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

• Published in: Biochimica et biophysica acta. General subjects Vol. 1863; no. 11; p. 129396
• Main Authors: Shao, Kankai, Wang, Qin, Wang, Ning, Gao, Xiao-Dong, Nakanishi, Hideki
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2019
• Subjects: Botulinum neurotoxin serotype C; botulinum toxin; Botulinum Toxins - biosynthesis; Botulinum Toxins - genetics; chimerism; exocytosis; Humans; mutants; mutation; Qa-SNARE Proteins - biosynthesis; Qa-SNARE Proteins - genetics; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - biosynthesis; Saccharomyces cerevisiae Proteins - genetics; serotypes; SNARE; SNARE proteins; Sso1; substrate specificity; synaptic transmission; Syntaxin 1 - biosynthesis; Syntaxin 1 - genetics; Syntaxin-1A; therapeutics; yeasts; BF; Sso1; BoNT/C; BoNT; SNARE; GFP; Botulinum neurotoxin serotype C; SNAREs; 5’-FOA; Saccharomyces cerevisiae; Syntaxin-1A; RFP; STX1A; SNAP-25; S. cerevisiae
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2019.07.005

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. 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. 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). 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. The yeast cell-based BoNT/C assay would be useful to characterize and engineer BoNT/C. •BoNT/C is expected to be applied to cosmetic and therapeutic uses.•Yeast cells are not sensitive to BoN/C.•Human syntaxin-1A is not functional in yeast cells.•Functional yeast and human syntaxin-1A chimeric SNAREs were produced.•Using the yeast/human chimeric SNARE, yeast cell-based BoNT/C assay was constructed.