Botulinum Neurotoxin Devoid of Receptor Binding Domain Translocates Active Protease

D. Borden Lacy,Mauricio Montal,Darren J. Mushrush,Audrey Fischer

doi:10.1371/journal.ppat.1000245

Abstract

Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The ∼50 kDa light chain (LC) protease is translocated into the cytosol by the ∼100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication.

Highlights

Botulinum neurotoxin (BoNT) inhibits synaptic exocytosis in peripheral cholinergic synapses causing botulism, a severe disease characterized by descending flaccid paralysis
We generated several truncation constructs of BoNT/A holotoxin, purified them and examined their channel activity; these include the heavy chain (HC), HN 2 the N-terminal half of the HC called the translocation domain (TD) [26], and the light chain (LC) and TD linked by a disulfide bridge (LC-TD) [33] (Figure 1)
The LC-TD was expressed as a single polypeptide chain of,100 kDa with a disulfide bridge; the functionally relevant di-chain protein was generated by trypsin cleavage of the linker between the disulfide bridge cysteine residues

Summary

Introduction

Botulinum neurotoxin (BoNT) inhibits synaptic exocytosis in peripheral cholinergic synapses causing botulism, a severe disease characterized by descending flaccid paralysis. The mature toxin consists of three modules [1,4,5,6]: a 50 kDa light chain (LC) Zn2+-metalloprotease, and the 100 kDa heavy chain (HC) which encompasses the N-terminal ,50 kDa translocation domain (TD), denoted HN and the C-terminal ,50 kDa receptor-binding domain (RBD) termed HC. This tri-modular architecture has a physiological counterpart. Exposure of the BoNTreceptor complex to the acidic milieu of endosomes [12,13,14,15,16] induces a conformational change leading to the insertion of the HC into the endosomal bilayer membrane, thereby forming transmembrane channels [17,18,19,20]

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Results

Discussion

Conclusion