Newton D L, Boque L, Wlodawer A, Huang C Y, Rybak S M
Intramural Research Support Program, SAIC Frederick, Macromolecular Structure Laboratory, ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA.
Biochemistry. 1998 Apr 14;37(15):5173-83. doi: 10.1021/bi972147h.
Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibitor, PRI (onconase is refractory to PRI inhibition). [Met-(-1)]rOnc(E1S) and [Met-(-1)]rOnc(E1Y) inhibited protein synthesis in intact SF539 neuroblastoma cells with IC50's very similar to that of onconase (IC50 3.5, 10, and 10 microg/mL after 1 day and 0.16, 0.35, and 2.5 microg/mL after 5 days for onconase, [Met-(-1)]rOnc(E1S), and [Met-(-1)]rOnc(E1Y), respectively). Similar to that of onconase, cytotoxic activity of the recombinant derivatives was potentiated by monensin, NH4Cl, and retinoic acid. Brefeldin A completely blocked the enhancement of cytotoxicity caused by retinoic acid with all three proteins. Thus, drug-induced alterations of the intracellular trafficking of the recombinant derivatives also resembles that of onconase. Stability studies as assessed in serum-containing medium in the presence or absence of cells at 37 degreesC showed that the recombinant proteins were as stable to temperature and cell culture conditions as the native protein. Therefore, exchanging the Glu amino acid residue at the amino terminus of onconase with an amino acid residue containing a hydroxyl group produces recombinant proteins with ribonuclease and cytotoxic properties similar to native onconase.
抑癌酶是一种具有抗肿瘤特性的细胞毒性核糖核酸酶。通过将编码104个氨基酸中前15个和后6个氨基酸的寡核苷酸与编码其余氨基酸残基的基因组克隆融合，构建了一个编码整个蛋白质序列的半合成基因[牛顿，D.L.等人（1997年）《蛋白质工程》10，463 - 470]。由于未加工的N端甲硫氨酸氨基酸残基，在大肠杆菌中表达的所得蛋白质产物表现出很少的酶活性和细胞毒性活性。在本研究中，我们证明将基因的5'区域修饰为编码[甲硫氨酸 - (-1)]丝氨酸或[甲硫氨酸 - (-1)]酪氨酸而非天然焦谷氨酸，会产生具有恢复活性的重组抑癌酶衍生物。在所有测试的实验中，[甲硫氨酸 - (-1)]rOnc(E1S)比[甲硫氨酸 - (-1)]rOnc(E1Y)更具活性。与天然抑癌酶的作用一致，[甲硫氨酸 - (-1)]rOnc(E1S)在无细胞兔网织红细胞裂解物实验中是蛋白质合成的有效抑制剂，在与蛋白质合成抑制相关的浓度下降解tRNA。重组抑癌酶衍生物与天然蛋白质之间一个有趣的差异是它们对主要细胞内核糖核酸酶抑制剂PRI抑制的敏感性（抑癌酶对PRI抑制具有抗性）。[甲硫氨酸 - (-1)]rOnc(E1S)和[甲硫氨酸 - (-1)]rOnc(E1Y)在完整的SF539神经母细胞瘤细胞中抑制蛋白质合成，其IC50与抑癌酶非常相似（抑癌酶、[甲硫氨酸 - (-1)]rOnc(E1S)和[甲硫氨酸 - (-1)]rOnc(E1Y)在1天后的IC50分别为3.5、10和10μg/mL，在5天后分别为0.16、0.35和2.5μg/mL）。与抑癌酶相似，重组衍生物的细胞毒性活性被莫能菌素、氯化铵和视黄酸增强。布雷菲德菌素A完全阻断了视黄酸对所有三种蛋白质引起的细胞毒性增强作用。因此，药物诱导的重组衍生物细胞内运输改变也类似于抑癌酶。在含有血清的培养基中，在37℃有或无细胞存在的情况下进行的稳定性研究表明，重组蛋白在温度和细胞培养条件下与天然蛋白一样稳定。因此，将抑癌酶氨基末端的谷氨酸氨基酸残基与含羟基的氨基酸残基交换，会产生具有与天然抑癌酶相似的核糖核酸酶和细胞毒性特性的重组蛋白。
