Tyrosine Phosphorylation of Botulinum Neurotoxin Protease Domains

Abstract

Botulinum neurotoxins are most potent of all toxins. Their N-terminal light chain domain (Lc) translocates into peripheral cholinergic neurons to exert its endoproteolytic action leading to muscle paralysis. Therapeutic development against these toxins is a major challenge due to their in vitro and in vivo structural differences. Although three-dimensional structures and reaction mechanisms are very similar, the seven serotypes designated A through G vastly vary in their intracellular catalytic stability. To investigate if protein phosphorylation could account for this difference, we employed Src-catalyzed tyrosine phosphorylation of the Lc of six serotypes namely LcA, LcB, LcC1, LcD, LcE, and LcG. Very little phosphorylation was observed with LcD and LcE but LcA, LcB, and LcG were maximally phosphorylated by Src. Phosphorylation of LcA, LcB, and LcG did not affect their secondary and tertiary structures and thermostability significantly. Phosphorylation of Y250 and Y251 made LcA resistant to autocatalysis and drastically reduced its kcat/Km for catalysis. A tyrosine residue present near the essential cysteine at the C-terminal tail of LcA, LcB, and LcG was readily phosphorylated in vitro. Inclusion of a competitive inhibitor protected Y426 of LcA from phosphorylation, shedding light on the role of the C-terminus in the enzyme’s substrate or product binding.

Botulinum Neurotoxins A and E Undergo Retrograde Axonal Transport in Primary Motor Neurons

“…This analysis is however complicated by the methodology used for the detection of the activity of these neurotoxins, which is based on antibodies recognizing the cleaved SNARE proteins. Indeed, this approach is highly dependent on variations in the enzymatic activity of the L chains of different serotypes, their efficiency of translocation into the cytoplasm, their post-translational modifications and the relative intracellular stability of both the L chain and the cleaved substrates, parameters for which our present understanding is very limited [6], [41], [61], [62].…”

Cellular Protection of SNAP-25 against Botulinum Neurotoxin/A: Inhibition of Thioredoxin Reductase through a Suicide Substrate Mechanism

“…(14) To probe whether or not 6 prevents BoNT/A’s phosphorylation, we screened the inhibitor against a panel of 50 kinases including Src at 20 μM (see the Supporting Information). Unfortunately, 6 presented no activity against all kinases examined.…”

“…(11) Furthermore, it has been suggested that the biologically relevant form of the LC enzyme is distinct from recombinant LCs used in current assays, due to cellular modifications by host proteins. (14)…”

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).…”