Fluorescent derivatives of diphtheria toxin's subunit B and their interaction with Vero cells

A. A. Kabemiuk, A. J. Labyntsev, D. V. Kolibo, O. S. Oliinyk, T. A. Redchuk, N. V. Korotkevich, V. F. Gorchev, S. O. Karahim, S. V. Komisarenko

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Abstract

Diphtheria toxin's B subunit provides toxin interaction with its receptor on the cell surface and translocation of toxin's A subunit from endosome to cytozole of sensitive cells. Functional analogues of B subunit with fluorescent label are considered as perspective tools for studying the above mentioned processes. The aim of the work was to obtain fluorescent B subunit analogues and to detect the specificity of their interaction with Vero line cells. B subunit fluorescent analogues were obtained in two different ways. The first one was B subunit chemical conjugation with fluorescein isothiocyanate and the second one was genetic fusion of recombinant B subunit chain with enhanced green fluorescent protein chain. Specific interaction of B subunit fluorescent derivatives with Vero cells was studied by flow cytometry and confocal microscopy. Using competitive analysis it was shown that B subunit fluorescent analogues possessed different affinity for cells. The affinity of EGFP-SbB was higher than FITC-SbB. Our results indicate the possibility to use the fluorescent derivatives of B subunit as tools for identification of diphtheria toxin's receptor (HB-EGF) expression on the cell surface as well as for studying the interaction and penetration of diphtheria toxin to the cell.

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Fluorescent derivatives of diphtheria toxin's subunit B and their interaction with Vero cells. / Kabemiuk, A. A.; Labyntsev, A. J.; Kolibo, D. V.; Oliinyk, O. S.; Redchuk, T. A.; Korotkevich, N. V.; Gorchev, V. F.; Karahim, S. O.; Komisarenko, S. V.

In: Ukrain'skyi Biokhimichnyi Zhurnal, Vol. 81, No. 1, 27.04.2009, p. 67-77.

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