The in-depth, high-sensitivity characterization of the glycome from complex biological samples, such as biofluids and tissues, is of utmost importance in basic biological research and biomarker ...discovery. Major challenges often arise from the vast structural diversity of glycans in combination with limited sample amounts. Here, we present a method for the highly sensitive characterization of released N-glycans by combining a capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) approach with linkage-specific derivatization of sialic acids and uniform cationic reducing end labelling of all glycans. This method allows the analysis of glycans at the attomole level, provides information on sialic acid isomers and enables the in-depth characterization of complex samples, even when available in minute amounts.
Monomeric serum immunoglobulin A (IgA) can contribute to the development of various autoimmune diseases, but the regulation of serum IgA effector functions is not well defined. Here, we show that the ...two IgA subclasses (IgA1 and IgA2) differ in their effect on immune cells due to distinct binding and signaling properties. Whereas IgA2 acts pro-inflammatory on neutrophils and macrophages, IgA1 does not have pronounced effects. Moreover, IgA1 and IgA2 have different glycosylation profiles, with IgA1 possessing more sialic acid than IgA2. Removal of sialic acid increases the pro-inflammatory capacity of IgA1, making it comparable to IgA2. Of note, disease-specific autoantibodies in patients with rheumatoid arthritis display a shift toward the pro-inflammatory IgA2 subclass, which is associated with higher disease activity. Taken together, these data demonstrate that IgA effector functions depend on subclass and glycosylation, and that disturbances in subclass balance are associated with autoimmune disease.
Glycosylation is a common posttranslational modification on membrane-associated and secreted proteins that is of pivotal importance for regulating cell functions. Aberrant glycosylation can lead to ...uncontrolled cell proliferation, cell-matrix interactions, migration and differentiation, and has been shown to be involved in cancer and other diseases. The epithelial-to-mesenchymal transition is a key step in the metastatic process by which cancer cells gain the ability to invade tissues and extravasate into the bloodstream. This cellular transformation process, which is associated by morphological change, loss of epithelial traits and gain of mesenchymal markers, is triggered by the secreted cytokine transforming growth factor-β (TGF-β). TGF-β bioactivity is carefully regulated, and its effects on cells are mediated by its receptors on the cell surface. In this review, we first provide a brief overview of major types of glycans, namely, N-glycans, O-glycans, glycosphingolipids and glycosaminoglycans that are involved in cancer progression. Thereafter, we summarize studies on how the glycosylation of TGF-β signaling components regulates TGF-β secretion, bioavailability and TGF-β receptor function. Then, we review glycosylation changes associated with TGF-β-induced epithelial-tomesenchymal transition in cancer. Identifying and understanding the mechanisms by which glycosylation affects TGF-β signaling and downstream biological responses will facilitate the identification of glycans as biomarkers and enable novel therapeutic approaches.
Proteins can undergo glycosylation during and/or after translation to afford glycoconjugates, which are often secreted by a cell or populate cell surfaces. Changes in the glycan portion can have a ...strong influence on a glycoconjugate and are associated with a multitude of human pathologies. Of particular interest are sialylated glycoconjugates, which exist as constitutional isomers that differ in their linkages (α2,3, α2,6, α2,8 or α2,9) between sialic acids and their neighbouring monosaccharides. In general, mass spectrometry enables the rapid and sensitive characterization of glycosylation, but there are challenges specific to identifying and (relatively) quantifying sialic acid isomers. These challenges can be addressed using linkage-specific methodologies for sialic acid derivatization, after which mass spectrometry can enable product identification. This Review is concerned with the new and important derivatization approaches reported in the past decade, which have been implemented in various mass-spectrometry-glycomics workflows and have found clinical glycomics applications. The convenience and wide applicability of the approaches make them attractive for studies of sialylation in different types of glycoconjugate.
Glycans on proteins and lipids are known to alter with malignant transformation. The study of these may contribute to the discovery of biomarkers and treatment targets as well as understanding of ...cancer biology. We here describe the change of glycosylation specifically defining colorectal cancer with view on N-glycans, O-glycans, and glycosphingolipid glycans in colorectal cancer cells and tissues as well as patient sera. Glycan alterations observed in colon cancer include increased β1,6-branching and correlating higher abundance of (poly-)N-acetyllactosamine extensions of N-glycans as well as an increase in (truncated) high-mannose type glycans, while bisected structures decrease. Colorectal cancer-associated O-glycan changes are predominated by reduced expression of core 3 and 4 glycans, whereas higher levels of core 1 glycans, (sialyl) T-antigen, (sialyl) Tn-antigen, and a generally higher density of O-glycans are observed. Specific changes for glycosphingolipid glycans are lower abundances of disialylated structures as well as globo-type glycosphingolipid glycans with exception of Gb3. In general, alterations affecting all discussed glycan types are increased sialylation, fucosylation as well as (sialyl) Lewis-type antigens and type-2 chain glycans. As a consequence, interactions with glycan-binding proteins can be affected and the biological function and cellular consequences of the altered glycosylation with regard to tumorigenesis, metastasis, modulation of immunity, and resistance to antitumor therapy will be discussed. Finally, analytical approaches aiding in the field of glycomics will be reviewed with focus on binding assays and mass spectrometry.
The analysis of N- and O-glycopeptides remains challenging due to the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of the ...glycoprotein. Trypsin is by far the most commonly used protease in glycoproteomic studies; however, it often results in long peptides that can harbor more than one glycan which may hamper site identification. The use of unspecific proteases such as Pronase can largely overcome this problem by generating glycopeptides with a small peptide portion. While the resulting glycopeptides are very useful for tandem mass spectrometric investigation, the analysis with conventional 1D-LC-ESI-MS/MS approaches can lead to incomplete glycosylation coverage because of the very heterogeneous physicochemical properties of the glycopeptides depending on the peptide sequence as well as the size and charges of the glycan moiety. Here, we describe a universal workflow for site-specific N- and O-glycopeptide analysis of Pronase treated glycoproteins with integrated, sequential C18 reverse phase and porous graphitized carbon-LC-ESI-QTOF-MS/MS employing a combination of lower- and enhanced-energy collision-induced dissociation. The approach was evaluated on glycoprotein standards and also applied to investigate the glycosylation of human IgG3 providing details on the hitherto uncharacterized glycosylation site Asn392 of the CH3 domain. This analytical tool can be applied to a variety of glycoproteins for site-specific N- and O-glycopeptide analysis, resulting in a good glycopeptide coverage within a single sample run and, thus, requiring only small amounts of sample.
Protein glycosylation is an important post-translational modification associated, among others, with diseases and the efficacy of biopharmaceuticals. Matrix-assisted laser desorption/ionization ...(MALDI) time-of-fight (TOF) mass spectrometry (MS) can be performed to study glycosylation in a high-throughput manner, but is hampered by the instability and ionization bias experienced by sialylated glycan species. Stabilization and neutralization of these sialic acids can be achieved by permethylation or by specific carboxyl group derivatization with the possibility of discrimination between α2,3- and α2,6-linked sialic acids. However, these methods typically require relatively pure glycan samples, show sensitivity to side reactions, and need harsh conditions or long reaction times. We established a rapid, robust and linkage-specific high-throughput method for sialic acid stabilization and MALDI-TOF-MS analysis, to allow direct modification of impure glycan-containing mixtures such as PNGase F-released human plasma N-glycome. Using a combination of carboxylic acid activators in ethanol achieved near-complete ethyl esterification of α2,6-linked sialic acids and lactonization of α2,3-linked variants, in short time using mild conditions. Glycans were recovered by hydrophilic interaction liquid chromatography solid phase extraction and analyzed by MALDI-TOF-MS in reflectron positive mode with 2,5-dihydroxybenzoic acid as the matrix substance. Analysis of the human plasma N-glycome allowed high-throughput detection and relative quantitation of more than 100 distinct N-glycan compositions with varying sialic acid linkages.
The thick mucus layer of the gut provides a barrier to infiltration of the underlying epithelia by both the normal microbiota and enteric pathogens. Some members of the microbiota utilise mucin ...glycoproteins as a nutrient source, but a detailed understanding of the mechanisms used to breakdown these complex macromolecules is lacking. Here we describe the discovery and characterisation of endo-acting enzymes from prominent mucin-degrading bacteria that target the polyLacNAc structures within oligosaccharide side chains of both animal and human mucins. These O-glycanases are part of the large and diverse glycoside hydrolase 16 (GH16) family and are often lipoproteins, indicating that they are surface located and thus likely involved in the initial step in mucin breakdown. These data provide a significant advance in our knowledge of the mechanism of mucin breakdown by the normal microbiota. Furthermore, we also demonstrate the potential use of these enzymes as tools to explore changes in O-glycan structure in a number of intestinal disease states.
Antibody glycosylation has received considerable attention in coronavirus disease 2019 (COVID-19) infections and recently also in vaccination. Antibody glycosylation and in particular immunoglobulin ...G1 fucosylation levels influence effector functions and are therefore key parameters for assessing the efficacy and safety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directed immune responses. This review article summarizes and interprets recent research into antibody glycosylation in COVID-19. Experimental approaches for analyzing the glycosylation of SARS-CoV-2-directed antibody responses are evaluated. The pronounced dynamics, effector functions, clinical utility, and regulation of antibody glycosylation in COVID-19 are assessed. Future research on the role of antibody glycosylation in COVID may cover the glycosylation of other antibody classes beyond immunoglobulin G, the regulation of antibody glycosylation, and the role of non-canonical antibody receptors in determining effector functions.
Graphical abstract
A subset of histo-blood group antigens including ABO and Lewis are oligosaccharide structures which may be conjugated to lipids or proteins. They are known to be important recognition motifs not only ...in the context of blood transfusions, but also in infection and cancer development.
Current knowledge on the molecular background and the implication of histo-blood group glycans in the prevention and therapy of infectious and non-communicable diseases, such as cancer and cardiovascular disease, is presented.
Glycan-based histo-blood groups are associated with intestinal microbiota composition, the risk of various diseases as well as therapeutic success of, e.g., vaccination. Their potential as prebiotic or anti-microbial agents, as disease biomarkers and vaccine targets should be further investigated in future studies. For this, recent and future technological advancements will be of particular importance, especially with regard to the unambiguous structural characterization of the glycan portion in combination with information on the protein and lipid carriers of histo-blood group-active glycans in large cohorts.
Histo-blood group glycans have a unique linking position in the complex network of genes, oncodevelopmental biological processes, and disease mechanisms. Thus, they are highly promising targets for novel approaches in the field of personalized medicine. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.
•Glycans are well-known for their function as recognition molecules.•Histo-blood group glycans are derived from allelic glycosyltransferase genes.•Histo-blood group glycans are associated with disease risk and therapeutic success.•Pathogens mimic human glycans that are recognized by the host's immune defense.•Soluble histo-blood group glycans serve as decoy receptors for microbes.
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