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.

Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

Botulinum neurotoxins (BoNT) are therapeutic proteins that are specific, potent, and effective. They are highly specific in binding to motor neurons but do not bind to other non-neuronal cells. These proteins are zinc-dependent endopeptidases that inhibit exocytosis by specific cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins involved in vesicle docking and fusion. The therapeutic effect of BoNT/A in humans lasts from 3 to 12 months, depending upon the condition treated. Data from animal and cell culture models suggests that the long-lasting duration of inhibition of neurotransmitter release induced by BoNT/A maybe due to the persistence of the endopeptidase activity of the light chain (LC/A) in cells, interactions of the cleaved substrates, and/or the response of the nerve to the temporary disruption of communication with its target tissue. We have analyzed the subcellular localization of the light chains from serotypes A, B, and E and have demonstrated that each light chain displays a distinct distribution within cells. LC/A localizes at the plasma membrane, LC/B is dispersed throughout the cell including the nucleus, and LC/E is mainly cytosolic. Localization is similar in non-neuronal cell lines, suggesting that the signals involved in proper subcellular localization are within the LC sequences and the moiety the light chain interacts with is present in both neuronal and non-neuronal cells.

Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild-type LCA was concentrated in plasma-membrane-associated clusters, colocalizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity.

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