NR ATOJ
AU Boetel,T.; Bade,S.; Schmidt,M.A.; Frey,A.
TI Identification and evolution of (poly-)peptidic PrP-ligands and their implementation in a novel preventive strategy against food-borne TSE
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Human prions, risk of blood products, and therapy HUMAN-33
PT Konferenz-Poster
AB
Being misfolded isoforms of a self-antigen prions are likely to evade mucosal immune defense. Thus, passively protecting intestinal mucosae against the entry of prions by trapping the infectious material would be a promising tool to prevent food-borne TSE. In order to develop prion-specific enterosorbent bacteria exposing a prion protein(PrP)-ligand as part of an outer membrane protein, we are searching for digestion-resistant, high-affinity PrP-binding peptides and polypeptides.
Putative PrP-binding proteins were chosen from the literature and their linear binding sites were identified and characterized with respect to core binding motif and essential amino acids by epitope mapping with fluorophor-labeled recombinant murine PrP fragment 90-231. The most promising motifs descending from laminin receptor precursor (LRP), neural cell adhesion molecule (N-CAM) and plasminogen (PLG) were tested for resistance to intestinal proteases and inserted into a permissive site of the E.coli AIDA autotransporter. All peptides were degraded by intestinal juice in milliseconds and PrP-capturing by ligand-expressing bacteria in pull-down-assays was observed for the AIDA-N-CAM chimera only. It bound 3-times more PrP than control cells. In contrast to that, a 5-fold raise in avidity was achieved when kringle-domain 3 (K3) of murine PLG was used as binding site. It also displayed a half-life of 1 min after incubation of whole bacteria in murine small intestinal lavage. As performance of discontinuous binders like K3 is difficult to improve, we are aiming to increase the affinity and intestinal stability of the N-CAM peptide using an evolutionary molecular breeding technology. Already after a first round of breeding candidates with 3-fold improved affinity could be detected by the in vitro assays. We conclude that autotransporter-mediated display of PrP-binding (poly-)peptides is feasible, but to confer effective protection, affinity and stability of current ligands must be enhanced.
AD T.Boetel, S.Bade, A.Frey, Division of Mucosal Immunology, Research Center Borstel, Parkallee 1-40, D-23845 Borstel, Germany; M.A.Schmidt, Institute of Infectiology, Center for Molecular Biology of Inflammation, University of Münster, Von-Esmarch-Straße 56, D-48149 Münster, Germany
SP englisch
PO Deutschland