Effect of rate of intracellular transport and diacytosis on cytotoxicity of hybrid toxins. Study with hybrids using hepatic asialoglycoprotein receptor-mediated endocytosis

Abstract

The effects of diacytosis and intracellular transport rate on cytotoxicity of hybrid toxins were studied with conjugates of diphtheria toxin fragment A (DTA) to asialoorosomucoid (ASOR) and its reduced and carboxymethylated cyanogen bromide fragment I (RC-ASCNBr-I) in cultured rat hepatocytes. In the hepatocytes the kinetics of uptake of the conjugate of asialoorosomucoid (DTA-ASOR) and that of the conjugate of the cyanogen bromide fragment (DTA-RC-ASCNBr-I) were quite similar, but the rate of accumulation of DTA moiety into the lysosomes, as determined by Percoll density gradient centrifugation, was found to be greater for the latter than the former. However, after internalization, DTA-RC-ASCNBr-I was diacytosed to a lesser extent than that of DTA-ASOR, particularly when colchicine was present during internalization. Analysis of the subunits of DTA-ASOR internalized by the hepatocytes indicated that they were accumulated disproportionately in a time-dependent manner so that the glycoprotein moiety was accumulated progressively more than the toxin moiety. Cytotoxicity of DTA-ASOR toward the hepatocytes was 2-times as much as that of DTA-RC-ASCNBr-I. Colchicine enhanced the toxicity of DTA-RC-ASCNBr-I (33-fold) to a greater extent than that of DTA-ASOR (12-fold). The difference in enhancement by colchicine was also observed in the rate of cell intoxication by the conjugates. Both conjugates were more toxic to the hepatocytes after incubation with the cells at 18°C than at 37°C. In the presence of vanadate (0.2 mM), which enhanced diacytosis, toxicity of DTA-ASOR decreased by 5-fold. After incubation with the hepatocytes, a partial dissociation of DTA-ASOR was found to occur independently of the receptor-mediated endocytosis. Taken together, these results indicate that diacytosis, subunit dissociation and rapid transport of conjugate toward lysosomes affect kinetically the rate of accumulation of the conjugate into a yet unidentified compartment of toxin translocation.