Clostridial neurotoxins and substrate proteolysis in intact neurons: botulinum neurotoxin C acts on synaptosomal-associated protein of 25 kDa.
Clostridial neurotoxins are zinc endopeptidases that block neurotransmission and have been shown to cleave, in vitro, specific proteins involved in synaptic vesicle docking and/or fusion. We have used immunohistochemistry and immunoblotting to demonstrate alterations in toxin substrates in intact neurons under conditions of toxin-induced blockade of neurotransmitter release. Vesicle-associated membrane protein, which colocalizes with synaptophysin, is not detectable in tetanus toxin-blocked cultures. Syntaxin, also concentrated in synaptic sites, is cleaved by botulinum neurotoxin C. Similarly, the carboxyl terminus of the synaptosomal-associated protein of 25 kDa (SNAP-25) is not detectable in botulinum neurotoxin A-treated cultures. Unexpectedly, tetanus toxin exposure causes an increase in SNAP-25 immunofluorescence, reflecting increased accessibility of antibodies to antigenic sites rather than increased expression of the protein. Furthermore, botulinum neurotoxin C causes a marked loss of the carboxyl terminus of SNAP-25 when the toxin is added to living cultures, whereas it has no action on SNAP-25 in vitro preparations. This study is the first to demonstrate in functioning neurons that the physiologic response to these toxins is correlated with the proteolysis of their respective substrates. Furthermore, the data demonstrate that botulinum neurotoxin C, in addition to cleaving syntaxin, exerts a secondary effect on SNAP-25.
Williamson
L C
LC
Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
Halpern
J L
JL
Montecucco
C
C
Brown
J E
JE
Neale
E A
EA
D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism* / Q000633:toxicity*; D002478:Cells, Cultured; D010450:Endopeptidases / Q000378:metabolism / Q000633:toxicity; D005333:Fetus; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D007150:Immunohistochemistry; D008565:Membrane Proteins; D051379:Mice; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009498:Neurotoxins / Q000378:metabolism* / Q000633:toxicity*; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism; D016708:Synaptophysin / Q000032:analysis / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D013744:Tetanus Toxin / Q000378:metabolism* / Q000633:toxicity*
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; C494209:Snap25 protein, mouse; D016708:Synaptophysin; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D018698:Glutamic Acid; D010450:Endopeptidases; D001905:Botulinum Toxins; D005998:Glycine