Abstract
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.
Highlights
botulinum neurotoxin (BoNT)/E enters cultured neurons more quickly than BoNT/A [15], has a higher potency, and acts faster to inhibit transmission at the neuromuscular junction (16 –18)
Chimera AE was less potent than EA or BoNT/E and much slower to enter neurons, but produced prolonged muscle weakness in mouse, matching that induced by clinical preparations of type A toxin-hemagglutinin complex
Chimera EA is composed of light chain (LC)-HN/E, two foreign amino acids, heavy chain (HC)/A, and 16 foreign residues including two trypsin cleavage sites before a His6 tag
Summary
Materials—The following materials were obtained as indicated; the pET29a expression vector, Novagen; E. coli BL21(DE3), Stratagene; restriction enzymes, New England Biolabs; TALON Superflow resin, Clontech; and UNO S1 and Q1 columns, Bradford protein assay kit, and Prescision PlusTM all blue protein standards, Bio-Rad. For constructing chimera EA, a fragment encoding the binding domain of BoNT/A (HC/A) was likewise generated, and nucleotides were added to encode a trypsin cleavage site at its C terminus This fragment, as well as a separate DNA sequence encoding the LC plus the translocation domain of BoNT/E (LC-HN/E), was amplified by PCR and cloned into pET29a to create an expression vector containing the EA insert (see Fig. 1). At the end of each experiment, all cell types were solubilized in lithium dodecyl sulfate sample buffer and heated to 80 °C for 5 min before SDS-PAGE and Western blotting; SNAP-25 was visualized with a monoclonal antibody (SMI-81, which recognizes intact and BoNT/A- or BoNT/E-cleaved products) and quantified as described [30, 32]. Statistical Analysis—Data were calculated and graphs generated using GraphPad Prism 4.0. p values were calculated by Student’s t test
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