Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting

Abstract

The long half-life of botulinum neurotoxin serotype A (BoNT/A) in cells poses a challenge in developing post-exposure therapeutics complementary to existing antitoxin strategies. Delivery vehicles consisting of the toxin heavy chain (HC), including the receptor-binding domain and translocation domain, connected to an inhibitory cargo offer a possible solution for rescuing intoxicated neurons in victims paralyzed from botulism. Here, we report the expression and purification of soluble recombinant prototype green fluorescent protein (GFP) cargo proteins fused to the entire BoNT/A-HC (residues 544-1295) in Escherichia coli with up to a 40 amino acid linker inserted between the cargo and BoNT/A-HC vehicle. We show that these GFP-HC fusion proteins are functionally active and readily taken up by cultured neuronal cells as well as by neuronal cells in mouse motor nerve endings.

Fig. 1.

Purification of GFP-BoNT/A-BD(875–1295). Shown are Coomassie blue-stained 10% SDS–PAGE gels of protein fractions during various steps in the purification of GFP-BoNT/A-BD(875–1295). Left panel: Ni²⁺-chelation column flow through (F), wash (W) and SP column elution fractions 15–24. Right panel: pooled SP column fractions (16–20) after desalting, lane 1–1.25 µg, lane 2–2.5 µg.

Fig. 2.

Purification of GFP-DE4-BoNT/A-HC(544–1295). Shown are Coomassie blue-stained 10% SDS–PAGE gels of protein fractions during various steps in the purification of GFP-DE4-BoNT/A-HC(544–1295). (A) Cell-free extract (C), Ni²⁺-chelation column flow through (HF), wash (HW), 100 mM imidazole elution (H) and ANX column elution peak fraction (AN). (B) Cell-free extract (C), Ni²⁺-chelation column flow through (HF), 100 mM imidazole elution (H), SP column flow through (SF) and SP column elution peak fraction (SP).

Fig. 3.

Purification of GFP-BoNT/A-HC(544–1295) and DE fusion proteins. Shown are Coomassie blue-stained 10% SDS–PAGE gels of protein fractions during various steps in the purification. (A) GFP-BoNT/A-HC(544–1295), cell-free extract (C), Ni²⁺-chelation column flow through (HF), wash (HW), 100 mM imidazole elution (H), pooled elution fractions from first SP column (SP1), pooled elution fractions from ANX column (AN) and pooled and desalted elution fractions from the second SP column (SP2). (B) Similarly purified fusion proteins after desalting, lane 1, GFP-BoNT/A-BD(875–1295); lane 2, GFP-BoNT/A-HC(544–1295); lane 3, GFP-DE4-BoNT/A-HC(544–1295); lane 4, GFP-DE8-BoNT/A-HC(544–1295); lane 5, GFP-DE12-BoNT/A-HC(544–1295); lane 6, GFP-DE16-BoNT/A-HC(544–1295).

Fig. 4.

Binding and entering of GFP-BoNT/A-BD(875–1295) and GFP-BoNT/A-HC(544–1295) into cultured neuronal cells. Shown are fluorescence micrographs of NG108-15 cells treated for 1 h with either 0.25 µM GFP-BoNT/A-HC(544–1295) (top panels) or 0.62 µM GFP-BoNT/A-BD(875–1295) (bottom panels).

Fig. 5.

Forty millimolar KCl-stimulated uptake of GFP-BoNT/A-HC(544–1295), GFP-BoNT/A-BD(875–1295) and BoNT/A into mouse motor nerve endings. Left panels: fluorescence micrographs illustrating representative targeting of Triangularis sterni endplate acetylcholine receptors with α-BnTx labeled with Alexa-647 (A and B) or Alexa-488 (C). Middle panels: fluorescence micrographs illustrating representative targeting of corresponding motor nerve endings with GFP-BoNT/A-HC(544–1295) (A), GFP-BoNT/A-BD(875–1295) (B) or BoNT/A labeled with Alexa-647 (C). Right panels: overlap of fluorescence micrographs from left and middle panels.

Fig. 6.

In vivo uptake of GFP-BoNT/A-HC(544–1295) and GFP-BoNT/A-BD(875–1295) into mouse motor nerve endings. Shown are fluorescence micrographs illustrating representative targeting of mouse motor nerve endings 3 days after injection into the peroneal nerve's entry into the EDL muscle with GFP-BoNT/A-BD(875–1295) or GFP-BoNT/A-HC(544–1295). (A, E) Targeting of Triangularis sterni endplate acetylcholine receptors with Alexa-647-labeled α-BnTx. (B, F) Targeting of corresponding motor nerve endings with GFP-BoNT/A-BD(875–1295) (B) or GFP-BoNT/A-HC(544–1295) (F). (C) Overlay of A and B. (G) Overlay of E and F. (D, H) Corresponding EDL nerve-muscle preparations stained for skeletal muscle using anti-troponin I antibodies.