Inhibition of autoproteolytic activation of interstitial procollagenase by recombinant metalloproteinase inhibitor MI/TIMP-2.
The purification and cloning of a novel metalloproteinase inhibitor (MI or TIMP-2) related to tissue inhibitor of metalloproteinases (TIMP) has been recently described by our laboratory (DeClerck, Y.A., Yean, T. D., Ratzkin, B.J., Lu, H.S., and Langley, K.E. (1989) J. Biol. Chem. 264, 17445-17453; Boone, T.C., Johnson, M.J., DeClerck, Y.A., and Langley, K.E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2800-2804). We have transfected Chinese hamster ovary cells with a vector containing human MI/TIMP-2 cDNA and purified recombinant-derived MI/TIMP-2 (rMI/rTIMP-2) from the conditioned medium of such cells. We have investigated the inhibitory activity of rMI/rTIMP-2 toward rabbit fibroblast interstitial collagenase. The inhibition of activated collagenase by rMI/rTIMP-2 is stoichiometric and consistent with the formation of a 1:1 molar ratio complex. In addition to blocking the activated enzyme, rMI/rTIMP-2 inhibits the conversion of 52-kDa procollagenase to the 42-kDa active enzyme initiated by organomercurials. When plasmin is used as activator, rMI/rTIMP-2 does not inhibit the plasmin-mediated conversion of the 52-kDa proenzyme to the 46-kDa inactive intermediate but blocks further conversion of the 46-kDa intermediate to the 42-kDa active enzyme. The data indicate that rMI/rTIMP-2 blocks the autoproteolytic activation of procollagenase. Also, rMI/rTIMP-2 forms complexes with the 52-kDa procollagenase, the 46-kDa intermediate, and with the 42-kDa activated enzyme which are stable to sodium dodecyl sulfate (SDS), such that the complexes can be visualized by SDS-polyacrylamide gel electrophoresis. It appears that the formation of a SDS-stable complex with procollagenase requires an initial conformational change of the procollagenase brought about by organomercurials or by plasmin cleavage. The data suggest that MI/TIMP-2 may be able to control the extracellular action of certain metalloproteinases not only at the level of the activated enzyme but also at the level of proenzyme activation.
DeClerck
Y A
YA
Division of Hematology/Oncology, Children's Hospital of Los Angeles, California 90027.
Yean
T D
TD
Lu
H S
HS
Ting
J
J
Langley
K E
KE
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D017364:Collagenases; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D003012:Microbial Collagenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000494:pharmacology*; D010957:Plasmids; D011817:Rabbits; D019716:Tissue Inhibitor of Metalloproteinase-2
D002352:Carrier Proteins; D004792:Enzyme Precursors; D009363:Neoplasm Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D017364:Collagenases; D008666:Metalloendopeptidases; C022440:procollagenase; D003012:Microbial Collagenase