Conde F P, Orlandi R, Canevari S, Mezzanzanica D, Ripamonti M, Muñoz S M, Jorge P, Colnaghi M I
Departamento de Investigación, Centro Ramón y Cajal, Madrid, Spain.
Eur J Biochem. 1989 Jan 2;178(3):795-802. doi: 10.1111/j.1432-1033.1989.tb14511.x.
The protein toxin restriction, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28-S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non-glycosylated polypeptide of Mr 16836, exhibiting in cell-free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restriction toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later. After derivatization with N-succinimidyl 3-(2-pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell-free system. This derivatized toxin was then coupled to monoclonal antibodies (MBr1, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites interanalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.
从局限曲霉中分离出的蛋白毒素局限菌素，通过阻断核糖体延伸循环使真核细胞中的蛋白质合成失活。这种蛋白质作为一种特异性核酸酶，从28 - S rRNA上切下一小段片段。该毒素的生化和生物学特性表明，它是一种Mr为16836的非糖基化多肽，在无细胞系统中表现出与蓖麻毒素A链相似的蛋白质合成抑制能力。通过其低体外细胞毒性测定，这种多肽似乎无法穿透大多数测试的癌细胞系。此外，在BALB/c小鼠中的体内研究表明，局限菌素的毒性非常低，在兔子中，静脉注射后24小时，仍有15%的毒素存在于血流中。用N - 琥珀酰亚胺基3 - (2 - 吡啶二硫基)丙酸进行衍生化并用二硫苏糖醇还原后，局限菌素在无细胞系统中测定时仍保持其蛋白质合成抑制活性。然后将这种衍生化毒素与单克隆抗体（MBr1、MLuC1、MLuC2、MOv17、MOv18、MOv19）偶联，这些单克隆抗体对人癌的反应谱有限。免疫缀合物的生化和生物学特性表明：（a）当局限菌素与单克隆抗体以约2的平均摩尔比偶联时，免疫缀合物保持了抗体的结合活性和毒素的蛋白质合成抑制活性；（b）对四种免疫缀合物进行了细胞毒性测试，其中三种与MBr1、MLuC1和MOv17单克隆抗体制备的免疫缀合物对相关靶细胞表现出良好的细胞毒性水平，对无关细胞系毒性低或无毒性。产生完全无效免疫缀合物的MLuC2单克隆抗体诱导内化的抗原位点不到十分之一，而MBr1、MLuC1和MOv17单克隆抗体表现出约三分之一的抗原位点被内化。从这些数据可以得出结论，只要选择合适的靶抗原和偶联程序，局限菌素可被认为是一种适合用于生成免疫缀合物的毒素。
