Manske J M, Buchsbaum D J, Vallera D A
Department of Therapeutic Radiology, University of Minnesota, Minneapolis 55455.
J Immunol. 1989 Mar 1;142(5):1755-66.
The mAb anti-CD5 was linked to purified ricin A chain (RTA) or intact ricin (Rc) containing B chain to determine the role of ricin B chain in the intracellular trafficking of anti-CD5 immunotoxins (IT). IT were radiolabeled with iodine-125 and then studied for their subcellular compartmentalization in an acute lymphoblastic leukemia T cell line, CEM. Ricin A chain IT was not as toxic to CEM cells as Rc-IT in protein synthesis inhibition assays. This difference was not attributed to differential binding or modulation of the CD5 determinant from the cell surface as measured by FACS analysis. However, we found a relationship between the toxicity of anti-CD5-Rc and anti-CD5-RTA and their ability to traffic to CEM lysosomes. Kinetic analysis of the transfer of radioimmunotoxin to the lysosomes showed that anti-CD5-Rc was trafficked significantly more slowly than anti-CD5-RTA, perhaps due to an extended period of time in the Golgi compartment. The possibility of a Golgi interaction was tested by adding monensin, a carboxylic ionophore that interrupts trafficking through the Golgi, to cells treated with anti-CD5-RTA. The addition of monensin caused anti-CD5-RTA to traffic in a manner identical to anti-CD5-Rc. We conclude that 1) B chain slows trafficking of anti-CD5-Rc to the lysosomes; 2) the rate-limiting step in the toxicity difference between anti-CD5-Rc and anti-CD5-RTA is the rate of transfer to the lysosomes; and 3) trafficking through the Golgi may be important for anti-CD5-IT toxicity.
将抗CD5单克隆抗体与纯化的蓖麻毒蛋白A链（RTA）或含有B链的完整蓖麻毒素（Rc）相连，以确定蓖麻毒素B链在抗CD5免疫毒素（IT）细胞内运输中的作用。IT用碘-125进行放射性标记，然后在急性淋巴细胞白血病T细胞系CEM中研究其亚细胞定位。在蛋白质合成抑制试验中，蓖麻毒蛋白A链IT对CEM细胞的毒性不如Rc-IT。通过荧光激活细胞分选分析测量，这种差异并非归因于CD5决定簇从细胞表面的差异结合或调节。然而，我们发现抗CD5-Rc和抗CD5-RTA的毒性与其运输到CEM溶酶体的能力之间存在关联。放射性免疫毒素向溶酶体转移的动力学分析表明，抗CD5-Rc的运输速度明显慢于抗CD5-RTA，这可能是由于在高尔基体区室中的时间延长。通过向用抗CD5-RTA处理的细胞中添加莫能菌素（一种中断通过高尔基体运输的羧酸离子载体）来测试高尔基体相互作用的可能性。添加莫能菌素使抗CD5-RTA的运输方式与抗CD5-Rc相同。我们得出结论：1）B链减缓了抗CD5-Rc向溶酶体的运输；2）抗CD5-Rc和抗CD5-RTA毒性差异的限速步骤是向溶酶体的转移速率；3）通过高尔基体的运输可能对抗CD5-IT的毒性很重要。
