Kathrin Stavenhagen

Fc-Glycosylation of IgG1 is Modulated by B-cell Stimuli

We have recently shown that IgG1 directed against antigens thought to be involved in the pathogenesis of rheumatoid arthritis harbor different glycan moieties on their Fc-tail, as compared with total sera IgG1. Given the crucial roles of Fc-linked N-glycans for the structure and biological activity of IgG, Fc-glycosylation of antibodies is receiving considerable interest. However, so far little is known about the signals and factors that could influence the composition of these carbohydrate structures on secreted IgG produced by B lymphocytes. Here we show that both “environmental” factors, such as all-trans retinoic acid (a natural metabolite of vitamin A), as well as factors stimulating the innate immune system (i.e. CpG oligodeoxynucleotide, a ligand for toll-like receptor 9) or coming from the adaptive immune system (i.e. interleukin-21, a T-cell derived cytokine) can modulate IgG1 Fc-glycosylation. These factors affect Fc-glycan profiles in different ways. CpG oligodeoxynucleotide and interleukin-21 increase Fc-linked galactosylation and reduce bisecting N-acetylglucosamine levels, whereas all-trans retinoic acid significantly decreases galactosylation and sialylation levels. Moreover, these effects appeared to be stable and specific for secreted IgG1 as no parallel changes of the corresponding glycans in the cellular glycan pool were observed. Interestingly, several other cytokines and molecules known to affect B-cell biology and antibody production did not have an impact on IgG1 Fc-coupled glycan profiles. Together, these data indicate that different stimuli received by B cells during their activation and differentiation can modulate the Fc-linked glycosylation of secreted IgG1 without affecting the general cellular glycosylation machinery. Our study, therefore, furthers our understanding of the regulation of IgG1 glycosylation at the cellular level.

N-glycosylation of colorectal cancer tissues: a liquid chromatography and mass spectrometry-based investigation

Colorectal cancer is the third most common cancer worldwide with an annual incidence of ∼1 million cases and an annual mortality rate of ∼655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers.

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro-heterogeneity) and evaluate the molar site occupancy (macro-heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N-glycans was chemically synthesised by solid-phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N-acetylglucosamine-linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well-defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI-IT, ESI-Q-TOF, MALDI-TOF, ESI/MALDI-FT-ICR-MS). Depending on the ion source/mass analyser, glycopeptides carrying complex-type N-glycans exhibited clearly lower signal strengths (10-50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano-ESI and medium-pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro-heterogeneity and macro-heterogeneity by label-free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics.

Clinical Glycomics Employing Graphitized Carbon Liquid Chromatography-Mass Spectrometry

Glycoconjugates and free glycan are involved in a variety of biological processes such as cell–cell interaction and cell trafficking. Alterations in the complex glycosylation machinery have been correlated with various pathological processes including cancer progression and metastasis. Mass Spectrometry (MS) has evolved as one of the most powerful tools in glycomics and glycoproteomics and in combination with porous graphitized carbon–liquid chromatography (PGC–LC) it is a versatile and sensitive technique for the analysis of glycans and to some extent also glycopeptides. PGC–LC–ESI–MS analysis is characterized by a high isomer separation power enabling a specific glycan compound analysis on the level of individual structures. This allows the investigation of the biological relevance of particular glycan structures and glycan features. Consequently, this strategy is a very powerful technique suitable for clinical research, such as cancer biomarker discovery, as well as in-depth analysis of recombinant glycoproteins. In this review, we will focus on how PGC in conjunction with MS detection can deliver specific structural information for clinical research on protein-bound N-glycans and mucin-type O-glycans. In addition, we will briefly review PGC analysis approaches for glycopeptides, glycosaminoglycans (GAGs) and human milk oligosaccharides (HMOs). The presented applications cover systems that vary vastly with regard to complexity such as purified glycoproteins, cells, tissue or body fluids revealing specific glycosylation changes associated with various biological processes including cancer and inflammation.

Investigations on Aberrant Glycosylation of Glycosphingolipids in Colorectal Cancer Tissues Using Liquid Chromatography and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)

Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.

Site-Specific Protein N- and O-Glycosylation Analysis by a C18-Porous Graphitized Carbon-Liquid Chromatography-Electrospray Ionization Mass Spectrometry Approach Using Pronase Treated Glycopeptides.

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.

Skewed Fc glycosylation profiles of anti-proteinase 3 immunoglobulin G1 autoantibodies from granulomatosis with polyangiitis patients show low levels of bisection, galactosylation, and sialylation

Granulomatosis with polyangiitis (GPA) is associated with circulating immunoglobulin (Ig) G anti-proteinase 3 specific (anti-PR3) anti-neutrophil cytoplasm antibodies (ANCA), which activate cytokine primed neutrophils via Fcgamma receptors. ANCA are class switched IgG antibodies implying T cell help in their production. Glycosylation of IgG Fc, under the control of T cell cytokines, determines the interaction between IgG and its receptors. Previous studies have reported aberrant glycosylation of Ig Fc in GPA patients. We investigated whether aberrant Fc glycosylation was present on anti-PR3 ANCA as well as whole IgG subclass preparations compared to healthy controls and whether this correlated with Birmingham vasculitis activity scores (BVAS), serum cytokines, and time to remission. Here, IgG Fc glycosylation of GPA patients and controls and anti-PR3 ANCA Fc glycosylation were determined by mass spectrometry of glycopeptides. IgG1 and IgG2 subclasses from GPA patients showed reduced galactosylation, sialylation, and bisection compared to healthy controls. Anti-PR3 IgG1 ANCA Fc galactosylation, sialylation, and bisection were reduced compared to total IgG1 in GPA. Galactosylation of anti-PR3 ANCA Fc correlated with inflammatory cytokines and time to remission but not BVAS. Bisection of anti-PR3 ANCA Fc correlated with BVAS. Total IgG1 and anti-PR3 IgG1 Fc galactosylation were weakly correlated, while bisection of IgG1 and anti-PR3 showed no correlation. Our data indicate that aberrant ANCA galactosylation may be driven in an antigen-specific manner.

Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines : HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples.

The whipworm (Trichuris suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells

Clinical trials have shown that administration of the nematode Trichuris suis can be beneficial in treating various immune disorders. To provide insight into the mechanisms by which this worm suppresses inflammatory responses, an active component was purified from T. suis soluble products (TsSPs) that suppress TNF and IL‐12 secretion from LPS‐activated human dendritic cells (DCs). Analysis by liquid chromatography tandem mass spectrometry identified this compound as prostaglandin (PG)E2. The purified compound showed similar properties compared with TsSPs and commercial PGE2 in modulating LPS‐induced expression of many cytokines and chemokines and in modulating Rab7B and P2RX7 expression in human DCs. Furthermore, the TsSP‐induced reduction of TNF secretion from DCs is reversed by receptor antagonists for EP2 and EP4, indicating PGE2 action. T. suis secretes extremely high amounts of PGE2 (45–90 ng/mg protein) within their excretory/secretory products but few related lipid mediators as established by metabololipidomic analysis. Culture of T. suis with several cyclooxygenase (COX) inhibitors that inhibit mammalian prostaglandin synthesis affected the worms motility but did not inhibit PGE2 secretion, suggesting that the worms can synthesize PGE2 via a COX‐independent pathway. We conclude that T. suis secretes PGE2 to suppress proinflammatory responses in human DCs, thereby modulating the hosts immune response.—Laan, L. C., Williams, A. R., Stavenhagen, K., Giera, M., Kooij, G., Vlasakov, I., Kalay, H., Kringel, H., Nejsum, P., Thamsborg, S. M., Wuhrer, M., Dijkstra, C. D., Cummings, R. D., van Die, I. The whipworm (Trichuris suis) secretes prostaglandin E2 to suppress proinflammatory properties in human dendritic cells. FASEB J. 31, 719–731 (2017). http://www.fasebj.org

Longitudinal monitoring of immunoglobulin A glycosylation during pregnancy by simultaneous MALDI-FTICR-MS analysis of N- and O-glycopeptides.

Immunoglobulin A (IgA) is a glycoprotein of which altered glycosylation has been associated with several pathologies. Conventional methods for IgA N- and O-glycosylation analysis are tedious, thus limiting such analyses to small sample sizes. Here we present a high-throughput strategy for the simultaneous analysis of serum-derived IgA1 N- and O-glycopeptides using matrix-assisted laser/desorption ionisation Fourier transform ion cyclotron resonance (MALDI-FTICR) mass spectrometry (MS). Six non-fucosylated diantennary complex type glycoforms were detected on the Asn144-containing glycopeptide. Thirteen distinct glycoforms were identified for the Asn340-containing tailpiece glycopeptide, mainly of the diantennary complex type, and low amounts of triantennary glycoforms. Simultaneously with these N-glycopeptides, 53 compositional glycoforms of the hinge region O-glycopeptide were profiled in a single high resolution MALDI-FTICR spectrum. Since many pregnancy associated changes have been recognized for immunoglobulin G, we sought to demonstrate the clinical applicability of this method in a cohort of 29 pregnant women, from whom samples were collected at three time points during pregnancy and three time points after delivery. Pregnancy associated changes of N-glycan bisection were different for IgA1 as compared to IgG-Fc described earlier. We foresee further applications of the developed method for larger patient cohorts to study IgA N- and O-glycosylation changes in pathologies.