Targeting Inhibitory Siglec-3 to Suppress IgE-Mediated Human Basophil Degranulation.

BACKGROUND: Siglec-3 (CD33) is a major Siglec expressed on human mast cells and basophils and engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs (ITIMs). OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BAT) were used to assess both antigen-specific (peanut) and antigen non-specific (polyclonal anti-IgE) stimulation. Whole blood from allergic donors was used for direct BAT, whereas non-food allergic donor blood was passively sensitized with peanut-allergic plasma in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for one hour or overnight, then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine (fMLP) for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for one hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to fMLP, providing evidence that this inhibition is IgE-pathway specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. CONCLUSIONS: Pre-treating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L prior to antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

Advances in understanding and exploiting Siglec-glycan interactions.

Sialic-acid-binding immunoglobulin-type lectins (Siglecs) are a family of cell-surface immunomodulatory receptors that recognize sialic-acid-containing glycans. The majority of Siglecs have an inhibitory motif in their intercellular domain and can regulate the cellular activation of immune cells. Importantly, the immunomodulatory role of Siglecs is regulated by engagement with distinct sialoglycan ligands. However, there are still many unanswered questions about the precise ligand(s) recognized by individual Siglec family members. New tools and approaches to study Siglec-ligand interactions are rapidly filling this knowledge gap. This review provides an overview of recent advances in discovering Siglec ligands as well as the development of approaches to modulate the function of Siglecs. In both aspects, chemical biology approaches are emphasized with a discussion on how these are complementing biochemical and genetic strategies.

Molecular Insights into O-Linked Sialoglycans Recognition by the Siglec-Like SLBR-N (SLBRUB10712) of Streptococcus gordonii.

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3'SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections.

Dissecting the Ability of Siglecs To Antagonize Fcγ Receptors.

Fcγ receptors (FcγRs) play key roles in the effector function of IgG, but their inappropriate activation plays a role in several disease etiologies. Therefore, it is critical to better understand how FcγRs are regulated. Numerous studies suggest that sialic acid-binding immunoglobulin-type lectins (Siglecs), a family of immunomodulatory receptors, modulate FcγR activity; however, it is unclear of the circumstances in which Siglecs can antagonize FcγRs and which Siglecs have this ability. Using liposomes displaying selective ligands to coengage FcγRs with a specific Siglec, we explore the ability of Siglec-3, Siglec-5, Siglec-7, and Siglec-9 to antagonize signaling downstream of FcγRs. We demonstrate that Siglec-3 and Siglec-9 can fully inhibit FcγR activation in U937 cells when coengaged with FcγRs. Cells expressing Siglec mutants reveal differential roles for the immunomodulatory tyrosine-based inhibitory motif (ITIM) and immunomodulatory tyrosine-based switch motif (ITSM) in this inhibition. Imaging flow cytometry enabled visualization of SHP-1 recruitment to Siglec-3 in an ITIM-dependent manner, while SHP-2 recruitment is more ITSM-dependent. Conversely, both cytosolic motifs of Siglec-9 contribute to SHP-1/2 recruitment. Siglec-7 poorly antagonizes FcγR activation for two reasons: masking by cis ligands and differences in its ITIM and ITSM. A chimera of the Siglec-3 extracellular domains and Siglec-5 cytosolic tail strongly inhibits FcγR when coengaged, providing evidence that Siglec-5 is more like Siglec-3 and Siglec-9 in its ability to antagonize FcγRs. Additionally, Siglec-3 and Siglec-9 inhibited FcγRs when coengaged by cells displaying ligands for both the Siglec and FcγRs. These results suggest a role for Siglecs in mediating FcγR inhibition in the context of an immunological synapse, which has important relevance to the effectiveness of immunotherapies.

Genetically encoded discovery of perfluoroaryl macrocycles that bind to albumin and exhibit extended circulation in vivo.

Peptide-based therapeutics have gained attention as promising therapeutic modalities, however, their prevalent drawback is poor circulation half-life in vivo. In this paper, we report the selection of albumin-binding macrocyclic peptides from genetically encoded libraries of peptides modified by perfluoroaryl-cysteine SNAr chemistry, with decafluoro-diphenylsulfone (DFS). Testing of the binding of the selected peptides to albumin identified SICRFFC as the lead sequence. We replaced DFS with isosteric pentafluorophenyl sulfide (PFS) and the PFS-SICRFFCGG exhibited KD = 4-6 µM towards human serum albumin. When injected in mice, the concentration of the PFS-SICRFFCGG in plasma was indistinguishable from the reference peptide, SA-21. More importantly, a conjugate of PFS-SICRFFCGG and peptide apelin-17 analogue (N3-PEG6-NMe17A2) showed retention in circulation similar to SA-21; in contrast, apelin-17 analogue was cleared from the circulation after 2 min. The PFS-SICRFFC is the smallest known peptide macrocycle with a significant affinity for human albumin and substantial in vivo circulation half-life. It is a productive starting point for future development of compact macrocycles with extended half-life in vivo.

Chemoenzymatic synthesis of genetically-encoded multivalent liquid N-glycan arrays.

Cellular glycosylation is characterized by chemical complexity and heterogeneity, which is challenging to reproduce synthetically. Here we show chemoenzymatic synthesis on phage to produce a genetically-encoded liquid glycan array (LiGA) of complex type N-glycans. Implementing the approach involved by ligating an azide-containing sialylglycosyl-asparagine to phage functionalized with 50-1000 copies of dibenzocyclooctyne. The resulting intermediate can be trimmed by glycosidases and extended by glycosyltransferases yielding a phage library with different N-glycans. Post-reaction analysis by MALDI-TOF MS allows rigorous characterization of N-glycan structure and mean density, which are both encoded in the phage DNA. Use of this LiGA with fifteen glycan-binding proteins, including CD22 or DC-SIGN on cells, reveals optimal structure/density combinations for recognition. Injection of the LiGA into mice identifies glycoconjugates with structures and avidity necessary for enrichment in specific organs. This work provides a quantitative evaluation of the interaction of complex N-glycans with GBPs in vitro and in vivo.

Disrupting Protein Expression with Double-Clicked sgRNA-Cas9 Complexes: A Modular Approach to CRISPR Gene Editing.

CRISPR-Cas9 is currently the most versatile technique to perform gene editing in living organisms. In this approach, the Cas9 endonuclease is guided toward its DNA target sequence by the guide RNA (gRNA). Chemical synthesis of a functional single gRNA (sgRNA) is nontrivial because of the length of the RNA strand. Recently we demonstrated that a sgRNA can be stitched together from three smaller fragments through a copper-catalyzed azide-alkyne cycloaddition, making the process highly modular. Here we further advance this approach by leveraging this modulator platform by incorporating chemically modified nucleotides at both ends of the modular sgRNA to increase resistance against ribonucleases. Modified nucleotides consisted of a 2'-O-Me group and a phosphorothioate backbone in varying number at both the 5'- and 3'-ends of the sgRNA. It was observed that three modified nucleotides at both ends of the sgRNA significantly increased the success of Cas9 in knocking out a gene of interest. Using these chemically stabilized sgRNAs facilitates multigene editing at the protein level, as demonstrated by successful knockout of both Siglec-3 and Siglec-7 using two fluorophores in conjunction with fluorescence-activated cell sorting. These results demonstrate the versatility of this modular platform for assembling sgRNAs from small, chemically modified strands to simultaneously disrupt the gene expression of two proteins.

Absolute Affinities from Quantitative Shotgun Glycomics Using Concentration-Independent (COIN) Native Mass Spectrometry.

Native mass spectrometry (nMS) screening of natural glycan libraries against glycan-binding proteins (GBPs) is a powerful tool for ligand discovery. However, as the glycan concentrations are unknown, affinities cannot be measured directly from natural libraries. Here, we introduce Concentration-Independent (COIN)-nMS, which enables quantitative screening of natural glycan libraries by exploiting slow mixing of solutions inside a nanoflow electrospray ionization emitter. The affinities (Kd) of detected GBP-glycan interactions are determined, simultaneously, from nMS analysis of their time-dependent relative abundance changes. We establish the reliability of COIN-nMS using interactions between purified glycans and GBPs with known Kd values. We also demonstrate the implementation of COIN-nMS using the catch-and-release (CaR)-nMS assay for glycosylated GBPs. The COIN-CaR-nMS results obtained for plant, fungal, viral, and human lectins with natural libraries containing hundreds of N-glycans and glycopeptides highlight the assay's versatility for discovering new ligands, precisely measuring their affinities, and uncovering "fine" specificities. Notably, the COIN-CaR-nMS results clarify the sialoglycan binding properties of the SARS-CoV-2 receptor binding domain and establish the recognition of monosialylated hybrid and biantennary N-glycans. Moreover, pharmacological depletion of host complex N-glycans reduces both pseudotyped virions and SARS-CoV-2 cell entry, suggesting that complex N-glycans may serve as attachment factors.

Flow Cytometry-Based Detection of Siglec Ligands.

Siglecs are a family of immunomodulatory cell surface receptors present on white blood cells. Binding to cell surface sialic acid-containing glycans modulates the proximity of Siglecs to other receptors that they regulate. This proximity is key to enabling signaling motifs on the cytosolic domain of Siglecs to modulate immune responses. Given the important roles that Siglecs play in helping to maintain immune homeostasis, a better understanding of their glycan ligands is needed to elucidate their roles in health and disease. A common approach for probing Siglec ligands on cells is to use soluble versions of the recombinant Siglecs in conjunction with flow cytometry. Flow cytometry has many advantages in enabling the relative levels of Siglec ligands between cell types to be rapidly quantified. Here, a step-by-step protocol is described on how to detect Siglec ligands most sensitively and accurately on cells by flow cytometry.

Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket.

Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands. Siglec-glycolipid interactions are often studied outside the context of a lipid bilayer, missing the complex behaviors of glycolipids in a membrane. Through optimizing a liposomal formulation to dissect Siglec-glycolipid interactions, it is shown that Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. A panel of synthetic neoglycolipids is used to probe the specificity of this glycolipid binding pocket on Siglec-6, leading to the development of a neoglycolipid with higher avidity for Siglec-6 compared to natural glycolipids. This neoglycolipid facilitates the delivery of liposomes to Siglec-6 on human mast cells, memory B-cells and placental syncytiotrophoblasts. A physiological relevance for glycolipid recognition by Siglec-6 is revealed for the binding and internalization of extracellular vesicles. These results demonstrate a unique and physiologically relevant ability of Siglec-6 to recognize glycolipids in a membrane.

CD33 isoforms in microglia and Alzheimer's disease: Friend and foe.

Alzheimer's disease (AD) is the most common form of neurodegenerative disease and is considered the main cause of dementia worldwide. Genome-wide association studies combined with integrated analysis of functional datasets support a critical role for microglia in AD pathogenesis, identifying them as important potential therapeutic targets. The ability of immunomodulatory receptors on microglia to control the response to pathogenic amyloid-ß aggregates has gained significant interest. Siglec-3, also known as CD33, is one of these immunomodulatory receptors expressed on microglia that has been identified as an AD susceptibility factor. Here, we review recent advances made in understanding the multifaceted roles that CD33 plays in microglia with emphasis on two human-specific CD33 isoforms that differentially correlate with AD susceptibility. We also describe several different therapeutic approaches for targeting CD33 that have been advanced for the purpose of skewing microglial cell responses.

Neuraminidase is a host-directed approach to regulate neutrophil responses in sepsis and COVID-19.

BACKGROUND AND PURPOSE: Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Because pathogen-derived neuraminidase (NEU) stimulates neutrophils, we investigated whether host NEU can be targeted to regulate the neutrophil dysregulation observed in severe infections. EXPERIMENTAL APPROACH: The effects of NEU inhibitors on lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients also was carried out. The effects of oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models. KEY RESULTS: Oseltamivir and zanamivir constrained host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with matrix metalloproteinase 9 (MMP-9). Inhibition of MMP-9 prevented LPS-induced NEU activity and neutrophil response. In vivo, treatment with oseltamivir fine-tuned neutrophil migration and improved infection control as well as host survival in peritonitis and pneumonia sepsis. NEU1 also is highly expressed in neutrophils from COVID-19 patients, and treatment of whole-blood samples from these patients with either oseltamivir or zanamivir reduced neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage. CONCLUSION AND IMPLICATIONS: These findings suggest that interplay of NEU1-MMP-9 induces neutrophil overactivation. In vivo, NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.

How Choice of Model Membrane Affects Protein-Glycosphingolipid Interactions: Insights from Native Mass Spectrometry.

Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) are involved in numerous physiological and pathophysiological processes. Many model membrane systems are available for studying GBP-GSL interactions, but a systematic investigation has not been carried out on how the nature of the model membrane affects binding. In this work, we use electrospray ionization mass spectrometry (ESI-MS), both direct and competitive assays, to measure the binding of cholera toxin B subunit homopentamer (CTB5) to GM1 ganglioside in liposomes, bilayer islands [styrene maleic acid lipid particles (SMALPs), nanodiscs (NDs), and picodiscs (PDs)], and micelles. We find that direct ESI-MS analysis of CTB5 binding to GM1 is unreliable due to non-uniform response factors, incomplete extraction of bound GM1 in the gas phase, and nonspecific CTB5-GM1 interactions. Conversely, indirect proxy ligand ESI-MS measurements show that the intrinsic (per binding site) association constants of CTB5 for PDs, NDs, and SMALPs are similar and comparable to the affinity of soluble GM1 pentasaccharide (GM1os). The observed affinity decreases with increasing GM1 content due to molecular crowding stemming from GM1 clustering. Unlike the smaller model membranes, the observed affinity of CTB5 toward GM1 liposomes is ∼10-fold weaker than GM1os and relatively insensitive to the GM1 content. GM1 glycomicelles exhibit the lowest affinity, ∼35-fold weaker than GM1os. Together, the results highlight experimental design considerations for quantitative GBP-GSL binding studies involving multisubunit GBPs and factors to consider when comparing results obtained with different membrane systems. Notably, they suggest that bilayer islands with a low percentage of GSL, wherein clustering is minimized, are ideal for assessing intrinsic strength of GBP-GSL interactions in a membrane environment, while binding to liposomes, which is sub-optimal due to extensive clustering, may be more representative of authentic cellular environments.

Targeting CD22 on memory B cells to induce tolerance to peanut allergens.

BACKGROUND: Circulating IgE and subsequent severe allergic reactions to peanut are sustained and propagated by recall of peanut allergen-specific memory B cells. OBJECTIVES: This study aimed to determine whether targeting mouse and human CD22 on peanut-specific memory B cells induces tolerance to peanut allergens. METHODS: Siglec-engaging tolerance-inducing antigenic liposomes (STALs) codisplaying peanut allergens (Ara h 1, Ara h 2, or Ara h 3) and high-affinity CD22 ligand (CD22L-STALs) were employed in various mouse models (BALB/cJ, C57BL/6, human CD22 transgenic, and NSG) of peanut allergy. To investigate memory B cells, a conferred memory model was used in which splenocytes from peanut-sensitized mice were transferred into naive animals. Reconstituted mice received either CD22L-STALs or an immunogenic liposome control, followed by a peanut allergen boost and later a challenge with individual peanut allergens. To assess the effects of CD22L-STALs on human B cells, PBMCs were injected into NSG mice, followed by administration of human CD22L-STALs (hCD22L-STALs) and later a whole peanut extract boost. Blood was collected to quantify WPE- and Ara h 1-, 2-, and 3-specific immunoglobulins. RESULTS: Mouse CD22L-STALs (mCD22L-STALs) significantly suppressed systemic memory to Ara h 1, Ara h 2, and Ara h 3 in BALB/cJ and C57BL/6 mice, as demonstrated by reduced allergen-specific IgE, IgG1, and anaphylaxis on challenge. Importantly, 2 doses of mCD22L-STALs led to prolonged tolerance for at least 3 months. hCD22L-STALs displayed similar suppression in mice expressing human CD22 on B cells. Finally, human B cells were tolerized in vivo in NSG mice by hCD22L-STALs. CONCLUSIONS: Antigen-specific exploitation of CD22 on memory B cells can induce systemic immune tolerance.

CRISPR-Click Enables Dual-Gene Editing with Modular Synthetic sgRNAs.

Gene-editing systems such as CRISPR-Cas9 readily enable individual gene phenotypes to be studied through loss of function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR-Cas9 to disrupt individual loci, technical limitations in single gRNA (sgRNA) synthesis hinder the expansion of this approach to multicolor cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-activated cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 5'-azide and 3'-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNAs in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single-color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating double-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.

Transgenic mouse models to study the physiological and pathophysiological roles of human Siglecs.

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are important immunomodulatory receptors. Due to differences between human and mouse Siglecs, defining the in vivo roles for human Siglecs (hSiglecs) can be challenging. One solution is the development and use of hSiglec transgenic mice to assess the physiological roles of hSiglecs in health and disease. These transgenic mice can also serve as important models for the pre-clinical testing of immunomodulatory approaches that are based on targeting hSiglecs. Four general methods have been used to create hSiglec-expressing transgenic mice, each with associated advantages and disadvantages. To date, transgenic mouse models expressing hSiglec-2 (CD22), -3 (CD33), -7, -8, -9, -11, and -16 have been created. This review focuses on both the generation of these hSiglec transgenic mice, along with the important findings that have been made through their study. Cumulatively, hSiglec transgenic mouse models are providing a deeper understanding of the differences between human and mice orthologs/paralogs, mechanisms by which Siglecs regulate immune cell signaling, physiological roles of Siglecs in disease, and different paradigms where targeting Siglecs may be therapeutically advantageous.

Mass Spectrometry-Based Shotgun Glycomics Using Labeled Glycan Libraries.

Mass spectrometry-based shotgun glycomics (MS-SG) is a rapid, sensitive, label-, and immobilization-free approach for the discovery of natural ligands of glycan-binding proteins (GBPs). To perform MS-SG, natural libraries of glycans derived from glycoconjugates in cells or tissues are screened against a target GBP using catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS). Because glycan concentrations are challenging to determine, ligand affinities cannot be directly measured. In principle, relative affinities can be ranked by combining CaR-ESI-MS data with relative concentrations established by hydrophilic interaction liquid chromatography (HILIC) performed on the fluorophore-labeled glycan library. To validate this approach, as well as the feasibility of performing CaR-ESI-MS directly on labeled glycans, libraries of labeled N-glycans extracted from the human monocytic U937 cells or intestinal tissues were labeled with 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA), or procainamide (proA). The libraries were screened against plant and human GBPs with known specificities for α2-3- and α2-6-linked sialosides and quantified by HILIC. Dramatic differences, in some cases, were found for affinity rankings obtained with libraries labeled with different fluorophores, as well as those produced using the combined unlabeled/labeled library approach. The origin of these differences could be explained by differential glycan labeling efficiencies, the impact of specific labels on glycan affinities for the GBPs, and the relative efficiency of release of ligands from GBPs in CaR-ESI-MS. Overall, the results of this study suggest that the 2-AB(CaR-ESI-MS)/2-AB(HILIC) combination provides the most reliable description of the binding specificities of GBPs for N-glycans and is recommended for MS-SG applications.