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@PHDTHESIS{Heine:822226,
author = {Heine, Viktoria},
othercontributors = {Elling, Lothar and Schwaneberg, Ulrich},
title = {{S}ynthesis of neo-glycoproteins for binding studies and
scavenging of {C}lostridium difficile toxin {A} - of
microgels and biosensors},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {RWTH Aachen University},
reportid = {RWTH-2021-06608},
pages = {1 Online-Ressource : Illustrationen},
year = {2021},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2021},
abstract = {Clostridium difficile infections cause enormous costs in
the health care sector. The bacterium colonizes the human
intestinal tract and secretes toxins that destroy the
epithelial cell layer of the colon. The toxins bind to
cell-surface glycans on human intestinal cells and induce
processes that eventually lead to cell death. Since toxin A
comprises a carbohydrate recognition domain with combined
repetitive oligopeptide domains, it can bind multiple glycan
ligands at once. This feature can be exploited for
increasing the interaction strength between ligand and toxin
by multivalency and can be transferred to other toxins
comprising carbohydrate recognition domains. To find the
best possible toxin ligand for C. difficile toxin A, we
established an in-plate ligand library. Multivalent
neo-glycoproteins served as scaffold for complex
glycosylation patterns to assemble a variety of 40 glycan
structures. Screening of this library with the receptor
domain of toxin A presented Lewisy-Lewisx-decorated BSA as
promising toxin scavenger. Tests with the holotoxin
confirmed the findings and the ligand was produced for in
vitro applications. For the production of the glycan
(Lewisy-Lewisx), a set of efficient fucosyltransferases was
established and tested with a range of new substrates
(N-acetyllactosamine tetrasaccharides), revealing unexpected
fucosylation patterns. The toxin A scavenger was produced
and coupled to BSA. They were further utilized for coupling
to microgels. Neo-glycoproteins and
neo-glycoprotein-presenting microgels were able to protect
human cells from toxin A. Furthermore, the neo-glycoproteins
showed a good affinity to the Cholera toxin. Hence,
Lewisy-Lewisx-BSA is considered a one-for-two scavenger. To
enable a deeper understanding of the binding behavior, a
neo-glycoprotein biosensor was established.
BSA-neo-glycoproteins carrying N-acetyllactosamine were
bound to the biosensor and glycosylated on-chip. Online
measurements enabled the real-time investigation of kinetic
binding processes between glycan and enzyme or glycan and
lectin via electrochemical impedance spectroscopy for the
first time. This approach could be transferred to a variety
of interaction partners. Summarizing, this work paves the
way towards the treatment of bacterial-associated diseases
by establishing toxin scavengers and developing novel
analytical methods for toxin-ligand interactions.},
cin = {162820 / 160000},
ddc = {570},
cid = {$I:(DE-82)162820_20140620$ / $I:(DE-82)160000_20140620$},
pnm = {SFB 985 C03 - Zielgerichtete multi-funktionalisierte
Mikrogele für entzündliche Darmerkrankungen (C03)
(221477736) / DFG project 191948804 - SFB 985: Funktionelle
Mikrogele und Mikrogelsysteme (191948804)},
pid = {G:(GEPRIS)221477736 / G:(GEPRIS)191948804},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2021-06608},
url = {https://publications.rwth-aachen.de/record/822226},
}
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