Resolution of Two Steps in Botulinum Neurotoxin Serotype A1 Light Chain Localization to the Intracellular Plasma Membrane

Abstract

Botulinum neurotoxin serotype A (BoNT/A) is the most potent protein toxin to humans. BoNT/A light chain (LC/A) cleavage of the membrane-bound SNAP-25 has been well-characterized, but how LC/A traffics to the plasma membrane to target SNAP-25 is unknown. Of the eight BoNT/A subtypes (A1–A8), LC/A3 has a unique short duration of action and low potency that correlate to the intracellular steady state of LC/A, where LC/A1 is associated with the plasma membrane and LC/A3 is present in the cytosol. Steady-state and live imaging of LC/A3-A1 chimeras identified a two-step process where the LC/A N terminus bound intracellular vesicles, which facilitated an internal α-helical-rich domain to mediate LC/A plasma membrane association. The propensity of LC/A variants for membrane association correlated with enhanced BoNT/A potency. Understanding the basis for light chain intracellular localization provides insight to mechanisms underlying BoNT/A potency, which can be extended to applications as a human therapy.

How Botulinum Neurotoxin Light Chain A1 Maintains Stable Association with the Intracellular Neuronal Plasma Membrane

“…LC/A3V proved to be a platform to resolve the role of the N terminus as a facilitator of LC/A1-intracellular vesicle interactions and the LHD as a facilitator of LC/A-plasma membrane interactions. Overall, the N-terminal-and LHD-mediated interactions were independent, sequential, and additive for the movement of LC/A1 from the cytosol to the plasma membrane [35].…”

“…BoNT/A LC (LC/A) then cleaves the substrate SyNaptosomal Associated Protein of 25 kDa (SNAP-25) [32] on the intracellular face of the plasma membrane. While previous studies have demonstrated LC/A1 localizes to the plasma membrane [33][34][35], the molecular mechanisms of intracellular LC trafficking to the membrane-bound SNAP- 25 have not yet been elucidated. Deciphering molecular interactions involved in intracellular BoNT LC trafficking is fundamental to our understanding of the neuronal cell intoxication pathways and pathology caused by these toxins.…”

“…Since BoNT/A potency correlates with LC trafficking along vesicles and association with the plasma membrane [35,41], we have further characterized LC/A1 localization with the plasma membrane in this study. Initial experiments localized the membrane targeting capacity to the Low Homology Domain of LC/A1 (amino acids 268-357), which was divided into three structural regions (R1-R3) based upon a co-crystal of LC/A1 bound to SNAP-25 (PDB: 1XTG) [42].…”

Botulinum Neurotoxin A4 Has a 1000-Fold Reduced Potency Due to Three Single Amino Acid Alterations in the Protein Receptor Binding Domain

“…Past research has revealed that subtypes of one serotype can have distinct characteristics. BoNT/A subtypes differ in neuronal cell entry, enzymatic activity of the LC, intracellular trafficking, and potency [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. The gene encoding BoNT/A4 is localized on a plasmid that also carries the gene encoding BoNT/B in the bivalent toxin producing Clostridium botulinum strain 657, showing the potential diverse genetic organization of these potent pathogens [ 30 ].…”

Advances in Clostridial and Related Neurotoxins

“…This study by Fabris et al demonstrates that this BoNT/B and tetanus toxin cleavage-specific anti-VAMP antibody uniquely detects the cleavage fragment but not intact VAMP [22], and with that provides a novel tool enabling much-needed future studies based on the detection of substrate cleavage in cultured cells and in vivo. The study by Gardner et al utilized the natural divergence of functional and structural characteristics of BoNT/A1 and BoNT/A3 to shed new light on intracellular neuronal localization and trafficking of the light chains of these two toxins [23]. This study provides intriguing data towards our goal of elucidating the molecular mechanisms determining intracellular light chain action and structure-function-specific differences between the various BoNT seroand subtypes.…”