Intracellular Pathways of Ricin A Chain Cytotoxins

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Abstract
Ricin A chain, a potent ribosomal poison, was disulfide linked either to the iron transport protein, transferrin, or to anti-transferrin receptor antibodies to produce highly specific derivative toxins, Tf-A and TfR-A, respectively. The ability of these agents to gain access to and damage ribosomes within the cell was accelerated in the presence of carboxylic ionophores. Their effectiveness for killing clonogenic target cells was correspondingly enhanced by 5 logs after a brief treatment with Tf-A plus ionophore. Intracellular trafficking of Tf-A and TfR-A was monitored by a variety of methods to better understand their mechanism of action. Data obtained with 125I-labeled A chain and 59Fe3+-labeled toxin probes indicated that the natural iron delivery pathway was initially followed. This was characterized by specific attachment to surface receptors, internalization, entry into low-density acidic vesicles, uncoupling of iron, an absence of lysosomal degradation, and sustained cycling. Ultrastructural studies using a colloidal gold-labeled anti-A chain probe confirmed the presence of these toxins within the structural elements associated with endocytosis. Toxic Tf-A molecules, however, diverged from this pathway (t1/2 = 88 min) to eventually kill cells as witnessed by a gradual loss in the ability to rescue cells using excess transferrin. Potentiating agents, such as carboxylic ionophores or B chain, seem to act by speeding the divergence of Tf-A and TfR-A from the normal endocytotoxic cycle.