Metabolic Labeling of Caenorhabditis elegans Primary Embryonic Cells with Azido-Sugars as a Tool for Glycoprotein Discovery 英文参考文献.docVIP

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Metabolic Labeling of Caenorhabditis elegans Primary Embryonic Cells with Azido-Sugars as a Tool for Glycoprotein Discovery 英文参考文献.doc

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MetabolicLabelingofCaenorhabditiselegansPrimary EmbryonicCellswithAzido-SugarsasaToolfor GlycoproteinDiscovery AmandaR.Burnham-Marusich,CaseyJ.Snodgrass,AnnaM.Johnson,ConradM.Kiyoshi,SarahE.Buzby, MattR.Gruner,PatriciaM.Berninsone* DepartmentofBiology,UniversityofNevadaReno,Reno,Nevada,UnitedStatesofAmerica Abstract GlycobiologyresearchwithCaenorhabditiselegans(C.elegans)hasbenefittedfromthenumerousgeneticandcellbiology toolsavailableinthissystem.However,thelackofacelllineandtherelativeinaccessibilityofC.eleganssomaticcellsinvivo havelimitedthebiochemicalapproachesavailableinthismodel.HerewereportthatC.elegansprimaryembryoniccellsin cultureincorporateazido-sugaranalogsofN-acetylgalactosamine(GalNAc)andN-acetylglucosamine(GlcNAc),andthatthe labeledglycoproteinscanbeanalyzedbymassspectrometry.Byusingthismetaboliclabelingapproach,wehaveidentified a set of novel C. elegans glycoprotein candidates, which include several mitochondrially-annotated proteins. This observationwasunexpectedgiventhatmitochondrialglycoproteinshaveonlyrarelybeenreported,anditsuggeststhat glycosylation of mitochondrially-annotated proteins might occur more frequently than previously thought. Using independentexperimentalstrategies,wevalidatedasubsetofourglycoproteincandidates.Theseincludeamitochondrial, atypical glycoprotein (ATP synthase a-subunit), a predicted glycoprotein (aspartyl protease, ASP-4), and a protein family withestablishedglycosylationinotherspecies(actin).Additionally,weobservedaglycosylatedisoformofATPsynthasea- subunitinbovinehearttissueandaprimatecellline(COS-7).Overall,ourfindingthatC.elegansprimaryembryoniccellsare amenabletometaboliclabelingdemonstratesthatbiochemicalstudiesinC.elegansarefeasible,whichopensthedoorto labelingC.eleganscellswithotherradioactiveorazido-substratesandshouldenabletheidentificationofadditionalpost- translationallymodifiedtargetsandanalysisofthegenesrequiredfortheirmodificationusingC.elegansmutantlibraries. Citation:Burnham-MarusichAR,SnodgrassCJ,