Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A-G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.
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.
Fluorescence spectroscopy is a powerful biophysical technique for studying protein structure, function, dynamics, and intermolecular interactions. Such studies are often conducted using intrinsic probes, such as tryptophan residues, or extrinsic probes introduced by post-translational modification, such as dansyl. Specificity, however, is often a concern since many proteins contain more than one tryptophan and chemical modification often will occur at more than one site. Herein we report the in vitro, site-specific incorporation of three fluorescent amino acid analogues, 5-hydroxytryptophan, 7-azatryptophan, and ε-dansyllysine, each of which was incorporated into β-galactosidase at a single designated site.
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