Modulation of botulinum neurotoxin A catalytic domain stability by tyrosine phosphorylation

Abstract

Botulinum neurotoxin A (BoNT A) is a substrate of the Src family of tyrosine kinases. Here, we report that the BoNT A light chain (LC) is phosphorylated in the tyrosine-71 located at N-terminus. Covalent modification of this residue notably increases the thermal stability of the endopeptidase activity, without affecting its catalytic efficacy. Similarly, mutation of this residue specifically affected the protein stability but not its endopeptidase function. Fusion of the Tat-translocating domain to the N-terminus of the enzyme produced a cell permeable, functional enzyme, as evidenced by immunocytochemistry and by the cleavage of cytosolic SNAP25 in intact PC12 cells. Noteworthy, truncation of cellular SNAP25 was reduced in cells when the Src kinase activity was inhibited with a specific antagonist, implying that tyrosine phosphorylation of BoNT A LC modulates the in vivo proteolytic activity of the neurotoxin. Taken together, these findings substantiate the tenet that tyrosine phosphorylation of BoNT A LC could be an important modulatory strategy of the neurotoxin stability and suggest that the phosphorylated neurotoxin may be a relevant molecule in vivo.

Lipid and cationic polymer based transduction of botulinum holotoxin, or toxin protease alone, extends the target cell range and improves the efficiency of intoxication

“…These receptors are generally present in significant amounts on primary neuronal cells but are often poorly expressed or even lacking in neuronal cell lines. Neuroblastoma cell lines are available that can become intoxicated but often require high doses of toxin to produce evidence of intoxication if it occurs at all (Ibanez et al, 2004; Purkiss et al, 2001). Primary neuronal cells are sensitive to most BoNT serotypes although these are difficult and costly to prepare and to maintain (Keller et al, 2004).…”

Src Family Kinase Inhibitors Antagonize the Toxicity of Multiple Serotypes of Botulinum Neurotoxin in Human Embryonic Stem Cell-Derived Motor Neurons

“…One hypothesis is that the examined SFK inhibitors may be preventing the Src-mediated phosphorylation of BoNT LCs. Previous studies have reported that BoNT LCs are phosphorylated by Src kinase, and such phosphorylation events might be critical for toxins’ activities and stability (Ibanez et al 2004; Blanes-Mira et al 2001; Encinar et al 1998; Ferrer-Montiel et al 1996), but there are contradictions regarding the phosphorylation sites and their functional effects (Toth et al 2012). Additionally, it is not known if the LCs are phosphorylated in the physiological target of BoNTs, i.e., motor neurons.…”

“…However, our mechanistic understanding of the host signaling pathways involved in BoNT intoxication and/or recovery remains minimal. Some studies have suggested that BoNT activity depends on its phosphorylation by Src (Ibanez et al 2004; Blanes-Mira et al 2001; Encinar et al 1998; Ferrer-Montiel et al 1996; Toth et al 2012). Src family kinases (SFKs) play critical roles in many cellular functions in the nervous system, including the development and maintenance of neurons, synaptic plasticity, axon guidance, and neurotransmission (Ohnishi et al 2011).…”

The Zinc-Dependent Protease Activity of the Botulinum Neurotoxins

“…What is not known to any great extent are other possible covalent modifications in which low molecular weight groups are added. The existence of phosphorylation of tyrosines of the type A, B, E and TeNT LCs [100], which may enhance its thermal stability while retaining its catalytic properties has been reported but is of uncertain functional significance within neurons [101]. Within the environment of the nerve terminal, rate constants (Section 3) may be affected by molecular crowding or confinement [102].…”