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

Abstract

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.

Transport Motility of Phagosomes on Actin and Microtubules Regulates Timing and Kinetics of Their Maturation

“…14). The observation that the biotin moiety promotes uptake of BSA by cells is consistent with previous reports 38, 39 .…”

Restoring functional neurofibromin by protein transduction

“…Fahrer et al . 38 for example were able to transfer endosomally trapped proteins to the cytoplasm using a fusion complex consisting of core streptavidin and clostridial C2 toxin. Nonetheless, using this reagent shows a limitation to the proteins’ ability to being unfolded and refolded again correctly in the target cells 39 .…”

Pasteurella multocida Toxin as a Transporter of Non-Cell-Permeating Proteins

“…In a large array of toxins, the receptor binding domain induces binding and endocytosis into vesicular compartments, and the translocation domain allows the transport of the A domain from inside the vesicle into the cytosol. Studies from recent years have shown that it is possible to utilize the receptor binding/translocation domain of exotoxins to transport a cargo protein into eukaryotic cells (41)(42)(43)(44)(45). Here we analyzed the transport of a non-cell-permeating toxin fragment (DTa) by using PMT as a transporter.…”