Formstabile Peptidepitope für die personalisierte Diagnostik der rheumatoiden Arthritis

Die molekularen Grundlagen der Autoimmunität wurden mit Hilfe von Filaggrin-abgeleiteten β-hairpin-Peptiden erforscht. Als Modellerkrankung diente die rheumatoide Arthritis (RA), in deren Krankheitsverlauf sich eine Vielzahl von Autoantikörpersubtypen ausbildet. Es wurden Antworten auf die Frage gef...

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Bibliographic Details
Main Author: Schrimpf, Andreas
Contributors: Geyer, Armin (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Published: Philipps-Universität Marburg 2018
Online Access:PDF Full Text
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Table of Contents: The molecular basis of autoimmunity was researched by utilising filaggrin derived β-hairpin peptides. Rheumatoid arthritis (RA) served as a role model. During its disease course, a wide variety of autoantibody subtypes emerges. In this context, answers to the question have been found, what actually makes a peptide an epitope, that is able to bind the aforementioned subtypes and ultimately distinguish them. Such peptide epitopes were synthesised via solid phase peptide synthesis and could be used as diagnostic tools for the early identification of rheumatoid arthritis (RA). Their conformational space was systematically varied by the correlated introduction of β-hairpin design elements (hydrophobic cluster, D-amino acids, disulfides, deletions etc.) and investigated in detail by NMR spectroscopy. The peptides’ diverse, unusual conformations and dynamics were then correlated with their biological activity against commercially available RA autoantibodies in ELISA tests. Based on these, a preselection of mutually complementary peptide antigens (regarding their conformation as well as their antibody affinities) were utilised in consecutive assays against blood sera. A barcode of measured absorptions in ELISA was thus obtained for every RA patient, which led to (indirect) identification of autoantibody profiles depending on the combinations of absolute and relative intensities among the employed antigens. These first tests deliver an important contribution towards a personalised RA diagnosis. At the same time, all applied methodologies can be transferred to other autoimmune diseases and their associated paratope-epitope-pairs. The deletion of amino acid pairs from the β-hairpin peptides did not only lead to highly affine epitopes, but also to valuable insights into the periodicity of this secondary structure. In this context, a disulfide-rich (every third amino acid of the 12mer hairpin constitutes a cysteine), antiparallel hinge peptide could be synthesised without elaborate protecting group chemistry. In a seminal bottom up approach, this biomolecule served as dimerisation domain in proteins and antibodies (the latter contain a parallel hinge region in their native state) by means of recombinatorial methods. This thesis provides a decisive contribution to the design, preparation and structural characterisation of β-hairpin peptide epitopes, their systematic and broadly applicable synthetic variation as well as their utilisation in autoimmune diagnostics. Beyond that, a reliably folding dimerisation domain has been created for the de novo design of proteins.