Assessment of ligand effects in intracellular trafficking of ricin A chain using anti-ricin hybridomas.
Intracellular ricin and immunotoxin trafficking has been difficult to study as only one to two cytosolic ricin A chain (RTA) molecules are sufficient to cause cell death. Previous studies (R.J. Youle and M. Colombatti, J. Biol. Chem., 262: 4676-4882, 1987) using anti-ricin hybridomas identified the secretory pre-Golgi as a critical site for RTA intoxication. We used ricin and RTA immunotoxins constructed with transferrin (TF) or anti-murine TF receptor antibody (RI7/217) to compare patterns of cytotoxicity and intracellular trafficking in anti-ricin hybridomas. Anti-RTA and anti-ricin B chain (RTB) hybridomas bound similar amounts of ricin and secreted comparable amounts of anti-ricin immunoglobulin. Anti-RTA hybridomas were 50- to 500-fold more resistant to ricin than nonsecretory and anti-RTB hybridomas, defining a ricin-resistant phenotype. All hybridomas expressed similar levels of surface TF receptors. RTA immunotoxins were constructed using human TF or RI7/217 and a disulfide linker. In protein synthesis inhibition assays, ricin-resistant hybridomas were manyfold more resistant to RI7/217-RTA than were ricin-sensitive hybridomas. In contrast, all hybridomas were equally sensitive to TF-RTA. Monensin increased ricin cytotoxicity minimally against all hybridomas, but dramatically increased RI7/217-RTA cytotoxicity in ricin-resistant and ricin-sensitive hybridomas in a way that abrogated the ricin-resistant phenotype. In contrast, monensin increased TF-RTA cytotoxicity equally in all hybridomas. Ammonium chloride had little effect on ricin or RI7/217-RTA cytotoxicity, but increased TF-RTA cytotoxicity against all hybridomas. Taken together, these results suggest that RTA molecules mediating cytotoxicity pass through an anti-RTA antibody-containing pre-Golgi compartment when bound to RTB or RI7/217, but not when bound to TF. Monensin abrogates the ricin-resistant phenotype when RTA is linked to RI7/217, but not RTB. This suggests that monensin alters RI7/217-RTA processing proximal to the pre-Golgi and that passage through the pre-Golgi may not be necessary for translocation of RTA to the cytoplasm. Ammonium chloride alters toxin cytotoxicity only when RTA is linked to TF, suggesting that only TF trafficks RTA through an acid-sensitive compartment prior to cytoplasmic translocation. With the addition of potentiating agents, each toxin studied showed a unique cytotoxicity profile against the anti-ricin hybridomas, demonstrating a dominant role of the cell binding ligand in intracellular toxin trafficking.
Kornfeld
S B
SB
Department of Medicine, University of California, San Diego.
Leonard
J E
JE
Mullen
M D
MD
Taetle
R
R
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D004351:Drug Resistance; D006825:Hybridomas / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D000922:Immunotoxins / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D014176:Protein Biosynthesis; D011990:Receptors, Transferrin / Q000276:immunology; D012276:Ricin / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology
D000922:Immunotoxins; D011990:Receptors, Transferrin; D000643:Ammonium Chloride; D012276:Ricin; D008985:Monensin