抗还原并烷化的AChE单克隆抗体识别的抗原决定基

抗还原并烷化的AChE单克隆抗体识别的抗原决定基 抗还原并烷化的AChE单克隆抗体识别的 抗原决定基 BIBLID.LSSN02539756ActaPh0l口Sica9iniea中国药理学植 1994May}15(3):193—196 Epitopesr . ecognizedbyanti—reducedandalkylatedacetylcholinesterase antibodies WANGYu—Xia,X1NYan—Bin.,SUNMan—Ji (IsstitutePhaemacolo99dToxicologg,,l”fPMicrobiologyand却 idemiolo99’ AcademyMilitaryMedicalclPncP,Beifin9100850,China) ABRACTPeptidesofthereducedand alkylatedacetylcholinesterase(RA—AChE) fromtheelectricorganofTotpediniformes Torpedof.edosubjectedtobromo—cynogen (CNBr)cleavageor/andpepticdigestionCOn— Serredwelltheantigen—antibodyreactivity withanti—rRA?AChEmonoclonalantibodies E9,F6,andF12,whereaspeptidesproduced byCNBrandtryptictreatmentslostal1there— activity.PeriodateoxidationoftheRA— AChEorglycopeptidasedigestionoftheCNBr cleavedRA—AChEdidnotchangetheantigen— antibodyreactivity-Itimpliedthattheepi— topesrecognizedbythe3anti—RA-AChEmon— oclonalantibodiesRreallpeptidedeterminants ratherthancarbohydratedeterminants. KEYW0RDSacetylcho1inestefas0;mono? clonalantibodies;antigenicdeterminants Acetylcholinesterase(acety1choIineace— tylhydrolase,EC3,l,1,7,AChE)isoneof theessentialenzymesinthenervoussystem. 1mmunologicalpropertiesoftheenzymepro— teinhavebeeninvestigated.However. 1ittleisknownaboutitsantigenicdetermi— nants(epitopes)whichisreckonedasanother importantdomainoftheenzymenexttothe catalyticcenter.ItbecomesamajorjSSUein thestudyofthestructureofenzymeprotein. Inthispaper,AChEpurifiedfromtheelectric organofTorpediniformesTorpedo,Dedo Received1993—06—03Accepted1994—0118 ProjectsupportedbytheNatJonaJNaturalScienCeFounds tionotChina.3F2.389702l1. wasreducedandalkylatedtounfoldtheen- zymemoleculeforthoroughexposureofthe continuousantigenicdeterminantsburiedin theproteinmolecule.Monoclonalantibodies directedtothereducedandalkylatedacetyl— cho1inesterase(RA—AChE)weremadeuseof inexplorationoftheepitopeproperty? MATERlALSANDMETH0DS o-Phenylenediamine,purchasedfromMerck} trypsin,pepsinandglycopeplidaseF,fromSirma; CNBractivatedSepharose4B,fromPharmaciaLKB; horse-rad~hperoxidaselabeledgoat—anti-mouseIgG, productofInstiluteofMicrobiologyandEpidemiolo— gY}otherreagentswereallchemicallypure-ELISA microplales,producedbyTlanjinFlexiglassFactory; TitertekMultiskanTypeMcc/340MKII?Flowprod- uct- RA—AChEandanti—RA—AChEmonoclonalanti- bodyascitesw船prepared- ELISAofsmailmolecularpeptidew船carried out.Themicroplateswerepretreatedwith200ul/ wellofthecoatingbuffer(phosphatebuffer50mmol ?L,,pH8.0)containing0.Il,5-pentadialfor3 h,washedoncewiththecoatingbuffer(200~l/wel1), andthenwentonforEL1SA. CleavageofRA-AChEbyCNBrAllquots(5 mg)oflyophllizedRA-ACHEwe托dissolvedin0?2mI CNBrsolution(10mg?mr,in70formicacid), incubatedat25?innitrogenfor4,l2,or24h,and mixedwith1.8m】delonlzedwaIefandlyophil~ed again? TrFptlcdige2ttlonofCNBr—Clea~edpeptideCN— Br—Cleavedpeptides(5mg)ofRA—AChEweredis— solvedin2mlofNH’HCOs50mmol?L(pH8.0), andmixedwith0-2mIofpackedSepharose4Bimmo— bilizedwithtrypsin.Aftershakingat37?for16h. thefltratewaslyophilt2ed? 194.BIBLID.ISSN0253—9759ActaPharmaco]ogicaSinica中国药理学 植1994M4I5(3) PepticdigestionofCNBr-Clea~edpeptldeCN— Br-Cleavedpeptides(5mg)ofRA-AChEweredis— solvedjn2.5mIHAc50mmol?L,,andincubated with50Jagpepsin(in50tzideion~edwater)at37? for18h?Thehydrolysatewasheftedfor5rainand thenlyophilized. GlycopeptldueFtreatmentCNBr—Cleaved RA-AChEpept~es(1mg)wereincubatedwith0.2U glycop~ptidaseFjn0.5mtphosphatebuffer50mmol ?L,(pH7.0)at37?for18handheatedat100C for3raintoterminatethereaction. Periodateo~ldatlon”.T0theant~en(RA— AChE)-coatedmicropIate,200~l/weilofwashing buffer(phosphatebuffer10mmel?L,,0.05 Tweed20,ph7.2)wasadded.Themicroplatewas washedthrice30minlater.Afterwashingwithac— etatebuffer50mmol?L(pH4.5),theperiodate (20mmol?L—inacetatebuffer,200~l/wel1)was addedtoeachwelltTheplatewasJeffindarkforl h,andthenwashedwithacetatebuffer3times. Glycine(1,in50mmol?Lphosphatebuffer,pH 8.0,freshlyprepared)wasusedforblockage(30 min).ELISAwasParriedoutafter5cyclesofwash ins.Ac~tatebufferwasusedinthenod—oxidizedon— troIinsteadoftheperiodate. RESULTSANDDISCUSSIoN CleavageandcuttingofRA—AChERA— AChEwaschemicallytieftbyCNBrat25? for24handthencutwithtrypsinorpepsin. AsshowninSDS—PAGE,thecleavage0fRA AChEwasnearlYCOTEIpletedafterCNBrtteat— mentfor24h(Fig1b,c)andtheenzymatic CUttingrenderedthecleavageevenmorether— oughly(Fig1d?e). ImmUllOreactlvityofantibodieswithRA— AChEanditscleavedpepItidesImmunoreac— tivitYofmofloclona1antibodiesE9,F6.and Fl2inmouseasciticfluid(1:102,1{l0di— lution)wastestedwithRA—AChE,CNBr— RA—AChE,trypsin—CNBT—RA—AChEor pepsin—CNBr—RA—AChE(0.5pg/welbyCNBranddisestedbypepzln.b) RA?AChEclovebyCNBranddigestedbytrypsin.c) RA?AChEclo~ebycNBrfor24h.d)RA—AchE clel1.~edbyCNBrfor4h.e)Marker:pholphorKse B,9?kDa’beneserumIlburnin,B7kDIlactIn.43 kDaIcarbonicanhydrase,30kDaIrMVcoatprotein, 17.5kDa. antigenicity(Tab1).Theresultsimplied thatepitopesdirectedbyE9,F6,andF12 mightnotcontainmethionine,phenyIaIanine, tryptophan,andleucineresidues.Butlysine and/orarginineresidueprobablyconstituted themostimportantconstituentsoftheepi— topes? Propertyofepitopesrecognizedbyanti— RA—AChEmono~lonaIantibodiesAChE妞a glyeoproteinofknownmolecularstrUCture. Thespecificsequencesofthepolypeptides and/oritscarbohydratesidechainsmightcon— stitutetheepitopes?Changesintheantigen— antibodyreactionsbetweenanti—RA—AChE BIBLID;1SSN02539756Ac妇Pharmae0JogicaSiniea中国药理学担 1994May;15(3)?195? Dilut-on Absorbaneeat(ELISA) RAAChE—CNBrRA-A”E=cNB卜RA—A”E_cNB tryps111pepsin Tab2?InfluenceofperlodlteoxidationorglycopeptldaseFtreatmentonantig enieityofreducedindalkylJted AChEanditsdelvedpeptlde$.,l一3,i土s. mortoclonalantibodiesandAChEoritscleaved peptidesbeforeand/Orafterthemodiflcation ofearbohydratesidechainsbyg1yc.peptidase hydrolysisorperiodateoxidationwouldrefleet somehowthefeaturesoftheepitopes.The resultsshowedthatthemodificationsdidnst influencethereactivitybetweenantigensand monoclonalantibodies(Tab2).The3eoi— topesdirectedbyE9,F6,andF12allbelong tothepeptide—typeratherthanthecarbohy— drate—type.Theyareburiedinsidethenative enzymemolecule,andtheglycosylationmight occuronlyattheglutamineresidueseXpOSed t0thesurfaceoftheprotein. ACKNOWLEDGMENTSWearegratefulto ZHANGHan,LIFeng—Zen.andWANGFei forthehelpinexperiments. REFERENCES lSakaiM,SaisuH?KoshlsoeN.AbeT.Deltergen solubleform0fae*tyteholiaesteTaseintheelectricotg~,nof electricrays-EurJBiochem1985:1’B:197—206. 2DortorBP?CampS,GentryMK.TaylorSS.TaylorP. AntlgeniecadstrueturaldIfkrtinthecatalvtub units0fthemolecularf0rmsofacetylcholinester~e Pr.cNatlAcadSeiUSAI983;aD:5767—71. 弓年,D7 BIBLID:...一.枢;.. 8SakaiM+K~higoeN.SaisuH,AbeT.Monoclonal &ntibodiesspecifictoasymmetricformsofa~ty[ eholin~t?efromtheelectricorganofeleetTl~rays? BiomedRes1986’7t1—5 4LappinRI,RubEnLL+LieberburgIM?Generationof subunit—sl~eificantibo4yprobesforTorpPdoacety】一 Cholinesterase:Cross—speciesreaetivhyanduin】I_free tray.slatIon.JNeurobioi1987I18:75—99. 5XJnYB,WangYX+SunMJ.Anti—unfoidedacetyi— eholinester~,eocj0naIantjbodies. ChinJPharmaeo】Toxieao】1993’7j56—9. 6WangXN?MaJ,GaoJM?LiWJ,MaXK,Wan8MX. Bio]ogJcalchsracteristicsofpolyclonalandmonocinnal aatibodlesag~nsthumaninter]eukin一2. JMonoclona]Antibody1990’?:5—10. 7LeeSL,CampSJ,TaylorP.Characterizationofa hydrophobic,dimericformofae*tytchotin~tetasefrom To.JBlotChem1982F257{12302—9. 8GrossE?WitkopB.NonenzymatIccleavageofpeptide bonds:themethioniueresiduesinbovin0pancreatic r.bo…】ease.JBlotChem1962’257:1856—60. 9Gibney0.MacPhee—Qu吨leyK,ThompsonB.Vedviek T?LowM0?TayiorSS-PdfDivergenceinprimary socturebetweenthemo]ecaIarformsoftyl cho]inesterJBioJChem1988;285:1140--5. 10W.odwardMP.YoangWWJr+B]oodgoodRA.Detec— lionofmono~]otmlantibodiesspecificforcarbohydrate epitop~usingperindateoxidation- JImm…IMethods1985’78:】43—53. 抗还原并烷化的AChE单克隆抗体识别的 抗原决定基 ,主生坐剥懂霁罗7 (军事医学科学院毒物药物研究所}.微生物与流行府 研究所,北京100850,中国) 摘要电器官还原并烷化的己酰胆碱酯酶 (RA—AChE)的溴化氰裂解肽或又经胃蛋白酶 切割的肽与抗RA—AChE单克隆抗体E9,F6 及F12仍有抗原抗体反应.经溴化氰及胰蛋 白酶割的肽与3者反应消失.氧化破坏RA— AChE糖侧链,溴化氰裂解的RA—AChE又经 酶切除糖侧链后,与3者反应无变化.E9, F6及F12所识别的抗原决定簇是多肽型,不 是多糖型. 关键词睦堕堡塑墼;单克隆抗体 《医学文摘》一医学世界之窗 《医学文摘》已于1993年9月扩大服务范围至中国读者. 着已建立的国际资讯网络,将传运给读者实用,精选的讯息. 英文编写并注有中文摘要. 发行于超过四十多国家的医学文摘,一本初衷,藉 以简便易读的形式,提供丰富的医学新知.全书以 本月刊现选择性地提供给国内专业医师阅读.其他医务人员及药业 人才如欲订购,可直接来信邮购 一 年港币......$120(12期) 二年港币......$192(24期) 出版爱发行:《医学文摘》 地址:香港湾仔骆克道200号东新商业中心1501室 电话;(852)5199303传真:(852)50738l7