EXTRACELLULAR MATRIX-ASSOCIATED SERINE-PROTEASE INHIBITORS (M(R)-33,000, M(R)-31,000, AND M(R)-27,000) ARE SINGLE-GENE PRODUCTS WITH DIFFERENTIAL GLYCOSYLATION - CDNA CLONING OF THE 33-KDA INHIBITOR REVEALS ITS IDENTITY TO TISSUE FACTOR PATHWAY INHIBITOR-2

Recently, we reported the identification and partial characterization of three serine protease inhibitors (M(r) 33,000, 31,000, and 27,000) from the extracellular matrix (ECM) of human umbilical vein endothelia l cells and skin cells. Here, we report that a full-length cDNA clone for the 33-kDa inhibitor from SV-40 transformed human skin fibroblasts (tl2FB) is identical to a recombinant trypsin/tissue factor pathway i nhibitor called TFPI-2 from placenta. By immunoblotting, the three inh ibitors from ECM and cell lysates demonstrated cross-reactivity with a n antiTFPI-2 IgG. To further elucidate how these inhibitors are relate d, pulse-chase labeling of T12FB with [S-35]methionine followed by imm unoprecipitation with antiTFPI-2 IgG was performed on ECM and cytosoli c proteins. A precursor-product relationship did not exist between the three inhibitors from ECM. In contrast, the various species of inhibi tors from cytosolic fractions demonstrated a precursor-product relatio nship. Within the cytosolic fraction, 26-, 29-, and 30-kDa inhibitors were detected in the early chases (0 and 15 min) but they form precurs ors to the synthesis of the 33-kDa inhibitor which accumulated in the later chases (30 min to 1 h). When pulse-chase experiments were perfor med in the presence of tunicamycin, synthesis as well as sequestration of the three inhibitors into ECM was completely inhibited. In the pre sence of tunicamycin, the cells synthesized and sequestered a single 2 5.5-kDa inhibitor into ECM. Peak quantities of the 25.5-kDa inhibitor appeared in the ECM after 6 h chase while they were 1 h for the 27- an d 31-kDa inhibitors and 3 h for the 33-kDa inhibitor. To further suppo rt that the three inhibitors are related but only differ in the extent of glycosylation, the 33-kDa inhibitor from the t12FB ECM was deglyco sylated with N-glycosidase F and the products were identified by immun oblotting with anti-TFPI-2 IgG. The enzyme released the 31-, 27-, and 25.5-kDa inhibitors from the 33-kDa inhibitor. Collectively, these res ults demonstrate that the ECM-associated 33-, 31-, and 27-kDa inhibito rs are biosynthetic products of a single gene with differential glycos ylation. The 25.5-kDa inhibitor is unglycosylated, whereas 27- and 30- to 31-kDa inhibitors are partially glycosylated and the 33-kDa inhibit or is fully glycosylated. Inhibition of glycosylation significantly re tarded the rate of secretion of the inhibitor but did not prevent its association with ECM. Quantitation of the inhibitors with cell-conditi oned medium and ECM fractions reveals that 70-75% were ECM-associated and 25-30% cell-associated. None or very little of the inhibitors (0-2 %) remained in a conditioned medium. Because they are primarily associ ated with ECM, the inhibitors may play a major role in ECM remodeling and turnover. (C) 1996 Academic Press, Inc.