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.
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.
Knäuper
V
V
Department of Cell and Molecular Biology, Strangeways Research Laboratory, Worts' Causeway, Cambridge, CB1 4RN, United Kingdom.
Will
H
H
López-Otin
C
C
Smith
B
B
Atkinson
S J
SJ
Stanton
H
H
Hembry
R M
RM
Murphy
G
G
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D017364:Collagenases / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D004926:Escherichia coli; D005341:Fibrinolysin / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D053509:Matrix Metalloproteinase 13; D020778:Matrix Metalloproteinase 2; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D014443:Tyrosine
D017931:DNA Primers; D011994:Recombinant Proteins; D014443:Tyrosine; D007611:Aprotinin; D005341:Fibrinolysin; D017364:Collagenases; D018093:Gelatinases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2