Kobayashi H, Schmitt M, Goretzki L, Chucholowski N, Calvete J, Kramer M, Günzler W A, Jänicke F, Graeff H
Frauenklinik der Technischen Universität München im Klinikum rechts der Isar, Federal Republic of Germany.
J Biol Chem. 1991 Mar 15;266(8):5147-52.
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.
纯化的人组织蛋白酶B作用于重组单链尿激酶型纤溶酶原激活剂（pro-uPA），产生了具有酶活性的双链uPA（HMW-uPA），通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白质印迹法，其与纤溶酶产生的HMW-uPA以及弹性蛋白酶或凝血酶产生的无活性双链尿激酶型纤溶酶原激活剂无法区分。在添加pro-uPA之前，将组织蛋白酶B与E-64（反式环氧琥珀酰-L-亮氨酰胺-（4-胍基）丁烷，一种有效的组织蛋白酶B抑制剂）预孵育，可防止pro-uPA的激活。通过N端氨基酸序列分析确定，组织蛋白酶B处理的尿激酶型纤溶酶原激活剂（uPA）分子内的切割位点位于Lys158和Ile159之间。pro-uPA被组织蛋白酶B切割的肽键与被纤溶酶或激肽释放酶切割的肽键相同。组织蛋白酶B激活的pro-uPA与U937细胞上的uPA受体的结合与无酶活性的pro-uPA没有差异，这表明在组织蛋白酶B处理的uPA分子的生长因子样结构域内存在完整的受体结合区域。不仅可溶性的pro-uPA，而且肿瘤细胞受体结合型pro-uPA都可以被组织蛋白酶B有效切割，以产生具有酶活性的双链uPA。因此，在pro-uPA向具有酶活性的HMW-uPA的蛋白水解激活过程中，组织蛋白酶B可以替代纤溶酶。相比之下，未观察到组织蛋白酶D对pro-uPA有明显激活作用。由于肿瘤细胞可能同时产生pro-uPA和组织蛋白酶B，因此可以考虑细胞蛋白酶对肿瘤细胞衍生的pro-uPA激活的影响。
