Retargeting the Clostridium botulinum C2 toxin to the neuronal cytosol.

Benjamin J Pavlik,Elizabeth J Hruska,Paul H Blum,Kevin E Van Cott

doi:10.1038/srep23707

Benjamin J Pavlik, Elizabeth J Hruska + Show 2 more

Open Access

https://doi.org/10.1038/srep23707

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Journal: Scientific Reports	Publication Date: Mar 30, 2016
Citations: 7	License type: CC BY 4.0

Affiliation: University of Nebraska–Lincoln

Abstract

Many biological toxins are known to attack specific cell types, delivering their enzymatic payloads to the cytosol. This process can be manipulated by molecular engineering of chimeric toxins. Using toxins with naturally unlinked components as a starting point is advantageous because it allows for the development of payloads separately from the binding/translocation components. Here the Clostridium botulinum C2 binding/translocation domain was retargeted to neural cell populations by deleting its non-specific binding domain and replacing it with a C. botulinum neurotoxin binding domain. This fusion protein was used to deliver fluorescently labeled payloads to Neuro-2a cells. Intracellular delivery was quantified by flow cytometry and found to be dependent on artificial enrichment of cells with the polysialoganglioside receptor GT1b. Visualization by confocal microscopy showed a dissociation of payloads from the early endosome indicating translocation of the chimeric toxin. The natural Clostridium botulinum C2 toxin was then delivered to human glioblastoma A172 and synchronized HeLa cells. In the presence of the fusion protein, native cytosolic enzymatic activity of the enzyme was observed and found to be GT1b-dependent. This retargeted toxin may enable delivery of therapeutics to peripheral neurons and be of use in addressing experimental questions about neural physiology.

Highlights

Modification of toxin binding specificity is accomplished by the incorporation of a heterologous protein domain with concurrent ablation of native binding affinities by mutagenesis[7] or complete replacement of the binding domain[23]
C2 toxin with C-terminal deletion of domain 4 (C2IIΔD4)[24] and the C1 neurotoxin binding domain C1 HCC were produced for use as controls
Linked toxins consist of a single chain protein having both a toxin domain and a binding/translocation domain

Summary

Introduction

Modification of toxin binding specificity is accomplished by the incorporation of a heterologous protein domain with concurrent ablation of native binding affinities by mutagenesis[7] or complete replacement of the binding domain[23]. Blocker et al showed that truncating the C-terminal binding domain of C2II by seven amino acids or removing of the entire binding domain maintains the stability of C2IIa and allows for oligomerization, but prevents receptor binding[24]. This led here, to the proposal that the C2 binding domain could be engineered to confer a new target cell binding specificity by replacement of the C2 C-terminus with another toxin–derived C-terminal binding domain (Fig. 1b). Future engineering of payloads may enable development of new methods to study and visualize intracellular biology

Methods

Results

Conclusion