Binding and uptake of diphtheria toxin by toxin-resistant Chinese hamster ovary and mouse cells.

Abstract

We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally resistant mouse cells. All are resistant due to a defect in the process of internalization and delivery of toxin to its target in the cytosol, elongation factor 2. By cell hybridization studies, analysis of cross-resistance, and determination of specific binding sites for 125I-labeled diphtheria toxin, we showed that these cell strains fall into two distinct complementation groups. The Dipr group encompasses Chinese hamster strains that are resistant only to diphtheria toxin, as well as mouse LM cells. These strains possess a normal complement of high-affinity binding sites for diphtheria toxin, but these receptors are unable to deliver active toxin fragment A to the cytosol. Cells of the DPVr group have a broader spectrum of resistance, including Pseudomonas exotoxin A and several enveloped viruses as well as diphtheria toxin. In these studies, which investigate the resistance of these cells to diphtheria toxin, we demonstrate that they possess a reduced number of specific binding sites for this toxin and behave, phenotypically, like cells treated with the proton ionophore monensin. Their resistance is related to a defect in a mechanism required for release of active toxin from the endocytic vesicle.

Defective acidification of endosomes in Chinese hamster ovary cell mutants "cross-resistant" to toxins and viruses.

“…For both CHO-KI and RPE.28 cells, the plasma membrane marker is unimodal in distribution at 1.037 g/ml (fractions 3-8). The small amount of lysosomal enzyme activity applied to this gradient (<10% of total activity) is distributed within a broad area between 1.037 and 1.041 g/ml (fractions 3-17), as well as in a discrete peak at 1.045 g/ml (fractions [15][16][17][18][19][20][21][22][23][24][25][26][27]. Similar results were obtained for RPE.44 and RE.31 cells (data not shown).…”

“…We postulated that certain mutant cells that are cross-resistant to these agents would prove to be defective in acidification of the intracellular compartment(s) through which these agents gain access to the cytosol (19,20,28). The present studies on the DPVr mutants of CHO-Ki cells support this hypothesis.…”

“…These mutants were selected for resistance to DT but proved also to be resistant to a number of RNA viruses. More recently, Didsbury et aL (19) (19,20 (14).…”

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

“…Several groups have obtained CHO mutants pleiotropically defective in endocytosis by selecting cells that were resistant to diphtheria toxin (2,9,14,21). These mutants are also resistant to other toxins and certain viruses that require an acidic pH for toxicity or infectivity.…”

Impairment of podocyte function by diphtheria toxin—a new reversible proteinuria model in mice

“…There are reports from resistant mouse and rat cells, that these cells bind the toxin. [28][29][30] Irrespective of the molecular mechanism involved DTx-induced transient podocyte malfunction and proteinuria might be a supplemental new and convenient experimental model, which can also be applied to the great variety of genetically engineered C57BL/6 mice. Of note, BALB/c mice get sick as well but the magnitude of proteinuria is lower than in C57BL/6 mice (Supplementary Figure 1D).…”