Apolipoprotein-CIII (apo-CIII) is involved in triglyceride-rich lipoprotein metabolism and linked to beta-cell damage, insulin resistance, and cardiovascular disease. Apo-CIII exists in four main... Show moreApolipoprotein-CIII (apo-CIII) is involved in triglyceride-rich lipoprotein metabolism and linked to beta-cell damage, insulin resistance, and cardiovascular disease. Apo-CIII exists in four main proteoforms: non-glycosylated (apo-CIII0a), and glycosylated apo-CIII with zero, one, or two sialic acids (apo-CIII0c, apo-CIII1 and apo-CIII2). Our objective is to determine how apo-CIII glycosylation affects lipid traits and type 2 diabetes prevalence, and to investigate the genetic basis of these relations with a genome-wide association study (GWAS) on apo-CIII glycosylation. We conducted GWAS on the four apo-CIII proteoforms in the DiaGene study in people with and without type 2 diabetes (n = 2318). We investigated the relations of the identified genetic loci and apo-CIII glycosylation with lipids and type 2 diabetes. The associations of the genetic variants with lipids were replicated in the Diabetes Care System (n = 5409). Rs4846913-A, in the GALNT2-gene, was associated with decreased apo-CIII0a. This variant was associated with increased high-density lipoprotein cholesterol and decreased triglycerides, while high apo-CIII0a was associated with raised high-density lipoprotein-cholesterol and triglycerides. Rs67086575-G, located in the IFT172-gene, was associated with decreased apo-CIII2 and with hypertriglyceridemia. In line, apo-CIII2 was associated with low triglycerides. On a genome-wide scale, we confirmed that the GALNT2-gene plays a major role i O-glycosylation of apolipoprotein-CIII, with subsequent associations with lipid parameters. We newly identified the IFT172/NRBP1 region, in the literature previously associated with hypertriglyceridemia, as involved in apolipoprotein-CIII sialylation and hypertriglyceridemia. These results link genomics, glycosylation, and lipid metabolism, and represent a key step towards unravelling the importance of O-glycosylation in health and disease. Show less
Parasitic nematodes responsible for filarial diseases cause chronic disablement in humans worldwide. Elimination programs have substantially reduced the rate of infection in certain areas, but... Show moreParasitic nematodes responsible for filarial diseases cause chronic disablement in humans worldwide. Elimination programs have substantially reduced the rate of infection in certain areas, but limitations of current diagnostics for population surveillance have been pointed out and improved assays are needed to reach the elimination targets. While serological tests detecting antibodies to parasite antigens are convenient tools, those currently available are compromised by the occurrence of antibodies cross-reactive between nematodes, as well as by the presence of residual antibodies in sera years after treatment and clearance of the infection. We recently characterized the N-linked and glycosphingolipid derived glycans of the parasitic nematode Brugia malayi and revealed the presence of various antigenic structures that triggered immunoglobulin G (IgG) responses in infected individuals. To address the specificity of IgG binding to these glycan antigens, we screened microarrays containing Brugia malayi glycans with plasma from uninfected individuals and from individuals infected with Loa loa, Onchocerca volvulus, Mansonella perstans and Wuchereria bancrofti, four closely related filarial nematodes. IgG to a restricted subset of cross-reactive glycans was observed in infection plasmas from all four species. In plasma from Onchocerca volvulus and Mansonella perstans infected individuals, IgG binding to many more glycans was additionally detected, resulting in total IgG responses similar to the ones of Brugia malayi infected individuals. For these infection groups, Brugia malayi, Onchocerca volvulus and Mansonella perstans, we further studied the different IgG subclasses to Brugia malayi glycans. In all three infections, IgG1 and IgG2 appeared to be the major subclasses involved in response to glycan antigens. Interestingly, in Brugia malayi infected individuals, we observed a marked reduction in particular in IgG2 to parasite glycans post-treatment with anthelminthic, suggesting a promising potential for diagnostic applications. Thus, we compared the IgG response to a broad repertoire of Brugia malayi glycans in individuals infected with various filarial nematodes. We identified broadly cross-reactive and more specific glycan targets, extending the currently scarce knowledge of filarial nematode glycosylation and host anti-glycan antibody response. We believe that our initial findings could be further exploited to develop disease-specific diagnostics as part of an integrated approach for filarial disease control. Show less
Haan, N. de; Pucic-Bakovic, M.; Novokmet, M.; Falck, D.; Lageveen-Kammeijer, G.; Razdorov, G.; ... ; Human Glycome Project 2022
Glycans expand the structural complexity of proteins by several orders of magnitude, resulting in a tremendous analytical challenge when including them in biomedical research. Recent... Show moreGlycans expand the structural complexity of proteins by several orders of magnitude, resulting in a tremendous analytical challenge when including them in biomedical research. Recent glycobiological research is painting a picture in which glycans represent a crucial structural and functional component of the majority of proteins, with alternative glycosylation of proteins and lipids being an important regulatory mechanism in many biological and pathological processes. Since interindividual differences in glycosylation are extensive, large studies are needed to map the structures and to understand the role of glycosylation in human (patho)physiology. Driven by these challenges, methods have emerged, which can tackle the complexity of glycosylation in thousands of samples, also known as high-throughput (HT) glycomics. For facile dissemination and implementation of HT glycomics technology, the sample preparation, analysis, as well as data mining, need to be stable over a long period of time (months/years), amenable to automation, and available to non-specialized laboratories. Current HT glycomics methods mainly focus on protein N-glycosylation and allow to extensively characterize this subset of the human glycome in large numbers of various biological samples. The ultimate goal in HT glycomics is to gain better knowledge and understanding of the complete human glycome using methods that are easy to adapt and implement in (basic) biomedical research. Aiming to promote wider use and development of HT glycomics, here, we present currently available, emerging, and prospective methods and some of their applications, revealing a largely unexplored molecular layer of the complexity of life. Show less
The Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative to standardize the reporting of glycoanalytical methods and to assess their reproducibility. To date, the... Show moreThe Minimum Information Required for a Glycomics Experiment (MIRAGE) is an initiative to standardize the reporting of glycoanalytical methods and to assess their reproducibility. To date, the MIRAGE Commission has published several reporting guidelines that describe what information should be provided for sample preparation methods, mass spectrometry methods, liquid chromatography analysis, exoglycosidase digestions, glycan microarray methods, and nuclear magnetic resonance methods. Here, we present the first version of reporting guidelines for glyco(proteo)mics analysis by capillary electrophoresis (CE) for standardized and high-quality reporting of experimental conditions in the scientific literature. The guidelines cover all aspects of a glyco(proteo)mics CE experiment including sample preparation, CE operation mode (CZE, CGE, CEC, MEKC, cIEF, cITP), instrument configuration, capillary separation conditions, detection, data analysis, and experimental descriptors. Show less
Cells are covered with a dense layer of carbohydrates, some of which are solely present on neoplastic cells. The so-called tumor-associated carbohydrate antigens (TACAs) are increasingly recognized... Show moreCells are covered with a dense layer of carbohydrates, some of which are solely present on neoplastic cells. The so-called tumor-associated carbohydrate antigens (TACAs) are increasingly recognized as promising targets for immunotherapy. These carbohydrates differ from those of the surrounding non-cancerous tissues and contribute to the malignant phenotype of the cancer cells by promoting proliferation, metastasis, and immunosuppression. However, due to tumor tissue heterogeneity and technological limitations, TACAs are insufficiently explored.Methods: A workflow was established to decode the colorectal cancer (CRC)-associated O-linked glycans from approximately 20,000 cell extracts. Extracts were obtained through laser capture microdissection of formalin fixed paraffin embedded tissues of both primary tumors and metastatic sites, and compared to healthy colon mucosa from the same patients. The released O-glycans were analyzed by porous graphitized carbon liquid chromatography-tandem mass spectrometry in negative ion mode.Results: Distinctive O-glycosylation features were found in cancerous, stromal and normal colon mucosal regions. Over 100 O-linked glycans were detected in cancerous regions with absence in normal mucosa. From those, six core 2 O-glycans were exclusively found in more than 33% of the cancers, carrying the terminal (sialyl-)Lewis(X/A) antigen. Moreover, two O-glycans were present in 72% of the analyzed cancers and 94% of the investigated cancers expressed at least one of these two O-glycans. In contrast, normal colon mucosa predominantly expressed core 3 O-glycans, carrying alpha 2-6-linked sialylation, (sulfo-)Lewis(X/A) and Sda antigens.Conclusion: In this study, we present a novel panel of highly specific TACAs, based upon differences in the glycomic profiles between CRC and healthy colon mucosa. These TACAs are promising new targets for development of innovative cancer immune target therapies and lay the foundation for the targeted treatment of CRC. Show less
Lageveen-Kammeijer, G.S.M.; Kuster, B.; Reusch, D.; Wuhrer, M. 2021
Many analytical challenges in biomedicine arise from the generally high heterogeneity and complexity of glycan- and glycoconjugate-containing samples, which are often only available in minute... Show moreMany analytical challenges in biomedicine arise from the generally high heterogeneity and complexity of glycan- and glycoconjugate-containing samples, which are often only available in minute amounts. Therefore, highly sensitive workflows and detection methods are required. In this review mass spectrometric workflows and detection methods are evaluated for glycans and glycoproteins. Furthermore, glycomic methodologies and innovations that are tailored for enzymatic treatments, chemical derivatization, purification, separation, and detection at high sensitivity are highlighted. The discussion is focused on the analysis of mammalian N-linked and GalNAc-type O-linked glycans. Show less
With 28 potential N-glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N-linked glycosylation, and approximately 60% of its molecular mass can be attributed to its... Show moreWith 28 potential N-glycosylation sites, human carcinoembryonic antigen (CEA) bears an extreme amount of N-linked glycosylation, and approximately 60% of its molecular mass can be attributed to its carbohydrates. CEA is often overexpressed and released by many solid tumors, including colorectal carcinomas. CEA displays an impressive heterogeneity and variability in sugar content; however, site-specific distribution of carbohydrate structures has not been reported so far. The present study investigated CEA samples purified from human colon carcinoma and human liver metastases and enabled the characterization of 21 out of 28 potential N-glycosylation sites with respect to their occupancy. The coverage was achieved by a multienzymatic digestion approach with specific enzymes, such as trypsin, endoproteinase Glu-C, and the nonspecific enzyme, Pronase, followed by analysis using sheathless CE-MS/MS. In total, 893 different N-glycopeptides and 128 unique N-glycan compositions were identified. Overall, a great heterogeneity was found both within (micro) and in between (macro) individual N-glycosylation sites. Moreover, notable differences were found on certain N-glycosylation sites between primary adenocarcinoma and metastatic tumor in regard to branching, bisection, sialylation, and fucosylation. Those features, if further investigated in a targeted manner, may pave the way toward improved diagnostics and monitoring of colorectal cancer progression and recurrence. Raw mass spectrometric data and Skyline processed data files that support the findings of this study are available in the MassIVE repository with the identifier MSV000086774 [DOI: 10.25345/C5Z50X]. Show less
Hanna Sawires, R.G.; Schiphuis, J.H.; Wuhrer, M.; Vasen, H.F.A.; Leerdam, M.E. van; Bonsing, B.A.; ... ; Tollenaar, R.A.E.M. 2021
Pancreatic ductal adenocarcinoma (PDAC) is known as a highly aggressive malignant disease. Prognosis for patients is notoriously poor, despite improvements in surgical techniques and new (neo... Show morePancreatic ductal adenocarcinoma (PDAC) is known as a highly aggressive malignant disease. Prognosis for patients is notoriously poor, despite improvements in surgical techniques and new (neo)adjuvant chemotherapy regimens. Early detection of PDAC may increase the overall survival. It is furthermore foreseen that precision medicine will provide improved prognostic stratification and prediction of therapeutic response. In this review, omics-based discovery efforts are presented that aim for novel diagnostic and prognostic biomarkers of PDAC. For this purpose, we systematically evaluated the literature published between 1999 and 2020 with a focus on protein- and protein-glycosylation biomarkers in pancreatic cancer patients. Besides genomic and transcriptomic approaches, mass spectrometry (MS)-based proteomics and glycomics of blood- and tissue-derived samples from PDAC patients have yielded new candidates with biomarker potential. However, for reasons discussed in this review, the validation and clinical translation of these candidate markers has not been successful. Consequently, there has been a change of mindset from initial efforts to identify new unimarkers into the current hypothesis that a combination of biomarkers better suits a diagnostic or prognostic panel. With continuing development of current research methods and available techniques combined with careful study designs, new biomarkers could contribute to improved detection, prognosis, and prediction of pancreatic cancer. Show less
Ederveen, A.L.H.; Haan, N. de; Baerenfaenger, M.; Lefeber, D.J.; Wuhrer, M. 2020
Protein N-glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in... Show moreProtein N-glycosylation is a multifactorial process involved in many biological processes. A broad range of congenital disorders of glycosylation (CDGs) have been described that feature defects in protein N-glycan biosynthesis. Here, we present insights into the disrupted N-glycosylation of various CDG patients exhibiting defects in the transport of nucleotide sugars, Golgi glycosylation or Golgi trafficking. We studied enzymatically released N-glycans of total plasma proteins and affinity purified immunoglobulin G (IgG) from patients and healthy controls using mass spectrometry (MS). The applied method allowed the differentiation of sialic acid linkage isomers via their derivatization. Furthermore, protein-specific glycan profiles were quantified for transferrin and IgG Fc using electrospray ionization MS of intact proteins and glycopeptides, respectively. Next to the previously described glycomic effects, we report unprecedented sialic linkage-specific effects. Defects in proteins involved in Golgi trafficking (COG5-CDG) and CMP-sialic acid transport (SLC35A1-CDG) resulted in lower levels of sialylated structures on plasma proteins as compared to healthy controls. Findings for these specific CDGs include a more pronounced effect for alpha 2,3-sialylation than for alpha 2,6-sialylation. The diverse abnormalities in glycomic features described in this study reflect the broad range of biological mechanisms that influence protein glycosylation. Show less
Benedetti, E.; Gerstner, N.; Pucic-Bakovic, M.; Keser, T.; Reiding, K.R.; Ruhaak, L.R.; ... ; Krumsiek, J. 2020
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... Show moreGlycomics 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. Show less
Changes in the abundance of antennary fucosylated glycans in human total plasma N-glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However... Show moreChanges in the abundance of antennary fucosylated glycans in human total plasma N-glycome (TPNG) have been associated with several diseases ranging from diabetes to various forms of cancer. However, it is challenging to address this important part of the human glycome. Most commonly, time-consuming chromatographic separations are performed to differentially quantify core and antenna fucosylation. Obtaining sufficient resolution for larger, more complex glycans can be challenging. We introduce a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) assay for the relative quantitation of antennary fucosylation in TPNG. N-linked glycans are released from plasma by PNGase F and further treated with a core fucosidase before performing a linkage-informative sialic acid derivatization. The core fucosylated glycans are thus depleted while the remaining antennary fucosylated glycans are quantitated. Simultaneous quantitation of alpha 2,3-linked sialic acids and antennary fucosylation allows an estimation of the sialyl-Lewis x motif. The approach is feasible using either ultrahigh-resolution Fourier-transform ion cyclotron resonance mass spectrometry or time-of-flight mass spectrometry. The assay was used to investigate changes of antennary fucosylation as clinically relevant marker in 14 colorectal cancer patients. In accordance with a previous report, we found elevated levels of antennary fucosylation pre-surgery which decreased after tumor resection. The assay has the potential for revealing antennary fucosylation signatures in various conditions including diabetes and different types of cancer. Show less
Most human proteins are glycosylated. Attachment of complex oligosaccharides to the polypeptide part of these proteins is an integral part of their structure and function and plays a central role... Show moreMost human proteins are glycosylated. Attachment of complex oligosaccharides to the polypeptide part of these proteins is an integral part of their structure and function and plays a central role in many complex disorders. One approach towards deciphering this human glycan code is to study natural variation in experimentally well characterized samples and cohorts. High-throughput capable large-scale methods that allow for the comprehensive determination of blood circulating proteins and their glycans have been recently developed, but so far, no study has investigated the link between both traits. Here we map for the first time the blood plasma proteome to its matching N-glycome by correlating the levels of 1116 blood circulating proteins with 113 N-glycan traits, determined in 344 samples from individuals of Arab, South-Asian, and Filipino descent, and then replicate our findings in 46 subjects of European ancestry. We report protein-specific N-glycosylation patterns, including a correlation of core fucosylated structures with immunoglobulin G (IgG) levels, and of trisialylated, trigalactosylated, and triantennary structures with heparin cofactor 2 (SERPIND2). Our study reveals a detailed picture of protein N-glycosylation and suggests new avenues for the investigation of its role and function in the associated complex disorders. Show less
Houwing-Duistermaat, J.J.; Uh, H.W.; Gusnanto, A. 2017
