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Antimicrobialpropertiesofavianeggshell-specificC-typelectin-likeproteinsOlivierWellman-Labadiea,RajamaniLakshminarayananb,1,MaxwellT.Hinckea,*aDepartmentofCellularandMolecularMedicine,UniversityofOttawa,451SmythRoad,Ottawa,Ontario,CanadaK1H8M5bDepartmentofChemistry,NationalUniversityofSingapore,3ScienceDrive3,Singapore117543,SingaporeReceived16January2008;revised24January2008;accepted25January2008Availableonline5February2008EditedbyPeterBrzezinskiAbstractC-typelectin-likeproteinsaremajorcomponentsofthecalcifiedeggshellofmultipleavianspecies.Inthisstudy,tworepresentativeavianC-typelectin-likeproteins,ovocleidin-17andansocalcin,werepurifiedfromdecalcifiedchickenandgooseeggshellproteinextractsandinvestigatedforcarbohydratebindingactivityaswellasantimicrobialactivity.Purifiedovo-cleidin-17andansocalcinwerefoundtobindbacterialpolysac-charides,andwerebactericidalagainstBacillussubtilis,StaphylococcusaureusandPseudomonaaeruginosa.Bacterici-dalactivitywasfoundtobeenhancedinthepresenceofcalciumbutwasnotdependentonitspresence.TheresultssuggestthatavianC-typelectin-likeproteinsmayplayanimportantantimi-crobialroleindefenceoftheavianembryo.2008FederationofEuropeanBiochemicalSocieties.PublishedbyElsevierB.V.Allrightsreserved.Keywords:Antibiotics;Antibacterial;Avianeggshell;Bacterialpolysaccharides;C-typelectin1.IntroductionOvocleidin-17(OC-17)andansocalcinarethemajoregg-shell-specificmatrixproteinsfromchickenandgoose,respec-tively[1–5].TheysharesignificantidentitywithC-typelectin-like(CTL)proteins,butlacktheconsensusQPDmotifthatisrequiredforbindingtosimplesugars.SimilarCTLpro-teinsarepresentineggshellofvariousdomesticatedbirds[6].Interestingly,twofamiliesofCTLproteinshavebeenidenti-fiedwithineggshellsofratitespecies;theaminoacidsequenceofoneformalignsbetterwithansocalcin(groupI)whiletheotherismoresimilartoOC-17(groupII)[7–9].ThepresenceofCTLproteinsintheeggshellsofmultipleavianspeciessug-gestsacommonbiologicallyimportantrole.TheC-typelectinsuperfamilyisalargegroupofextracellu-larproteinswithdiversefunctionsinmulticellularorganisms[10].Recently,RegIIIc,amouseepithelialC-typelectin(GroupVIICTL)anditshumancounterpart,HIP/PAP,wereshowntoinhibitthegrowthofGram-positivebacteria[11].Ansocalcin(33%)andOC-17(28%)possesssignificantidentitytotheRegfamilyofC-typelectins.Giventheirwidespreadpresenceandabundancewithintheeggshellmatricesofvari-ousspecies,wesoughttoinvestigatetheantimicrobialproper-tiesofavianeggshellCTLproteins.OurstrategywastostudythebindingofansocalcinandOC-17tobacterialcellwallpoly-saccharides,andinvestigatetheirantimicrobialpropertiesagainstGram-positiveandGram-negativebacteria.There-sultsshowthatavianeggshellCTLproteinshaveexcellentantimicrobialaswellasbacterialpolysaccharidebindingprop-erties.Thisisthefirstreportontheantimicrobialpropertiesofeggshell-specificCTLproteinsandhighlightsthemultiplerolesplayedbytheseproteins.2.Materialsandmethods2.1.ProteinextractionandpurificationEggshellsweregroundintoafinepowderanddecalcifiedusing20%aceticacid(10mlofaceticacid/grameggshell)accordingtothemethodofReyes-Grajedaetal.[5].PurificationofansocalcinandOC-17wasperformedaspreviouslydescribedbyLakshminarayananetal.[3,4].TheconcentrationofproteinsusedforSDS–PAGE,carbohydratebindingandantimicrobialassayswasdeterminedbybicinchoninicacid(BCA)proteinassay(Pierce,Rockford,IL)usingBSA(Bioshop,Bur-lington,ON)asastandard.2.2.CarbohydratebindingactivityTheabilityofansocalcinandOC-17tobindtovariouscomplexcar-bohydrateswasanalyzedbyapull-downassayadaptedfromCashetal.[11].Dryproteinsamplesweresuspendedin0.01%aceticacidandfurtherdilutedwith0.01%aceticacidand0.1%bovineserumalbumen(BSA).SampleswereincubatedinthepresenceorabsenceofMicrococcuslysodeikticusATCC4698cellwalls(Sigma–Aldrich,Oakville,ON),Bacillussubtilispeptidoglycan(Sigma–Aldrich),crabshellchitin(Sigma–Aldrich),cornstarch(BestFoodsCanada,Etobi-coke,ON)orEscherichiacoli0127:B8lipopolysaccharide(Sigma–Al-drich)suspendedin0.01%aceticacidat4Cwithgentleshakingfor24h.Afterincubation,sampleswerecentrifugedfor5min(13000rpm)at4C,andthesupernatantcarefullyremoved.Superna-tantandpelletwereanalyzedbySDS–PAGE.2.3.AntimicrobialactivityofpurifiedansocalcinandOC-17Antimicrobialactivitywasevaluatedbythemicro-brothdilutionas-sayadaptedfromSteinbergandLehrer[12].VehiclewasBSA(0.1%)inaceticacid(0.01%),andpositivecontrolwas100lg/mlbovinelacto-ferricinB(Sigma–Aldrich).M.lysodeikticusATCC4698cellwallsorE.coli0127:B8lipopolysaccharide(0.5–5mg/ml)inthepresenceandabsenceofansocalcinorOC-17wasalsoinvestigatedforpotentialinhibitionofantimicrobialactivity.Antimicrobialactivitywasevalu-atedagainsttwoGram-positive(B.subtilisATCC19659andStaphy-lococcusaureusATCC6538)andtwoGram-negative(PseudomonaaeruginosaATCC15442andE.coliD31)bacteria.Eachantimicrobialassaywasconductedintriplicatefortwoindependenttrials(N=2).*Correspondingauthor.Fax:+16135625687.E-mailaddress:mhincke@uottawa.ca(M.T.Hincke).1Presentaddress:CenterforCraniofacialMolecularBiology,2250AlcazarStreet,CSA103,UniversityofSouthernCalifornia,LosAngeles,CA90033,USA.0014-