ISSN 1068 1620, Russian Journal of Bioorganic Chemistry, 2012, Vol. 38, No. 6, pp. 565–577. © Pleiades Publishing, Ltd., 2012. Original Russian Text © S.I. Romaniuk, D.V. Kolybo, S.V. Komisarenko, 2012, published in Bioorganicheskaya Khimiya, 2012, Vol. 38, No. 6, pp. 639–652.
Recombinant Diphtheria Toxin Derivatives: Perspectives of Application S. I. Romaniuk1, D. V. Kolybo, and S. V. Komisarenko Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv Received October 12, 2011; in final form, November 25, 2011
Abstract—Diphtheria toxin (DT) is a unique bacterial protein which consists of three domains with various biological functions. Using genetic engineering for the creation of various recombinant constructions of DT with definite features, it is possible to create unique tools for cellular biology and toxins with efficient and selective action on certain populations of cells. The review highlights the structural and functional aspects of the DT molecule, its fragments and domains, as well as the major areas of application of its recombinant derivatives. In particular, the perspectives for practical use of recombinant DT derivatives are discussed for creating immunobiological preparations, cytotoxins, blockers of the heparin binding epidermal growth fac tor like growth factor (HB EGF), protein constructions for direct delivery of substances into the cell, and also the possibility to use DT recombinant derivatives for therapy and prevention of a number of diseases.
Keywords: diphtheria toxin, fragments and domains of diphtheria toxin, recombinant proteins, heparin binding epidermal growth factor like growth factor (HB EGF), vaccines, cytotoxins DOI: 10.1134/S106816201206012X
1 INTRODUCTION medicine, since they did not satisfy the thermostability requirements and the process to obtain them was not Diphtheria toxin (DT) is a globular protein that is standardized completely. classified to the family of the AB type bacterial exo toxins [1]. It is the major factor of pathogenicity of the The development of genetic engendering tech Сorynebacterium diphtheriae, diphtheria causative niques made it possible to obtain recombinant diph agent [2]; nevertheless, the toxin is encoded in the theria toxoids with mutations in various enzymes that genome of a corynephage, which infects this bacte are currently more often used in both fundamental rium [3]. DT is the most toxic toxin among protein research and in medical practice (Fig. 1). The fast pace toxins in relation to sensitive cells in vitro (one mole of development in this area, and progress in the field of cule of this toxin is sufficient to kill a cell [4]), molecular biological techniques, make it urgent to although DT yields to the botulism and tetanus neuro estimate the prospects for the use of DT recombinant toxins in its influence on the organism [5]. The high derivatives in various areas of human activity. toxicity of DT opened perspectives on the use of its derivatives in medical practice and attracted the atten 1. Structure of the DT Molecule. Application tion of researchers to the study of the structure and of Recombinant Derivatives of the Toxin mechanism of action of this toxin. for Studying the Mechanism of Its Action When studying the antigenic features of DT, The DT is encoded by the nucleotide sequence of mutant forms of the toxin were found that exhibit no the tox gene (1683 bp) which is absolutely identical in cytotoxicity and were called CRM (from English— corynephages β [7], γ [8], and ω [9], which evidences cross reacting material) due to their ability of cross the high conservatism of this gene and the importance reactivity in a precipitation reaction with immune sera of all of the elements of the toxin molecule to fulfill its to DT [6]. Nevertheless, the chemically conjugated cytotoxic function. CRM based chimeric proteins were not widely used in The DT polypeptide has a molecular weight of Abbreviations: DT is diphtheria toxin, CRM is cross reacting 58358 Da and consists of 560 amino acid residues, 25 material (mutant diphtheria toxoid is similar in its antigenic fea of which comprise the leader peptide [10]. Two disul tures to the diphtheria toxin), HB EGF is heparin binding epi fide bonds are formed between cysteines at the posi dermal growth factor like growth factor, IL is interleukin, and scFv is a single chain fragment variable. tions 186–201 and 461–471 [11]. Figure 2 shows a 1 Corresponding author: phone: +38(044)234 33 54; fax: scheme of the DT molecular structure, which consists +38(044)279 63 65; [email protected]. of two fragments: the fragment A (from English–
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Studying the functions of cell receptors Studying the mechanisms Studying the role of DT action of cells in biological processes
Fundamental
Areas for use for the recombinant DT analogues
Vaccines Direct delivery (against of drugs, Applied diphtheria and vaccines, other etc into cells infections)
HB EGF blockers Diagnostic (antitumor and test systems and antidiphtheria) immune therapeutic Cytotoxins for cancer, preparations virus, autoimmune, and other disease treatment
Fig. 1. The areas of recombinant DT derivatives application.
Active) (21164 Da) which presents the catalytic C of the eukaryotic translation elongation factor eEF2 domain, and the fragment B (from English–Binding) that results in protein synthesis arrest in the cell [14] (37194 Da) which consists of a transport T domain and cell death via apoptosis [15]. and a receptor