Cellular Glycans Research Articles

Altered Glycan Expression on Breast Cancer Cells Facilitates Infection by T3 Seroptype Oncolytic Reovirus.

Breast cancer is the most common cancer in women. Although current therapies have increased survival rates for some breast cancer types, other aggressive invasive breast cancers remain difficult to treat. As the onset of breast cancer is often associated with the appearance of extracellular markers, these could be used to better target therapeutic agents. Here, we demonstrated by nanobiophysical approaches that overexpression of α-sialylated glycans in breast cancer provides an opportunity to combat cancer cells with oncolytic reoviruses. Notably, a correlation between cellular glycan expression and the mechanical properties of reovirus attachment and infection is observed in a serotype-dependent manner. Furthermore, we enhance the infectivity of reoviruses in malignant cells by the coinjection of α-sialylated glycans. In conclusion, this study supports both the use of reoviruses as an oncolytic agent in nanomedicine and the role of α-sialylated glycans as adjuvants in oncolysis, offering new perspective in oncolytic cancer therapy.

Glycan Labeling and Analysis in Cells and In Vivo.

As one of the major types of biomacromolecules in the cell, glycans play essential functional roles in various biological processes. Compared with proteins and nucleic acids, the analysis of glycans in situ has been more challenging. Herein we review recent advances in the development of methods and strategies for labeling, imaging, and profiling of glycans in cells and in vivo. Cellular glycans can be labeled by affinity-based probes, including lectin and antibody conjugates, direct chemical modification, metabolic glycan labeling, and chemoenzymatic labeling. These methods have been applied to label glycans with fluorophores, which enables the visualization and tracking of glycans in cells, tissues, and living organisms. Alternatively, labeling glycans with affinity tags has enabled the enrichment of glycoproteins for glycoproteomic profiling. Built on the glycan labeling methods, strategies enabling cell-selective and tissue-specific glycan labeling and protein-specific glycan imaging have been developed. With these methods and strategies, researchers are now better poised than ever to dissect the biological function of glycans in physiological or pathological contexts.

Hepatitis C Virus Glycan-Dependent Interactions and the Potential for Novel Preventative Strategies.

Chronic hepatitis C virus (HCV) infections continue to be a major contributor to liver disease worldwide. HCV treatment has become highly effective, yet there are still no vaccines or prophylactic strategies available to prevent infection and allow effective management of the global HCV burden. Glycan-dependent interactions are crucial to many aspects of the highly complex HCV entry process, and also modulate immune evasion. This review provides an overview of the roles of viral and cellular glycans in HCV infection and highlights glycan-focused advances in the development of entry inhibitors and vaccines to effectively prevent HCV infection.

Chemical reporters to study mammalian O-glycosylation.

Glycans play essential roles in a range of cellular processes and have been shown to contribute to various pathologies. The diversity and dynamic nature of glycan structures and the complexities of glycan biosynthetic pathways make it challenging to study the roles of specific glycans in normal cellular function and disease. Chemical reporters have emerged as powerful tools to characterise glycan structures and monitor dynamic changes in glycan levels in a native context. A variety of tags can be introduced onto specific monosaccharides via the chemical modification of endogenous glycan structures or by metabolic or enzymatic incorporation of unnatural monosaccharides into cellular glycans. These chemical reporter strategies offer unique opportunities to study and manipulate glycan functions in living cells or whole organisms. In this review, we discuss recent advances in metabolic oligosaccharide engineering and chemoenzymatic glycan labelling, focusing on their application to the study of mammalian O-linked glycans. We describe current barriers to achieving glycan labelling specificity and highlight innovations that have started to pave the way to overcome these challenges.

Application of the Inverse-Electron-Demand Diels-Alder Reaction for Metabolic Glycoengineering.

The inverse electron-demand Diels-Alder (IEDDA or DAinv) reaction is an emerging bioorthogonal ligation reaction that finds application in all areas of chemistry and chemical biology. In this review we highlight its application in metabolic glycoengineering (MGE). MGE is a versatile tool to introduce unnatural sugar derivatives that are modified with a chemical reporter group into cellular glycans. The IEDDA reaction can then be used to modify the chemical reporter group allowing, for instance, the visualization or isolation of glycoconjugates. During the last years, many different sugar derivatives as well as reporter groups have been published. These probes are summarized, and their chemical and biological properties are discussed. Furthermore, we discuss examples of MGE and subsequent IEDDA reaction that highlight its suitability for application within living systems.

Infection of porcine small intestinal enteroids with human and pig rotavirus A strains reveals contrasting roles for histo-blood group antigens and terminal sialic acids.

Rotaviruses (RVs) are a leading cause of acute viral gastroenteritis in young children and livestock worldwide. Growing evidence suggests that host cellular glycans, such as histo-blood group antigens (HBGAs) and sialic acids (SA), are recognized by the RV surface protein VP4. However, a mechanistic understanding of these interactions and their effects on RV infection and pathogenesis is lacking. Here, we established a porcine crypt-derived 3D intestinal enteroids (PIEs) culture system which contains all intestinal epithelial cells identified in vivo and represents a unique physiologically functional model to study RV-glycan interactions in vitro. PIEs expressing different HBGAs (A+, H+, and A+/H+) were established and isolation, propagation, differentiation and RV infection conditions were optimized. Differentiated PIEs were infected with human RV (HRV) G1P[8] Wa, porcine RV (PRV) G9P[13], PRV Gottfried G4P[6] or PRV OSU G5P[7] virulent and attenuated strains and virus replication was measured by qRT-PCR. Our results indicated that virulent HRV G1P[8] Wa replicated to the highest titers in A+ PIEs, while a distinct trend was observed for PRV G9P[13] or G5P[7] with highest titers in H+ PIEs. Attenuated Wa and Gottfried strains replicated poorly in PIEs while the replication of attenuated G9P[13] and OSU strains in PIEs was relatively efficient. However, the replication of all 4 attenuate strains was less affected by the PIE HBGA phenotypes. HBGA synthesis inhibitor 2-F-Peracetyl-Fucose (2F) treatment demonstrated that HBGAs are essential for G1P[8] Wa replication; however, they may only serve as a cofactor for PRVs G9P[13] and OSU G5P[7]. Interestingly, contrasting outcomes were observed following sialidase treatment which significantly enhanced G9P[13] replication, but inhibited the growth of G5P[7]. These observations suggest that some additional receptors recognized by G9P[13] become unmasked after removal of terminal SA. Overall, our results confirm that differential HBGAs-RV and SA-RV interactions determine replication efficacy of virulent group A RVs in PIEs. Consequently, targeting individual glycans for development of therapeutics may not yield uniform results for various RV strains.

Metabolic Glycoengineering with Azide‐ and Alkene‐Modified Hexosamines: Quantification of Sialic Acid Levels

Metabolic glycoengineering (MGE) is an established method to incorporate chemical reporter groups into cellular glycans for subsequent bioorthogonal labeling. The method has found broad application for the visualization and isolation of glycans allowing their biological roles to be probed. Furthermore, targeting of drugs to cancer cells that present high concentrations of sialic acids on their surface is an attractive approach. We report the application of a labeling reaction using 1,2‐diamino‐4,5‐methylenedioxybenzene for the quantification of sialic acid derivates after MGE with various azide‐ and alkene‐modified ManNAc, GlcNAc, and GalNAc derivatives. We followed the time course of sialic acid production and were able to detect sialic acids modified with the chemical reporter group – not only after addition of ManNAc derivatives to the cell culture. A cyclopropane‐modified ManNAc derivative, being a model for the corresponding cyclopropene analog, which undergoes fast inverse‐electron‐demand Diels‐Alder reactions with 1,2,4,5‐tetrazines, resulted in the highest incorporation efficiency. Furthermore, we investigated whether feeding the cells with natural and unnatural ManNAc derivative results in increased levels of sialic acids and found that this is strongly dependent on the investigated cell type and cell fraction. For HEK 293T cells, a strong increase in free sialic acids in the cell interior was found, whereas cell‐surface sialic acid levels are only moderately increased.

Thermally Triggered, Cell-Specific Enzymatic Glyco-Editing: In Situ Regulation of Lectin Recognition and Immune Response on Target Cells.

In situ glyco-editing on the cell surface can endow cellular glycoforms with new structures and properties; however, the lack of cell specificity and dependence on cells' endogenous functions plague the revelation of cellular glycan recognition properties and hamper the application of glyco-editing in complicated authentic biosystems. Herein, we develop a thermally triggered, cell-specific glyco-editing method for regulation of lectin recognition on target live cells in both single- and cocultured settings. The method relies on the aptamer-mediated anchoring of microgel-encapsulated neuraminidase on target cells and subsequent thermally triggered enzyme release for localized sialic acid (Sia) trimming. This temperature-based enzyme accessibility modulation strategy exempts genetic or metabolic engineering operations and, thus for the first time, enables tumor-specific desialylation on complicated tissue slices. The proposed method also provides an unprecedented opportunity to potentiate the innate immune response of natural killer cells toward target tumor cells through thermally triggered cell-specific desialylation, which paves the way for in vivo glycoimmune-checkpoint-targeted cancer therapeutic intervention.

JCPyV VP1 Mutations in Progressive MultifocalLeukoencephalopathy: Altering Tropismor Mediating Immune Evasion?

Polyomaviruses are ubiquitous human pathogens that cause lifelong, asymptomatic infections in healthy individuals. Although these viruses are restrained by an intact immune system, immunocompromised individuals are at risk for developing severe diseases driven by resurgent viral replication. In particular, loss of immune control over JC polyomavirus can lead to the development of the demyelinating brain disease progressive multifocal leukoencephalopathy (PML). Viral isolates from PML patients frequently carry point mutations in the major capsid protein, VP1, which mediates virion binding to cellular glycan receptors. Because polyomaviruses are non-enveloped, VP1 is also the target of the host’s neutralizing antibody response. Thus, VP1 mutations could affect tropism and/or recognition by polyomavirus-specific antibodies. How these mutations predispose susceptible individuals to PML and other JCPyV-associated CNS diseases remains to be fully elucidated. Here, we review the current understanding of polyomavirus capsid mutations and their effects on viral tropism, immune evasion, and virulence.

Differential Labeling of Glycoproteins with Alkynyl Fucose Analogs.

Fucosylated glycans critically regulate the physiological functions of proteins and cells. Alterations in levels of fucosylated glycans are associated with various diseases. For detection and functional modulation of fucosylated glycans, chemical biology approaches using fucose (Fuc) analogs are useful. However, little is known about how efficiently each unnatural Fuc analog is utilized by enzymes in the biosynthetic pathway of fucosylated glycans. We show here that three clickable Fuc analogs with similar but distinct structures labeled cellular glycans with different efficiency and protein specificity. For instance, 6-alkynyl (Alk)-Fuc modified O-Fuc glycans much more efficiently than 7-Alk-Fuc. The level of GDP-6-Alk-Fuc produced in cells was also higher than that of GDP-7-Alk-Fuc. Comprehensive in vitro fucosyltransferase assays revealed that 7-Alk-Fuc is commonly tolerated by most fucosyltransferases. Surprisingly, both protein O-fucosyltransferases (POFUTs) could transfer all Fuc analogs in vitro, likely because POFUT structures have a larger space around their Fuc binding sites. These findings demonstrate that labeling and detection of fucosylated glycans with Fuc analogs depend on multiple cellular steps, including conversion to GDP form, transport into the ER or Golgi, and utilization by each fucosyltransferase, providing insights into design of novel sugar analogs for specific detection of target glycans or inhibition of their functions.

Development of smart anti-glycan reagents using immunized lampreys

Studies on the expression of cellular glycans are limited by a lack of sensitive tools that can discriminate specific structural features. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus), which secrete variable lymphocyte receptors called VLRBs as antibodies, for generating libraries of anti-glycan reagents. We identified a wide variety of glycan-specific VLRBs detectable in lamprey plasma after immunization with whole fixed cells, tissue homogenates, and human milk. The cDNAs from lamprey lymphocytes were cloned into yeast surface display (YSD) libraries for enrichment by multiple methods. We generated VLRB-Ig chimeras, termed smart anti-glycan reagents (SAGRs), whose specificities were defined by microarray analysis and immunohistochemistry. 15 VLRB antibodies were discovered that discriminated between linkages, functional groups and unique presentations of the terminal glycan motif. The development of SAGRs will enhance future studies on glycan expression by providing sequenced, defined antibodies for a variety of research applications.

Various Tastes of Sugar: The Potential of Glycosylation in Targeting and Modulating Human Immunity via C-Type Lectin Receptors.

C-type lectin receptors (CLRs) are important in several immune regulatory processes. These receptors recognize glycans expressed by host cells or by pathogens. Whereas pathogens are recognized through their glycans, which leads to protective immunity, aberrant cellular glycans are now increasingly recognized as disease-driving factors in cancer, auto-immunity, and allergy. The vast variety of glycan structures translates into a wide spectrum of effects on the immune system ranging from immune suppression to hyper-inflammatory responses. CLRs have distinct expression patterns on antigen presenting cells (APCs) controlling their role in immunity. CLRs can also be exploited to selectively target specific APCs, modulate immune responses and enhance antigen presentation. Here we will discuss the role of glycans and their receptors in immunity as well as potential strategies for immune modulation. A special focus will be given to different dendritic cell subsets as these APCs are crucial orchestrators of immune responses in infections, cancer, auto-immunity and allergies. Furthermore, we will highlight the potential use of nanoscale lipid bi-layer structures (liposomes) in targeted immunotherapy.

A metabolically engineered spin-labeling approach for studying glycans on cells.

Metabolic glycan engineering (MGE) coupled with nitroxide spin-labeling (SL) was utilized to investigate the heterogeneous environment of cell surface glycans in select cancer and normal cells. This approach exploited the incorporation of azides into cell surface glycans followed by a click reaction with a new nitroxide spin label. Both sialic acid and N-acetylglucosamine (GlcNAc) were targeted for spin labelling. Although each of these moieties experiences a diverse and heterogeneous glycan environment, their EPR spectra and hence mobility are both characterized as a linear combination of two distinct spectra where one component reflects a highly mobile or uncrowded micro-environment with the second component reflecting more restricted motion, reflective of increased crowding and packing within the glycocalyx. What differs among the spectra of the targeted glycans is the relative percentage of each component, with sialic acid moieties experiencing on average an ∼80% less crowded environment, where conversely GlcNAc/GalNAz labeled sites reported on average a ∼50% more crowded environment. These distinct environments are consistent with the organization of sugar moieties within cellular glycans where some residues occur close to the cell membrane/protein backbone (i.e. more restricted) and others are more terminal in the glycan (i.e. more mobile). Strikingly, different cell lines displayed varied relative populations of these two components, suggesting distinctive glycan packing, organization, and composition of different cells. This work demonstrates the capability of SDSL EPR to be a broadly useful tool for studying glycans on cells, and interpretation of the results provides insights for distinguishing the differences and changes in the local organization and heterogeneity of the cellular glycocalyx.

Directed evolution of a remarkably efficient Kdnase from a bacterial neuraminidase.

N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid), which is the principal sialic acid family member of the non-2-ulosonic acids and their various derivatives, is often found at the terminal position on the glycan chains that adorn all vertebrate cells. This terminal position combined with subtle variations in structure and linkage to the underlying glycan chains between humans and other mammals points to the importance of this diverse group of nine-carbon sugars as indicators of the unique aspects of human evolution and is relevant to understanding an array of human conditions. Enzymes that catalyze the removal N-acetylneuraminic acid from glycoconjugates are called neuraminidases. However, despite their documented role in numerous diseases, due to the promiscuous activity of many neuraminidases, our knowledge of the functions and metabolism of many sialic acids and the effect of the attachment to cellular glycans is limited. To this end, through a concerted effort of generation of random and site-directed mutagenesis libraries, subsequent screens and positive and negative evolutionary selection protocols, we succeeded in identifying three enzyme variants of the neuraminidase from the soil bacterium Micromonospora viridifaciens with markedly altered specificity for the hydrolysis of natural Kdn (3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid) glycosidic linkages compared to those of N-acetylneuraminic acid. These variants catalyze the hydrolysis of Kdn-containing disaccharides with catalytic efficiencies (second-order rate constants: kcat/Km) of greater than 105M-1s-1; the best variant displayed an efficiency of >106M-1s-1 at its optimal pH.

Next-generation unnatural monosaccharides reveal that ESRRB O-GlcNAcylation regulates pluripotency of mouse embryonic stem cells

Unnatural monosaccharides such as azidosugars that can be metabolically incorporated into cellular glycans are currently used as a major tool for glycan imaging and glycoproteomic profiling. As a common practice to enhance membrane permeability and cellular uptake, the unnatural sugars are per-O-acetylated, which, however, can induce a long-overlooked side reaction, non-enzymatic S-glycosylation. Herein, we develop 1,3-di-esterified N-azidoacetylgalactosamine (GalNAz) as next-generation chemical reporters for metabolic glycan labeling. Both 1,3-di-O-acetylated GalNAz (1,3-Ac2GalNAz) and 1,3-di-O-propionylated GalNAz (1,3-Pr2GalNAz) exhibit high efficiency for labeling protein O-GlcNAcylation with no artificial S-glycosylation. Applying 1,3-Pr2GalNAz in mouse embryonic stem cells (mESCs), we identify ESRRB, a critical transcription factor for pluripotency, as an O-GlcNAcylated protein. We show that ESRRB O-GlcNAcylation is important for mESC self-renewal and pluripotency. Mechanistically, ESRRB is O-GlcNAcylated by O-GlcNAc transferase at serine 25, which stabilizes ESRRB, promotes its transcription activity and facilitates its interactions with two master pluripotency regulators, OCT4 and NANOG.

Emerging structural insights into glycosyltransferase-mediated synthesis of glycans.

Glycans linked to proteins and lipids play key roles in biology; thus, accurate replication of cellular glycans is crucial for maintaining function following cell division. The fact that glycans are not copied from genomic templates suggests that fidelity is provided by the catalytic templates of glycosyltransferases that accurately add sugars to specific locations on growing oligosaccharides. To form new glycosidic bonds, glycosyltransferases bind acceptor substrates and orient a specific hydroxyl group, frequently one of many, for attack of the donor sugar anomeric carbon. Several recent crystal structures of glycosyltransferases with bound acceptor substrates reveal that these enzymes have common core structures that function as scaffolds upon which variable loops are inserted to confer substrate specificity and correctly orient the nucleophilic hydroxyl group. The varied approaches for acceptor binding site assembly suggest an ongoing evolution of these loop regions provides templates for assembly of the diverse glycan structures observed in biology.

Alteration of the Total Cellular Glycome during Late Differentiation of Chondrocytes

In normal articular cartilage, chondrocytes do not readily proliferate or terminally differentiate, and exhibit a low level of metabolism. Hypertrophy-like changes of chondrocytes have been proposed to play a role in the pathogenesis of osteoarthritis by inducing protease-mediated cartilage degradation and calcification; however, the molecular mechanisms underlying these changes are unclear. Glycans are located on the outermost cell surface. Dynamic cellular differentiation can be monitored and quantitatively characterized by profiling the glycan structures of total cellular glycoproteins. This study aimed to clarify the alterations in glycans upon late differentiation of chondrocytes, during which hypertrophy-like changes occur. Primary mouse chondrocytes were differentiated using an insulin-induced chondro-osteogenic differentiation model. Comprehensive glycomics, including N-glycans, O-glycans, free oligosaccharides, glycosaminoglycan, and glycosphingolipid, were analyzed for the chondrocytes after 0-, 10- and 20-days cultivation. The comparison and clustering of the alteration of glycans upon hypertrophy-like changes of primary chondrocytes were performed. Comprehensive glycomic analyses provided complementary alterations in the levels of various glycans derived from glycoconjugates during hypertrophic differentiation. In addition, expression of genes related to glycan biosynthesis and metabolic processes was significantly correlated with glycan alterations. Our results indicate that total cellular glycan alterations are closely associated with chondrocyte hypertrophy and help to describe the glycophenotype by chondrocytes and their hypertrophic differentiation. our results will assist the identification of diagnostic and differentiation biomarkers in the future.

Hyperuricemia is Associated with Immunoglobulin G N-Glycosylation: A Community-Based Study of Glycan Biomarkers.

The increasing prevalence of hyperuricemia has been recognized as an emerging public health concern in both developed and developing countries. Hyperuricemia is a metabolic condition characterized by an elevated serum uric acid, and associated with renal damage, diabetes, autoimmune disorders, and cardiovascular diseases. Although human genetic variation has been recognized as a factor, posttranslational cellular processes and glycan biomarkers have not been studied extensively for susceptibility to hyperuricemia. We evaluated whether immunoglobulin (Ig)G N-glycans play a role in hyperuricemia in the general population. This cross-sectional study enrolled 635 participants (208 men and 427 women), ages ≥18 years, from a community-based population in Beijing, China. The IgG N-glycan composition of serum was analyzed by an ultraperformance liquid chromatography method. The prevalence of hyperuricemia observed in this sample was 5.98% (14.9% in men and 1.6% in women). Serum uric acid level was positively correlated with glycan peaks (GP)1, GP2, GP4, GP6, GP10, and GP11, whereas it was negatively correlated with GP12, GP13, GP14, GP15, GP18, and GP20. The combination of GP9, GP10, body mass index, and gender distinguished individuals with hyperuricemia from subjects without hyperuricemia, with an area under the curve value of 0.849 (95% confidence interval: 0.784-0.915). These findings collectively suggest a possible link between hyperuricemia and IgG N-glycans, which might be potentially mediated through inflammation-related mechanisms. Additional research on glycan biomarkers in independent and community-based population samples might allow the development of glycan diagnostics for hyperuricemia and gout in the future.

Analysis of UDP-Sugars from Cultured Cells and Small Tissue Samples.

UDP-sugars are important substrates for the synthesis of various cellular glycans and glycoconjugates, many of which play essential roles in the pathobiology of diseases associated with deranged glucose metabolism, such as cancer and type 2 diabetes. Hence, their analysis from cultured cells and especially from tissue samples can give valuable information. This chapter describes a method for UDP-sugar isolation from various sources utilizing ion-pair solid-phase extraction with graphitized carbon cartridges, and their analysis using anion-exchange high-performance liquid chromatography.

Understanding cellular glycan surfaces in the central nervous system.

Glycosylation, the enzymatic process by which glycans are attached to proteins and lipids, is the most abundant and functionally important type of post-translational modification associated with brain development, neurodegenerative disorders, psychopathologies and brain cancers. Glycan structures are diverse and complex; however, they have been detected and targeted in the central nervous system (CNS) by various immunohistochemical detection methods using glycan-binding proteins such as anti-glycan antibodies or lectins and/or characterized with analytical techniques such as chromatography and mass spectrometry. The glycan structures on glycoproteins and glycolipids expressed in neural stem cells play key roles in neural development, biological processes and CNS maintenance, such as cell adhesion, signal transduction, molecular trafficking and differentiation. This brief review will highlight some of the important findings on differential glycan expression across stages of CNS cell differentiation and in pathological disorders and diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, schizophrenia and brain cancer.