Two Protein Trafficking Processes at Motor Nerve Endings Unveiled by Botulinum Neurotoxin E

Abstract

The unique ability of a family of botulinum neurotoxins to block neuroexocytosis specifically-by selective interaction with peripheral cholinergic nerve endings, endocytotic uptake, translocation to the cytosol, and enzymic cleavage of essential proteins-underlies their increasing therapeutic applications. Although clinical use of type A is most widespread due to its prolonged inactivation of the synaptosomal-associated protein of 25 kDa, botulinum neurotoxin E cleaves this same target but at a different bond and exhibits faster onset of neuromuscular paralysis. Herein, insights were gained into the different dynamics of action of types A and E toxins, which could help in designing variants with new pharmacological profiles. Natural and recombinant type E dichain forms showed similar proteolytic and neuromuscular paralytic activities. The neuroparalysis induced by type E toxin was accelerated between 21 and 35°C and attenuated by bafilomycin A1. Temperature elevation also revealed an unanticipated bipartite dose response indicative of two distinct internalization processes, one being independent of temperature and the other dependent. Although elevating the temperature also hastened intoxication by type A, a second uptake mechanism was not evident. Increasing the frequency of nerve stimulation raised the uptake of type E via both processes, but the enhanced trafficking through the temperaturedependent pathway was only seen at 35°C. These novel observations reveal that two membrane retrieval mechanisms are operative at motor nerve terminals which type E toxin exploits to gain entry via an acidification-dependent step, whereas A uses only one.Seven serotypes (termed A-G) of botulinum neurotoxin (BoNT), produced by the bacteria Clostridium botulinum, cause the disorder botulism that is characterized by flaccid neuromuscular paralysis (Dolly et al., 2002). BoNTs are proteins (mol. wt. 150 ϫ 10 3 ) with a heavy chain (ϳ100 ϫ 10 3 ) and light chain (50 ϫ 10 3 ) linked by a disulfide bond and noncovalent interactions. They are synthesized as singlechain (SC) precursors that require proteolytic nicking to yield the potent dichain (DC) forms. Each acts by inhibiting acetylcholine release via a complex multiphasic mechanism (Simpson, 1980(Simpson, , 2004: binding to high-affinity ectoacceptors on cholinergic nerve endings (Dolly et al., 1984), endocytotic uptake (Black and Dolly, 1986a,b), translocation of a toxic moiety, and specific cleavage of proteins essential for the release of transmitters from vesicles (Schiavo et al., 2000). Their heavy chain contributes to binding (Poulain et al., 1989) plus internalization (Dolly et al., 1994). Synaptic vesicle proteins have been identified as acceptors for some serotypes: SV2 for BoNT/A (Dong et al., 2006; Mahrhold et al., 2006) and synaptotagmins I and II for BoNT/B and BoNT/G (Nishiki et al., 1994(Nishiki et al., , 1996 Dong et al., 2003;Rummel et al., 2004a). An intact DC is needed for translocation (de Paiva et al., 1993) of the light chain to the presynaptic c...

Excitatory Cholinergic and Purinergic Signaling in Bladder Are Equally Susceptible to Botulinum Neurotoxin A Consistent with Co-Release of Transmitters from Efferent Fibers

“…It is noteworthy that the relationships between concentration and paralysis time for BoNT/E and BoNT/C were fit well by a power function ( Fig. 1E; Table 1), a property well established for the action of BoNTs at the neuromuscular junction (Simpson, 1980;Lawrence et al, 2007). Although BoNT/A showed more variation, the data could also be fitted with a power function (Table 1).…”

“…Because of limitations in the amounts of BoNTs available, a large static bath was used, as done previously by us (Lawrence et al, 2007) and others (Simpson, 1980) to study the action of these proteins on innervated skeletal muscle. This recording format gives a lower efficiency of EFS for nerve (and muscle) because of reduced shunt resistance; hence, the pulse frequencies and widths required to elicit muscle contractions (see figure legends for details) are larger than those reported in studies using smaller baths (e.g., MacKenzie et al, 1982).…”

Novel Chimeras of Botulinum Neurotoxins A and E Unveil Contributions from the Binding, Translocation, and Protease Domains to Their Functional Characteristics

“…Muscle contractions were elicited by electrical stimulation (50-ms square pulses of 5-10 V at 0.2 Hz) of the nerve and measured using FORT25 isometric force transducers (World Precision Instruments) as described previously (18). BoNTs were added directly to the Krebs-Ringer buffer, and the time taken for a 90% reduction in muscle tension was recorded.…”

A Dileucine in the Protease of Botulinum Toxin A Underlies Its Long-lived Neuroparalysis

“…Accordingly, neuromuscular paralysis induced by BoNT A can be reversed by elevating [Ca 2ϩ ] i but not in the case of BoNT E treatment (13)(14)(15). Although BoNT E blocks neurotransmission more quickly and more potently than BoNT A (16), the clinical applications of BoNT E are restricted by its neuromuscular paralytic action being transient: less than 4 weeks in contrast to more than 4 months for BoNT A (17,18).…”