Exchanging the minimal cell binding fragments of tetanus neurotoxin in botulinum neurotoxin A and B impacts their toxicity at the neuromuscular junction and central neurons

Abstract

The modular four domain structure of clostridial neurotoxins supports the idea to reassemble individual domains from tetanus and botulinum neurotoxins to generate novel molecules with altered pharmacological properties. To treat disorders of the central nervous system drug transporter molecules based on catalytically inactive clostridial neurotoxins circumventing the passage of the blood-brain-barrier are desired. Such molecules can be produced based on the highly effective botulinum neurotoxin serotype A incorporating the retrograde axonal sorting property of tetanus neurotoxin which is supposed to be encoded within its C-terminal cell binding domain HC. The corresponding exchange of the tetanus neurotoxin HC-fragment in botulinum neurotoxin A yielded the novel hybrid molecule AATT which displayed decreased potency at the neuromuscular junction like tetanus neurotoxin but exerted equal activity in cortical neurons compared to botulinum neurotoxin A wild-type. Minimizing the tetanus neurotoxin cell binding domain to its N- or C-terminal half drastically reduced the potencies of AATA and AAAT in cortical neurons indicating that the structural motif mediating sorting of tetanus neurotoxin is predominantly encoded within the entire HC-fragment. However, the reciprocal exchange resulted in TTAA which showed a similar potency as tetanus neurotoxin at the neuromuscular junction indicating that the tetanus neurotoxin portion prevents a high potency as observed for botulinum neurotoxins. In conclusion, clostridial neurotoxin based inactivated drug transporter for targeting central neurons should contain the cell binding domain of tetanus neurotoxin to exert its tropism for the central nervous system.

Keywords: 100 kDa heavy chain; 25 kDa C-terminal half of H(C); 25 kDa N-terminal half of H(C); 50 kDa C-terminal half of HC of CNTs; 50 kDa N-terminal half of HC; 50 kDa light chain; BBB; BoNT; BoNT/A hydrolyzed SNAP-25 comprising aa 1–197; Botulinum neurotoxin; CD; CNS; CNT; Cell binding domain; Central neuron; GBS; H(C)X; H(CC); H(CN); H(N); HC; Hybrid; LC; MPN; NMJ; Neuromuscular junction; SDS-PAGE; SNAP-25; SNAP-25*A; SNARE; SV; SV2; Syt-X; TeNT; Tetanus neurotoxin; blood–brain-barrier; botulinum neurotoxin; central nervous system; circular dichroism; clostridial neurotoxin; ganglioside binding site; isoform X of synaptotagmin; mouse phrenic nerve; neuromuscular junction; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; soluble NSF attachment protein receptor; synaptic vesicle; synaptic vesicle glycoprotein 2; synaptosomal associated protein of 25 kDa; tetanus neurotoxin.