Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties

J Biol Chem. 1992 Oct 25;267(30):21712-9.

Abstract

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Blotting, Western
  • Cathepsin G
  • Cathepsins / pharmacology
  • Chymotrypsin / pharmacology
  • Collagenases / genetics
  • Collagenases / isolation & purification*
  • Collagenases / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation
  • Enzyme Precursors / isolation & purification*
  • Enzyme Precursors / metabolism
  • Fibrosarcoma
  • Glycoproteins / pharmacology
  • Humans
  • Kinetics
  • Matrix Metalloproteinase 3
  • Matrix Metalloproteinase 9
  • Matrix Metalloproteinase Inhibitors
  • Metalloendopeptidases / pharmacology
  • Molecular Sequence Data
  • Phenylmercuric Acetate / analogs & derivatives
  • Phenylmercuric Acetate / pharmacology
  • Serine Endopeptidases
  • Substrate Specificity
  • Tissue Inhibitor of Metalloproteinases
  • Trypsin / pharmacology
  • Tumor Cells, Cultured

Substances

  • Enzyme Precursors
  • Glycoproteins
  • Matrix Metalloproteinase Inhibitors
  • Tissue Inhibitor of Metalloproteinases
  • 4-aminophenylmercuriacetate
  • Cathepsins
  • Serine Endopeptidases
  • Chymotrypsin
  • CTSG protein, human
  • Cathepsin G
  • Trypsin
  • Collagenases
  • Metalloendopeptidases
  • Matrix Metalloproteinase 3
  • Matrix Metalloproteinase 9
  • Phenylmercuric Acetate