Engineering botulinum neurotoxin domains for activation by toxin light chain

• Published in: The FEBS journal 2012-02, Vol.279 (3), p.515-523
• Main Authors: Stancombe, Patrick R., Masuyer, Geoffrey, Birch‐Machin, Ian, Beard, Matthew, Foster, Keith A., Chaddock, John A., Acharya, K. Ravi
• Format: Article
• Language: English
• Subjects: Biochemistry; Bioengineering; botulinum toxin scaffold; Botulinum Toxins; Botulinum Toxins, Type A - chemistry; Botulinum Toxins, Type A - genetics; Botulinum Toxins, Type A - therapeutic use; Endopeptidases - metabolism; LC endopeptidase domain; Models, Molecular; Neurotoxins; Peptide Hydrolases - metabolism; Peptides; Pharmacology; Protein Engineering - methods; Protein Structure, Tertiary; Proteins; self‐activation; SNARE peptides; SNARE Proteins - antagonists & inhibitors; targeted secretion inhibitors
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2011.08444.x

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. Self‐activating LHN/A consists of the botulinum toxin light chain (LC/A) endopeptidase and translocation domains (HN/A) engineered with a SNAP25 peptide linker. LC/A cleaves the SNAP25 linker enabling the protein to assemble into a di‐sulphide bonded dichain. SNAP25 engineered LHN/A acts as a self‐processing protease that assembles into activated, fully functional protein capable of endosome translocation and cleavage of intracellular SNAP25