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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Celiac Disease: Synthesis of Overlapping Linear PeptideEpitopes of tTG[Aa(1-230)]Margherita Di Pisa 1,2 , Giuseppina Sabatino 1,3 , Mario Chelli 1,2 ,Paolo Rovero 1,5 , Claudio Tiberti 6 , and Anna Maria Papini 1,2,41 Laboratory of Peptide & Protein Chemistry & Biology, University of Florence, I-50019,Sesto Fiorentino, Italy; 2 Department of Chemistry “Ugo Schiff” and CNR-ICCOM,University of Florence, I-50019, Sesto Fiorentino, Italy; 3 Espikem Srl, I-59100, Prato,Italy; 4 Laboratoire SOSCO – EA4505 Université de Cergy-Pontoise, Neuville-sur-Oise,F-95031, Cergy-Pontoise, France; 5 Department of Pharmaceutical Sciences, University ofFlorence, I-50019, Sesto Fiorentino, Italy; 6 Department of Clinical Sciences, PoliclinicoUmberto I, Sapienza, University of Rome, ItalyIntroductionCeliac Disease (CD) is considered an autoimmune disease characterized by villous atrophyand inflammatory cell infiltration of the lamina propria triggered by the gliadin fraction ofwheat gluten and similar alcohol soluble proteins called prolamines. CD has a complex andmultifactorial aetiology and occurs in genetically susceptible individuals in association withenvironmental factors (gliadin), leading to a chronic inflammatory condition [1]. The glutenintake leads to a complex immune response, both T cell mediated and humoral responseleading to the secretion of autoantibodies. The endomysial protein tissual Transglutaminase(tTG) is considered the main autoantigen in CD playing a key role in the pathogenesis [2].Untreated CD patients have high levels of circulating IgA and IgG directed to differentautoantigens, i.e. tTG, gliadin, and endomysium that are directly involved in mucosalinjury [3]. Up to now the “gold standard” for the diagnosis of CD is the bowel biopsy eventhough simple serological tests could have a high impact as non invasive diagnostic tools.Reliable assays are necessary not only for an early diagnosis but also for monitoring thedisease activity evaluating antibodies as specific biomarkers [4]. The improvement of thesensitivity of serological tests could be obtained by substituting extracted or recombinantantigens with synthetic probes based on peptide epitopes [5]. The screening of overlappingpeptides libraries is an excellent strategy to identify the minimal epitope recognised as anantigen [6]. Therefore, the characterization of tTG antigenic domains is a crucial step inunderstanding onset and developement of CD. Sera from patients with CD at first diagnosiswere demonstrated to have high levels of auto-antibodies recognising distinct tTG’functional domains. In particular, Tiberti et al. showed that there is an evidence of aspecific epitope loss of anti-transglutaminase immunoreactivity in gluten free diet celiacsera (6 and 24 months after the diagnosis), which is supposed to be only against protein N-terminal portion [7]. Our aim was to characterize linear autoantigenic epitopes by testing inceliac patients’ sera the reactivity of different overlapping synthetic peptide fragments oftTG [aa(1-230)]. We performed an epitope mapping based on the “Chemical ReverseApproach”, synthesizing 23 overlapping peptides of tTG(1-230) by SPPS. The pentadecapeptideswill be useful for RIA, ELISA, and SPR to evaluate the IgA response againsttTG specific epitopes in gluten free diet celiac patients’ sera.Results and DiscussionThe activity of peptide fragments of tTG(1-230) (Table1) was qualitatively andquantitatively evaluated in inhibition experiments of the 35 S tTG-antibody binding inpatients’ sera. The gluten free diet patients’ sera used in the assay were selected becausethey recognise in RIA antibodies to full length tTG protein but not of the protein fragmentstTG(227-687) and tTG(473-687).492