Autolytic activation of recombinant human 72 kilodalton type IV collagenase.
Human 72 kDa type IV collagenase (gelatinase A, MMP-2) was expressed in a baculovirus/insect cell system. The enzyme was produced in the wild-type form and in two mutant forms, where the active site Glu375 was substituted by Asp or Gln. The mutated proteins had strongly reduced or no detectable activity, respectively, allowing detailed analysis of rapid autoactivation reactions. MMP-2 was readily degraded to a proenzyme form lacking the first four amino acid residues. This cleavage was shown to be an autolytic process, although enzyme activity was apparently not affected by this truncation. Conversion to the active enzyme form was achieved without external activator in a concentration-dependent manner at 37 degrees C. The activation of MMP-2 was shown to be a stepwise process, probably via a delta 1-50 form as a highly unstable intermediate. The C-terminal hemopexin-like domain is removed rather early at two cleavage sites, and degradation within the Zn-binding site inactivates the enzyme. The fibronectin- and hemopexin-like domains are stable, although the autodegradation pattern did not show any sequence specificity, except for charged residues in the P1' position. The results indicate that a specific activator may not be essential for MMP-2.
Bergmann
U
U
Biocenter Oulu, Finland.
Tuuttila
A
A
Stetler-Stevenson
W G
WG
Tryggvason
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001329:Autolysis; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D017364:Collagenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D020780:Matrix Metalloproteinase 9; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
D010446:Peptide Fragments; D011994:Recombinant Proteins; D017364:Collagenases; D020780:Matrix Metalloproteinase 9