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Mutations in the FUT2 gene that result in a lack of expression of histo-blood group antigens on secreted glycoproteins may shape the vaginal microbiota with consequences for birth outcome. To test this, we analysed the relationship between secretor status, vaginal microbiota and gestational length in an ethnically diverse cohort of 302 pregnant women, including 82 who delivered preterm. Lactobacillus gasseri and L. jensenii were found to have distinct co-occurrence patterns with other microbial taxa in non-secretors. Moreover, non-secretors with Lactobacillus spp. depleted high diversity vaginal microbiota in early pregnancy had significantly shorter gestational length than Lactobacillus spp. dominated non-secretors (mean of 241.54 days (sd=47.14) versus 266.21 (23.61); P -value=0.0251). Similar gestational length differences were observed between non-secretors with high vaginal diversity and secretors with Lactobacillus spp. dominance (mean of 262.52 days (SD=27.73); p - value =0.0439) or depletion (mean of 266.05 days (SD=20.81); p - value =0.0312). Our data highlight secretor status and blood-group antigen expression as being important mediators of vaginal microbiota-host interactions in the context of preterm birth risk.

During pregnancy the immune system needs to maintain immune tolerance of the foetus while also responding to infection, which can cause premature activation of the inflammatory pathways leading to the onset of labour and preterm birth. The vaginal microbiome is an important modifier of preterm birth risk, with Lactobacillus dominance during pregnancy associated with term delivery while high microbial diversity is associated with an increased risk of preterm birth. Glycans on glycoproteins along the lower female reproductive tract are fundamental to microbiota-host interactions and the mediation of inflammatory responses. However, the specific glycan epitopes involved in these processes are not well understood. To address this, we conducted glycomic analyses of cervicovaginal fluid (CVF) from 36 pregnant women at high risk of preterm birth and 4 non-pregnant women. Our analysis of N- and O-glycans revealed a rich CVF glycome. While O-glycans were shown to be the main carriers of ABO blood group epitopes, the main features of N-glycans were the presence of abundant paucimannose and high mannose glycans, and a remarkable diversity of complex bi-, tri-, and tetra-antennary glycans decorated with fucose and sialic acid. We identified immuno-regulatory epitopes, such as Lewis antigens, and found that fucosylation was negatively correlated to pro-inflammatory factors, such as IL-1β, MMP-8, C3a and C5a, while glycans with only sialylated antennae were mainly positively correlated to those. Similarly, paucimannose glycans showed a positive correlation to pro-inflammatory factors. We revealed a high abundance of glycans which have previously been identified as hallmarks of cancer and viral glycosylation, such as Man8 and Man9 high mannose glycans. Although each pregnant woman had a unique glycomic profile, longitudinal studies showed that the main glycosylation features were consistent throughout pregnancy in women who delivered at term, whereas women who experienced extreme preterm birth exhibited sharp changes in the CVF glycome shortly before delivery. These findings shed light on the processes underlying the role of glycosylation in maintaining a healthy vaginal microbiome and associated host immune responses. In addition, these discoveries facilitate our understanding of the lower female reproductive tract which has broad implications for women's health., (© 2024. The Author(s).)

Evolution of human H3N2 influenza viruses driven by immune selection has narrowed the receptor specificity of the hemagglutinin (HA) to a restricted subset of human-type (Neu5Acα2-6 Gal) glycan receptors that have extended poly-LacNAc (Galβ1-4GlcNAc) repeats. This altered specificity has presented challenges for hemagglutination assays, growth in laboratory hosts, and vaccine production in eggs. To assess the impact of extended glycan receptors on virus binding, infection, and growth, we have engineered N-glycan extended (NExt) cell lines by overexpressing β3-Ν-acetylglucosaminyltransferase 2 in MDCK, SIAT, and hCK cell lines. Of these, SIAT-NExt cells exhibit markedly increased binding of H3 HAs and susceptibility to infection by recent H3N2 virus strains, but without impacting final virus titers. Glycome analysis of these cell lines and allantoic and amniotic egg membranes provide insights into the importance of extended glycan receptors for growth of recent H3N2 viruses and relevance to their production for cell- and egg-based vaccines., (© 2023. Springer Nature Limited.)

Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N-oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N-OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Placental abnormalities cause impaired fetal growth and poor pregnancy outcome (e.g. preeclampsia [PE]) with long-lasting consequences for the mother and offspring. The molecular dialogue between the maternal niche and the developing placenta is critical for the function of this organ. Galectin-1 (gal-1), a highly expressed glycan-binding protein at the maternal-fetal interface, orchestrates the maternal adaptation to pregnancy and placenta development. Down-regulation or deficiency of gal-1 during pregnancy is associated with the development of PE; however, the maternal- and placental-derived gal-1 contributions to the disease onset are largely unknown. We demonstrate that lack of gal-1 imposes a risk for PE development in a niche-specific manner, and this is accompanied by a placental dysfunction highly influenced by the absence of maternal-derived gal-1. Notably, differential placental glycosylation through the Sda-capped N-glycans dominates the invasive trophoblast capacity triggered by maternal-derived gal-1. Our findings show that gal-1 derived from the maternal niche is essential for healthy placenta development and indicate that impairment of the gal-1 signaling pathway within the maternal niche could be a molecular cause for maternal cardiovascular maladaptation during pregnancy., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Protein N-linked glycosylation is a structurally diverse post-translational modification that stores biological information in a larger order of magnitude than other post-translational modifications such as phosphorylation, ubiquitination and acetylation. This gives N-glycosylated proteins a diverse range of properties and allows glyco-codes (glycan-related information) to be deciphered by glycan-binding proteins (GBPs). The intervillous space of the placenta is richly populated with membrane-bound and secreted glycoproteins. Evidence exists to suggest that altering the structural nature of their N-glycans can impact several trophoblast functions, which include those related to interactions with decidual cells. This review summarizes trophoblast-related activities influenced by N-glycan-GBP recognition, exploring how different subtypes of trophoblasts actively adapt to characteristics of the decidualized endometrium through cell-specific expression of N-glycosylated proteins, and how these cells receive decidua-derived signals via N-glycan-GBP interactions. We highlight work on how changes in N-glycosylation relates to the success of trophoblast infiltration, interactions of immunomodulators, and uterine angiogenesis. We also discuss studies that suggest aberrant N-glycosylation of trophoblasts may contribute to the pathogenesis of pregnancy complications (e.g. pre-eclampsia, early spontaneous miscarriages and hydatidiform mole). We propose that a more in-depth understanding of how N-glycosylation shapes trophoblast phenotype during early pregnancy has the potential to improve our approach to predicting, diagnosing and alleviating poor maternal/fetal outcomes associated with placental dysfunction., (© 2023 The Author(s).)

Glycoengineering of recombinant glycans and glycoconjugates is a rapidly evolving field. However, the production and exploitation of glycans has lagged behind that of proteins and nucleic acids. Biosynthetic glycoconjugate production requires the coordinated cooperation of three key components within a bacterial cell: a substrate protein, a coupling oligosaccharyltransferase, and a glycan biosynthesis locus. While the acceptor protein and oligosaccharyltransferase are the products of single genes, the glycan is a product of a multigene metabolic pathway. Typically, the glycan biosynthesis locus is cloned and transferred en bloc from the native organism to a suitable Escherichia coli strain. However, gene expression within these pathways has been optimized by natural selection in the native host and is unlikely to be optimal for heterologous production in an unrelated organism. In recent years, synthetic biology has addressed the challenges in heterologous expression of multigene systems by deconstructing these pathways and rebuilding them from the bottom up. The use of DNA assembly methods allows the convenient assembly of such pathways by combining defined parts with the requisite coding sequences in a single step. In this study, we apply combinatorial assembly to the heterologous biosynthesis of the Campylobacter jejuni  N-glycosylation (pgl) pathway in E. coli. We engineered reconstructed biosynthesis clusters that faithfully reproduced the C. jejuni heptasaccharide glycan. Furthermore, following a single round of combinatorial assembly and screening, we identified pathway clones that outperform glycan and glycoconjugate production of the native unmodified pgl cluster. This platform offers a flexible method for optimal engineering of glycan structures in E. coli., (© The Author(s) 2023. Published by Oxford University Press.)

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TICs) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines through the loss of I-branching enzyme, β1,6 N-acetylglucosaminyltransferase 2. Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. In this study, we explored hypoxia-dependent alterations in MM glycome‒associated genes and found that β1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8-ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low β1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed β1,6 N-acetylglucosaminyltransferase 2 in MM cells upregulated TIC marker, NGFR/CD271, whereas loss of MM cell‒intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity in vivo. Extracellular Gal-8 bound preferentially to i-linear poly-N-acetyllactosamines on N-glycans of the TIC marker and prometastatic molecule CD44, among other receptors, and activated prosurvival factor protein kinase B. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-N-acetyllactosamine and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-N-acetyllactosamine and Gal-8 as biomarkers and therapeutic targets of MM., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Melanoma is a highly aggressive skin cancer with poor outcomes associated with distant metastasis. Intrinsic properties of melanoma cells alongside the crosstalk between melanoma cells and surrounding microenvironment determine the tumor behavior. Galectin-3 (Gal-3), a ß-galactoside-binding lectin, has emerged as a major effector in cancer progression, including melanoma behavior. Data from melanoma models and patient studies reveal that Gal-3 expression is dysregulated, both intracellularly and extracellularly, throughout the stages of melanoma progression. This review summarizes the most recent data and hypotheses on Gal-3 and its tumor-modulating functions, highlighting its role in driving melanoma growth, invasion, and metastatic colonization. It also provides insight into potential Gal-3-targeted strategies for melanoma diagnosis and treatment., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest concerning this review article., (Copyright © 2023. Published by Elsevier Inc.)

Placental hormones orchestrate maternal metabolic adaptations to support pregnancy. We hypothesized that placental ER stress, which characterizes early-onset pre-eclampsia (ePE), compromises glycosylation, reducing hormone bioactivity and these maladaptations predispose the mother to metabolic disease in later life. We demonstrate ER stress reduces the complexity and sialylation of trophoblast protein N-glycosylation, while aberrant glycosylation of vascular endothelial growth factor reduced its bioactivity. ER stress alters the expression of 66 of the 146 genes annotated with "protein glycosylation" and reduces the expression of sialyltransferases. Using mouse placental explants, we show ER stress promotes the secretion of mis-glycosylated glycoproteins. Pregnant mice carrying placentas with junctional zone-specific ER stress have reduced blood glucose, anomalous hepatic glucose metabolism, increased cellular stress and elevated DNA methyltransferase 3A. Using pregnancy-specific glycoproteins as a readout, we also demonstrate aberrant glycosylation of placental proteins in women with ePE, thus providing a mechanistic link between ePE and subsequent maternal metabolic disorders., Competing Interests: Authors declare no competing interests., (© 2022 The Author(s).)

Human cervicovaginal fluid (CVF) is a complex, functionally important and glycan rich biological fluid, fundamental in mediating physiological events associated with reproductive health. Using a comprehensive glycomic strategy we reveal an extremely rich and complex N-glycome in CVF of pregnant and non-pregnant women, abundant in paucimannose and high mannose glycans, complex glycans with 2-4 N-Acetyllactosamine (LacNAc) antennae, and Poly-LacNAc glycans decorated with fucosylation and sialylation. N-glycosylation profiles were observed to differ in relation to pregnancy status, microbial composition, immune activation, and pregnancy outcome. Compared to CVF from women experiencing term birth, CVF from women who subsequently experienced preterm birth showed lower sialylation, which correlated to the presence of a diverse microbiome, and higher fucosylation, which correlated positively to pro-inflammatory cytokine concentration. This study is the first step towards better understanding the role of cervicovaginal glycans in reproductive health, their contribution to the mechanism of microbial driven preterm birth, and their potential for preventative therapy., (© 2022. The Author(s).)

Red blood cells (RBCs) lose plasma membrane in the spleen as they age, but the cells and molecules involved are yet to be identified. Sickle cell disease and infection by Plasmodium falciparum cause oxidative stress that induces aggregates of cross-linked proteins with N-linked high-mannose glycans (HMGs). These glycans can be recognised by mannose-binding lectins, including the mannose receptor (CD206), expressed on macrophages and specialised phagocytic endothelial cells in the spleen to mediate the extravascular haemolysis characteristic of these diseases. We postulated this system might also mediate removal of molecules and membrane in healthy individuals. Surface expression of HMGs on RBCs from patients who had previously undergone splenectomy was therefore assessed: high levels were indeed observable as large membrane aggregates. Glycomic analysis by mass spectrometry identified a mixture of Man 5-9 GlcNAc 2 structures. HMG levels correlated well with manual pit counts (r = 0.75-0.85). To assess further whether HMGs might act as a splenic reticuloendothelial function test, we measured levels on RBCs from patients with potential functional hyposplenism, some of whom exhibited high levels that may indicate risk of complications., (© 2022 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Glycosylation can be a critical quality attribute in biologic manufacturing. In particular, it has implications on the half-life, immunogenicity, and pharmacokinetics of therapeutic monoclonal antibodies (mAbs), and must be closely monitored throughout drug development and manufacturing. To address this, advances have been made primarily in upstream processing, including mammalian cell line engineering, to yield more predictably glycosylated mAbs and the addition of media supplements during fermentation to manipulate the metabolic pathways involved in glycosylation. A more robust approach would be a conjoined upstream-downstream processing strategy. This could include implementing novel downstream technologies, such as the use of Fc γ-based affinity ligands for the separation of mAb glycovariants. This review highlights the importance of controlling therapeutic antibody glycosylation patterns, the challenges faced in terms of glycosylation during mAb biosimilar development, current efforts both upstream and downstream to control glycosylation and their limitations, and the need for research in the downstream space to establish holistic and consistent manufacturing processes for the production of antibody therapies., (© 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Brucellosis is a global disease and the world's most prevalent zoonosis. All cases in livestock and most cases in humans are caused by members of the genus Brucella that possess a surface O-polysaccharide (OPS) comprised of a rare monosaccharide 4-deoxy-4-formamido-D-mannopyranose assembled with α1,2 and α1,3 linkages. The OPS of the bacterium is the basis for serodiagnostic tests for brucellosis. Bacteria that also contain the same rare monosaccharide can induce antibodies that cross-react in serological tests. In previous work we established that synthetic oligosaccharides, representing elements of the Brucella A and M polysaccharide structures, were excellent antigens to explore the antibody response in the context of infection, immunisation and cross reaction. These studies suggested the existence of antibodies that are specific to the tip of the Brucella OPS. Sera from naturally and experimentally Brucella abortus -infected cattle as well as from cattle experimentally infected with the cross-reactive bacterium Yersinia enterocolitica O:9 and field sera that cross react in conventional serological assays were studied here with an expanded panel of synthetic antigens. The addition of chemical features to synthetic antigens that block antibody binding to the tip of the OPS dramatically reduced their polyclonal antibody binding capability providing conclusive evidence that the OPS tip (non-reducing end) is a potent epitope. Selected short oligosaccharides, including those that were exclusively α1,2 linked, also demonstrated superior specificity when evaluated with cross reactive sera compared to native smooth lipopolysaccharide (sLPS) antigen and capped native OPS. This surprising discovery suggests that the OPS tip epitope, even though common to both Brucella and Y. enterocolitica O:9, has more specific diagnostic properties than the linear portion of the native antigens. This finding opens the way to the development of improved serological tests for brucellosis.

Intravenous immunoglobulin (IVIG) is an established treatment for numerous autoimmune conditions. Although Fc fragments derived from IVIG have shown efficacy in controlling immune thrombocytopenia in children, the mechanisms of action are unclear and controversial. The aim of this study was to dissect IVIG effector mechanisms using further adapted Fc fragments on demyelination in an ex vivo model of the central nervous system-immune interface. Using organotypic cerebellar slice cultures (OSCs) from transgenic mice, we induced extensive immune-mediated demyelination and oligodendrocyte loss with an antibody specific for myelin oligodendrocyte glycoprotein (MOG) and complement. Protective effects of adapted Fc fragments were assessed by live imaging of green fluorescent protein expression, immunohistochemistry and confocal microscopy. Cysteine- and glycan-adapted Fc fragments protected OSC from demyelination in a dose-dependent manner where equimolar concentrations of either IVIG or control Fc were ineffective. The protective effects of the adapted Fc fragments are partly attributed to interference with complement-mediated oligodendroglia damage. Transcriptome analysis ruled out signatures associated with inflammatory or innate immune responses. Taken together, our findings show that recombinant biomimetics can be made that are at least two hundred-fold more effective than IVIG in controlling demyelination by anti-MOG antibodies., (© 2021 The Authors. Immunology published by John Wiley & Sons Ltd.)

Bacteria use carbohydrate-binding proteins (CBPs), such as lectins and carbohydrate-binding modules (CBMs), to anchor to specific sugars on host surfaces. CBPs in the gut microbiome are well studied, but their roles in the vagina microbiome and involvement in sexually transmitted infections, cervical cancer and preterm birth are largely unknown. We established a classification system for lectins and designed Hidden Markov Model (HMM) profiles for data mining of bacterial genomes, resulting in identification of >100,000 predicted bacterial lectins available at unilectin.eu/bacteria. Genome screening of 90 isolates from 21 vaginal bacterial species shows that those associated with infection and inflammation produce a larger CBPs repertoire, thus enabling them to potentially bind a wider array of glycans in the vagina. Both the number of predicted bacterial CBPs and their specificities correlated with pathogenicity. This study provides new insights into potential mechanisms of colonisation by commensals and potential pathogens of the reproductive tract that underpin health and disease states.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423 ∗ ), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423 ∗ ) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background : Siglec-1 is a macrophage lectin-like receptor that mediates sialic acid-dependent cellular interactions. Its upregulation on macrophages in autoimmune disease was shown previously to promote inflammation through suppressing the expansion of regulatory T cells (Tregs). Here we investigate the molecular basis for Siglec-1 binding to Tregs using in vitro -induced cells as a model system. Methods : Glycosylation changes that affect Siglec‑1 binding were studied by comparing activated and resting Tregs using RNA-Seq, glycomics, proteomics and binding of selected antibodies and lectins. A proximity labelling and proteomics strategy was used to identify Siglec-1 counter-receptors expressed on activated Tregs. Results : Siglec-1 binding was strongly upregulated on activated Tregs, but lost under resting conditions. Glycomics revealed changes in N-glycans and glycolipids following Treg activation and we observed changes in expression of multiple 'glycogenes' that could lead to the observed increase in Siglec-1 binding. Proximity labelling of intact, living cells identified 49 glycoproteins expressed by activated Tregs that may function as Siglec-1 counter-receptors. These represent ~5% of the total membrane protein pool and were mainly related to T cell activation and proliferation. We demonstrate that several of these counter-receptors were upregulated following activation of Tregs and provide initial evidence that their altered glycosylation may also be important for Siglec-1 binding. Conclusions : We provide the first comprehensive analysis of glycan changes that occur in activated Tregs, leading to recognition by the macrophage lectin, Siglec-1 and suppression of Treg expansion. We furthermore provide insights into glycoprotein counter-receptors for Siglec-1 expressed by activated Tregs that are likely to be important for suppressing Treg expansion., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Wu G et al.)

There is a critical need to develop small-molecule inhibitors of mucin-type O-linked glycosylation. The best-known reagent currently is benzyl-GalNAc, but it is effective only at millimolar concentrations. This article demonstrates that Ac 5 GalNTGc, a peracetylated C-2 sulfhydryl-substituted GalNAc, fulfills this unmet need. When added to cultured leukocytes, breast cells, and prostate cells, Ac 5 GalNTGc increased cell-surface VVA binding by ∼10-fold, indicating truncation of O-glycan biosynthesis. Cytometry, mass spectrometry, and western blot analysis of HL-60 promyelocytes demonstrated that 50-80 μM Ac 5 GalNTGc prevented elaboration of 30%-60% of the O-glycans beyond the Tn-antigen (GalNAcα1-Ser/Thr) stage. The effect of the compound on N-glycans and glycosphingolipids was small. Glycan inhibition induced by Ac 5 GalNTGc resulted in 50%-80% reduction in leukocyte sialyl-Lewis X expression and L-/P-selectin-mediated rolling under flow conditions. Ac 5 GalNTGc was pharmacologically active in mouse. It reduced neutrophil infiltration to sites of inflammation by ∼60%. Overall, Ac 5 GalNTGc may find diverse applications as a potent inhibitor of O-glycosylation., Competing Interests: Declaration of interests We do not have competing financial or non-financial interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In healthy joints, synovial fibroblasts (SFs) provide the microenvironment required to mediate homeostasis, but these cells adopt a pathological function in rheumatoid arthritis (RA). Carbohydrates (glycans) on cell surfaces are fundamental regulators of the interactions between stromal and immune cells, but little is known about the role of the SF glycome in joint inflammation. Here we study stromal guided pathophysiology by mapping SFs glycosylation pathways. Combining transcriptomic and glycomic analysis, we show that transformation of fibroblasts into pro-inflammatory cells is associated with glycan remodeling, a process that involves TNF-dependent inhibition of the glycosyltransferase ST6Gal1 and α2-6 sialylation. SF sialylation correlates with distinct functional subsets in murine experimental arthritis and remission stages in human RA. We propose that pro-inflammatory cytokines remodel the SF-glycome, converting the synovium into an under-sialylated and highly pro-inflammatory microenvironment. These results highlight the importance of glycosylation in stromal immunology and joint inflammation.

The novel coronavirus SARS-CoV-2, the infective agent causing COVID-19, is having a global impact both in terms of human disease as well as socially and economically. Its heavily glycosylated spike glycoprotein is fundamental for the infection process, via its receptor-binding domains interaction with the glycoprotein angiotensin-converting enzyme 2 on human cell surfaces. We therefore utilized an integrated glycomic and glycoproteomic analytical strategy to characterize both N- and O- glycan site-specific glycosylation within the receptor-binding domain. We demonstrate the presence of complex-type N-glycans with unusual fucosylated LacdiNAc at both sites N331 and N343 and a single site of O-glycosylation on T323., (© The Author(s) 2020. Published by Oxford University Press.)

In both sickle cell disease and malaria, red blood cells (RBCs) are phagocytosed in the spleen, but receptor-ligand pairs mediating uptake have not been identified. Here, we report that patches of high mannose N-glycans (Man 5-9 GlcNAc 2 ), expressed on diseased or oxidized RBC surfaces, bind the mannose receptor (CD206) on phagocytes to mediate clearance. We find that extravascular hemolysis in sickle cell disease correlates with high mannose glycan levels on RBCs. Furthermore, Plasmodium falciparum-infected RBCs expose surface mannose N-glycans, which occur at significantly higher levels on infected RBCs from sickle cell trait subjects compared to those lacking hemoglobin S. The glycans are associated with high molecular weight complexes and protease-resistant, lower molecular weight fragments containing spectrin. Recognition of surface N-linked high mannose glycans as a response to cellular stress is a molecular mechanism common to both the pathogenesis of sickle cell disease and resistance to severe malaria in sickle cell trait.

Pregnancy-specific beta 1 glycoprotein (PSG1) is secreted from trophoblast cells of the human placenta in increasing concentrations as pregnancy progresses, becoming one of the most abundant proteins in maternal serum in the third trimester. PSG1 has seven potential N-linked glycosylation sites across its four domains. We carried out glycomic and glycoproteomic studies to characterize the glycan composition of PSG1 purified from serum of pregnant women and identified the presence of complex N-glycans containing poly LacNAc epitopes with α2,3 sialyation at four sites. Using different techniques, we explored whether PSG1 can bind to galectin-1 (Gal-1) as these two proteins were previously shown to participate in processes required for a successful pregnancy. We confirmed that PSG1 binds to Gal-1 in a carbohydrate-dependent manner with an affinity of the interaction of 0.13 μM. In addition, we determined that out of the three N-glycosylation-carrying domains, only the N and A2 domains of recombinant PSG1 interact with Gal-1. Lastly, we observed that the interaction between PSG1 and Gal-1 protects this lectin from oxidative inactivation and that PSG1 competes the ability of Gal-1 to bind to some but not all of its glycoprotein ligands., (Published by Oxford University Press 2020. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Lipocalins are a family of secreted proteins. They are capable of binding small lipophilic compounds and have been extensively studied for their role in chemosignalling in rodent urine. Urine of the common brushtail possum ( Trichosurus vulpecula ) contains a prominent glycoprotein of 20 kDa, expressed in both sexes. We have isolated this protein and determined its primary sequence by mass spectrometry, including the use of metabolic labelling to resolve the leucine/isoleucine isobaric ambiguity. The protein sequence was identified as a lipocalin, and phylogenetic analysis grouped the protein with other marsupial lipocalin sequences in a phylogenetic clade distinct from established cross-species lipocalin sub-families. The pattern of expression in possum urine and the similarity in sequence and structure to other lipocalins suggests this protein may have a role in brushtail possum chemosignalling.

Glycosylation is a fundamental cellular process that has a dramatic impact on the functionality of glycoconjugates such as proteins or lipids and mediates many different biological interactions including cell migration, cellular signaling, and synaptic interactions in the nervous system. In spinal cord injury (SCI), all of these cellular processes are altered, but the potential contributions of glycosylation changes to these alterations has not been thoroughly investigated. We studied the glycosylation of injured spinal cord tissue from rats that received a contusion SCI. The N- and O-linked glycosylation was assessed at 3 and 14 days post-injury (DPI), and compared with uninjured control and time-matched sham spinal tissue. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS/MS) were performed to analyze carbohydrate structures. Results revealed diverse and abundant glycosylation in all groups, with some carbohydrate structures differentially produced in SCI animals compared with uninjured controls and shams. One such change occurred in the abundance of the Sda structure, Neu5Ac-α-(2,3)-[GalNAc-β-(1,4)-]Gal-β-(1,4)-GlcNAc, which was increased in SCI samples compared with shams and non-injured controls. Immunohistochemistry (IHC) and western blot were performed on SCI and sham samples using the CT1 antibody, which recognizes the terminal trisaccharide of Sda with high specificity. Both of these metrics confirmed elevated Sda structure in SCI tissue, where IHC further showed that Sda is expressed mainly by microglia. The results of these studies suggest that SCI causes a significant alteration in N- and O-linked glycosylation.

Alzheimer disease (AD) is a devastating disease and a global health problem, and current treatments are only symptomatic. A wealth of clinical studies support that the disease starts to develop decades before the first symptoms appear, emphasizing the importance of studying early changes for improving early diagnosis and guiding toward novel treatment strategies. Protein glycosylation is altered in AD but it remains to be clarified why these alterations occur and how they affect the disease development. Here, we used a glycomics approach to search for alterations in protein glycosylation in cerebrospinal fluid (CSF) in AD compared with nondemented controls. Using both matrix-assisted laser desorption ionization-time of flight and liquid chromatography-electrospray mass spectrometry, we observed an increase in N-glycans carrying bisecting N-acetylglucosamine in AD. Based on those findings, we designed an enzyme-linked multiwell plate assay to quantify N-glycans binding to the lectin Phaseolus vulgaris Erythroagglutinin (PHA-E), which is specific for N-glycans containing bisecting N-acetylglucosamine. Using this assay, we found a similar increase in CSF in AD compared with controls. Further analysis of CSF from 242 patients with subjective cognitive impairment (SCI), mild cognitive impairment (MCI), or AD dementia revealed significantly increased binding to PHA-E in MCI and AD compared to SCI. Interestingly, PHA-E binding correlated with CSF levels of phosphorylated tau and total tau and this correlation was most prominent in the SCI group (R = 0.53-0.54). This study supports a link between N-glycosylation, neurodegeneration, and tau pathology in AD and suggests that glycan biomarkers have potential to identify SCI cases at risk of developing AD., (© 2019 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Growing evidence suggests that galectins, an evolutionarily conserved family of glycan-binding proteins, fulfill key roles in pregnancy including blastocyst implantation, maternal-fetal immune tolerance, placental development, and maternal vascular expansion, thereby establishing a healthy environment for the growing fetus. In this review, we comprehensively present the function of galectins in shaping cellular circuits that characterize a healthy pregnancy. We describe the current understanding of galectins in term and preterm labor and discuss how the galectin-glycan circuits contribute to key immunological pathways sustaining maternal tolerance and preventing microbial infections. A deeper understanding of the glycoimmune pathways regulating early events in preterm birth could offer the broader translational potential for the treatment of this devastating syndrome.

GNE Myopathy is a recessive neuromuscular disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, and a typical muscle pathology. Although GNE, which is the mutated gene in the disease, is well known as the key enzyme in the biosynthesis pathway of sialic acid, the pathophysiological pathway leading from GNE mutations to the muscle phenotype in GNE Myopathy is still unclear. The obvious hypothesis of impaired sialylation in patients' skeletal muscle as the cause of the disease is still controversial. In the present study we have investigated whether a distinctive altered pattern of sialylation in GNE Myopathy cultured muscle cells could be attributed to a specific glycoconjugate. Mass spectrometry based glycomic methodologies have been utilized to assess the sialylation level of protein N- and O-linked glycans and glycolipid derived glycans from patient and matched control samples. No consistent change in sialylation was detected in glycoconjugates. These results suggest potential additional roles for GNE that could account for the disease pathology., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Glycodelin is a major glycoprotein expressed in reproductive tissues, like secretory and decidualized endometrium. It has several reproduction related functions that are dependent on specific glycosylation, but it has also been found to drive differentiation of endometrial carcinoma cells toward a less malignant phenotype. Here we aimed to elucidate whether the glycosylation and function of glycodelin is altered in endometrial carcinoma as compared with a normal endometrium. We carried out glycan structure analysis of glycodelin expressed in HEC-1B human endometrial carcinoma cells (HEC-1B Gd) by mass spectrometry glycomics strategies. Glycans of HEC-1B Gd were found to comprise a typical mixture of high-mannose, hybrid, and complex-type N-glycans, often containing undecorated LacNAc (Galβ1-4GlcNAc) antennae. However, several differences, as compared with previously reported glycan structures of normal human decidualized endometrium-derived glycodelin isoform, glycodelin-A (GdA), were also found. These included a lower level of sialylation and more abundant poly-LacNAc antennae, some of which are fucosylated. This allowed us to select lectins that showed different binding to these classes of glycodelin. Despite the differences in glycosylation between HEC-1B Gd and GdA, both showed similar inhibitory activity on trophoblast cell invasion and peripheral blood mononuclear cell proliferation. For the detection of cancer associated glycodelin, we established a novel in situ proximity-ligation based histochemical staining method using a specific glycodelin antibody and UEAI lectin. We found that the UEAI reactive glycodelin was abundant in endometrial carcinoma, but virtually absent in normal endometrial tissue even when glycodelin was strongly expressed. In conclusion, we established a histochemical staining method for the detection of endometrial carcinoma-associated glycodelin and showed that this specific glycodelin is exclusively expressed in cancer, not in normal endometrium. Similar methods can be used for studies of other glycoproteins.

Abs are glycoproteins that carry a conserved N -linked carbohydrate attached to the Fc whose presence and fine structure profoundly impacts on their in vivo immunogenicity, pharmacokinetics, and functional attributes. The host cell line used to produce IgG plays a major role in this glycosylation, as different systems express different glycosylation enzymes and transporters that contribute to the specificity and heterogeneity of the final IgG-Fc glycosylation profile. In this study, we compare two panels of glycan-adapted IgG1-Fc mutants expressed in either the human endothelial kidney 293-F or Chinese hamster ovary-K1 systems. We show that the types of N -linked glycans between matched pairs of Fc mutants vary greatly and in particular, with respect, to sialylation. These cell line effects on glycosylation profoundly influence the ability of the engineered Fcs to interact with either human or pathogen receptors. For example, we describe Fc mutants that potently disrupted influenza B-mediated agglutination of human erythrocytes when expressed in Chinese hamster ovary-K1, but not in human endothelial kidney 293-F cells., (Copyright © 2020 The Authors.)

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that is essential for the maintenance of pregnancy. Glycosylation of hCG is known to be essential for its biological activity. "Hyperglycosylated" variants secreted during early pregnancy have been proposed to be involved in initial implantation of the embryo and as a potential diagnostic marker for gestational diseases. However, what constitutes "hyperglycosylation" is not yet fully understood. In this study, we perform comparative N-glycomic analysis of hCG expressed in the same individuals during early and late pregnancy to help provide new insights into hCG function, reveal new targets for diagnostics and clarify the identity of hyperglycosylated hCG. hCG was isolated in urine collected from women at 7 weeks and 20 weeks' gestation. hCG was also isolated in urine from women diagnosed with gestational trophoblastic disease (GTD). We used glycomics methodologies including matrix assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and MS/MS methods to characterise the N-glycans associated with hCG purified from the individual samples. The structures identified on the early pregnancy (EP-hCG) and late pregnancy (LP-hCG) samples corresponded to mono-, bi-, tri-, and tetra-antennary N-glycans. A novel finding was the presence of substantial amounts of bisected type N-glycans in pregnancy hCG samples, which were present at much lower levels in GTD samples. A second novel observation was the presence of abundant LewisX antigens on the bisected N-glycans. GTD-hCG had fewer glycoforms which constituted a subset of those found in normal pregnancy. When compared to EP-hCG, GTD-hCG samples had decreased signals for tri- and tetra-antennary N-glycans. In terms of terminal epitopes, GTD-hCG had increased signals for sialylated structures, while LewisX antigens were of very minor abundance. hCG carries the same N-glycans throughout pregnancy but in different proportions. The N-glycan repertoire is more diverse than previously reported. Bisected and LewisX structures are potential targets for diagnostics. hCG isolated from pregnancy urine inhibits NK cell cytotoxicity in vitro at nanomolar levels and bisected type glycans have previously been implicated in the suppression of NK cell cytotoxicity, suggesting that hCG-related bisected type N-glycans may directly suppress NK cell cytotoxicity., Competing Interests: Affiliation with Mologic LTD UK I can confirm that this commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in guide for authors http://journals.plos.org/plosone/s/competing-interests).

Approximately 50% of the mass of the Envelope (Env) glycoprotein surface subunit (gp120) of human immunodeficiency virus type 1 (HIV-1) is composed of N-linked carbohydrate. Until now, the dogma has been that HIV-1 lacks O-linked carbohydrate on Env. Here we show that a subset of patient-derived HIV-1 isolates contain O-linked carbohydrate on the variable 1 (V1) domain of Env gp120. We demonstrate the presence of this O-glycosylation both on virions and on gp120 expressed as a secreted protein. Further, we establish that these O-linked glycans can confer a more than 1,000-fold decrease in neutralization sensitivity (IC 50 ) to V3-glycan broadly neutralizing antibodies. These findings uncover a structural modification to the HIV-1 Env and suggest a functional role in promoting viral escape from one category of broadly neutralizing antibodies., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The lipopolysaccharide O-antigen structure expressed by the European Helicobacter pylori model strain G27 encompasses a trisaccharide, an intervening glucan-heptan and distal Lewis antigens that promote immune escape. However, several gaps still remain in the corresponding biosynthetic pathway. Here, systematic mutagenesis of glycosyltransferase genes in G27 combined with lipopolysaccharide structural analysis, uncovered HP0102 as the trisaccharide fucosyltransferase, HP1283 as the heptan transferase, and HP1578 as the GlcNAc transferase that initiates the synthesis of Lewis antigens onto the heptan motif. Comparative genomic analysis of G27 lipopolysaccharide biosynthetic genes in strains of different ethnic origin revealed that East-Asian strains lack the HP1283/HP1578 genes but contain an additional copy of HP1105 and JHP0562. Further correlation of different lipopolysaccharide structures with corresponding gene contents led us to propose that the second copy of HP1105 and the JHP0562 may function as the GlcNAc and Gal transferase, respectively, to initiate synthesis of the Lewis antigen onto the Glc-Trio-Core in East-Asian strains lacking the HP1283/HP1578 genes. In view of the high gastric cancer rate in East Asia, the absence of the HP1283/HP1578 genes in East-Asian H. pylori strains warrants future studies addressing the role of the lipopolysaccharide heptan in pathogenesis., Competing Interests: BJM is the founder and shareholder of Ondek Pty. Ltd. MB, HN are former employees of Ondek Pty. Ltd. The remaining authors disclose no conflicts.

ES-62 is the major secreted product of the parasitic filarial nematode Acanthocheilonema viteae and has potent anti-inflammatory activities as a consequence of posttranslational decoration by phosphorylcholine (PC). Previously, we showed that ES-62's PC was attached to N-linked glycans, and using fast atom bombardment mass spectrometry, we characterized the structure of the glycans. However, it was unknown at this time which of ES-62's four potential N-glycosylation sites carries the PC-modified glycans. In the present study, we now employ more advanced analytical tools-nano-flow liquid chromatography with high-definition electrospray mass spectrometry-to show that PC-modified glycans are found at all four potential N-glycosylation sites. Also, our earlier studies showed that up to two PC groups were detected per glycan, and we are now able to characterize N-glycans with up to five PC groups. The number per glycan varies in three of the four glycosylation sites, and in addition, for the first time, we have detected PC on the N-glycan chitobiose core in addition to terminal GlcNAc. Nevertheless, the majority of PC is detected on terminal GlcNAc, enabling it to interact with the cells and molecules of the immune system. Such expression may explain the potent immunomodulatory effects of a molecule that is considered to have significant therapeutic potential in the treatment of certain human allergic and autoimmune conditions., (© The Author(s) 2019. Published by Oxford University Press.)

In eukaryotes, glycosylation plays a role in proteome stability, protein quality control, and modulating protein function; however, similar studies in bacteria are lacking. Here, we investigate the roles of general protein glycosylation systems in bacteria using the enteropathogen Campylobacter jejuni as a well-defined example. By using a quantitative proteomic strategy, we were able to monitor changes in the C. jejuni proteome when glycosylation is disrupted. We demonstrate that in C. jejuni , N- glycosylation is essential to maintain proteome stability and protein quality control. These findings guided us to investigate the role of N- glycosylation in modulating bacterial cellular activities. In glycosylation-deficient C. jejuni , the multidrug efflux pump and electron transport pathways were significantly impaired. We demonstrate that in vivo , fully glycosylation-deficient C. jejuni bacteria were unable to colonize its natural avian host. These results provide the first evidence of a link between proteome stability and complex functions via a bacterial general glycosylation system. IMPORTANCE Advances in genomics and mass spectrometry have revealed several types of glycosylation systems in bacteria. However, why bacterial proteins are modified remains poorly defined. Here, we investigated the role of general N- linked glycosylation in a major food poisoning bacterium, Campylobacter jejuni The aim of this study is to delineate the direct and indirect effects caused by disrupting this posttranslational modification. To achieve this, we employed a quantitative proteomic strategy to monitor alterations in the C. jejuni proteome. Our quantitative proteomic results linked general protein N- glycosylation to maintaining proteome stability. Functional analyses revealed novel roles for bacterial N- glycosylation in modulating multidrug efflux pump, enhancing nitrate reduction activity, and promoting host-microbe interaction. This work provides insights on the importance of general glycosylation in proteins in maintaining bacterial physiology, thus expanding our knowledge of the emergence of posttranslational modification in bacteria., (Copyright © 2019 Abouelhadid et al.)

The lectin Helix pomatia agglutinin (HPA) recognizes altered glycosylation in solid cancers and the identification of HPA binding partners in tumour tissue and serum is an important aim. Among the many HPA binding proteins, IgA1 has been reported to be the most abundant in liver metastases. In this study, the glycosylation of IgA1 was evaluated using serum samples from patients with breast cancer (BCa) and the utility of IgA1 glycosylation as a biomarker was assessed. Detailed mass spectrometric structural analysis showed an increase in disialo-biantennary N- linked glycans on IgA1 from BCa patients ( p < 0.0001: non-core fucosylated; p = 0.0345: core fucosylated) and increased asialo-Thomsen-Friedenreich antigen (TF) and disialo-TF antigens in the O- linked glycan preparations from IgA1 of cancer patients compared with healthy control individuals. An increase in Sambucus nigra binding was observed, suggestive of increased α 2,6-linked sialic acid on IgA1 in BCa. Logistic regression analysis showed HPA binding to IgA1 and tumour size to be significant independent predictors of distant metastases ( χ 2 13.359; n = 114; p = 0.020) with positive and negative predictive values of 65.7% and 64.6%, respectively. Immunohistochemical analysis of tumour tissue samples showed IgA1 to be detectable in BCa tissue. This report provides a detailed analysis of serum IgA1 glycosylation in BCa and illustrates the potential utility of IgA1 glycosylation as a biomarker for BCa prognostication., Competing Interests: We declare we have no competing interests.

In therapeutic applications in which the Fc of IgG is critically important, the receptor binding and functional properties of the Fc are lost after deglycosylation or removal of the unique Asn 297 N-X-(T/S) sequon. A population of Fcs bearing sialylated glycans has been identified as contributing to this functionality, and high levels of sialylation also lead to longer serum retention times advantageous for therapy. The efficacy of sialylated Fc has generated an incentive to modify the unique N-linked glycosylation site at Asn 297 , either through chemical and enzymatic methods or by mutagenesis of the Fc, that disrupts the protein-Asn 297 carbohydrate interface. In this study, we took an alternative approach by inserting or deleting N-linked attachment sites into the body of the Fc to generate a portfolio of mutants with tailored effector functions. For example, we describe mutants with enhanced binding to low-affinity inhibitory human Fcγ and glycan receptors that may be usefully incorporated into existing Ab engineering approaches to treat or vaccinate against disease. The IgG1 Fc fragments containing complex sialylated glycans attached to the N-terminal Asn 221 sequon bound influenza virus hemagglutinin and disrupted influenza A-mediated agglutination of human erythrocytes., (Copyright © 2019 The Authors.)

Metabolic decoys are synthetic analogs of naturally occurring biosynthetic acceptors. These compounds divert cellular biosynthetic pathways by acting as artificial substrates that usurp the activity of natural enzymes. While O-linked glycosides are common, they are only partially effective even at millimolar concentrations. In contrast, we report that N-acetylglucosamine (GlcNAc) incorporated into various thioglycosides robustly truncate cell surface N- and O-linked glycan biosynthesis at 10-100 μM concentrations. The >10-fold greater inhibition is in part due to the resistance of thioglycosides to hydrolysis by intracellular hexosaminidases. The thioglycosides reduce β-galactose incorporation into lactosamine chains, cell surface sialyl Lewis-X expression, and leukocyte rolling on selectin substrates including inflamed endothelial cells under fluid shear. Treatment of granulocytes with thioglycosides prior to infusion into mouse inhibited neutrophil homing to sites of acute inflammation and bone marrow by ∼80%-90%. Overall, thioglycosides represent an easy to synthesize class of efficient metabolic inhibitors or decoys. They reduce N-/O-linked glycan biosynthesis and inflammatory leukocyte accumulation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the CEL gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined CEL allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly O- glycosylated, mucin-like C terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-α1,3(Fuc-α1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF MS to characterize the released O- glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the O- glycome of CEL consisted mainly of core 1/core 2 structures with a composition depending on the subject's FUT2 and ABO gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum., (© 2018 El Jellas et al.)

Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-β1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.

Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide. It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. In many bacterial and archaeal species, the S-layer is glycosylated, but no such modifications have been demonstrated in C. difficile. Here, we show that a C. difficile strain of S-layer cassette type 11, Ox247, has a complex glycan attached via an O -linkage to Thr-38 of the S-layer low-molecular-weight subunit. Using MS and NMR, we fully characterized this glycan. We present evidence that it is composed of three domains: (i) a core peptide-linked tetrasaccharide with the sequence -4-α-Rha-3-α-Rha-3-α-Rha-3-β-Gal-peptide; (ii) a repeating pentasaccharide with the sequence -4-β-Rha-4-α-Glc-3-β-Rha-4-(α-Rib-3-)β-Rha-; and (iii) a nonreducing end-terminal 2,3 cyclophosphoryl-rhamnose attached to a ribose-branched sub-terminal rhamnose residue. The Ox247 genome contains a 24-kb locus containing genes for synthesis and protein attachment of this glycan. Mutations in genes within this locus altered or completely abrogated formation of this glycan, and their phenotypes suggested that this S-layer modification may affect sporulation, cell length, and biofilm formation of C. difficile In summary, our findings indicate that the S-layer protein of SLCT-11 strains displays a complex glycan and suggest that this glycan is required for C. difficile sporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2018 Richards et al.)

Classically, the unfolded protein response (UPR) safeguards secretory pathway proteostasis. The most ancient arm of the UPR, the IRE1-activated spliced X-box binding protein 1 (XBP1s)-mediated response, has roles in secretory pathway maturation beyond resolving proteostatic stress. Understanding the consequences of XBP1s activation for cellular processes is critical for elucidating mechanistic connections between XBP1s and development, immunity, and disease. Here, we show that a key functional output of XBP1s activation is a cell type-dependent shift in the distribution of N-glycan structures on endogenous membrane and secreted proteomes. For example, XBP1s activity decreased levels of sialylation and bisecting GlcNAc in the HEK293 membrane proteome and secretome, while substantially increasing the population of oligomannose N-glycans only in the secretome. In HeLa cell membranes, stress-independent XBP1s activation increased the population of high-mannose and tetraantennary N-glycans, and also enhanced core fucosylation. mRNA profiling experiments suggest that XBP1s-mediated remodeling of the N-glycome is, at least in part, a consequence of coordinated transcriptional resculpting of N-glycan maturation pathways by XBP1s. The discovery of XBP1s-induced N-glycan structural remodeling on a glycome-wide scale suggests that XBP1s can act as a master regulator of N-glycan maturation. Moreover, because the sugars on cell-surface proteins or on proteins secreted from an XBP1s-activated cell can be molecularly distinct from those of an unactivated cell, these findings reveal a potential new mechanism for translating intracellular stress signaling into altered interactions with the extracellular environment., Competing Interests: The authors declare no conflict of interest.

Activation of invariant natural killer T lymphocytes (iNKT cells) by α-galactosylceramide (α-GC) elicits a range of pro-inflammatory or anti-inflammatory immune responses. We report the synthesis and characterization of a series of α-GC analogues with acyl chains of varying length and a terminal benzophenone. These bound efficiently to the glycolipid antigen presenting protein CD1d, and upon photoactivation formed stable CD1d-glycolipid covalent conjugates. Conjugates of benzophenone α-GCs with soluble or cell-bound CD1d proteins retained potent iNKT cell activating properties, with biologic effects that were modulated by acyl chain length and the resulting affinities of conjugates for iNKT cell antigen receptors. Analysis by mass spectrometry identified a unique covalent attachment site for the glycolipid ligands in the hydrophobic ligand binding pocket of CD1d. The creation of covalent conjugates of CD1d with α-GC provides a new tool for probing the biology of glycolipid antigen presentation, as well as opportunities for developing effective immunotherapeutics.

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.

The arabinan-containing polysaccharides, arabinogalactan (AG) and lipoarabinomannan (LAM), are key cell wall components of the Corynebacterineae , which include Corynebacteria, Norcadia and Mycobacteria. Both AG and LAM contain elaborate arabinan domains composed of distinct structural motifs. Mycobacterial EmbA, EmbB and EmbC, collectively known as the Emb proteins, have been identified as arabinosyltransferases (Ara f Ts), which are targeted by the front-line anti-tubercular drug ethambutol. Previous studies have established that EmbA and EmbB play a role in the synthesis of the characteristic terminal hexa-arabinosuranosyl motif, whilst EmbC is involved exclusively in the biosynthesis of LAM. Herein, we have investigated the role of the singular Emb protein from Corynebacterium glutamicum through the detailed biochemical and chemical analysis of a double ΔaftAΔemb mutant, where the priming Cg-AftA protein, which generates the substrate for Cg-Emb has been deleted. Analysis of its cell wall revealed a complete absence of arabinose resulting in a truncated cell wall containing only a galactan backbone accompanied with complete loss of cell wall bound mycolates. In vitro cell-free assays using C. glutamicumΔaftA, C. glutamicumΔemb, C. glutamicumΔaftAΔemb and C. glutamicumΔaftBΔaftD and two synthetic acceptors, which mimick the arabinofuranose (Ara f ) "primed" galactan chain, demonstrated that Cg-Emb is able to transfer an Ara f residue to the C5 of the Ara f positioned on the synthetic acceptor(s). These results indicate that Cg-Emb acts as an α(1 → 5) Ara f T and elongates the arabinan core during the early stages of arabinan biosynthesis in C. glutamicum .

Glycosylation is considered one of the most complex and structurally diverse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious sample preparation steps. Therefore, in this study, we aim to improve sample preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein/protein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of samples proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.

A highly conserved threonine near the C terminus of gp120 of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) was investigated for its contributions to envelope protein function and virion infectivity. When this highly conserved Thr residue was substituted with anything other than serine (the other amino acid that can accept O-glycosylation), the resulting virus was noninfectious. We found that this Thr was critical for the association of gp120 with the virion and that amino acid substitution increased the amount of dissociated gp120 in the cell culture supernatant. When HIV virions were generated in cells overexpressing polypeptide N -acetylgalactosaminyltransferase 1 (GalNAcT1), viral infectivity was increased 2.5-fold compared to that of virus produced in wild-type HEK293T cells; infectivity was increased 8-fold when the Thr499Ser mutant was used. These infectivity enhancements were not observed when GalNAcT3 was used. Using HEK293T knockout cell lines totally devoid of the ability to perform O-linked glycosylation, we demonstrated production of normal levels of virions and normal levels of infectivity in the complete absence of O-linked carbohydrate. Our data indicate that O-glycosylation is not necessary for the natural replication cycle of HIV and SIV. Nonetheless, it remains theoretically possible that the repertoire of GalNAc transferase isoforms in natural target cells for HIV and SIV in vivo could result in O-glycosylation of the threonine residue in question and that this could boost the infectivity of virions beyond the levels seen in the absence of such O-glycosylation. IMPORTANCE Approximately 50% of the mass of the gp120 envelope glycoprotein of both HIV and SIV is N-linked carbohydrate. One of the contributions of this N-linked carbohydrate is to shield conserved peptide sequences from recognition by humoral immunity. This N-linked glycosylation is one of the reasons that primary isolates of HIV and SIV are so heavily resistant to antibody-mediated neutralization. Much less studied is any potential contribution from O-linked glycosylation. The literature on this topic to date is somewhat confusing and ambiguous. Our studies described in this report demonstrate unambiguously that O-linked glycosylation is not necessary for the natural replication cycle of HIV and SIV. However, the door is not totally closed because of the diversity of numerous GalNAc transferase enzymes that initiate O-linked carbohydrate attachment and the theoretical possibility that natural target cells for HIV and SIV in vivo could potentially complete such O-linked carbohydrate attachment to further increase infectivity., (Copyright © 2017 American Society for Microbiology.)

Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE), galactokinase 1 (GALK1), and galactokinase 2 (GALK2) genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc) to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2), O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.