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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.
Insights
Two Chinese hamster cell mutants and mouse cells exhibit resistance to diphtheria toxin due to impaired toxin delivery. These cells fall into two groups based on their resistance mechanisms and toxin binding.
Area of Science:
- Cell Biology
- Toxicology
- Genetics
Background:
- Diphtheria toxin (DT) exerts its cytotoxic effect by inhibiting protein synthesis.
- Cellular resistance to DT can arise from defects in toxin internalization, processing, or translocation to the cytosol.
Purpose of the Study:
- To characterize the mechanisms of DT resistance in two phenotypically distinct Chinese hamster cell mutants and compare them to naturally resistant mouse cells.
- To determine the complementation groups and molecular basis for DT resistance in these cell lines.
Main Methods:
- Cell hybridization studies to assess complementation.
- Analysis of cross-resistance to other toxins and viruses.
- Quantification of specific binding sites for 125I-labeled DT.
Main Results:
- Two distinct complementation groups (Dipr and DPVr) were identified.
- Dipr cells are resistant only to DT, possessing normal binding sites but impaired toxin delivery to the cytosol.
- DPVr cells show broader resistance, reduced DT binding sites, and a defect in toxin release from endocytic vesicles.
Conclusions:
- DT resistance in these cell lines is primarily due to defects in toxin internalization and delivery pathways.
- The Dipr group has a defect in intracellular toxin transport, while the DPVr group has issues with toxin release from endosomes.