Engineering botulinum neurotoxin domains for activation by toxin light chain

Abstract

Targeted secretion inhibitors (TSI) are a new class of biopharmaceuticals designed from a botulinum neurotoxin protein scaffold. The backbone consists of the 50-kDa endopeptidase light chain and translocation domain (N-terminal portion of the heavy chain), lacks neuronal toxicity, but retains the ability to target cytoplasmic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. TSI are produced as single-chain proteins and then cleaved post-translationally to generate functional heterodimers. Precise proteolytic cleavage is essential to activate the protein to a dichain form. TSI are themselves highly specific proteases. We have exploited this activity to create self-activating enzymes by replacing the native proteolytic site with a substrate SNARE peptide for the TSI protease. We have also created cross-activating backbones. By replacing the proteolytic activation site in one backbone with the substrate SNARE peptide for another serotype, controlled activation is achieved. SNARE peptides encompassing the whole of the coiled-coil region enabled complete activation and assembly of the dichain backbone. These engineered TSI backbones are capable of translocating their enzymatic domains to target intracellular SNARE proteins. They are also investigative tools with which to further the understanding of endopeptidase activity of light chain in SNARE interactions.

Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function

“…Proteins of choices have been tethered to enzymatically inactive BoNT and demonstrated to translocate and function within the neuronal cell (Bade et al, 2004). While these studies have limited themselves to modulation of the enzymatic domain, replacement of the neuronal targeting receptor binding domain with one that recognizes a unique cellular surface protein could make BoNT an attractive delivery system for fold permissive cargo proteins to the scientist's target tissue of choice (Edupuganti et al, 2012; Stancombe et al, 2012). …”

Cargo-Delivery Platforms for Targeted Delivery of Inhibitor Cargos Against Botulism

“…In this fashion the range of BoNT therapeutic potential can be extended to non-neuronal cells as well, in particular secretory cells and sensory neurons [72–73]. Additionally, engineered chimeric BoNT toxins, where domains displaying selective properties are swapped among the BoNT serotypes, are being developed as anti-nociceptive therapeutics to treat chronic pain and other secretory disorders [50].…”

“…It has been shown that a single mutation (E244Q) in the active site of BoNT/A still cleaves SNAP25, whereas a double mutant (E224Q/Y366F) is catalytically inactive and forms a stable complex with SNAP25(141–204) peptide substrate [131]. Additional enzymatically inactive BoNTs have been reported, including BoNT/A RYM (R363A/Y365F) [132], ci-BoNT/A1 (H223A/E224A/H227A) [133], BoTIM/B (E231A/H234Y) [109], LH N /A (E224Q and H227Y) [73], and TeTIM (E234A) [134]. All these inactive mutants could have dual use for both developing vaccines as well as cargo-delivery platforms.…”

“…For the purpose of selectively activating the engineered holotoxin proteins into dichain products to facilitate delivery, selective cleavage sites have been incorporated into the recombinant toxin proteins, including substrate sequences of factor Xa or enterokinase [135], TEV protease [136], and even the SNARE substrate sequences for BoNTs [73]. It was demonstrated that using the piggybacked mutant holotoxin approach, a core streptavidin (CS) moiety was delivered into cultured spinal cord neurons by coupling catalytically inactive CS-BoTIM/B holotoxin fusion protein with biotinylated lentiviral vectors [109] or biotinylated liposomes [137].…”

Molecular dissection of botulinum neurotoxin reveals interdomain chaperone function

“…Proteins of choices have been tethered to enzymatically inactive BoNT and demonstrated to translocate and function within the neuronal cell (Bade et al, 2004). While these studies have limited themselves to modulation of the enzymatic domain, replacement of the neuronal targeting receptor binding domain with one that recognizes a unique cellular surface protein could make BoNT an attractive delivery system for fold permissive cargo proteins to the scientist's target tissue of choice (Edupuganti et al, 2012;Stancombe et al, 2012 A -BoNT/A HC (Fischer and Montal, 2006), B -TD (Fischer et al, 2008), and C -BTD (Fischer et al, 2012) A -BoNT/A TD and B -BTD channel activity is independent of a pH gradient across the membrane. Top panels illustrate representative channel activity monitored with pH 5 cis/7 trans, middle panel shows pH 6/7 and bottom panel shows no pH, symmetric pH 7, all measured at an applied voltage of −100 mV.…”