Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA).
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.
Kobayashi
H
H
Frauenklinik der Technischen Universität München im Klinikum rechts der Isar, Federal Republic of Germany.
Schmitt
M
M
Goretzki
L
L
Chucholowski
N
N
Calvete
J
J
Kramer
M
M
Günzler
W A
WA
Jänicke
F
F
Graeff
H
H
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*
D004792:Enzyme Precursors; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; D002401:Cathepsin B