Abstract

Many disease states are caused by miss-regulated neurotransmission. A small fraction of these
diseases can currently be treated with botulinum neurotoxin type A (BoNT/A). BoNT/A is
composed of three functional domains – the light chain (Lc) is a zinc metalloprotease that
cleaves intracellular SNAP25 which inhibits exocytosis, the translocation domain (Td) that
enables the export of the light chain from the endosome to the cytosol, and the receptor binding
domain (Rbd) that binds to extracellular gangliosides and synaptic vesicle glycoproteins while
awaiting internalisation [1]. Current endeavours are directed towards retargeting Bont/A as well
as finding safer methods of preparation and administration. Recently, our laboratory has
developed a SNARE based linking strategy to recombine non-toxic BoNT/A fragments into a
functional protein by simple mixing [2]. This SNARE based linking strategy permits the stepwise
assembly of highly stable macromolecular complexes [2,3]. Onto these three SNARE
peptides, diverse functional groups can be attached to the N- or C- terminus by direct synthesis
and/or by genetic design. To enhance the therapeutic potential of BoNT/A, this method enables
the rapid assembly of a large array of neuropeptide-SNAREs to their cognate LcTd-SNARE. A
substitution of the Rbd with various neuropeptide sequences permits a large throughput
combinatorial assay of LcTd to target new cell types. In this study, we have fused LcTd to 3
different Synaptobrevin sequences; we also use a small protein staple, and 26 different
Syntaxin-neuropeptide fusions (permitting the assay of 78 new chimeric LcTd proteins with
modified targeting domains). These neuropeptides such as, but not exclusively, somatostatin
(SS), vasoactive intestinal peptide,
substance P, opioid peptide analogues,
Gonadotropin releasing hormone,
and Arginine Vasopressin,
which natively function through G
protein coupled receptors (GPCR)
can undergo agonist induced
internalisation upon activation.
The ability of our new constructs,
once endocytosed, to inhibit
neurotransmitter release was tested
on different neuronal cell lines
with immunoblotting of endogenous
SNAP25. This cleavage by
Lc reflects the ultimate readout of
the enzyme’s efficacy, which
incorporates the cell surface
binding, internalisation kinetics, translocation of the Lc to the cytosol, and finally the enzymatic
cleavage of SNAP25. Internalisation of the toxins can also be monitored with confocal
microscopy and FACS by the substitution of the staple peptide for a fluorescent homologue.
Figure 1 shows that whole boNT/A (upper left) can have its Rbd replaced with SNARE
peptides, which will fuse together to form highly stable chimeric proteins with an altered
targeting domain (right). Figure 1 also shows 4 different neuropeptide synthaxins in complex,
resolved on SDS-PAGE gel (bottom left lanes 1-4, boiled 1’-4’).
Fig. 1. SNARE-linked botulinum neurotoxins used for the
retargeting of Bont/A.
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