Purification and Characterization of Recombinant Botulinum Neurotoxin Serotype FA, Also Known as Serotype H

Botulinum neurotoxins (BoNTs) are categorised into immunologically distinct serotypes BoNT/A to /G). Each serotype can also be further divided into subtypes based on differences in amino acid sequence. BoNTs are ~150 kDa proteins comprised of three major functional domains: an N-terminal zinc metalloprotease light chain (LC), a translocation domain (HN), and a binding domain (HC). The HC is responsible for targeting the BoNT to the neuronal cell membrane, and each serotype has evolved to bind via different mechanisms to different target receptors. Most structural characterisations to date have focussed on the first identified subtype within each serotype (e.g., BoNT/A1). Subtype differences within BoNT serotypes can affect intoxication, displaying different botulism symptoms in vivo, and less emphasis has been placed on investigating these variants. This review outlines the receptors for each BoNT serotype and describes the basis for the highly specific targeting of neuronal cell membranes. Understanding receptor binding is of vital importance, not only for the generation of novel therapeutics but also for understanding how best to protect from intoxication.

Section: Receptor-binding Domain Variationmentioning

confidence: 99%

Variations in the Botulinum Neurotoxin Binding Domain and the Potential for Novel Therapeutics

Davies

Liu

Acharya

2018

“…The individual assessment of novel new subtype toxin FA (H) when expressed in a bivalent Clostridial strain B2/FA was facilitated by a KO of the B2 component of host strain [ 67 ]. However, an engineered recombinant form of the FA provided valuable information about the role of the activation loop length and characterization of the highly purified toxin [ 69 ].…”

Section: Discussionmentioning

confidence: 99%

“…A 2018 study detailed the production and characterization of a fully recombinant /FA toxin in E. coli [ 69 ]. A codon optimized /FA ORF was synthesized with an engineered BoNT/F1 (K 430 -L 444 ) activation loop in place of the native BoNT/FA activation loop (S 429 -L 437 ).…”

Section: Recombinant Functional Bontsmentioning

confidence: 99%

Engineering of Botulinum Neurotoxins for Biomedical Applications

Webb

2018

Botulinum neurotoxins (BoNTs) have been used as therapeutic agents in the clinical treatment of a wide array of neuromuscular and autonomic neuronal transmission disorders. These toxins contain three functional domains that mediate highly specific neuronal cell binding, internalization and cytosolic delivery of proteolytic enzymes that cleave proteins integral to the exocytosis of neurotransmitters. The exceptional cellular specificity, potency and persistence within the neuron that make BoNTs such effective toxins, also make them attractive models for derivatives that have modified properties that could potentially expand their therapeutic repertoire. Advances in molecular biology techniques and rapid DNA synthesis have allowed a wide variety of novel BoNTs with alternative functions to be assessed as potential new classes of therapeutic drugs. This review examines how the BoNTs have been engineered in an effort to produce new classes of therapeutic molecules to address a wide array of disorders.

“…Light Chain of Tetanus toxin (LC/T), LC/B, LC/D, LC/F, LC/FA and LC/G ( Figure 2 ) cleave vesicle-associated membrane protein (VAMP)/Synaptobrevin, a V-SNARE [ 32 , 35 , 36 , 37 , 38 ]. BoNT/FA, previously named as a new serotype, BoNT/H, is a chimera between BoNT/F and BoNT/A [ 39 ]. LC/FA shares 81% homology with LC/F5, while the HCC/FA has 93% identity to BoNT/A1 [ 40 ].…”

Section: Bont Structure and Functionmentioning

confidence: 99%

Light Chain Diversity among the Botulinum Neurotoxins

Gardner

Barbieri

2018

Botulinum neurotoxins (BoNT) are produced by several species of clostridium. There are seven immunologically unique BoNT serotypes (A–G). The Centers for Disease Control classifies BoNTs as ‘Category A’ select agents and are the most lethal protein toxins for humans. Recently, BoNT-like proteins have also been identified in several non-clostridia. BoNTs are di-chain proteins comprised of an N-terminal zinc metalloprotease Light Chain (LC) and a C-terminal Heavy Chain (HC) which includes the translocation and receptor binding domains. The two chains are held together by a disulfide bond. The LC cleaves Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The cleavage of SNAREs inhibits the fusion of synaptic vesicles to the cell membrane and the subsequent release of acetylcholine, which results in flaccid paralysis. The LC controls the catalytic properties and the duration of BoNT action. This review discusses the mechanism for LC catalysis, LC translocation, and the basis for the duration of LC action. Understanding these properties of the LC may expand the applications of BoNT as human therapies.