title: 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.affiliation: Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Handacho 3600, Hamamatsu, 431-3192, Shizuoka, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Suzuki
authors.forename: H
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Y
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Y
authors.initials: Y
authors.lastname: Hirashima
authors.forename: T
authors.initials: T
authors.lastname: Terao
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D019765:Chondroitin ABC Lyase; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008666:Metalloendopeptidases; D009439:Neuraminidase; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology; D015398:Signal Transduction; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D010455:Peptides; D014361:Trypsin Inhibitors; D009439:Neuraminidase; D014568:Urokinase-Type Plasminogen Activator; D008666:Metalloendopeptidases; D019765:Chondroitin ABC Lyase; C028665:urinastatin