Cellular mechanisms for human procollagenase-3 (MMP-13) activation. Evidence that MT1-MMP (MMP-14) and gelatinase a (MMP-2) are able to generate active enzyme

J Biol Chem. 1996 Jul 19;271(29):17124-31. doi: 10.1074/jbc.271.29.17124.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Aprotinin / pharmacology
  • Base Sequence
  • Cloning, Molecular
  • Collagenases / metabolism*
  • DNA Primers
  • Enzyme Activation
  • Escherichia coli
  • Fibrinolysin / metabolism
  • Gelatinases / metabolism*
  • Humans
  • Kinetics
  • Matrix Metalloproteinase 13
  • Matrix Metalloproteinase 2
  • Matrix Metalloproteinases, Membrane-Associated
  • Metalloendopeptidases / metabolism*
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagenesis, Site-Directed
  • Point Mutation
  • Protein Processing, Post-Translational*
  • Recombinant Proteins / metabolism
  • Tyrosine

Substances

  • DNA Primers
  • Recombinant Proteins
  • Tyrosine
  • Aprotinin
  • Fibrinolysin
  • Collagenases
  • Gelatinases
  • MMP13 protein, human
  • Matrix Metalloproteinase 13
  • Matrix Metalloproteinases, Membrane-Associated
  • Metalloendopeptidases
  • Matrix Metalloproteinase 2