Fabbrini M S, Carpani D, Bello-Rivero I, Soria M R
Dibit-Department of Biological and Technological Research, San Raffaele Scientific Institute, Milano, Italy.
FASEB J. 1997 Nov;11(13):1169-76. doi: 10.1096/fasebj.11.13.9367352.
In contrast to two-chain urokinase (uPA), a chemical conjugate between uPA and native saporin (a cytotoxic plant seed ribosome-inactivating protein) did not require plasminogen activator inhibitors to be internalized. To dissect this pathway, we constructed a chimera consisting of the amino-terminal fragment (ATF) of human urokinase fused to a saporin isoform (SAP-3). The chimeric ATF-SAP toxin was expressed in Escherichia coli, purified, and characterized for its ribosome-inactivating activity. Besides being a potent inhibitor of protein synthesis in cell-free assays, ATF-SAP was specifically cytotoxic toward cells expressing human uPAR. Competition experiments indicated that both the human uPAR and the LDL-related receptor protein are involved in mediating the cell killing ability of ATF-SAP. We conclude that neither plasminogen activator inhibitors nor the catalytic moiety of urokinase are necessary to initiate these internalization pathways. Thus, saporin may play a role similar to plasminogen activator inhibitors in its ability to trigger internalization of uPAR-bound ligands through endocytic receptors.
与双链尿激酶（uPA）不同，uPA与天然皂草素（一种具有细胞毒性的植物种子核糖体失活蛋白）的化学偶联物在被内化时不需要纤溶酶原激活物抑制剂。为了剖析这一途径，我们构建了一种嵌合体，它由与人尿激酶氨基末端片段（ATF）融合的一种皂草素异构体（SAP-3）组成。嵌合的ATF-SAP毒素在大肠杆菌中表达、纯化，并对其核糖体失活活性进行了表征。除了在无细胞试验中是一种有效的蛋白质合成抑制剂外，ATF-SAP对表达人uPAR的细胞具有特异性细胞毒性。竞争实验表明，人uPAR和低密度脂蛋白相关受体蛋白都参与介导ATF-SAP的细胞杀伤能力。我们得出结论，纤溶酶原激活物抑制剂和尿激酶的催化部分都不是启动这些内化途径所必需的。因此，皂草素在通过内吞受体触发uPAR结合配体内化的能力方面可能发挥与纤溶酶原激活物抑制剂类似的作用。
