Effects of membrane properties on the binding activities of the HNand HCheavy-chain domains of botulinum neurotoxin A.
Binding behaviors of the HNand the HCdomains 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 HNand HCregions were prepared (HN519-845 and HC967-1296) 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 HN519-845 to Neuro 2a cells without affecting HC967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both HC967-1296 and HN519-845 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 HC967-1296 and HN519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K+) depletion buffer lowered the binding of both HC967-1296 and HN519-845 to the cells, but seemed to exert a more pronounced effect on the binding of HC967-1296 than on the binding of HN519-845. Results indicate that while both the HNand HCdomains 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.
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
B Vijayalakshmi
BV
Ayyar
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address: matassi@bcm.edu.
M Zouhair
MZ
Atassi
D000818:Animals; D019274:Botulinum Toxins, Type A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021941:Caveolae / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D008958:Models, Molecular; D009474:Neurons / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D013572:Synaptic Vesicles / Q000378:metabolism
D002966:Clathrin; D009498:Neurotoxins; D010446:Peptide Fragments; D019274:Botulinum Toxins, Type A
Botulinum neurotoxin A; Endocytosis; H(C) domain; H(N) domain; Membrane properties; Neuronal cell binding