Generation of a Potent Chimeric Toxin by Replacement of Domain III of Pseudomonas Exotoxin with Ricin A Chain-KDEL

Abstract

Following cellular uptake, Pseudomonas exotoxin (PE) is cleaved by cellular protease which generates an enzymatically active C-terminal fragment (amino acids 280-613). This 37 kD fragment translocates to the cell cytosol where it ADP-ribosylates elongation factor 2 and inhibits protein synthesis. A recombinant hybrid toxin (designated PE-RTA) in which the ADP-ribosylation domain (domain 111) was replaced by the RNA N-glycosidase domain of ricin (the A chain or RTA) has been produced in E. coli. The hybrid toxin effectively and specifically depurinated 28S ribosomal RNA, indicating that the ricin A moiety folded into its native conformation. The cytotoxicity of PE-RTA for L929 cells was approximately 100-fold less than either native PE or whole ricin. However, the addition of the tetrapeptide KDEL to the C-terminus of PE-RTA (producing PE-RTA KDEL) increased cytotoxicity to the level of the native toxins. By analogy to PE, both PE-RTA and PE-RTA KDEL would be proteolytically cleaved within PE domain II during cell entry. A single amino acid substitution, believed to disrupt an essential step in the transport of the catalytically active PE fragment to the cell cytosol (Trp281 to Ala: Zdanovsky, A.G., Chiron, M., Pastan, I., and FitzGerald, D. J. (1993) J. Biol. Chem. 268, 21791-21799), reduced the cytotoxicities of both PE and PE-RTA KDEL by approximately 100-fold. Taken together, these data show that the ricin A chain component of the hybrid toxin requires essential PE-derived sequences at both the N- and C-termini of the translocating fragment. Clearly, in the context of this fusion protein, ricin A chain cannot effect its own transfer to the cytosol.

Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

“…The latter chimera has been used essentially to investigate the intracellular pathway(s) of these toxins, demonstrating that cytotoxicity of PE-RTA was approximately two orders of magnitude less than either native PE or the ricin holotoxin, but that the addition of the KDEL tetrapeptide to its C -terminus (producing PE-RTA-Lys-Asp-Glu-Leu) increased cytotoxicity to the same level of the native toxins [127]. …”

Engineering of bacterial toxins for research and medicine

“…Hence, C2IN has been fused to the virulence factor SpvB from Salmonella enterica to trigger its internalization into mammalian cells [91], to the biotin-binding protein streptavidin, generating a delivery system for biotin-labeled molecules [92], and to the tumor suppressor protein p53 [93]. Also, the B chains of DT or Pseudomonas exotoxin A have been linked with the enzymatic component from ricin and the resulting chimeric toxins efficiently translocate the ricin enzymatic moiety [94,95]. DT can translocate proteins if they are highly unstable or unfolded.…”

The Pseudomonas aeruginosa exotoxin A translocation domain facilitates the routing of CPP–protein cargos to the cytosol of eukaryotic cells

“…1A). ETA 253-412 has been frequently used in the design of immunotoxins in the past with domain Ib acting as a spacer between domains II and III [21][22][23]. Importantly, the assignment of the ETA translocation function to only domain II (residues 253-364) remains speculative as this minimal structural domain has never been analyzed to provide this function in isolation from both domains I and III.…”

“…Specifically, domain I has been replaced with different ligands including transforming growth factor type alpha (TGF-α), interleukin 4, as well as antibody domains [14,[16][17][18][19][20] in an effort to re-direct these chimeric toxins to specific cell types. In other instances, domain III has been swapped with other cytotoxic moieties, such as barnase and the ricin A chain [21][22][23]. Interestingly, the translocating capacity of ETA has never been tested to assess the cytosolic delivery of CPPs or molecular cargos such as polypeptides, drugs or nucleic acids.…”

“…Since a limiting step in achieving the full potential of CPP-delivered agents is to facilitate their endosomal escape, we hypothesized that fusing a cationic CPP to the translocation domain of ETA used in designing immunotoxins (residues 253 to 412) [21][22][23] would favor the efficient internalization of a large molecular cargo and allow it to reach the cytosol. Accordingly, a series of CPP-containing fusion proteins were generated and comparatively tested for their ability to efficiently deliver a large reporter protein, namely the enhanced green fluorescent protein (eGFP), to the cytosol of treated cells.…”