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Construction of functional chimeras of syntaxin-1A and its yeast orthologue, and their application to the yeast cell-based assay for botulinum neurotoxin serotype C.
- Source :
-
Biochimica et biophysica acta. General subjects [Biochim Biophys Acta Gen Subj] 2019 Nov; Vol. 1863 (11), pp. 129396. Date of Electronic Publication: 2019 Jul 11. - Publication Year :
- 2019
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Abstract
- Background: Botulinum neurotoxins (BoNTs) prevent synaptic transmission because they hydrolyze synaptic N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). BoNT serotype C (BoNT/C) targets syntaxin-1A and SNAP-25, and is expected to be applied to cosmetic and therapeutic uses. SNAREs are evolutionally conserved proteins and in yeast a syntaxin-1A orthologue Sso1 is involved in exocytosis. The substrate specificity of BoNT/C is strict and it cannot cleave Sso1.<br />Methods: Domain swapping and mutational screenings were performed to generate functional chimeras SNAREs of syntaxin-1A and Sso1. Such chimeras are expressed in yeast cells and assessed whether they are susceptible to BoNT/C digestion.<br />Results: The Sso1 and syntaxin-1A chimera (Sso1/STX1A), in which the SNARE domain in Sso1 was replaced with that of syntaxin-1A, was not functional in yeast. The functional incompatibility of Sso1/STX1A was attributable to its accumulation in the ER. We found several mutations that could release Sso1/STX1A from the ER to make the chimera functional in yeast. Yeast cells harboring the mutant chimeras grew similarly to wild-type cells. However, unlike wild-type, yeast harboring the mutant chimeras exhibited a severe growth defect upon expression of BoNT/C. Results of further domain swapping analyses suggest that Sso1 is not digested by BoNT/C because it lacks a binding region to BoNT/C (α-exosite-binding region).<br />Conclusions: We obtained functional Sso1/STX1A chimeras, which can be applied to a yeast cell-based BoNT/C assay. BoNT/C can recognize these chimeras in a similar manner to syntaxin-1A.<br />General Significance: The yeast cell-based BoNT/C assay would be useful to characterize and engineer BoNT/C.<br /> (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Subjects :
- Humans
Botulinum Toxins biosynthesis
Botulinum Toxins genetics
Qa-SNARE Proteins biosynthesis
Qa-SNARE Proteins genetics
Recombinant Fusion Proteins biosynthesis
Recombinant Fusion Proteins genetics
Saccharomyces cerevisiae genetics
Saccharomyces cerevisiae metabolism
Saccharomyces cerevisiae Proteins biosynthesis
Saccharomyces cerevisiae Proteins genetics
Syntaxin 1 biosynthesis
Syntaxin 1 genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1872-8006
- Volume :
- 1863
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- Biochimica et biophysica acta. General subjects
- Publication Type :
- Academic Journal
- Accession number :
- 31302181
- Full Text :
- https://doi.org/10.1016/j.bbagen.2019.07.005