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Over the past decades, the genome and proteome have been widely explored for biomarker discovery and personalized medicine. However, there is still a large need for improved diagnostics and stratification strategies for a wide range of diseases. Post-translational modification of proteins by glycosylation affects protein structure and function, and glycosylation has been implicated in many prevalent human diseases. Numerous proteins for which the plasma levels are nowadays evaluated in clinical practice are glycoproteins. While the glycosylation of these proteins often changes with disease, their glycosylation status is largely ignored in the clinical setting. Hence, the implementation of glycomic markers in the clinic is still in its infancy. This is for a large part caused by the high complexity of protein glycosylation itself and of the analytical techniques required for their robust quantification. Mass spectrometry-based workflows are particularly suitable for the quantification of glycans and glycoproteins, but still require advances for their transformation from a biomedical research setting to a clinical laboratory. In this review, we describe why and how glycomics is expected to find its role in clinical tests and the status of current mass spectrometry-based methods for clinical glycomics. (C) 2020 The Authors. Published by Elsevier B.V. on behalf of The Association for Mass Spectrometry: Applications to the Clinical Lab (MSACL).

SummaryAntibody-mediated rejection (AMR) is the leading cause of graft failure. While donor-specific antibodies (DSA) are associated with a higher risk of AMR, not all patients with DSA develop rejection suggesting that the characteristics of alloantibodies that determine their pathogenicity remain undefined. Using human leukocyte antigen (HLA)-A2-specific antibodies as a model, we applied systems serology tools to investigate qualitative features of immunoglobulin G (IgG) alloantibodies including Fc-glycosylation patterns and FcγR binding properties. The levels of afucosylation of anti-A2 antibodies were elevated in all seropositive patients and were significantly higher in AMR patients, suggesting potential cytotoxicity via FcγRIII-mediated mechanisms. Afucosylation of both glycoengineered monoclonal and naturally glycovariant polyclonal serum IgG specific to HLA-A2 exhibited potentiated binding to, slower dissociation from, and enhanced signaling through FcγRIII, a receptor widely expressed on innate effector cells. Collectively, these results suggest that afucosylated DSA may be a biomarker of AMR and could contribute to its pathogenesis. Graphical Abstract.Potential influence of HLA-A2-specific IgG1 afucosylation, FcγRIIIa binding and activation on ADCC and graft rejection.Illustration created with https://BioRender.com.

HLA class I (HLA-I) drives immune responses by presenting antigen-derived peptides to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Since therapeutic options for restoring HLA-I antigen presentation are limited, we aimed to identify new HLA-I pathway targets. By iterative genome-wide screens we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augments B3GNT5 enzyme activity, resulting in upregulated levels of surface (neo)lacto-series GSLs. These GSLs sterically impede molecular interactions with HLA-I and diminish CD8+ T cell activation. In accordance, a disturbed SPPL3-B3GNT5 pathway in glioma associates with decreased patient survival. Importantly, we show that this immunomodulatory effect can be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that functionally inhibits antigen presentation and represents a potential therapeutic target in cancer, infection and autoimmunity.

The binding strength between IgG and Fc gamma R is influenced by the composition of the N-linked glycan at position N297 in the Fc-domain of IgG. Particularly, afucosylation increases the binding affinity of human IgG1 to human Fc gamma RIIIa up to similar to 20 fold, and additional galactosylation of the afucosylated IgG increases the affinity up to similar to 40 fold. The increase in affinity for afucosylated IgG has previously been shown to depend on direct carbohydrate carbohydrate interactions between the IgG-Fc glycan with an N-linked glycan at position 162 unique to hFc gamma RIIIa and hFc gamma RIIIb. Here we report that the N162 glycosylation site is also found in the orthologous mouse Fc gamma R, mFc gamma RIV. The N162-glycan in mFc gamma RIV was also responsible for enhancing the binding to mouse IgG with reduced fucose similar to hFc gamma RIIIa. However, unlike hFc gamma RIIIa, mFc gamma RIV did not bind more avidly to IgG with increased galactose and reduced fucose. Overall, these results suggest the N162-glycan in the human Fc gamma RIII family and its orthologous mouse Fc gamma RIV to be functionally conserved

The humanGb3/CD77 synthase, encoded by theA4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Gala1 -> 4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, P-k) and the P1 antigen. Gb3 is themajor receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid sub-stitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise toNOR1 andNOR2GSLs. HumanGb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Gal-alpha 1 -> 4Gal beta 1 -> 4GlcNAc-R) on a complex type N-glycan and a syntheticN-glycoprotein (saposinD). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminatedN-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays showthat Stx1 can useP1N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonicalGSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

Background: Effector functions of IgG Abs are regulated by their Fc N-glycosylation pattern. IgG Fc glycans that lack galactose and terminal sialic acid residues correlate with the severity of inflammatory (auto)immune disorders and have also been linked to protection against viral infection and discussed in the context of vaccine-induced protection. In contrast, sialylated IgG Abs have shown immunosuppressive effects.Objective: We sought to investigate IgG glycosylation programming during the germinal center (GC) reaction following immunization of mice with a foreign protein antigen and different adjuvants.Methods: Mice were analyzed for GC T-cell, B-cell, and plasma cell responses, as well as for antigen-specific serum IgG subclass titers and Fc glycosylation patterns.Results: Different adjuvants induce distinct IgG(+) GC B-cell responses with specific transcriptomes and expression levels of the alpha 2,6-sialyltransferase responsible for IgG sialylation that correspond to distinct serum IgG Fc glycosylation patterns. Low IgG Fc sialylation programming in GC B cells was overall highly dependent on the Foxp3(-) follicular helper T (TFH) cell-inducing cytokine IL-6, here in particular induced by water-inoil adjuvants and Mycobacterium tuberculosis. Furthermore, low IgG Fc sialylation programming was dependent on adjuvants that induced IL-27 receptor-dependent IFN-gamma(+) TFH1 cells, IL-6/IL-23-dependent IL-17A(+) T-FH17 cells, and high ratios of TFH cells to Foxp31 follicular regulatory T cells. Here, the 2 latter were dependent on M tuberculosis and its cord factor.Conclusion: This study's findings regarding adjuvant-dependent GC responses and IgG glycosylation programming may aid in the development of novel vaccination strategies to induce IgG Abs with both high affinity and defined Fc glycosylation patterns in the GC.

Biopharmaceuticals are drugs of biotechnological origin used as vaccines or for the treatment of non-communicable diseases, such as cancer or anemia. Due to their high efficacy and specificity, the market for novel and biosimilar biopharmaceuticals is growing immensely. This growth is accompanied by new challenges in quality control and analytical characterization during drug development and production. Glycosylation is one of the structural modifications that occur during production of many protein-based drugs and can have significant effects on their pharmacological properties. Mass spectrometry (MS) is a promising technique for high-quality analytical characterization of glycosylation, starting from early drug development through to final lot release. Here, we review the most recent trends in biopharmaceutical glycosylation analysis by MS with and without coupling to liquid chromatography or capillary electrophoresis, and draw comparisons with established, non-MS methods. We discuss future prospects for the emerging MS approaches for the biotech industry and biopharmaceutical research.

Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins. (C) 2010 Elsevier Inc. All rights reserved.

Schistosomiasis is a parasitic infection caused by Schistosoma flatworms, prime examples of multicellular parasites that live in the mammalian host for many years. Glycoconjugates derived from the parasite have been shown to play an important role in many aspects of schistosomiasis, and some of them are present in the circulation of the host. The aim of this study was to identify novel glycoconjugates related to schistosomiasis in urine of Schistosoma mansoni-infected individuals using a combination of glycopeptide separation techniques and in-depth mass spectrometric analysis. Surprisingly, we characterized a heterogeneous population of novel aberrantly O-glycosylated peptides derived from the C terminus of human apolipoprotein C-III (apoC-III) in urine of S. mansoni-infected individuals that were not detected in urine of non-infected controls. The glycan composition of these glycopeptides is completely different from what has been described previously for apoC-III. Most importantly, they lack sialylation and display a high degree of fucosylation. This study exemplifies the potential of mass spectrometry for the identification and characterization of O-glycopeptides without prior knowledge of either the glycan or the peptide sequence. Furthermore, our results indicate for the first time that as a result of S. mansoni infection the glycosylation of a host protein is altered. Molecular & Cellular Proteomics 9: 667-681, 2010.

The major immunopathological consequences of infection with Schistosoma mansoni, a T helper type 2 response and granuloma formation leading to fibrotic tissue damage, are caused by the egg stage of the parasite. Three antigens of S. mansoni eggs, termed IPSE/alpha-1, omega-1 and kappa-5, have been found to be the primary targets of the egg-directed antibody response of the host. Here, we report on the isolation, cloning and characterisation of kappa-5. Apart from an uncharacterised mRNA sequence in S. japonicum, no significant similarities of kappa-5 to known sequences from other species were found. In contrast to IPSE/alpha-1 and omega-1, which have been found only in eggs, kappa-5 was present in miracidia as well as in eggs at the mRNA and protein levels. In eggs, isoforms of kappa-5 were observed with both three and four fully occupied N-glycosylation sites, while in miracidia only one isoform with four N-glycans could be detected. Interestingly, in Western blots sera from S. mansoni-infected Africans were reactive against kappa-5 with IgE and IgG isotype antibodies, but against IPSE/alpha-1 and omega-1 only with IgG antibodies. The further characterisation of kappa-5 as one of the three major egg antigens should help to better understand the immunology and immunopathology of schistosomiasis.

A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS.

Keyhole limpet hemocyanin (KLH) of the mollusc Megathura crenulata is known to serologically cross-react with Schistosoma mansoni glycoconjugates in a carbohydrate-dependent manner. To elucidate the structural basis for this cross-reactivity, KLH glycans were released from tryptic glycopeptides and fluorescently labeled. Cross-reacting glycans were identified using a polyclonal antiserum reacting with soluble S. mansoni egg antigens, isolated by a three-dimensional fractionation scheme and analyzed by different mass spectrometric techniques as well as linkage analysis and exoglycosidase treatment. The results revealed that cross-reacting species comprise approximately 4.5% of released glycans. They all represent novel types of N-glycans with a Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc motif, which is known to occur also in schistosomal glycoconjugates. The tetrasaccharide unit is attached to the 3-linked antenna of a trimannosyl core, which can be further decorated by galactosyl residues, a xylose residue in 2-position of the central mannose and/or a fucose at the innermost N-acetylglucosamine. This study provides for the first time detailed structural data on the KLH carbohydrate entities responsible for cross-reactivity with glycoconjugates from S. mansoni.

Fitzsimmons, C. M., Schramm, G., Jones, F. M., Chalmers, I. W., Hoffmann, K. F., Grevelding, C. G., Wuhrer, M., Hokke, C. H., Haas, H., Doenhoff, M. J., Dunne, D. W. (2005). Molecular characterization of omega-1: A hepatotoxic ribonuclease from Schistosoma mansoni eggs. Molecular & Biochemical Parasitology, 144(1), pp. 123-7.

Conventional normal-phase high performance liquid chromatography of fluorescently labeled oligosaccharides with or without on-line mass spectrometry is an established tool for the structure characterization of protein and lipid derived glycans. Here we describe the miniaturization of such a system to the nano-scale using a 75 μm internal diameter normal-phase amide column on-line with electrospray ionization ion-trap mass spectrometry. 2-Aminobenzamide-labeled oligosaccharides have a predictable retention on this normal-phase column that can be expressed as glucose units by comparison to the retention of a standard 2-aminobenzamide glucose polymer mixture. Isobaric compounds are separated on the basis of their structural differences, and by on-line electrospray ionization ion-trap mass spectrometry, the sequence of the monosaccharides can be deduced. The major improvement of the on-line nano-LC-MS system in comparison to conventional systems is the gain in sensitivity with detection of low femtomole amounts of glycans. This implies that LC-MS of 2-aminobenzamide-labeled oligosaccharides can now be performed at higher sensitivity than their analysis with fluorescence detection.

Polysialylated neural cell adhesion molecule (NCAM) was immunoaffinity-purified from the brains of newborn calves. A degree of polymerization of up to 40 was chromatographically determined for released polysialic acid (PSA) chains. For characterization of N-glycan structures and attachment sites, PSA-NCAM was digested with trypsin, and the generated glycopeptides were fractionated by serial immunoaffinity chromatography using immobilized monoclonal antibodies specific for PSA or the HNK1 epitope, i.e., HSO(3)-3GlcA(beta 1-3)Gal(beta 1-4)GlcNAc(beta 1-, yielding PSA-glycopeptides, HNK-glycopeptides and non-PSA/HNK1-(glyco) peptides. Using a combination of enzymatic deglycosylation, peptide fractionation, mass spectrometry and Edman degradation, HNK1-N-glycans could be assigned to glycosylation sites 2, 4, 5 and 6. Non-PSA/HNK1-glycans were assigned to glycosylation site 2, whereas PSA-N-glycans of bovine NCAM had been already previously shown to be restricted to glycosylation sites 5 and 6 (Glycobiology 12 (2002) 47). Respective oligosaccharides were enzymatically released, labeled with 2-aminopyridine and characterized by linkage analysis and mass spectrometry. Carbohydrate chains bearing PSA or the HNK1 epitope comprised mainly fucosylated, partially sulfated diantennary, triantennary or tetraantennary glycans without bisecting GlcNAc or fucosylated diantennary and triantennary species carrying, in part, bisecting GlcNAc residues, respectively. Some N-glycans simultaneously contained both the HNK1-epitope and PSA. Non-PSA/HNK1-glycans exhibited a heterogeneous pattern of partially truncated, mostly diantennary structures with one to three fucose residues, bisecting GlcNAc and/or sulfate residues. In addition, they were demonstrated to carry, to some extent, the Lewis X epitope. When compared with previous data on murine NCAM glycosylation, our results indicate a conservation of structural features and attachment sites for the different types of NCAM N-glycans.

Glycosphingolipids of Schistosoma mansoni adults, cercariae and eggs comprise ceramide monohexosides (CMH) with glucose or galactose and ceramide dihexosides (CDH) with the schistosome-specific structure GalNAc(beta1-4)Glc(1-1)ceramide. Ceramide analysis revealed C18- and C20-phytosphingosines in egg CMH, C18-sphinganine as well as C18-, C19- and C20-phytosphingosines in cercarial CMH, and C18- and C20-phytosphingosines as well as C18-sphingosine and C18-sphinganine in adult CMH. For all three life cycle stages, the predominant fatty acid was C16h:0. As a characteristic feature, a range of saturated, unsaturated and hydroxylated long-chain fatty acids with 24-28 carbon atoms were additionally found in minor cercarial CMH species. The corresponding ceramides represented major constituents in cercarial CDH, while adult and egg CDH were dominated by ceramides with short fatty acid chains. The resultant ceramide patterns could be correlated with the differential expression of carbohydrate antigens on schistosomal glycolipids at various stages. A possible impact of ceramide structure on the biosynthesis of the carbohydrate moieties is discussed.