Characterization of binding properties of urinary trypsin inhibitor (UTI) to cell-associated binding sites on human chondrosarcoma cell line HCS-2/8

Urinary trypsin inhibitor (UTI) forms membrane complexes with UTI-binding proteins (UTI-BPs) and initiates modulation of urokinase-type plasminogen activator (uPA) expression, which results in UTI-mediated suppression of cell invasiveness. It has been established that suppression of uPA expression and invasiveness by UTI is mediated through inhibition of protein kinase Cdependent signaling pathways and that human chondrosarcoma cell line HCS2/8 expresses two types of UTI-binding proteins (UTI-BPs); a 40 kDa UTI-BP (UTI-BP40), which is identical to link protein (LP), and a 45 kDa UTI-BP (UTIBP45). Here we characterize binding properties of UTI-BPs—UTI complexes in the cells. In vitro ligand-blot, cell-binding and competition assays, and Scatchard analyses demonstrate that both UTI-BP40 and UTI-BP45 bind 125I-UTI. A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been removed, binds only to UTI-BP40. Additional experiments, using various reagents to block binding of 125I-UTI and NG-UTI to the UTI-BP40 and UTI-BP45 confirm that the chondroitin-sulfate side chain of UTI is required for its binding to UTI-BP45. Analysis of binding of 125I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP40 (which can bind NGUTI) and the high affinity sites are the UTI-BP45. In addition, UTI-induced suppression of phorbol ester stimulated up-regulation of uPA is inhibited by reagents that were shown to prevent binding of UTI to the 40 kDa and 45 kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation. by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from