Abstract
Botulinum neurotoxin A (BoNT/A) is the deadliest of all known biological substances. Although its toxicity makes BoNT/A a biological warfare threat, its biologic activity makes it an increasingly useful therapeutic agent for the treatment of muscular disorders. However, almost 200 years after its discovery, the neuronal cell components required for the activity of this deadly toxin have not been unequivocally identified. In this work, neuroblastoma cells expressing synaptotagmin I, a protein shown to be bound by BoNT/A, were used to determine whether specific gangliosides were necessary for BoNT/A activity as measured by synaptosomal-associated protein of 25 kDa (SNAP-25) cleavage. Ganglioside GT1b was found to support BoNT/A activity significantly more effectively than GD1a, which was far more effective than GM1 when added to ganglioside-deficient murine cholinergic Neuro 2a or to human adrenergic SK-N-SH neuroblastoma cells. Whereas both cell lines expressed synaptotagmin I, SNAP-25 cleavage was not observed in the absence of complex gangliosides. These results indicate that 1) gangliosides are required for BoNT/A activity, 2) synaptotagmin I in the absence of gangliosides does not support BoNT/A activity, and 3) Neuro 2a cells are an efficient model system for studying the biological activity of BoNT/A.
Highlights
Botulinum neurotoxin A (BoNT/A),1 one of seven serotypes of BoNT, is the deadliest of all known biological substances, being approximately one million times more poisonous than cobra toxin [1]
Neuroblastoma cells expressing synaptotagmin I, a protein shown to be bound by BoNT/A, were used to determine whether specific gangliosides were necessary for BoNT/A activity as measured by synaptosomal-associated protein of 25 kDa (SNAP-25) cleavage
These results indicate that 1) gangliosides are required for BoNT/A activity, 2) synaptotagmin I in the absence of gangliosides does not support BoNT/A activity, and 3) Neuro 2a cells are an efficient model system for studying the biological activity of BoNT/A
Summary
BoNT/A, botulinum neurotoxin serotype A; GM1, Gal(1–3)GalNAc(1– 4)[NeuAc(␣2–3)]Gal(1– 4)Glc1cer; GM2, GalNAc(1– 4)[NeuAc(␣2–3)]Gal(1– 4)Glc1-cer; GM3, NeuAc(␣2–3)Gal(1– 4)Glc1-cer; GD3, NeuAc(␣2– 8)NeuAc(␣2– 3)Gal(1– 4)Glc1-cer; GD1a, NeuAc(␣2–3)Gal(1–3)GalNAc(1– 4)[NeuAc(␣2–3)]Gal(1– 4)Glc1-cer; GT1b, NeuAc(␣2–3)Gal(1– 3)GalNAc(1– 4)[NeuAc(␣2– 8)NeuAc(␣2–3)]Gal(1– 4)Glc1-cer; PPMP, D,L-threo-l-phenyl-2-hexadecanoylamino-3-morpholino-propanol-HCl; HPTLC, high performance thin layer chromatography; PVDF, polyvinylidenefluoride; PBS, phosphate buffered saline; N2a, Neuro 2a; HRP, horseradish peroxidase; TBST, tris buffered saline with Tween 20 (0.1%), pH 7.4; SNAP-25, synaptosomal-associated protein of 25 kDa; DMEM, Dulbecco’s modified Eagle’s medium; RIPA, radioimmune precipitation assay buffer. Whereas the enzymatic activity of the neurotoxin has been well defined, questions still exist regarding the molecular interactions needed for its cellular binding, internalization, and transport to its protein substrate Identification of these interactions will provide guidance for developing potential inhibitors of its action. The question of whether specific gangliosides were needed for BoNT/A to act on SNAP-25 in synaptotagmin I expressing murine cholinergic Neuro 2a and human adrenergic SK-N-SH neuroblastoma cells was addressed. Both cell types were found to be susceptible to BoNT/A only when they contained specific gangliosides. In their absence, BoNT/A activity was not observed, even though synaptotagmin I was present
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