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Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B.

ABSTRACT: Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.

SUBMITTER: Buzzatto MV

PROVIDER: S-EPMC11292472 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B.

Buzzatto Micaela Vanina MV Benegas Guerrero Fabiana Cristina FC Álvarez Pablo Ariel PA Zizzias María Paz MP Polo Luis Mariano LM Tomes Claudia Nora CN

Bioscience reports 20240701 7

Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We ha ...[more]

PMID: 39011584

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Project description: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.

Dataset Information

Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B.

Publications

Expression, purification and application of a recombinant, membrane permeating version of the light chain of botulinum toxin B.

Similar Datasets

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