Plasma membrane localization signals in the light chain of botulinum neurotoxin

Abstract

Botulinum neurotoxin (BoNT) is a potent biological substance used to treat neuromuscular and pain disorders. Both BoNT type A and BoNT type E display high-affinity uptake into motor neurons and inhibit exocytosis through cleavage of the synaptosome-associated protein of 25 kDa (SNAP25). The therapeutic effects of BoNT͞A last from 3 to 12 months, whereas the effects of BoNT͞E last less than 4 weeks. Using confocal microscopy and site-specific mutagenesis, we have determined that the protease domain of BoNT͞A light chain (BoNT͞ A-LC) localizes in a punctate manner to the plasma membrane, colocalizing with the cleaved product, SNAP25 197. In contrast, the short-duration BoNT͞E serotype is cytoplasmic. Mutations in the BoNT͞A-LC have revealed sequences at the N terminus necessary for plasma membrane localization, and an active dileucine motif in the C terminus that is likely involved in trafficking and interaction with adaptor proteins. These data support sequence-specific signals as determinants of intracellular localization and as a basis for the different durations of action in these two BoNT serotypes.otulinum neurotoxins (BoNTs) are the most potent of all biological substances (1). Although BoNTs are well publicized as a potential biological weapon and as the causative agents in clinical botulism, the potency and myorelaxant actions of BoNTs have been exploited clinically in more than 100 indications, including muscle hyperactivity in cerebral palsy and cervical dystonia, migraines, myofacial pain, and focal hyperhidrosis (2-5). These toxins are specific endoproteases that, collectively, target several distinct proteins in nerve terminals. Motor nerve terminals at neuromuscular junctions are particularly sensitive to these neurotoxins, resulting in a transient and reversible muscle relaxation through inhibition of acetylcholine release. The Clostridium neurotoxin family includes seven serotypes of BoNT (A-G), and a single form of toxin produced by Clostridium tetani (TeNT). These toxins consist of a heavy chain (HC, 100 kDa) and light chain (LC, 50 kDa) linked by a disulfide bond (6, 7). The three-dimensional crystal structures of BoNT͞A (8) and BoNT͞B (9) have been resolved, providing a basis for understanding the structure͞function mechanism of BoNT action. The BoNT-LCs are zinc-dependent endoproteases that specifically cleave one of three soluble Nethylmaleimide-sensitive factor-attachment protein-receptor (SNARE) proteins (10) involved in synaptic vesicle docking and fusion at the nerve terminal (11). The synaptosome-associated protein of 25 kDa (SNAP25) is cleaved at distinct sites near the C terminus by ) and BoNT͞E (R 180 -I 181 ), generating truncated SNAP25 197 (12) and SNAP25 180 (13), respectively.

Drug Insight: biological effects of botulinum toxin A in the lower urinary tract

“…8 The cleavage of SNAP-25 by BTX-A is extremely specific because BTX-A recognizes a highly conserved, nine-residue binding motif that occurs four times within the SNAP-25 molecule. [38][39][40][41] The differences in SNARE-binding profiles between the BTX serotypes may explain the different amount of time it takes for each protein to inhibit acetylcholine exocytosis. 42,43 Alternatively, the intraneuronal localization may affect the duration of inhibition.…”

“…42,43 Alternatively, the intraneuronal localization may affect the duration of inhibition. 39 BTX-A has the longest duration of activity when it is used for the treatment of dystonias, inducing clinical effects on neuromuscular activity for longer than 4 months, compared with about 2 months for BTX-B and less than 4 weeks for BTX-E. 44,45 Cell-culture studies that calculated the inhibitory half-life of BTX-A provided further evidence for a prolonged effect of BTX-A; the inhibitory half-life of BTX-A was more than 31 days, compared with 10 days for BTX-B, 2 days for BTX-F, and 19-20 h for BTX-E. 42 Recovery of cholinergic neurotransmission is dependent on removal of the BTX protease and restoration of intact SNARE proteins. 19,42,46 …”

A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate

“…Second, the chimeric SNARE substrate must retain function to support yeast cell growth, minimizing appearance of unwanted SNARE mutations that dramatically alter substrate conformation. Third, B-LC͞SNARE interactions can be probed in the presence of cellular membranes, which are essential for SNARE protein function and thought to be important for efficient LC activity (2,11,12). As such, our approach utilizes a convenient eukaryotic cell model to examine a protease whose activity underlies the flaccid paralysis produced by BoNT intoxication of human motor neurons.…”

“…For example, the assay could be used in a genetic search for mutations within B-LC endoprotease. Such mutations are likely to provide insights into substrate binding and LC͞membrane interactions (12,23). Growth selection against LC activity also offers the potential to select B-LC endoprotease inhibitors or rapidly screen existing inhibitors (24,25) in a eukaryotic cell model where membrane permeability and cytotoxicity are relevant assay parameters.…”

“…In addition to these apparent conflicts, both approaches are limited by their use of soluble SNARE substrates, which lack membrane-binding domains that are essential for SNARE protein function inside cells (1,2). Biological membranes are also thought to interact directly with LC endoproteases (11,12), thus revealing a need for cell-based assays to probe BoNT͞LC substrate specificity.…”