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Particular features of diphtheria toxin internalization by resistant and sensitive mammalian cells

Manoilov K.Y., Labyntsev A.Ju., Korotkevych N.V., Maksymovych I.S., Kolybo D.V., Komisarenko S.V.

SUMMARY. Several mammalian species are resistant to diphtheria toxin (DT). The DT receptor, proHB-EGF in resistant and sensitive species have different primary structure due to amino acid substitutions, however there is no a definite opinion about how the differences in primary receptor structure alter 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 development of DT resistance of mammalian cells. It was shown that resistant to toxin L929 cells derived from the strain C3H mouse, adsorb the recombinant fluorescent subunit B of DT with the value of binding constant K_b that was close to that of highly sensitive to toxin African green monkey Vero cells (0,269 µM and 0,372 µM respectively). Endoсytosis 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. Obtained results suggest that at the initial stages, DT internalizes by resistant cells as rapidly as by sensitive cells. Nevertheless, at further stages of toxin uptake, its amount in cells can sufficiently vary depending on receptor expression level and physiological features of cell culture. It was concluded that internalisation and therefore 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.

Key words: absorption, binding, diphtheria toxin, endocytosis, enhanced green fluorescent protein EGFP, internalization

Tsitologiya i Genetika 2018, vol. 52, no. 5, pp. 41-50

E-mail: manoilov.inbox gmail.com; lab.andrey gmail.com; gnr.nata gmail.com; slavamax gmail.com; kolibo biochem.kiev.ua; svk biochem.kiev.ua

Manoilov K.Y., Labyntsev A.Ju., Korotkevych N.V., Maksymovych I.S., Kolybo D.V., Komisarenko S.V. Particular features of diphtheria toxin internalization by resistant and sensitive mammalian cells, Tsitol Genet., 2018, vol. 52, no. 5, pp. 41-50.

In "Cytology and Genetics":
K. Y. Manoilov, A. J, u. Labyntsev, N. V. Korotkevych, I. S. Maksymovych, D. V. Kolybo, S. V. Komisarenko Particular Features of Diphtheria Toxin Internalization by Resistant and Sensitive Mammalian Cells, Cytol Genet., 2018, vol. 52, no. 5, pp. 353–359
DOI: 10.3103/S0095452718050080