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The Light Chain Defines the Duration of Action of Botulinum Toxin Serotype A Subtypes.

ABSTRACT: Botulinum neurotoxin (BoNT) is the causative agent of botulism and a widely used pharmaceutical to treat a variety of neurological diseases. BoNTs are 150-kDa protein toxins organized into heavy chain (HC) and light chain (LC) domains linked by a disulfide bond. The HC selectively binds to neurons and aids cell entry of the enzymatically active LC. There are seven immunological BoNT serotypes (A to G); each serotype includes genetic variants, termed subtypes. Only two subtypes, BoNT/A1 and BoNT/B1, are currently used as therapeutics. BoNT serotype A (BoNT/A) subtypes A2 to A8 show distinct potency, duration of action, and pathology relative to BoNT/A1. Specifically, BoNT/A3 possesses shorter duration of action and elicits distinct symptoms in mice at high toxin doses. In this report, we analyzed the roles of LC and HC of BoNT/A3 for duration of action, neuronal cell entry, and mouse pathology by using clostridium-derived recombinant hybrid BoNTs consisting of reciprocal LC and HC (BoNTA1/A3 and BoNTA3/A1). Hybrid toxins were processed in their expression host to a dichain BoNT consisting of LC and HC linked via a disulfide bond. The LC and HC defined BoNT potency in mice and BoNT toxicity for cultured neuronal cells, while the LC defined the duration of BoNT action in cell and mouse models. Protein alignment identified a previously unrecognized region within the LC subtype A3 (LC/A3) relative to the other LC serotype A (LC/A) subtypes (low primary acid homology [LPH]) that correlated to intracellular LC localization. This study shows the utility of recombinant hybrid BoNTs with new therapeutic potential, while remaining sensitive to antitoxins and therapies to native BoNT.IMPORTANCE Botulinum neurotoxins are the most potent protein toxins for humans and potential bioterrorism threats, but they are also widely used as pharmaceuticals. Within the large family of BoNTs, only two subtypes are currently used as pharmaceuticals, with a large number of BoNT subtypes remaining as untapped potential sources for unique pharmaceuticals. Here, two recombinant hybrid toxins were engineered, consisting of domains from two BoNT subtypes that possess distinct duration of action and activity in human neurons and mice. We define the functional domains responsible for BoNT action and demonstrate creation of functional hybrid BoNTs with new therapeutic potential, while remaining sensitive to antitoxins and therapies to native BoNT.

SUBMITTER: Pellett S

PROVIDER: S-EPMC5874905 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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The Light Chain Defines the Duration of Action of Botulinum Toxin Serotype A Subtypes.

Pellett Sabine S Bradshaw Marite M Tepp William H WH Pier Christina L CL Whitemarsh Regina C M RCM Chen Chen C Barbieri Joseph T JT Johnson Eric A EA

mBio 20180327 2

Botulinum neurotoxin (BoNT) is the causative agent of botulism and a widely used pharmaceutical to treat a variety of neurological diseases. BoNTs are 150-kDa protein toxins organized into heavy chain (HC) and light chain (LC) domains linked by a disulfide bond. The HC selectively binds to neurons and aids cell entry of the enzymatically active LC. There are seven immunological BoNT serotypes (A to G); each serotype includes genetic variants, termed subtypes. Only two subtypes, BoNT/A1 and BoNT/ ...[more]

PMID: 29588398

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Project description:Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2-7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2-7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2-7 and BLC3) are close to the human germline sequences, which suggest that they may be well tolerated in potential clinical development.

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The Light Chain Defines the Duration of Action of Botulinum Toxin Serotype A Subtypes.

Publications

The Light Chain Defines the Duration of Action of Botulinum Toxin Serotype A Subtypes.

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