Search

Rheumatoid arthritis (RA) Is a highly prevalent autoimmune disease that affects the joints but also various other organs. The disease is characterized by autoantibodies that are often already observed pre-disease. Since the 1980s, it has been known that antibody glycosylation is different in RA as compared to control individuals. While the literature on glycosylation changes in RA is dominated by reports on serum or plasma immunoglobulin G (IgG), our recent studies have indicated that the glycosylation changes observed for immunoglobulin A (IgA) and total serum N-glycome (TSNG) may be similarly prominent, and useful in differentiating between the RA patients and controls, or as a proxy of the disease activity. In this study, we integrated and compared the RA glycosylation signatures of IgG, IgA and TSNG, all determined in the pregnancy-induced amelioration of rheumatoid arthritis (PARA) cohort. We assessed the association of the altered glycosylation patterns with the disease, autoantibody positivity and disease activity. Our analyses indicated a common, composite glycosylation signature of RA that was independent of the autoantibody status.

A newly developed analytical strategy was applied to profile the total serum N-glycome of 64 colorectal cancer (CRC) patients before and after surgical intervention. In this cohort, it was previously found that serum N-glycome alterations in CRC were associated with patient survival. Here, fluorescent labeling of serum N-glycans was applied using procainamide and followed by sialic acid derivatization specific for α2,6- and α2,3-linkage types via ethyl esterification and amidation, respectively. This strategy allowed efficient separation of specific positional isomers on reversed-phase liquid chromatography–fluorescence detection–mass spectrometry (RPLC-FD-MS) and complemented the previous glycomics data based on matrix-assisted laser desorption/ionization (MALDI)-MS that did not include such separations. The results from comparing pre-operative CRC to post-operative samples were in agreement with studies that identified a decrease in di-antennary structures with core fucosylation and an increase in sialylated tri- and tetra-antennary N-glycans in CRC patient sera. Pre-operative abundances of N-glycans showed good performance for the classification of adenocarcinoma and led to the revisit of the previous MALDI-MS dataset with regard to histological and clinical data. This strategy has the potential to monitor patient profiles before, during, and after clinical events such as treatment, therapy, or surgery and should also be further explored.

Reception of Wnt signals by cells is predominantly mediated by Frizzled receptors in conjunction with a co-receptor, the latter being LRP6 or LRP5 for the Wnt/β-catenin signalling pathway. It is important that cells maintain precise control of receptor activation events in order to properly regulate Wnt/β-catenin signalling as aberrant signalling can result in disease in humans. Phosphorylation of the intracellular domain (ICD) of LRP6 is well known to regulate Wntβ-catenin signalling; however, less is known for regulatory post-translational modification events within the extracellular domain (ECD). Using a cell culture-based expression screen for functional regulators of LRP6, we identified a glycosyltransferase, B3GnT2-like, from a teleost fish (medaka) cDNA library, that modifies LRP6 and regulates Wnt/β-catenin signalling. We provide both gain-of-function and loss-of-function evidence that the single human homolog, B3GnT2, promotes extension of polylactosamine chains at multiple N-glycans on LRP6, thereby enhancing trafficking of LRP6 to the plasma membrane and promoting Wnt/β-catenin signalling. Our findings further highlight the importance of LRP6 as a regulatory hub in Wnt signalling and provide one of the few examples of how a specific glycosyltransferase appears to selectively target a signalling pathway component to alter cellular signalling events.

Acute myeloid leukemia (AML) is characterized by a dysregulated expansion of poorly differentiated myeloid cells. Although patients are usually treated effectively by chemotherapy, a high rate of relapsed or refractory disease poses a major hurdle in its treatment. Recently, several studies have proposed implications of protein glycosylation in the pathobiology of AML including chemoresistance. Accordingly, associations have been found between specific glycan epitopes and the outcome of the disease. To advance this poorly studied field, we performed an exploratory glycomics study characterizing 21 widely used AML cell lines. Exploiting the benefits of porous graphitized carbon chromatography coupled to tandem mass spectrometry (PGC nano-LC-MS2), we qualitatively and quantitatively profiled N- and O-linked glycans. AML cell lines exhibited distinct glycan fingerprints differing in relevant glycan traits correlating with their cellular phenotype as classified by the FAB system. By implementing transcriptomics data, specific glycosyltransferases and hematopoietic transcription factors were identified, which are candidate drivers of the glycan phenotype of these cells. In conclusion, we report the varying expression of glycan structures across a high number of AML cell lines, including those associated with poor prognosis, identified underlying glycosyltransferases and transcription factors, and provide insights into the regulation of the AML glycan repertoire.

Meningiomas are the most common non-malignant intracranial tumors and prefer, like most tumors, anaerobic glycolysis for energy production (Warburg effect). This anaerobic glycolysis leads to an increased synthesis of the metabolite methylglyoxal (MGO) or glyoxal (GO), which is known to react with amino groups of proteins. This reaction is called glycation, thereby building advanced glycation end products (AGEs). In this study, we investigated the influence of glycation on sialylation in two meningioma cell lines, representing the WHO grade I (BEN-MEN-1) and the WHO grade III (IOMM-Lee). In the benign meningioma cell line, glycation led to differences in expression of sialyltransferases (ST3GAL1/2/3/5/6, ST6GAL1/2, ST6GALNAC2/6, and ST8SIA1/2), which are known to play a role in tumor progression. We could show that glycation of BEN-MEN-1 cells led to decreased expression of ST3Gal5. This resulted in decreased synthesis of the ganglioside GM3, the product of ST3Gal5. In the malignant meningioma cell line, we observed changes in expression of sialyltransferases (ST3GAL1/2/3, ST6GALNAC5, and ST8SIA1) after glycation, which correlates with less aggressive behavior.

Correct glycosylation of proteins is essential for production of therapeutic proteins as glycosylation is important for protein solubility, stability, half-life and immunogenicity. The heavily glycosylated plasma protein C1-inhibitor (C1-INH) is used in treatment of hereditary angioedema attacks. In this study, we used C1-INH as a model protein to propose an approach to develop recombinant glycoproteins with the desired glycosylation. We produced fully functional recombinant C1-INH in Chinese hamster ovary (CHO) cells. In vivo we observed a biphasic clearance, indicating different glycosylation forms. N-glycan analysis with mass spectrometry indeed demonstrated heterogeneous glycosylation for recombinant C1-INH containing terminal galactose and terminal sialic acid. Using a Ricinus Communis Agglutinin I (RCA120) column, we could reduce the relative abundance of terminal galactose and increase the relative abundance of terminal sialic acid. This resulted in a fully active protein with a similar in vivo clearance rate to plasmaderived C1-INH. In summary, we describe the development of a recombinant human glycoprotein using simple screening tools to obtain a product that is similar in function and in vivo clearance rate to its plasma-derived counterpart. The approach used here is of potential use in the development of other therapeutic recombinant human glycoproteins.

Glycomics measurements, like all other high-throughput technologies, are subject to technical variation due to fluctuations in the experimental conditions. The removal of this non-biological signal from the data is referred to as normalization. Contrary to other omics data types, a systematic evaluation of normalization options for glycomics data has not been published so far. In this paper, we assess the quality of different normalization strategies for glycomics data with an innovative approach. It has been shown previously that Gaussian Graphical Models (GGMs) inferred from glycomics data are able to identify enzymatic steps in the glycan synthesis pathways in a data-driven fashion. Based on this finding, here, we quantify the quality of a given normalization method according to how well a GGM inferred from the respective normalized data reconstructs known synthesis reactions in the glycosylation pathway. The method therefore exploits a biological measure of goodness. We analyzed 23 different normalization combinations applied to six large-scale glycomics cohorts across three experimental platforms: Liquid Chromatography-ElectroSpray Ionization-Mass Spectrometry (LC-ESI-MS), Ultra High Performance Liquid Chromatography with Fluorescence Detection (UHPLC-FLD), and Matrix Assisted Laser Desorption Ionization-Furier Transform Ion Cyclotron Resonance-Mass Spectrometry (MALDI-FTICR-MS). Based on our results, we recommend normalizing glycan data using the ‘Probabilistic Quotient’ method followed by log-transformation, irrespective of the measurement platform. This recommendation is further supported by an additional analysis, where we ranked normalization methods based on their statistical associations with age, a factor known to associate with glycomics measurements.

A new mass spectrometry imaging approach to simultaneously map the two-dimensional distribution of N-glycans in tissues has been recently developed. The method uses Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS) to spatially profile the location and distribution of multiple N-linked glycan species released by peptide N-glycosidase F in frozen or formalin-fixed tissues. Multiple formalin-fixed human hepatocellular carcinoma tissues were evaluated with this method, resulting in a panel of over 30 N-glycans detected. An ethylation reaction of extracted N-glycans released from adjacent slides was done to stabilize sialic acid containing glycans, and these structures were compared to N-glycans detected directly from tissue profiling. In addition, the distribution of singly fucosylated N-glycans detected in tumor tissue microarray cores were compared to the histochemistry staining pattern of a core fucose binding lectin. As this MALDI-IMS workflow has the potential to be applied to any formalin-fixed tissue block or tissue microarray, the advantages and limitations of the technique in context with other glycomic methods are also summarized.

The human glycome is characterized by a high degree of sialylation, affecting, amongst others, cell−cell interactions and protein half-life. An established method for the linkage isomer-specific characterization of N-glycan sialylation is based on the linkage-specific derivatization of sialylated glycoconjugates, inducing ethyl esterification of α2,6-linked sialic acids and lactonization of α2,3-linked sialic acids. While the carboxylic acid activator and nucleophile used in this reaction received extensive investigation, the role of the catalyst was never thoroughly explored. A frequently used catalyst for the linkage-specific esterification of sialic acids is 1-hydroxybenzotriazole (HOBt). Here, a systematic evaluation was performed of five HOBt alternatives in combination with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in ethanol for the linkage-specific derivatization of sialic acids. Derivatized glycans were analyzed by MALDI-TOF-MS and the catalyst performance was evaluated based on the completeness of the reactions and the linkage-specificity obtained. The use of both 6-Cl-HOBt and 6-CF3-HOBt resulted in high linkage-specificity and minimal byproduct formation, similar to the benchmark method using HOBt. Performing the reaction with these catalysts at neutral or acidic pH showed comparable efficiencies on both sialyllactose and complex-type N-glycans. The reported investigations resulted in an expansion of the reaction space for linkage-specific sialic acid derivatization.

The caudal-related homeobox protein 1 (CDX1) is a transcription factor, which is important in the development, differentiation, and homeostasis of the gut. Although the involvement of CDX genes in the regulation of the expression levels of a few glycosyltransferases has been shown, associations between glycosylation phenotypes and CDX1 mRNA expression have hitherto not been well studied. Triggered by our previous study, we here characterized the N-glycomic phenotype of 16 colon cancer cell lines, selected for their differential CDX1 mRNA expression levels. We found that high CDX1 mRNA expression associated with a higher degree of multi-fucosylation on N-glycans, which is in line with our previous results and was supported by up-regulated gene expression of fucosyltransferases involved in antenna fucosylation. Interestingly, hepatocyte nuclear factors (HNF)4A and HNF1A were, among others, positively associated with high CDX1 mRNA expression and have been previously proven to regulate antenna fucosylation. Besides fucosylation, we found that high CDX1 mRNA expression in cancer cell lines also associated with low levels of sialylation and galactosylation and high levels of bisection on N-glycans. Altogether, our data highlight a possible role of CDX1 in altering the N-glycosylation of colorectal cancer cells, which is a hallmark of tumor development.

Fimbriae are long, proteinaceous adhesion organelles expressed on the bacterial envelope, evolutionarily adapted by Escherichia coli strains for the colonization of epithelial linings. Using glycan arrays of the Consortium for Functional Glycomics (CFG), the lectin domains were screened of the fimbrial adhesins F17G and FedF from enterotoxigenic E. coli (ETEC) and of the FimH adhesin from uropathogenic E. coli. This has led to the discovery of a more specific receptor for F17G, GlcNAcb1,3Gal. No significant differences emerged from the glycan binding profiles of the F17G lectin domains from five different E. coli strains. However, strain-dependent amino acid variations, predominantly towards the positively charged arginine, were indicated by sulfate binding in FedF and F17G crystal structures. For FedF, no significant binders could be observed on the CFG glycan array. Hence, a shotgun array was generated from microvilli scrapings of the distal jejunum of a 3-week old piglet about to be weaned. On this array, the blood group A type 1 hexasaccharide emerged as a receptor for the FedF lectin domain and remarkably also for F18-fimbriated E. coli. F17G was found to selectively recognize glycan species with a terminal GlcNAc, typifying intestinal mucins. In conclusion, F17G and FedF recognize long glycan sequences that could only be identified using the shotgun approach. Interestingly, ETEC strains display a large capacity to adapt their fimbrial adhesins to ecological niches via charge-driven interactions, congruent with binding to thick mucosal surfaces displaying an acidic gradient along the intestinal tract.