Abstract
Gelatinase A and membrane-type metalloproteinase (MT1-MMP) were able to process human procollagenase-3 (Mr 60,000) to the fully active enzyme (Tyr85 N terminus; Mr 48,000). MT1-MMP activated procollagenase-3 via a Mr 56,000 intermediate (Ile36 N terminus) to 48,000 which was the result of the cleavage of the Glu84-Tyr85 peptide bond. We have established that the activation rate of procollagenase-3 by MT1-MMP was enhanced in the presence of progelatinase A, thereby demonstrating a unique new activation cascade consisting of three members of the matrix metalloproteinase family. In addition, procollagenase-3 can be activated by plasmin, which cleaved the Lys38-Glu39 and Arg76-Cys77 peptide bonds in the propeptide domain. Autoproteolysis then resulted in the release of the rest of the propeptide domain generating Tyr85 N-terminal active collagenase-3. However, plasmin cleaved the C-terminal domain of collagenase-3 which results in the loss of its collagenolytic activity. Concanavalin A-stimulated fibroblasts expressing MT1-MMP and fibroblast-derived plasma membranes were able to process human procollagenase-3 via a Mr 56,000 intermediate form to the final Mr 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 A-stimulated 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.
Highlights
Gelatinase A and membrane-type metalloproteinase (MT1-matrix metalloproteinases (MMPs)) were able to process human procollagenase-3 (Mr 60,000) to the fully active enzyme (Tyr85 N terminus; Mr 48,000)
(MMP-2)—Human procollagenase-3 was processed by active gelatinase A (23:1 molar ratio) which resulted in the rapid generation of enzymatic activity within 60 min (Fig. 1) with a concomitant loss of the propeptide domain, thereby reducing the molecular mass of the proenzyme (Mr 60,000) to 48,000 (Fig. 1, lanes 2– 4)
N-terminal amino acid sequence analysis revealed that the Gly35–Ile36 peptide bond within the propeptide domain was initially hydrolyzed by MT1MMP which was followed by the cleavage of the Glu84–Tyr85 peptide bond (Fig. 2)
Summary
Gelatinase A and membrane-type metalloproteinase (MT1-MMP) were able to process human procollagenase-3 (Mr 60,000) to the fully active enzyme (Tyr N terminus; Mr 48,000). We have established that progelatinase A can considerably potentiate the activation rate of procollagenase-3 by crude plasma membrane preparations from concanavalin Astimulated fibroblasts, 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. 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 collagenase3.2 Collagenase-3 is a powerful collagenolytic and gelatinolytic enzyme that preferentially cleaves type II collagen, and it can be implied that this enzyme may play a considerable role in connective tissue turnover [5]. We have recently shown that procollagenase-3 can be directly activated by stromelysin [5]; other mechanisms may be of physiological and pathophysiological significance
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