The soluble catalytic domain of membrane type 1 matrix metalloproteinase cleaves the propeptide of progelatinase A and initiates autoproteolytic activation. Regulation by TIMP-2 and TIMP-3.
It has been proposed that the cell-mediated activation of progelatinase A requires binding of the C-terminal domain of the proenzyme to a membrane-associated complex of the membrane type matrix metalloproteinase MT1-MMP and TIMP-2. Subsequent sequential proteolysis of the propeptide by MT1-MMP and gelatinase A is thought to generate the active form of gelatinase A. We have prepared the proform of the catalytic domain of the MT1-MMP and demonstrated that this may be activated in vitro by trypsin proteolysis to yield a functional proteinase capable of cleaving typical metalloproteinase peptide substrates, gelatin and casein. The active catalytic domain of MT1-MMP was also shown to activate progelatinase A to a fully active form. Using the inactive mutant pro-E375A gelatinase A, we dissected the propeptide processing events that occur. MT1-MMP cleaves the propeptide at the sequence Asn37-Leu38 only. Further cleavage of the mutant enzyme propeptide at Asn80-Tyr81, equivalent to that of the active wild type gelatinase A, could only be effected by addition of gelatinase A to the system. TIMP-1 was essentially unable to prevent MT1-MMP processing of wild type or E375A progelatinase A, whereas TIMP-2 and TIMP-3 were good inhibitors of these events. Analysis of the rate of binding of TIMPs to the catalytic domain of MT1-MMP using kinetic methods showed that TIMP-1 is an extremely poor inhibitor of MT1-MMP. In comparison, TIMP-2 and TIMP-3 are excellent inhibitors, binding more rapidly to the catalytic domain of MT1-MMP than to the catalytic domain of gelatinase A. These data demonstrate the basic mechanism of MT1-MMP action on progelatinase A and the reason for the lack of inhibition by TIMP-1 previously demonstrated in cell-based activation studies.
Will
H
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In Vitek GmbH, Robert-Roessle-Strasse 10, D-13125 Berlin-Buch, Federal Republic of Germany.
Atkinson
S J
SJ
Butler
G S
GS
Smith
B
B
Murphy
G
G
D000595:Amino Acid Sequence; D001216:Asparagine; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D017364:Collagenases / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D018093:Gelatinases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007930:Leucine; D020781:Matrix Metalloproteinase 1; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D019716:Tissue Inhibitor of Metalloproteinase-2; D019717:Tissue Inhibitor of Metalloproteinase-3; D014443:Tyrosine
D004792:Enzyme Precursors; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D019717:Tissue Inhibitor of Metalloproteinase-3; D019716:Tissue Inhibitor of Metalloproteinase-2; D014443:Tyrosine; D001216:Asparagine; D017364:Collagenases; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase; D020781:Matrix Metalloproteinase 1; D007930:Leucine