Cell Surface Glycans Research Articles

The green tea catechin EGCG provides proof-of-concept for a pan-coronavirus attachment inhibitor

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has emphasized the serious threat to human health posed by emerging coronaviruses. Effective broadly-acting antiviral countermeasures are urgently needed to prepare for future emerging CoVs, as vaccine development is not compatible with a rapid response to a newly emerging virus. The green tea catechin, epigallocatechin gallate (EGCG), has broad-spectrum antiviral activity, although its mechanisms against coronavirus (CoV) infection have remained unclear. Here, we show that EGCG prevents human and murine CoV infection and blocks the entry of lentiviral particles pseudotyped with spike proteins from bat or highly pathogenic CoVs, including SARS-CoV-2 variants of concern, in lung epithelial cells. Mechanistically, EGCG treatment reduces CoV attachment to target cell surfaces by interfering with attachment to cell-surface glycans. Heparan sulfate proteoglycans are a required attachment factor for SARS-CoV-2 and are shown here to be important in endemic HCoV-OC43 infection. We show that EGCG can compete with heparin, a heparan sulfate analog, for virion binding. Our results highlight heparan sulfate as a conserved cell attachment factor for CoVs, and demonstrate the potential for the development of pan-coronavirus attachment inhibitors, which may be useful to protect against future emerging CoVs.

The Glycan-Binding Trait of the Sarbecovirus Spike N-Terminal Domain Reveals an Evolutionary Footprint.

ABSTRACTThe spike protein on sarbecovirus virions contains two external, protruding domains: an N-terminal domain (NTD) with unclear function and a C-terminal domain (CTD) that binds the host receptor, allowing for viral entry and infection. While the CTD is well studied for therapeutic interventions, the role of the NTD is far less well understood for many coronaviruses. Here, we demonstrate that the spike NTD from SARS-CoV-2 and other sarbecoviruses binds to unidentified glycans in vitro similarly to other members of the Coronaviridae family. We also show that these spike NTD (S-NTD) proteins adhere to Calu3 cells, a human lung cell line, although the biological relevance of this is unclear. In contrast to what has been shown for Middle East respiratory syndrome coronavirus (MERS-CoV), which attaches sialic acids during cell entry, sialic acids present on Calu3 cells inhibited sarbecovirus infection. Therefore, while sarbecoviruses can interact with cell surface glycans similarly to other coronaviruses, their reliance on glycans for entry is different from that of other respiratory coronaviruses, suggesting sarbecoviruses and MERS-CoV have adapted to different cell types, tissues, or hosts during their divergent evolution. Our findings provide important clues for further exploring the biological functions of sarbecovirus glycan binding and adds to our growing understanding of the complex forces that shape coronavirus spike evolution.IMPORTANCE Spike N-terminal domains (S-NTD) of sarbecoviruses are highly diverse; however, their function remains largely understudied compared with the receptor-binding domains (RBD). Here, we show that sarbecovirus S-NTD can be phylogenetically clustered into five clades and exhibit various levels of glycan binding in vitro. We also show that, unlike some coronaviruses, including MERS-CoV, sialic acids present on the surface of Calu3, a human lung cell culture, inhibit SARS-CoV-2 and other sarbecoviruses. These results suggest that while glycan binding might be an ancestral trait conserved across different coronavirus families, the functional outcome during infection can vary, reflecting divergent viral evolution. Our results expand our knowledge on the biological functions of the S-NTD across diverse sarbecoviruses and provide insight on the evolutionary history of coronavirus spike.

Cell surface glycan engineering reveals that matriglycan alone can recapitulate dystroglycan binding and function

α-Dystroglycan (α-DG) is uniquely modified on O-mannose sites by a repeating disaccharide (-Xylα1,3-GlcAβ1,3-)n termed matriglycan, which is a receptor for laminin-G domain-containing proteins and employed by old-world arenaviruses for infection. Using chemoenzymatically synthesized matriglycans printed as a microarray, we demonstrate length-dependent binding to Laminin, Lassa virus GP1, and the clinically-important antibody IIH6. Utilizing an enzymatic engineering approach, an N-linked glycoprotein was converted into a IIH6-positive Laminin-binding glycoprotein. Engineering of the surface of cells deficient for either α-DG or O-mannosylation with matriglycans of sufficient length recovers infection with a Lassa-pseudovirus. Finally, free matriglycan in a dose and length dependent manner inhibits viral infection of wildtype cells. These results indicate that matriglycan alone is necessary and sufficient for IIH6 staining, Laminin and LASV GP1 binding, and Lassa-pseudovirus infection and support a model in which it is a tunable receptor for which increasing chain length enhances ligand-binding capacity.

Pathogen-sugar interactions revealed by universal saturation transfer analysis.

Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily modified pathogen proteins can be confounded by overlapping sugar signals and/or compounded with known experimental constraints. Universal saturation transfer analysis (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin-lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an "end-on" manner. uSTA-guided modeling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar binding in SARS-CoV-2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins deep in the human lung as potentially relevant to virulence and/or zoonosis.

Glycan expression profile of signet ring cell gastric cancer cells and potential applicability of rBC2LCN-targeted lectin drug conjugate therapy.

Signet ring cell carcinoma (SRC) is a distinct subtype of gastric cancer (GC); however, the specific characteristics of cancer cell surface glycans and glycosylation remain unclear. In this study, we investigated SRC-specific glycans using lectin microarray and evaluated the potential applicability of a glycan-targeting therapy. SRC cell lines (NUGC-4 and KATO-III) and non-SRC (NSRC) cell lines (NCI-N87, SNU-1, and MKN-45) were subjected to lectin microarray analysis to identify the SRC-specific glycans. Additionally, we performed immunohistochemical lectin staining and evaluated the anti-tumor effects of lectin drug conjugates (LDCs) using high-affinity lectins for SRC. Among the 96 lectins tested, 11 high-affinity and 8 low-affinity lectins were identified for SRC. Glycan-binding motifs varied in the high-affinity lectins, but 5 (62.5%) low-affinity lectins bound the same glycan structure, α2-6-linked sialic acids. The ratio of signal intensity in SRC to NSRC (SRC/NSRC) was highest in the rBC2LCN lectin (1.930-fold), followed by the BPL lectin (1.786-fold). rBC2LCN lectin showed high affinity for both SRC cell lines and one of the three NSRC cell lines (NCI-N87). The therapeutic effects of the LDC, rBC2LCN-PE38 (rBC2LCN, and Pseudomonas exotoxin A), showed cytocidal effects in vitro and tumor regression in in vivo mouse xenograft models. We reported specific glycan profiles in SRC cells, showing reduced α2-6-linked sialic acids. Additionally, we found a targeted therapy using rBC2LCN lectin might be applicable as an alternative treatment option for patients with SRC.

Abstract 3300: Glycoimmune checkpoint and tumor stroma-targeted oncolytic vaccinia virus for the treatment of solid tumors

Abstract Oncolytic virotherapy represents a promising therapeutic strategy for cancers that do not respond well to immune checkpoint inhibitors. Oncolytic viruses (OV) preferentially replicate in and lyse tumor cells, leading to systemic immune stimulation. Studies have shown that hypersialylation promotes tumor growth and metastasis, as well as the suppression of immune cells. Cancer-associated fibroblasts (CAFs) are known to facilitate tumor invasion and angiogenesis and maintain immunosuppressive microenvironment in solid tumors. We are developing vvDD-Sial-FAP/CD3, an oncolytic vaccinia virus expressing a membrane-bound sialidase, to remove sialic acids from the cell surface glycans, and fibroblast activation protein (FAP)-targeted T cell engager, to eliminate glycoimmune checkpoint and tumor stroma, respectively. vvDD-Sial-FAP/CD3 is an engineered vaccinia virus of Western Reserve (WR) strain with: (1) an insertional disruption of the viral thymidine kinase (TK) gene with the sialidase and FAP/CD3 transgenes. TK is an essential enzyme for the pyrimidine synthesis pathway; viral TK gene deletion thus results in selective replication of virus in rapidly dividing cancerous cells with high intracellular nucleotide pools, and (2) a deletion of the vaccinia growth factor (VGF) genes for greater dependence on the cell cycling status of the cancer cells. Our results showed that sialidase expressed from vvDD-Sial-FAP/CD3 efficiently cleaves the sialic acids from the cell surface and the Fc fused to the sialidase induced antibody-dependent cell-mediated cytotoxicity (ADCC) using an ADCC reporter assay. In vitro efficacy studies were conducted using HCT-116 human colon cancer cells mixed with FAP-expressing normal human dermal fibroblasts or FAP-positive HCC1143 human breast cancer cells in the presence of human peripheral blood mononuclear cells. In both tumor models, vvDD-Sial-FAP/CD3 induced activation of both CD4+ and CD8+ T cells, as measured by the upregulation of CD69 and CD25 markers and increased granzyme B release, which resulted in enhanced cell killing. Using A549 co-cultured with CAFs microtissues, we showed that vvDD-Sial-FAP/CD3 spread efficiently within the tumor spheroids. In addition, vvDD-Sial-FAP/CD3 increased tumor-infiltrated lymphocytes, leading to enhanced cell killing. Taken together, our results have demonstrated potent anti-tumor and -stroma effects induced by vvDD-Sial-FAP/CD3 in multiple cancer models. In vivo efficacy of vvDD-Sial-FAP/CD3, either alone or in combination with checkpoint inhibitor or NK cell therapy, is being evaluated. Citation Format: Winnie M. Chan, Mariya Viskovska, Micaela McArthur, Xue Mary Yu, Wei Liang, Zane Norman, Xiaotong Song, Nancy T. Chang, Haiyan Jiang. Glycoimmune checkpoint and tumor stroma-targeted oncolytic vaccinia virus for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3300.

Abstract 5328: Near-infrared photoimmunotherapy for pancreatic cancer: Targeting cell surface glycans using lectin-photoabsorber conjugate (LPC)

Abstract Introduction: Since protein targets in cancer therapy largely have been explored, glycans would be attractive alternative targets. We had previously discovered a fucosylated glycan epitope highly expressed on pancreatic cancer cells, and a lectin probe called rBC2LCN, which can be used as a specific targeting bullet to this glycan (Shimomura O, Oda T, et al: Mol Cancer Ther 2018). We now then hypothesized that, this lectin-based cancer-targeting strategies should be reasonably applicable to near-infrared photoimmunotherapy (NIR-PIT). NIR-PIT have already developed using a conjugate of a photoabsorber, IRdye700DX (IR700), and a monoclonal antibody which recognizes protein antigens on the target cells. In this study, we firstly developed a lectin-photoabsorber conjugate (BC2-IR700) and investigated a new cancer therapy targeting cancer cell surface glycans. Material and Methods: We developed BC2-IR700 by conjugation of rBC2LCN lectin with IR700 NHS ester. A pancreatic cancer cell line Capan-1 was used as a model which is bound by rBC2LCN. The cytotoxic effect of NIR-PIT with BC2-IR700 was first evaluated in vitro. We then evaluated the anti-cancer effect of BC2-IR700 in vivo using pancreatic cancer xenograft mice including subcutaneous tumor model. Results: The product of BC2-IR700 was confirmed by SDS-PAGE, that showed reasonable 16KDa sharp band with clear fluorescence intensity. After incubation with BC2-IR700, Capan-1 cells showed high fluorescence signal, which was confirmed with flow cytometry and fluorescence microscopy. BC2-IR700 was detected on the cell surface of Capan-1 after incubation for 1h. Immediately after exposure to NIR light, the cell death of Capan-1 was induced and the cytotoxicity significantly increased in a light-dose-dependent manner. After intravenous administration of BC2-IR700 into pancreatic cancer xenograft mice, BC2-IR700 was observed in vivo fluorescence imaging to be accumulated in tumor. NIR-PIT was performed by exposing to NIR light (100 J/cm2) at 6 h following BC2-IR700 intravenous administration (20 μg/mice). Tumor growth was significantly inhibited in the NIR-PIT treatment group compared with the other control groups such as no treatment, BC2-IR700 only, and NIR light exposure only (P < 0.05). Conclusions: The lectin-photoabsorber conjugate, BC2-IR700, was able to be sufficiently applied for NIR-PIT in in vivo mouse model. The novel NIR-PIT targeting cell surface glycans could be a promising therapeutic strategy for the treatment of pancreatic cancer. Citation Format: Yukihito Kuroda, Osamu Shimomura, Hiromitsu Nakahashi, Tomoaki Furuta, Yoshihiro Miyazaki, Takahito Nakajima, Hiroaki Tateno, Tatsuya Oda. Near-infrared photoimmunotherapy for pancreatic cancer: Targeting cell surface glycans using lectin-photoabsorber conjugate (LPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5328.

Extracellular sialyltransferase st6gal1 in breast tumor cell growth and invasiveness

The sialyltransferase ST6GAL1 that adds α2–6 linked sialic acids to N-glycans of cell surface and secreted glycoproteins is prominently associated with many human cancers. Tumor-native ST6GAL1 promotes tumor cell behaviors such as invasion and resistance to cell stress and chemo- and radio-treatments. Canonically, ST6GAL1 resides in the intracellular secretory apparatus and glycosylates nascent glycoproteins in biosynthetic transit. However, ST6GAL1 is also released into the extracellular milieu and extracellularly remodels cell surface and secreted glycans. The impact of this non-canonical extrinsic mechanism of ST6GAL1 on tumor cell pathobiology is not known. We hypothesize that ST6GAL1 action is the combined effect of natively expressed sialyltransferase acting cell-autonomously within the ER-Golgi complex and sialyltransferase from extracellular origins acting extrinsically to remodel cell-surface glycans. We found that shRNA knockdown of intrinsic ST6GAL1 expression resulted in decreased ST6GAL1 cargo in the exosome-like vesicles as well as decreased breast tumor cell growth and invasive behavior in 3D in vitro cultures. Extracellular ST6GAL1, present in cancer exosomes or the freely soluble recombinant sialyltransferase, compensates for insufficient intrinsic ST6GAL1 by boosting cancer cell proliferation and increasing invasiveness. Moreover, we present evidence supporting the existence novel but yet uncharacterized cofactors in the exosome-like particles that potently amplify extrinsic ST6GAL1 action, highlighting a previously unknown mechanism linking this enzyme and cancer pathobiology. Our data indicate that extracellular ST6GAL1 from remote sources can compensate for cellular ST6GAL1-mediated aggressive tumor cell proliferation and invasive behavior and has great clinical potential for extracellular ST6GAL1 as these molecules are in the extracellular space should be easily accessible targets.

Structural Characterization and Evaluation of an Epitope at the Tip of the A-Band Rhamnan Polysaccharide of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen often termed A-band PS that was composed of a neutral d-rhamnan trisaccharide repeating unit as a relatively conserved cell surface carbohydrate. However, nuclear magnetic resonance (NMR) spectra and chemical analysis of A-PS preparations showed the presence of several additional components. Here, we report the characterization of the carbohydrate component responsible for these signals. The carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the nonreducing end of the A-band PS. Initial studies performed with the isolated antigen permitted the production of conjugates that were used to immunize mice and rabbits and generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and three monoclonal antibodies were generated, one of which was able to recognize and facilitate opsonophagocytic killing of a majority of P. aeruginosa strains. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection that includes clinical and serotype strains. Synthetic oligosaccharides (mono- to pentasaccharides) representing the terminal 3-O-methyl d-rhamnan were prepared, and the trisaccharide was identified as the antigenic determinant required to effectively mimic the natural antigen recognized by the broadly cross-reactive monoclonal antibody. These data suggest that there is considerable promise in this antigen as a vaccine or therapeutic target.

Glycan Profiling by Sequencing to Uncover Multicellular Communication: Launching Glycobiology in Single Cells and Microbiomes.

Glycans are essential building blocks of life that are located at the outermost surface of all cells from mammals to bacteria and even viruses. Cell surface glycans mediate multicellular communication in diverse biological processes and are useful as “surface markers” to identify cells. Various single-cell sequencing technologies have already emerged that enable the high-throughput analysis of omics information, such as transcriptome and genome profiling on a cell-by-cell basis, which has advanced our understanding of complex multicellular interactions. However, there has been no robust technology to analyze the glycome in single cells, mainly because glycans with branched and heterogeneous structures cannot be readily amplified by polymerase chain reactions like nucleic acids. We hypothesized that the generation of lectins conjugated with DNA barcodes (DNA-barcoded lectins) would enable the conversion of glycan information to gene information, which may be amplified and measured using DNA sequencers. This technology will enable the simultaneous analysis of glycan and RNA in single cells. Based on this concept, we developed a technology to analyze glycans and RNA in single cells, which was referred to as scGR-seq. Using scGR-seq, we acquired glycan and gene expression profiles of individual cells constituting heterogeneous cell populations, such as tissues. We further extended Glycan-seq to the profiling of the surface glycans of bacteria and even gut microbiota. Glycan-seq and scGR-seq are new technologies that enable us to elucidate the function of glycans in cell–cell and cell–microorganism communication, which extends glycobiology to the level of single cells and microbiomes.

Enhanced anti-tumor immunity in ST8Sia6 knockout mice

Abstract Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells, which recognize specific sialic acid modifications on cell surface glycans. Sialyltransferases are the enzymes that catalyzing the addition of sialic acids onto glycoproteins and glycolipids. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6 on O-linked glycoproteins and functions to inhibit activation of innate immune cells. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered macrophage polarization and increased tumor growth through engagement of ligands generated by ST8Sia6 with Siglec-E. Here, we examine the role of ST8Sia6 on immune cells in regulating anti-tumor immunity. The loss of ST8Sia6 in tumor bearing mice reduced tumor growth, increased survival, and altered the immune response as compared to ST8Sia6 sufficient tumor bearing mice. Using MC38, B16, and B16-OVA flank tumor mouse models, tumor growth was decreased in ST8Sia6 KO mice as compared to C57Bl/6 mice. Within the tumor microenvironment of tumors present in ST8Sia6 KO mice compared to tumors present in C57Bl/6 mice, macrophages and dendritic cells altered their phenotype towards enhanced immune activation. Tregs from tumors in ST8Sia6 KO mice also displayed an increase in RORγt and a reduction in TNFRII, suggesting a possible shift to a less suppressive Treg. These alterations in the immune response to tumors were due to the loss of ST8Sia6 in macrophages and dendritic cells and not T-cells. Thus, ST8Sia6 generates ligands for Siglecs that dampen anti-tumor immunity. Supported by grants from NIH (R01CA243545, 2T32AI007425) and Mayo Clinic Center of Biomedical Discovery

Chemical synthesis of a synthetically useful L-galactosaminuronic acid building block.

Most bacterial cell surface glycans are structurally unique, and have been considered as ideal target molecules for the developments of detection and diagnosis techniques, as well as vaccines. Chemical synthesis has been a promising approach to prepare well-defined oligosaccharides, facilitating the structure-activity relationship exploration and biomedical applications of bacterial glycans. L-Galactosaminuronic acid is a rare sugar that has been only found in cell surface glycans of gram-negative bacteria. Here, an orthogonally protected L-galactosaminuronic acid building block was designed and chemically synthesized. A synthetic strategy based on glycal addition and TEMPO/BAIB-mediated C6 oxidation served well for the transformation of commercial L-galactose to the corresponding L-galactosaminuronic acid. Notably, the C6 oxidation of the allyl glycoside was more efficient than that of the selenoglycoside. In addition, a balance between the formation of allyl glycoside and the recovery of selenoglycoside was essential to improve efficiency of the NIS/TfOH-catalyzed allylation. This synthetically useful L-galactosaminuronic acid building block will provide a basis for the syntheses of complex bacterial glycans.

Genetic and small molecule strategies to edit the glycocalyx

The glycocalyx covers all living cells and is a key regulator of cell communication with the extracellular milieu/matrix, other cells, foreign pathogens and the microbiome. A variety of glycoconjugate types decorate mammalian cells including N‐ and O‐linked glycans attached to proteins, glycolipids, and glycosaminoglycans. A complete understanding of the molecular and cellular features controlling living systems is not possible without delineating the structural and functional contributions of the glycocalyx. My laboratory studies this aspect by developing systems based experimental and computational tools to characterize the role of the glycocalyx in normal physiological and selected pathophysiological contexts. For such investigations, we have developed both novel CRISPR‐Cas9 based tools to perturb cell surface glycans and small molecule inhibitors of the same. Additionally, we have developed a Synthetic Glycan toolkit and liquid‐chromatography mass‐spectrometry (LC‐MS) workflow to longitudinally track glycan changes in living cells in a non‐destructive manner. My talk will present the development and application of these technologies in two contexts. First, I will discuss studies of human inflammatory diseases which will present some new molecular players regulating glycosylation and leukocyte‐endothelial cell adhesion mechanics under fluid shear flow. Second, I will present our studies on the role of glycans in regulating SARS‐CoV‐2 viral entry into human cells expressing ACE2. The data demonstrate a critical role for N‐glycans and O‐linked glycans in viral entry and propagation.

Cell Surface Glycan Engineering to Examine Receptor Specificities of Influenza and Other Viruses

The transmission of viruses from animal reservoirs to humans poses great threats to public health. Preparedness for future zoonotic outbreaks requires a fundamental understanding how viruses of animal origin have adapted to replication in humans. In particular, it necessitates insight how viruses have evolved to binding to a cell surface component, or receptor, of the new host. We have developed glycan arrays and glycan cell surface engineering strategies to examine receptor specificities of viruses. The approaches were employed to examine receptor specificities of evolutionary distinct A/H3N2 influenza viruses. Recent A/H3N2 influenza viruses exhibit altered receptor specificities, and as a result they have lost the ability to agglutinate erythrocytes critical for antigenic characterization and give low yields and acquire adaptive mutations when cultured in eggs and cells, contributing to recent vaccine challenges. Glycan array technology showed that these viruses have compensated for decreased binding of the prototypic human receptor by recognizing α2,6‐sialosides on extended LacNAc moieties. Erythrocyte glycomics showed an absence of extended glycans, providing a rationale for lack of agglutination by recent A/H3N2 viruses. A glycan remodeling approach installed functional receptors on erythrocytes, allowing antigenic characterization of recent A/H3N2 viruses and confirming the cocirculation of antigenically different viruses in humans. Computational studies of HAs in complex with sialosides having extended LacNAc moieties reveal that mutations distal to the RBD reoriented the Y159 side chain, resulting in an extended receptor binding site. The approach was extended to examining the receptor specificity of Lass Virus.

Cutting Edge: Enhanced Antitumor Immunity in ST8Sia6 Knockout Mice.

Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells that recognize specific sialic acid modifications on cell surface glycans. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered immune responses and increased tumor growth. In this study, we examined the role of ST8Sia6 on immune cells in regulating antitumor immunity. ST8Sia6 knockout mice had an enhanced immune response to tumors. The loss of ST8Sia6 promoted an enhanced intratumoral activation of macrophages and dendritic cells, including upregulation of CD40. Intratumoral regulatory T cells exhibited a more inflammatory phenotype in ST8Sia6 knockout mice. Using adoptive transfer studies, the change in regulatory T cell phenotype was not cell intrinsic and depended on the loss of ST8Sia6 expression in APCs. Thus, ST8Sia6 generates ligands for Siglecs that dampen antitumor immunity.

Exo-Enzymatic Cell-Surface Glycan Labeling for Capturing Glycan-Protein Interactions through Photo-Cross-Linking.

Tools to interrogate glycoconjugate-protein interactions in the context of living cells are highly attractive for the identification of critically important functional binding partners of glycan-binding proteins. These interactions are challenging to study due to the low affinity and rapid dissociation rates of glycan-protein binding events. The use of photo-cross-linkers to capture glycan-protein interaction complexes has shown great promise for identifying binding partners involved in these interactions. Current methodologies use metabolic oligosaccharide engineering (MOE) to incorporate photo-cross-linking sugars. However, these MOE strategies are not amenable to all cell types and can result in low incorporation and cell-surface display of the photo-cross-linking probe, limiting their utility for studying many types of interactions. We describe here an exo-enzymatic strategy for selectively introducing photo-cross-linking probes into cell-surface glycoconjugates using the recombinant human sialyltransferase ST6GAL1 and a diazirine-linked CMP-Neu5Ac derivative. Probe introduction is highly efficient, amenable to different cell types, and resulted in improved cross-linking when compared to MOE. This exo-enzymatic labeling approach can selectively introduce the photo-cross-linking sugar onto specific glycan epitopes and subclasses by harnessing the specificity of the sialyltransferase employed, underscoring its potential as a tool to interrogate and identify glycoconjugate ligands for diverse glycan-binding proteins.

Sialoglycan-binding patterns of bacterial AB5 toxin B subunits correlate with host range and toxicity, indicating evolution independent of A subunits

Many pathogenic bacteria secrete AB5 toxins that can be virulence factors. Cytotoxic A subunits are delivered to the cytosol following B subunit binding to specific host cell surface glycans. Some B subunits are not associated with A subunits, for example, YpeB of Yersinia pestis, the etiologic agent of plague. Plague cannot be eradicated because of Y. pestis' adaptability to numerous hosts. We previously showed selective binding of other B5 pentamers to a sialoglycan microarray, with sialic acid (Sia) preferences corresponding to those prominently expressed by various hosts, for example, N-acetylneuraminic acid (Neu5Ac; prominent in humans) or N-glycolylneuraminic acid (Neu5Gc; prominent in ruminant mammals and rodents). Here, we report that A subunit phylogeny evolved independently of B subunits and suggest a future B subunit nomenclature based on bacterial species names. We also found via phylogenetic analysis of B subunits, which bind Sias, that homologous molecules show poor correlation with species phylogeny. These data indicate ongoing lateral gene transfers between species, including mixing of A and B subunits. Consistent with much broader host range of Y. pestis, we show that YpeB recognizes all mammalian Sia types, except for 4-O-acetylated ones. Notably, YpeB alone causes dose-dependent cytotoxicity, which is abolished by a mutation (Y77F) eliminating Sia recognition, suggesting that cell proliferation and death are promoted via lectin-like crosslinking of cell surface sialoglycoconjugates. These findings help explain the host range of Y. pestis and could be important for pathogenesis. Overall, our data indicate ongoing rapid evolution of both host Sias and pathogen toxin-binding properties.

Synthesis of structurally defined nitroxide spin-labeled glycolipids as useful probes for electron paramagnetic resonance (EPR) spectroscopy studies of cell surface glycans.

Four glycolipids carrying different glycans and a nitroxide free radical spin at the glycan non-reducing end were designed and synthesized from free glucose and maltooligosaccharides by an efficient and streamlined synthetic strategy. The main features of this synthetic strategy include regioselective functionalization of the free carbohydrates and coupling of the radical spin label with functionalized free glycans as the last synthetic step. These glycolipids are useful probes for the study of cell surface glycans by electron paramagnetic resonance spectroscopy. Moreover, the key synthetic intermediates, free glycolipids carrying a flexible azido group at the glycan non-reducing end, are widely useful platforms for accessing glycolipids with other molecular labels.

Glycoconjugate Nanoparticle-Based Systems in Cancer Immunotherapy: Novel Designs and Recent Updates.

For many years, cell-surface glycans (in particular, Tumor-Associated Carbohydrate Antigens, TACAs) have been the target of both passive and active anticancer immunotherapeutic design. Recent advances in immunotherapy as a treatment for a variety of malignancies has revolutionized anti-tumor treatment regimens. Checkpoint inhibitors, Chimeric Antigen Receptor T-cells, Oncolytic virus therapy, monoclonal antibodies and vaccines have been developed and many approvals have led to remarkable outcomes in a subset of patients. However, many of these therapies are very selective for specific patient populations and hence the search for improved therapeutics and refinement of techniques for delivery are ongoing and fervent research areas. Most of these agents are directed at protein/peptide epitopes, but glycans–based targets are gaining in popularity, and a handful of approved immunotherapies owe their activity to oligosaccharide targets. In addition, nanotechnology and nanoparticle-derived systems can help improve the delivery of these agents to specific organs and cell types based on tumor-selective approaches. This review will first outline some of the historical beginnings of this research area and subsequently concentrate on the last 5 years of work. Based on the progress in therapeutic design, predictions can be made as to what the future holds for increasing the percentage of positive patient outcomes for optimized systems.

Coordinated changes in glycosylation regulate the germinal center through CD22.

SUMMARYGerminal centers (GCs) are essential for antibody affinity maturation. GC B cells have a unique repertoire of cell surface glycans compared with naive B cells, yet functional roles for changes in glycosylation in the GC have yet to be ascribed. Detection of GCs by the antibody GL7 reflects a downregulation in ligands for CD22, an inhibitory co-receptor of the B cell receptor. To test a functional role for downregulation of CD22 ligands in the GC, we generate a mouse model that maintains CD22 ligands on GC B cells. With this model, we demonstrate that glycan remodeling plays a critical role in the maintenance of B cells in the GC. Sustained expression of CD22 ligands induces higher levels of apoptosis in GC B cells, reduces memory B cell and plasma cell output, and delays affinity maturation of antibodies. These defects are CD22 dependent, demonstrating that downregulation of CD22 ligands on B cells plays a critical function in the GC.