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

Abstract

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 C-dependent signaling pathways and that human chondrosarcoma cell line HCS-2/8 expresses two types of UTI-BPs; a 40-kDa UTI-BP (UTI-BP(40)), which is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP(45)). 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-BP(40) and UTI-BP(45) bind (125)I-UTI. A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been removed, binds only to UTI-BP(40). Additional experiments, using various reagents to block binding of (125)I-UTI and NG-UTI to the UTI-BP(40) and UTI-BP(45) confirm that the chondroitin sulfate side chain of UTI is required for its binding to UTI-BP(45). Analysis of binding of (125)I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP(40) (which can bind NG-UTI), and the high affinity sites are the UTI-BP(45). 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- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.

• Authors: Y Hirashima, H Kobayashi, M Suzuki, Y Tanaka, N Kanayama, M Fujie, T Nishida, M Takigawa, T Terao
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 276 Issue 17 Pg. 13650-6 (Apr 27 2001) ISSN: 0021-9258 [Print] United States
• PMID: 11278581 (Publication Type: Journal Article)

Chemical References

• Glycoproteins
• Ligands
• Phorbol Esters
• Chondroitin Sulfates
• Hyaluronoglucosaminidase
• Plasminogen Activators
• Chondroitin ABC Lyase
• urinastatin

Topics

• Binding Sites
• Binding, Competitive
• Blotting, Western
• Chondroitin ABC Lyase (pharmacology)
• Chondroitin Sulfates (chemistry)
• Chondrosarcoma (metabolism)
• Electrophoresis, Polyacrylamide Gel
• Glycoproteins (chemistry, metabolism)
• Glycosylation
• Humans
• Hyaluronoglucosaminidase (pharmacology)
• Kinetics
• Ligands
• Models, Biological
• Phorbol Esters (pharmacology)
• Plasminogen Activators (metabolism)
• Protein Binding
• Signal Transduction
• Time Factors
• Tumor Cells, Cultured
• Up-Regulation (drug effects)