Is the light chain subcellular localization an important factor in botulinum toxin duration of action?

Abstract

Botulinum neurotoxins (BoNT) are therapeutic proteins that are specific, potent, and effective. They are highly specific in binding to motor neurons but do not bind to other non-neuronal cells. These proteins are zinc-dependent endopeptidases that inhibit exocytosis by specific cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins involved in vesicle docking and fusion. The therapeutic effect of BoNT/A in humans lasts from 3 to 12 months, depending upon the condition treated. Data from animal and cell culture models suggests that the long-lasting duration of inhibition of neurotransmitter release induced by BoNT/A maybe due to the persistence of the endopeptidase activity of the light chain (LC/A) in cells, interactions of the cleaved substrates, and/or the response of the nerve to the temporary disruption of communication with its target tissue. We have analyzed the subcellular localization of the light chains from serotypes A, B, and E and have demonstrated that each light chain displays a distinct distribution within cells. LC/A localizes at the plasma membrane, LC/B is dispersed throughout the cell including the nucleus, and LC/E is mainly cytosolic. Localization is similar in non-neuronal cell lines, suggesting that the signals involved in proper subcellular localization are within the LC sequences and the moiety the light chain interacts with is present in both neuronal and non-neuronal cells.

Association of Botulinum Neurotoxin Serotype A Light Chain with Plasma Membrane-bound SNAP-25

“…Although earlier studies reported the intracellular localization of the LC of BoNT/A to the plasma membrane (21,22), the basis for plasma membrane association was not determined. Identification of a SNAP-25 interaction with the N terminus of LC/A was not expected because BoNT/A or LC/A was previously to bind and utilize SNAP-25(146 -206) as an efficient substrate for cleavage (13,15,28).…”

“…Membrane-inserted HCT facilitates LC translocation into the cytosol, where LC cleaves plasma membraneassociated SNAP-25 (18 -20). Although earlier studies reported the intracellular localization of ectopic expressed BoNT/A LC to the host plasma membrane (21,22), there is limited understanding of the intracellular events that lead to plasma membrane localization. In this study, a new interaction between LC/A and SNAP-25 is identified that facilitates high affinity binding of LC/A to SNAP-25 on the plasma membrane of neurons.…”

Persistence of Botulinum Neurotoxin Inactivation of Nerve Function

“…Both of these proteases appeared to be dispersed throughout the cell, not localized to the plasma membrane [41,46]. These results suggest that localization to the plasma membrane is not required for protease persistence in these other long-lasting BoNT serotypes and their persistence depends on other mechanisms.…”

“…BoNT protease subcellular localization to persistence-Because of the difficulties in detecting cytosolic BoNT protease following natural intoxication, several labs have employed DNA transfection methods to promote expression of the proteases within cells to study persistence [26,[39][40][41]. Fernandez-Salas et al over-expressed either recombinant BoNT/A or BoNT/E proteases within rat PC12 neuroblastoma cells as fusions to green fluorescent protein (GFP) and monitored protease localization by fluorescence microscopy.…”

Deubiquitinating enzyme VCIP135 dictates the duration of botulinum neurotoxin type A intoxication

“…This has necessitated the transfection of plasmids encoding BoNT LCs that have been codon-optimized for mammalian expression to study BoNT persistence (12,(40)(41)(42). It is natural to ask whether discoveries based on transfection of BoNT LCs can be extended to intoxication with the holotoxin.…”