Decreased fucosylation impacts epithelial integrity and increases risk for COPD.

COPD causes significant morbidity and mortality worldwide. Epithelial damage is fundamental to disease pathogenesis, although the mechanisms driving disease remain undefined. Published evidence from a COPD cohort (SPIROMICS) and confirmed in a second cohort (COPDgene) demonstrate a polymorphism in Fucosyltransferese-2 (FUT2) is a trans-pQTL for E-cadherin, which is critical in COPD pathogenesis. We found by MALDI-TOF analysis that FUT2 increased terminal fucosylation of E-cadherin. Using atomic force microscopy, we found that FUT2-dependent fucosylation enhanced E-cadherin-E-cadherin bond strength, mediating the improvement in monolayer integrity. Tracheal epithelial cells from Fut2-/- mice have reduced epithelial integrity, which is recovered with reconstitution of Fut2. Overexpression of FUT2 in COPD derived epithelia rescues barrier function. Fut2-/- mice show increased susceptibility in an elastase model of disease developing both emphysema and fibrosis. We propose this is due to the role of FUT2 in proliferation and cell differentiation. Overexpression of FUT2 significantly increased proliferation. Loss of Fut2 results in accumulation of Spc+ cells suggesting a failure of alveolar type 2 cells to undergo transdifferentiation to alveolar type 1. Using a combination of population data, genetically manipulated mouse models, and patient-derived cells, we present a novel mechanism by which post-translational modifications modulate tissue pathology and serve as a proof of concept for the development of a disease-modifying target in COPD.

Glyco-engineered MDCK cells display preferred receptors of H3N2 influenza absent in eggs used for vaccines.

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 (Galß1-4GlcNAc) 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 ß3-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.

Development of a novel glycoengineering platform for the rapid production of conjugate vaccines.

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.

Maternal-derived galectin-1 shapes the placenta niche through Sda terminal glycosylation: Implication for preeclampsia.

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.

Roles of N-linked glycosylation and glycan-binding proteins in placentation: trophoblast infiltration, immunomodulation, angiogenesis, and pathophysiology.

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.

The pleiotropic role of galectin-3 in melanoma progression: Unraveling the enigma.

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.

Perturbation of placental protein glycosylation by endoplasmic reticulum stress promotes maladaptation of maternal hepatic glucose metabolism.

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.

A combinatorial DNA assembly approach to biosynthesis of N-linked glycans in E. coli.

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.

Hypoxia Controls the Glycome Signature and Galectin-8-Ligand Axis to Promote Protumorigenic Properties of Metastatic Melanoma.

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, ß1,6 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 ß1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8-ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low ß1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed ß1,6 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.

N-glycosylation of cervicovaginal fluid reflects microbial community, immune activity, and pregnancy status.

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.

The Tip of Brucella O-Polysaccharide Is a Potent Epitope in Response to Brucellosis Infection and Enables Short Synthetic Antigens to Be Superior Diagnostic Reagents.

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.

Measurement of erythrocyte membrane mannoses to assess splenic function.

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 Man5-9 GlcNAc2 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.

Strategies to control therapeutic antibody glycosylation during bioprocessing: Synthesis and separation.

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.

Proteome-wide prediction of bacterial carbohydrate-binding proteins as a tool for understanding commensal and pathogen colonisation of the vaginal microbiome.

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.

A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

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.

Loss of α2-6 sialylation promotes the transformation of synovial fibroblasts into a pro-inflammatory phenotype in arthritis.

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.

Modified recombinant human IgG1-Fc is superior to natural intravenous immunoglobulin at inhibiting immune-mediated demyelination.

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.

Red blood cell mannoses as phagocytic ligands mediating both sickle cell anaemia and malaria resistance.

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 (Man5-9GlcNAc2), 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.

Efficient inhibition of O-glycan biosynthesis using the hexosamine analog Ac5GalNTGc.

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 Ac5GalNTGc, a peracetylated C-2 sulfhydryl-substituted GalNAc, fulfills this unmet need. When added to cultured leukocytes, breast cells, and prostate cells, Ac5GalNTGc 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 Ac5GalNTGc 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 Ac5GalNTGc resulted in 50%-80% reduction in leukocyte sialyl-Lewis X expression and L-/P-selectin-mediated rolling under flow conditions. Ac5GalNTGc was pharmacologically active in mouse. It reduced neutrophil infiltration to sites of inflammation by ∼60%. Overall, Ac5GalNTGc may find diverse applications as a potent inhibitor of O-glycosylation.

Activation of regulatory T cells triggers specific changes in glycosylation associated with Siglec-1-dependent inflammatory responses.

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 Siglec­1 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.