Prostate cancer (PCa) is the second most prevalent cancer among men worldwide when assessing age-standardized incidence rates. The primary method for early PCa diagnosis involves measuring the... Show moreProstate cancer (PCa) is the second most prevalent cancer among men worldwide when assessing age-standardized incidence rates. The primary method for early PCa diagnosis involves measuring the serum concentration of prostate-specific antigen (PSA), with elevated levels (> 3 ng/mL in the Netherlands) indicating the potential presence of PCa. However, the conventional PSA test exhibits a low specificity. Thus, clinical challenges persist, including the differentiation between PCa and benign prostatic hyperplasia and distinguishing indolent PCa from aggressive forms. This underscores the need for a more specific biomarker for early PCa detection and stratification. Previous studies have reported altered glycosylation features in two prostate-secreted glycoproteins, PSA and prostatic acid phosphatase (PAP) in PCa patients, e.g. variation in sialylation, fucosylation and the level of LacdiNAc . The aim of this thesis was to identify PCa biomarkers for early detection and to improve patient stratification, focusing specifically on the glycomic profiles of PSA and PAP. In addition, as PSA plays an important role with regard to fertility, its glycosylation -in relation to male infertility- was also touched upon. For this purpose, mass spectrometry (MS) based glycoproteomic methods were established to map the glycoprofiles of PSA and PAP derived from various biofluids. Show less
In a typical glycosylation reaction, a donor is activated to form a (variety of) electrophilic species which can react with a nucleophilic acceptor, following a reaction mechanism having both SN1... Show moreIn a typical glycosylation reaction, a donor is activated to form a (variety of) electrophilic species which can react with a nucleophilic acceptor, following a reaction mechanism having both SN1 and SN2 character. On the SN1-side of the spectrum oxocarbenium ions partake in the mechanism while covalent intermediates act as the product forming intermediates on the SN2-side. The result of a glycosylation reaction is influenced by the properties of both donor and acceptor and can be affected by external factors as well. Because the challenges in carbohydrate synthesis are typically solved for a specific synthetic problem, a general understanding of what effect changing a single variable has on the mechanistic pathway, and thus the (stereochemical) outcome is not available. This in turn means that synthesis of glycosidic bonds typically requires a considerable amount of optimisation at the expense of a significant amount of time and resources. The goal of this thesis is to systematically investigate how changes in stereochemistry and protecting group patterns on the donor and acceptor affect the mechanism and outcome of glycosylation reactions, to enable a more rational design of synthesis routes. Show less
The research described in this thesis focused on the use of bioorthogonal antigens to investigate immunological processes in antigen presenting cells. Bioorthogonal antigens are antigenic proteins... Show moreThe research described in this thesis focused on the use of bioorthogonal antigens to investigate immunological processes in antigen presenting cells. Bioorthogonal antigens are antigenic proteins produced through recombinant expression in a methionine auxotrophic E. coli strain. This allows for the replacement of methionine residues with the bioorthogonal non-canonical amino acid, azidohomoalanine (Aha), that resembles methionine. Aha contains an azide group that enables the selective and rapid visualization or enrichment of the antigen after a biological experiment using alkyne-modified fluorophores or alkyne-containing resins, respectively, via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The research involved studying the effects of post-translational modifications (PTMs), antigen complexation and glycosylation of antibodies in immune complexes on the uptake, proteolysis, and T cell activation by dendritic cells (DCs) of Aha-containing antigens. Additionally, a new method was developed to enrich low abundant bioorthogonal antigenic fragments from complex mixtures. This method can be used in future studies to identify processed Aha-containing fragments from immune cells that are preserved for T cell presentation. Show less
Protein glycosylation has profound implications in a wide range of molecular and biological processes occurring in cancer, where specific changes in the glycan structures have shown to be... Show moreProtein glycosylation has profound implications in a wide range of molecular and biological processes occurring in cancer, where specific changes in the glycan structures have shown to be associated with the development and progression of the disease paving the way for the development of new clinical biomarkers as well as providing specific targets for therapeutic intervention, patient stratification and personalized medicine. Protein glycosylation is also critical for the development of biopharmaceuticals, as even minor shifts in manufacturing procedures can substantially impact the bioactivity, safety, and efficacy of therapeutic proteins. Although a variety of mass spectrometric and chromatographic methods are available for the identification and characterization of glycans from complex sample mixtures, the lack of standardized protocols across platforms often results in inconsistent results, making data integration and comparison challenging. Furthermore, most of the current technology for the study of intact glycans would not be suitable for the rapid analysis of large sample sets, mainly due to limitations in sample throughput. The scope of this thesis is to establish standardized, high-throughput glycomics technologies for the quantitative analysis of protein N- and O-glycosylation and improve current methodologies in order to facilitate the characterization of intact oligosaccharides from in vitro established model systems. Show less
Proteins are widely known as key players that fulfill crucial roles at the molecular level in the human body but also for their involvement in many processes in everyday life. For example, proteins... Show moreProteins are widely known as key players that fulfill crucial roles at the molecular level in the human body but also for their involvement in many processes in everyday life. For example, proteins can be used as medicine in health care or for their enzymatic function in the food industry. All these proteins do not exist as a single species but rather as a complex mixture of structural variants, so-called proteoforms. This heterogeneity results mainly from the presence of post-translational modifications (PTMs), such as glycosylation and glycation. To further complicate this matter, these PTMs can induce structural as well as functional changes. To allow in-depth structural and functional characterization of these proteoforms, novel analytical approaches are required to resolve proteoform heterogeneity while persevering protein nativity. The hyphenation of native separation techniques with mass spectrometry has emerged as a powerful approach to reliably study these aspects. The work in this thesis describes the (further) development and application of such methodologies for biopharmaceutical and biotechnological products. Show less
Immunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19.... Show moreImmunoglobulin G (IgG) antibodies can exert their functions via both Fab-mediated neutralization and Fc-mediated effector functions, both of which are crucial for protective immunity in COVID-19. Importantly, effector functions and resulting inflammatory responses are impacted by the structure of N-glycans linked to the Fc-tail of IgG. Studying antibody glycosylation in emerging infectious diseases such as SARS-CoV-2 allows to gain insight into specific glycan signatures at the early stages of infection, and to investigate whether these reflect how the disease would progress. For example, low fucosylation is a common glyco-phenotypic signature of IgG1 produced against the spike (S) protein of severely ill SARS-CoV-2 infected patients early on in their disease course, but has likewise been described in other disease settings, where the antigen is presented in the context of host-cell membranes (Chapter 2). In this thesis, antibody glycomics signatures of SARS-CoV-2 infection and vaccination have been explored using an established liquid chromatography – mass spectrometry-based method relying on affinity-isolation and proteolytic digestion of both total and anti-S IgG. In Chapter 3, the glycosylation of SARS-CoV-2 anti-S IgG antibodies were found to be vastly skewed relative to total IgG and to change in a highly dynamic fashion. Moreover, IgG glycosylation was shown to be an early severity marker and showed patient stratification potential, with predicting power for intensive care admission within a hospitalized patient population. Early detection of a pro-inflammatory glycosylation pattern may provide a broader intervention window and decrease the number of ICU-admissions. Furthermore, anti-S IgG1 glycosylation levels obtained with LC-MS show promise to supplement clinical parameters and biomarkers of inflammation, that have together been used for the severity score calculation of hospitalized COVID-19 patients. Similarly to SARS-CoV-2 infection, antibodies generated against the spike protein upon BNT162b2 mRNA vaccination also induced a transient afucosylated anti-S IgG1 response in antigen naïve individuals, albeit to a lower extent than in severely ill patients, exemplifying the influence of the type of immunization on antibody glycosylation (Chapter 4). Upon vaccination, the observed initial, mild afucosylated response was additionally accompanied by low fucosyltransferase (FUT8) expression in antigen-specific plasma cells. Furthermore, the observed initial anti-S IgG afucosylation signature may aided mounting a stronger immune response, as indicated by its correlation with antibody amounts following the second vaccination dose. Given the impact of glycosylation on antibody function, deciphering theunderlying regulatory mechanisms influencing IgG glycosylation will be of great importance to better understand the inflammatory potential, vaccine efficacy and protective capacity of vaccine- or pathogen-induced IgG in both body fluids and tissues in the future.In Chapter 5 and 6, the reaction steps of a previously developed linkage-specific sialic acid derivatization workflow were studied in more detail. Key players in such reactions are catalyst, of which novel types with different physico-chemical properties were introduced in Chapter 5. In Chapter 6, prior lactone formation was found to be a prerequisite for subsequent amidation of α2,3-linked sialic acids, which proceeds via direct aminolysis of the C2 lactone. Together, these new insights will be beneficial for the rational optimization of high-throughput (MALDI-)MS-based glycomics and glycoproteomics workflows relying on linkage-specific sialic acid derivatization. Show less
Het onderzoek beschreven in dit proefschrift is gericht op de identificatie en karakterisering van regulatoren en/of effectoren van TGF-β-signalering en TGF-β-geïnduceerde EMT in pancreas-, long-... Show moreHet onderzoek beschreven in dit proefschrift is gericht op de identificatie en karakterisering van regulatoren en/of effectoren van TGF-β-signalering en TGF-β-geïnduceerde EMT in pancreas-, long- en borstkankertypes. Deze nieuw geïdentificeerde componenten en mechanismen kunnen worden onderzocht voor de ontwikkeling van geneesmiddelen voor kankertherapie. Show less
The surface of eukaryotic cells contains a very dense layer of oligosaccharides called glycans that are linked to protein and lipid carriers and play an important role in cell-cell and cell... Show moreThe surface of eukaryotic cells contains a very dense layer of oligosaccharides called glycans that are linked to protein and lipid carriers and play an important role in cell-cell and cell-extracellular matrix interactions. Cancer-induced changes in glycosylation have an impact on the function of major glycoproteins in the human colon, therefore studies focused on colorectal cancer (CRC)-specific glycosylation signatures can provide novel insights into onset and progression of this disease. The major focus of this thesis was to investigate mucin type O-glycosylation signatures of CRC. For this purpose, a protocol for in-depth analysis of N- and O-glycans obtained from cell lines was developed (Chapter 2) using nanoscale porous graphitized carbon liquid chromatography coupled to mass spectrometry (PGC-nano-LC-MS). In Chapter 3 additional conditions were optimized in the MS methodology by using polar protic dopant (methanol and isopropanol) enriched nitrogen gas to increase sensitivity on the MS and tandem MS level. In Chapter 4 we applied the methodology developed in Chapter 2 to the analysis of O-glycosylation signatures of 26 different CRC cell lines. This analysis resulted in the characterization of more than 150 O-glycan structures and increased our understanding of glycan expression in the analyzed cell lines. To gain further understanding in the mechanisms underlying glycomic changes with colon cell differentiation, we explored changes in the cell line glycome and proteome upon spontaneous and butyrate-stimulated differentiation in in vitro cell culture (Chapter 5). By performing an integrative approach, we generated hypotheses about glycosylation signatures of specific cell adhesion proteins, which may play an important role in cancer progression. The localization of glycans on the cell surface and their role in biological processes are important in cancer pathogenesis, making them potential candidates for glycan targeting immunotherapy. Therefore, we further optimized the methodology to enable comprehensive analysis of N- and O-glycans from specific regions of formalin-fixed, paraffin-embedded tissues using laser capture microdissections and applied it for the analysis of selected regions of CRC tissues and their patient-matched colon mucosa controls (Chapter 6). We identified specific tumor-associated carbohydrate antigens (TACAs) that show expression only in the tumor samples, with no or limited expression in the normal colon mucosa. Since TACAs are present in high abundance on the surface of cancer cells which are linked to many different proteins, these are very promising targets for the development of tumor-specific immunotherapy. Show less
Therapeutic proteins have been successfully developed for advancing medical treatments. They are usually large molecules produced by host cells and have a high degree of complexity compared to... Show moreTherapeutic proteins have been successfully developed for advancing medical treatments. They are usually large molecules produced by host cells and have a high degree of complexity compared to synthetic small molecule-based therapeutics. The complexity is mainly attributed to the heterogenic nature of post-translational modifications (PTMs). Glycosylation is one of the main drivers of protein heterogeneity. Since each modification may potentially impact the safety and efficacy, analyticalmethods for the structural and functional characterization of protein-based therapeutics are highly demanded. This thesis presents novel mass spectrometry (MS)-based methods for the analysis of therapeutic glycoproteins. It covers aspects of glycobioinformatics and sample preparation for improved bottom-up glycoproteomic analysis. Further, the profiling of complex intact glycoproteins by matrix-assisted laser desorption ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) MS is demonstrated. Finally, Fc gamma receptor III affinity chromatography combined with online MS detection is presented for novel proteoform-resolved structure-function insights of monoclonal antibodies. Show less
Biomarker molecules are analyzed in clinical tests to diagnose a disease, but often these test lack sensitivity or specificity. Also, for many diseases there is not even a blood based test... Show moreBiomarker molecules are analyzed in clinical tests to diagnose a disease, but often these test lack sensitivity or specificity. Also, for many diseases there is not even a blood based test available, while blood collection is relatively low invasive. For breast- and pancreatic cancer, there are several proteins that could potentially serve as biomarkers in blood, but these are not yet specific enough to use for diagnostic testing. Further research on other types of biomarkers may therefore be a valuable addition to eventually be able to develop a blood test. Methods for glycosylation profiling from serum and dried bloodspots with mass spectrometry were developed and applied to pancreatic- and breast cancer biomarker studies. Differences were found between profiles of healthy and sick persons for pancreatic cancer, but no clear differences were seen for breast cancer. This is probably due to the many different forms of breast cancer which result in different profiles. In the future, combining different types of markers from serum might ensure that differences between healthy and sick, between different diseases and between types of disease can be identified. This could lead to the development of a blood test for the early detection of cancer and other diseases. Show less
Schistosomiasis is an acute and chronic disease caused by blood dwelling parasitic trematodes of the genus Schistosoma, and it is classified as the second most socioeconomically devastating... Show moreSchistosomiasis is an acute and chronic disease caused by blood dwelling parasitic trematodes of the genus Schistosoma, and it is classified as the second most socioeconomically devastating parasitic disease, second only to malaria. Currently the wormload is determined by the Kato-Katz method, which is not always reliable. In order to prepare diagnostic tools able to capture specific anti-carbohydrate antibodies or develop conjugate vaccines targeting these carbohydrate structures, sufficient amounts of well-defined fragments are needed. This thesis describes the synthesis of several glycans of these glycans present on the S. mansoni parasite, focusing mainly on the Circulating Anodic Antigen (CAA) and glycans bearing the unique α-(1-2)-L-Fucose-α-(1-2)-L-Fucose motifs. These glycans have been attached to gold nanoparticles and these particles were screened against several monoclonal antibodies and sera of individuals suffering from schistosomiasis. Show less
Rheumatoid arthritis (RA) is an auto-inflammatory disease, affecting ~1% of the world population. RA is hallmarked by the presence of autoantibodies, one of the well-known autoantibodies in RA are... Show moreRheumatoid arthritis (RA) is an auto-inflammatory disease, affecting ~1% of the world population. RA is hallmarked by the presence of autoantibodies, one of the well-known autoantibodies in RA are the anti-citrullinated protein antibodies (ACPA). ACPA is highly specific for RA and about 70-80% of the RA patients are positive for ACPA. Previously, we discovered that ACPA-IgG are extensively glycosylated in the variable (V) region. In this thesis, we determined that over 90% of the ACPA-IgG carries glycans in the v-domain were as only 17% of the conventional IgG carries additional glycans. Additionally, the glycans were highly sialylated. Intriguingly, we also showed that the glycans are introduced via somatic hyper mutations in the germinal center reaction. Furthermore, we discovered that ACPA-IgG v-domain glycosylation is a predictive marker for the development of ACPA positive RA. Lastly, we found that the binding strength is not influenced by the glycans but it influences the amount of available binding sites. Nevertheless, the ACPA-BCR still overcome negative selection which might suggest that the glycans can trigger an alternative way for positive selection in the germinal center. This will be subject for further studies regarding the role of ACPA-IgG v-domain glycosylation. Show less
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic and persistent inflammation of the joints. Around 50-80% of the RA patients harbour either one or multiple types... Show moreRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by systemic and persistent inflammation of the joints. Around 50-80% of the RA patients harbour either one or multiple types of autoantibodies, under which also Anti-Citrullinated Protein Antibodies (ACPA). ACPA are highly specific for RA and are strongly associated with the severity of arthritis. The studies described in this thesis aim to explore the structure and biological function of ACPA and the B cells producing these ACPA.The studies highlight the importance of the synovial environment for the survival of ACPA B cells. Moreover, we demonstrate that the variable region of ACPA-IgG differs from that of ACPA-IgM by the introduction of glycans, and that glycans in the constant part of antibodies can be modulated by influences from the micro-environment. This may affect the biological functions of the antibodies. Furthermore, we show that ACPA can bind to so-called Fc gamma receptor I on immune cells which could be a potential effector mechanisms of ACPA. These findings demonstrate that the structure and environment of ACPA play an important role in the ACPA immune response and provide multiple arguments for the active contribution of ACPA in the chronic inflammation of RA. Show less
This Thesis focuses on the design and synthesis of ADP-ribosylated compounds that can be applied in biological studies.The limitations of the contemporary methods of chemical ADP-ribosylation and a... Show moreThis Thesis focuses on the design and synthesis of ADP-ribosylated compounds that can be applied in biological studies.The limitations of the contemporary methods of chemical ADP-ribosylation and a relative scarcity of the well-defined synthetic ADP-ribosylated derivative was an incentive to undertake synthetic studies to further advance the methodologies in the bioorganic chemistry of ADP-ribosylated molecules. This Thesis aims specifically at the developing of new and improved synthetic methodologies and to synthesize advanced mono- or oligo-ADP-ribosylated biomolecules. The target compounds that are described in this Thesis are not only represent a synthetic challenge but also have great value in biology for a better understanding of ADP-ribosylation. Show less
The aim of this thesis is to explore the glycosylation of PSA as well as to study if alterations can be observed between patients with indolent and malignant PCa. For this purpose the powerful... Show moreThe aim of this thesis is to explore the glycosylation of PSA as well as to study if alterations can be observed between patients with indolent and malignant PCa. For this purpose the powerful analytical platform CE-ESI-MS(/MS) was explored with a special focus on the analysis of glycans and glycopeptides (Chapter 1). The first section of the thesis describes the different method developments implemented for the analysis of PSA with CE-ESI-MS. Namely, Chapter 2 describes that CE-ESI-MS enables to separate glycopeptides with differently linked sialic acids without any additional sample treatment, Chapter 3 shows that an introduction of a dopant enriched nitrogen gas improves the limit of detection (sensitivity) of glycopeptides and Chapter 4 introduces a novel labeling procedure of total plasma N-glycome with the hydrazide Girard’s reagent P. Chapter 5 describes the development of a PSA Glycomics Assay which allows the capture of intact PSA from patients’ urine followed by analysis with the optimized CE-ESI-MS platform (Chapters 2 and 3). Finally, Chapter 6 offers a general discussion about future developments, the potential of PSA glycosylation in the clinical setting, showing the relevance of our results and how these may contribute to further clinical applications towards personalized medicine. Show less
Glycosylation of immunoglobulins is suspected to play a key role in the regulation of the immune system. In this thesis, mass spectrometry-based glycoproteomics methods were used to characterize... Show moreGlycosylation of immunoglobulins is suspected to play a key role in the regulation of the immune system. In this thesis, mass spectrometry-based glycoproteomics methods were used to characterize the glycosylation of various immunoglobulins. In Chapter 2 we describe the development of a glycoproteomics method to analyze IgE glycosylation. In Chapter 3 we reported partial O-glycosylation of IgG3. In addition to structural glycosylation research, we also analyzed antibody glycosylation in population cohorts. In Chapter 4 and Chapter 5, IgG Fc glycopeptide analysis was performed on blood samples using LC-MS(/MS). In a cohort of 76 ANCA vasculitis patients, low galactosylation and sialylation of IgG was associated with a higher chance of future relapse. Furthermore, in the approximately 1800 participants of the Leiden Longevity study (LLS), low galactosylation and sialylation of IgG, together with high fucosylation, showed association with markers of inflammation. We hope that the novel data presented in this thesis may contribute to the elucidation of the role of antibody glycosylation in the immune system, of which the understanding is currently still very limited. Show less
The post-translational modification of proteins known as adenosine diphosphate ribosylation (ADPr) is involved in a wide variety of important biological processes and is also associated with... Show moreThe post-translational modification of proteins known as adenosine diphosphate ribosylation (ADPr) is involved in a wide variety of important biological processes and is also associated with carcinogenesis and the process of aging. Therefore, a better understanding of the biology of ADP-ribosylation is crucial for the development of novel therapeutics. To facilitate this research, the availability of well-defined fragments of mono- and poly-ADP-ribose is essential. This Thesis describes the development of new synthetic methodologies to achieve this and the successful synthesis of well-defined ADP-ribosylated biomolecules. Show less
Glycan modifications of proteins and lipids form an integral part of the cell’s outermost layer and an array of ligands, adding a high degree of complexity to the cellular phenotype. While... Show more Glycan modifications of proteins and lipids form an integral part of the cell’s outermost layer and an array of ligands, adding a high degree of complexity to the cellular phenotype. While this complexity is an analytical challenge, it also offers a wide range of opportunities for biomarkers and treatment targets. This thesis deals with the analysis of colorectal cancer (CRC)-associated glycomic changes. Current knowledge on CRC-associated glycan changes and their biological role have been reviewed in Chapter 1. In Chapters 2, 3, and 6, we developed novel, high-end methodologies for the glycomic analysis of tissues and cell lines to be able to expand our knowledge on cancer glycomics and to overcome some limitations of current techniques. By applying these new methods, this thesis also covers the characterization of changes in glycosylation in CRC tissues as well as cell lines, thereby contributing to the understanding of CRC biology while identifying cancer-specific signatures underlying CRC development. These signatures can be further explored as potential markers to improve patient care. Additionally, in Chapter 5, we extended our research to pancreatic duct adenocarcinoma and characterized the N-glycome of PDAC cells with different metastatic potential and of a normal pancreatic duct cell line. Show less
The glycosylation, the reaction which forms a bond between sugar molecules (the donor and the acceptor), is the central reaction in carbohydrate chemistry. Despite tremendous advances in... Show more The glycosylation, the reaction which forms a bond between sugar molecules (the donor and the acceptor), is the central reaction in carbohydrate chemistry. Despite tremendous advances in the past decades, however, the glycosylation reaction remains relatively poorly understood. Especially the formation of 1,2-cis glycosidic linkages remains a significant challenge. This thesis describes an investigation of the influence of reactivity and selectivity of several classes of carbohydrate donors and –acceptors on the selectivity in glycosylation reactions. Special emphasis was placed on the influence of protecting groups on the donor, and nucleophilicity of the acceptor, two major factors that play a tremendous role in the outcome of a glycosylation reaction. The obtained knowledge has been applied in the synthesis of complex carbohydrate molecules, native to pathogens such as Staphylococcus aureus, of which antibiotic-resistant forms such as MRSA present significant danger in hospitals, and the parasite Schistosoma mansoni, causative agent of the neglected tropical disease schistosomiasis. The synthesis of these molecules can play a part in the development of vaccines targeted against these pathogens. Show less
Bioconjugates are involved in many biological processes. Glycoconjugates can bind to lectin receptors based on their specific carbohydrate structure. The mannose receptor, a C-type lectin, makes an... Show moreBioconjugates are involved in many biological processes. Glycoconjugates can bind to lectin receptors based on their specific carbohydrate structure. The mannose receptor, a C-type lectin, makes an interesting target to induce selective uptake by dendritic cells and macrophages. This thesis focus on the design and synthesis glycoconjugates linked with oligomannosides and artificial mannose clusters as homing device for the mannose receptor and other mannose binding lectins. Show less
