The 25 kDa HCN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity
Abstract
:1. Introduction
2. Results
2.1. Generation of HCN Deletion Mutants and Hybrids of BoNT/A, B and TeNT
2.2. Determination of Residual Biological Activity Using the Mouse Phrenic Nerve Hemidiaphragm (MPN) Assay
2.3. Pull-Down Assays Employing GST-rSV2C 454-579, GST-rSyt-II 1-61 and ghSV2CLD-Fc
2.4. Ganglioside Binding of CNT Deletion Mutants and Hybrids in Liposome Binding Assay
2.5. Secondary Structure Analyses of the Deletion Mutant AA-A
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Plasmid Construction
5.2. Expression and Purification of Recombinant Proteins
5.3. Potency of CNT Deletion Mutants and Hybrids at MPN Hemidiaphragm Assay
5.4. GST-Pull-Down Assay
5.5. Liposome Binding Assay
5.6. Circular Dichroism Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
AA | amino acid |
BoNT | botulinum neurotoxin |
CD | circular dichroism |
CNTs | clostridial neurotoxins |
Rhod-DHPE | sulforhodamine 1,2-dihexadecanoyl-SN-glycero-3-phosphoethanolLamine, triethylammonium salt |
Egg-PC | egg phosphatidylcholine |
GBS | ganglioside binding site |
GST | glutathion-S-transferase |
h | human |
HC | heavy chain |
HC | carboxyl-terminal half of HC; HCNA, HCNB, HCNT, HCN domain of BoNT serotype A, B, and TeNT, respectively; HCN and HCC: 25 kDa halves of HC |
HN | amino-terminal half of HC |
HSA | human serum albumin |
LC | light chain |
LD | luminal domain |
MPN assay | mice phrenic nerve hemidiaphragm assay |
MW | molecular weight |
PS | polystyro |
S | C-terminal Streptag |
SNAP-25 | synaptosomal associated protein of 25 kDa |
SNARE | soluble N-ethylmaleimide-sensitive factor attachment protein receptor |
SV | synaptic vesicle |
rSV2A-C | rat synaptic vesicle glycoprotein 2 isoform A, B, or C |
Syt | synaptotagmin |
TMD | transmembrane domain |
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Deppe, J.; Weisemann, J.; Mahrhold, S.; Rummel, A. The 25 kDa HCN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity. Toxins 2020, 12, 743. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12120743
Deppe J, Weisemann J, Mahrhold S, Rummel A. The 25 kDa HCN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity. Toxins. 2020; 12(12):743. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12120743
Chicago/Turabian StyleDeppe, Julian, Jasmin Weisemann, Stefan Mahrhold, and Andreas Rummel. 2020. "The 25 kDa HCN Domain of Clostridial Neurotoxins Is Indispensable for Their Neurotoxicity" Toxins 12, no. 12: 743. https://0-doi-org.brum.beds.ac.uk/10.3390/toxins12120743