@phdthesis{Buchholz2021,
  author    = {Ina S. L. Buchholz},
  title     = {Impact of beta2-Glycoprotein I Post-translational Modifications on Protein Conformational Dynamics and Immunogenicity in the Context of the Antiphospholipid Syndrome},
  journal    = {Auswirkungen der posttranslationalen Modifikationen von beta2-Glykoprotein I auf die Proteinkonformationsdynamik und Immunogenit{\"a}t im Zusammenhang mit dem Antiphospholipid-Syndrom},
  url       = {https://nbn-resolving.org/urn:nbn:de:gbv:9-opus-58271},
  pages     = {142},
  year      = {2021},
  abstract  = {The soluble blood protein beta2-glycoprotein I (beta2GPI; 326 aa, MW: 48 kDa, 5 domains) is one of the most abundant proteins in human serum and exhibits two main conformational states: the circular or closed conformation, where the first domain (DI) is bound to the last domain (DV) of the protein, and the linear or open conformation. The defined physiological function of beta2GPI is still unknown, though several roles in pro- and anticoagulation as well as oxidative stress protection were discovered. The open form is considered to play a crucial role in the systemic autoimmune disease antiphospholipid syndrome (APS), which is an acquired thrombophilia characterized by recurring thrombotic events and pregnancy morbidity. Beta2GPI constitutes the main antigen for APS autoantibodies which are supposed to bind a cryptic epitope within DI after a conformational change from closed to open form. However, the pathophysiological mechanism of APS is poorly understood. Therefore, investigating the structural dynamics of this protein in relation to its antigenicity is of high interest. Post-translational modifications (PTM) of a target protein often show an impact on the formation of neoantigens, for instance in the autoimmune-mediated diseases type 1 diabetes mellitus, rheumatoid arthritis, or multiple sclerosis. Such modified antigens may lead to immune tolerance breakdown as they are unknown to the immune system, which therefore could mistakes self for non-self proteins. In this thesis, two frequently occurring PTM were introduced to beta2GPI and their impact on the protein conformation was studied by biophysical tools (i.e. atomic force microscopy (AFM) imaging, transmission electron microscopy (TEM) imaging, dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy). In order to examine immunopathophysiological relevance of these PTM, additional insights were gained from ELISA which was used to examine binding of anti-DI autoantibodies purified from the blood of APS patients to the modified beta2GPI species. A characteristic feature of beta2GPI is the high content of lysine residues. Previously, opening of beta2GPI was found to be triggered by a drastic shift in pH and salt concentration (pH 11.5 and 1.15 M NaCl), which results in reversible uncharging of the lysine residues. The aim of this study was to investigate the beta2GPI conformation after lysine acetylation as a model system, to elucidate the role of lysine residues on the conformational dynamics of this protein, and to examine anti-DI autoantibody binding to both the untreated as well as acetylated species. A strategy to permanently open up the closed form under physiological conditions by chemical acetylation of lysine residues utilizing the sensitive acetylation agent acetic acid N-hydroxysuccinimide ester (NHS-Ac) was established. Complete and specific lysine acetylation was verified by quantification of primary amines exerting a fluoraldehyde o-phthaldialdehyde (OPA) reagent assay, as well as by native PAGE and western blot analysis with an anti-acetylated lysine antibody. Beta2GPI acetylation revealed a partial opening of beta2GPI molecules. Compared to untreated, i.e. native beta2GPI which exhibited 93\% of the molecules in closed and 7\% in open form, complete lysine residue acetylation generated 39\% of beta2GPI in closed and 61\% in open conformation as shown by AFM high-resolution imaging. pH 11.5-treated beta2GPI was used as a reference in the applied methods and revealed 38\% of the protein in closed and 62\% in open conformation. Thus, a significant shift in beta2GPI conformation occurred upon lysine residue acetylation as well as basic pH-treatment. The data indicate that lysine residue acetylation destabilizes the closed form, leading to a facilitated opening of the structure. The closed conformation might be predominantly stabilized by electrostatic interactions of lysine residues, which potentially control the conformational dynamics of this glycoprotein. ELISA confirmed that anti-DI autoantibodies do not bind to untreated (closed) beta2GPI. Although acetylated beta2GPI was shown to have a substantial portion of open proteins, no binding of anti-DI autoantibodies to the acetylated species was found either. Hence, acetylated lysine residues may disrupt the immunorelevant epitope in DI which prevents antibody binding. This finding reveals a new hint for epitope organization. However, further detailed epitope mapping has to be performed. Beta2GPI carries two structural disulfide bonds per domain, whereas an additional disulfide bond Cys288/Cys326 is located at the C-terminus of DV near the putative contact interface of DI and DV in the closed conformation. It was previously shown that beta2GPI is a substrate of thiol oxidoreductases, including human thioredoxin-1 (Trx-1) generating different redox states of disulfide bond Cys288/Cys326, which might serve as a scavenger in oxidative stress protection in the blood stream. In APS patients, anti-DI antibody titers as well as an enhanced risk for thrombotic events are associated with an increase in the oxidized state of the protein. Hitherto, no structural study has been performed in order to prove a correlation of the redox state and the conformation of beta2GPI. Therefore, investigations of beta2GPI conformation in different redox states of disulfide bond Cys288/Cys326 were carried out. In addition, binding of anti-DI autoantibodies to the untreated (native) as well as reduced protein should be explored. At first, cysteine residues of untreated, i.e. native beta2GPI were confirmed to be completely in oxidized state using Ellman’s reagent assay and the absence of binding of a thiol-specific agent. Statistical analyses of AFM images revealed that untreated beta2GPI was mainly in closed conformation (80\% in closed and 20\% in open conformation) in the respective system. In this study, an optimized protocol for enzymatic reduction of disulfide bond Cys288/Cys326 was established. The agent TCEP was used to reduce human Trx-1, which in turn enzymatically reduced beta2GPI. To block reoxidation of free thiols and to facilitate product analysis, cysteine residues of reduced beta2GPI were subsequently labeled with the sensitive and thiol-specific reagent 3-(N-maleimidopropionyl) biocytin (MPB), which carries a biotin function. During protocol establishment, complete and specific reduction of disulfide bond Cys288/Cys326 was confirmed utilizing SDS-PAGE, streptavidin western blot, mass spectrometry (MS) analyses, and a biotin quantification assay. Protocol improvements constituted a homogenous system with remarkable decrease of unspecifically reduced beta2GPI. Upon beta2GPI reduction, AFM imaging revealed no significant shift in protein conformation (75\% in closed and 25\% in open conformation). These results were qualitatively confirmed by TEM imaging. Therefore, reduction of beta2GPI disulfide bond Cys288/Cys326 did not result in a major conformational change of the protein. Upon in vitro reduction, the closed form is still the main conformation and a direct correlation of beta2GPI redox state and conformation must be refused. Furthermore, beta2GPI reduction led to a strong and statistically highly significant increase in anti-DI autoantibody binding compared to untreated beta2GPI. Thus, the reduced form might be the antigenic form of the protein. In contrast to previous knowledge, these findings suggest that anti-DI autoantibodies may also bind to the closed conformation under certain conditions. Hypothetically, reduction of beta2GPI could induce a minor structural change in DV that might facilitate the binding of APS autoantibodies. Overall, this study reveals that PTM of beta2GPI may lead to a critical level of destabilization of the closed conformation (as in the case of acetylated beta2GPI) or significantly increase the binding of APS autoantibodies (as in the case of reduced beta2GPI), both of which could have a large impact on APS disease. However, further investigations are necessary to put these new findings in the context of APS immunopathophysiology.},
  language  = {en}
}