Siegall C B, Chaudhary V K, FitzGerald D J, Pastan I
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
J Biol Chem. 1989 Aug 25;264(24):14256-61.
Pseudomonas exotoxin is composed of three structural domains that are responsible for cell recognition, membrane translocation, and ADP-ribosylation. The substitution of the cell recognition domain (domain Ia) with a growth factor such as transforming growth factor alpha (TGF alpha), creates a cell-specific cytotoxic agent, TGF alpha-PE40, which kills cells bearing epidermal growth factor (EGF) receptors. We have used TGF alpha-PE40 to define the role of sequences in domains II, Ib, and III. Various mutations were made in these domains and mutant forms of TGF alpha-PE40 expressed in Escherichia coli. Mutant proteins were then tested for their ADP-ribosylation, EGF receptor-binding, and cell-killing activities. Additionally, the amino boundary of domain III, which contains the ADP-ribosylation activity, was determined by deletion analysis. Data indicate that (i) the functional amino terminus of domain III is near amino acid 400; (ii) deletion of various regions in domain II or conversion of cysteines 265 and 268 to serines results in a loss of cytotoxicity which ranged from 10-fold to more than 150-fold, indicating that domain II is essential for full expression of cytotoxicity; (iii) deletion of the amino terminus of domain Ib results in a molecule with somewhat increased cytotoxic activity, indicating that domain Ib is not essential for the cytotoxic effect of TGF alpha-PE40; and (iv) TGF alpha-PE40, produced by denaturing and refolding of insoluble material from inclusion bodies, binds better to EGF receptors and is about 10-fold more cytotoxic to cells bearing EGF receptors than is the secreted form of soluble TGF alpha-PE40.
铜绿假单胞菌外毒素由三个结构域组成，分别负责细胞识别、膜转位和ADP核糖基化。用诸如转化生长因子α（TGFα）等生长因子替代细胞识别结构域（结构域Ia），可产生一种细胞特异性细胞毒剂，即TGFα-PE40，它能杀死携带表皮生长因子（EGF）受体的细胞。我们利用TGFα-PE40来确定结构域II、Ib和III中序列的作用。在这些结构域中进行了各种突变，并在大肠杆菌中表达了TGFα-PE40的突变形式。然后测试突变蛋白的ADP核糖基化、EGF受体结合和细胞杀伤活性。此外，通过缺失分析确定了包含ADP核糖基化活性的结构域III的氨基边界。数据表明：（i）结构域III的功能性氨基末端靠近氨基酸400；（ii）结构域II中各个区域的缺失或半胱氨酸265和268突变为丝氨酸会导致细胞毒性丧失10倍至150倍以上，这表明结构域II对于细胞毒性的充分表达至关重要；（iii）结构域Ib氨基末端的缺失导致一种细胞毒性活性略有增加的分子，这表明结构域Ib对于TGFα-PE40的细胞毒性作用并非必不可少；（iv）由包涵体中的不溶性物质变性和复性产生的TGFα-PE40与EGF受体的结合更好，对携带EGF受体的细胞的细胞毒性比可溶性TGFα-PE40的分泌形式高约10倍。
