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Thebotulinumneurotoxinsareamongthemosttoxicsubstancesknowntoman.Fouroftheseven immunologicallydistinctserotypes,A,B,E,andF,causebotulisminhumans.Azincendoproteaseonthe50 kDalightchainofthe150kDaholotoxin,cleavesasingletargetproteinessentialforsynapticvesicle membranefusion.Thisinhibitsneurotransmitterreleaseandleadstomuscularparalysis.Measurementof theproteolyticactivityprovidesapotentiallysensitiveanddirectmeansfordetectingthesepotenttoxins. Also,theneurotoxinlightchainspresentanidealtargetforthedevelopmentofpotentialtherapeuticinhibitors. Theclassicapproachformonitoringenzymaticactivityutilizesshortpeptidescontainingachromophoreor fluorophoremoietyatthesiteofcleavage.However,forallthebotulinumserotypes,efficientcleavage requireslargerpeptidesubstrates.Also,hydrolysisoccursonlyforpeptidesthatspanbothsidesofthe cleavagesite.Analternativeapproachistousefluorescenceresonanceenergytransfer(FRET)peptides. Thesefluorogenicpeptidescontainafluorescentgroupatoneendandasuitablechromogenicacceptor groupattheother.Thisallowsfortheinclusionofaminoacidsoneithersideoftheenzymaticallycleaved bond.Thefluorescenceisquenchedinitiallybyintramolecularenergytransferbetweenthedonor/acceptor pair.Uponcleavage,thefluorescenceisrecovered. PeptidesubstratesspecificforbotulinumtoxintypesA,B,C,andEhavebeendesignedtakingadvantageof thefactthateachserotypeoftheneurotoxinselectivelycleavesoneoftheSNAREmembranefusionproteins atanuniquesite.ThesubstratesfortypeA,SNAPtide,andtypeE,SNAPEtide,arebasedonthenative synaptosomalsubstrateforthesetoxins,SNAP-25.ThesubstratesfortypeB,VAMPtide,andtypeC, SYNTAXtidearebasedonthenativetargetSNAREproteins,VAMPandsyntaxin,respectively.The peptidescontainFRETpairsinvariedpositionsinthesequence.Datademonstratingtheefficiencyof cleavageofthesepeptidesbytheirrespectiveneurotoxinlightchainsaswellasstudiesoftheminimum concentrationofenzymedetectablebyeachpeptidesubstrate,ispresented.