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. 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. 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. 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. This project received funding from the European Commission's Horizon2020 research and innovation program for H2020-MSCA-ITN IMforFUTURE, under grant agreement number 721815, and supported by Crowdfunding Wake Up To Corona, organized by the Leiden University Fund.

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 FcγRIIIa 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. 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). 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. 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. LSBR1721, 1908; 10.13039/501100001826 ZonMW 10430012010021, 09150161910033, 10430012010008; DFG398859914, 400912066, 390884018; PMI; DOI4-Nr. 3; H2020-MSCA-ITN 721815.

Most of the proteins in the circulation are N-glycosylated, shaping together the total blood N-glycome (TBNG). Glycosylation is known to affect protein function, stability, and clearance. The TBNG is influenced by genetic, environmental, and metabolic factors, in part epigenetically imprinted, and responds to a variety of bioactive signals including cytokines and hormones. Accordingly, physiological and pathological events are reflected in distinct TBNG signatures. Here, we assess the specificity of the emerging disease-associated TBNG signatures with respect to a number of key glycosylation motifs including antennarity, linkage-specific sialylation, fucosylation, as well as expression of complex, hybrid-type and oligomannosidic N-glycans, and show perplexing complexity of the glycomic dimension of the studied diseases. Perspectives are given regarding the protein- and site-specific analysis of N-glycosylation, and the dissection of underlying regulatory layers and functional roles of blood protein N-glycosylation.

Antibody glycosylation has received considerable attention in coronavirus disease 2019 (COVID-19) infections and recently also in vaccination. Antibody glycosylation and in particular immunoglobulin G1 fucosylation levels influence effector functions and are therefore key parameters for assessing the efficacy and safety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directed immune responses. This review article summarizes and interprets recent research into antibody glycosylation in COVID-19. Experimental approaches for analyzing the glycosylation of SARS-CoV-2-directed antibody responses are evaluated. The pronounced dynamics, effector functions, clinical utility, and regulation of antibody glycosylation in COVID-19 are assessed. Future research on the role of antibody glycosylation in COVID may cover the glycosylation of other antibody classes beyond immunoglobulin G, the regulation of antibody glycosylation, and the role of non-canonical antibody receptors in determining effector functions. Graphical abstract

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 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 non-cleaved 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.

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