How Botulinum Neurotoxin Light Chain A1 Maintains Stable Association with the Intracellular Neuronal Plasma Membrane
Abstract
:1. Introduction
2. Results
2.1. Structural Analysis of the Low Homology Domain of LC/A1 Reveals Three Distinct Structural Regions
2.2. Regions R1 and R2 of the A1 LHD Target LC/A3V to the Plasma Membrane
2.3. The A1 MLD and N Terminus Have Additive Functions in the Transition of LC to Vesicles, and the Plasma Membrane
2.4. Role of A1 R1:R2 in MLD Transition of LC/A3V from the Cytosol to the Plasma Membrane
2.5. The LC MLD and Membrane Localization Are Conserved amongst BoNT/A Subtypes Other Than A3
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Light Chain Structural Alignment
5.2. BoNT-LC/A1 and /A3V Sequence Alignment
5.3. Engineering of GFP-LC/A3V (A1 LHD) Chimera Expression Plasmids
5.4. Expression, Capture, and Analysis of Immunofluorescent EGFP-LC/A Plasmids
5.5. Live-Cell Imaging
5.6. Western Blotting Confirming Chimera Molecular Weight
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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LC/A3V(A1-LHD) Chimeras * | Designation | Chimera Schematic |
---|---|---|
EGFP-LC/A3V(A1 275–300) | LC/A3V(R1) | |
EGFP-LC/A3V(A1 302–334) | LC/A3V(R2) | |
EGFP-LC/A3V(A1 335–357) | LC/A3V(R3) | |
EGFP-LC/A3V(A1 275–334) | LC/A3V(R1:R2) [MLD] | |
EGFP-LC/A3V(A1 302–357) | LC/A3V(R2:R3) | |
EGFP-LC/A3V(A1 275–300, 335–357) | LC/A3V(R1:R3) | |
EGFP-LC/A3V (A1 268–357) | LC/A3V(LHD) | |
EGFP-LC/A3V (A1 1–17, 275–334) | LC/A3V(A1 N, A1 R1:R2) [MLD] | |
EGFP-LC/A3V (A1 287–334) | LC/A3V(A1-287-334) | |
EGFP-LC/A3V (A1 275–334) T306A, T307A, Y312A | LC/A3V(A1MLD-AAA) |
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Gardner, A.P.; Barbieri, J.T.; Pellett, S. How Botulinum Neurotoxin Light Chain A1 Maintains Stable Association with the Intracellular Neuronal Plasma Membrane. Toxins 2022, 14, 814. https://doi.org/10.3390/toxins14120814
Gardner AP, Barbieri JT, Pellett S. How Botulinum Neurotoxin Light Chain A1 Maintains Stable Association with the Intracellular Neuronal Plasma Membrane. Toxins. 2022; 14(12):814. https://doi.org/10.3390/toxins14120814
Chicago/Turabian StyleGardner, Alexander P., Joseph T. Barbieri, and Sabine Pellett. 2022. "How Botulinum Neurotoxin Light Chain A1 Maintains Stable Association with the Intracellular Neuronal Plasma Membrane" Toxins 14, no. 12: 814. https://doi.org/10.3390/toxins14120814