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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, 2010Recognition of Cytoskeletal Proteins Monoclonal IgGs byCSF114(Glc), the Synthetic Probe of Multiple SclerosisShashank Pandey 1,2 , Duccio Lambardi 1,3 , Feliciana Real-Fernández 1,2 ,Maria Claudia Alcaro 4 , Elisa Peroni 1,5 , Mario Chelli 1,2 ,Anna M Papini 1,2,5 , Francesco Lolli 1,6 , and Paolo Rovero 1,3,41 Laboratory of Peptide & Protein Chemistry & Biology, Polo Scientifico e Tecnologico,University of Florence, Sesto Fiorentino (FI), Italy; 2 Department of Chemistry and CNRICCOM, Via della Lastruccia 3/13, University of Florence, I-50019, Sesto Fiorentino (FI),Italy; 3 Department of Pharmaceutical Sciences Via Ugo Schiff 6, University of Florence,I-50019, Sesto Fiorentino (FI), Italy; 4 Toscana Biomarkers Srl, via Fiorentina 1, I-53100,Siena, Italy; 5 Laboratoire SOSCO-EA4505, Universitè de Cergy-Pontoise, Neuville-sur-Oise, F-95031, Cergy-Pontoise, France; 6 Department of Neurological Sciences & AziendaOspedaliera Careggi, Viale Morgagni 34, University of Florence, I-50134, Firenze, ItalyIntroductionMultiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervoussystem (CNS), the pathogenesis of the disease has not been yet elucidated. In our previousstudy, we demonstrated for the first time that CSF114(Glc) (Figure 1) is able to recognizethe presence of specific autoantibodies (94%)in the sera of a statistically significant numberof Multiple Sclerosis patients with highN7G 8Fig. 1. Ribbon diagram of the lowestenergy conformer of 200 calculatedstructures of CSF114(Glc) derived fromNMR data.specificity. No false positive data wereobtained with sera from patients affected byother autoimmune diseases or otherneurological diseases. Therefore, CSF114(Glc)is a simple, reliable, and efficient tool, whichclearly shows that an aberrant N-glucosylationis involved in autoantibody recognition inMultiple Sclerosis [1-5].We report herein for the first time therecognition of specific cytoskeletal proteinsmonoclonal IgGs antibodies and itscrossreactivity with the artificially N-glucosylated peptide sequence.Results and DiscussionFrozen rat brain was homogenized andsolubilized in tritonX-100. Solubilizedfractions were analysed in 12% SDS-PAGE.Extracted proteins were transferred ontoNitrocellulose membrane and western blotswere performed using affinity purified anti-CSF114(Glc) IgGs from Multiple Sclerosispatients’ sera and normal blood donors ascontrols. We constantly found in Multiple Sclerosis three positive bands recognizingaffinity purified anti-CSF114(Glc) IgGs and corresponding to 130kDa, 98kDa, and 47kDa.On the contrary, normal blood donors’ sera displayed no relevant recognition.Bands of interest from SDS-PAGE were excised and trypsin digested. Digestedsamples were analyzed by MALDI-TOF followed by MS/MS analysis. We succeeded inidentifying 2’,3’-Cyclic-Nucleotide 3’ Phosphodiesterase, Creatine kinase BB, Alphaactinin, and Alpha fodrin corresponding to positions 47kDa, 98Da, and 130kDarespectively (Table 1).290