title: Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA).
abstract: Action of purified human cathepsin B on recombinant single-chain urokinase-type plasminogen activator (pro-uPA) generated enzymatically active two-chain uPA (HMW-uPA), which was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot from plasmin-generated HMW-uPA and from elastase- or thrombin-generated inactive two-chain urokinase-type plasminogen activator. Preincubation of cathepsin B with E-64 (transepoxysuccinyl-L-leucylamino- (4-guanidino)butane, a potent inhibitor for cathepsin B) prior to the addition of pro-uPA prevented the activation of pro-uPA. The cleavage site within the cathepsin B-treated urokinase-type plasminogen activator (uPA) molecule, determined by N-terminal amino acid sequence analysis, is located between Lys158 and Ile159. Pro-uPA is cleaved by cathepsin B at the same peptide bond that is cleaved by plasmin or kallikrein. Binding of cathepsin B-activated pro-uPA to the uPA receptor on U937 cells did not differ from that of enzymatically inactive pro-uPA, indicating an intact receptor-binding region within the growth factor-like domain of the cathepsin B-treated uPA molecule. Not only soluble but also tumor cell receptor-bound pro-uPA could be efficiently cleaved by cathepsin B to generate enzymatically active two-chain uPA. Thus, cathepsin B can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. In contrast, no significant activation of pro-uPA by cathepsin D was observed. As tumor cells may produce both pro-uPA and cathepsin B, implications for the activation of tumor cell-derived pro-uPA by cellular proteases may be considered.
authors.affiliation: Frauenklinik der Technischen Universität München im Klinikum rechts der Isar, Federal Republic of Germany.
authors.forename: H
authors.initials: H
authors.lastname: Kobayashi
authors.forename: M
authors.initials: M
authors.lastname: Schmitt
authors.forename: L
authors.initials: L
authors.lastname: Goretzki
authors.forename: N
authors.initials: N
authors.lastname: Chucholowski
authors.forename: J
authors.initials: J
authors.lastname: Calvete
authors.forename: M
authors.initials: M
authors.lastname: Kramer
authors.forename: W A
authors.initials: WA
authors.lastname: Günzler
authors.forename: F
authors.initials: F
authors.lastname: Jänicke
authors.forename: H
authors.initials: H
authors.lastname: Graeff
mesh_terms: D015153:Blotting, Western; D002401:Cathepsin B / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D006868:Hydrolysis; D010960:Plasminogen Activators / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; D002401:Cathepsin B