Calcein permeability of liposomes mediated by type A botulinum neurotoxin and its light and heavy chains.
The mode of botulinum neurotoxin action involves binding of its heavy chain for internalization into the presynaptic end of a nerve cell through endocytosis. The low-pH conditions of endosomes trigger translocation of the light chain across the endosomal membrane to the cytosol, where the light chain cleaves specific target proteins involved in the docking and fusion of synaptic vesicles for acetylcholine release. In an effort to model the interaction of botulinum neurotoxin and its subunit chains with lipid bilayer at low pH during the translocation process, we have examined type A botulinum neurotoxin-mediated calcein release from asolectin liposomes. At equimolar concentration (0.1 microM), the neurotoxin and its heavy and light chains evoked 23%, 58%, and 28% calcein release, respectively. Calcein release was observed only when the cis-side (the side to which neurotoxin samples were added) pH was lowered to 4. Calcein release activity of the heavy chain was mostly blocked (76%) by a polyclonal antibody raised against the neurotoxin. Additionally, two peptide-specific polyclonal antibodies derived from the N-terminal and C-terminal halves of the heavy chain were also able to block the calcein release activity by 15-20%. In summary, these results suggest that calcein release from liposomes is specifically mediated by the heavy chain, and the light chain also integrates into the membrane. Implications of these results for the molecular mode of neurotoxin light-chain translocation across the endosomal membrane are discussed.
Fu
F N
FN
Department of Chemistry and Biochemistry, and Center for Marine Science and Technology, University of Massachusetts, Dartmouth 02747, USA.
Singh
B R
BR
D000595:Amino Acid Sequence; D001905:Botulinum Toxins / Q000737:chemistry / Q000378:metabolism*; D002463:Cell Membrane Permeability; D005452:Fluoresceins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000378:metabolism; D008081:Liposomes; D008969:Molecular Sequence Data
D005452:Fluoresceins; D007473:Ion Channels; D008081:Liposomes; D001905:Botulinum Toxins; C007740:fluorexon