Chemoproteomics reveals immunogenic and tumor-associated cell surface substrates of ectokinase CK2α.

New epitopes for immune recognition provide the basis of anticancer immunity. Due to the high concentration of extracellular adenosine triphosphate in the tumor microenvironment, we hypothesized that extracellular kinases (ectokinases) could have dysregulated activity and introduce aberrant phosphorylation sites on cell surface proteins. We engineered a cell-tethered version of the extracellular kinase CK2α, demonstrated it was active on cells under tumor-relevant conditions, and profiled its substrate scope using a chemoproteomic workflow. We then demonstrated that mice developed polyreactive antisera in response to syngeneic tumor cells that had been subjected to surface hyperphosphorylation with CK2α. Interestingly, these mice developed B cell and CD4+ T cell responses in response to these antigens but failed to develop a CD8+ T cell response. This work provides a workflow for probing the extracellular phosphoproteome and demonstrates that extracellular phosphoproteins are immunogenic even in a syngeneic system.

Microglia Mediate Contact-Independent Neuronal Network Remodeling via Secreted Neuraminidase-3 Associated with Extracellular Vesicles.

Neurons communicate with each other through electrochemical transmission at synapses. Microglia, the resident immune cells of the central nervous system, modulate this communication through a variety of contact-dependent and -independent means. Microglial secretion of active sialidase enzymes upon exposure to inflammatory stimuli is one unexplored mechanism of modulation. Recent work from our lab showed that treatment of neurons with bacterial sialidases disrupts neuronal network connectivity. Here, we find that activated microglia secrete neuraminidase-3 (Neu3) associated with fusogenic extracellular vesicles. Furthermore, we show that Neu3 mediates contact-independent disruption of neuronal network synchronicity through neuronal glycocalyx remodeling. We observe that NEU3 is transcriptionally upregulated upon exposure to inflammatory stimuli and that a genetic knockout of NEU3 abrogates the sialidase activity of inflammatory microglial secretions. Moreover, we demonstrate that Neu3 is associated with a subpopulation of extracellular vesicles, possibly exosomes, that are secreted by microglia upon inflammatory insult. Finally, we demonstrate that Neu3 is necessary and sufficient to both desialylate neurons and decrease neuronal network connectivity. These results implicate Neu3 in remodeling of the glycocalyx leading to aberrant network-level activity of neurons, with implications in neuroinflammatory diseases such as Parkinson's disease and Alzheimer's disease.

Microglia mediate contact-independent neuronal pruning via secreted Neuraminidase-3 associated with extracellular vesicles.

Neurons communicate with each other through electrochemical transmission at synapses. Microglia, the resident immune cells of the central nervous system, can prune these synapses through a variety of contact-dependent and -independent means. Microglial secretion of active sialidase enzymes upon exposure to inflammatory stimuli is one unexplored mechanism of pruning. Recent work from our lab showed that treatment of neurons with bacterial sialidases disrupts neuronal network connectivity. Here, we find that activated microglia secrete Neuraminidase-3 (Neu3) associated with fusogenic extracellular vesicles. Furthermore, we show Neu3 mediates contact-independent pruning of neurons and subsequent disruption of neuronal networks through neuronal glycocalyx remodeling. We observe that NEU3 is transcriptionally upregulated upon exposure to inflammatory stimuli, and that a genetic knock-out of NEU3 abrogates the sialidase activity of inflammatory microglial secretions. Moreover, we demonstrate that Neu3 is associated with a subpopulation of extracellular vesicles, possibly exosomes, that are secreted by microglia upon inflammatory insult. Finally, we demonstrate that Neu3 is both necessary and sufficient to both desialylate neurons and decrease neuronal network connectivity. These results implicate Neu3 in remodeling of the glycocalyx leading to aberrant network-level activity of neurons, with implications in neuroinflammatory diseases such as Parkinson's disease and Alzheimer's disease.

MYC-driven synthesis of Siglec ligands is a glycoimmune checkpoint.

The Siglecs (sialic acid-binding immunoglobulin-like lectins) are glycoimmune checkpoint receptors that suppress immune cell activation upon engagement of cognate sialoglycan ligands. The cellular drivers underlying Siglec ligand production on cancer cells are poorly understood. We find the MYC oncogene causally regulates Siglec ligand production to enable tumor immune evasion. A combination of glycomics and RNA-sequencing of mouse tumors revealed the MYC oncogene controls expression of the sialyltransferase St6galnac4 and induces a glycan known as disialyl-T. Using in vivo models and primary human leukemias, we find that disialyl-T functions as a "don't eat me" signal by engaging macrophage Siglec-E in mice or the human ortholog Siglec-7, thereby preventing cancer cell clearance. Combined high expression of MYC and ST6GALNAC4 identifies patients with high-risk cancers and reduced tumor myeloid infiltration. MYC therefore regulates glycosylation to enable tumor immune evasion. We conclude that disialyl-T is a glycoimmune checkpoint ligand. Thus, disialyl-T is a candidate for antibody-based checkpoint blockade, and the disialyl-T synthase ST6GALNAC4 is a potential enzyme target for small molecule-mediated immune therapy.

Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19.

Severe cases of coronavirus disease 2019 (COVID-19), caused by infection with SARS-CoV-2, are characterized by a hyperinflammatory immune response that leads to numerous complications. Production of proinflammatory neutrophil extracellular traps (NETs) has been suggested to be a key factor in inducing a hyperinflammatory signaling cascade, allegedly causing both pulmonary tissue damage and peripheral inflammation. Accordingly, therapeutic blockage of neutrophil activation and NETosis, the cell death pathway accompanying NET formation, could limit respiratory damage and death from severe COVID-19. Here, we demonstrate that synthetic glycopolymers that activate signaling of the neutrophil checkpoint receptor Siglec-9 suppress NETosis induced by agonists of viral toll-like receptors (TLRs) and plasma from patients with severe COVID-19. Thus, Siglec-9 agonism is a promising therapeutic strategy to curb neutrophilic hyperinflammation in COVID-19.

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.

Membrane-tethered mucin-like polypeptides sterically inhibit binding and slow fusion kinetics of influenza A virus.

The mechanism(s) by which cell-tethered mucins modulate infection by influenza A viruses (IAVs) remain an open question. Mucins form both a protective barrier that can block virus binding and recruit IAVs to bind cells via the sialic acids of cell-tethered mucins. To elucidate the molecular role of mucins in flu pathogenesis, we constructed a synthetic glycocalyx to investigate membrane-tethered mucins in the context of IAV binding and fusion. We designed and synthesized lipid-tethered glycopolypeptide mimics of mucins and added them to lipid bilayers, allowing chemical control of length, glycosylation, and surface density of a model glycocalyx. We observed that the mucin mimics undergo a conformational change at high surface densities from a compact to an extended architecture. At high surface densities, asialo mucin mimics inhibited IAV binding to underlying glycolipid receptors, and this density correlated to the mucin mimic's conformational transition. Using a single virus fusion assay, we observed that while fusion of virions bound to vesicles coated with sialylated mucin mimics was possible, the kinetics of fusion was slowed in a mucin density-dependent manner. These data provide a molecular model for a protective mechanism by mucins in IAV infection, and therefore this synthetic glycocalyx provides a useful reductionist model for studying the complex interface of host-pathogen interactions.

Synthetic Siglec-9 Agonists Inhibit Neutrophil Activation Associated with COVID-19.

Severe cases of coronavirus disease 2019 (COVID-19), caused by infection with SARS-Cov-2, are characterized by a hyperinflammatory immune response that leads to numerous complications. Production of proinflammatory neutrophil extracellular traps (NETs) has been suggested to be a key factor in inducing a hyperinflammatory signaling cascade, allegedly causing both pulmonary tissue damage and peripheral inflammation. Accordingly, therapeutic blockage of neutrophil activation and NETosis, the cell death pathway accompanying NET formation, could limit respiratory damage and death from severe COVID-19. Here, we demonstrate that synthetic glycopolymers that activate the neutrophil checkpoint receptor Siglec-9 suppress NETosis induced by agonists of viral toll-like receptors (TLRs) and plasma from patients with severe COVID-19. Thus, Siglec-9 agonism is a promising therapeutic strategy to curb neutrophilic hyperinflammation in COVID-19.
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N-Carboxyanhydride Polymerization of Glycopolypeptides That Activate Antigen-Presenting Cells through Dectin-1 and Dectin-2.

The C-type lectins dectin-1 and dectin-2 contribute to innate immunity against microbial pathogens by recognizing their foreign glycan structures. These receptors are promising targets for vaccine development and cancer immunotherapy. However, currently available agonists are heterogeneous glycoconjugates and polysaccharides from natural sources. Herein, we designed and synthesized the first chemically defined ligands for dectin-1 and dectin-2. They comprised glycopolypeptides bearing mono-, di-, and trisaccharides and were built through polymerization of glycosylated N-carboxyanhydrides. Through this approach, we achieved glycopolypeptides with high molecular weights and low dispersities. We identified structures that elicit a pro-inflammatory response through dectin-1 or dectin-2 in antigen-presenting cells. With their native proteinaceous backbones and natural glycosidic linkages, these agonists are attractive for translational applications.