Domain structure of human 72-kDa gelatinase/type IV collagenase. Characterization of proteolytic activity and identification of the tissue inhibitor of metalloproteinase-2 (TIMP-2) binding regions.
The 72-kDa gelatinase/type IV collagenase, a metalloproteinase thought to play a role in metastasis and in angiogenesis, forms a noncovalent stoichiometric complex with the tissue inhibitor of metalloproteinase-2 (TIMP-2), a potent inhibitor of enzyme activity. To define the regions of the 72-kDa gelatinase responsible for TIMP-2 binding, a series of NH2- and COOH-terminal deletions of the enzyme were constructed using the polymerase chain reaction technique. The full-length and the truncated enzymes were expressed in a recombinant vaccinia virus mammalian cell expression system (Vac/T7). Two truncated enzymes ending at residues 425 (delta 426-631) and 454 (delta 455-631) were purified. Like the full-length recombinant 72-kDa gelatinase, both COOH-terminally truncated enzymes were activated with organomercurial and digested gelatin and native collagen type IV. In contrast to the full-length enzyme, delta 426-631 and delta 455-631 enzymes were less sensitive to TIMP-2 inhibition requiring 10 mol of TIMP-2/mol of enzyme to achieve maximal inhibition of enzymatic activity. The activated but not the latent forms of the delta 426-631 and delta 455-631 proteins formed a complex with TIMP-2 only when excess molar concentrations of inhibitor were used. We also expressed the 205-amino acid COOH-terminal fragment, delta 1-426, and found that it binds TIMP-2. In addition, a truncated version of the 72-kDa gelatinase lacking the NH2-terminal 78 amino acids (delta 1-78) of the proenzyme retained the ability to bind TIMP-2. These studies demonstrate that 72-kDa gelatinases lacking the COOH-terminal domain retain full enzymatic activity but acquire a reduced sensitivity to TIMP-2 inhibition. These data suggest that both the active site and the COOH-terminal tail of the 72-kDa gelatinase independently and cooperatively participate in TIMP-2 binding.
Fridman
R
R
Molecular Oncology Inc., Gaithersburg, Maryland 20878.
Fuerst
T R
TR
Bird
R E
RE
Hoyhtya
M
M
Oelkuct
M
M
Kraus
S
S
Komarek
D
D
Liotta
L A
LA
Berman
M L
ML
Stetler-Stevenson
W G
WG
D001483:Base Sequence; D001665:Binding Sites; D018093:Gelatinases; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D003012:Microbial Collagenase / Q000235:genetics* / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010434:Pepsin A / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D015183:Restriction Mapping; D019716:Tissue Inhibitor of Metalloproteinase-2
D007527:Isoenzymes; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; D019716:Tissue Inhibitor of Metalloproteinase-2; D010434:Pepsin A; D018093:Gelatinases; D003012:Microbial Collagenase