Dealing with the Ambiguity of Glycan Substructure Search

The level of ambiguity in describing glycan structure has significantly increased with the upsurge of large-scale glycomics and glycoproteomics experiments. Consequently, an ontology-based model appears as an appropriate solution for navigating these data. However, navigation is not sufficient and the model should also enable advanced search and comparison. A new ontology with a tree logical structure is introduced to represent glycan structures irrespective of the precision of molecular details. The model heavily relies on the GlycoCT encoding of glycan structures. Its implementation in the GlySTreeM knowledge base was validated with GlyConnect data and benchmarked with the Glycowork library. GlySTreeM is shown to be fast, consistent, reliable and more flexible than existing solutions for matching parts of or whole glycan structures. The model is also well suited for painless future expansion.

Production of galactosylated complex-type N-glycans in glycoengineered Saccharomyces cerevisiae

N-glycosylation is an important posttranslational modification affecting the properties and quality of therapeutic proteins. Glycoengineering in yeast aims to produce proteins carrying human-compatible glycosylation, enabling the production of therapeutic proteins in yeasts. In this work, we demonstrate further development and characterization of a glycoengineering strategy in a Saccharomyces cerevisiae Δalg3 Δalg11 strain where a truncated Man3GlcNAc2 glycan precursor is formed due to a disrupted lipid-linked oligosaccharide synthesis pathway. We produced galactosylated complex-type and hybrid-like N-glycans by expressing a human galactosyltransferase fusion protein both with and without a UDP-glucose 4-epimerase domain from Schizosaccharomyces pombe. Our results showed that the presence of the UDP-glucose 4-epimerase domain was beneficial for the production of digalactosylated complex-type glycans also when extracellular galactose was supplied, suggesting that the positive impact of the UDP-glucose 4-epimerase domain on the galactosylation process can be linked to other processes than its catalytic activity. Moreover, optimization of the expression of human GlcNAc transferases I and II and supplementation of glucosamine in the growth medium increased the formation of galactosylated complex-type glycans. Additionally, we provide further characterization of the interfering mannosylation taking place in the glycoengineered yeast strain. Key points • Glycoengineered Saccharomyces cerevisiae can form galactosylated N-glycans. • Genetic constructs impact the activities of the expressed glycosyltransferases. • Growth medium supplementation increases formation of target N-glycan structure.

Oxygen-Dependent Changes in the N-Glycome of Murine Pulmonary Endothelial Cells

Supplemental oxygen is frequently used together with mechanical ventilation to achieve sufficient blood oxygenation. Despite the undoubted benefits, it is vigorously debated whether too much oxygen can also have unpredicted side-effects. Uncertainty is also due to the fact that the molecular mechanisms are still insufficiently understood. The lung endothelium is covered with an exceptionally broad glycocalyx, carrying N- and O-glycans, proteoglycans, glycolipids and glycosaminoglycans. Glycan structures are not genetically determined but depend on the metabolic state and the expression level and activity of biosynthetic and glycan remodeling enzymes, which can be influenced by oxygen and the redox status of the cell. Altered glycan structures can affect cell interactions and signaling. In this study, we investigated the effect of different oxygen conditions on aspects of the glycobiology of the pulmonary endothelium with an emphasis on N-glycans and terminal sialylation using an in vitro cell culture system. We combined a proteomic approach with N-glycan structure analysis by LC-MS, qRT-PCR, sialic acid analysis and lectin binding to show that constant and intermittent hyperoxia induced time dependent changes in global and surface glycosylation. An siRNA approach identified St6gal1 as being primarily responsible for the early transient increase of α2-6 sialylated structures in response to hyperoxia.

SugarDrawer: A Web-Based Database Search Tool with Editing Glycan Structures

In life science fields, database integration is progressing and contributing to collaboration between different research fields, including the glycosciences. The integration of glycan databases has greatly progressed collaboration worldwide with the development of the international glycan structure repository, GlyTouCan. This trend has increased the need for a tool by which researchers in various fields can easily search glycan structures from integrated databases. We have developed a web-based glycan structure search tool, SugarDrawer, which supports the depiction of glycans including ambiguity, such as glycan fragments which contain underdetermined linkages, and a database search for glycans drawn on the canvas. This tool provides an easy editing feature for various glycan structures in just a few steps using template structures and pop-up windows which allow users to select specific information for each structure element. This tool has a unique feature for selecting possible attachment sites, which is defined in the Symbol Nomenclature for Glycans (SNFG). In addition, this tool can input and output glycans in WURCS and GlycoCT formats, which are the most commonly-used text formats for glycan structures.

Synthesis of N-Glycans Implicated in Asthma and Allergy

<p><b>Asthma and allergies affect a large number of people, with over 300 million people worldwide suffering from asthma alone. Although, on the ‟macroscopic‟ level, it is known how allergens trigger allergic reactions, it is not known how an allergen's ‟micro‟ structure causes such a profound allergic response in sensitised individuals. A review of inter-species carbohydrate motifs revealed a striking similarity between carbohydrate moieties (N-glycans) present on antigens derived from species known to give an allergic T helper (Th) 2 response in humans (such as pollen, schistosomes, and food allergens). Preliminary studies on mixtures of allergen extracts have suggested that these carbohydrate motifs (glycoproteins) bias the immune response to an allergic (Th2) response.</b></p> <p>This project presents work conducted towards the synthesis of three fragments of a larger N-glycan found on allergens. The synthesis of these N-glycans will allow the first detailed study regarding the relationship between N-glycan structure and Th2 bias to be performed and thereby aid in our understanding of the molecular triggers of asthma. Ultimately, this could lead to the elucidation of the mechanisms of the allergic Th2 immune response.</p>

Synthesis of N-Glycans Implicated in Asthma and Allergy

<p><b>Asthma and allergies affect a large number of people, with over 300 million people worldwide suffering from asthma alone. Although, on the ‟macroscopic‟ level, it is known how allergens trigger allergic reactions, it is not known how an allergen's ‟micro‟ structure causes such a profound allergic response in sensitised individuals. A review of inter-species carbohydrate motifs revealed a striking similarity between carbohydrate moieties (N-glycans) present on antigens derived from species known to give an allergic T helper (Th) 2 response in humans (such as pollen, schistosomes, and food allergens). Preliminary studies on mixtures of allergen extracts have suggested that these carbohydrate motifs (glycoproteins) bias the immune response to an allergic (Th2) response.</b></p> <p>This project presents work conducted towards the synthesis of three fragments of a larger N-glycan found on allergens. The synthesis of these N-glycans will allow the first detailed study regarding the relationship between N-glycan structure and Th2 bias to be performed and thereby aid in our understanding of the molecular triggers of asthma. Ultimately, this could lead to the elucidation of the mechanisms of the allergic Th2 immune response.</p>

Synthesis of N-glycans implicated in asthma and allergy

<p>Asthma and allergies affect a large number of people, with over 300 million people worldwide suffering from asthma alone. Although, on the ‟macroscopic‟ level, it is known how allergens trigger allergic reactions, it is not known how an allergen's ‟micro‟ structure causes such a profound allergic response in sensitised individuals. A review of inter-species carbohydrate motifs revealed a striking similarity between carbohydrate moieties (N-glycans) present on antigens derived from species known to give an allergic T helper (Th) 2 response in humans (such as pollen, schistosomes, and food allergens). Preliminary studies on mixtures of allergen extracts have suggested that these carbohydrate motifs (glycoproteins) bias the immune response to an allergic (Th2) response. This project presents work conducted towards the synthesis of three fragments of a larger N-glycan found on allergens. The synthesis of these N-glycans will allow the first detailed study regarding the relationship between N-glycan structure and Th2 bias to be performed and thereby aid in our understanding of the molecular triggers of asthma. Ultimately, this could lead to the elucidation of the mechanisms of the allergic Th2 immune response.</p>

Synthesis of N-glycans implicated in asthma and allergy

<p>Asthma and allergies affect a large number of people, with over 300 million people worldwide suffering from asthma alone. Although, on the ‟macroscopic‟ level, it is known how allergens trigger allergic reactions, it is not known how an allergen's ‟micro‟ structure causes such a profound allergic response in sensitised individuals. A review of inter-species carbohydrate motifs revealed a striking similarity between carbohydrate moieties (N-glycans) present on antigens derived from species known to give an allergic T helper (Th) 2 response in humans (such as pollen, schistosomes, and food allergens). Preliminary studies on mixtures of allergen extracts have suggested that these carbohydrate motifs (glycoproteins) bias the immune response to an allergic (Th2) response. This project presents work conducted towards the synthesis of three fragments of a larger N-glycan found on allergens. The synthesis of these N-glycans will allow the first detailed study regarding the relationship between N-glycan structure and Th2 bias to be performed and thereby aid in our understanding of the molecular triggers of asthma. Ultimately, this could lead to the elucidation of the mechanisms of the allergic Th2 immune response.</p>

Dealing with the Ambiguity of Glycan Substructure Search

The level of ambiguity in describing glycan structure has significantly increased with the upsurge of large scale glycomics and glycoproteomics experiments. Consequently, an ontology-based model appears as an appropriate solution for navigating this data. However, navigation is not sufficient and the model should also enable advanced search and comparison. A new ontology with a tree logical structure is introduced to represent glycan structures irrespective of the precision of molecular details. The model heavily relies on the GlycoCT encoding of glycan structures. Its implementation in the GlySTreeM knowledge base was validated with GlyConnect data and benchmarked with the Glycowork library. GlySTreeM is shown to be fast, consistent, reliable and more flexible than existing solutions for matching parts of or whole glycan structures. The model is also well suited to painless future expansion. Availability:https://glyconnect.expasy.org/glystreem/wiki