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Title: A study of the interaction between botulinum neurotoxin and cellular membranes
Author: Harris, Iain John
Awarding Body: University of Bath
Current Institution: University of Bath
Date of Award: 2013
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Eight genetically distinct serotypes (A-H) of Botulinum neurotoxin (BoNT) cause flaccid muscular paralysis through inhibition of neurotransmission at cholinergic nerve terminals. BoNT binds with specificity to the neuromuscular junction and enters through endocytosis. It translocates its catalytic moiety through the endosomal membrane into the cell cytosol and finally proteolyses soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) inside the neuromuscular junction, thereby preventing synaptic vesicle fusion with the plasma membrane. The action of BoNT is facilitated by its tri-domain structure which includes: the receptor binding domain (HC), the translocation domain (HN) and the catalytic domain (LC). Removal of BoNT’s HC domain leaves a stable, catalytically active molecule (LHN), which lacks specificity for the neuromuscular junction. The least understood step in the mechanism of BoNT action is the translocation of the LC domain through the endosomal membrane into the cell cytosol; this action is thought to be preceded by the formation of an HN domain pore following acidification of the endosomal compartment. In this study we set out to investigate this step. We investigated the interaction of BoNT and LHN proteins with cellular and artificial membranes. Firstly, the interaction of LHN serotype D (LHD) with Chinese Hamster Ovary cells (CHO-K1 cells) was investigated by measuring an apparent LHD induced increase in CHO-K1 cell intracellular Ca2+ levels. The increase was affected by extracellular Ca2+ concentration suggesting that LHD may permeabilise the membrane of CHO-K1 cells at near neutral pH. Secondly, we developed novel expression and purification methods for BoNT HN domains (serotypes A, C and D). This allowed us to compare the lipophilicity of the HN domains from multiple serotypes, further supporting a role for this domain as a pore forming moiety. Thirdly, vesicle leakage assays were explored as a method for determining protein or peptide pore formation in an attempt to further define the pore forming potential of BoNT and LHN proteins of various serotypes. However, in these experiments, control proteins with no known pore forming ability also caused vesicle leakage at acidic pH; the ability of a vesicle leakage assay to sufficiently demonstrate protein or peptide pore formation has not therefore been confirmed in this study. Investigation into LHN and BoNT induced pore formation in lipid vesicles via electron microscopy was similarly inconclusive. Together the results presented in this thesis show that the HN region of BoNT serotypes A, C and D can be purified as isolated lipophilic domains, furthermore, the interaction of BoNT constructs containing the HN domain with membranes is consistent with a function in transmembrane protein translocation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available