Relevance of the Diversity among Members of the Trypanosoma Cruzi Trans-Sialidase Family Analyzed with Camelids Single-Domain Antibodies 英文参考文献.docVIP

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Relevance of the Diversity among Members of the Trypanosoma Cruzi Trans-Sialidase Family Analyzed with Camelids Single-Domain Antibodies 英文参考文献.doc

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Relevance of the Diversity among Members of the Trypanosoma Cruzi Trans-Sialidase Family Analyzed with Camelids Single-Domain Antibodies 英文参考文献

RelevanceoftheDiversityamongMembersofthe TrypanosomaCruziTrans-SialidaseFamilyAnalyzedwith CamelidsSingle-DomainAntibodies LauraRatier1.,MarielaUrrutia2.,Gasto′nParis2,LauraZarebski2¤,AlbertoC.Frasch1*,FernandoA. Goldbaum2* 1Instituto de Investigaciones Biotecnolo′gicas-Instituto Tecnolo′gico de Chascomu′s (IIB-INTECH), Universidad Nacional de General San Mart?′n-CONICET, Buenos Aires, Argentina,2Fundacio′nInstitutoLeloir,InstitutodeInvestigacionesBioqu?′micasBuenosAires-CONICET,BuenosAires,Argentina Abstract ThesialicacidpresentintheprotectivesurfacemucincoatofTrypanosomacruziisaddedbyamembraneanchoredtrans- sialidase(TcTS),amodifiedsialidasethatisexpressedfromalargegenefamily.Inthiswork,weanalyzedsingledomain camelid antibodies produced against trans-sialidase. Llamas were immunized with a recombinant trans-sialidase and inhibitorysingle-domainantibodyfragmentswereobtainedbyphagedisplayselection,takingadvantageofascreening strategyusinganinhibitiontestinsteadoftheclassicbindingassay.Foursingledomainantibodiesdisplayingstrongtrans- sialidase inhibition activity against the recombinant enzyme were identified. They share the same complementarity- determiningregion3length(17residues)andhaveverysimilarsequences.Thisresultindicatesthattheylikelyderivedfrom auniqueclone.ProbablythereisonlyonestructuralsolutionfortightbindinginhibitoryantibodiesagainsttheTcTSused for immunization. To our surprise, this single domain antibody that inhibits the recombinant TcTS, failed to inhibit the enzymatic activity present in parasite extracts. Analysis of individual recombinant trans-sialidases showed that enzymes expressed from different genes were inhibited to different extents (from 8 to 98%) by the llama antibodies. Amino acid changes at key positions are likely to be responsible for the differences in inhibition found among the recombinant enzymes.Theseresultssuggestthatthepresenceofalargeanddiversetrans-sialidasefamilymightberequiredtoprevent theinhibitoryresponseagainstthisess