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Receptor-mediated internalization and degradation of diphtheria toxin by monkey kidney cells
Abstract:
The receptor-mediated internalization and degradation of radiolabeled diphtheria toxin by cultured monkey kidney cells was studied. The ability of a number of enzymes and chemicals to remove cell surface-bound toxin was tested; the combination of pronase and inositol hexaphosphate (PIHP) proved most effective. Using PIHP, the kinetics of toxin-cell association at 37 degrees C was resolved into two compounds: surface binding and internalization. The PIHP assay also allowed estimation of the half-time of toxin internalization (about 25 min). An assay involving precipitation of culture supernatants with trichloroacetic acid was developed and used to measure the rate of degradation and excretion of cell-associated toxin. Agents which markedly inhibited toxin internalization similarly prevented degradation, implying an intracellular location for the degradative process. The primary radioactive product excreted by Vero cells was monoiodotyrosine. The extent and rate of toxin degradation indicated lysosomal involvement. Finally, agents which blocked internalization or degradation, or both, (e.g. antibody and concanavalin A), protected cells from the cytotoxin action of diphtheria toxin, suggesting that these processes are necessary for expression of biological effect.
Insights
Researchers studied how cultured monkey kidney cells internalize and degrade diphtheria toxin. A pronase and inositol hexaphosphate (PIHP) treatment effectively removed cell-bound toxin, revealing internalization kinetics and lysosomal involvement in degradation.
Area of Science:
- Cell Biology
- Toxicology
- Biochemistry
Background:
- Diphtheria toxin exerts its cytotoxic effect after entering target cells.
- Understanding the mechanisms of toxin internalization and degradation is crucial for developing antitoxin strategies.
Purpose of the Study:
- To investigate the receptor-mediated internalization and degradation pathways of diphtheria toxin in cultured monkey kidney cells.
- To identify effective methods for removing cell surface-bound toxin and to characterize the kinetics of toxin-cell interactions.
- To elucidate the cellular location and mechanisms involved in diphtheria toxin degradation.
Main Methods:
- Utilized radiolabeled diphtheria toxin to study cellular uptake and degradation.
- Employed enzymes and chemicals, notably pronase and inositol hexaphosphate (PIHP), to assess cell surface-bound toxin removal.
- Developed an assay using trichloroacetic acid precipitation to quantify toxin degradation and excretion.
- Investigated the effects of various agents on toxin internalization, degradation, and cellular protection.
Main Results:
- Pronase and PIHP combination proved most effective in removing cell surface-bound diphtheria toxin.
- The PIHP assay resolved toxin-cell association into surface binding and internalization, with an internalization half-time of approximately 25 minutes.
- Inhibition of toxin internalization also prevented degradation, suggesting an intracellular degradative process.
- Degradation products included monoiodotyrosine, and the rate and extent of degradation indicated lysosomal involvement.
- Agents blocking internalization or degradation, such as antibody and concanavalin A, protected cells from diphtheria toxin's cytotoxic effects.
Conclusions:
- Receptor-mediated internalization and subsequent intracellular degradation, likely lysosomal, are essential for diphtheria toxin's cytotoxic activity.
- The developed PIHP assay is valuable for studying toxin internalization kinetics.
- Blocking these cellular processes offers a potential strategy for protecting cells against diphtheria toxin.