| Botulinum neurotoxins(Bo NTs)can specificly hydrolyze soluble N-ethylmaleimidesensitive factor attachment protein receptor(SNARE)in neurons,then inhibit the release of neurotransmitters during membrane fusion.It is widely used in clinical and aesthetic fields.With the development of gene sequencing and bioinformatics,there are increasing amount of new Bo NTs and Bo NT-Like genes,but there still lack a simple,rapid and economical detection system for Bo NTs activity.The membrane fusion mechanism is highly conserved among eukaryotes,this study successfully constructed a Bo NT-LC yeast detection system using Saccharomyces cerevisiae as a model organism,and detected the activities of various Bo NTLCs using the sporulation rate of yeast as a characterization.The main results are as follow:(1)Construction of a Bo NT-LC yeast assay system.Wild-type yeast is not sensitive to Bo NT-LC,but Bo NT-LC can hydrolyze SNARE region of SNAP25.Therefore,in this study,the SNARE region of SNAP25 was used to replace corresponding region of SPO20,its homologous gene in yeast.We get Spo20/SNAP25(C)and Spo20/SNAP25(N)chimeras.SPO20 is a vital gene in the sporulation process in yeast.In this study,spo20Δ yeast with lost sporulation function was obtained by homologous recombination.The two chimeras were then integrated into the spo20Δ yeast genome by homologous recombination.Thus,we obtain a Bo NT-LC yeast assay system that can compensate for the sporulation defects of yeast.(2)Validation of Bo NT-LC yeast assay system.The dityrosine in the outermost layer of the spore wall is a fluorescent molecule,and we found that the sporulation rate of yeast is proportional to the fluorescence intensity.First,Bo NT-LCs have no effect on sporulation of wild-type yeast.Then,Bo NT-LCs were overexpressed in two Bo NT-LC yeast detection systems.The results show that Bo NT/A,C and E-LCs can reduce the sporulation rate of Spo20/SNAP25(C)yeast system.Meanwhile,Bo NT/En-LC can reduce the sporulation rate of Spo20/SNAP25(N)yeast system.Western blotting results show that Bo NT-LCs can hydrolyze Spo20/SNAP25(C)and Spo20/SNAP25(N)chimeric proteins,thereby reducing the sporulation rate of yeast.Therefore,the yeast system could be used to detect the activity of Bo NT-LC.(3)The activity of unknown Bo NT-LC was detected using the above Bo NT-LC yeast assay system.Through sequence alignment,a new undetected Bo NT,Bo NT/E12-LC,was identified.Bo NT/E12-LC was overexpressed in two yeast systems,by detecting the sporulation rate of the yeast assay system and western blotting test,we determined that the Spo20/SNAP25(C)chimera protein could be specifically hydrolyzed by this new type of Bo NT,and thus reducing the sporulation rate of Spo20/SNAP25(C)yeast system.The above results indicate that the yeast system constructed in this study can be used to verify the activity of unknown Bo NT-LC.This yeast assay system greatly simplifies the screening process of unknown Bo NTs,and provides technical reserves for the medical and cosmetic applications of Bo NTs.(4)Expression and purification of Bo NT/A-HC.Bo NTs are composed of a light chain and a heavy chain(HC).In neuron cells,HC promotes the internalization of Bo NTs by specifically recognizing glycosylated receptors on the cell membrane.To detect the activity of full-length Bo NTs,Bo NT/A-HC proteins were purified by protein affinity chromatography method. |
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