The role of ricin B chain in the intracellular trafficking of anti-CD5 immunotoxins.
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.
Manske
J M
JM
Department of Therapeutic Radiology, University of Minnesota, Minneapolis 55455.
Buchsbaum
D J
DJ
Vallera
D A
DA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000633:toxicity*; D000943:Antigens, Differentiation / Q000276:immunology*; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D018956:CD5 Antigens; D002460:Cell Line; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D004705:Endocytosis / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D000922:Immunotoxins / Q000633:toxicity*; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D011500:Protein Synthesis Inhibitors / Q000633:toxicity; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D012276:Ricin / Q000502:physiology* / Q000633:toxicity
D000911:Antibodies, Monoclonal; D000943:Antigens, Differentiation; D018956:CD5 Antigens; D000922:Immunotoxins; D011500:Protein Synthesis Inhibitors; D011948:Receptors, Antigen, T-Cell; D012276:Ricin