Diversity Of Glycans Research Articles

Glycans as shapers of tumour microenvironment: A sweet driver of T-cell-mediated anti-tumour immune response.

Essentially all cells are covered with a dense coat of different glycan structures/sugar chains, giving rise to the so-called glycocalyx. Changes in cellular glycosylation are a hallmark of cancer, affecting most of the pathophysiological processes associated with malignant transformation, including tumour immune responses. Glycans are chief macromolecules that define T-cell development, differentiation, fate, activation and signalling. Thus, the diversity of glycans expressed at the surface of T cells constitutes a fundamental molecular interface with the microenvironment by regulating the bilateral interactions between T-cells and cancer cells, fine-tuning the anti-tumour immune response. In this review, we will introduce the power of glycans as orchestrators of T-cell-mediated immune response in physiological conditions and in cancer. We discuss how glycans modulate the glyco-metabolic landscape in the tumour microenvironment, and whether glycans can synergize with immunotherapy as a way of rewiring T-cell effector functions against cancer cells.

High Diversity of Glycosphingolipid Glycans of Colorectal Cancer Cell Lines Reflects the Cellular Differentiation Phenotype.

Colorectal cancer (CRC)–associated changes of protein glycosylation have been widely studied. In contrast, the expression of glycosphingolipid (GSL) patterns in CRC has, hitherto, remained largely unexplored. Even though GSLs are major carriers of cell surface carbohydrates, they are understudied due to their complexity and analytical challenges. In this study, we provide an in-depth analysis of GSL glycans of 22 CRC cell lines using porous graphitized carbon nano–liquid chromatography coupled with electrospray ionization–mass spectrometry. Our data revealed that the GSL expression varies among different cell line classifications, with undifferentiated cell lines showing high expression of blood group A, B, and H antigens while for colon-like cell lines the most prominent GSL glycans contained (sialyl)-LewisA/X and LewisB/Y antigens. Moreover, the GSL expression correlated with relevant glycosyltransferases that are involved in their biosynthesis as well as with transcription factors (TFs) implicated in colon differentiation. Additionally, correlations between certain glycosyltransferases and TFs at mRNA expression level were found, such as FUT3, which correlated with CDX1, ETS2, HNF1A, HNF4A, MECOM, and MYB. These TFs are upregulated in colon-like cell lines pointing to their potential role in regulating fucosylation during colon differentiation. In conclusion, our study reveals novel layers of potential GSL glycans regulation relevant for future research in colon differentiation and CRC.

Turkeys possess diverse Siaα2-3Gal glycans that facilitate their dual susceptibility to avian influenza viruses isolated from ducks and chickens

Avian influenza viruses (AIVs) circulating in wild ducks are rarely transmitted directly to chickens. Previous studies demonstrated that chickens possess fucosylated and/or sulfated α2,3 sialosides on their tracheal epithelia, whereas intestinal epithelia of ducks express canonical α2,3 sialosides. Turkeys, the third major poultry species in the world, are known to show broad susceptibility to various avian influenza viruses. To elucidate the molecular basis of the broad susceptibility of turkeys to duck and chicken AIVs, we characterized various receptors for AIVs on their tissues. The experimental infection of turkeys demonstrated their dual susceptibility to duck and chicken AIVs. Further, comprehensive histochemical analyses using lectins, anti-glycan antibodies, and recombinant hemagglutinins, combined with glycosidase digestions, identified the presence of fucosylated and/or sulfated in addition to canonical α2,3 sialosides on their respiratory epithelia. The receptor distributions in turkeys were consistent with their dual susceptibility to duck and chicken AIVs. Also, our findings suggested the potential roles of turkeys in interspecies transmission of AIVs from ducks to chickens.

CarbArrayART: a new software tool for carbohydrate microarray data storage, processing, presentation, and reporting.

Glycan microarrays are essential tools in glycobiology and are being widely used for assignment of glycan ligands in diverse glycan recognition systems. We have developed a new software, called Carbohydrate microArray Analysis and Reporting Tool (CarbArrayART), to address the need for a distributable application for glycan microarray data management. The main features of CarbArrayART include: (i) Storage of quantified array data from different array layouts with scan data and array-specific metadata, such as lists of arrayed glycans, array geometry, information on glycan-binding samples, and experimental protocols. (ii) Presentation of microarray data as charts, tables, and heatmaps derived from the average fluorescence intensity values that are calculated based on the imaging scan data and array geometry, as well as filtering and sorting functions according to monosaccharide content and glycan sequences. (iii) Data export for reporting in Word, PDF, and Excel formats, together with metadata that are compliant with the guidelines of MIRAGE (Minimum Information Required for A Glycomics Experiment). CarbArrayART is designed for routine use in recording, storage, and management of any slide-based glycan microarray experiment. In conjunction with the MIRAGE guidelines, CarbArrayART addresses issues that are critical for glycobiology, namely, clarity of data for evaluation of reproducibility and validity.

Synthetic glycans control gut microbiome structure and mitigate colitis in mice

Relative abundances of bacterial species in the gut microbiome have been linked to many diseases. Species of gut bacteria are ecologically differentiated by their abilities to metabolize different glycans, making glycan delivery a powerful way to alter the microbiome to promote health. Here, we study the properties and therapeutic potential of chemically diverse synthetic glycans (SGs). Fermentation of SGs by gut microbiome cultures results in compound-specific shifts in taxonomic and metabolite profiles not observed with reference glycans, including prebiotics. Model enteric pathogens grow poorly on most SGs, potentially increasing their safety for at-risk populations. SGs increase survival, reduce weight loss, and improve clinical scores in mouse models of colitis. Synthetic glycans are thus a promising modality to improve health through selective changes to the gut microbiome.

Exploring the Candidate Terminal Glycan Profile in Neural Regeneration of the Sea Urchin Paracentrotus lividus, Using Lectin Blotting and Mass Spectrometry.

Glycans are expressed as conjugates of glycoproteins, glycolipids, and proteoglycans. The huge diversity of glycans on glycoconjugates contributes to many biological processes, from glycan-based molecular recognition to developmental events, such as regeneration in the nervous system. Echinoderms, which have a close phylogenetic relationship with chordates, are an important group of marine invertebrates for body regeneration. Although many major roles of glycans on glycoconjugates are known, their role in the glycosylation profile of the nervous system in sea urchins is poorly understood. In this study, we aimed to determine the terminal glycan profile by lectin blotting and to quantify sialic acids by the capillary liquid chromatography electrospray ionization tandem mass spectrometry system in the nervous tissue of the sea urchin Paracentrotus lividus. We determined the N-acetyl-D-glucosamine, mannose, and sialic acids (mainly α2,3 linked) by lectin blotting and five types of sialic acids (N-glycolylneuraminic acid, N-acetylneuraminic acid, 9-O-acetyl-N-alycolylneuraminic acid, 5-N-acetyl-9-O-acetyl-N-acetylneuraminic acid, and di-O-acetylated-N-alycolylneuraminic acid) by capillary liquid chromatography electrospray ionization tandem mass spectrometry. This potential first description of the terminal glycan profile in the nervous system of the sea urchin is expected to help us understand its role in nervous system development and regeneration.

Review] Studies on Structural and Functional Diversity of Mucin Glycans and Glycan Synthesis by Applying Microbial Enzymes

Review] Studies on Structural and Functional Diversity of Mucin Glycans and Glycan Synthesis by Applying Microbial Enzymes

N-Glycoprofiling of immunoglobulin G and lactoferrin with site-specificity from goat milk using RP-UHPLC MS/MS

Glycans present in glycoproteins are structurally diverse and contribute to the carbohydrate pool of the milk. Goat milk is a leading non-bovine milk source, wherein glycan diversity of several glycoproteins remains unexplored. Herein, site-specific N-glycoprofiling of two major glycoproteins – immunoglobulin G (IgG) and lactoferrin (Lf) from goat milk was performed through RP-UHPLC Q-Tof MS/MS approach. IgG revealed diverse complex glycans that were predominantly biantennary type with differential core fucosylation, bisecting GlcNAc, and mono/di- sialylation (NeuAc/NeuGc). The N-glycan repertoire of Lf at four sites indicated the range of high mannose, complex and hybrid types with varying abundances. High mannose glycans were specifically observed at N252NT and N564DT sites. Majorly complex glycans with fully sialylated were found at N387VT site. While N495QT site revealed complex and hybrid types with differential core fucosylation and sialylation. The glycan features observed in these glycoproteins would pave way for effective utilization as bioactive ingredients.

Simultaneously Identifying and Distinguishing Glycoproteins with O-GlcNAc and O-GalNAc (the Tn Antigen) in Human Cancer Cells.

Glycoproteins with diverse glycans are essential to human cells, and subtle differences in glycan structures may result in entirely different functions. One typical example is proteins modified with O-linked β-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc) (the Tn antigen), in which the two glycans have very similar structures and identical chemical compositions, making them extraordinarily challenging to be distinguished. Here, we developed an effective method benefiting from selective enrichment and the enzymatic specificity to simultaneously identify and distinguish glycoproteins with O-GlcNAc and O-GalNAc. Metabolic labeling was combined with bioorthogonal chemistry for enriching glycoproteins modified with O-GlcNAc and O-GalNAc. Then, the enzymatic reaction with galactose oxidase was utilized to specifically oxidize O-GalNAc, but not O-GlcNAc, generating the different tags between glycopeptides with O-GlcNAc and O-GalNAc that can be easily distinguishable by mass spectrometry (MS). Among O-GlcNAcylated proteins commonly identified in three types of human cells, those related to transcription and RNA binding are highly enriched. Cell-specific features are also revealed. Among glycoproteins exclusively in Jurkat cells, those involved in human T-lymphotropic virus type 1 (HTLV-1) infection are overrepresented, which is consistent with the cell line source and suggests that protein O-GlcNAcylation participated in the response to the virus infection. Furthermore, glycoproteins with the Tn antigen have different subcellular distributions in different cells, which may be attributed to the distinct mechanisms for the formation of protein O-GalNAcylation.

Assessment of Surface Glycan Diversity on Extracellular Vesicles by Lectin Microarray and Glycoengineering Strategies for Drug Delivery Applications (Small Methods 2/2022)

Back Cover In article number 2100785, Akiyoshi and co-workers assessed surface glycan diversity on extracellular vesicles (EVs) using an evanescent field fluorescence-assisted lectin microarray. This is a useful technique not only for highly sensitive determination of EV surface glycans but also profiling EV heterogeneity. Moreover, glycoengineered EVs can control cell-EV interactions for drug delivery applications.

Role of sialylation of N-linked glycans in prion pathogenesis.

Mammalian prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of the prion protein or PrPC. PrPC and PrPSc are posttranslationally modified with N-linked glycans, which are sialylated at the terminal positions. More than 30years have passed since the first characterization of the composition and structural diversity of N-linked glycans associated with the prion protein, yet the role of carbohydrate groups that constitute N-glycans and, in particular, their terminal sialic acid residues in prion disease pathogenesis remains poorly understood. A number of recent studies shed a light on the role of sialylation in the biology of prion diseases. This review article discusses several mechanisms by which terminal sialylation dictates the spread of PrPSc across brain regions and the outcomes of prion infection in an organism. In particular, relationships between the sialylation status of PrPSc and important strain-specific features including lymphotropism, neurotropism, and neuroinflammation are discussed. Moreover, emerging evidence pointing out the roles of sialic acid residues in prion replication, cross-species transmission, strain competition, and strain adaptation are reviewed. A hypothesis according to which selective, strain-specified recruitment of PrPC sialoglycoforms dictates unique strain-specific disease phenotypes is examined. Finally, the current article proposes that prion strains evolve as a result of a delicate balance between recruiting highly sialylated glycoforms to avoid an "eat-me" response by glia and limiting heavily sialylated glycoforms for enabling rapid prion replication.

Nanoglycocluster based diagnostic platform for colorimetric detection of bacteria; A comparative study analysing the effect of AuNPs size, linker length, and glycan diversity

Nanoglycoclusters, an upcoming class of functional nanomaterial are known to drive various processes like detection, imaging, targeting proteins, cells, and bacteria. Nanoglycoclusters are a type of nanomaterial functionalized with various glycans. The array of glycan in multiple copies enhances binding affinity with proteins. Selective and sensitive bacteria/lectin interactions using nanomaterials are an emerging area of research. The measurement of different ligand receptor interactions require sophisticated analytical tools that limit the application in biosensor domain. Recently, colorimetric biosensors gained importance in the field of the biosensor for the detection of bacteria/lectins. Herein we have demonstrated that different size of gold nanoparticles (AuNPs) along with various polyethylene glycol (PEG) linkers, functionalized with synthesized monopod and tripod of mannose and galactose that have different bacteria/lectins specificity. The newly synthesized nanoglycoclusters were able to discriminate between different lectins and bacteria. The aggregation of specific nanoglycocluster upon interaction with specific bacteria/lectins revealed that mannose monopod (MM) and mannose tripod (MT) are specific to Escherichia coli and concanavalin A (ConA) lectin, while galactose monopod (GM) and galactose tripod (GT) are specific to Pseudomonas aeruginosa and Peanut agglutinin (PNA) lectin. Further, the binding events depict the affinity of tripod glycans is more with respect to its corresponding monopod glycans. Our findings explored the potential of colorimetric sensing depending upon the size of AuNPs, linker length, specificity, along with glycans density to develop user friendly diagnostic system for the detection of bacteria.

Lactobacillus, glycans and drivers of health in the vaginal microbiome.

A microbiome consists of microbes and their genomes, encompassing bacteria, viruses, fungi, protozoa, archaea, and eukaryotes. These elements interact dynamically in the specific environment in which they reside and evolve. In the past decade, studies of various microbiomes have been prevalent in the scientific literature, accounting for the shift from culture-dependent to culture-independent identification of microbes using new high-throughput sequencing technologies that decipher their composition and sometimes provide insights into their functions. Despite tremendous advances in understanding the gut microbiome, relatively little attention has been devoted to the vaginal environment, notably regarding the ubiquity and diversity of glycans which denote the significant role they play in the maintenance of homeostasis. Hopefully, emerging technologies will aid in the determination of what is a healthy vaginal microbiome, and provide insights into the roles of Lactobacillus, glycans and microbiome-related drivers of health and disease.

Nutritional strategies for mucosal health: the interplay between microbes and mucin glycans.

Many aspects of the mechanisms underlying the symbiosis between humans and gut microbes remain unknown and encompass some of the most intriguing questions in microbiome research. An important factor in this symbiosis is the interplay between microbes and human-produced glycans in mucin and breast milk. In this Opinion paper, I propose a synergy between the structural diversity of human mucin glycans and the enzymatic repertoire of the gut microbiome. The contribution of microbes to mucosal health is discussed, and the role of breast milk glycans in mucosal colonization by microbes is explained. The use of prebiotic mucin glycans in general, and specialized infant and medical nutrition in particular, should be considered as the field of interest to modulate the microbiota and improve mucosal health.

Determining the metabolic fate of human milk oligosaccharides: it may just be more complex than you think?

Human milk oligosaccharides (HMOs) are a class of structurally diverse and complex unconjugated glycans present in breast milk, which act as selective substrates for several genera of select microbes and inhibit the colonisation of pathogenic bacteria. Yet, not all infants are breastfed, instead being fed with formula milks which may or may not contain HMOs. Currently, formula milks only possess two HMOs: 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT), which have been suggested to be similarly effective as human breast milk in supporting age-related growth. However, the in vivo evidence regarding their ability to beneficially reduce respiratory infections along with altering the composition of an infant's microbiota is limited at best. Thus, this review will explore the concept of HMOs and their metabolic fate, and summarise previous in vitro and in vivo clinical data regarding HMOs, with specific regard to 2'FL and LNnT.

Method for Identifying Galectin Ligands on Lymphocyte Membrane Glycoproteins.

Glycosylation is one of the most common protein posttranslational modifications. Most human lymphocyte membrane receptors are modified by diverse glycan structures, and functional studies have indicated that a family of glycan-binding proteins, galectins, can significantly modulate lymphocyte development and function by interacting with these glycans. Several galectins have a varying degree of affinity for the N-acetyllactosamine (LacNAc) disaccharide, and some critical lymphocyte receptors can be modified by glycan structures carrying this motif. However, the site-specific glycan composition on primary lymphocyte membrane receptors in healthy individuals is largely limited. The main reason for the limitation is low abundance of available material from a single donor and compositional heterogeneity in glycan structures that can potentially modify a protein. Donor-dependent variability in N-glycan structures on CD16a isolated from primary NK cells of healthy human donors was recently reported. NK cell CD16a is glycosylated at five N-glycosylation sites, and two of the five sites are modified, almost exclusively, by N-glycans with multiple LacNAc repeats which can serve as ligands for endogenous galectins. Thus, the protocol described in this section can be utilized to identify galectin ligands at specific glycosylation sites of endogenous membrane receptor from circulating primary human lymphocytes.

Sir Dai Rees (28 April 1936 to 10 June 2021)

Sir Dai Rees (28 April 1936 to 10 June 2021)

Differentiation of disaccharide isomers via a combination of IR and UV photodissociation mass spectrometry.

The challenge of glycan identification due to their structural complexity and diversity has profited enormously from recent developments in mass spectrometry (MS)-related methods. For photodissociation MS, infrared (IR) and ultraviolet (UV) lasers can generate complementary fragment ions, so an effective combination of the two methods may provide rich and valuable fragmentation patterns for glycan analysis. A 7.0T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer equipped with a double-beam laser system was applied for the experiments. 3,5-Diiodo-L-tyrosine was selected as the assistant molecule to form complex ions with ten isomeric disaccharides through electrospray ionization. The complex ions were further isolated and irradiated by IR and UV lasers separately or continuously in the FTICR cell. By combining the two complementary fragment spectra generated from the IR and UV lasers, a clear identification of all the ten isomers was achieved using their binary codes based on their fragmentation patterns. The double-beam method simplifies the experiment by introducing the two lasers sequentially in one experiment, providing richer fragmentation patterns and making the full discrimination easier. This study demonstrates the capabilities of the combination of IR and UV photodissociation MS in the identification of diverse glycan isomers. The double-beam photodissociation method described here distinguished compositional, configurational and connectivity disaccharide isomers successfully. Compared with the data accumulation method based on separate IR and UV experiments, this method is simpler, faster, more flexible and also characterized by richer fragmentation patterns.

Mechanistic insights into the digestion of complex dietary fibre by the rumen microbiota using combinatorial high-resolution glycomics and transcriptomic analyses

There is a knowledge gap regarding the factors that impede the ruminal digestion of plant cell walls or if rumen microbiota possess the functional activities to overcome these constraints. Innovative experimental methods were adopted to provide a high-resolution understanding of plant cell wall chemistries, identify higher-order structures that resist microbial digestion, and determine how they interact with the functional activities of the rumen microbiota. We characterized the total tract indigestible residue (TTIR) from cattle fed a low-quality straw diet using two comparative glycomic approaches: ELISA-based glycome profiling and total cell wall glycosidic linkage analysis. We successfully detected numerous and diverse cell wall glycan epitopes in barley straw (BS) and TTIR and determined their relative abundance pre- and post-total tract digestion. Of these, xyloglucans and heteroxylans were of higher abundance in TTIR. To determine if the rumen microbiota can further saccharify the residual plant polysaccharides within TTIR, rumen microbiota from cattle fed a diet containing BS were incubated with BS and TTIR ex vivo in batch cultures. Transcripts coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences encoded CAZymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan.This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can be extended to other feed-host systems and inform next-generation solutions to improve the performance of ruminants fed low-quality forages.

Assessment of Surface Glycan Diversity on Extracellular Vesicles by Lectin Microarray and Glycoengineering Strategies for Drug Delivery Applications.

Extracellular vesicles (EVs) are released by all types of mammalian cells for cell-cell communication. In this study, surface glycans on EVs are compared in terms of their cell type, size, and isolation method to examine whether EV glycan profiles by lectin microarray can be used to define EV subpopulations. Moreover, EVs are glycoengineered with four distinctive surface glycan patterns and evaluated their cellular uptake efficiencies for potential drug delivery applications. Both similarities and differences in glycan patterns are identified on EVs obtained under each experimental condition. EV size- and isolation method-dependent lectin-binding patterns are observed. Moreover, cellular uptake behaviors of EVs are affected by EV glycan profiles and acceptor cells. The in vivo biodistribution of EVs is also dependent on their glycan profile. These results suggest that EV surface glycans are a potential novel indicator of EV heterogeneity, and glycoengineering is a useful approach to regulate cell-EV interactions for biomedical applications.