Characterization of Binding Properties of Urinary Trypsin Inhibitor to Cell-associated Binding Sites on Human Chondrosarcoma Cell Line HCS-2/8

Yasuyuki Hirashima, Hiroshi Kobayashi, Mika Suzuki, Yoshiko Tanaka, Naohiro Kanayama, Michio Fujie, Takashi Nishida, Masaharu Takigawa, Toshihiko Terao

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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-BP40), which is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP45). 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-BP 40. Additional experiments, using various reagents to block binding of 125I-UTI and NG-UTI to the UTI-BP40 and UTI-BP 45 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 NG-UTI), 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- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.

Original languageEnglish
Pages (from-to)13650-13656
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number17
Publication statusPublished - Apr 27 2001

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Chondrosarcoma
Binding Sites
Cells
Cell Line
urinastatin
Urokinase-Type Plasminogen Activator
Chondroitin Sulfates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Hirashima, Y., Kobayashi, H., Suzuki, M., Tanaka, Y., Kanayama, N., Fujie, M., ... Terao, T. (2001). Characterization of Binding Properties of Urinary Trypsin Inhibitor to Cell-associated Binding Sites on Human Chondrosarcoma Cell Line HCS-2/8. Journal of Biological Chemistry, 276(17), 13650-13656.

Characterization of Binding Properties of Urinary Trypsin Inhibitor to Cell-associated Binding Sites on Human Chondrosarcoma Cell Line HCS-2/8. / Hirashima, Yasuyuki; Kobayashi, Hiroshi; Suzuki, Mika; Tanaka, Yoshiko; Kanayama, Naohiro; Fujie, Michio; Nishida, Takashi; Takigawa, Masaharu; Terao, Toshihiko.

In: Journal of Biological Chemistry, Vol. 276, No. 17, 27.04.2001, p. 13650-13656.

Research output: Contribution to journalArticle

Hirashima, Yasuyuki ; Kobayashi, Hiroshi ; Suzuki, Mika ; Tanaka, Yoshiko ; Kanayama, Naohiro ; Fujie, Michio ; Nishida, Takashi ; Takigawa, Masaharu ; Terao, Toshihiko. / Characterization of Binding Properties of Urinary Trypsin Inhibitor to Cell-associated Binding Sites on Human Chondrosarcoma Cell Line HCS-2/8. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 17. pp. 13650-13656.
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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-BP40), which is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP45). 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-BP 40. Additional experiments, using various reagents to block binding of 125I-UTI and NG-UTI to the UTI-BP40 and UTI-BP 45 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 NG-UTI), 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- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.",
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AU - Terao, Toshihiko

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