Abstract
The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3 Å, and that of SXN101959 at 3.1 Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D. Taken together, these results demonstrate the robustness of the ‘LHn fold’ across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.
Highlights
Recent outbreaks of botulism in cattle[5]
The arrangement of the functional domains within SXN101959 involves a novel orientation in which the growth hormone releasing hormone (GHRH) ligand is located centrally between the LC and HC in the single chain polypeptide expressed in E. coli and with a protease cleavage site located between the GHRH ligand and the LC, such that following protease activation to generate the active di-chain targeted secretion inhibitor (TSI) the GHRH ligand is at the amino-terminus of the HC domain
Botulinum neurotoxins (BoNTs) are some of the most potent protein toxins and their modular architecture have made them molecules of choice for the design of a novel class of biopharmaceuticals labelled Targeted Secretion Inhibitors
Summary
Recent outbreaks of botulism in cattle[5]. This has raised some interest in this serotype and understanding its precise mechanism of action. Coffield et al.[6] investigated the impact of serotype D on human tissues and demonstrated its inability to block neuromuscular transmission when tested at a level 10 times higher than that of serotype A This limited activity was recently confirmed in vivo by electrophysiological study on human muscles[7] and may be linked to differences in the receptor binding domain[8,9]. A targeted secretion inhibitor (TSI) combining a growth hormone releasing hormone (GHRH) receptor targeting domain with the LHn/D fragment, called qGHRH-LHn/D, was shown to inhibit pituitary somatotroph growth hormone release[13] This molecule demonstrated efficient intracellular activity on VAMP-3 in rat pituicytes and encouraging potential in the treatment of Acromegaly[14]. This study should provide the structural basis for the development of BoNT/D-based Targeted Secretory Inhibitor (TSI) which could prove useful in the treatment of hypersecretory disorders
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