Studies of the mechanism of cell intoxication by diphtheria toxin fragment A-asialoorosomucoid hybrid toxins. Evidence for utilization of an alternative receptor-mediated transport pathway.

Abstract

In order to study the variables important for determining the cytotoxicity of protein-protein hybrid toxins, the disulfide and thioiether conjugates between diphtheria toxin fragment A (DTA) and asialoorosomucoid (ASOR) were synthesized, purified, and tested for cytotoxicity toward isolated rat hepatocytes as monitored by the inhibition of protein synthesis. After 4-h incubation at 37 degrees C, both DTA-ASOR conjugates were nontoxic, but were highly toxic in the presence of an inhibitor of 125I-ASOR degradation. At concentrations inhibiting 125I-ASOR degradation to the same extent, the efficiency of inhibitors to enhance the toxicity of both conjugates varied with the following rank order: colchicine greater than chloroquine greater than NH4Cl greater leupeptin greater than cytochalasin B. In the absence of an inhibitor, both conjugates, after 10-h incubation, also exhibited measurable toxicity at concentrations as low as 10(-9) M, and DTA-S-S-ASOR was 60 times more toxic than DTA-S-ASOR. In a physiological salt solution containing energy source and bovine serum albumin, both conjugates also exhibited partial inhibition of protein synthesis after 4-h incubation and were enhanced by 2 microM colchicine toward complete inhibition with similar C0.5 of 2 X 10(-10) M. The toxicity of the conjugates were prevented in the presence of excess ASOR. These results not only indicate that intracellular degradation is an important factor restricting the toxicity of the conjugates, but also indicate that the toxic entry of DTA activity from these hybrids is independent of lysosomal degradation. Consequently, it appears that conjugate toxicity is dependent on the release of DTA activity from an extralysosomal compartment whose pool size is increased by the inhibitors and the change of medium. The effects of the inhibitors to inhibit 125I-ASOR uptake and degradation were also studied to facilitate interpretation of their enhancement of conjugate toxicity. Specifically, colchicine inhibited both internalization and degradation of the ligand in a saturable fashion with a C0.5 of 0.5 microM and a maximum inhibition of 65%. The data of the present study are interpreted so as to support a hypothesis that two receptor-mediated pathways are involved in the internalization and transport to lysosomes of ASOR and DTA-ASOR conjugates, only one of which is colchicine-sensitive.