A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by... Show moreA group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by its substrate specificity,methods that can extensively characterize substrate specificity are valuabletools for protease research. Previously, we achieved an in-depth characteri-zation of PPEP prime-side specificity. However, PPEP specificity is alsodetermined by the non-prime-side residues in the substrate. To gain a morecomplete insight into the determinants of PPEP specificity, we character-ized the non-prime- and prime-side specificity of various PPEPs using acombination of synthetic combinatorial peptide libraries and mass spec-trometry. With this approach, we deepened our understanding of the P3-P30 specificities of PPEP-1 and PPEP-2, while identifying the endogenoussubstrate of PPEP-2 as the most optimal substrate in our library data. Fur-thermore, by employing the library approach, we investigated the alteredspecificity of mutants of PPEP-1 and PPEP-2. Additionally, we character-ized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displaysa PPEP-1-like prime-side specificity, which was substantiated by the experi-mental data. Intriguingly, another putative PPEP from Clostridioides diffi-cile, CD1597, did not display Pro-Pro endoproteolytic activity.Collectively, we characterized PPEP specificity in detail using our robustpeptide library method and, together with additional structural informa-tion, provide more insight into the intricate mechanisms that govern prote-ase specificity. Show less
Kissel, T.; Derksen, V.F.A.M.; Bentlage, A.E.H.; Koeleman, C.; Hafkenscheid, L.; Woude, D. van der; ... ; Toes, R.E.M. 2024
IgG secreted by B cells carry asparagine N(297)-linked glycans in the fragment crystallizable (Fc) region. Changes in Fc glycosylation are related to health or disease and are functionally relevant... Show moreIgG secreted by B cells carry asparagine N(297)-linked glycans in the fragment crystallizable (Fc) region. Changes in Fc glycosylation are related to health or disease and are functionally relevant, as IgG without Fc glycans cannot bind to Fcɣ receptors or complement factors. However, it is currently unknown whether ɣ-heavy chain (ɣHC) glycans also influence the function of membrane-bound IgG-B-cell receptors (BCR) and thus the outcome of the B-cell immune response. Here, we show in a germinal center (GC)-derived human B-cell line that ɣHC glycans do not affect membrane expression of IgG-BCRs. Furthermore, antigen binding or other BCR-facilitated mechanisms appear unaffected, including BCR downmodulation or BCR-mediated signaling. As expected, secreted IgG lacking Fc glycosylation is unable to carry out effector functions. Together, these observations indicate that IgG-Fc glycosylation serves as a mechanism to control the effector functions of antibodies, but does not regulate the activation of IgG-switched B cells, as its absence had no apparent impact on BCR function. Show less
Graaf, E.L. de; Larsen, M.D.; Bolt, N. van der; Visser, R.; Verhagen, O.J.H.M.; Ederveen, A.L.H.; ... ; Vidarsson, G. 2023
The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and... Show moreThe type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies. Show less
Wang, W.; Nier, C.R. de; Wuhrer, M.; Lageveen-Kammeijer, G.S.M. 2023
Prostate-specific antigen (PSA) is a well-known clinical biomarker in prostate cancer (PCa) diagnosis, but a better test is still needed, as the serum-level-based PSA quantification exhibits... Show moreProstate-specific antigen (PSA) is a well-known clinical biomarker in prostate cancer (PCa) diagnosis, but a better test is still needed, as the serum-level-based PSA quantification exhibits limited specificity and comes with poor predictive value. Prior to PSA, prostatic acid phosphatase (PAP) was used, but it was replaced by PSA because PSA improved the early detection of PCa. Upon revisiting PAP and its glycosylation specifically, it appears to be a promising new biomarker candidate. Namely, previous studies have indicated that PAP glycoforms differ between PCa and non-PCa individuals. However, an in-depth characterization of PAP glycosylation is still lacking. In this study, we established an in-depth glycoproteomic assay for urinary PAP by characterizing both the micro- and macroheterogeneity of the PAP glycoprofile. For this purpose, PAP samples were analyzed by capillary electrophoresis coupled to mass spectrometry after affinity purification from urine and proteolytic digestion. The developed urinary PAP assay was applied on a pooled DRE (digital rectal examination) urine sample from nine individuals. Three glycosylation sites were characterized, namely N-94, N-220, and N-333, via N-glycopeptide analysis. Taking sialic acid linkage isomers into account, a total of 63, 27, and 4 N-glycan structures were identified, respectively. The presented PAP glycoproteomic assay will enable the determination of potential glycomic biomarkers for the early detection and prognosis of PCa in cohort studies. Show less
Glycans play a pivotal role in biology. However, because of the low-affinity of glycan-protein interactions, many interaction pairs remain unknown. Two important glycoproteins involved in B-cell... Show moreGlycans play a pivotal role in biology. However, because of the low-affinity of glycan-protein interactions, many interaction pairs remain unknown. Two important glycoproteins involved in B-cell biology are the B-cell receptor and its secreted counterpart, antibodies. It has been indicated that glycans expressed by these B-cell-specific molecules can modulate immune activation via glycan-binding proteins. In several autoimmune diseases, an increased prevalence of variable domain glycosylation of IgG autoantibodies has been observed. Especially, the hallmarking autoantibodies in rheumatoid arthritis, anti-citrullinated protein antibodies, carry a substantial amount of variable domain glycans. The variable domain glycans expressed by these autoantibodies are N-linked, complex-type, and alpha 2-6 sialylated, and B-cell receptors carrying variable domain glycans have been hypothesized to promote selection of autoreactive B cells via interactions with glycan-binding proteins. Here, we use the anti-citrullinated protein antibody response as a prototype to study potential in solution and in situ B-cell receptor-variable domain glycan interactors. We employed SiaDAz, a UV-activatable sialic acid analog carrying a diazirine moiety that can form covalent bonds with proximal glycan-binding proteins. We show, using oligosaccharide engineering, that SiaDAz can be readily incorporated into variable domain glycans of both antibodies and B-cell receptors. Our data show that antibody variable domain glycans are able to interact with inhibitory receptor, CD22. Interestingly, although we did not detect this interaction on the cell surface, we captured CD79 beta glycan-B-cell receptor interactions. These results show the utility of combining photoaffinity labeling and oligosaccharide engineering for identifying antibody and B-cell receptor interactions and indicate that variable domain glycans appear not to be lectin cis ligands in our tested conditions. Show less
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease... Show moreFetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available. Show less
Naber, A.; Demus, D.; Slieker, R.; Nicolardi, S.; Beulens, J.W.J.; Elders, P.J.M.; ... ; Hoek, M. van 2023
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
Wojcik, I.; Wuhrer, M.; Heeringa, P.; Stegeman, C.A.; Rutgers, A.; Falck, D. 2023
Introduction: Immunoglobulin G (IgG) contains a conserved N-glycan in the fragment crystallizable (Fc), modulating its structure and effector functions. In anti-neutrophil cytoplasmic antibody ... Show moreIntroduction: Immunoglobulin G (IgG) contains a conserved N-glycan in the fragment crystallizable (Fc), modulating its structure and effector functions. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) alterations of IgG Fc-glycosylation have been observed to correlate with the disease course. Here, we examined longitudinal changes in N-linked Fc glycans of IgG in an AAV patient cohort and their relationship with disease flares.Methods: Using liquid chromatography coupled with mass spectrometry, we analysed IgG Fc-glycosylation in 410 longitudinal samples from 96 individuals with AAV.Results: Analysis of the cross-sectional differences as well as longitudinal changes demonstrated that IgGs of relapsing PR3-ANCA patients have higher ΔFc-bisection at diagnosis (P = 0.004) and exhibit a decrease in Fc-sialylation prior to the relapse (P = 0.0004), discriminating them from non-relapsing patients. Most importantly, PR3-ANCA patients who experienced an ANCA rise and relapsed shortly thereafter, exhibit lower IgG Fc-fucosylation levels compared to non-relapsing patients already 9 months before relapse (P = 0.02).Discussion: Our data indicate that IgG Fc-bisection correlates with long-term treatment outcome, while lower IgG Fc-fucosylation and sialylation associate with impending relapse. Overall, our study replicated the previously published reduction in total IgG Fc-sialylation at the time of relapse, but showed additionally that its onset precedes relapse. Furthermore, our findings on IgG fucosylation and bisection are entirely new. All these IgG Fc-glycosylation features may have the potential to predict a relapse either independently or in combination with known risk factors, such as a rise in ANCA titre. Show less
Marco, F. di; Blöchl, C.; Esser-Skala, W.; Schäpertöns, V.; Zhang, T.; Wuhrer, M.; ... ; Huber, C.G. 2023
Characterization of highly glycosylated biopharmaceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an... Show moreCharacterization of highly glycosylated biopharmaceuticals by mass spectrometry is challenging because of the huge chemical space of coexistent glycoforms present. Here, we report the use of an array of HPLC-mass spectrometry-based approaches at different structural levels of released glycan, glycopeptide, and hitherto unexplored intact glycoforms to scrutinize the biopharmaceutical Myozyme, containing the highly complex lysosomal enzyme recombinant acid alpha-glucosidase. The intrinsic heterogeneity of recombinant acid alpha-glucosidase glycoforms was unraveled using a novel strong anion exchange HPLC-mass spectrometry approach involving a pH-gradient of volatile buffers to facilitate chromatographic separation of glycoforms based on their degree of sialylation, followed by the acquisition of native mass spectra in an Orbitrap mass spectrometer. Upon considering the structures of 60 different glycans attached to seven glycosylation sites in the intact protein, the large set of interdependent data acquired at different structural levels was integrated using a set of bioinformatic tools and allowed the annotation of intact glycoforms unraveling more than 1,000,000 putative intact glycoforms. Detectable isoforms also included several mannose-6-phosphate variants, which are essential for directing the drug toward its target, the lysosomes. Finally, for the first time, we sought to validate the intact glycoform annotations by integrating experimental data on the enzymatically dissected proteoforms, which reduced the number of glycoforms supported by experimental evidence to 42,104. The latter verification clearly revealed the strengths but also intrinsic limitations of this approach for fully characterizing such highly complex glycoproteins by mass spectrometry. Show less
Svecla, M.; Nour, J.; Bladergroen, M.R.; Nicolardi, S.; Zhang, T.; Beretta, G.; ... ; Falck, D. 2023
The asialoglycoprotein receptor (ASGPR) and the mannose receptor C -type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N... Show moreThe asialoglycoprotein receptor (ASGPR) and the mannose receptor C -type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and Nglycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor. Show less
Coillie, J. van; Pongracz, T.; Sustic, T.; Wang, W.J.; Nouta, J.; Gars, M. le; ... ; COUGH1 Study Grp 2023
IgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linke... Show moreIgG antibodies are important mediators of vaccine-induced immunity through complement- and Fc receptor-dependent effector functions. Both are influenced by the composition of the conserved N-linked glycan located in the IgG Fc domain. Here, we compared the anti-Spike (S) IgG1 Fc glycosylation profiles in response to mRNA, adenoviral, and protein-based COVID-19 vaccines by mass spectrometry (MS). All vaccines induced a transient increase of antigen-specific IgG1 Fc galactosylation and sialylation. An initial, transient increase of afucosylated IgG was induced by membrane-encoding S protein formulations. A fucose-sensitive ELISA for antigen-specific IgG (FEASI) exploiting FcγRIIIa affinity for afucosylated IgG was used as an orthogonal method to confirm the LC-MS-based afucosylation readout. Our data suggest that vaccine-induced anti-S IgG glycosylation is dynamic, and although variation is seen between different vaccine platforms and individuals, the evolution of glycosylation patterns display marked overlaps. Show less
Visceral leishmaniasis (VL) is a clinical form of leishmaniasis with high mortality rates when not treated. Diagnosis suffers from invasive techniques and sub-optimal sensitivities. The current ... Show moreVisceral leishmaniasis (VL) is a clinical form of leishmaniasis with high mortality rates when not treated. Diagnosis suffers from invasive techniques and sub-optimal sensitivities. The current (affordable) treatment with pentavalent antimony as advised by the WHO is possibly harmful to the patient. There is need for an improved diagnosis to prevent possibly unnecessary treatment. N-glycan analysis may aid in diagnosis. We evaluated the N-glycan profiles from active VL, asymptomatic infections (ASYMP) and controls from non-endemic (NC) and endemic (EC) areas. Active VL has a distinct N-glycome profile that associates with disease severity. Our study suggests that the observed glycan signatures could be a valuable additive to diagnosis and assist in identifying possible markers of disease and understanding the pathogenesis of VL. Further studies are warranted to assess a possible future role of blood glycome analysis in active VL diagnosis and should aim at disease specificity. Show less
Memarian, E.; Heijmans, R.; Slieker, R.C.; Sierra, A.; Gornik, O.; Beulens, J.W.J.; ... ; Hoek, M. van 2023
Aims/HypothesisInflammation is important in the development of type 2 diabetes complications. The N-glycosylation of IgG influences its role in inflammation. To date, the association of plasma IgG N-. Show moreAims/HypothesisInflammation is important in the development of type 2 diabetes complications. The N-glycosylation of IgG influences its role in inflammation. To date, the association of plasma IgG N-glycosylation with type 2 diabetes complications has not been extensively investigated. We hypothesised that N-glycosylation of IgG may be related to the development of complications of type 2 diabetes.MethodsIn three independent type 2 diabetes cohorts, plasma IgG N-glycosylation was measured using ultra performance liquid chromatography (DiaGene n = 1815, GenodiabMar n = 640) and mass spectrometry (Hoorn Diabetes Care Study n = 1266). We investigated the associations of IgG N-glycosylation (fucosylation, galactosylation, sialylation and bisection) with incident and prevalent nephropathy, retinopathy and macrovascular disease using Cox- and logistic regression, followed by meta-analyses. The models were adjusted for age and sex and additionally for clinical risk factors.ResultsIgG galactosylation was negatively associated with prevalent and incident nephropathy and macrovascular disease after adjustment for clinical risk factors. Sialylation was negatively associated with incident diabetic nephropathy after adjustment for clinical risk factors. For incident retinopathy, similar associations were found for galactosylation, adjusted for age and sex.ConclusionsWe showed that IgG N-glycosylation, particularly galactosylation and to a lesser extent sialylation, is associated with a higher prevalence and future development of macro- and microvascular complications of diabetes. These findings indicate the predictive potential of IgG N-glycosylation in diabetes complications and should be analysed further in additional large cohorts to obtain the power to solidify these conclusions. Show less
Marco, F. di; Blümel, G.; Blöchl, C.; Wuhrer, M.; Huber, C.G. 2023
BackgroundGonadotropins are a class of heavily glycosylated protein hormones, thus extremely challenging to characterize by mass spectrometry. As biopharmaceuticals, gonadotropins are prescribed... Show moreBackgroundGonadotropins are a class of heavily glycosylated protein hormones, thus extremely challenging to characterize by mass spectrometry. As biopharmaceuticals, gonadotropins are prescribed for the treatment of infertility and are derived from different sources: either from pooled urine of pregnant women or upon production in genetically modified Chinese Hamster Ovary cells. Human chorionic gonadotropin (hCG) is sold as a biopharmaceutical under the name Pregnyl® (urinary hCG, u-hCG) and Ovitrelle® (recombinant hCG, r-hCG), and recombinant human follicle stimulating hormone (r-hFSH) is marketed as Gonal-f®. Recently, we reported the exhaustive characterization of r-hCG at different structural levels.ResultsWe implement size exclusion (SE) HPLC-MS to automatize the acquisition of native mass spectra of r-hCG dimer, but also u-hCG and r-hFSH, comparing the drug products up to intact heterodimer level. A hybrid HPLC-MS approach was employed for the characterization of r-hCG, u-hCG and r-hFSH drug products at different structural levels. Released glycans were analyzed by porous graphitized carbon (PGC)-HPLC-MS/MS, glycopeptides by reversed-phase (RP)-HPLC-MS/MS, subunits by RP-HPLC-MS and finally the intact native heterodimers by semi-automated online buffer exchange SE-HPLC-MS. The data were integrated using bioinformatic tools, to finally unravel the composition of 1481 co-existing dimeric glycoforms for r-hCG, 1167 glycoforms for u-hCG, and 1440 glycoforms for r-hFSH, and to compare critical quality attributes of the different drug products such as their degree of sialylation and O-glycosylation.Significance and noveltyThe strong alliance of bioanalytics and bioinformatics data integration at the different structural levels allowed the identification of more than thousand different glycoforms of r-hCG, u-hCG, and r-hFSH. The results showed that these biopharmaceuticals differ considerably in their glycosylation patterns and highlight the importance of in-depth characterization of biopharmaceuticals for quality control. Show less
Mayboroda, O.A.; Lageveen-Kammeijer, G.S.M.; Wuhrer, M.; Dolhain, R.J.E.M. 2023
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... Show moreRheumatoid 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. Show less
Ketodeoxynononic acid (Kdn) is a rather uncommon class of sialic acid in mammals. However, associations have been found between elevated concentrations of free or conjugated Kdn in relation to... Show moreKetodeoxynononic acid (Kdn) is a rather uncommon class of sialic acid in mammals. However, associations have been found between elevated concentrations of free or conjugated Kdn in relation to human cancer progression. Hitherto, there has been a lack of conclusive evidence that Kdn occurs on (specific) human glycoproteins (conjugated Kdn). Here, we report for the first time that Kdn is expressed on prostate-specific antigen (PSA) N-linked glycans derived from human seminal plasma and urine. Interestingly, Kdn was found only in an α2,3-linkage configuration on an antennary galactose, indicating a highly specific biosynthesis. This unusual glycosylation feature was also identified in a urinary PSA cohort in relation to prostate cancer (PCa), although no differences were found between PCa and non-PCa patients. Further research is needed to investigate the occurrence, biosynthesis, biological role, and biomarker potential of both free and conjugated Kdn in humans. Show less
Aberrant glycosylation is considered to be a hallmark of colorectal cancer (CRC), as demonstrated by various studies. While the N-glycosylation of cell lines and serum has been widely examined, the... Show moreAberrant glycosylation is considered to be a hallmark of colorectal cancer (CRC), as demonstrated by various studies. While the N-glycosylation of cell lines and serum has been widely examined, the analysis of cancer-associated N-glycans from tissues has been hampered by the heterogeneity of tumors and the complexity of N-glycan structures. To overcome these obstacles, we present a study using laser capture microdissection that makes it possible to largely deconvolute distinct N-glycomic signatures originating from different regions of heterogeneous tissues including cancerous, stromal, and healthy mucosa cells. N-glycan alditols were analyzed by means of porous graphitized carbon liquid chromatography-electrospray ionization tandem mass spectrometry, enabling the differentiation and structural characterization of isomeric species. In total, 116 N-glycans were identified that showed profound differences in expression among cancer, stroma, and normal mucosa. In comparison with healthy mucosa, the cancer cells showed an increase in α2-6 sialylation and monoantennary N-glycans, as well as a decrease in bisected N-glycans. Moreover, specific sialylated and (sialyl-)LewisA/X antigen-carrying N-glycans were exclusively expressed in cancers. In comparison with cancer, the stroma showed lower levels of oligomannosidic and monoantennary N-glycans, LewisA/X epitopes, and sulfation, as well as increased expression of (core-)fucosylation and α2-3 sialylation. Our study reveals the distinct N-glycomic profiles of different cell types in CRC and control tissues, proving the necessity of their separate analysis for the discovery of cancer-associated glycans. Show less
Despite the success of COVID-19 vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern have emerged that can cause breakthrough infections. Although protection... Show moreDespite the success of COVID-19 vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern have emerged that can cause breakthrough infections. Although protection against severe disease has been largely preserved, the immunological mediators of protection in humans remain undefined. We performed a substudy on the ChAdOx1 nCoV-19 (AZD1222) vaccinees enrolled in a South African clinical trial. At peak immunogenicity, before infection, no differences were observed in immunoglobulin (Ig)G1-binding antibody titers; however, the vaccine induced different Fc-receptor-binding antibodies across groups. Vaccinees who resisted COVID-19 exclusively mounted FcγR3B-binding antibodies. In contrast, enhanced IgA and IgG3, linked to enriched FcγR2B binding, was observed in individuals who experienced breakthrough. Antibodies unable to bind to FcγR3B led to immune complex clearance and resulted in inflammatory cascades. Differential antibody binding to FcγR3B was linked to Fc-glycosylation differences in SARS-CoV-2-specific antibodies. These data potentially point to specific FcγR3B-mediated antibody functional profiles as critical markers of immunity against COVID-19. Show less
Schaick, G. van; Wuhrer, M.; Domínguez-Vega, E. 2023
Proteins carry a plethora of post-translational modifications (PTMs), such as glycosylation or phosphorylation, which may affect stability and activity. Analytical strategies are needed to... Show moreProteins carry a plethora of post-translational modifications (PTMs), such as glycosylation or phosphorylation, which may affect stability and activity. Analytical strategies are needed to investigate these PTMs in their native state to determine the link between structure and function. The coupling of native separation techniques with mass spectrometry (MS) has emerged as a powerful tool for in-depth protein characterization. Yet obtaining high ionization efficiency still can be challenging. Here, we explored the potential of dopant-enriched nitrogen (DEN) gas to improve nano-electrospray ionization (nano-ESI)-MS of native proteins after anion exchange chromatography. The dopant gas was enriched with different dopants (acetonitrile, methanol, and isopropanol) and the effects were compared with the use of solely nitrogen gas for six proteins covering a wide range of physicochemical properties. The use of DEN gas resulted generally in lower charge states, independent of the selected dopant. Moreover, less adduct formation was observed, particularly for the acetonitrile-enriched nitrogen gas. Importantly, striking differences in MS signal intensity and spectral quality were observed for extensively glycosylated proteins, where isopropanol- and methanol-enriched nitrogen appeared to be most beneficial. Altogether, the use of DEN gas improved nano-ESI of native glycoproteins and increased spectral quality for highly glycosylated proteins that normally suffer from low ionization efficiency. Show less
Schaick, G. van; Pot, S.; Schouten, O.; Hartog, J. den; Akeroyd, M.; Hoeven, R.V.; ... ; Dominguez-Vega, E. 2023
Protein glycation may occur naturally when reducing sugars and proteins coexist, which is often the case for industrial enzymes. The impact of post-translational modifications on enzyme... Show moreProtein glycation may occur naturally when reducing sugars and proteins coexist, which is often the case for industrial enzymes. The impact of post-translational modifications on enzyme performance (e.g., stability or function) is often not predictable, highlighting the importance of having appropriate analytical methodologies to monitor the influence of glycation on performance. Here, a boronate affinity chromatography method was developed to enrich glycated species followed by mass spectrometry for structural characterization and activity assays for functional assessment. This approach was applied to a (temperature-stressed) lipase used for food applications revealing that storage at -20 °C and 4 °C resulted in minor glycation (below 9%), whereas storage at 25 °C led to a higher glycation level with up to four sugars per lipase molecule. Remarkably, activity measurements revealed that glycation did not reduce lipase activity or stability. Altogether, this novel strategy is a helpful extension to the current analytical toolbox supporting development of enzyme products. Show less
