Functions Of Glycans Research Articles

Rewiring the pneumococcal capsule pathway for investigating glycosyltransferase specificity and genetic glycoengineering.

Virtually all living cells are covered with glycans. Their structures are primarily controlled by the specificities of glycosyltransferases (GTs). GTs typically adopt one of the three folds, namely, GT-A, GT-B, and GT-C. However, what defines their specificities remain poorly understood. Here, we developed a genetic glycoengineering platform by reprogramming the capsular polysaccharide pathways in Streptococcus pneumoniae to interrogate GT specificity and manipulate glycan structures. Our findings suggest that the central cleft of GT-B enzymes is important for determining acceptor specificity. The constraint of the glycoengineering platform was partially alleviated when the specificity of the precursor transporter was reduced, indicating that the transporter contributes to the overall fidelity of glycan synthesis. We also modified the pneumococcal capsule to produce several medically important mammalian glycans, as well as demonstrated the importance of regiochemistry in a glycosidic linkage on binding lung epithelial cells. Our work provided mechanistic insights into GT specificity and an approach for investigating glycan functions.

Cholera intoxication of human enteroids reveals interplay between decoy and functional glycoconjugate ligands.

Prior research on cholera toxin (CT) binding and intoxication has relied on human colonic- cancer-derived epithelial cells. While these transformed cell lines have been beneficial, they neither derive from small intestine where intoxication occurs, nor represent the diversity of small-intestinal-epithelial-cells (SI-ECs) and variation in glycoconjugate expression among individuals. Here, we used human enteroids, derived from jejunal biopsies of multiple donors to study CT binding and intoxication of human non-transformed SI-ECs. We modulated surface expression of glycosphingolipids, glycoproteins and specific glycans to distinguish the role of each glycan/glycoconjugate. Cholera-toxin-subunit-B (CTB) mutants were generated to decipher the preference of each glycoconjugate to different binding sites and the correlation between CT binding and intoxication. Human enteroids contain trace amounts of GM1, but other glycosphingolipids may be contributing to CT intoxication. We discovered that inhibition of either fucosylation or O-glycosylation sensitize enteroids to CT-intoxication. This can either be a consequence of the removal of fucosylated "decoy-like-ligands" binding to CTB's non-canonical site and/or increase in the availability of Gal/GalNAc-terminating glycoconjugates binding to the canonical site. Furthermore, simultaneous inhibition of fucosylation and O-glycosylation increased the availability of additional Gal/GalNAc-terminating glycoconjugates but counteracts the sensitization in CT intoxication caused by inhibiting O-glycosylation because of reduction in fucose. This implies a dual role of fucose as a functional glycan and a decoy, the interplay of which influences CT binding and intoxication. Finally, while the results were similar for enteroids from different donors, they were not identical, pointing to a role for human genetic variation in determining sensitivity to CT.

Brain-specific glycosylation enzyme GnT-IX maintains levels of protein tyrosine phosphatase receptor PTPRZ, thereby mediating glioma growth

Gliomas are the most prevalent primary tumor of the CNS. Despite advances in imaging technologies, neurosurgical techniques, and radiotherapy, a cure for high-grade glioma remains elusive. Several groups have reported that protein tyrosine phosphatase receptor Z (PTPRZ) is highly expressed in glioblastoma, and that targeting PTPRZ attenuates tumor growth in mice. PTPRZ is modified with diverse glycan, including the PTPRZ-unique human natural killer-1 (HNK-1) capped O-Man core M2 glycans. However, the regulation and function of these unique glycans are unclear. Using CRISPR genome-editing technology we first demonstrated that disruption of the PTPRZ gene in human glioma LN-229 cells resulted in profoundly reduced tumor growth in xenografted mice, confirming the potential of PTPRZ as a therapeutic target for glioma. Furthermore, multiple glycan analyses revealed that PTPRZ derived from glioma patients and from xenografted glioma expressed abundant levels of HNK-1 capped O-Man glycans via extrinsic signals. Finally, since deficiency of O-Man core M2 branching enzyme N-acetylglucosaminyltransferase IX (GnT-IX) was reported to reduce PTPRZ protein levels, we disrupted the GnT-IX gene in LN-229 cells and found a significant reduction of glioma growth both in vitro and in the xenograft model. These results suggest that the PTPR glycosylation enzyme GnT-IX may represent a promising therapeutic target for glioma.

Practical Antibody Recruiting by Metabolic Labeling with Caged Glycans

AbstractWe propose a de novo glycan display approach that combines metabolic labeling and a glycan‐caging strategy as a facile editing method for cell‐surface glycans. This method enables the introduction of antigen glycans onto cancer cells to induce immune responses through antibody recruiting. The caging strategy prevents the capture of α‐rhamnose (an antigen glycan) by endogenous antibodies during the introduction of the glycan to the targeted cell surface, and subsequent uncaging successfully induces immune responses. Therefore, this study proposes a practical method for editing the cell‐surface glycocalyx under promiscuous conditions, such as those in vivo, which paves the way for the development of glycan function analysis and regulation.

Practical Antibody Recruiting by Metabolic Labeling with Caged Glycans.

We propose a de novo glycan display approach that combines metabolic labeling and a glycan-caging strategy as a facile editing method for cell-surface glycans. This method enables the introduction of antigen glycans onto cancer cells to induce immune responses through antibody recruiting. The caging strategy prevents the capture of α-rhamnose (an antigen glycan) by endogenous antibodies during the introduction of the glycan to the targeted cell surface, and subsequent uncaging successfully induces immune responses. Therefore, this study proposes a practical method for editing the cell-surface glycocalyx under promiscuous conditions, such as those in vivo, which paves the way for the development of glycan function analysis and regulation.

Recent Advances in Protein-Specific Glycan Imaging and Editing.

Glycosylation in live cell and animal modulate a constellation of biological functions. The advent of Chemical Biology has revolutionized the analysis and tailoring of glycans, by introducing myriads of glycan engineering methods. However, the ideal scenario to achieve glycan monitoring and structural manipulation at any hierarchical levels is unmet yet. Herein we review recent advances in the methodological innovation and the versatile applications of the protein-specific glycan visualization and editing in deciphering the biological functions of glycans. An outlook for future directions toward specific sugar-chain editing is also included.

Many Roles of Carbohydrates: A Computational Spotlight on the Coronavirus S Protein Binding.

Glycosylation is one of the post-translational modifications with more than 50% of human proteins being glycosylated. The exact nature and chemical composition of glycans are inaccessible to X-ray or cryo-electron microscopy imaging techniques. Therefore, computational modeling studies and molecular dynamics must be used as a "computational microscope". The spike (S) protein of SARS-CoV-2 is heavily glycosylated, and a few glycans play a more functional role "beyond shielding". In this mini-review, we discuss computational investigations of the roles of specific S-protein and ACE2 glycans in the overall ACE2-S protein binding. We highlight different functions of specific glycans demonstrated in myriad computational models and simulations in the context of the SARS-CoV-2 virus binding to the receptor. We also discuss interactions between glycocalyx and the S protein, which may be utilized to design prophylactic polysaccharide-based therapeutics targeting the S protein. In addition, we underline the recent emergence of coronavirus variants and their impact on the S protein and its glycans.

User-friendly bioorthogonal reactions click to explore glycan functions in complex biological systems.

User-friendly bioorthogonal reactions click to explore glycan functions in complex biological systems.

The 2022 Nobel Prize in Chemistry-sweet!

On 2022 October 5, The Royal Swedish Academy of Sciences announced its decision to award the Nobel Prize in Chemistry 2022 to Carolyn R. Bertozzi (Stanford University), Morten Meldal (University of Copenhagen), and K. Barry Sharpless (Scripps Research) “for the development of click chemistry and bioorthogonal chemistry.” The glycobiology community was exultant! To understand why, one must appreciate how challenges in glycoscience research motivated the development of these transformative chemical tools. Glycoscience has a storied history contributing significant discoveries to hematology, metabolism, microbiology, and other fields of biomedical research throughout much of the 20th century. But when the 1970s and 80s ushered in new molecular biology methods that accelerated studies of nucleic acid and protein function, studies of glycans began to lag behind (National Research Council 2012; Agre et al. 2016). Fortunately, since then, vast improvements have been made in areas such as carbohydrate synthesis and glycan arrays (Rillahan and Paulson 2011; Li and Bennett 2022), availability of glyco-enzymes and small molecule inhibitors (Moremen et al. 2018; Almahayni et al. 2022), mass spectrometry analysis of glycans and glycopeptides (Ruhaak et al. 2018; Suttapitugsakul et al. 2020; Bagdonaite et al. 2022), as well understanding of biological systems (Varki 2017), all of which have facilitated studies of glycan function. Notwithstanding these and other important advances, glycan detection has presented a recurrent challenge. Indeed, much of the modern practice of biological research hinges on the ability to specifically address a biomolecule of interest so that it can be isolated, visualized, or quantified. For nucleic acids, this can often be achieved via hybridization, whereas specific proteins can be detected and tracked using antibodies or protein tagging methods. But genetically encoded tags cannot be used to track glycans, which are not primary gene products. For the glycobiologist, lectins and antibodies have been essential tools but can suffer from limitations relating to specificity and affinity (Cummings et al. 2022).

Biomimetic Tweezers for N-Glycans: Selective Recognition of the Core GlcNAc2 Disaccharide of the Sialylglycopeptide SGP.

In recent years, glycomics have shown how pervasive the role of carbohydrates in biological systems is and how chemical tools are essential to investigate glycan function and modulate carbohydrate-mediated processes. Biomimetic receptors for carbohydrates can carry out this task but, although significant affinities and selectivities toward simple saccharides have been achieved, targeting complex glycoconjugates remains a goal yet unattained. In this work we report the unprecedented recognition of a complex biantennary sialylglycopeptide (SGP) by a tweezers-shaped biomimetic receptor, which selectively binds to the core GlcNAc2 disaccharide of the N-glycan with an affinity of 170 μM. Because of the simple structure and the remarkable binding ability, this biomimetic receptor can represent a versatile tool for glycoscience, opening the way to useful applications.

Live-Cell Glycocalyx Engineering.

A heavy layer of glycans forms a brush matrix bound to the outside of all the cells in our bodies; it is referred to as the "sugar forest" or glycocalyx. Beyond the increased appreciation of the glycocalyx over the past two decades, recent advances in engineering the glycocalyx on live cells have spurred the creation of cellular drugs and novel medical treatments. The development of new tools and techniques has empowered scientists to manipulate the structures and functions of cell-surface glycans on target cells and endow target cells with desired properties. Herein, we provide an overview of live-cell glycocalyx engineering strategies for controlling the cell-surface molecular repertory to suit therapeutic applications, even though the realm of this field remains young and largely unexplored.

Identification of global inhibitors of cellular glycosylation

Small molecule inhibitors of glycosylation enzymes are valuable tools for dissecting glycan functions and potential drug candidates. Screening for inhibitors of glycosyltransferases are mainly performed by in vitro enzyme assays with difficulties moving candidates to cells and animals. Here, we circumvent this by employing a cell-based screening assay using glycoengineered cells expressing tailored reporter glycoproteins. We focused on GalNAc-type O-glycosylation and selected the GalNAc-T11 isoenzyme that selectively glycosylates endocytic low-density lipoprotein receptor (LDLR)-related proteins as targets. Our screen of a limited small molecule compound library did not identify selective inhibitors of GalNAc-T11, however, we identify two compounds that broadly inhibited Golgi-localized glycosylation processes. These compounds mediate the reversible fragmentation of the Golgi system without affecting secretion. We demonstrate how these inhibitors can be used to manipulate glycosylation in cells to induce expression of truncated O-glycans and augment binding of cancer-specific Tn-glycoprotein antibodies and to inhibit expression of heparan sulfate and binding and infection of SARS-CoV-2.

Chemical Biology Study for Elucidating and Regulating Emergent Glycan Functions

Chemical Biology Study for Elucidating and Regulating Emergent Glycan Functions

N-acetylglucosaminyltransferase-V (GnT-V)-enriched small extracellular vesicles mediate N-glycan remodeling in recipient cells

Small extracellular vesicles (sEVs) secreted from cancer cells play pivotal roles in cancer metastasis and malignancy by transferring biomolecules and conditioning future metastatic sites. Studies have elucidated structures and functions of glycans on sEVs; however, whether sEVs remodel glycans in recipient cells remains poorly understood. Here, we examined the enzyme activity of glycosyltransferases for complex N-glycan biosynthesis in cancer-derived sEVs and discovered that cancer-related glycosyltransferase, N-acetylglucosaminyltransferase-V (GnT-V, a.k.a. MGAT5), is selectively enriched in sEVs among various glycosyltransferases. GnT-V in sEVs is a cleaved form, and cleavage by SPPL3 protease is necessary for loading GnT-V in sEVs. Fractionation experiments and single-particle imaging further revealed that GnT-V was enriched in non-exosomal sEVs. Strikingly, we found that enzymatically active GnT-V in sEVs was transferred to recipient cells and the N-glycan structures of recipient cells were remodeled to express GnT-V-produced glycans. Our results suggest GnT-V-enriched sEVs' role in glycan remodeling in cancer metastasis.

Target acquisition of anti-aging manno-oligosaccharide that triggers ECM process via TGF-β/Smads-SIRT1 signalling pathway

Glycans play a crucial role in a variety of physiological and pathological processes. In terms of skin, the sugar chain length, monosaccharide composition and structure of glycans change with age, and thus the changes in glycogens in skin cells are a potential biomarker of aging. The exogenous addition of structurally defined glycans is of great importance for delaying the skin aging process. Fortunately, a functional glycan named manno-oligosaccharide (DOMOS) from Dendrobium officinale was obtained herein by efficient enzymatic depolymerization and exerts anti-aging effects on human skin in vitro and in vivo together with human clinical studies. Further studies show that DOMOS exerts anti-aging effects by triggering the ECM process through a TGF-β/Smad-SIRT1 signalling pathway. This is the first study to concentrate on the beneficial effects of glycan degradation by a highly specific method on skin aging and provides an all-new solution to the skin aging problem that people are most concerned about.

Structural remodeling of SARS-CoV-2 spike protein glycans reveals the regulatory roles in receptor-binding affinity.

Glycans of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein are speculated to play functional roles in the infection processes as they extensively cover the protein surface and are highly conserved across the variants. The spike protein has been the principal target for vaccine and therapeutic development while the exact effects of its glycosylation remain elusive. Analytical reports have described the glycan heterogeneity of the spike protein. Subsequent molecular simulation studies provided a knowledge basis of the glycan functions. However, experimental data on the role of discrete glycoforms on the spike protein pathobiology remains scarce. Building an understanding of their roles in SARS-CoV-2 is important as we continue to develop effective medicines and vaccines to combat the disease. Herein, we used designed combinations of glycoengineering enzymes to simplify and control the glycosylation profile of the spike protein receptor-binding domain (RBD). Measurements of the receptor-binding affinity revealed opposite regulatory effects of the RBD glycans with and without sialylation, which presents a potential strategy for modulating the spike protein behaviors through glycoengineering. Moreover, we found that the reported anti-SARS-CoV-(2) antibody, S309, neutralizes the impact of different RBD glycoforms on the receptor-binding affinity. In combination with molecular dynamics simulation, this work reports the regulatory roles that glycosylation plays in the interaction between the viral spike protein and host receptor, providing new insights into the nature of SARS-CoV-2. Beyond this study, enzymatic glycan remodeling offers the opportunity to understand the fundamental role of specific glycoforms on glycoconjugates across molecular biology.

Characterization and Function of Glycans on the Spike Proteins of SARS-CoV-2 Variants of Concern.

SARS-CoV-2 variants of concern (VOCs) pose a great challenge to viral prevention and treatment owing to spike (S) protein mutations, which enhance their infectivity and capacity for immune evasion. However, whether these S protein mutations affect glycosylation patterns and thereby influence infectivity and immunogenicity remains unclear. In this study, four VOC S proteins-S-Alpha, S-Beta, S-Delta, and S-Omicron-were expressed and purified. Lectin microarrays were performed to characterize their glycosylation patterns. Several glycans were differentially expressed among the four VOC S proteins. Furthermore, the functional examination of glycans differentially expressed on S-Omicron revealed a higher expression of fucose-containing glycans, which modestly increased the binding of S-Omicron to angiotensin converting enzyme 2 (ACE2). A higher abundance of sialic acid and galactose-containing glycan was observed on S-Omicron, which significantly reduced its sensitivity against broad S protein-neutralizing antibodies. These findings contribute to the further understanding of SARS-CoV-2 infection mechanisms and provide novel glycan targets for emerging and future variants of SARS-CoV-2. IMPORTANCE Though glycosylation sites of SARS-CoV-2 S protein remain highly conserved, we confirmed that mutations in the Spike gene affect the S protein glycan expression pattern in different variants. More importantly, we found that glycans were differentially expressed on the S protein of the Omicron variant, enabling different forms of receptor binding and neutralization resistance. This study improves our understanding of SARS-CoV-2 glycomics and glycobiology and provides novel therapeutic and preventive strategies for SARS-CoV-2 VOCs.

Use of a glycomics array to establish the anti-carbohydrate antibody repertoire in type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease, characterized by the presence of autoantibodies to protein and non-protein antigens. Here we report the identification of specific anti-carbohydrate antibodies (ACAs) that are associated with pathogenesis and progression to T1D. We compare circulatory levels of ACAs against 202 glycans in a cross-sectional cohort of T1D patients (n = 278) and healthy controls (n = 298), as well as in a longitudinal cohort (n = 112). We identify 11 clusters of ACAs associated with glycan function class. Clusters enriched for aminoglycosides, blood group A and B antigens, glycolipids, ganglio-series, and O-linked glycans are associated with progression to T1D. ACAs against gentamicin and its related structures, G418 and sisomicin, are also associated with islet autoimmunity. ACAs improve discrimination of T1D status of individuals over a model with only clinical variables and are potential biomarkers for T1D.

Selective Exoenzymatic Labeling of Lipooligosaccharides of Neisseria gonorrhoeae with α2,6-Sialoside Analogues.

The interactions between bacteria and their host often rely on recognition processes that involve host or bacterial glycans. Glycoengineering techniques make it possible to modify and study the glycans on the host's eukaryotic cells, but only a few are available for the study of bacterial glycans. Here, we have adapted selective exoenzymatic labeling (SEEL), a chemical reporter strategy, to label the lipooligosaccharides of the bacterial pathogen Neisseria gonorrhoeae, using the recombinant glycosyltransferase ST6Gal1, and three synthetic CMP-sialic acid derivatives. We show that SEEL treatment does not affect cell viability and can introduce an α2,6-linked sialic acid with a reporter group on the lipooligosaccharides by Western blot, flow cytometry and fluorescent microscopy. This new bacterial glycoengineering technique allows for the precise modification, here with α2,6-sialoside derivatives, and direct detection of specific surface glycans on live bacteria, which will aid in further unravelling the precise biological functions of bacterial glycans.

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.