Edwards G M, DeFeo-Jones D, Tai J Y, Vuocolo G A, Patrick D R, Heimbrook D C, Oliff A
Department of Cancer Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Mol Cell Biol. 1989 Jul;9(7):2860-7. doi: 10.1128/mcb.9.7.2860-2867.1989.
TGF-alpha-PE40 is a hybrid protein composed of transforming growth factor-alpha (TGF-alpha) fused to a 40,000-dalton segment of Pseudomonas exotoxin A (PE40). This hybrid protein possesses the receptor-binding activity of TGF-alpha and the cell-killing properties of PE40. These properties enable TGF-alpha-PE40 to bind to and kill tumor cells that possess epidermal growth factor (EGF) receptors. Unexpectedly, TGF-alpha-PE40 binds approximately 100-fold less effectively to EGF receptors than does native TGF-alpha (receptor-binding inhibition IC50 = 540 and 5.5 nM, respectively). To understand the factors governing receptor binding, deletions and site-specific substitutions were introduced into the PE40 domain of TGF-alpha-PE40. Removal of the N-terminal 59 or 130 amino acids from the PE40 domain of TGF-alpha-PE40 improved receptor binding (IC50 = 340 and 180 nM, respectively) but decreased cell-killing activity. Substitution of alanines for cysteines at positions 265 and 287 within the PE40 domain dramatically improved receptor binding (IC50 = 37 nM) but also decreased cell-killing activity. Similar substitutions of alanines for cysteines at positions 372 and 379 within the PE40 domain did not significantly affect receptor-binding or cell-killing activities. These studies indicate that the PE40 domain of TGF-alpha-PE40 interferes with EGF receptor binding. The cysteine residues at positions 265 and 287 of PE40 are responsible for a major part of this interference.
转化生长因子α-绿脓杆菌外毒素A 40kDa片段融合蛋白（TGF-α-PE40）是一种杂合蛋白，由转化生长因子α（TGF-α）与绿脓杆菌外毒素A（PE40）的一个40,000道尔顿片段融合而成。这种杂合蛋白具有TGF-α的受体结合活性和PE40的细胞杀伤特性。这些特性使TGF-α-PE40能够结合并杀死具有表皮生长因子（EGF）受体的肿瘤细胞。出乎意料的是，TGF-α-PE40与EGF受体的结合效率比天然TGF-α低约100倍（受体结合抑制IC50分别为540和5.5 nM）。为了了解影响受体结合的因素，对TGF-α-PE40的PE40结构域进行了缺失和位点特异性替换。从TGF-α-PE40的PE40结构域中去除N端的59或130个氨基酸可改善受体结合（IC50分别为340和180 nM），但会降低细胞杀伤活性。将PE40结构域内第265和287位的半胱氨酸替换为丙氨酸可显著改善受体结合（IC50 = 37 nM），但也会降低细胞杀伤活性。在PE40结构域内第372和379位进行类似的半胱氨酸替换为丙氨酸的操作，对受体结合或细胞杀伤活性没有显著影响。这些研究表明，TGF-α-PE40的PE40结构域会干扰EGF受体结合。PE40第265和287位的半胱氨酸残基是这种干扰的主要原因。
