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Recombinant hybrid toxin with dual enzymatic activities. Potential use in preparing highly effective immunotoxins
1Department of Pharmacology, University of Minnesota, Minneapolis 55455.
Abstract:
Bacterial toxins and ribosomal inhibitory proteins isolated from plants are used to prepare tumor-specific cytotoxic conjugates. The ability of these conjugates to kill tumor cells depends on binding, internalization, translocation to cytoplasm, and translation inhibition. Modulation of any one of these processes can improve cytotoxicity. Since bacterial and plant toxins act at a distinct step in translation, a combination of their activities could be more effective. Therefore, a chimeric protein was prepared by genetically fusing the coding region of the ricin A chain (RTA) and the fragment A of diphtheria toxin (DTA). The hybrid protein (RTA-DTA) expressed in bacteria retained the N-glycosidase activity of the RTA and ADP-ribosylation activity of the DTA. The hybrid toxin was more potent than the ricin A chain (11-fold) and the diphtheria toxin (50-fold) in inhibiting cell-free translation. Immunotoxin made with the hybrid toxin was about 100- and 1000-fold more effective than RTA or DTA conjugate, respectively, in inhibiting tumor cell growth in vitro. These results indicate that the hybrid toxin with dual activities could be useful in preparing potent immunotoxins with better anti-tumor cell activity.
Insights
A novel hybrid toxin combining ricin A chain (RTA) and diphtheria toxin A fragment (DTA) shows enhanced anti-tumor activity. This dual-action toxin is more effective in immunoconjugates for cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Tumor-specific cytotoxic conjugates utilize bacterial toxins and plant proteins to target cancer cells.
- Conjugate efficacy relies on binding, internalization, translocation, and translation inhibition.
- Combining toxins acting at different translation steps may enhance cytotoxicity.
Purpose of the Study:
- To create a chimeric protein by fusing ricin A chain (RTA) and diphtheria toxin A fragment (DTA).
- To evaluate the cytotoxic potential of the hybrid RTA-DTA toxin in inhibiting tumor cell growth.
Main Methods:
- Genetically fusing the coding regions of RTA and DTA to create a hybrid protein (RTA-DTA).
- Expressing the hybrid protein in bacteria.
- Assessing cell-free translation inhibition and in vitro tumor cell growth inhibition of immunoconjugates.
Main Results:
- The hybrid RTA-DTA protein retained both N-glycosidase activity (from RTA) and ADP-ribosylation activity (from DTA).
- RTA-DTA was significantly more potent than RTA or DTA individually in inhibiting cell-free translation (11-fold and 50-fold, respectively).
- Immunoconjugates with RTA-DTA demonstrated superior efficacy in inhibiting tumor cell growth in vitro (100-fold for RTA and 1000-fold for DTA).
Conclusions:
- The dual-activity hybrid toxin RTA-DTA exhibits enhanced potency compared to its individual components.
- This hybrid toxin holds promise for developing more effective immunotoxins for cancer therapy.