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HighpurityCM-Pachymanforuseinresearch,biochemicalenzymeassaysandinvitrodiagnosticanalysis.
Carboxymethylated,(DS~0.2)highlypurifiedpachyman.Asoluble/gelatinoussubstratefortheassayofendo-1,3-β-D-glucanase.
Identificationofnovelβ-mannan-andβ-glucan-bindingmodules:evidenceforasuperfamilyofcarbohydrate-bindingmodules.
Sunna,A.,Gibbs,M.D.&Bergquist,P.L.(2001).Biochem.J,356(3),791-798.
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Manyglycosidehydrolases,whichdegradelong-chaincarbohydratepolymers,possessdistinctcatalyticmodulesandnon-catalyticcarbohydrate-bindingmodules(CBMs).Onthebasisofconservedproteinsecondarystructure,wedescribeheretheidentificationandexperimentalcharacterizationofnoveltypeofmannanase-associatedmannan-bindingmoduleandalsocharacterizationoftwoCBMfamily4laminarinase-associatedβ-glucan-bindingmodules.ThesemodulesarepredictedtobelongtoasuperfamilyofCBMswhichincludefamilies4,16,17,22andaproposednewfamily,family27.
Lentinulaedodestlg1encodesathaumatin-likeproteinthatisinvolvedinlentinandegradationandfruitingbodysenescence.
Sakamoto,Y.,Watanabe,H.,Nagai,M.,Nakade,K.,Takahashi,M.&Sato,T.(2006).PlantPhysiology,141(2),793-801.
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LentinanisanantitumorproductthatispurifiedfromfreshLentinulaedodesfruitingbodies.Itisacellwallcomponent,comprisingβ-1,3-glucanwithβ-1,6-linkedbranches,whichbecomesdegradedduringpostharvestpreservationasaresultofincreasedglucanaseactivity.Inthisstudy,weusedN-terminalaminoacidsequencetoisolatetlg1,ageneencodingathaumatin-like(TL)proteininL.edodes.TheCDNAclonewasapproximately1.0kbwhereasthegenomicsequencewas2.1kb,andcomparisonofthetwoindicatedthattlg1contains12introns.Thetlg1geneproduct(TLG1)waspredictedtocomprise240aminoacids,withamolecularmassof25kDandisoelectricpointvalueof3.5.Theputativeaminoacidsequenceexhibitsapproximately40%identitywithplantTLproteins,andafungalgenomedatabasesearchrevealedthattheseTLproteinsareconservedinmanyfungiincludingthebasidiomycotaandascomycota.Transcriptionoftlg1wasnotdetectedinvegetativemyceliumoryoungandfreshmushrooms.However,transcriptionincreasedfollowingharvest.Western-blotanalysisdemonstratedariseinTLG1levelsfollowingharvestandsporediffusion.TLG1expressedinEscherichiacoliandAspergillusoryzaeexhibitedβ-1,3-glucanaseactivityand,whenpurifiedfromtheL.edodesfruitingbody,demonstratedlentinandegrADIngactivity.Thus,wesuggestthatTLG1isinvolvedinlentinanandcellwalldegradationduringsenescencefollowingharvestandsporediffusion.
Biochemical,molecularandstructuralanalysisofmultiplethaumatin-likeproteinsfromtheelderberrytree(SambucusnigraL.).
VanDamme,E.J.,Charels,D.,Menu-Bouaouiche,L.,Proost,P.,Barre,A.,Rougé,P.&Peumans,W.J.(2002).Planta,214(6),853-862.
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Thaumatin-likeproteins(TLPs)wereisolatedandcharacterizedfromfruitsandleavesofelderberry(Sambucusnigra)andtheircorrespondinggenescloned.Inaddition,thedevelopmentalregulationandinductionofthedifferentTLPswasfollowedinsomedetail.Ripeningberriesaccumulatedafruit-specificTLPduringthefinalstagesofmaturation.Thisfruit-specificTLPhadnoantifungalactivityandwasdevoidofβ-glucanaseactivity.LeavesconstitutivelyexpressedaTLPthatcloselyresembledthefruit-specifichomologue.TreatmentwithjasmonatemethylesterinducedtwoadditionalTLPsinleavesbutdidnotinduceorenhancetheexpressionofTLPsinimmatureberries.Incontrasttojasmonatemethylester,bothethephonandgarlicextractinducedtheexpressionofaTLPinunripeberriesthatnormallydonotexpressanyTLP.Sequenceanalysisandmolecularmodellingindicatedthatallelderberrythaumatin-likeproteinsshareahighsequencesimilaritywithgroup-5pathogenesis-relatedproteins.However,theproteinsencodedbythedifferentsequencesdifferedfromeachotherinisoelectricpointandthedistributionofthechargesonthesurfaceofthemolecule.
Amolecularbasisfortheendo-β1,3-glucanaseactivityofthethaumatin-likeproteinsfromedIBLefruits.
Menu-Bouaouiche,L.,Vriet,C.,Peumans,W.J.,Barre,A.,VanDamme,E.J.M.&Rougé,P.(2003).Biochimie,85(1),123-131.
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Fruit-specificthaumatin-likeproteinswereisolatedfromcherry,appleandbanana,andtheirenzymaticandantifungalactivitiescompared.Boththeappleandcherrypossessamoderateendo-β1,3-glucanaseactivitybutaredevoidofantifugalactivity.Incontrast,thebananathaumatin-likeproteininhibitstheinvitrohyphalgrowthofVerticilliumalbo-atrumbutisvirtuallydevoidofendo-β1,3-glucanaseactivity.Bothstructuralandmolecularmodelingstudiesshowedthatallthreethaumatin-likeproteinspossessanextendedelectronegativelychargedcleftattheirsurface,whichisbelievedtobeaprerequisiteforendo-β1,3-glucanaseactivity.Dockingexperimentsshowedthatthepositioningoflinear(1,3)-β-D-glucansinthecleftoftheappleandcherryproteinsallowsaninteractionwiththeglutamicacidresiduesthatareresponsibleforthehydrolyticcleavageoftheglucan.Duetoadifferentpositioninginthecleftofthebananathaumatin-likeprotein,thelinearβ-glucanscannotproperlyinteractwiththecatalyticglutamicacidresiduesandasaresulttheproteinpossessesnoenzymaticactivity.Thepossiblefunctionofthefruit-specificthaumatin-likeproteinsisdiscussedinviewoftheobservedBIOLOGicalactivitiesandstructuralfeatures.
Somethaumatin‐likeproteinshydrolysepolymericβ‐1,3‐glucans.
Grenier,J.,Potvin,C.,Trudel,J.&Asselin,A.(1999).ThePlantJournal,19(4),473-480.
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Thaumatinand12purifiedthaumatin-like(TL)proteinsweresurveyedfortheircapacitytohydrolyseβ-1,3-glucansbyusinganin-gelglucanaseassay.SixTLproteinsidentifiedbyN-terminalaminoacidmicrosequencingwerefoundtobeactiveoncarboxymethyl(CM)-pachyman:abarleyleafstress-relatedpermatin,twotomatofruitosmotins,acherryfruitandtwotobaccostigmaproteins.TLenzymesrangedinspecificactivityfrom0.07to89nkatmg-1withCM-pachymanassubstrate.HydrolyticactivitieswerenotrestrictedtoTLproteinsstronglybindingtowater-insolubleβ-1,3-glucanssincethetwoosmotinswereactivewithouttightbindingtopachyman.SomeTLproteinshydrolysedcrudefungalwallsandonebarleyTLenzymeevenlysedfungalspores.Noactivitywasobservedonlaminarininthein-gelhydrolaseassay.Thin-layerchromatographyrevealedthatthesixenzymesactedasendo-β-1,3-glucanasesleadingtotheformationofvariousoligoglucosides.Thusfar,theTLenzymes(EC3.2.1.x)appeareddifferentfromthewell-knownβ-1,3-glucanases(EC3.2.1.39).Noactivitywasfoundwiththaumatin,zeamatin,tobaccoleafPR-Rproteinandfourstress-relatedTLproteinsfrombarleyandpea.ThisisthefirstdemonstrationthatdiverseTLproteinsareenzymaticallyactive.ThefunctionsofsomeTLproteinsmustbereassessedbecausetheydisplayendo-β-1,3-glucanaseactivityonpolymericβ-1,3-glucans.
Characterizationofpeachthaumatin‐likeproteinsandtheiridentificationasmajorpeachallergens.
Palacin,A.,Tordesillas,L.,Gamboa,P.,Sanchez‐Monge,R.,Cuesta‐Herranz,J.,Sanz,M.L.,Barber,D.,SalcedoG.&Díaz‐Perales,A.(2010).Clinical&ExperimentalAllergy,40(9),1422-1430.
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BackgroundPeachisthemostimportantfruitrelatedtofoodallergyintheMediterraneanarea.Prup3,itslipidtransferprotein,hasbeendescribedastheprincipalallergenresponsibleforcross-reactivitieswithotherfoodsandpollenandtheseverityofclinicalsymptoms.However,theinvolvementofotherallergenicfamiliescannotberuledout.Thaumatin-likeproteins(TLPs)havebeendescribedasfoodallergeninseveralfruits,suchasapple,cherry,kiwiandbanana,andpollen.ObjectiveToidentifymembersoftheTLPfamilyinpeachfruitandtocharacterizeputativeallergens.MethodsThroughtwo-dimensional(2D)electrophoresisofpeachextractandimmunodetectionswithapoolofpeach-allergicpatients,IgE-bindingspotswereidentifiedandthecorrespondingproteinspurifiedandcharacterizedasallergensbyinvitroandinvivoassays.Threeisoforms,belongingtotheTLPfamily,werepurifiedbydifferentchromatographicsystemsandcharacterizedbyN-terminalaminoacidsequences,molecularweightdetermination(MALDI)andenzymaticactivityanalysis(β-1,3-gluconasetestandinhibitiongrowthoffungi).Inthesameway,theirIgE-bindingcapacityandallergenicactivityweretestedbyELISAassays,basophilactivationtestsandskinpricktests(SPT).ResultsTwopeach-TLPs,Prup2.0101andPrup2.0201,wereidentifiedasIgE-bindingspotsby2Delectrophoresis.Anotherpeach-TLP,Prup2.0301,wasclonedandproducedasrecombinantproteininayeastsystem.ThethreeisoformswerepurifiedandcharacterizedasTLPsbyimmunoblottingwithanti-chestnutTLPantibodiesandanti-plantN-asparaginecomplexglycan(anti-cross-reactivecarbohydratedeterminant).Allofthemshowedβ-1,3-glucanaseactivityandinhibitionoffungalgrowth.ThethreeTLPswererecognizedbyaround50%oftheserafrom31patientsanalysedinELISAexperiments.AllthreegaveapositiveresponsetoanSPTand/orinbasophilactivationexperiments.ConclusionThreeisoforms,belongingtotheTLPfamily,wereidentifiedinpeachasprincipalallergens.Theirprevalence,observedininvitro,exvivoandinvivoanalyses,suggeststhattheyareimportantallergensandshouldthereforebeincludedintheroutinediagnosisofpeachallergy,atleastintheMediterraneanarea.
Endo-β-1,3-glucanaseGLU1,fromthefruitingbodyofLentinulaedodes,belongstoanewglycosidehydrolasefamily.
Sakamoto,Y.,Nakade,K.&Konno,N.(2011).AppliedandEnvironmentalMicrobiology,77(23),8350-8354.
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ThecellwallofthefruitingbodyofthemushroomLentinulaedodesisdegradedafterharvestingbyenzymessuchasβ-1,3-glucanase.Inthisstudy,anovelendo-typeβ-1,3-glucanase,GLU1,waspurifiedfromL.edodesfruitingbodiesafterharvesting.Thegeneencodingit,glu1,wasisolatedbyrapidamplificationofcDNAends(RACE)-PCRusingprimersdesignedfromtheN-terminalaminoacidsequenceofGLU1.Theputativeaminoacidsequenceofthematureproteincontained247aminoacidresidueswithamolecularmassof26kDaandapIof3.87,andrecombinantGLU1expressedinPichiapastorisexhibitedβ-1,3-glucanaseactivity.GLU1catalyzeddepolymerizationofglucanscomposedofβ-1,3-linkedmainchains,andreactionproductanalysisbythin-layerchromatography(TLC)clearlyindicatedthattheenzymehadanendolyticmode.However,theaminoacidsequenceofGLU1showednosignificantsimilaritytoknownglycosidehydrolases.GLU1hassimilaritytoseveralhypotheticalproteinsinfungi,andGLU1andhighlysimilarproteinsshouldbeclassifiedasanovelglycosidehydrolasefamily(GH128).
Identification,cloning,andcharacterizationofβ-glucosidasefromUstilagoesculenta.
Nakajima,M.,Yamashita,T.,Takahashi,M.,Nakano,Y.&Takeda,T.(2012).AppliedMicrobiologyandBiotechnology,93(5),1989-1998.
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HydrolyticenzymesresponsibleforlaminarindegradationwerefoundtobesecretedduringgrowthofUstilagoesculentaonlaminarin.Anenzymeinvolvedinlaminarindegradationwaspurifiedbyassayingreleaseofglucosefromlaminaribiose.Ion-exchangechromatographyoftheculturefiltratefollowedbysize-exclusionchromatographyyieldeda110-kDaproteinassociatedwithlaminaribiosehydrolysis.LC/MS/MSanalysisofthe110-kDaproteinidentifiedthreepeptidesequencesthatsharedsignificantsimilaritywithaputativeglucosidehydrolasefamily(GH)3β-glucosidaseinUstilagomaydis.BasedontheDNAsequenceoftheU.maydisGH3β-glucosidase,ageneencodingaputativeGH3β-glucosidaseinU.esculenta(Uebgl3A)wasclonedbyPCR.Basedonthededucedaminoacidsequence,theproteinencodedbyUebgl3Ahasamolecularmassof91kDaandshares90%identitywithU.maydisGH3β-glucosidase.RecombinantUeBgl3AexpressedinAspergillusoryzaereleasedglucosefromβ-1,3-,β-1,4-,andβ-1,6-linkedoligosaccharides,andfrom1,3-1,4-β-glucanandlaminarinpolysaccharides,indicatingthatUeBgl3Aisaβ-glucosidase.KineticanalysisshowedthatUeBgl3Apreferentiallyhydrolyzedlaminaritrioseandlaminaritetraose.TheseresultssuggestthatUeBgl3AisakeyenzymethatproducesglucosefromlaminarioligosaccharidesduringgrowthofU.esculentaonlaminarin.
Detectionofenzymesactiveonvariousβ‐1,3‐glucansafterdenaturingpolyacrylamidegelelectrophoresis.
Trudel,J.,Grenier,J.&Asselin,A.(1998).Electrophoresis,19(10),1788-1792.
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Enzymeswereassayedforglucanaseactivityafterdenaturingsodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)ingelscontainingβ-1,3-glucansembeddedassubstrate.Lentinan,curdlan,paramylon,baker"syeastalkali-insolubleglucan,baker"syeastalkali-solubleglucanandcarboxymethyl(CM)-pachymanwerecomparedtooligomericlaminarin,whichistheusualsubstrateforassayingβ-1,3-glucanaseactivities.Detectingenzymeactivitiesbyanilinebluefluorescentstainingwasalsocomparedwiththestainingofreleasedreducingsugarsby2,3,5-triphenyltetrazoliumchloride(TTC).Forthenonreducedproteins,theDriselaseextractexhibitedonemajorbandat32.5kDaandonelessintensebandat23kDaformostsubstrateswiththetwodetectionprocedures.NoLyticaseenzymewasdetectedineitherdetectionproceduresforalltestedsubstrates.Forbarleyenzymes,noactivitywasrevealedafteranilinebluestainingwhileoneundescribed19kDaglucanaseactivitywasbestshownafterTTCstainingwithcurdlan,paramylonandCM-pachymanassubstrates.Inthecaseofreducedproteins,theLyticaseextractyieldedthreebands(33,36and46kDa)onseveralsubstrateswithbothdetectionprocedures.Thiswasthesameforthebarleyleafextract(32,36and39kDa).TheDriselaseextractshowedone42kDaband.Manyenzymesactiveonβ-1,3-glucansarethusbestrevealedwhenproteinsaredenaturedandreducedandwhenproteinrenaturationafterSDS-PAGEinvolvesapH8.0treatmentandtheinclusionof1mMcysteineinbuffers.However,someenzymesareonlydetectedwhenproteinsaredenaturedwithoutreduction.Finally,theuseofvariouspolymericβ-1,3-glucansubstratesdifferentfromoligomericlaminarinisnecessarytodetectnewtypesofenzymessuchasthe19kDabarleyglucanase.
Developmentofβ‐1,3‐glucanaseactivityingerminatedtomatoseeds.
Morohashi,Y.&Matsushima,H.(2000).JournalofExperimentalBotany,51(349),1381-1387.
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Laminarin‐hydrolysingactivitydevelopedintheendospermoftomato(Lycopersiconesculentum)seedsfollowinggermination.Theenzymewasbasic(pI>10)andtheapparentmolecularmasswasestimatedtobe35 kDabySDS‐PAGE.Itwasspecificforlinearβ‐1,3‐glucansubstrates.Laminarinwashydrolysedbytheenzymetoyieldamixtureofoligoglucosides,indicatingthattheenzymehadanendo‐actionpattern.Thus,theenzymewasidentifiedasβ‐1,3‐endoglucanase(EC3.2.1.39).Theactivityoftheenzymedevelopedintheendospermafterradicleprotrusion(germination)hadoccurredandtheenzymeactivitywaslocalizedexclusivelyinthemicropylarregionoftheendospermwheretheradiclehadpenetrated.Whenthelateralendospermregion,wherenoinductionoftheenzymeoccurred,waswounded(cutorpunctured),therewasamarkedenhancementofβ‐1,3‐glucanaseactivity.Thusthepost‐germinativeβ‐1,3‐glucanaseactivityinthemicropylarendospermportionmightbebroughtaboutbywoundingresultingfromendospermrupturebyradiclepenetration.
