation cascade may be significant in human breast cancer pathology, where all three enzymes have been implicated as playing important roles.Degradation of the extracellular matrix during tumor invasion is thought to result from a combined action of several proteolytic enzyme systems, including the collagenases and other matrix metalloproteinases (MMPs) 1 (1, 2) and serine proteases, such as plasmin generated by the urokinase pathway of plasminogen activation (3).Human collagenase-3 (MMP-13), a new member of the MMP family, is expressed by breast tumors and is likely to play a crucial role in the modulation of extracellular matrix degradation and cell-matrix interactions involved in metastasis (4). Procollagenase-3 comprises three distinct domains which include an 85-amino acid residue propeptide that is lost during activation (5), and in which the conserved sequence PRCGVPD is responsible for the latency of the MMPs (6). This sequence is followed by the catalytic domain containing the active site of the enzyme linked via a short hinge sequence motif to the third, C-terminal domain, that shows homology to vitronectin and which is essential for the collagenolytic activity of collagenase-3.2 Collagenase-3 is a powerful collagenolytic and gelatinolytic enzyme that preferentially cleaves type II collagen, and it can therefore be implied that this enzyme may play a considerable role in connective tissue turnover (5). One of the key events in the regulation of extracellular collagenolytic activity is the activation of procollagenase-3, but there are currently only limited data available on how this may occur in vivo. We have recently shown that procollagenase-3 can be directly activated by stromelysin (5); however, other mechanisms may be of physiological and pathophysiological significance.Increasing evidence is accumulating that the newly discovered membrane associated MMPs (MT-MMPs) act as cell surface activator(s) of progelatinase A (proMMP-2) under physiological or pathophysiological conditions (7-12). This mechanism was thought to be specific for progelatinase A, since other MMPs such as progelatinase B, fibroblast procollage...