ISSN 10681620, 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 heparinbinding epidermal growth fac torlike growth factor (HBEGF), 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, heparinbinding epidermal growth factorlike growth factor (HBEGF), 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 ABtype 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 crossreacting 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. CRMbased chimeric proteins were not widely used in The DT polypeptide has a molecular weight of Abbreviations: DT is diphtheria toxin, CRM is crossreacting 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), HBEGF is heparinbinding epi fide bonds are formed between cysteines at the posi dermal growth factorlike growth factor, IL is interleukin, and scFv is a singlechain fragment variable. tions 186–201 and 461–471 [11]. Figure 2 shows a 1 Corresponding author: phone: +38(044)2343354; fax: scheme of the DT molecular structure, which consists +38(044)2796365; [email protected]. of two fragments: the fragment A (from English– 565 566 ROMANIUK et al. 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) HBEGF 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 Tdomain and cell death via apoptosis [15]. and a receptorbinding Rdomain [11]. Each of the Using mutant diphtheria toxoids, the hydrophobic three domains of DT performs a specific biological Cterminal fragment (482–535) of the Rdomain of function for cytotoxic action. The Cterminal DT was established to be responsible for interaction Rdomain (386–535) binds with a receptor on the sur with the receptor of the target cell [12] (Fig. 3). The face of the cell [12], the Tdomain (205–378) provides spacious structure of the Rdomain is formed by two the translocation of the Cdomain into the cytosol βsheets which lie close to one another: the fourfold [13], and the Nterminal Cdomain (1–193) catalyzes βsheet is formed by the RB2, RB3, RB5, and RB8 the reaction of NAD+dependent ADPribosylation folds and the fivefold βsheet by the RB4, RB6, RB7, RB9, and RB10 folds [11]. The formation of hydrogen bonds between the RB6 fold and both βsheets results in the formation of the βbarrel with a jellyrolllike A B topology which is found for many proteins which С R interact with carbohydrate residues. The 461–471 dis ulfide bond holds a loop of nine amino acid residues by СRT which the phosphatebinding Psite (456–458–460– 472–474) with unknown function is located [11]. The S SSSLys516 and Phe530 residues, and also possibly the T Tyr514, Val523, Asn524, and Lys526, play an essential role in receptor recognition and are located in the exposed region of the Rdomain [16]. The DT is activated via splitting the peptide bond Fig. 2. Scheme of DT domain structure. C is the catalytic domain, T is the transmembrane domain, R is the recep between the fragments A and B in the region 186–201 torbinding domain, A is fragment A, B is fragment B, and under the action of proteinases, e.g., furin [17] in the –S–S– is disulfide bonds. presence of disulfide bond reducers. The uptake of RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38 No. 6 2012 RECOMBINANT DIPHTHERIA TOXIN DERIVATIVES 567 Glu148 Tyr54 Active center His21 Tyr65 Thr23 Fragment Hairpin loop Т8/9 Lys516 482–535 Asp352 Glu349 Fig. 3. Structural elements of the DT molecule that are especially essential for catalytic, transport, and receptorbinding functions of the toxin. activated toxin complex with a cellular receptor by the fusion of early endosomes [24], and possibly to endosome with lower pH values (4.5–5.5) provides cause a delay in their maturation, which may be essen conformational rearrangements in the Tdomain [18], tial for the translocation process. which is a specialized pHdependent chaperone that Data on the participation of some cytosol proteins provides Cdomain translocation through the endo in the translocation of Cdomain have recently been some membrane into the cytosol [19]. obtained, e.g., vesicular transport proteins COPI, The Tdomain consists of nine helices organized which interact with Lys residues of the TH1 helix [25], into three layers. The first layer forms two long hydro actin [26], Hsp 90 chaperone, and thioredoxin reduc phobic Cterminal TH8 and TH9 helices; the second tase [27]. Nevertheless, a definite concept of the trans layer, three hydrophobic TH5–TH7 helices; and the location mechanism has not been formulated thus far, third, four TH1–TH4 helices with hydrophilic fea which makes it necessary and promising to further tures [11]. The construction of diphtheria toxoids study this process, including the application of recom which are able to bind with the receptor and exhibit binant DT derivatives. catalytic activity, but which would be unable to trans The Cdomain which carries out the NAD+ locate, e.g., the CRM503 [20], was useful for detecting dependent ADPribosylation of the eEF2 elongation the regions of the toxin molecule essential for translo factor consists of two βsheet subdomains, whose folds cation. Protonation of the Glu349 and Asp352 resi are oriented almost perpendicularly and form the core dues at low pH values (Fig. 3) leads to the TH8/9 hair of the domain. The first subdomain consists of the pin loop insertion into the membrane [21]. At a deep CB2, CB4, and CB8 folds which are surrounded by insertion, the TH5–TH7 helices are also inserted into the CH2, CH3, CH6, and CH7 helices. The second the membrane, and the TH5 helix, along with the TH8 subdomain consists of the CB1, CB3, CB5, CB6, and and TH9 helices, participates in pore formation. The CB7 folds which are surrounded by the CH1, CH4, TH6 and TH7 helices may form a “cork”, which par and CH5 helices. Four helices CL1–CL4 connect two tially blocks the transmembrane pore [22].