Frankel A E, Fu T, Burbage C, Tagge E, Harris B, Vesely J, Willingham M C
Department of Medicine, Medical University of South Carolina, Charleston, 29425, USA.
Carbohydr Res. 1997 May 16;300(3):251-8. doi: 10.1016/s0008-6215(97)00048-7.
Ricin toxin with genetic or chemical modification of lectin sites has been previously reported to show markedly reduced cytotoxicity to cells following uptake by several receptors including the mannose receptor. Investigators have hypothesized that an intracellular galactoside-binding function was required for optimal intracellular targeting of ricin for these receptors. We have prepared insect-derived mutant ricin toxin B chain (RTB) with modifications of three lectin side domains (1 alpha, 1 beta, and 2 gamma) yielding a 1000-fold reduced galactoside avidity. After reassociation with plant RTA, the recombinant heterodimer and plant ricin were tested for cytotoxicity on mammalian cells expressing (mouse peritoneal macrophages, J774E cells, and MMR61 cells) or not expressing (KB cells) the D-mannose receptor. Receptor expression was confirmed by immunofluorescence microscopy. Lactose was included in the media to block cell-surface galactoside binding, and mannan was added as a control in each experiment to confirm mannose receptor-specific targeting. Plant ricin A chain (RTA) and E. coli-derived RTA were also tested for cytotoxicity on J774E and KB cells. Both wild-type and lectin-deficient ricin displayed mannose-receptor mediated cell cytotoxicity. This is the first report of a genetically modified ricin showing that RTB intracellular galactose binding activity is not required for ricin cytotoxicity. Sensitivity of mannose-receptor bearing cells, but not control cells, to mannosylated RTA, but not unglycosylated RTA, confirmed these observations. These results imply fusion toxins employing ricin can be prepared with maximal reductions in normal tissue binding.
先前有报道称，对凝集素位点进行基因或化学修饰的蓖麻毒素，在被包括甘露糖受体在内的几种受体摄取后，对细胞的细胞毒性显著降低。研究人员推测，这些受体要实现蓖麻毒素在细胞内的最佳靶向作用，需要细胞内半乳糖苷结合功能。我们制备了昆虫源突变型蓖麻毒素B链（RTB），对三个凝集素侧域（1个α、1个β和2个γ）进行了修饰，使其半乳糖苷亲和力降低了1000倍。与植物RTA重新结合后，对重组异二聚体和植物蓖麻毒素在表达D - 甘露糖受体的哺乳动物细胞（小鼠腹腔巨噬细胞、J774E细胞和MMR61细胞）或不表达该受体的细胞（KB细胞）上的细胞毒性进行了测试。通过免疫荧光显微镜确认受体表达。培养基中加入乳糖以阻断细胞表面半乳糖苷结合，每次实验中加入甘露聚糖作为对照以确认甘露糖受体特异性靶向。还对植物蓖麻毒素A链（RTA）和大肠杆菌源RTA在J774E和KB细胞上的细胞毒性进行了测试。野生型和凝集素缺陷型蓖麻毒素均表现出甘露糖受体介导的细胞毒性。这是关于基因修饰蓖麻毒素的首次报道，表明RTB细胞内半乳糖结合活性对于蓖麻毒素的细胞毒性并非必需。表达甘露糖受体的细胞对甘露糖基化RTA敏感，而对照细胞不敏感，对未糖基化RTA则不然，证实了这些观察结果。这些结果表明，可以制备出在正常组织结合方面最大程度降低的蓖麻毒素融合毒素。
