Reactive site-modified tissue inhibitor of metalloproteinases-2 inhibits the cell-mediated activation of progelatinase A.
Tissue inhibitor of metalloproteinases-2 (TIMP-2) is supposed to play a regulatory role in the cell-mediated activation of progelatinase A. To investigate the mechanism of the regulation, we prepared and characterized a chemically modified TIMP-2, and examined its effects on the activation of progelatinase A. We found that treatment of TIMP-2 with cyanate ion led to loss of inhibitory activity toward matrilysin or gelatinase A. Structural and functional analyses of the modified TIMP-2 showed that carbamylation of the alpha-amino group of the NH2-terminal Cys1 of TIMP-2 led to complete loss of the inhibitory activity. When the reactive-site modified TIMP-2 was added to culture medium of concanavalin A-stimulated HT1080 cells, the conversion of endogenous progelatinase A to the intermediate form was partially inhibited, whereas that of the intermediate form to the mature one was strongly inhibited. The reactive site-modified TIMP-2 also prevented an accumulation of active gelatinase A on the cell surface. We speculate that occupation of the hemopexin-like domain of gelatinase A by the reactive site-modified TIMP-2 makes it unable for gelatinase A to be retained on the cell surface, thus preventing the autocatalytic conversion of the intermediate form of gelatinase A to its mature form.
Higashi
S
S
Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Maioka-cho 641-12, Totsuka-ku, Yokohama 244, Japan. shigashi@cserv2.yokohama-cu.ac.jp
Miyazaki
K
K
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D018093:Gelatinases / Q000378:metabolism*; D006466:Hemopexin / Q000378:metabolism; D006801:Humans; D020783:Matrix Metalloproteinase 7; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D004792:Enzyme Precursors; D019716:Tissue Inhibitor of Metalloproteinase-2; D006466:Hemopexin; D018093:Gelatinases; D008666:Metalloendopeptidases; C069860:progelatinase; D020783:Matrix Metalloproteinase 7