Binding, Uptake and Degradation of the Toxic Proteins Abrin and Ricin by Toxin‐Resistant Cell Variants

Abstract

Ricin‐resistant variants derived from HeLa cells and baby hamster kidney (BHK) cells had a lower number of toxin‐binding sites than the sensitive parent lines, while the apparent association constants between the toxins and the surface receptors were almost the same. In the ricin‐resistant cell lines a much greater number of bound toxin molecules was required to inhibit protein synthesis by 50% after 3 h than in the parent lines, indicating that in addition to a reduced number of toxin‐binding sites other factors are involved in the reduced sensitivity of the variants to the toxins. Apparently, the toxin resistance involves an impairment of the transfer of the toxin A‐chain from the cell surface to the target, the ribosomes. Neuraminidase treatment of the various cell lines increased the number of binding sites for abrin and ricin, particularly in the resistant variants having originally a reduced number of binding sites and increased amount of sialic acid. The sensitivity of the neuraminidase‐treated cells to protein synthesis inhibition by abrin and ricin increased concomitantly with the increase in the number of binding sites. In most of the cell lines the number of toxin molecules bound to the cells when protein synthesis was decreased by 50% was about the same as before the neuraminidase treatment. The results indicate that in these cells the new binding sites appearing after neuraminidase treatment are as efficient as the normally occurring ones in mediating the uptake of the toxins. Most of the toxin bound to the cells may be washed off with 0.1 M lactose. However, after incubation at 37°C a certain fraction could not be released by this treatment. Subsequent treatment of these cells with 125I‐labelled antibodies to the toxins showed that the lactose‐resistant toxins were not accessible to the antibody and probably were internalized. Studies with 125I‐labelled abrin and ricin showed that the cell‐bound toxins are slowly degraded by the cells and that they are partly excreted in an apparently intact form into the medium. No evidence was obtained that the degradation of toxin was greater in resistant than in sensitive cell lines. The data indicate that the uptake of toxins by pinocytosis in two ricin‐resistant variants occurs at the same rate as the parent HeLa cells. In a ricin‐resistant variant of BHK cells it was reduced to about half of the control value. The possible role of pinocytosis in the intoxication process is discussed. Copyright © 1978, Wiley Blackwell. All rights reserved