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
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
Oskam, N.; Damelang, T.; Streutker, M.; Heer, P.; Nouta, J.; Koeleman, C.; ... ; Rispens, T. 2023
Of the four human immunoglobulin G (IgG) subclasses, IgG4 is considered the least inflammatory, in part because it poorly activates the complement system. Regardless, in IgG4 related disease (IgG4... Show moreOf the four human immunoglobulin G (IgG) subclasses, IgG4 is considered the least inflammatory, in part because it poorly activates the complement system. Regardless, in IgG4 related disease (IgG4-RD) and in autoimmune disorders with high levels of IgG4 autoantibodies, the presence of these antibodies has been linked to consumption and deposition of complement components. This apparent paradox suggests that conditions may exist, potentially reminiscent of in vivo deposits, that allow for complement activation by IgG4. Furthermore, it is currently unclear how variable glycosylation and Fab arm exchange may influence the ability of IgG4 to activate complement. Here, we used well-defined, glyco-engineered monoclonal preparations of IgG4 and determined their ability to activate complement in a controlled system. We show that IgG4 can activate complement only at high antigen and antibody concentrations, via the classical pathway. Moreover, elevated or reduced Fc galactosylation enhanced or diminished complement activation, respectively, with no apparent contribution from the lectin pathway. Fab glycans slightly reduced complement activation. Lastly, we show that bispecific, monovalent IgG4 resulting from Fab arm exchange is a less potent activator of complement than monospecific IgG4. Taken together, these results imply that involvement of IgG4-mediated complement activation in pathology is possible but unlikely. Show less
Background: The new types of mRNA-containing lipid nanoparticle vaccines BNT162b2 and mRNA-1273 and the adenovirus-based vaccine AZD1222 were developed against SARS-CoV-2 and code for its spike (S)... Show moreBackground: The new types of mRNA-containing lipid nanoparticle vaccines BNT162b2 and mRNA-1273 and the adenovirus-based vaccine AZD1222 were developed against SARS-CoV-2 and code for its spike (S) protein. Several studies have investigated short-term antibody (Ab) responses after vaccination. Objective: However, the impact of these new vaccine formats with unclear effects on the long-term Ab response - including isotype, subclass, and their type of Fc glycosylation - is less explored. Methods: Here, we analyzed anti-S Ab responses in blood serum and the saliva of SARS-CoV-2 naive and non-hospitalized pre-infected subjects upon two vaccinations with different mRNA- and adenovirus-based vaccine combinations up to day 270. Results: We show that the initially high mRNA vaccine-induced blood and salivary anti-S IgG levels, particularly IgG1, markedly decrease over time and approach the lower levels induced with the adenovirus-based vaccine. All three vaccines induced, contrary to the short-term anti-S IgG1 response with high sialylation and galactosylation levels, a long-term anti-S IgG1 response that was characterized by low sialylation and galactosylation with the latter being even below the corresponding total IgG1 galactosylation level. Instead, the mRNA, but not the adenovirus-based vaccines induced long-term IgG4 responses - the IgG subclass with inhibitory effector functions. Furthermore, salivary anti-S IgA levels were lower and decreased faster in naive as compared to pre-infected vaccinees. Predictively, age correlated with lower long-term anti-S IgG titers for the mRNA vaccines. Furthermore, higher total IgG1 galactosylation, sialylation, and bisection levels correlated with higher long-term anti-S IgG1 sialylation, galactosylation, and bisection levels, respectively, for all vaccine combinations. Conclusion: In summary, the study suggests a comparable "adjuvant" potential of the newly developed vaccines on the anti-S IgG Fc glycosylation, as reflected in relatively low long-term anti-S IgG1 galactosylation levels generated by the long-lived plasma cell pool, whose induction might be driven by a recently described T-H1-driven B cell response for all three vaccines. Instead, repeated immunization of naive individuals with the mRNA vaccines increased the proportion of the IgG4 subclass over time which might influence the long-term Ab effector functions. Taken together, these data shed light on these novel vaccine formats and might have potential implications for their long-term efficacy. Show less
Coillie, J. van; Pongracz, T.; Rahmoller, J.; Chen, H.J.; Geyer, C.E.; Vught, L.A. van; ... ; UMC COVID-19 S HCW Study Grp 2022
Background: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through... Show moreBackground: Afucosylated IgG1 responses have only been found against membrane-embedded epitopes, including anti-S in SARS-CoV-2 infections. These responses, intrinsically protective through enhanced Fc gamma RIIIa binding, can also trigger exacerbated pro-inflammatory responses in severe COVID-19. We investigated if the BNT162b2 SARS-CoV-2 mRNA also induced afucosylated IgG responses. Methods: Blood from vaccinees during the first vaccination wave was collected. Liquid chromatography-Mass spectrometry (LC-MS) was used to study anti-S IgG1 Fc glycoprofiles. Responsiveness of alveolar-like macrophages to produce proinflammatory cytokines in presence of sera and antigen was tested. Antigen-specific B cells were characterized and glycosyltransferase levels were investigated by Fluorescence-Activated Cell Sorting (FACS). Findings: Initial transient afucosylated anti-S IgG1 responses were found in naive vaccinees, but not in antigen -experienced ones. All vaccinees had increased galactosylated and sialylated anti-S IgG1. Both naive and antigen -experienced vaccinees showed relatively low macrophage activation potential, as expected, due to the low antibody levels for naive individuals with afucosylated IgG1, and low afucosylation levels for antigen-experienced individuals with high levels of anti-S. Afucosylation levels correlated with FUT8 expression in antigen-specific plasma cells in naive individuals. Interestingly, low fucosylation of anti-S IgG1 upon seroconversion correlated with high anti-S IgG levels after the second dose. Interpretation: Here, we show that BNT162b2 mRNA vaccination induces transient afucosylated anti-S IgG1 responses in naive individuals. This observation warrants further studies to elucidate the clinical context in which potent afucosylated responses would be preferred. Copyright (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Show less
Siekman, S.L.; Pongracz, T.; Wang, W.J.; Nouta, J.; Kremsner, P.G.; Silva-Neto, P.V. da; ... ; Wuhrer, M. 2022
Immunoglobulin G (IgG) antibodies play an important role in the immune response against viruses such as SARS-CoV-2. As the effector functions of IgG are modulated by N-glycosylation of the Fc... Show moreImmunoglobulin G (IgG) antibodies play an important role in the immune response against viruses such as SARS-CoV-2. As the effector functions of IgG are modulated by N-glycosylation of the Fc region, the structure and possible function of the IgG N-glycome has been under investigation in relation to divergent COVID-19 disease courses. Through LC-MS analysis we studied both total IgG1 and spike protein-specific IgG1 Fc glycosylation of 129 German and 163 Brazilian COVID-19 patients representing diverse patient populations. We found that hospitalized COVID-19 patients displayed decreased levels of total IgG1 bisection and galactosylation and lowered anti-S IgG1 fucosylation and bisection as compared to mild outpatients. Anti-S IgG1 glycosylation was dynamic over the disease course and both anti-S and total IgG1 glycosylation were correlated to inflammatory markers. Further research is needed to dissect the possible role of altered IgG glycosylation profiles in (dys)regulating the immune response in COVID-19. Show less
Sustic, T.; Coillie, J. van; Larsen, M.D.; Derksen, N.I.L.; Szittner, Z.; Nouta, J.; ... ; Vidarsson, G. 2022
Background: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (Fc gamma R). Absence of fucose on the highly conserved N-linked glycan in the... Show moreBackground: Immunoglobulin G (IgG) antibodies serve a crucial immuno-protective function mediated by IgG Fc receptors (Fc gamma R). Absence of fucose on the highly conserved N-linked glycan in the IgG Fc domain strongly enhances IgG binding and activation of myeloid and natural killer (NK) cell Fc gamma Rs. Although afucosylated IgG can provide increased protection (malaria and HIV), it also boosts immunopathologies in alloimmune diseases, COVID-19 and dengue fever. Quantifying IgG fucosylation currently requires sophisticated methods such as liquid chromatography-mass spectrometry (LC-MS) and extensive analytical skills reserved to highly specialized laboratories. Methods: Here, we introduce the Fucose-sensitive Enzyme-linked immunosorbent assay (ELISA) for Antigen-Specific IgG (FEASI), an immunoassay capable of simultaneously quantitating and qualitatively determining IgG responses. FEASI is a two-tier immunoassay; the first assay is used to quantify antigen-specific IgG (IgG ELISA), while the second gives Fc gamma RIIIa binding-dependent readout which is highly sensitive to both the IgG quantity and the IgG Fc fucosylation (Fc gamma R-IgG ELISA). Findings: IgG Fc fucosylation levels, independently determined by LC-MS and FEASI, in COVID-19 responses to the spike (S) antigen, correlated very strongly by simple linear regression (R-2=0.93, p < 0.0001). The FEASI method was then used to quantify IgG levels and fucosylation in COVID-19 convalescent plasma which was independently validated by LC-MS. Interpretation: FEASI can be reliably implemented to measure relative and absolute IgG Fc fucosylation and quantify binding of antigen-specific IgG to Fc gamma R in a high-throughput manner accessible to all diagnostic and research laboratories. Copyright (c) 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Show less
Background: Immunoglobulin G1 (IgG1) effector functions are impacted by the structure of fragment crystallizable (Fc) tail-linked N-glycans. Low fucosylation levels on severe acute respiratory... Show moreBackground: Immunoglobulin G1 (IgG1) effector functions are impacted by the structure of fragment crystallizable (Fc) tail-linked N-glycans. Low fucosylation levels on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein-specific IgG1 has been described as a hallmark of severe coronavirus disease 2019 (COVID-19) and may lead to activation of macrophages via immune complexes thereby promoting inflammatory responses, altogether suggesting involvement of IgG1 Fc glycosylation modulated immune mechanisms in COVID-19. Methods: In this prospective, observational single center cohort study, IgG1 Fc glycosylation was analyzed by liquid chromatography-mass spectrometry following affinity capturing from serial plasma samples of 159 SARS-CoV-2 infected hospitalized patients. Findings: At baseline close to disease onset, anti-S IgG1 glycosylation was highly skewed when compared to total plasma IgG1. A rapid, general reduction in glycosylation skewing was observed during the disease course. Low anti S IgG1 galactosylation and sialylation as well as high bisection were early hallmarks of disease severity, whilst high galactosylation and sialylation and low bisection were found in patients with low disease severity. In line with these observations, anti-S IgG1 glycosylation correlated with various inflammatory markers. Interpretation: Association of low galactosylation, sialylation as well as high bisection with disease severity and inflammatory markers suggests that further studies are needed to understand how anti-S IgG1 glycosylation may contribute to disease mechanism and to evaluate its biomarker potential. Copyright (c) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Show less
Immune-mediated platelet refractoriness (PR) remains a significant problem in the setting of platelet transfusion and is predominantly caused by the presence of alloantibodies directed against... Show moreImmune-mediated platelet refractoriness (PR) remains a significant problem in the setting of platelet transfusion and is predominantly caused by the presence of alloantibodies directed against class I human leukocyte antigens (HLA). Opsonization of donor platelets with these alloantibodies can result in rapid clearance after transfusion via multiple mechanisms, including antibody dependent cellular phagocytosis (ADCP). Interestingly, not all alloimmunized patients develop PR to unmatched platelet transfusions, suggesting variation in HLA-specific IgG responses between patients. Previously, we observed that the glycosylation profile of anti-HLA antibodies was highly variable between PR patients, especially with respect to Fc galactosylation, sialylation and fucosylation. In the current study, we investigated the effect of different Fc glycosylation patterns, with known effects on complement deposition and FcγR binding, on phagocytosis of opsonized platelets by monocyte-derived human macrophages. We found that the phagocytosis of antibody- and complement-opsonized platelets, by monocyte derived M1 macrophages, was unaffected by these qualitative IgG-glycan differences. Show less
A subset of patients with coronavirus disease 2019 (COVID-19) become critically ill, suffering from severe respiratory problems and also increased rates of thrombosis. The causes of thrombosis in... Show moreA subset of patients with coronavirus disease 2019 (COVID-19) become critically ill, suffering from severe respiratory problems and also increased rates of thrombosis. The causes of thrombosis in severely ill patients with COVID-19 are still emerging, but the coincidence of critical illness with the timing of the onset of adaptive immunity could implicate an excessive immune response. We hypothesized that platelets might be susceptible to activation by anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) antibodies and might contribute to thrombosis. We found that immune complexes containing recombinant SARS CoV 2 spike protein and anti-spike immunoglobulin G enhanced platelet-mediated thrombosis on von Willebrand factor in vitro, but only when the glycosylation state of the Fc domain was modified to correspond with the aberrant glycosylation previously identified in patients with severe COVID-19. Furthermore, we found that activation was dependent on Fc gamma RIIA, and we provide in vitro evidence that this pathogenic platelet activation can be counteracted by the therapeutic small molecules R406 (fostamatinib) and ibrutinib, which inhibit tyrosine kinases Syk and Btk, respectively, or by the P2Y(12) antagonist cangrelor. Show less
Larsen, M.D.; Lopez-Perez, M.; Dickson, E.K.; Ampomah, P.; Ndam, N.T.; Nouta, J.; ... ; Vidarsson, G. 2021
Here, Larsen et al. describe differences in Fc fucosylation of P. falciparum PfEMP1-specific IgG produced in response to natural infection versus VAR2CSA-type subunit vaccination, which leads to... Show moreHere, Larsen et al. describe differences in Fc fucosylation of P. falciparum PfEMP1-specific IgG produced in response to natural infection versus VAR2CSA-type subunit vaccination, which leads to differences in the ability to induce Fc gamma RIIIa-dependent natural killer cell degranulation.Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family members mediate receptor- and tissue-specific sequestration of infected erythrocytes (IEs) in malaria. Antibody responses are a central component of naturally acquired malaria immunity. PfEMP1-specific IgG likely protects by inhibiting IE sequestration and through IgG-Fc Receptor (Fc gamma R) mediated phagocytosis and killing of antibody-opsonized IEs. The affinity of afucosylated IgG to Fc gamma RIIIa is up to 40-fold higher than fucosylated IgG, resulting in enhanced antibody-dependent cellular cytotoxicity. Most IgG in plasma is fully fucosylated, but afucosylated IgG is elicited in response to enveloped viruses and to paternal alloantigens during pregnancy. Here we show that naturally acquired PfEMP1-specific IgG is strongly afucosylated in a stable and exposure-dependent manner, and efficiently induces Fc gamma RIIIa-dependent natural killer (NK) cell degranulation. In contrast, immunization with a subunit PfEMP1 (VAR2CSA) vaccine results in fully fucosylated specific IgG. These results have implications for understanding protective natural- and vaccine-induced immunity to malaria. Show less
Osch, T.L.J. van; Nouta, J.; Derksen, N.I.L.; Mierlo, G. van; Schoot, C.E. van der; Wuhrer, M.; ... ; Vidarsson, G. 2021
Human IgG contains one evolutionarily conserved N-linked glycan in its Fc region at position 297. This glycan is crucial for Fc-mediated functions, including its induction of the classical... Show moreHuman IgG contains one evolutionarily conserved N-linked glycan in its Fc region at position 297. This glycan is crucial for Fc-mediated functions, including its induction of the classical complement cascade. This is induced after target recognition through the IgG-Fab regions, allowing neighboring IgG-Fc tails to associate through Fc:Fc interaction, ultimately leading to hexamer formation. This hexamerization seems crucial for IgG to enable efficient interaction with the globular heads of the first complement component C1q and subsequent complement activation. In this study, we show that galactose incorporated in the IgG1-Fc enhances C1q binding, C4, C3 deposition, and complement-dependent cellular cytotoxicity in human erythrocytes and Raji cells. IgG1-Fc sialylation slightly enhanced binding of C1q, but had little effect on downstream complement activation. Using various mutations that decrease or increase hexamerization capacity of IgG1, we show that IgG1-Fc galactosylation has no intrinsic effect on C1q binding to IgG1, but enhances IgG1 hexamerization potential and, thereby, complement activation. These data suggest that the therapeutic potential of Abs can be amplified without introducing immunogenic mutations, by relatively simple glycoengineering. Show less
Hoepel, W.; Chen, H.J.; Geyer, C.E.; Allahverdiyeva, S.; Manz, X.D.; Taeye, S.W. de; ... ; Dunnen, J. den 2021
Patients diagnosed with coronavirus disease 2019 (COVID-19) become critically ill primarily around the time of activation of the adaptive immune response. Here, we provide evidence that antibodies... Show morePatients diagnosed with coronavirus disease 2019 (COVID-19) become critically ill primarily around the time of activation of the adaptive immune response. Here, we provide evidence that antibodies play a role in the worsening of disease at the time of seroconversion. We show that early-phase severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) spike protein-specific immunoglobulin G (IgG) in serum of critically ill COVID-19 patients induces excessive inflammatory responses by human alveolar macrophages. We identified that this excessive inflammatory response is dependent on two antibody features that are specific for patients with severe COVID-19. First, inflammation is driven by high titers of anti-spike IgG, a hallmark of severe disease. Second, we found that anti-spike IgG from patients with severe COVID-19 is intrinsically more proinflammatory because of different glycosylation, particularly low fucosylation, of the antibody Fc tail. Low fucosylation of anti-spike IgG was normalized in a few weeks after initial infection with SARS-CoV-2, indicating that the increased antibody-dependent inflammation mainly occurs at the time of seroconversion. We identified Fc gamma receptor (Fc gamma R) Ila and FeyRIII as the two primary IgG receptors that are responsible for the induction of key COVID-19-associated cytokines such as interleukin-6 and tumor necrosis factor. In addition, we show that anti-spike IgG-activated human macrophages can subsequently break pulmonary endothelial barrier integrity and induce microvascular thrombosis in vitro. Last, we demonstrate that the inflammatory response induced by anti-spike IgG can be specifically counteracted by fostamatinib, an FDA- and EMA-approved therapeutic small-molecule inhibitor of Syk kinase. Show less
Apolipoprotein-CIII (apo-CIII) is a glycoprotein involved in lipid metabolism and its levels are associated with cardiovascular disease risk. Apo-CIII sialylation is associated with improved plasma... Show moreApolipoprotein-CIII (apo-CIII) is a glycoprotein involved in lipid metabolism and its levels are associated with cardiovascular disease risk. Apo-CIII sialylation is associated with improved plasma triglyceride levels and its glycosylation may have an effect on the clearance of triglyceride-rich lipoproteins by directing these particles to different metabolic pathways. Large-scale sample cohort studies are required to fully elucidate the role of apo-CIII glycosylation in lipid metabolism and associated cardiovascular disease. In this study, we revisited a high-throughput workflow for the analysis of intact apo-CIII by ultrahigh-resolution MALDI FT-ICR MS. The workflow includes a chemical oxidation step to reduce methionine oxidation heterogeneity and spectrum complexity. Sinapinic acid matrix was used to minimize the loss of sialic acids upon MALDI. MassyTools software was used to standardize and automate MS data processing and quality control. This method was applied on 771 plasma samples from individuals without diabetes allowing for an evaluation of the expression levels of apo-CIII glycoforms against a panel of lipid biomarkers demonstrating the validity of the method. Our study supports the hypothesis that triglyceride clearance may be regulated, or at least strongly influenced by apo-CIII sialylation. Interestingly, the association of apo-CIII glycoforms with triglyceride levels was found to be largely independent of body mass index. Due to its precision and throughput, the new workflow will allow studying the role of apo-CIII in the regulation of lipid metabolism in various disease settings. Show less
Early detection of prostate cancer may lead to the overdiagnosis and overtreatment of patients as well as missing significant cancers. The current diagnostic approach uses elevated serum... Show moreEarly detection of prostate cancer may lead to the overdiagnosis and overtreatment of patients as well as missing significant cancers. The current diagnostic approach uses elevated serum concentrations of prostate-specific antigen (PSA) as an indicator of risk. However, this test has been widely criticized as it shows poor specificity and sensitivity. In order to improve early detection and diagnosis, several studies have investigated whether different PSA proteoforms are correlated to prostate cancer. Until now, studies and methodologies for the comprehensive characterization of PSA proteoforms from biofluids are scarce. For this purpose, we developed an intact protein assay to analyze PSA by capillary electrophoresis-electrospray ionization-mass spectrometry after affinity purification from patients? urine. Here, we determined six proteolytic cleavage variants. In regard to glycosylation, tri-, di-, mono- and non-sialylated complex-type N-glycans were found on non-cleaved PSA, as well as the non-glycosylated variant. The performance of the intact protein assay was assessed using a pooled sample, obtaining an inter-day variability of 15%. Furthermore, urinary patient samples were analyzed by intact protein analysis and a bottom-up approach (glycopeptide analysis). This combined approach revealed complimentary information on both levels, demonstrating the benefit of using two orthogonal techniques to provide a thorough profile of urinary PSA.Significance: The detection of clinically relevant prostate cancer requires a more specific and sensitive biomarker and, in this case, several PSA proteoforms may be able to aid or improve the current PSA test. However, a comprehensive analysis of the intact PSA proteoform profile is still lacking. This study investigated the PSA proteoforms present in urine and, in particular, determined the relative contribution of cleaved PSA and noncleaved PSA forms to the total glycosylation profile. Importantly, intact protein analysis did not require further sample treatment before being measured by CE-ESI-MS. Furthermore, its glycosylation was also assessed in a bottom-up approach to provide complementary information. Overall, these results represent an important basis for future characterization and biomarker studies. Show less
Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows... Show moreImmunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (Fc gamma RIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger Fc gamma RIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19. Show less
Wang, W.; Kaluza, A.; Nouta, J.; Nicolardi, S.; Ferens-Sieczkowska, M.; Wuhrer, M.; ... ; Haan, N. de 2021
An altered total seminal plasma glycosylation has been associated with male infertility, and the highly abundant seminal plasma glycoprotein prostate-specific antigen (PSA) plays an important role... Show moreAn altered total seminal plasma glycosylation has been associated with male infertility, and the highly abundant seminal plasma glycoprotein prostate-specific antigen (PSA) plays an important role in fertilization. However, the exact role of PSA glycosylation in male fertility is not clear. To understand the involvement of PSA glycosylation in the fertilization process, analytical methods are required to study the glycosylation of PSA from seminal plasma with a high glycoform resolution and in a protein-specific manner. In this study, we developed a novel, high-throughput PSA glycopeptide workflow, based on matrix-assisted laser desorption/ionization-mass spectrometry, allowing the discrimination of sialic acid linkage isomers via the derivatization of glycopeptides. The method was successfully applied on a cohort consisting of seminal plasma from infertile and fertile men (N = 102). Forty-four glycopeptides were quantified in all samples, showing mainly complex-type glycans with high levels of fucosylation and sialylation. In addition, N,N-diacetyllactosamine (LacdiNAc) motives were found as well as hybrid-type and high mannose-type structures. Our method showed a high intra- and interday repeatability and revealed no difference in PSA glycosylation between fertile and infertile men. Next to seminal plasma, the method is also expected to be of use for studying PSA glycopeptides derived from other biofluids and/or in other disease contexts. Show less
Lippold, S.; Ru, A.H. de; Nouta, J.; Veelen, P.A. van; Palmblad, M.; Wuhrer, M.; Haan, N. de 2020
Glycoproteomic data are often very complex, reflecting the high structural diversity of peptide and glycan portions. The use of glycopeptide-centered glycoproteomics by mass spectrometry is rapidly... Show moreGlycoproteomic data are often very complex, reflecting the high structural diversity of peptide and glycan portions. The use of glycopeptide-centered glycoproteomics by mass spectrometry is rapidly evolving in many research areas, leading to a demand in reliable data analysis tools. In recent years, several bioinformatic tools were developed to facilitate and improve both the identification and quantification of glycopeptides. Here, a selection of these tools was combined and evaluated with the aim of establishing a robust glycopeptide detection and quantification workflow targeting enriched glycoproteins. For this purpose, a tryptic digest from affinity-purified immunoglobulins G and A was analyzed on a nano-reversed-phase liquid chromatography-tandem mass spectrometry platform with a high-resolution mass analyzer and higher-energy collisional dissociation fragmentation. Initial glycopeptide identification based on MS/MS data was aided by the Byonic software. Additional MS1-based glycopeptide identification relying on accurate mass and retention time differences using Glycopeptide-GraphMS considerably expanded the set of confidently annotated glycopeptides. For glycopeptide quantification, the performance of LaCyTools was compared to Skyline, and GlycopeptideGraphMS. All quantification packages resulted in comparable glycosylation profiles but featured differences in terms of robustness and data quality control. Partial cysteine oxidation was identified as an unexpectedly abundant peptide modification and impaired the automated processing of several IgA glycopeptides. Finally, this study presents a semiautomated workflow for reliable glyco-proteomic data analysis by the combination of software packages for MS/MS- and MS1-based glycopeptide identification as well as the integration of analyte quality control and quantification. Show less
Drouin, N.; Mever, M. van; Zhang, W.; Tobolkina, E.; Ferre, S.; Servais, A.C.; ... ; Ramautar, R. 2020
Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison... Show moreCapillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (mu(eff)) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The mu(eff) was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the mu(eff) for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics. Show less
