Tag Archives: diphtheria toxin

Enhancement of internalization of diphtheria toxin recombinant fragments in sensitive cells mediated by toxin’s T-domain

K. Yu. Manoilov, A. J. Labyntsev, N. V. Korotkevych, D. V. Kolybo

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: manoilovmail@gmail.com

Subunit B of diphtheria toxin (DT) and its R-domain differ by the presence of T-domain. The aim of the present work was to analyze the interaction of these toxin fragments with mammalian cells in order to evaluate the T-domain’s influence on endocytosis in resistant cells. Internalization of recombinant fluorescent subunit B and R-domain was characterized in toxin-resistant L929 cells derived from mouse connective tissue and toxin-sensitive Vero cells from African green monkey kidney. It was found that during incubation of cells in the presence of both subunit B and R-domain in the culture medium, Vero cells internalize more molecules of subunit B than of R-domain. Under the same conditions, L929 cells internalize more molecules of R-domain than of subunit B. Colocalization of fluorescent subunit B and R-domain in L929 was rapid and proceeded almost completely at the early period of incubation compared to Vero cells in which it was slow and occurred gradually. The obtained data suggest that T-domain affects internalization and endosomal transport of DT in cells indirectly correlated with their toxin sensitivity. It was concluded that T-domain participates in intracellular endosomal transport and sorting of DT only in toxin-sensitive cells by enhancing the internalization of toxin molecules.

Effect of diphtheria toxin T-domain on endosomal pH

A. J. Labyntsev, N. V. Korotkevych, D. V. Kolybo, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: lab.andrey@gmail.com

A key step in the mode of cytotoxic action of diphtheria toxin (DT) is the transfer of its catalytic domain (Cd) from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td), but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT. The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT, which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demons­trated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol.

Cell model for the study of receptor and regulatory functions of human proHB-EGF

N. V. Korotkevych, A. Ju. Labyntsev, K. Yu. Manoilov, O. I. Krynina,
L. V. Dyachenko, D. V. Kolibo, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: gnr.nata@gmail.com

Developing of new models and approaches, particularly with fluorescent techniques, for investigation of intracellular transport of proHB-EGF and its ligand-receptor complexes is strongly required.  In order to create a model for studying proHB-EGF functions the genetic construction pEGFP-N1-proHB-EGF, encoding proHB-EGF-EGFP which is fluorescent-labeled form of proHB-EGF with enhanced green fluorescent protein EGFP in the cytoplasmic terminus of the molecule, was obtained. Eukaryotic cells expressing fusion protein proHB-EGF-EGFP on the cell surface were obtained by transfection with pEGFP-N1-proHB-EGF. Expressed in the Vero cells proHB-EGF-EGFP could bind fluorescent derivative of nontoxic receptor-binding subunit B of diphtheria toxin mCherry-SubB. After stimulation of transfected cells with TPA (12-O-Tetradecanoylphorbol-13-acetate), proHB-EGF-EGFP formed a fluorescentl-labeled C-terminal fragment of the molecule – CTF-EGFP. Thus, the obtained genetic construction pEGFP-N1-proHB-EGF could be helpful in visualization of molecules proHB-EGF and CTF in cells, may open new possibilities for the studying of their functions, such as receptor function of proHB-EGF for diphtheria toxin, intracellular translocation of CTF and provide possibilities for natural proHB-EGF ligands search.

Effect of the T-domain on intracellular transport of diphtheria toxin

А. J. Labyntsev, D. V. Kolybo, E. S. Yurchenko,
A. A. Kaberniuk, N. V. Korotkevych, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: lab.andrey@gmail.com

Subunit B of diphtheria toxin (DT), which consists of two domains: R (receptor-binding) and T (transmembrane), plays an important role in toxin-receptor binding on the cell-targets and in transportation of catalytic subunit A to the cell cytosol. Recombinant analogues of the subunit B are promising representatives in the unique class of transporting proteins, able to deliver different types of biologically active molecules to cell cytosol. In the development of these protein constructs understanding of the role of each of the DT fragments in determination of transporting pathways of endocytosed complex toxin-receptor is urgently required.
We have studied in this work the T-domain effect on intracellular transport of recombinant fragments of DT. We have compared intracellular transport of the R-domain and the subunit B, the last one consisted of both R-domain and T-domain. Recombinant fragments of DT used in this work were labeled with fluorescent proteins, which allowed applying colocalization technique for our study. Application of confocal microscopy technique revealed differences in transportation of recombinant derivates of DT in Vero cells: R domain moved faster than subunit B to tubular compartments. Analysis of R-domain and subunit B transportation confirmed almost linear increase of their colocalization with the time regarding to Pearsons correlation coefficient (PCC). However, amount of colocalized with R-domain subunit B were not linearly increased with time according to Manders coefficient (M1), this could indicate the ability of subunit B to transport to such compartments that R-domain do not reach. Possible role of the T-domain in intracellular transportation and compartmentalization of the toxin may be associated with the ability of the T-domain to form a proton channels and its ability to interact with COPI complex.