An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic, and structural basis for glycan binding in a natural context. The cell-based glycan array is self-renewable and reports glycosyltransferase genes required (or blocking) for interactions through logical sequential biosynthetic steps, which is predictive of structural glycan features involved and provides instructions for synthesis, recombinant production, and genetic dissection strategies. Broad utility of the cell-based glycan array is demonstrated, and we uncover higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.

Tumor cell-intrinsic and tumor microenvironmental conditions co-determine signaling by the glycoimmune checkpoint receptor Siglec-7.

Tumors create an immunosuppressive tumor microenvironment by altering protein expression, but also by changing their glycosylation status, like altered expression of sialoglycans. Sialoglycans are capped with sialic acid sugar residues and are recognized by Siglec immune receptors. Siglec-7 is an inhibitory immune receptor similar to PD-1, and is emerging as glycoimmune checkpoint exploited by cancer cells to evade the immune system. However, the exact cellular and molecular conditions required for Siglec-7-mediated immune cell inhibition remain largely unknown. Here, we report on the development of a chimeric Siglec-7 cell system that enables dissection of Siglec-7 signaling, rather than Siglec-7 binding. Antibody-induced clustering, sialic acid-containing polymers, and highly sialylated erythrocytes effectively induced Siglec-7 signaling, thereby validating functionality of this reporter system. Moreover, the system reveals tumor cell-dependent Siglec-7 signaling. Tumor-associated conditions important for Siglec-7 signaling were defined, such as Siglec-7 ligand expression levels, presence of the known Siglec-7 ligand CD43, and sialic acid availability for sialylation of glycans. Importantly, therapeutic targeting of the Siglec-7/sialic acid axis using a sialyltransferase inhibitor resulted in strong reduction of Siglec-7 signaling. In conclusion, using a newly established cellular tool, we defined a set of tumor-associated conditions that influence Siglec-7 signaling. Moreover, the system allows to assess the efficacy of novel cancer drugs interfering with the Siglec-7/sialic acid axis as immunotherapy to treat cancer.

Probing the binding specificities of human Siglecs by cell-based glycan arrays.

Siglecs are a family of sialic acid-binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2-3(6-O-sulfo)Galß1-4GlcNAc (6'-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer's disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid-binding proteins.

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins.

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid-binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galß1-3GalNAcα1-O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.

Targeting aberrant sialylation and fucosylation in prostate cancer cells using potent metabolic inhibitors.

Aberrant glycosylation is a hallmark of cancer and is not just a consequence, but also a driver of a malignant phenotype. In prostate cancer, changes in fucosylated and sialylated glycans are common and this has important implications for tumor progression, metastasis, and immune evasion. Glycans hold huge translational potential and new therapies targeting tumor-associated glycans are currently being tested in clinical trials for several tumor types. Inhibitors targeting fucosylation and sialylation have been developed and show promise for cancer treatment, but translational development is hampered by safety issues related to systemic adverse effects. Recently, potent metabolic inhibitors of sialylation and fucosylation were designed that reach higher effective concentrations within the cell, thereby rendering them useful tools to study sialylation and fucosylation as potential candidates for therapeutic testing. Here, we investigated the effects of global metabolic inhibitors of fucosylation and sialylation in the context of prostate cancer progression. We find that these inhibitors effectively shut down the synthesis of sialylated and fucosylated glycans to remodel the prostate cancer glycome with only minor apparent side effects on other glycan types. Our results demonstrate that treatment with inhibitors targeting fucosylation or sialylation decreases prostate cancer cell growth and downregulates the expression of genes and proteins important in the trajectory of disease progression. We anticipate our findings will lead to the broader use of metabolic inhibitors to explore the role of fucosylated and sialylated glycans in prostate tumor pathology and may pave the way for the development of new therapies for prostate cancer.

Sialoglycans and Siglecs Can Shape the Tumor Immune Microenvironment.

Sialic acid sugar-carrying glycans, sialoglycans, are aberrantly expressed on many tumor cells and have emerged as potent regulatory molecules involved in creating a tumor-supportive microenvironment. Sialoglycans can be recognized by sialic acid-binding immunoglobulin-like lectins (Siglecs), a family of immunomodulatory receptors. Most mammalian Siglecs transmit inhibitory signals comparable with the immune checkpoint inhibitor programmed death protein 1 (PD-1), but some are activating. Recent studies have shown that tumor cells can exploit sialoglycan-Siglec interactions to modulate immune cell function, contributing to an immunosuppressive tumor microenvironment (TME). Interference with sialoglycan synthesis or sialoglycan-Siglec interactions might improve antitumor immunity. Many questions regarding specificity, signaling, and regulatory function of sialoglycan-Siglec interactions remain. We posit that sialoglycans and Siglecs present as potential glyco-immune 'checkpoints' for cancer immunotherapy.

Genetic glycoengineering in mammalian cells.

Advances in nuclease-based gene-editing technologies have enabled precise, stable, and systematic genetic engineering of glycosylation capacities in mammalian cells, opening up a plethora of opportunities for studying the glycome and exploiting glycans in biomedicine. Glycoengineering using chemical, enzymatic, and genetic approaches has a long history, and precise gene editing provides a nearly unlimited playground for stable engineering of glycosylation in mammalian cells to explore and dissect the glycome and its many biological functions. Genetic engineering of glycosylation in cells also brings studies of the glycome to the single cell level and opens up wider use and integration of data in traditional omics workflows in cell biology. The last few years have seen new applications of glycoengineering in mammalian cells with perspectives for wider use in basic and applied glycosciences, and these have already led to discoveries of functions of glycans and improved designs of glycoprotein therapeutics. Here, we review the current state of the art of genetic glycoengineering in mammalian cells and highlight emerging opportunities.

Sialic acid O-acetylation: From biosynthesis to roles in health and disease.

Sialic acids are nine-carbon sugars that frequently cap glycans at the cell surface in cells of vertebrates as well as cells of certain types of invertebrates and bacteria. The nine-carbon backbone of sialic acids can undergo extensive enzymatic modification in nature and O-acetylation at the C-4/7/8/9 position in particular is widely observed. In recent years, the detection and analysis of O-acetylated sialic acids have advanced, and sialic acid-specific O-acetyltransferases (SOATs) and O-acetylesterases (SIAEs) that add and remove O-acetyl groups, respectively, have been identified and characterized in mammalian cells, invertebrates, bacteria, and viruses. These advances now allow us to draw a more complete picture of the biosynthetic pathway of the diverse O-acetylated sialic acids to drive the generation of genetically and biochemically engineered model cell lines and organisms with altered expression of O-acetylated sialic acids for dissection of their roles in glycoprotein stability, development, and immune recognition, as well as discovery of novel functions. Furthermore, a growing number of studies associate sialic acid O-acetylation with cancer, autoimmunity, and infection, providing rationale for the development of selective probes and inhibitors of SOATs and SIAEs. Here, we discuss the current insights into the biosynthesis and biological functions of O-acetylated sialic acids and review the evidence linking this modification to disease. Furthermore, we discuss emerging strategies for the design, synthesis, and potential application of unnatural O-acetylated sialic acids and inhibitors of SOATs and SIAEs that may enable therapeutic targeting of this versatile sialic acid modification.

Dynamic tracing of sugar metabolism reveals the mechanisms of action of synthetic sugar analogs.

Synthetic sugar analogs are widely applied in metabolic oligosaccharide engineering (MOE) and as novel drugs to interfere with glycoconjugate biosynthesis. However, mechanistic insights on their exact cellular metabolism over time are mostly lacking. We combined ion-pair ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry mass spectrometry using tributyl- and triethylamine buffers for sensitive analysis of sugar metabolites in cells and organisms and identified low abundant nucleotide sugars, such as UDP-arabinose in human cell lines and CMP-sialic acid (CMP-NeuNAc) in Drosophila. Furthermore, MOE revealed that propargyloxycarbonyl (Poc)-labeled ManNPoc was metabolized to both CMP-NeuNPoc and UDP-GlcNPoc. Finally, time-course analysis of the effect of antitumor compound 3Fax-NeuNAc by incubation of B16-F10 melanoma cells with N-acetyl-D-[UL-13C6]glucosamine revealed full depletion of endogenous ManNAc 6-phosphate and CMP-NeuNAc within 24 h. Thus, dynamic tracing of sugar metabolic pathways provides a general approach to reveal time-dependent insights into the metabolism of synthetic sugars, which is important for the rational design of analogs with optimized effects.

Cellular Fucosylation Inhibitors Based on Fluorinated Fucose-1-phosphates*.

Fucosylation of glycans impacts a myriad of physiological and pathological processes. Inhibition of fucose expression emerges as a potential therapeutic avenue for example in cancer, inflammation, and infection. In this study, we found that protected 2-fluorofucose 1-phosphate efficiently inhibits cellular fucosylation with a four to seven times higher potency than known inhibitor 2FF, independently of the anomeric stereochemistry. Nucleotide sugar analysis revealed that both the α- and ß-GDP-2FF anomers are formed inside the cell. In conclusion, we developed A2FF1P and B2FF1P as potent new tools for studying the role of fucosylation in health and disease and they are potential therapeutic candidates.

Sialic Acid Mimetics to Target the Sialic Acid-Siglec Axis.

Sialic acid sugars are vital regulators of the immune system through binding to immunosuppressive sialic acid-binding immunoglobulin-like lectin (Siglec) receptors on immune cells. Aberrant sialic acid-Siglec interactions are associated with an increasing number of pathologies including infection, autoimmunity, and cancer. Therefore, the sialic acid-Siglec axis is an emerging target to prevent or affect the course of several diseases. Chemical modifications of the natural sialic acid ligands have led to sialic acid mimetics (SAMs) with improved binding affinity and selectivity towards Siglecs. Recent progress in glycobiotechnology allows the presentation of these SAMs on nanoparticles, polymers, and living cells via bioorthogonal synthesis. These developments now enable the detailed study of the sialic acid-Siglec axis including its therapeutic potential as an immune modulator.

Combined sialic acid and histone deacetylase (HDAC) inhibitor treatment up-regulates the neuroblastoma antigen GD2.

Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is only sparsely expressed on healthy tissue. GD2 is a primary target for the development of immunotherapy for neuroblastoma. Immunotherapy with monoclonal anti-GD2 antibodies has proven safety and efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. Strategies to modulate GD2 expression in neuroblastoma could further improve anti-GD2-targeted immunotherapy. Here, we report that the cellular sialylation pathway, as well as epigenetic reprogramming, strongly modulates GD2 expression in human and mouse neuroblastoma cell lines. Recognition of GD2 by the 14G2a antibody is sialic acid-dependent and was blocked with the fluorinated sialic acid mimetic Ac53FaxNeu5Ac. Interestingly, sialic acid supplementation using a cell-permeable sialic acid analogue (Ac5Neu5Ac) boosted GD2 expression without or with minor alterations in overall cell surface sialylation. Furthermore, sialic acid supplementation with Ac5Neu5Ac combined with various histone deacetylase (HDAC) inhibitors, including vorinostat, enhanced GD2 expression in neuroblastoma cells beyond their individual effects. Mechanistic studies revealed that Ac5Neu5Ac supplementation increased intracellular CMP-Neu5Ac concentrations, thereby providing higher substrate levels for sialyltransferases. Furthermore, HDAC inhibitor treatment increased mRNA expression of the sialyltransferases GM3 synthase (ST3GAL5) and GD3 synthase (ST8SIA1), both of which are involved in GD2 biosynthesis. Our findings reveal that sialic acid analogues and HDAC inhibitors enhance GD2 expression and could potentially be employed to boost anti-GD2 targeted immunotherapy in neuroblastoma patients.

Sialic acid glycoengineering using N-acetylmannosamine and sialic acid analogs.

Sialic acids cap the glycans of cell surface glycoproteins and glycolipids. They are involved in a multitude of biological processes and aberrant sialic acid expression is associated with several pathologies. Sialic acids modulate the characteristics and functions of glycoproteins and regulate cell-cell as well as cell-extracellular matrix interactions. Pathogens such as influenza virus use sialic acids to infect host cells and cancer cells exploit sialic acids to escape from the host's immune system. The introduction of unnatural sialic acids with different functionalities into surface glycans enables the study of the broad biological functions of these sugars and presents a therapeutic option to intervene with pathological processes involving sialic acids. Multiple chemically modified sialic acid analogs can be directly utilized by cells for sialoglycan synthesis. Alternatively, analogs of the natural sialic acid precursor sugar N-Acetylmannosamine (ManNAc) can be introduced into the sialic acid biosynthesis pathway resulting in the intracellular conversion into the corresponding sialic acid analog. Both, ManNAc and sialic acid analogs, have been employed successfully for a large variety of glycoengineering applications such as glycan imaging, targeting toxins to tumor cells, inhibiting pathogen binding, or altering immune cell activity. However, there are significant differences between ManNAc and sialic acid analogs with respect to their chemical modification potential and cellular metabolism that should be considered in sialic acid glycoengineering experiments.

Expression profiling of immune inhibitory Siglecs and their ligands in patients with glioma.

Gliomas appear to be highly immunosuppressive tumors, with a strong myeloid component. This includes MDSCs, which are a heterogeneous, immature myeloid cell population expressing myeloid markers Siglec-3 (CD33) and CD11b and lacking markers of mature myeloid cells including MHC II. Siglec-3 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family and has been suggested to promote MDSC expansion and suppression. Siglecs form a recently defined family of receptors with potential immunoregulatory functions but only limited insight in their expression on immune regulatory cell subsets, prompting us to investigate Siglec expression on MDSCs. We determined the expression of different Siglec family members on monocytic-MDSCs (M-MDSCs) and polymorphnuclear-MDSCs (PMN-MDSCs) from blood of glioma patients and healthy donors, as well as from patient-derived tumor material. Furthermore, we investigated the presence of sialic acid ligands for these Siglecs on MDSCs and in the glioma tumor microenvironment. Both MDSC subsets express Siglec-3, -5, -7 and -9, with higher levels of Siglec-3, -7 and -9 on M-MDSCs and higher Siglec-5 levels on PMN-MDSCs. Similar Siglec expression profiles were found on MDSCs from healthy donors. Furthermore, the presence of Siglec-5 and -9 was also confirmed on PMN-MDSCs from glioma tissue. Interestingly, freshly isolated glioma cells predominantly expressed sialic acid ligands for Siglec-7 and -9, which was confirmed in situ. In conclusion, our data show a distinct Siglec expression profile for M- and PMN-MDSCs and propose possible sialic acid-Siglec interactions between glioma cells and MDSCs in the tumor microenvironment.

Desialylation of Platelets by Pneumococcal Neuraminidase A Induces ADP-Dependent Platelet Hyperreactivity.

Platelets are increasingly recognized to play a role in the complications of Streptococcus pneumoniae infections. S. pneumoniae expresses neuraminidases, which may alter glycans on the platelet surface. In the present study, we investigated the capability of pneumococcal neuraminidase A (NanA) to remove sialic acid (desialylation) from the platelet surface, the consequences for the platelet activation status and reactivity, and the ability of neuraminidase inhibitors to prevent these effects. Our results show that soluble NanA induces platelet desialylation. Whereas desialylation itself did not induce platelet activation (P-selectin expression and platelet fibrinogen binding), platelets became hyperreactive to ex vivo stimulation by ADP and cross-linked collagen-related peptide (CRP-XL). Platelet aggregation with leukocytes also increased. These processes were dependent on the ADP pathway, as inhibitors of the pathway (apyrase and ticagrelor) abrogated platelet hyperreactivity. Inhibition of NanA-induced platelet desialylation by neuraminidase inhibitors (e.g., oseltamivir acid) also prevented the platelet effects of NanA. Collectively, our findings show that soluble NanA can desialylate platelets, leading to platelet hyperreactivity, which can be prevented by neuraminidase inhibitors.

The Epstein-Barr Virus Glycoprotein gp150 Forms an Immune-Evasive Glycan Shield at the Surface of Infected Cells.

Cell-mediated immunity plays a key role in host control of viral infection. This is exemplified by life-threatening reactivations of e.g. herpesviruses in individuals with impaired T-cell and/or iNKT cell responses. To allow lifelong persistence and virus production in the face of primed immunity, herpesviruses exploit immune evasion strategies. These include a reduction in viral antigen expression during latency and a number of escape mechanisms that target antigen presentation pathways. Given the plethora of foreign antigens expressed in virus-producing cells, herpesviruses are conceivably most vulnerable to elimination by cell-mediated immunity during the replicative phase of infection. Here, we show that a prototypic herpesvirus, Epstein-Barr virus (EBV), encodes a novel, broadly acting immunoevasin, gp150, that is expressed during the late phase of viral replication. In particular, EBV gp150 inhibits antigen presentation by HLA class I, HLA class II, and the non-classical, lipid-presenting CD1d molecules. The mechanism of gp150-mediated T-cell escape does not depend on degradation of the antigen-presenting molecules nor does it require gp150's cytoplasmic tail. Through its abundant glycosylation, gp150 creates a shield that impedes surface presentation of antigen. This is an unprecedented immune evasion mechanism for herpesviruses. In view of its likely broader target range, gp150 could additionally have an impact beyond escape of T cell activation. Importantly, B cells infected with a gp150-null mutant EBV displayed rescued levels of surface antigen presentation by HLA class I, HLA class II, and CD1d, supporting an important role for iNKT cells next to classical T cells in fighting EBV infection. At the same time, our results indicate that EBV gp150 prolongs the timespan for producing viral offspring at the most vulnerable stage of the viral life cycle.

Disease mutations in CMP-sialic acid transporter SLC35A1 result in abnormal α-dystroglycan O-mannosylation, independent from sialic acid.

Binding of cellular α-dystroglycan (α-DG) to its extracellular matrix ligands is fully dependent on a unique O-mannose-linked glycan. Disrupted O-mannosylation is the hallmark of the muscular dystrophy-dystroglycanopathy (MDDG) syndromes. SLC35A1, encoding the transporter of cytidine 5'-monophosphate-sialic acid, was recently identified as MDDG candidate gene. This is surprising, since sialic acid itself is dispensable for α-DG-ligand binding. In a novel SLC35A1-deficient cell model, we demonstrated a lack of α-DG O-mannosylation, ligand binding and incorporation of sialic acids. Removal of sialic acids from HAP1 wild-type cells after incorporation or preventing sialylation during synthesis did not affect α-DG O-mannosylation or ligand binding but did affect sialylation. Lentiviral-mediated complementation with the only known disease mutation p.Q101H failed to restore deficient O-mannosylation in SLC35A1 knockout cells and partly restored sialylation. These data indicate a role for SLC35A1 in α-DG O-mannosylation that is distinct from sialic acid metabolism. In addition, human SLC35A1 deficiency can be considered as a combined disorder of α-DG O-mannosylation and sialylation, a novel variant of the MDDG syndromes.

Metabolic sialic acid blockade lowers the activation threshold of moDCs for TLR stimulation.

Sialic acid sugars cover the surface of dendritic cells (DCs) and have been suggested to impact several aspects of DC biology. Research into the role of sialic acids in DCs, however, is complicated by the limited number of tools available to modulate sialic acid expression. Here we report on a synthetic, fluorinated sialic acid mimetic, Ac53FaxNeu5Ac, which potently blocks sialic acid expression in human monocyte-derived DCs (moDCs). Sialic acid blockade enhanced the responsiveness of moDCs to Toll-like receptor (TLR) stimulation as measured by increased maturation marker expression and cytokine production. Consequently, the T-cell activation capacity of Ac53FaxNeu5Ac-treated moDCs was strongly increased. In addition to sialic acids, moDCs also expressed the sialic acid-binding immunoglobulin-like lectins (Siglecs) -3, -5, -7, -9 and -10, immune inhibitory receptors recognizing these sialic acids. Treatment with Ac53FaxNeu5Ac abrogated putative cis and trans interactions between sialic acids and Siglec-7/-9. Together, these data indicate that sialic acids limit the activation of moDCs via the TLR pathway, potentially by interacting with Siglec-7 or Siglec-9. Metabolic sialic acid blockade with Ac53FaxNeu5Ac could therefore potentially be used to generate more potent DC-based vaccines for induction of robust anti-viral or anti-tumor immune responses.

Metabolic Oligosaccharide Engineering with Alkyne Sialic Acids Confers Neuraminidase Resistance and Inhibits Influenza Reproduction.

Metabolic incorporation of azide- or alkyne-modified sialic acids into the cellular glycosylation pathway enables the study of sialoglycan expression, localization, and trafficking via bioorthogonal chemistry. Herein, we report that such modifications of the sialic acid sugar can have a profound influence on their hydrolysis by neuraminidases (sialidase). Azidoacetyl (Az)-modified sialic acids were prone to neuraminidase cleavage, whereas propargyloxycarbonyl (Poc)-modified sialic acids were largely resistant to cleavage. Because the influenza virus infection cycle depends on the hydrolysis of host-cell-surface sialic acids, influenza cell-to-cell transmission was strongly reduced in Poc sialic acid glycoengineered host cells. The use of Poc sialic acids may disturb biological processes involving neuraminidase cleavage but also provides perspective for use in applications in which sialic acid hydrolysis is not desired, such as antibody modification, viral infection, etc.

Steering Siglec-Sialic Acid Interactions on Living Cells using Bioorthogonal Chemistry.

Sialic acid sugars that terminate cell-surface glycans form the ligands for the sialic acid binding immunoglobulin-like lectin (Siglec) family, which are immunomodulatory receptors expressed by immune cells. Interactions between sialic acid and Siglecs regulate the immune system, and aberrations contribute to pathologies like autoimmunity and cancer. Sialic acid/Siglec interactions between living cells are difficult to study owing to a lack of specific tools. Here, we report a glycoengineering approach to remodel the sialic acids of living cells and their binding to Siglecs. Using bioorthogonal chemistry, a library of cells with more than sixty different sialic acid modifications was generated that showed dramatically increased binding toward the different Siglec family members. Rational design reduced cross-reactivity and led to the discovery of three selective Siglec-5/14 ligands. Furthermore, glycoengineered cells carrying sialic acid ligands for Siglec-3 dampened the activation of Siglec-3+ monocytic cells through the NF-κB and IRF pathways.