Effects of membrane properties on the binding activities of the H N and H C heavy-chain domains of botulinum neurotoxin A

• Published in: Biochimica et biophysica acta 2016-12, Vol.1864 (12), p.1678-1685
• Main Authors: Ayyar, B Vijayalakshmi, Atassi, M Zouhair
• Format: Article
• Language: English
• Subjects: Animals; Botulinum Toxins, Type A - chemistry; Botulinum Toxins, Type A - genetics; Botulinum Toxins, Type A - metabolism; Caveolae - metabolism; Cell Line; Cell Membrane - metabolism; Clathrin - metabolism; Endocytosis; Membrane Microdomains - metabolism; Mice; Microtubules - metabolism; Models, Molecular; Neurons - metabolism; Neurotoxins - chemistry; Neurotoxins - genetics; Neurotoxins - metabolism; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Binding; Protein Domains; Synaptic Vesicles - metabolism
• ISSN: 0006-3002; 1570-9639
• DOI: 10.1016/j.bbapap.2016.09.001

Binding behaviors of the H and the H domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A H and H regions were prepared (H 519-845 and H 967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of H 519-845 to Neuro 2a cells without affecting H 967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both H 967-1296 and H 519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both H 967-1296 and H 519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K ) depletion buffer lowered the binding of both H 967-1296 and H 519-845 to the cells, but seemed to exert a more pronounced effect on the binding of H 967-1296 than on the binding of H 519-845. Results indicate that while both the H and H domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells.