Mining the “glycocode”–exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy
- FASEB journal : official publication of the Federation of American Societies for Experimental Biology
- Published over 4 years ago
Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The binding specificity of lectins Ricinus communis agglutinin I (RCA), peanut (Arachis hypogaea) agglutinin (PNA), Maackia amurensis lectin II (MALII), and Ulex europaeus agglutinin I (UEA) was utilized in force spectroscopy measurements to quantify the affinity and spatial distribution of their cognate sugars at the molecular scale. Binding energy of 4, 1.6, and 26 aJ was determined on pPGM for RCA, PNA, and UEA. Binding was abolished by competition with free ligands, demonstrating the validity of the affinity data. The distributions of the nearest binding site separations estimated the number of binding sites in a 200-nm mucin segment to be 4 for RCA, PNA, and UEA, and 1.8 for MALII. Binding site separations were affected by partial defucosylation of pPGM. Furthermore, we showed that this new approach can resolve differences between gastric and jejunum mucins.-Gunning, A. P., Kirby, A. R., Fuell, C., Pin, C., Tailford L. E., Juge, N. Mining the “glycocode”-exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy.
The first step in influenza infection of the human respiratory tract is binding of the virus to sialic (Sia) acid terminated receptors. The binding of different strains of virus for the receptor is determined by the α linkage of the sialic acid to galactose and the adjacent glycan structure. In this study the N- and O-glycan composition of the human lung, bronchus and nasopharynx was characterized by mass spectrometry. Analysis showed that there was a wide spectrum of both Sia α2-3 and α2-6 glycans in the lung and bronchus. This glycan structural data was then utilized in combination with binding data from 4 of the published glycan arrays to assess whether these current glycan arrays were able to predict replication of human, avian and swine viruses in human ex vivo respiratory tract tissues. The most comprehensive array from the Consortium for Functional Glycomics contained the greatest diversity of sialylated glycans, but was not predictive of productive replication in the bronchus and lung. Our findings indicate that more comprehensive but focused arrays need to be developed to investigate influenza virus binding in an assessment of newly emerging influenza viruses.
- Current opinion in allergy and clinical immunology
- Published about 5 years ago
PURPOSE OF REVIEW: Siglec-8 and Siglec-F are single pass transmembrane inhibitory receptors found on the surface of human and mouse eosinophils, respectively, but very little is known about their physiologic glycan ligands. This article reviews the latest knowledge on this topic and outlines the strategies being used to further define the production and glycobiochemical nature of these molecules in the lung. RECENT FINDINGS: Both Siglec-8 and Siglec-F recognize the same glycan structure, namely 6'-sulfated sialyl Lewis X, as determined using glycan array technologies. Studies have identified α2,3-linked sialylated glycoprotein structures localized to mouse airway epithelium in tissue sections, where their constitutive expression requires the specific sialyltransferase St3gal3. Expression of these ligands in lung is enhanced during allergic inflammation and by cytokines such as IL-13, and is maintained in primary air-liquid interface cultures of mouse lung epithelium. Further characterization suggests that they are high molecular weight sialylated proteins, putatively mucins. By combining analytic glycomics, glycoproteomic mapping, and further in-vitro eosinophil experimentation including the ability of candidate structures to enhance eosinophil apoptosis, a finely detailed appreciation of the structural requirements for productive Siglec-8 and Siglec-F engagement should soon emerge. SUMMARY: An enhanced understanding of Siglec-F, Siglec-8, and their ligands should improve our understanding of endogenous lung pathways limiting the survival of eosinophils within the airway in diseases such as asthma. Knowledge of this biology may also result in novel opportunities for drug development involving glycans and glycomimetics that selectively bind to Siglec-8 and induce eosinophil death.
- Chembiochem : a European journal of chemical biology
- Published over 4 years ago
Seeing the sugar coating: N-Acetyl-glucosamine and mannosamine derivatives tagged with an isonitrile group are metabolically incorporated into cell-surface glycans and can be detected with a fluorescent tetrazine. This bioorthogonal isonitrile-tetrazine ligation is also orthogonal to the commonly used azide-cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars.
Seminal plasma aids sperm by inhibiting premature capacitation, helping in the intracervical transport and formation of oviductal sperm reservoir, all of which appear to be important in fertilization process. Epitopes such as Lewis x and y are known to be present on seminal plasma glycoproteins, which can modulate the maternal immune response. It is suggested by multiple studies that seminal plasma glycoproteins play, largely undiscovered, important roles in the process of fertilization. We have devised a strategy to analyze glycopeptides from a complex, unknown mixture of protease-digested proteins. This analysis provides identification for the glycoproteins, glycosylation sites, glycan compositions and proposed structures from the original sample. This strategy has been applied to human seminal plasma total glycoproteins. We have elucidated glycan compositions and proposed structures for 243 glycopeptides belonging to 73 N-glycosylation sites on 50 glycoproteins. Majority of the proposed glycan structures were complex type (83%) followed by high-mannose (10%) and then hybrid (7%). Most of the glycoproteins were either sialylated, fucosylated or both. Many Lewis x/a and y/b epitopes bearing glycans were found suggesting immune-modulating epitopes on multiple seminal plasma glycoproteins. The study also shows that large scale N-glycosylation mapping is achievable with current techniques and the depth of the analysis is roughly proportional to prefractionation and complexity of the sample.
The biological function of glycosphingolipids (GSLs) is largely determined by their glycan head group moiety. This has placed a renewed emphasis on detailed GSL head group structural analysis. Comprehensive profiling of GSL head groups in biological samples requires the use of endoglycoceramidases with broad substrate specificity and a robust workflow that enables their high-throughput analysis. We present here the first high-throughput glyco-analytical platform for GSL head group profiling. The workflow features enzymatic release of GSL glycans with a novel broad-specificity endoglycoceramidase I (EGCase I) from Rhodococcus triatomea, selective glycan capture on hydrazide beads on a robotics platform, 2AB-fluorescent glycan labelling and analysis by UPLC-HILIC-FLD. R. triatomea EGCase I displayed a wider specificity than known EGCases and was able to efficiently hydrolyze gangliosides, globosides, (n)Lc-type GSLs and cerebrosides. Our workflow was validated on purified GSL standard lipids and was applied to the characterization of GSLs extracted from several mammalian cell lines and human serum. This study should facilitate the analytical workflow in functional glycomics studies and biomarker discovery.
Despite sustained biomedical research effort, influenza A virus remains an imminent threat to the world population and a major healthcare burden. The challenge in developing vaccines against influenza is the ability of the virus to mutate rapidly in response to selective immune pressure. Hemagglutinin is the predominant surface glycoprotein and the primary determinant of antigenicity, virulence and zoonotic potential. Mutations leading to changes in the number of HA glycosylation sites are often reported. Such genetic sequencing studies predict at best the disruption or creation of sequons for N-linked glycosylation; they do not reflect actual phenotypic changes in HA structure. Therefore, combined analysis of glycan micro- and macro-heterogeneity and bioassays will better define the relationships among glycosylation, viral bioactivity and evolution. We present a study that integrates proteomics, glycomics and glycoproteomics of HA before and after adaptation to innate immune system pressure. We combined this information with glycan array and immune lectin binding data to correlate the phenotypic changes with biological activity. Underprocessed glycoforms predominated at the glycosylation sites found to be involved in viral evolution in response to selection pressures and interactions with innate immune-lectins. To understand the structural basis for site-specific glycan microheterogeneity at these sites, we performed structural modeling and molecular dynamics simulations. We observed that the presence of immature, high-mannose type glycans at a particular site correlated with reduced accessibility to glycan remodeling enzymes. Further, the high mannose glycans at sites implicated in immune lectin recognition were predicted to be capable of forming trimeric interactions with the immune-lectin surfactant protein-D.
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 1 year ago
Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions.
Glycans are known as the third major class of biopolymers, next to DNA and proteins. They cover the surfaces of many cells, serving as the ‘face’ of cells, whereby other biomolecules and viruses interact. The structure of glycans, however, differs greatly from DNA and proteins in that they are branched, as opposed to linear sequences of amino acids or nucleotides. Therefore, the storage of glycan information in databases, let alone their curation, has been a difficult problem. This has caused many duplicated efforts when integration is attempted between different databases, making an international repository for glycan structures, where unique accession numbers are assigned to every identified glycan structure, necessary. As such, an international team of developers and glycobiologists have collaborated to develop this repository, called GlyTouCan and is available at http://glytoucan.org/, to provide a centralized resource for depositing glycan structures, compositions and topologies, and to retrieve accession numbers for each of these registered entries. This will thus enable researchers to reference glycan structures simply by accession number, as opposed to by chemical structure, which has been a burden to integrate glycomics databases in the past.
Mapping posttranscriptional regulation of the human glycome uncovers microRNA defining the glycocode
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 3 years ago
Cell surface glycans form a critical interface with the biological milieu, informing diverse processes from the inflammatory cascade to cellular migration. Assembly of discrete carbohydrate structures requires the coordinated activity of a repertoire of proteins, including glycosyltransferases and glycosidases. Little is known about the regulatory networks controlling this complex biosynthetic process. Recent work points to a role for microRNA (miRNA) in the regulation of specific glycan biosynthetic enzymes. Herein we take a unique systems-based approach to identify connections between miRNA and the glycome. By using our glycomic analysis platform, lectin microarrays, we identify glycosylation signatures in the NCI-60 cell panel that point to the glycome as a direct output of genomic information flow. Integrating our glycomic dataset with miRNA data, we map miRNA regulators onto genes in glycan biosynthetic pathways (glycogenes) that generate the observed glycan structures. We validate three of these predicted miRNA/glycogene regulatory networks: high mannose, fucose, and terminal β-GalNAc, identifying miRNA regulation that would not have been observed by traditional bioinformatic methods. Overall, our work reveals critical nodes in the global glycosylation network accessible to miRNA regulation, providing a bridge between miRNA-mediated control of cell phenotype and the glycome.