Dissecting the abilities of murine Siglecs to interact with gangliosides.

Siglecs are cell surface receptors whose functions are tied to the binding of their sialoglycan ligands. Recently, we developed an optimized liposome formulation and used it to investigate the binding of human Siglecs (hSiglec) against a panel of gangliosides. Animal models, more specifically murine models, are used to understand human biology, however, species-specific differences can complicate the interpretation of the results. Herein, we used our optimized liposome formulation to dissect the interactions between murine Siglecs (mSiglecs) and gangliosides to assess the appropriateness of mSiglecs as a proxy to better understand the biological roles of hSiglec-ganglioside interactions. Using our optimized liposome formulation, we found that ganglioside binding is generally conserved between mice and humans with mSiglec-1, -E, -F, and -15 binding multiple gangliosides like their human counterparts. However, in contrast to the hSiglecs, we observed little to no binding between the mSiglecs and ganglioside GM1a. Detailed analysis of mSiglec-1 interacting with GM1a and its structural isomer, GM1b, suggests that mSiglec-1 preferentially binds α2-3-linked sialic acids presented from the terminal galactose residue. The ability of mSiglecs to interact or not interact with gangliosides, particularly GM1a, has implications for using mice to study neurodegenerative diseases, infections, and cancer, where interactions between Siglecs and glycolipids have been proposed to modulate these human diseases.

Alzheimer's disease associated isoforms of human CD33 distinctively modulate microglial cell responses in 5XFAD mice.

Microglia play diverse pathophysiological roles in Alzheimer's disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans. We address this challenge by studying transgenic mice expressing either of the human CD33 isoforms crossed with the 5XFAD mouse model of amyloidosis and find that human CD33 isoforms have opposing effects on the response of microglia to amyloid-ß (Aß) deposition. Mice expressing CD33M have increased Aß levels, more diffuse plaques, fewer disease-associated microglia, and more dystrophic neurites compared to 5XFAD control mice. Conversely, CD33m promotes plaque compaction and microglia-plaque contacts, and minimizes neuritic plaque pathology, highlighting an AD protective role for this isoform. Protective phenotypes driven by CD33m are detected at an earlier timepoint compared to the more aggressive pathology in CD33M mice that appears at a later timepoint, suggesting that CD33m has a more prominent impact on microglia cell function at earlier stages of disease progression. In addition to divergent roles in modulating phagocytosis, scRNAseq and proteomics analyses demonstrate that CD33m+ microglia upregulate nestin, an intermediate filament involved in cell migration, at plaque contact sites. Overall, our work provides new functional insights into how CD33, as a top genetic susceptibility factor for AD, modulates microglial cell function.

Sialic acid blockade inhibits the metastatic spread of prostate cancer to bone.

BACKGROUND: Bone metastasis is a common consequence of advanced prostate cancer. Bisphosphonates can be used to manage symptoms, but there are currently no curative treatments available. Altered tumour cell glycosylation is a hallmark of cancer and is an important driver of a malignant phenotype. In prostate cancer, the sialyltransferase ST6GAL1 is upregulated, and studies show ST6GAL1-mediated aberrant sialylation of N-glycans promotes prostate tumour growth and disease progression. METHODS: Here, we monitor ST6GAL1 in tumour and serum samples from men with aggressive prostate cancer and using in vitro and in vivo models we investigate the role of ST6GAL1 in prostate cancer bone metastasis. FINDINGS: ST6GAL1 is upregulated in patients with prostate cancer with tumours that have spread to the bone and can promote prostate cancer bone metastasis in vivo. The mechanisms involved are multi-faceted and involve modification of the pre-metastatic niche towards bone resorption to promote the vicious cycle, promoting the development of M2 like macrophages, and the regulation of immunosuppressive sialoglycans. Furthermore, using syngeneic mouse models, we show that inhibiting sialylation can block the spread of prostate tumours to bone. INTERPRETATION: Our study identifies an important role for ST6GAL1 and α2-6 sialylated N-glycans in prostate cancer bone metastasis, provides proof-of-concept data to show that inhibiting sialylation can suppress the spread of prostate tumours to bone, and highlights sialic acid blockade as an exciting new strategy to develop new therapies for patients with advanced prostate cancer. FUNDING: Prostate Cancer Research and the Mark Foundation For Cancer Research, the Medical Research Council and Prostate Cancer UK.

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.

Targeting inhibitory Siglec-3 to suppress IgE-mediated human basophil degranulation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs. OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for 1 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 N-formylmethionyl-leucyl-phenylalanine, 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: Pretreating 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 before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

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.

ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) synthesis of Siglec ligands mediates anti-tumour immunity in prostate cancer.

Immune checkpoint blockade has yet to produce robust anti-cancer responses for prostate cancer. Sialyltransferases have been shown across several solid tumours, including breast, melanoma, colorectal and prostate to promote immune suppression by synthesising sialoglycans, which act as ligands for Siglec receptors. We report that ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) levels negatively correlate with androgen signalling in prostate tumours. We demonstrate that ST3Gal1 plays an important role in modulating tumour immune evasion through the synthesises of sialoglycans with the capacity to engage the Siglec-7 and Siglec-9 immunoreceptors preventing immune clearance of cancer cells. Here, we provide evidence of the expression of Siglec-7/9 ligands and their respective immunoreceptors in prostate tumours. These interactions can be modulated by enzalutamide and may maintain immune suppression in enzalutamide treated tumours. We conclude that the activity of ST3Gal1 is critical to prostate cancer anti-tumour immunity and provide rationale for the use of glyco-immune checkpoint targeting therapies in advanced prostate cancer.

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.

Boolean Models of the Transport, Synthesis, and Metabolism of Tryptophan in Escherichia coli.

The tryptophan (trp) operon in Escherichia coli codes for the proteins responsible for the synthesis of the amino acid tryptophan from chorismic acid, and has been one of the most well-studied gene networks since its discovery in the 1960s. The tryptophanase (tna) operon codes for proteins needed to transport and metabolize it. Both of these have been modeled individually with delay differential equations under the assumption of mass-action kinetics. Recent work has provided strong evidence for bistable behavior of the tna operon. The authors of Orozco-Gómez et al. (Sci Rep 9(1):5451, 2019) identified a medium range of tryptophan in which the system has two stable steady-states, and they reproduced these experimentally. In this paper, we will show how a Boolean model can capture this bistability. We will also develop and analyze a Boolean model of the trp operon. Finally, we will combine these two to create a single Boolean model of the transport, synthesis, and metabolism of tryptophan. In this amalgamated model, the bistability disappears, presumably reflecting the ability of the trp operon to produce tryptophan and drive the system toward homeostasis. All of these models have longer attractors that we call "artifacts of synchrony", which disappear in the asynchronous automata. This curiously matches the behavior of a recent Boolean model of the arabinose operon in E. coli, and we discuss some open-ended questions that arise along these lines.

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.

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.