High-Throughput Glycomic Methods.

Glycomics aims to identify the structure and function of the glycome, the complete set of oligosaccharides (glycans), produced in a given cell or organism, as well as to identify genes and other factors that govern glycosylation. This challenging endeavor requires highly robust, sensitive, and potentially automatable analytical technologies for the analysis of hundreds or thousands of glycomes in a timely manner (termed high-throughput glycomics). This review provides a historic overview as well as highlights recent developments and challenges of glycomic profiling by the most prominent high-throughput glycomic approaches, with N-glycosylation analysis as the focal point. It describes the current state-of-the-art regarding levels of characterization and most widely used technologies, selected applications of high-throughput glycomics in deciphering glycosylation process in healthy and disease states, as well as future perspectives.

Altered IgG glycosylation at COVID-19 diagnosis predicts disease severity.

The nature of the immune responses associated with COVID-19 pathogenesis and disease severity, as well as the breadth of vaccine coverage and duration of immunity, is still unclear. Given the unpredictability for developing a severe/complicated disease, there is an urgent need in the field for predictive biomarkers of COVID-19. We have analyzed IgG Fc N-glycan traits of 82 SARS-CoV-2+ unvaccinated patients, at diagnosis, by nano-LC-ESI-MS. We determined the impact of IgG Fc glyco-variations in the induction of NK cells activation, further evaluating the association between IgG Fc N-glycans and disease severity/prognosis. We found that SARS-CoV-2+ individuals display, at diagnosis, variations in the glycans composition of circulating IgGs. Importantly, levels of galactose and sialic acid structures on IgGs are able to predict the development of a poor COVID-19 disease. Mechanistically, we demonstrated that a deficiency on galactose structures on IgG Fc in COVID-19 patients appears to induce NK cells activation associated with increased release of IFN-γ and TNF-α, which indicates the presence of pro-inflammatory immunoglobulins and higher immune activation, associated with a poor disease course. This study brings to light a novel blood biomarker based on IgG Fc glycome composition with capacity to stratify patients at diagnosis.

Robustness and repeatability of GlycoWorks RapiFluor-MS IgG N-glycan profiling in a long-term high-throughput glycomic study.

Protein glycosylation is the attachment of a carbohydrate moiety to a protein backbone affecting both structure and function of the protein. Abnormal glycosylation is associated with various diseases, and some of the changes in glycosylation are detectable even before symptom development. As such, glycans have emerged as compelling new biomarker candidates. A wide range of analytical methods exist for small-scale glycan analyses. However, there is a growing need for highly robust and reproducible high-throughput techniques that allow for large-scale glycoprofiling. Here, we describe the evaluation of robustness and repeatability of immunoglobulin G (IgG) N-glycan analysis using the GlycoWorks RapiFluor-MS N-Glycan Kit followed by hydrophilic interaction ultra-high-performance liquid chromatography (HILIC-UHPLC) from 335 technical replicates of human plasma randomly distributed across 67 96-well plates. The data was collected over a 5-month period using multiple UHPLC systems and chromatographic columns. Following relative IgG N-glycan quantification in acquired chromatograms, data analysis showed that the most abundant peaks that together made up for three-fourths of the detected IgG N-glycome all had coefficients of variation (CVs) lower than 2%. The highest CVs ranging from 16 to 29% accompanied low abundance glycan peaks with the individual relative peak area below 1% that together made up for <2% of the detected IgG N-glycome. These results show that the tested method is very robust and repeatable, making it suitable for the IgG N-glycan analysis of a large number of samples in a high-throughput manner over a longer period of time.

Composition of the immunoglobulin G glycome associates with the severity of COVID-19.

A large variation in the severity of disease symptoms is one of the key open questions in coronavirus disease 2019 (COVID-19) pandemics. The fact that only a small subset of people infected with severe acute respiratory syndrome coronavirus 2 develops severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the immunoglobulin G (IgG) glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of interindividual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting N-acetylglucosamine in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing.

SARS-CoV-2 S glycoprotein binding to multiple host receptors enables cell entry and infection.

The severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) infection displays a wide array of clinical manifestations. Although some risk factors for coronavirus disease 2019 (COVID-19) severity and outcomes have been identified the underlying biologic mechanisms are still not well understood. The surface SARS-CoV-2 proteins are heavily glycosylated enabling host cell interaction and viral entry. Angiotensin-converting enzyme 2 (ACE2) has been identified to be the main host cell receptor enabling SARS-CoV-2 cell entry after interaction with its S glycoprotein. However, recent studies report SARS-CoV-2 S glycoprotein interaction with other cell receptors, mainly C-type lectins which recognize specific glycan epitopes facilitating SARS-CoV-2 entry to susceptible cells. Here, we are summarizing the main findings on SARS-CoV-2 interactions with ACE2 and other cell membrane surface receptors and soluble lectins involved in the viral cell entry modulating its infectivity and potentially playing a role in subsequent clinical manifestations of COVID-19.

The Importance of Glycosylation in COVID-19 Infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently one of the major health problems worldwide. SARS-CoV-2 survival and virulence are shown to be impacted by glycans, covalently attached to proteins in a process of glycosylation, making glycans an area of interest in SARS-CoV-2 biology and COVID-19 infection. The SARS-CoV-2 uses its highly glycosylated spike (S) glycoproteins to bind to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) glycoprotein and facilitate host cell entry. Viral glycosylation has wide-ranging roles in viral pathobiology, including mediating protein folding and stability, immune evasion, host receptor attachment, and cell entry. Modification of SARS-CoV-2 envelope membrane with glycans is important in host immune recognition and interaction between S and ACE2 glycoproteins. On the other hand, immunoglobulin G, a key molecule in immune response, shows a distinct glycosylation profile in COVID-19 infection and with increased disease severity. Hence, further studies on the role of glycosylation in SARS-CoV-2 infectivity and COVID-19 infection are needed for its successful prevention and treatment. This chapter focuses on recent findings on the importance of glycosylation in COVID-19 infection.

IgG N-glycome changes during the course of severe COVID-19: An observational study.

BACKGROUND: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes a respiratory illness named coronavirus disease 2019 (COVID-19), which is one of the main global health problems since 2019. Glycans attached to the Fc portion of immunoglobulin G (IgG) are important modulators of IgG effector functions. Fc region binds to different receptors on the surface of various immune cells, dictating the type of immune response. Here, we performed a large longitudinal study to determine whether the severity and duration of COVID-19 are associated with altered IgG glycosylation. METHODS: Using ultra-high-performance liquid chromatography analysis of released glycans, we analysed the composition of the total IgG N-glycome longitudinally during COVID-19 from four independent cohorts. We analysed 77 severe COVID-19 cases from the HR1 cohort (74% males, median age 72, age IQR 25-80); 31 severe cases in the HR2 cohort (77% males, median age 64, age IQR 41-86), 18 mild COVID-19 cases from the UK cohort (17% males, median age 50, age IQR 26-71) and 28 mild cases from the BiH cohort (71% males, median age 60, age IQR 12-78). FINDINGS: Multiple statistically significant changes in IgG glycome composition were observed during severe COVID-19. The most statistically significant changes included increased agalactosylation of IgG (meta-analysis 95% CI [0.03, 0.07], adjusted meta-analysis P= <0.0001), which regulates proinflammatory actions of IgG via complement system activation and indirectly as a lack of sialylation and decreased presence of bisecting N-acetylglucosamine on IgG (meta-analysis 95% CI [-0.11, -0.08], adjusted meta-analysis P= <0.0001), which indirectly affects antibody-dependent cell-mediated cytotoxicity. On the contrary, no statistically significant changes in IgG glycome composition were observed in patients with mild COVID-19. INTERPRETATION: The IgG glycome in severe COVID-19 patients is statistically significantly altered in a way that it indicates decreased immunosuppressive action of circulating immunoglobulins. The magnitude of observed changes is associated with the severity of the disease, indicating that aberrant IgG glycome composition or changes in IgG glycosylation may be an important molecular mechanism in COVID-19. FUNDING: This work has been supported in part by Croatian Science Foundation under the project IP-CORONA-2020-04-2052 and Croatian National Centre of Competence in Molecular Diagnostics (The European Structural and Investment Funds grant #KK.01.2.2.03.0006), by the UKRI/MRC (Cov-0331 - MR/V027883/1) and by the National Institutes for Health Research Nottingham Biomedical Research Centre and by Ministry Of Science, Higher Education and Youth Of Canton Sarajevo, grant number 27-02-11-4375-10/21.

Immunoglobulin G glycome composition in transition from premenopause to postmenopause.

Gonadal hormones affect immunoglobulin G (IgG) glycosylation, and the more proinflammatory IgG glycome composition might be one of the molecular mechanisms behind the increased proinflammatory phenotype in perimenopause. Using ultra-high-performance liquid chromatography, we analyzed IgG glycome composition in 5,080 samples from 1940 pre-, peri-, and postmenopausal women. Statistically significant decrease in galactosylation and sialylation was observed in postmenopausal women. Furthermore, during the transition from pre- to postmenopausal period, the rate of increase in agalactosylated structures (0.051/yr; 95%CI = 0.043-0.059, p < 0.001) and decrease in digalactosylated (-0.043/yr; 95%CI = -0.050 to -0.037, p < 0.001) and monosialylated glycans (-0.029/yr; 95%CI = -0.034 to -0.024, p < 0.001) were significantly higher than in either pre- or postmenopausal periods. The conversion to the more proinflammatory IgG glycome and the resulting decrease in the ability of IgG to suppress low-grade chronic inflammation may be an important molecular mechanism mediating the increased health risk in perimenopause and postmenopause.

Genetic regulation of post-translational modification of two distinct proteins.

Post-translational modifications diversify protein functions and dynamically coordinate their signalling networks, influencing most aspects of cell physiology. Nevertheless, their genetic regulation or influence on complex traits is not fully understood. Here, we compare the genetic regulation of the same PTM of two proteins - glycosylation of transferrin and immunoglobulin G (IgG). By performing genome-wide association analysis of transferrin glycosylation, we identify 10 significantly associated loci, 9 of which were not reported previously. Comparing these with IgG glycosylation-associated genes, we note protein-specific associations with genes encoding glycosylation enzymes (transferrin - MGAT5, ST3GAL4, B3GAT1; IgG - MGAT3, ST6GAL1), as well as shared associations (FUT6, FUT8). Colocalisation analyses of the latter suggest that different causal variants in the FUT genes regulate fucosylation of the two proteins. Glycosylation of these proteins is thus genetically regulated by both shared and protein-specific mechanisms.

Differences in Immunoglobulin G Glycosylation Between Influenza and COVID-19 Patients.

The essential role of immunoglobulin G (IgG) in immune system regulation and combatting infectious diseases cannot be fully recognized without an understanding of the changes in its N-glycans attached to the asparagine 297 of the Fc domain that occur under such circumstances. These glycans impact the antibody stability, half-life, secretion, immunogenicity, and effector functions. Therefore, in this study, we analyzed and compared the total IgG glycome-at the level of individual glycan structures and derived glycosylation traits (sialylation, galactosylation, fucosylation, and bisecting N-acetylglucosamine (GlcNAc))-of 64 patients with influenza, 77 patients with coronavirus disease 2019 (COVID-19), and 56 healthy controls. Our study revealed a significant decrease in IgG galactosylation, sialylation, and bisecting GlcNAc (where the latter shows the most significant decrease) in deceased COVID-19 patients, whereas IgG fucosylation was increased. On the other hand, IgG galactosylation remained stable in influenza patients and COVID-19 survivors. IgG glycosylation in influenza patients was more time-dependent: In the first seven days of the disease, sialylation increased and fucosylation and bisecting GlcNAc decreased; in the next 21 days, sialylation decreased and fucosylation increased (while bisecting GlcNAc remained stable). The similarity of IgG glycosylation changes in COVID-19 survivors and influenza patients may be the consequence of an adequate immune response to enveloped viruses, while the observed changes in deceased COVID-19 patients may indicate its deviation.

Lectin and Liquid Chromatography-Based Methods for Immunoglobulin (G) Glycosylation Analysis.

Immunoglobulin (Ig) glycosylation has been shown to dramatically affect its structure and effector functions. Ig glycosylation changes have been associated with different diseases and show a promising biomarker potential for diagnosis and prognosis of disease advancement. On the other hand, therapeutic biomolecules based on structural and functional features of Igs demand stringent quality control during the production process to ensure their safety and efficacy. Liquid chromatography (LC) and lectin-based methods are routinely used in Ig glycosylation analysis complementary to other analytical methods, e.g., mass spectrometry and capillary electrophoresis. This chapter covers analytical approaches based on LC and lectins used in low- and high-throughput N- and O-glycosylation analysis of Igs, with the focus on immunoglobulin G (IgG) applications. General principles and practical examples of the most often used LC methods for Ig purification are described, together with typical workflows for N- and O-glycan analysis on the level of free glycans, glycopeptides, subunits, or intact Igs. Lectin chromatography is a historical approach for the analysis of lectin-carbohydrate interactions and glycoprotein purification but is still being used as a valuable tool in Igs purification and glycan analysis. On the other hand, lectin microarrays have found their application in the rapid screening of glycan profiles on intact proteins.

Seminal Plasma Protein N-Glycan Peaks Are Potential Predictors of Semen Pathology and Sperm Chromatin Maturity in Men.

BACKGROUND: Male infertility is increasingly becoming a health and demographic problem. While it may originate from congenital or acquired diseases, it can also result from environmental exposure. Hence, the complexity of involved molecular mechanisms often requires a multiparametric approach. This study aimed to associate semen parameters with sperm DNA fragmentation, chromatin maturity and seminal plasma protein N-glycosylation. METHODS: The study was conducted with 166 participants, 20-55 y old, 82 normozoospermic and 84 with pathological diagnosis. Sperm was analyzed by Halosperm assay and aniline blue staining, while seminal plasma total protein N-glycans were analyzed by ultra-high-performance liquid chromatography. RESULTS: Sperm DNA fragmentation was significantly increased in the pathological group and was inversely correlated with sperm motility and viability. Seminal plasma total protein N-glycans were chromatographically separated in 37 individual peaks. The pattern of seminal plasma N-glycan peaks (SPGP) showed that SPGP14 significantly differs between men with normal and pathological semen parameters (p < 0.001). The multivariate analysis showed that when sperm chromatin maturity increases by 10%, SPGP17 decreases by 14% while SPGP25 increases by 25%. CONCLUSION: DNA integrity and seminal plasma N-glycans are associated with pathological sperm parameters. Specific N-glycans are also associated with sperm chromatin maturity and have a potential in future fertility research and clinical diagnostics.