Modulation of immune cell reactivity with cis-binding Siglec agonists.

Inflammatory pathologies caused by phagocytes lead to numerous debilitating conditions, including chronic pain and blindness due to age-related macular degeneration. Many members of the sialic acid-binding immunoglobulin-like lectin (Siglec) family are immunoinhibitory receptors whose agonism is an attractive approach for antiinflammatory therapy. Here, we show that synthetic lipid-conjugated glycopolypeptides can insert into cell membranes and engage Siglec receptors in cis, leading to inhibitory signaling. Specifically, we construct a cis-binding agonist of Siglec-9 and show that it modulates mitogen-activated protein kinase (MAPK) signaling in reporter cell lines, immortalized macrophage and microglial cell lines, and primary human macrophages. Thus, these cis-binding agonists of Siglecs present a method for therapeutic suppression of immune cell reactivity.

Current Status on Therapeutic Molecules Targeting Siglec Receptors.

The sialic acid-binding immunoglobulin-type of lectins (Siglecs) are receptors that recognize sialic acid-containing glycans. In the majority of the cases, Siglecs are expressed on immune cells and play a critical role in regulating immune cell signaling. Over the years, it has been shown that the sialic acid-Siglec axis participates in immunological homeostasis, and that any imbalance can trigger different pathologies, such as autoimmune diseases or cancer. For all this, different therapeutics have been developed that bind to Siglecs, either based on antibodies or being smaller molecules. In this review, we briefly introduce the Siglec family and we compile a description of glycan-based molecules and antibody-based therapies (including CAR-T and bispecific antibodies) that have been designed to therapeutically targeting Siglecs.

Phosphoproteomics identifies microglial Siglec-F inflammatory response during neurodegeneration.

Alzheimer's disease (AD) is characterized by the appearance of amyloid-ß plaques, neurofibrillary tangles, and inflammation in brain regions involved in memory. Using mass spectrometry, we have quantified the phosphoproteome of the CK-p25, 5XFAD, and Tau P301S mouse models of neurodegeneration. We identified a shared response involving Siglec-F which was upregulated on a subset of reactive microglia. The human paralog Siglec-8 was also upregulated on microglia in AD. Siglec-F and Siglec-8 were upregulated following microglial activation with interferon gamma (IFNγ) in BV-2 cell line and human stem cell-derived microglia models. Siglec-F overexpression activates an endocytic and pyroptotic inflammatory response in BV-2 cells, dependent on its sialic acid substrates and immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation sites. Related human Siglecs induced a similar response in BV-2 cells. Collectively, our results point to an important role for mouse Siglec-F and human Siglec-8 in regulating microglial activation during neurodegeneration.

A versatile soluble siglec scaffold for sensitive and quantitative detection of glycan ligands.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. The connections between Siglecs and human disease motivate improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively. Using these Siglec-Fc proteins, Siglec ligands are systematically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. Additional features enable the production of small, homogenous Siglec fragments and development of a quantitative ligand-binding mass spectrometry assay. Using this assay, the ligand specificities of several Siglecs are clarified. For CD33 (Siglec-3), we demonstrate that it recognizes both α2-3 and α2-6 sialosides in solution and on cells, which has implications for its link to Alzheimer's disease susceptibility. These soluble Siglecs reveal the abundance of their glycan ligands on host cells as self-associated molecular patterns.

Targeted glycan degradation potentiates the anticancer immune response in vivo.

Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody-sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody-sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.

The Roles of Siglec7 and Siglec9 on Natural Killer Cells in Virus Infection and Tumour Progression.

The function of natural killer (NK) cells, defending against virus infection and tumour progression, is regulated by multiple activating and inhibiting receptors expressed on NK cells, among which sialic acid-bind immunoglobulin-like lectins (Siglecs) act as a vital inhibitory group. Previous studies have shown that Siglec7 and Siglec9 are expressed on NK cells, which negatively regulate the function of NK cells and modulate the immune response through the interaction of sialic acid-containing ligands. Siglec7 and Siglec9 are very similar in distribution, gene encoding, protein sequences, ligand affinity, and functions in regulating the immune system against virus and cancers, but differences still exist between them. In this review, we aim to discuss the similarities and differences between Siglec7 and Siglec9 and analyze their functions in virus infection and tumour progression in order to develop better anti-viral and anti-tumor immunotherapy in the future.

Potential Roles of Siglecs in the Regulation of Allo-Immune Reaction.

Siglecs are mammalian sialic acid (Sia) recognizing immuno-globulin-like receptors expressed across the major leukocyte lineages, and function to recognize ubiquitous Sia epitopes on the cell surface. Many Siglecs are inhibitory receptors expressed on innate immune cells, they also have a role in maintaining B cell tolerance as well as modulating the activation of conventional and plasmocytic dendritic cells. Through these and other roles they contribute directly and indirectly to the regulation of T cell function. Siglecs have been identified to play key roles in several forms of blood cancers, autoimmune and infection deceases. So far as we know, there's no Siglecs related research works on solid organ transplantation. In this review, we describe our understanding of the potential roles of Siglecs in the regulation of immune cell function, which may be crosslinked to allo-rejection and ischemia-reperfusion injury.

The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins.

Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.

Glycan Chains of Gangliosides: Functional Ligands for Tissue Lectins (Siglecs/Galectins).

Molecular signals on the cell surface are responsible for adhesion and communication. Of relevance in this respect, their chemical properties endow carbohydrates with the capacity to store a maximum of information in a minimum of space. One way to present glycans on the cell surface is their covalent conjugation to a ceramide anchor. Among the resulting glycosphingolipids, gangliosides are special due to the presence of at least one sialic acid in the glycan chains. Their spatial accessibility and the dynamic regulation of their profile are factors that argue in favor of a role of glycans of gangliosides as ligands (counterreceptors) for carbohydrate-binding proteins (lectins). Indeed, as discovered first for a bacterial toxin, tissue lectins bind gangliosides and mediate contact formation (trans) and signaling (cis). While siglecs have a preference for higher sialylated glycans, certain galectins also target the monosialylated pentasaccharide of ganglioside GM1. Enzymatic interconversion of ganglioside glycans by sialidase action, relevant for neuroblastoma cell differentiation and growth control in vitro, for axonogenesis and axon regeneration, as well as for proper communication between effector and regulatory T cells, changes lectin-binding affinity profoundly. The GD1a-to-GM1 "editing" is recognized by such lectins, for example, myelin-associated glycoprotein (siglec-4) losing affinity and galectin-1 gaining reactivity, and then translated into postbinding signaling. Orchestrations of loss/gain of affinity, of ganglioside/lectin expression, and of lectin presence in a network offer ample opportunities for fine-tuning. Thus glycans of gangliosides such as GD1a and GM1 are functional counterreceptors by a pairing with tissue lectins, an emerging aspect of ganglioside and lectin functionality.

Sialylation of N-glycans: mechanism, cellular compartmentalization and function.

Sialylated N-glycans play essential roles in the immune system, pathogen recognition and cancer. This review approaches the sialylation of N-glycans from three perspectives. The first section focuses on the sialyltransferases that add sialic acid to N-glycans. Included in the discussion is a description of these enzymes' glycan acceptors, conserved domain organization and sequences, molecular structure and catalytic mechanism. In addition, we discuss the protein interactions underlying the polysialylation of a select group of adhesion and signaling molecules. In the second section, the biosynthesis of sialic acid, CMP-sialic acid and sialylated N-glycans is discussed, with a special emphasis on the compartmentalization of these processes in the mammalian cell. The sequences and mechanisms maintaining the sialyltransferases and other glycosylation enzymes in the Golgi are also reviewed. In the final section, we have chosen to discuss processes in which sialylated glycans, both N- and O-linked, play a role. The first part of this section focuses on sialic acid-binding proteins including viral hemagglutinins, Siglecs and selectins. In the second half of this section, we comment on the role of sialylated N-glycans in cancer, including the roles of ß1-integrin and Fas receptor N-glycan sialylation in cancer cell survival and drug resistance, and the role of these sialylated proteins and polysialic acid in cancer metastasis.

Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E.

CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation. Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer.

Multivalent Interactions of Human Primary Amine Oxidase with the V and C22 Domains of Sialic Acid-Binding Immunoglobulin-Like Lectin-9 Regulate Its Binding and Amine Oxidase Activity.

Sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on leukocyte surface is a counter-receptor for endothelial cell surface adhesin, human primary amine oxidase (hAOC3), a target protein for anti-inflammatory agents. This interaction can be used to detect inflammation and cancer in vivo, since the labeled peptides derived from the second C2 domain (C22) of Siglec-9 specifically bind to the inflammation-inducible hAOC3. As limited knowledge on the interaction between Siglec-9 and hAOC3 has hampered both hAOC3-targeted drug design and in vivo imaging applications, we have now produced and purified the extracellular region of Siglec-9 (Siglec-9-EC) consisting of the V, C21 and C22 domains, modeled its 3D structure and characterized the hAOC3-Siglec-9 interactions using biophysical methods and activity/inhibition assays. Our results assign individual, previously unknown roles for the V and C22 domains. The V domain is responsible for the unusually tight Siglec-9-hAOC3 interactions whereas the intact C22 domain of Siglec-9 is required for modulating the enzymatic activity of hAOC3, crucial for the hAOC3-mediated leukocyte trafficking. By characterizing the Siglec-9-EC mutants, we could conclude that R120 in the V domain likely interacts with the terminal sialic acids of hAOC3 attached glycans whereas residues R284 and R290 in C22 are involved in the interactions with the active site channel of hAOC3. Furthermore, the C22 domain binding enhances the enzymatic activity of hAOC3 although the sialic acid-binding capacity of the V domain of Siglec-9 is abolished by the R120S mutation. To conclude, our results prove that the V and C22 domains of Siglec-9-EC interact with hAOC3 in a multifaceted and unique way, forming both glycan-mediated and direct protein-protein interactions, respectively. The reported results on the mechanism of the Siglec-9-hAOC3 interaction are valuable for the development of hAOC3-targeted therapeutics and diagnostic tools.

Glycome complexity of human seminal plasma high molecular mass components: Evaluation of the contribution of acid-soluble glycoproteins/mucins and extracellular vesicles.

This study was aimed at evaluation of the contribution of acid-soluble glycoproteins (ASG)/mucins and extracellular vesicles (EVs), yet unexplored components of human seminal plasma (hSP) to the complexity of its glycome. Gaining insight into the native presentation and distribution of glycans across hSP could help establish molecular environments supporting specific biological activities based on unique ligand capacities. Soluble and particulate fractions of hSP from healthy subjects were analyzed by gel filtration, electrophoresis, ion-exchange chromatography and a solid phase assay with immobilized charge-resolved glycospecies to test their reactivity with plant lectins, carbohydrate-binding antibodies and selected human lectins. Common O- and N-glycosylated species were detected on mixed or overlapped underlying protein scaffolds in both soluble and particulate fractions of hSP. Siaα2,6Gal and N-glycans were concentrated on EVs, whereas Siaα2,3Gal, T and Tn antigens were selectively associated with distinct glycospecies of ASG/mucins. Accessible ligands for the lectins, DC-SIGN and Siglec-9, were detected in all hSP components, but they preferentially bound to EVs glycospecies. Insight into the complexity of hSP glycans as recognition signals under normal physiological conditions could be of interest for regulation and possible modulation of its biological activity, as well as for biomarker potential related to male health.

Therapeutic Targeting of Siglecs using Antibody- and Glycan-Based Approaches.

The sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of immunomodulatory receptors whose functions are regulated by their glycan ligands. Siglecs are attractive therapeutic targets because of their cell type-specific expression pattern, endocytic properties, high expression on certain lymphomas/leukemias, and ability to modulate receptor signaling. Siglec-targeting approaches with therapeutic potential encompass antibody- and glycan-based strategies. Several antibody-based therapies are in clinical trials and continue to be developed for the treatment of lymphoma/leukemia and autoimmune disease, while the therapeutic potential of glycan-based strategies for cargo delivery and immunomodulation is a promising new approach. Here we review these strategies with special emphasis on emerging approaches and disease areas that may benefit from targeting the Siglec family.

A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D.

We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac)-galactose (Gal)/N-acetylgalactosamine (GalNAc) sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3)Gal, Neu5Ac(α2,6)GalNAc or Neu5Ac(α2,8)Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3)Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 µM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3)Gal or Neu5Ac(α2,6)Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C has characteristics of a checkpoint inhibitor.

Role of siglecs and related glycan-binding proteins in immune responses and immunoregulation.

Virtually all cells and extracellular material are heavily decorated by various glycans, yet our understanding of the structure and function of these moieties lags behind the understanding of nucleic acids, lipids, and proteins. Recent years have seen a tremendous acceleration of knowledge in the field of glycobiology, revealing many intricacies and functional contributions that were previously poorly appreciated or even unrecognized. This review highlights several topics relevant to glycoimmunology in which mammalian and pathogen-derived glycans displayed on glycoproteins and other scaffolds are recognized by specific glycan-binding proteins (GBPs), leading to a variety of proinflammatory and anti-inflammatory cellular responses. The focus for this review is mainly on 2 families of GBPs, sialic acid-binding immunoglobulin-like lectins (siglecs) and selectins, that are involved in multiple steps of the immune response, including distinguishing pathogens from self, cell trafficking to sites of inflammation, fine-tuning of immune responses leading to activation or tolerance, and regulation of cell survival. Importantly for the clinician, accelerated rates of discovery in the field of glycoimmunology are being translated into innovative medical approaches that harness the interaction of glycans and GBPs to the benefit of the host and might soon lead to novel diagnostics and therapeutics.

PILRα and PILRß have a siglec fold and provide the basis of binding to sialic acid.

Paired immunoglobulin-like type 2 receptor α (PILRα) and ß (PILRß) belong to the PILR family and are related to innate immune regulation in various species. Despite their high sequence identity, PILRα and PILRß are shown to have variant sialic acid (SA) binding avidities. To explore the molecular basis of this interaction, we solved the crystal structures of PILRα and PILRß at resolutions of 1.6 Å and 2.2 Å, respectively. Both molecules adopt a typical siglec fold but use a hydrophobic bond to substitute the siglec-specific disulfide linkage for protein stabilization. We further used HSV-1 glycoprotein B (gB) as a representative molecule to study the PILR-SA interaction. Deploying site-directed mutagenesis, we demonstrated that three residues (Y2, R95, and W108) presented on the surface of PILRα form the SA binding site equivalent to those in siglecs but are arranged in a unique linear mode. PILRß differs from PILRα in one of these three residues (L108), explaining its inability to engage gB. Mutation of L108 to tryptophan in PILRß restored the gB-binding capacity. We further solved the structure of this PILRß mutant complexed with SA, which reveals the atomic details mediating PILR/SA recognition. In comparison with the free PILR structures, amino acid Y2 oriented variantly in the complex structure, thereby disrupting the linear arrangement of PILR residues Y2, R95, and W108. In conclusion, our study provides significant implications for the PILR-SA interaction and paves the way for understanding PILR-related ligand binding.

Translating the concept of peptide labeling with 5-deoxy-5-[18F]fluororibose into preclinical practice: 18F-labeling of Siglec-9 peptide for PET imaging of inflammation.

Peptide glycosylation with 5-deoxy-5-[(18)F]fluororibose was translated into preclinical settings. The novel (18)F-labeled Siglec-9 peptide was produced using an automated synthesis procedure. The (18)F-labeled Siglec-9 peptide showed favorable binding in the animal model of inflammation in vivo.