A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate.
The seven functionally distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are dichains consisting of light chain (LC) with zinc-dependent endoprotease activity connected by one disulfide bond to heavy chain with neuronal-cell translocation and receptor-binding domains. LC-mediated proteolysis of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and consequent inhibition of synaptic vesicle fusion to the presynaptic membrane of human motor neurons are responsible for flaccid paralysis associated with botulism. LC endoproteolysis is complex, requiring highly extended SNARE sequences at the surface of intracellular membranes and prompting our development of a genetically amenable assay to monitor the interaction between BoNT/LC and its SNARE substrate. Using BoNT serotype B as a model, the assay employs a chimeric SNARE protein where a portion of neuronal synaptobrevin (Sb) is fused to Snc2p, a Sb ortholog required for protein secretion from yeast cells. Regulated expression of serotype B-LC in yeast leads to cleavage of the chimera and a conditional growth defect. To assess utility of this assay for monitoring SNARE protein cleavage, we growth-selected chimeric SNARE mutations that inhibited proteolysis. When these mutations were introduced into Sb and examined for cleavage, substrate residues located near and distal to the cleavage site were important, including residues positioned near the Sb transmembrane domain, an unexplored aspect of BoNT cell intoxication. Additional mutations were positioned in a nine-residue SNARE motif, supporting a previously assigned role for this motif in LC recognition and providing proof of principle for the application of yeast-based technology to study intracellular BoNT/LC endoproteases.
Fang
Hong
H
Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232-2363, USA. hong.fang@vanderbilt.edu
Luo
Wentian
W
Henkel
Jim
J
Barbieri
Joseph
J
Green
Neil
N
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D001905:Botulinum Toxins / Q000235:genetics / Q000378:metabolism*; D019274:Botulinum Toxins, Type A; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D022162:Cytoplasmic Vesicles / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009474:Neurons / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C096323:rimabotulinumtoxinB; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A