Particular Features of Diphtheria Toxin Internalization by Resistant and Sensitive Mammalian Cells
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Several mammalian species are resistant to diphtheria toxin (DT). The DT receptor, proHB-EGF in resistant and sensitive species, has a different primary structure due to the amino acid substitutions; however, there is no definite opinion regarding how the difference in primary receptor structure alters the process of DT internalization by resistant cells compared to sensitive. The aim of the present study was to evaluate the role of DT internalization in the development of DT resistance of mammalian cells. It was shown that L929 cells resistant to toxin and derived from the C3H mouse strain absorb the recombinant fluorescent subunit B of DT with the value of binding constant Kb that was close to that of African green monkey Vero cells highly sensitive to toxin (0.269 and 0.372 μM, respectively). Endocytosis dynamics analysis by confocal microscopy indicated that the amount of subunit B internalized by Vero varied from nearly equal (at early stages of the process) to approximately 2‒5 times bigger (after 30 min) compared to L929 cells. The obtained results suggest that, at the initial stages, DT is internalized by resistant cells as rapidly as by sensitive cells. Nevertheless, at further stages of uptake, toxin amount in cells sufficiently varies depending on the receptor expression level and physiological features of the cell culture. It was concluded that the internalization and, therefore, the resistance of cells to the DT depends insufficiently on receptor structure in resistant and sensitive species but may be dependent on subsequent endosomal transport and accumulation of DT molecules in cells at late stages of internalization.
Keywords:absorption binding diphtheria toxin endocytosis internalization enhanced green fluorescent protein EGFP
This work was supported by grants of National Academy of Sciences of Ukraine, nos. 0114U003216 and 0112U002624.
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