Abstract
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.
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REFERENCES
Collier, R.J., Diphtheria toxin: mode of action and structure, Bacteriol. Rev., 1975, vol. 39, no. 1, pp. 54–85.
Simpson, J.C., Smith, D.C., Roberts, L.M., and Lord, J.M., Expression of mutant dynamin protects cells against diphtheria toxin but not against ricin, Exp. Cell Res., 1998, vol. 239, no. 2, pp. 293–300.
Bennett, M.J. and Eisenberg, D., Refined structure of monomeric diphtheria toxin at 2.3 A resolution, Protein Sci., 1994, vol. 3, no. 9, pp. 1464–1475.
Abraham, J.A., Damm, D., Bajardi, A., Miller, J., Klagsbrun, M., and Ezekowitz, R.A., Heparin-binding EGF-like growth factor: characterization of rat and mouse cDNA clones, protein domain conservation across species, and transcript expression in tissues, B-iochem. Biophys. Res. Commun., 1993, vol. 190, no. 1, pp. 125–133. doi.org/10.1006/bbrc.1993.1020
Collier, R.J., Diphtheria toxin: mode of action and structure, Bacteriol. Rev., 1975, vol. 39, no. 1, pp. 54–85.
Morris, R.E. and Saelinger, C.B., Diphtheria toxin does not enter resistant cells by receptor-mediated endocytosis, Infect. Immun., 1983, vol. 42, no. 2, pp. 812–817.
Mitamura, T., Higashiyama, S., Taniguchi, N., Klagsbrun, M., and Mekada, E., Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor and inhibits specifically its mitogenic activity, J. Biol. Chem., 1995, vol. 270, no. 3, pp. 1015–1019.
Mitamura, T., Umata, T., Nakano, F., Shishido, Y., Toyoda, T., Itai, A., Kimura, H., and Mekada, E., Structure-function analysis of the diphtheria toxin receptor toxin binding site by site-directed mutagenesis, J. Biol. Chem., 1997, vol. 272, no. 43, pp. 27084–27090.
Naglich, J.G., Metherall, J.E., Russell, D.W., and Eidels, L., Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor, Cell, 1992, vol. 69, no. 6, pp. 1051–1061.
Moehring, T.J. and Moehring, J.M., Interaction of diphtheria toxin and its active subunit, fragment A, with toxin-sensitive and toxin-resistant cells, Infect. Immun., 1976, vol. 13, no. 5, pp. 1426–1432.
El Hage, T., Decottignies, P., and Authier, F., Endosomal proteolysis of diphtheria toxin without toxin translocation into the cytosol of rat liver in vivo, FEBS J., 2008, vol. 275, no. 8, pp. 1708–1722. doi 10.1111/j.1742-4658. 2008.06326.x
Heagy, W.E. and Neville, D.M.J., Kinetics of protein synthesis inactivation by diphtheria toxin in toxinresistant L cells. Evidence for a low efficiency receptor-mediated transport system, J. Biol. Chem., 1981, vol. 256, no. 24, pp. 12788–12792.
Didsbury, J.R., Moehring, J.M., and Moehring, T.J., Binding and uptake of diphtheria toxin by toxin-resistant Chinese hamster ovary and mouse cells, Mol. Cell. Biol., 1983, vol. 3, no. 7, pp. 1283–1294.
Labyntsev, A.J., Korotkevych, N.V., Manoilov, K.J., Kaberniuk, A.A., Kolybo, D.V., and Komisarenko, S.V., Recombinant fluorescent models for studying the diphtheria toxin, J. Bioorg. Chem., 2014, vol. 40, no. 4, pp. 401–409. doi 10.1134/S1068162014040086
Kaberniuk, A.A., Labyntsev, A.I., Kolybo, D.V., Oliinyk, O.S., Redchuk, T.A., Korotkevych, N.V., Horchev, V.F., Karakhim, S.O., and Komisarenko, S.V., Fluorescent derivatives of diphtheria toxin subunit B and their interaction with Vero cells, Ukr. Biokhim. Zh., 2009, vol. 81, no. 1, pp. 67–77.
Labyntsev, A.I., Korotkevich, N.V., Kaberniuk, A.A., Romaniuk, S.I., Kolybo, D.V., and Komisarenko, S.V., Interaction of diphtheria toxin B subunit with sensitive and insensitive mammalian cells, Ukr. Biokhim. Zh., 2010, vol. 82, no. 6, pp. 65–75.
Manoilov, K.Y., Gorbatiuk, O.B., Usenko, M.O., Shatursky, O.Y., Borisova, T.O., and Kolybo, D.V., The characterization of purified recombinant protein CRM197 as a tool to study diphtheria toxin, Dopov. Nac. Acad. Nauk Ukr., 2016, no. 9, pp. 124–133. doi.org/10.15407/dopovidi2016.09.124
Moehring, J.M. and Moehring, T.J., Comparison of diphtheria intoxication in human and nonhuman cell lines and their resistant variants, Infect. Immun., 1976, vol. 13, no. 1, pp. 221–228.
Gabliks, J. and Falconer, M., Interaction of diphtheria toxin with cell cultures from susceptible and resistant animals, J. Exp. Med., 1966, vol. 123, no. 4, pp. 723–732.
Piersma, S.J., van der Gun, J.W., Hendriksen, C.F.M., and Thalen, M., Decreased sensitivity to diphtheria toxin of Vero cells cultured in serum-free medium, Biologicals, 2005, vol. 33, no. 2, pp. 117–122. doi.org/ 10.1016/j.biologicals.2005.03.002
Rudolph, R. and Lilie, H., In vitro folding of inclusion body proteins, FASEB J., 1996, vol. 10, no. 1, pp. 49–56.
Basu, A., Li, X., and Leong, S.S.J., Refolding of proteins from inclusion bodies: rational design and recipes, Appl. Microbiol. Biotechnol., 2011, vol. 92, no. 2, pp. 241–251. doi 10.1007/s00253-011-3513-y
Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P., and Cardona, A., Fiji: an open-source platform for biological-image analysis, Nat. Methods, 2012, vol. 9, no. 7, pp. 676–682. doi 10.1038/nmeth.2019
Schaefer, E.M., Moehring, J.M., and Moehring, T.J., Binding of diphtheria toxin to CHO-K1 and Vero cells is dependent on cell density, J. Cell. Physiol., 1988, vol. 135, no. 3, pp. 407–415. doi.org/10.1002/ jcp.1041350307
Sanford, K.K., Earle, W.R., and Likely, G.D., The growth in vitro of single isolated tissue cells, J. Natl. Cancer Inst., 1948, vol. 9, no. 3, pp. 229–246.
Moehring, T.J. and Moehring, J.M., Interaction of diphtheria toxin and its active subunit, fragment A, with toxin-sensitive and toxin-resistant cells, Infect. Immun., 1976, vol. 13, no. 5, pp. 1426–1432.
Gibson, A.E., Noel, R.J., Herlihy, J.T., and Ward, W.F., Phenylarsine oxide inhibition of endocytosis: effects on asialofetuin internalization, Am. J. Physiol., 1989, vol. 257, no. 2, pt. 1, pp. C182–C184. doi 10.1152/ajpcell.1989.257.2.C182
ACKNOWLEDGMENTS
This work was supported by grants of National Academy of Sciences of Ukraine, nos. 0114U003216 and 0112U002624.
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Manoilov, K.Y., Labyntsev, A.J., Korotkevych, N.V. et al. Particular Features of Diphtheria Toxin Internalization by Resistant and Sensitive Mammalian Cells. Cytol. Genet. 52, 353–359 (2018). https://doi.org/10.3103/S0095452718050080
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DOI: https://doi.org/10.3103/S0095452718050080