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Study on the variables affecting toxicity of hybrid toxins. The effect of different target cell receptor distribution
1Biochemistry Laboratory, Isaac Gordon Center for Digestive Diseases and Nutrition, Genesee Hospital Rochester, NY 14607.
Abstract:
Disulfide conjugates of diphtheria toxin (DT) and its fragment A (DTA) to asialoorosomucoid (ASOR) were prepared. The toxicity of the conjugates were compared with DT in isolated rat, rabbit and guinea pig hepatocytes containing different concentration of asialoglycoprotein receptors (Biochim. Biophys, Acta 942, 57, 1988). In rat hepatocytes DTA-ASOR was highly toxic with half-maximal inhibitory concentration (IC50) of protein synthesis occurring at 4 +/- 3.10(-11) M (n = 7) which was much lower than that of DT DT (7.8 +/- 9.8.10(-9) M, n = 7). In rabbit hepatocytes toxicity of the conjugate (IC50 = 5.4 +/- 4.9.10(-10) M, n = 7) was higher than that of DT (IC50 = 5 +/- 4.10(-11) M, n = 7). In guinea pig hepatocytes, DTA-ASOR was not toxic at concentration below 10(-8) M, although DT was highly toxic (IC50 = 1.8 +/- 1.4.10(-10), n = 3). In the presence of 5 microM colchicine, the toxicity of DTA-ASOR in rat and rabbit hepatocytes increased by 10-fold, while in guinea pig hepatocytes it became detectable with an IC50 of 1.2 +/- 0.8.10(-9) M (n = 3). The toxicity of DT in the rat cells was also enhanced 10-fold by colchicine, but not at all in either the rabbit or the guinea pig cells. Addition of isolated diphtheria toxin fragment B (DTB) did not affect significantly the toxicity of DTA-ASOR in all three hepatocytes and that of DT in rat hepatocytes, but reduced toxicity of DT more than 20-fold in the rabbit and guinea pig cells. Toxicity of DT-ASOR in rat hepatocytes was the same as DTA-ASOR both in the absence and presence of colchicine, and abolished completely by excess ASOR, but not by DTB. Toxicity of DT-ASOR in rabbit hepatocytes was 40-times higher than DTA-ASOR, enhanced 10-fold by cochicine and reduced more than 30-fold by excess ASOR, but only slightly by DTB. These results indicate that entry of DTA from DTA-ASOR involve a DTB-independent translocation mechanism which can be as efficient as the DTB-dependent mechanism used by DT in the rabbit and guinea pig cells. The entry of both conjugates appeared to be mediated by the asialoglycoprotein receptors. However, the DTB moiety of DT-ASOR could function only in the DT-sensitive cells indicating the lack of a DTB-mediated translocation in the DT-resistant cells.
Insights
Diphtheria toxin conjugates (DT-ASOR and DTA-ASOR) show varying toxicity in liver cells, with DTA-ASOR utilizing a DTB-independent entry mechanism. Receptor-mediated uptake via asialoglycoprotein receptors is key for both conjugates.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Diphtheria toxin (DT) exerts its toxicity by inhibiting protein synthesis.
- DT utilizes its B fragment (DTB) for receptor binding and translocation into cells.
- Asialoorosomucoid (ASOR) is a ligand for asialoglycoprotein receptors (ASGPRs).
Purpose of the Study:
- To compare the toxicity of DT and its conjugates with ASOR in different liver cells.
- To investigate the mechanism of entry and translocation of DT and DTA-ASOR conjugates.
- To determine the role of DTB and ASGPRs in the cellular uptake and toxicity of these conjugates.
Main Methods:
- Preparation of disulfide conjugates of DT and DTA with ASOR.
- Assessment of protein synthesis inhibition (toxicity) in isolated rat, rabbit, and guinea pig hepatocytes.
- Evaluation of the effects of colchicine and excess ASOR or DTB on conjugate toxicity.
Main Results:
- DTA-ASOR demonstrated high toxicity in rat hepatocytes, exceeding that of DT, and showed varying toxicity in rabbit and guinea pig cells.
- Colchicine enhanced DTA-ASOR toxicity in rat and rabbit cells, and made it detectable in guinea pig cells.
- DT-ASOR exhibited higher toxicity than DTA-ASOR in rabbit cells, suggesting a role for DTB in this context.
- DTB-independent translocation of DTA from DTA-ASOR was observed, comparable in efficiency to DTB-dependent translocation by DT in some cell types.
- Entry of both conjugates was mediated by ASGPRs, but DTB function was limited to DT-sensitive cells.
Conclusions:
- DTA-ASOR employs a DTB-independent translocation mechanism, distinct from DT.
- ASGPRs mediate the cellular entry of both DT and DTA-ASOR conjugates.
- The DTB moiety of DT-ASOR is crucial for toxicity in DT-sensitive cells, indicating cell-specific translocation mechanisms.