IDENTIFICATION AND CHARACTERIZATION OF HUMAN TISSUE INHIBITOR OF METALLOPROTEINASE-3 AND DETECTION OF 3 ADDITIONAL METALLOPROTEINASE INHIBITOR ACTIVITIES IN EXTRACELLULAR-MATRIX

We have identified and characterized a novel human tissue inhibitor of metalloproteinase (TIMP). It is found exclusively in the extracellula r matrix of a large number of cultured human cells, including: primary embryonal kidney (293), neuroblastoma (SK-N-SH), normal whole embryo (FHs 173We), cervical carcinoma (HeLa S3), colon adenocarcinoma (Caco- 2), ileocecal adenocarcinoma (HCT-8), fibrosarcomas (SW 684 and Hs 913 T) and normal gingival fibroblasts (GF11 and 1292). It was not detecte d in the conditioned media from any of these cell lines. Its apparent molecular mass of 24-25 kDa, as determined by its migration on proteas e-substrate gels, is intermediate between TIMP-1 (28.5 kDa) and TIMP-2 (21 kDa). Like the latter two proteins, human TIMP-3 contains intrach ain disulfide bonds and displays altered electrophoretic mobility in t he presence of beta-mercaptoethanol. The N-terminal amino acid sequenc e of the protein is identical to that of chicken TIMP-3 (ChIMP-3), and its amino acid composition is similar. The protein is not N-glycosyla ted, as determined by treatment with N-glycosidase-F. Finally, it is r ecognized by antisera raised against pure ChIMP-3 but not by anti-huma n TIMP-1 or anti-human TIMP-2 antibodies. Based on these properties, w e propose that this protein is TIMP-3 and is the human counterpart of ChIMP-3 (Pavloff et al., J. Biol. Chem. 267: 17321-17326, 1992). Two a dditional inhibitors detected in the matrix of human cell lines, desig nated inhibitor of metalloproteinase (IMP)-a and IMP-b, migrate with a pparent masses of 29 kDa and 30 kDa. Both are N-glycosylated. A fourth inhibitor activity, which is smaller in mass than TIMP-3 and is also pecifically located in the matrix, is detectable in some cell lines.