A lectin microarray approach for the rapid analysis of bacterial glycans

Rapid evaluation of microbial cell-surface carbohydrates is essential to understanding the mechanisms by which bacteria use glycans to establish pathogenic or symbiotic relationships. Microbial glycan analysis is complicated both by the vast diversity of possible carbohydrate structures and by their dynamic nature. Bacteria can rapidly alter their glycan coats by switching the genes that are involved on and off in a phase-variable manner. Currently, there is a lack of appropriate tools for studying dynamic carbohydrate alterations. Here, we present a lectin microarray protocol for the high-throughput evaluation of cell-surface microbial sugars. The binding patterns of fluorescent bacteria to these arrays provide a simple means to fingerprint bacteria based on their surface carbohydrates. In addition, this method provides a rapid, parallel evaluation of glycans from multiple bacterial samples, allowing dynamic changes in carbohydrate structures to be studied. The entire procedure takes approximately 12 h but the printing of the microarray can be performed in advance.     

Chemistry-driven glycoscience

Carbohydrates are the most prominent features of the cell’s exterior—they are the cell’s “face” and serve as the cell’s identification card. The features of cell surface glycans (e.g. glycoproteins, glycolipids, polysaccharides) can be read by proteins, other cells, or organisms. In all of these contexts, glycan-binding proteins typically recognize (“read”) glycan identity. This recognition mediates important host-microbe interactions, as well as critical physiological functions, including fertilization, development, and immune system function. This article focuses on how proteins recognize glycans with an emphasis on three objectives: 1) to understand the molecular basis for carbohydrate recognition, 2) to implement that understanding to develop functional probes of protein-carbohydrate interactions, and 3) to apply those probes to elucidate and exploit the physiological consequences of protein–carbohydrate interactions. In this context, our group has focused on two key aspects of carbohydrate recognition: CH-π and multivalent interactions. We are applying the foundational knowledge gained from our studies for purposes ranging from illuminating host-microbe interactions to probing immune system function.     

Glycoengineering of host mimicking type‐2 LacNAc polymers and Lewis X antigens on bacterial cell surfaces

Bacterial carbohydrate structures play a central role in mediating a variety of host–pathogen interactions. Glycans can either elicit protective immune response or lead to escape of immune surveillance by mimicking host structures. Lipopolysaccharide (LPS), a major component on the surface of Gram‐negative bacteria, is composed of a lipid A‐core and the O‐antigen polysaccharide. Pathogens like Neisseria meningitidis expose a lipooligosaccharide (LOS), which outermost glycans mimick mammalian epitopes to avoid immune recognition. Lewis X (Galβ1–4(Fucα1–3)GlcNAc) antigens of Helicobacter pylori or of the helminth Schistosoma mansoni modulate the immune response by interacting with receptors on human dendritic cells. In a glycoengineering approach we generate human carbohydrate structures on the surface of recombinant Gram‐negative bacteria, such as Escherichia coli and Salmonella enterica sv. Typhimurium that lack O‐antigen. A ubiquitous building block in mammalian N‐linked protein glycans is Galβ1‐4GlcNAc, referred to as a type‐2 N‐acetyllactosamine, LacNAc, sequence. Strains displaying polymeric LacNAc were generated by introducing a combination of glycosyltransferases that act on modified lipid A‐cores, resulting in efficient expression of the carbohydrate epitope on bacterial cell surfaces. The poly‐LacNAc scaffold was used as an acceptor for fucosylation leading to polymers of Lewis X antigens. We analysed the distribution of the carbohydrate epitopes by FACS, microscopy and ELISA and confirmed engineered LOS containing LacNAc and Lewis X repeats by MALDI‐TOF and NMR analysis. Glycoengineered LOS induced pro‐inflammatory response in murine dendritic cells. These bacterial strains can thus serve as tools to analyse the role of defined carbohydrate structures in different biological processes.     

Perspectives for establishment and reorganization of carbohydrate-directed CD antibodies. Report of the carbohydrate section

Complex glycosylated glycoproteins, glycolipids and proteoglycans are expressed on the cell surface and are also found as constituents of the extracellular matrix (ECM). Interactions of the carbohydrate moiety of these macromolecules with specific receptors (lectins) are involved in many functions of immune cells such as cell-cell or cell-ECM adhesion, recognition, and neutralization of pathogens and regulation of apoptosis. For studies on live cells mAbs recognizing distinct oligosaccharide structures are useful tools because in contrast to other analytical methods of carbohydrate biochemistry they are able to react with glycans in the complex sterical context of the cell surface. In general expression patterns of carbohydrate mAbs depend on (i) the number and type of carriers to which the glycans are linked (glycoproteins, glycolipids), (ii) the steric situation on the cell surface, and (iii) modifications of the basic glycotope (different branching, chain length, masking by sialylation, sulphation or fucosylation).     

Recent progress in chemical synthesis of bacterial surface glycans

With the continuing advancement of carbohydrate chemical synthesis, bacterial glycomes have become increasingly attractive and accessible synthetic targets. Although bacteria also produce carbohydrate-containing secondary metabolites, our review here will cover recent chemical synthetic efforts on bacterial surface glycans. The obtained compounds are excellent candidates for the development of improved structurally defined glycoconjugate vaccines to combat bacterial infections. They are also important probes for investigating glycan–protein interactions. Glycosylation strategies applied for the formation of some challenging glycosidic bonds of various uncommon sugars in a number of recently synthesized bacterial surface glycans are highlighted.     

Dynamic responses of Bacteroides thetaiotaomicron during growth on glycan mixtures

Bacteroides thetaiotaomicron (Bt) is a human colonic symbiont that degrades many different complex carbohydrates (glycans), the identities and amounts of which are likely to change frequently and abruptly from meal‐to‐meal. To understand how this organism reacts to dynamic growth conditions, we challenged it with a series of different glycan mixtures and measured responses involved in glycan catabolism. Our results demonstrate that individual Bt cells can simultaneously respond to multiple glycans and that responses to new glycans are extremely rapid. The presence of alternative carbohydrates does not alter response kinetics, but reduces expression of some glycan utilization genes as well as the cell's sensitivity to glycans that are present in lower concentration. Growth in a mixture containing 12 different glycans revealed that Bt preferentially uses some before others. This metabolic hierarchy is not changed by prior exposure to lower priority glycans because re‐introducing high priority substrates late in culture re‐initiates repression of genes involved in degrading those with lower priority. At least some carbohydrate prioritization effects occur at the level of monosaccharide recognition. Our results provide insight into how a bacterial glycan generalist modifies its responses in dynamic glycan environments and provide essential knowledge to interpret related metabolic behaviour in vivo.     

Mass spectrometric strategies: providing structural clues for helminth glycoproteins

Here we review current knowledge of helminth glycans and introduce parasitologists to the power of the mass spectrometric techniques that have been largely responsible for defining their carbohydrate moieties. A brief overview of glycosylation in other eukaryotes is presented, with a focus on mammalian glycosylation, to facilitate understanding of how parasite structures might be recognized as 'self' or 'foreign' by the immune system of the host.     

Fine control of adenylate cyclase by the phosphoenolpyruvate:sugar phosphotransferase systems in Escherichia coli and Salmonella typhimurium.

Inhibition of cellular adenylate cyclase activity by sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system was reliant on the activities of the protein components of this enzyme system and on a gene designated crrA. In bacterial strains containing very low enzyme I activity, inhibition could be elicited by nanomolar concentrations of sugar. An antagonistic effect between methyl alpha-glucoside and phosphoenolpyruvate was observed in permeabilized Escherichia coli cells containing normal activities of the phosphotransferase system enzymes. In contrast, phosphoenolpyruvate could not overcome the inhibitory effect of this sugar in strains deficient for enzyme I or HPr. Although the in vivo sensitivity of adenylate cyclase to inhibition correlated with sensitivity of carbohydrate permease function to inhibition in most strains studied, a few mutant strains were isolated in which sensitivity of carbohydrate uptake to inhibition was lost and sensitivity of adenylate cyclase to regulation was retained. These results are consistent with the conclusions that adenylate cyclase and the carbohydrate permeases were regulated by a common mechanism involving phosphorylation of a cellular constituent by the phosphotransferase system, but that bacterial cells possess mechanisms for selectively uncoupling carbohydrate transport from regulation.     

The determination of the anomeric configuration of glycosyl-phosphoryl linkages of immunogenic phosphoglycans

Phosphoglycans from the cell wall of many strains of Streptococci contain terminal carbohydrate units linked by phosphodiester bridges to other residues of the glycans. In the immune response to phosphoglycans, the terminal carbohydrate-phosphate moieties function as antigenic determinants and induce the synthesis of antibodies with specificity for the glycosyl-phosphoryl units. It has now been found that such terminal carbohydrate units can be removed by treatment of the glycans with appropriate glycosidases. Thus, an almond beta-glucosidase releases glucose from a streptococcal Group D phosphoglycan with beta-glucosyl phosphate units, a jack bean N-acetyl-beta-glucosaminidase releases N-acetylglucosamine from a streptococcal Group L phosphoglycan with N-acetyl-beta-glucosaminyl phosphate units, and a rice alpha-glucosidase releases glucose from a yeast phosphoglycan with alpha-glucosyl phosphate units. The glycosidases also hydrolyze the hexose phosphates of the proper anomeric configuration and structure. The preparations of glycosidases used in this study exhibit specificity for single types of carbohydrate residues and are devoid of phosphatase and phosphodiesterase activities. The glycosidases act on glycosyl-phosphoryl linkages by a stereospecific mechanism and can therefore be used for the determination of the anomeric configuration of glycosyl-phosphoryl units of complex carbohydrates.     

Glycoprofiling with micro-arrays of glycoconjugates and lectins

To facilitate deciphering the information content in the glycome, thin film-coated photoactivatable surfaces were applied for covalent immobilization of glycans, glycoconjugates, or lectins in microarray formats. Light-induced immobilization of a series of bacterial exopolysaccharides on photoactivatable dextran-coated analytical platforms allowed covalent binding of the exopolysaccharides. Their specific galactose decoration was detected with fluorescence-labeled lectins. Similarly, glycoconjugates were covalently immobilized and displayed glycans were profiled for fucose, sialic acid, galactose, and lactosamine epitopes. The applicability of such platforms for glycan profiling was further tested with extracts of Caco2 epithelial cells. Following spontaneous differentiation or on pretreatment with sialyllactose, Caco2 cells showed a reduction of specific glycan epitopes. The changed glycosylation phenotypes coincided with altered enteropathogenic E. coli adhesion to the cells. This microarray strategy was also suitable for the immobilization of lectins through biotin-neutravidin-biotin bridging on platforms functionalized with a biotin derivatized photoactivatable dextran. All immobilized glycans were specifically and differentially detected either on glycoconjugate or lectin arrays. The results demonstrate the feasibility and versatility of the novel platforms for glycan profiling.     

A novel monoclonal antibody associated with glucoside kills gastric adenocarcinoma AGS cells based on glycosylation target

Glycosylation results in the production of glycans which are required for certain proteins to function. These glycans are also present on cell surfaces where they help maintain cell membrane integrity and are a key component of immune recognition. As such, cancer has been shown to alter glycosylation to promote tumour proliferation, invasion, angiogenesis, and immune envasion. Currently, there are few therapeutic monoclonal antibodies (mAb) which target glycosylation alterations in cancer. Here, we report a novel mAb associated with a glucoside, mAb 201E4, which is able induce cancer cell death and apoptosis based on a specific glycosylation target. This mAb evokes cancer cell death in vitro via caspase, fas, and mitochondrial associated apoptotic pathways. The efficacy of this mAb was further confirmed in vivo as treatment of mice with mAb 201E4 resulted in potent tumour shrinkage. Finally, the antibody was proven to be specific to glycosylation alterations in cancer and have no binding to normal tissues. This data indicates that mAb 201E4 successfully targets glycosylation alterations in neoplasms to induce cancer cell death, which may provide a new strategy for therapy in cancer.     

An engineered eukaryotic protein glycosylation pathway in Escherichia coli

We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins.     

Plant LysM proteins: modules mediating symbiosis and immunity

Microbial glycans, such as bacterial peptidoglycans, fungal chitin or rhizobacterial Nod factors (NFs), are important signatures for plant immune activation or for the establishment of beneficial symbioses. Plant lysin motif (LysM) domain proteins serve as modules mediating recognition of these different N-acetylglucosamine (GlcNAc)-containing ligands, suggesting that this class of proteins evolved from an ancient sensor for GlcNAc. During early plant evolution, these glycans probably served as immunogenic patterns activating LysM protein receptor-mediated plant immunity and stopping microbial infection. The biochemical potential of plant LysM proteins for sensing microbial GlcNAc-containing glycans has probably since favored the evolution of receptors facilitating microbial infection and symbiosis.     

Bacterial glycans: key mediators of diverse host immune responses

Recent studies have shown that the synthesis of various polysaccharides by bacteria can induce immune responses that are beneficial to the bacterium, the host, or both. Here, we discuss the diverse interactions between bacterial glycans and the host immune system.     

Novel glycan biomarkers for the detection of lung cancer

Lung cancer has a poor prognosis and a 5-year survival rate of 15%. Therefore, early detection is vital. Diagnostic testing of serum for cancer-associated biomarkers is a noninvasive detection method. Glycosylation is the most frequent post-translational modification of proteins and it has been shown to be altered in cancer. In this paper, high-throughput HILIC technology was applied to serum samples from 100 lung cancer patients, alongside 84 age-matched controls and significant alterations in N-linked glycosylation were identified. Increases were detected in glycans containing Sialyl Lewis X, monoantennary glycans, highly sialylated glycans and decreases were observed in core-fucosylated biantennary glycans, with some being detectable as early as in Stage I. The N-linked glycan profile of haptoglobin demonstrated similar alterations to those elucidated in the total serum glycome. The most significantly altered HILIC peak in lung cancer samples includes predominantly disialylated and tri- and tetra-antennary glycans. This potential disease marker is significantly increased across all disease groups compared to controls and a strong disease effect is visible even after the effect of smoking is accounted for. The combination of all glyco-biomarkers had the highest sensitivity and specificity. This study identifies candidates for further study as potential biomarkers for the disease.     

Dendritic cells and C-type lectin receptors: coupling innate to adaptive immune responses

Dendritic cells (DCs) have an important function in the initiation and differentiation of immune responses, linking innate information to tailored adaptive responses. Depending on the pathogen invading the body, specific immune responses are built up that are crucial for eliminating the pathogen from the host. Host recognition of invading microorganisms relies on evolutionarily ancient, germline-encoded pattern recognition receptors (PRRs) that are highly expressed on the cell surface of DCs, of which the Toll-like receptors (TLRs) are well characterized and recognize bacterial or viral components. Moreover, they bind a variety of self-proteins released from damaged tissues including several heat-shock proteins. The membrane-associated C-type lectin receptors (CLRs) recognize glycan structures expressed by host cells of the immune system or on specific tissues, which upon recognition allow cellular interactions between DCs and other immune or tissue cells. In addition, CLRs can function as PRRs. In contrast to TLRs, CLRs recognize carbohydrate structures present on the pathogens. Modification of glycan structures on pathogens to mimic host glycans can thereby alter CLR interactions that subsequently modifies DC-induced polarization. In this review, we will discuss in detail how specific glycosylation of antigens can dictate both the innate and adaptive interactions that are mediated by CLRs on DCs and how this balances immune activation and inhibition of DC function.     

Alterations in the Colonic Microbiota of Pigs Associated with Feeding Distillers Dried Grains with Solubles

In an effort to reduce feed costs, many pork producers have increased their use of coproducts of biofuel production in commercial pig diets, including increased feeding of distiller’s dried grains with solubles (DDGS). The inclusion of DDGS increases the insoluble fiber content in the ration, which has the potential to impact the colonic microbiota considerably as the large intestine contains a dynamic microenvironment with tremendous interplay between microorganisms. Any alteration to the physical or chemical properties of the colonic contents has the potential to impact the resident bacterial population and potentially favor or inhibit the establishment of pathogenic species. In the present study, colonic contents collected at necropsy from pigs fed either 30% or no DDGS were analyzed to examine the relative abundance of bacterial taxa associated with feeding this ingredient. No difference in alpha diversity (richness) was detected between diet groups. However, the beta diversity was significantly different between groups with feeding of DDGS being associated with a decreased Firmicutes:Bacteriodetes ratio (P = .004) and a significantly lower abundance of Lactobacillus spp. (P = .016). Predictive functional profiling of the microbiota revealed more predicted genes associated with carbohydrate metabolism, protein digestion, and degradation of glycans in the microbiota of pigs fed DDGS. Taken together, these findings confirm that alterations in dietary insoluble fiber significantly alter the colonic microbial profile of pigs and suggest the resultant microbiome may predispose to the development of colitis.     

Glycomics of human embryonic stem cells and human induced pluripotent stem cells

Most cells are coated by a dense glycocalyx composed of glycoconjugates such as glycosphingolipids, glycoproteins, and proteoglycans. The overall glycomic profile is believed to be crucial for the diverse roles of glycans, which are mediated by specific interactions that regulate cell-cell adhesion, the immune response, microbial pathogenesis, and other cellular events. Many cell surface markers were discovered and identified as glycoconjugates such as stage-specific embryonic antigen, Tra-1-60/81 and various other cell surface molecules (e.g., cluster of differentiation). Recent progress in the development of analytical methodologies and strategies has begun to clarify the cellular glycomics of various cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). The glycomic profiles of these cells are highly cell type-specific and reflect cellular alterations, such as development, differentiation and cancerous change. In this mini review, we briefly summarize the glycosylation spectra specific to hESCs and hiPSCs, which cover glycans of all major glycoconjugates (i.e., glycosphingolipids, N- and O-glycans of glycoproteins, and glycosaminoglycans) and free oligosaccharides.     

Solution NMR Analyses of the C-type Carbohydrate Recognition Domain of DC-SIGNR Protein Reveal Different Binding Modes for HIV-derived Oligosaccharides and Smaller Glycan Fragments

The C-type lectin DC-SIGNR (dendritic cell-specific ICAM-3-grabbing non-integrin-related; also known as L-SIGN or CD299) is a promising drug target due to its ability to promote infection and/or within-host survival of several dangerous pathogens (e.g. HIV and severe acute respiratory syndrome coronavirus (SARS)) via interactions with their surface glycans. Crystallography has provided excellent insight into the mechanism by which DC-SIGNR interacts with small glycans, such as (GlcNAc)₂Man₃; however, direct observation of complexes with larger, physiological oligosaccharides, such as Man₉GlcNAc₂, remains elusive. We have utilized solution-state nuclear magnetic resonance spectroscopy to investigate DC-SIGNR binding and herein report the first backbone assignment of its active, calcium-bound carbohydrate recognition domain. Direct interactions with the small sugar fragments Man₃, Man₅, and (GlcNAc)₂Man₃ were investigated alongside Man₉GlcNAc derived from recombinant gp120 (present on the HIV viral envelope), providing the first structural data for DC-SIGNR in complex with a virus-associated ligand, and unique binding modes were observed for each glycan. In particular, our data show that DC-SIGNR has a different binding mode for glycans on the HIV viral envelope compared with the smaller glycans previously observed in the crystalline state. This suggests that using the binding mode of Man₉GlcNAc, instead of those of small glycans, may provide a platform for the design of DC-SIGNR inhibitors selective for high mannose glycans (like those on HIV). ¹⁵N relaxation measurements provided the first information on the dynamics of the carbohydrate recognition domain, demonstrating that it is a highly flexible domain that undergoes ligand-induced conformational and dynamic changes that may explain the ability of DC-SIGNR to accommodate a range of glycans on viral surfaces.     

LacdiNAc-glycans constitute a parasite pattern for galectin-3-mediated immune recognition

Although Gal beta 1-4GlcNAc (LacNAc) moieties are the most common constituents of N-linked glycans on vertebrate proteins, GalNAc beta 1-4GlcNAc (LacdiNAc, LDN)-containing glycans are widespread in invertebrates, such as helminths. We postulated that LDN might be a molecular pattern for recognition of helminth parasites by the immune system. Using LDN-based affinity chromatography and mass spectrometry, we have identified galectin-3 as the major LDN-binding protein in macrophages. By contrast, LDN binding was not observed with galectin-1. Surface plasmon resonance (SPR) analysis and a solid phase binding assay demonstrated that galectin-3 binds directly to neoglycoconjugates carrying LDN glycans. In addition, galectin-3 bound to Schistosoma mansoni soluble egg Ags and a mAb against the LDN glycan inhibited this binding, suggesting that LDN glycans within S. mansoni soluble egg Ags contribute to galectin-3 binding. Immunocytochemistry demonstrated high levels of galectin-3 in liver granulomas of S. mansoni-infected hamsters, and a colocalization of galectin-3 and LDN glycans was observed on the parasite eggshells. Finally, we demonstrate that galectin-3 can mediate recognition and phagocytosis of LDN-coated particles by macrophages. These findings provide evidence that LDN-glycans constitute a parasite pattern for galectin-3-mediated immune recognition.