BINDING OF URINARY PROTEIN-C INHIBITOR TO CULTURED HUMAN EPITHELIAL KIDNEY TUMOR-CELLS (TCL-598) - THE ROLE OF GLYCOSAMINOGLYCANS PRESENT ON THE LUMINAL CELL-SURFACE

Binding of urinary protein C inhibitor (PCI) to cultured human epithel ial kidney tumor cells (TCL-598) was studied. Binding was dose-depende nt, time-dependent, and saturable. Heparin interfered in a dose-depend ent way with PCI binding to TCL-598 as did heparan sulfate and to a le sser degree also dermatan sulfate. Pre- treatment of TCL-598 with prot amine sulfate inhibited subsequent binding of PCI in a dose-dependent manner and >100 mu g/ml protamine sulfate reduced binding of PCI to <1 0% of the control. Binding of I-125-pCI was specific, and bound (125)- pCI was recovered from the cells by heparin treatment or detached toge ther with intact cells by EDTA treatment, migrated on sodium dodecyl s ulfate-polyacrylamide gel electrophoresis with the same mobility (M(r) = 57,000) as unbound I-125-pCI. Furthermore, cell-bound PCI was funct ionally active as judged from its ability to inhibit the amidolytic ac tivity of urokinase, and its inhibitory activity was stimulated approx imate to 3-4-fold as compared to fluid-phase PCI. Immunogold electron microscopy revealed that PCI-antigen presented to the cells from the l uminal side bound exclusively to that surface in native as well as in prefixed cells. This binding of PCI was abolished in the presence of h eparin (50 mu g/ml) and after pretreatment of the cells either with pr otamine sulfate (400 mu g/ml) or with heparinase III (0.5 unit/ml). A slight decrease in PCI binding was seen after pretreatment of the cell s with chondroitinase ABC and chondroitinase AC. In contrast, binding of PCI to extracellular matrices of TCL-598 was decreased to approxima te to 70% after chondroitinase ABC treatment of the extracellular matr ices, whereas both heparinase III or chondroitinase AC treatment only reduced matrix-bound PCI to approximate to 95%. These data suggest tha t heparan sulfate-containing proteoglycans are predominantly involved in binding of PCI to the luminal side of TCL-598, while dermatan sulfa te-containing proteoglycans, the overall predominant PCI-binding prote oglycans in TCL extracts, are responsible for PCI binding to the extra cellular matrix. Heparan sulfate, however, exposed to an environment c ontaining PCI under physiological conditions, might localize PCI and m odulate its target enzyme specificity in vivo.