Ukr.Biochem.J. 2015; Volume 87, Issue 4, Jul-Aug, pp. 13-23

doi: https://doi.org/10.15407/ubj87.04.013

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.

Keywords: , , , , ,


References:

  1. Kolibo DV, Labyntsev AJ, Romaniuk SI, Kaberniuk AA, Oliinyk ES, Korotkevich N V, Komisarenko SV. Immunobiology of diphtheria. Recent approaches for the prevention, diagnosis, and treatment of the disease. Biotechnol. Acta. 2013;6(4):43–62. CrossRef
  2. Huotari J, Helenius A. Endosome maturation. EMBO J. 2011 Aug 31;30(17):3481-500. Review. PubMed, PubMedCentral, CrossRef
  3. Schmidt O, Teis D. The ESCRT machinery. Curr Biol. 2012 Feb 21;22(4):R116-20. PubMed, PubMedCentral, CrossRef
  4. Fujita H, Yamanaka M, Imamura K, Tanaka Y, Nara A, Yoshimori T, Yokota S, Himeno M. A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments: class E vps phenotype in mammalian cells. J Cell Sci. 2003 Jan 15;116(Pt 2):401-14. PubMed, CrossRef
  5. Guha S, Padh H. Cathepsins: fundamental effectors of endolysosomal proteolysis. Indian J Biochem Biophys. 2008 Apr;45(2):75-90. Review. PubMed
  6. Labyntsev AJ, Kolybo DV, Yurchenko ES, Kaberniuk AA, Korotkevych NV, Komisarenko SV. Effect of the T-domain on intracellular transport of diphtheria toxin. Ukr Biochem J. 2014 May-Jun;86(3):77-87. PubMed, CrossRef
  7. Morimoto YV, Kojima S, Namba K, Minamino T. M153R mutation in a pH-sensitive green fluorescent protein stabilizes its fusion proteins. PLoS One. 2011 May 3;6(5):e19598. PubMed, PubMedCentral, CrossRef
  8. Miesenböck G, De Angelis DA, Rothman JE. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature. 1998 Jul 9;394(6689):192-5. PubMed, CrossRef
  9. Labyntsev AJ, Korotkevych NV, Manoilov KJ, Kaberniuk AA, Kolybo DV, Komisarenko SV. Recombinant fluorescent models for studying the diphtheria toxin. Russ J Bioorganic Chem. 2014 Jul;40(4):401-9. PubMed, CrossRef
  10. Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368-79. PubMed, CrossRef
  11. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012 Jun 28;9(7):676-82. PubMed, PubMedCentralCrossRef
  12. Bolte S, Cordelières FP. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc. 2006 Dec;224(Pt 3):213-32. PubMed, CrossRef
  13. Chudina T, Labyntsev A, Manoilov K, Kolybo D, Komisarenko S. Cellobiose-coated poly(lactide-co-glycolide) particles loaded with diphtheria toxoid for per os immunization. Croat Med J. 2015 Apr;56(2):85-93. PubMed, PubMedCentral, CrossRef
  14. Van Rossum G., de Boer J. Interactively Testing Remote Servers Using the Python Programming Language. CWI Quarterly. 1991;4(4):283-303.
  15. Bradski G. OpenCV. Dr Dobbs J. Softw. Tools 2000.
  16. Hunter JD. Matplotlib: A 2D graphics environment. Comput Sci Eng. 2007;9(3):90-5. CrossRef
  17. van der Walt S, Colbert SC, Varoquaux G. The NumPy Array: A Structure for Efficient Numerical Computation. Comput Sci Eng. 2011 Mar;13(2):22-30. CrossRef
  18. Katoh K, Shibata H, Suzuki H, Nara A, Ishidoh K, Kominami E, Yoshimori T, Maki M. The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting. J Biol Chem. 2003 Oct 3;278(40):39104-13. Epub 2003 Jul 14. PubMed, CrossRef
  19. Yorikawa C, Shibata H, Waguri S, Hatta K, Horii M, Katoh K, Kobayashi T, Uchiyama Y, Maki M. Human CHMP6, a myristoylated ESCRT-III protein, interacts directly with an ESCRT-II component EAP20 and regulates endosomal cargo sorting. Biochem J. 2005 Apr 1;387(Pt 1):17-26. PubMed, PubMedCentral, CrossRef
  20. Chenal A, Prongidi-Fix L, Perier A, Aisenbrey C, Vernier G, Lambotte S, Fragneto G, Bechinger B, Gillet D, Forge V, Ferrand M. Deciphering membrane insertion of the diphtheria toxin T domain by specular neutron reflectometry and solid-state NMR spectroscopy. J Mol Biol. 2009 Sep;391(5):872–883. PubMedCrossRef

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