Structure and function analysis of urinary trypsin inhibitor (UTI): identification of binding domains and signaling property of UTI by analysis of truncated proteins.

Abstract

The binding of urinary trypsin inhibitor (UTI) to its binding sites/receptors on tumor cells inhibits cell invasion in a number of experimental systems and that UTI downregulates constitutive and phorbol ester-induced urokinase production by certain tumor cells. To determine whether the carbohydrate moieties and core protein are required for urokinase suppression, we obtained UTI derivatives that contained O-glycoside-linked N-terminal glycopeptide (UTIm1), N-glycoside-linked C-terminal tandem Kunitz domains (UTIm2), UTI lacking O-glycoside (UTIc), asialo UTI (UTIa), UTI lacking N-glycoside (UTIn), purified Kunitz domain II of UTI (HI-8), and recombinant Kunitz domain II of UTI (R-020). The IC(50) of inhibiting binding of (125)I-labeled UTI to cells was indistinguishable for UTIa, UTIn and intact UTI, whereas the IC(50) for inhibiting binding of (125)I-labeled UTI to cells was 2.5-, 25- and 29-fold greater for UTIm1, UTIm2 and UTIc than for native UTI. We next looked at the suppression of the urokinase expression by UTI derivatives. An enzyme-linked immunosorbent assay was carried out to measure secreted and cell-associated urokinase. Intact UTI, UTIa, or UTIn effectively suppressed urokinase expression, but UTIm1, UTIm2, UTIc, HI-8 and R-020 had no significant effect. These data show that UTI requires either the N-terminal extension with the O-linked carbohydrate moiety (chondroitin 4-sulfate sugar side chain; Ala1 to Lys21 residues) or the Kunitz domain I (Lys22 to Arg77 residues) of UTI to bind to cells, but the urokinase expression was inhibited only by the O-glycoside-linked core protein without the N-glycoside side chain.

• Authors: M Suzuki, H Kobayashi, Y Tanaka, Y Hirashima, T Terao
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 1547 Issue 1 Pg. 26-36 (May 05 2001) ISSN: 0006-3002 [Print] Netherlands
• PMID: 11343788 (Publication Type: Journal Article)

Chemical References

• Glycoproteins
• Peptides
• Trypsin Inhibitors
• Neuraminidase
• Urokinase-Type Plasminogen Activator
• Metalloendopeptidases
• Chondroitin ABC Lyase
• urinastatin

Topics

• Binding Sites
• Binding, Competitive
• Cell Line
• Cell Membrane (chemistry, metabolism)
• Chondroitin ABC Lyase
• Down-Regulation
• Electrophoresis, Polyacrylamide Gel
• Glycoproteins (chemistry, isolation & purification, metabolism)
• Humans
• Metalloendopeptidases
• Neuraminidase
• Peptides (isolation & purification, pharmacology)
• Signal Transduction
• Trypsin Inhibitors (chemistry, metabolism)
• Tumor Cells, Cultured
• Urokinase-Type Plasminogen Activator (metabolism)