The conjugation of diphtheria toxin T domain to poly(ethylenimine) based vectors for enhanced endosomal escape during gene transfection

Kakimoto, Shinji; Hamada, Tsutomu; Komatsu, Yuuki; Takagi, Masahiro; Tanabe, Toshizumi; Azuma, Hideki; Shinkai, Seiji; Nagasaki, Takeshi

doi:10.1016/j.biomaterials.2008.09.042

Cited by 50 publications

(45 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A transferrin-DT conjugate has very recently been shown to promote tumor regression in vivo (Yoon et al 2010). In a novel approach, the transmembrane domain of DT was genetically fused to streptavidin and coupled with biotinylated poly(etyleneimine) to facilitate endosomal escape during non-viral gene delivery (Kakimoto et al 2009). …”

Section: Discussionmentioning

confidence: 99%

Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin

Fahrer

Funk

Lillich

et al. 2010

Naunyn-Schmied Arch Pharmacol

View full text Add to dashboard Cite

The C2 toxin produced by Clostridium botulinum is a binary AB-type exotoxin composed of the enzyme subunit C2I and the binding/translocation moiety C2II. After proteolytic activation, C2IIa mediates the subsequent internalization of C2I into the cytosol of mammalian target cells. The N-terminal domain of C2I (C2IN) is necessary for C2IIa-dependent uptake, but lacks the enzyme domain that is responsible for cytotoxicity. In the present study, we generated a delivery system building on C2IN and a truncated core streptavidin (Stv13) with enhanced solubility for the C2IIa-dependent internalization of biotinylated cargo molecules into mammalian cells. C2IN-Stv13 fusion protein expressed in Escherichia coli was obtained in high yields and purity. The affinity-purified protein formed tetramers and a defined higher order species in solution as shown by gel filtration and retained its biotin-binding properties, however with an obvious reduction in affinity. Uptake of C2IN-Stv13 into the cytosol of HeLa and other cancer cell lines was observed by immunoblot analysis, which was corroborated by confocal microscopy. In addition, the fusion protein was not cytotoxic and did not inhibit cell proliferation as determined by MTS assay. Finally, we demonstrated the C2IN-Stv13/C2IIa-mediated uptake of biocytin-Alexa 488 as cargo into HeLa cells, underscoring the functionality of the generated transport system.

show abstract

Section: Discussionmentioning

confidence: 99%

Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin

Fahrer

Funk

Lillich

et al. 2010

Naunyn-Schmied Arch Pharmacol

View full text Add to dashboard Cite

show abstract

“…It has also been suggested that it assists other partially unfolded proteins across the lipid bilayer [50], indicating a general, rather than specific translocation pathway. Recently, this membrane-translocating ability of the T-domain has been utilized to improve cellular delivery of poly(ethylenimine)-based vectors during gene transfection [51]. Diphtheria toxin has been utilized as a prospective anti-cancer agent for the targeted delivery of cytotoxic therapy to cancer cells [52,53,54,55,56,57,58,59,60,61,62,63,64,65].…”

Section: Perspectives and Applicationsmentioning

confidence: 99%

pH-Triggered Conformational Switching along the Membrane Insertion Pathway of the Diphtheria Toxin T-Domain

Ladokhin

2013

Toxins

View full text Add to dashboard Cite

The translocation (T)-domain plays a key role in the action of diphtheria toxin and is responsible for transferring the catalytic domain across the endosomal membrane into the cytosol in response to acidification. Deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the T-domain, which is considered to be a paradigm for cell entry of other bacterial toxins, reveals general physicochemical principles underlying membrane protein assembly and signaling on membrane interfaces. Structure-function studies along the T-domain insertion pathway have been affected by the presence of multiple conformations at the same time, which hinders the application of high-resolution structural techniques. Here, we review recent progress in structural, functional and thermodynamic studies of the T-domain archived using a combination of site-selective fluorescence labeling with an array of spectroscopic techniques and computer simulations. We also discuss the principles of conformational switching along the insertion pathway revealed by studies of a series of T-domain mutants with substitutions of histidine residues.

show abstract

“…Otherwise, rather than using a quencher molecule, self-quenching of certain fluorophores can be achieved when used at a sufficiently high concentration, which is relieved upon leakage resulting in increased fluorescence. For instance, calcein [58,59], carboxyfluorescein (CF) [45] and sulforhodamine B (SulfoB) [61] have been used in ex cellulo leakage assays to investigate membrane integrity of artificial endosomes by spectrofluorimetry. In cellular assays, these tracer molecules are loaded in the endosomes by constitutive endocytosis.…”

Section: Assays For Studying Pore Formationmentioning

confidence: 99%

“…Removing the influence of pH on endosomal escape is done by either altering the compound under investigation so it is no longer pH-reactive [44], or the pH of the endosomal compartment itself is altered. In a controlled ex cellulo environment, the pH can be modeled by using buffers with different pH [45,46]. In cellular assays, acidification can be blocked by the use of inhibitors.…”

Section: Verifying Ph-induced Membrane Destabilizationmentioning

confidence: 99%

Intracellular delivery of nanomaterials: How to catch endosomal escape in the act

et al. 2014

View full text Add to dashboard Cite

Successful cytosolic delivery of nanomaterials is becoming more and more important, given the increase in intracellular applications of quantum dots, gold nanoparticles, liposomal drug formulations and polymeric gene delivery vectors. Most nanomaterials are taken up by the cell via endocytosis, yet endosomal escape has long been recognized as a major bottleneck in cytosolic delivery. Although it is essential to detect and reliably quantify endosomal escape, no consensus has been reached so far on the methods to do so. This review will summarize and discuss for the first time the different assays used to investigate this elusive step to date.

show abstract

The conjugation of diphtheria toxin T domain to poly(ethylenimine) based vectors for enhanced endosomal escape during gene transfection

Cited by 50 publications

References 38 publications

Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin

Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin

pH-Triggered Conformational Switching along the Membrane Insertion Pathway of the Diphtheria Toxin T-Domain

Intracellular delivery of nanomaterials: How to catch endosomal escape in the act

Contact Info

Product

Resources

About