Glycoconjugate Journal

Separation and preparation of N -glycans based on ammonia-catalyzed release method Abstract The study of carbohydrates requires large amounts of glycans. N-Glycans can be synthesized but generating large quantities of N-glycans with diverse structures remains difficult. In this study, we aimed to obtain large amounts of glycans using an optimized procedure. Two types of reductive N-glycans were released from chicken egg albumin (ovalbumin) and soy protein using an ammonia catalysis method and labeled with benzenesulfonyl hydrazide (BSH). After preliminary separation by preparative HPLC, N-glycan-BSH components were de-labeled separately and reducing N-glycans were recovered. The de-labeled reducing N-glycans were derived with different labeling reagents and further separated and purified with two/multi-dimensional HPLC for various studies. We selected the bifunctional reagent 2-amino-N-(2-aminoethyl)-benzamide (AEAB) as a labeling reagent combined with C18 column for two-dimensional HPLC separation. A total of 21 and 8 N-glycan-AEAB conjugates were obtained from ovalbumin and soy protein, respectively. A reactive primary alkylamine of N-glycan-AEAB conjugates can be effectively immobilized on microarray surfaces, allowing for subsequent functional studies of glycans.

Advanced glycosylation end products (AGEs) controls proliferation, invasion and permeability through orchestrating ARHGAP18/RhoA pathway in human umbilical vein endothelial cells Abstract Diabetic vascular complications caused by endothelial dysfunction play an important role in the pathogenesis of diabetic foot. A well understanding of the role of endothelial dysfunction in diabetic foot vasculopathy will help to further reveal the pathogenesis of diabetic foot. This study aimed to assess whether the RhoA/ROCK signaling pathway is controlled by Rho GTPase-activating proteins (RhoGAP, ARHGAP) and advanced glycosylation end products (AGEs), and to clarify the roles of ARHGAP and AGEs in the RhoA/ROCK signaling pathway or the mechanism by which AGEs regulated RhoA. Real-time PCR was applied to detect gene expression. Manipulation of endothelial biological functions by ARHGAP18 and AGEs were studied via cell counting kit-8 (CCK-8), Western blot, transwell, FITC-Dextran and TEER permeability experiments. RhoA-specific inhibitor Y-27632 was used to silence the activity of RhoA. Dual Luciferase Reporter Assay, Western blot and ELISA assays were used to detect molecular mechanism of endothelial biological functions. In this study, we found that ARHGAP18 was negatively correlated with RhoA, and the expression of ARHGAP18 in human umbilical vein endothelial cells (HUVECs) was decreased with gradient-increased AGEs. Furthermore, AGEs and ARHGAP18 could orchestrate RhoA activity, then activate NF-κB signaling pathway, affect the structural and morphological of VE-cadherin and tight junction protein, and cause endothelial cell contraction, thereby increasing permeability of endothelial cells. In conclusion, AGEs and ARHGAP18 orchestrate cell proliferation, invasion and permeability by controlling the RhoA/ROCK signaling pathway, affecting NF-κB signaling pathway as well as the structure and morphology of VE-cadherin and tight junction protein, and regulating endothelial cell contraction.

Let’s talk about sexes: sex-related N-glycosylation in ecologically important invertebrates Abstract Parasitic helminths and pest insects are organisms with great ecological importance, having direct or indirect detrimental effects on people’s lives worldwide. Several reports in literature indicate that the glycan repertoire of parasites plays important roles in host-parasite interactions and modulation and evasion of the host immune system, while insect glycans are essential for their survival, growth and development. Although glycosylation is the result of a highly conserved machinery, differences between species and between different stages of one organism’s life cycle occur. This review provides insight into recent glycomics studies both for helminths and insects, focussing on sex differences and the role of carbohydrate structures in reproduction. Information on the differential N-glycosylation process between males and females can generate a better understanding of the biology and physiology of these economic important organisms, and can contribute to the discovery of novel anti-fecundity vaccine candidates and drug targets, as well as in the elaboration of innovative pest management strategies.

Relationship between heat treatment on structural properties and antitumor activity of the cold-water soluble polysaccharides from Grifola frondosa Abstract Grifola frondosa is a basidiomycete fungus with potential biomedical applications owing to the presence of bioactive polysaccharides. The activities of polysaccharides are influenced by many factors, particularly temperature; however, the optimal temperature and conditions for preparation of polysaccharides from this organism have not yet been determined. Therefore, in this study, cold-water soluble polysaccharides from Grifola frondosa were extracted at 4 °C (GFP-4) and purified. GFP-4-30, GFP-4-60 and GFP-4-90 were obtained from GFP-4 after treatment at 30 °C, 60 °C, or 90 °C, respectively, for 6 h. MTT results showed that GFP-4 had the highest inhibitory effects on the proliferation of SPC-A-1 cells in vitro. High-performance gel permeation chromatography results demonstrated that the molecular weight of GFP-4 was 1.05 × 106 Da and that GFP-4-30, GFP-4-60, and GFP-4-90 showed different levels of degradation and generated small molecule sugars. Fourier transform infrared spectroscopy, gas chromatography, and nuclear magnetic resonance results indicated that GFPs mainly consisted of α-d-Galp, α-d-Manp and α-d-Glcp. Periodate oxidation, Smith degradation, and methylation results showed that the backbones of the molecules consisted of 1,3-linked-Galp. After heat treatment, percentages of (1 → 3,4) α-d-Galp in heat-treated polysaccharides were obviously decreased, indicating their lower branching degree, and resulting in weaker antitumor effects. Overall, our findings demonstrated changes in the structure-activity relationships of GFP-4 after heat treatment and provided a theoretical basis for the application of GFP-4 in the food and drug industries.

Chemical structures of oligosaccharides in milks of the American black bear ( Ursus americanus americanus ) and cheetah ( Acinonyx jubatus ) Abstract The milk oligosaccharides were studied for two species of the Carnivora: the American black bear (Ursus americanus, family Ursidae, Caniformia), and the cheetah, (Acinonyx jubatus, family Felidae, Feliformia). Lactose was the most dominant saccharide in cheetah milk, while this was a minor saccharide and milk oligosaccharides predominated over lactose in American black bear milk. The structures of 8 neutral saccharides from American black bear milk were found to be Gal(β1–4)Glc (lactose), Fuc(α1–2)Gal(β1–4)Glc (2′-fucosyllactose), Gal(α1–3)Gal(β1–4)Glc (isoglobotriose), Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)Glc (B-tetrasaccharide), Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)[Fuc(α1–3)]Glc (B-pentasaccharide), Fuc(α1–2)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc (difucosyl lacto-N-neotetraose), Gal(α1–3)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc (monogalactosyl monofucosyl lacto-N-neotetraose) and Gal(α1–3)Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (Galili pentasaccharide). Structures of 5 acidic saccharides were also identified in black bear milk: Neu5Ac(α2–3)Gal(β1–4)Glc (3′-sialyllactose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Fuc(α1–2)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyl monofucosyl lacto-N-neohexaose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3)[Gal(α1–3)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (monosialyl monogalactosyl lacto-N-neohexaose), Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3){Gal(α1–3)Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–6)}Gal(β1–4)Glc (monosialyl monogalactosyl monofucosyl lacto-N-neohexaose), and Neu5Ac(α2–6)Gal(β1–4)GlcNAc(β1–3){Gal(α1–3)[Fuc(α1–2)]Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–6)}Gal(β1–4)Glc (monosialyl monogalactosyl difucosyl lacto-N-neohexaose). A notable feature of some of these milk oligosaccharides is the presence of B-antigen (Gal(α1–3)[Fuc(α1–2)]Gal), α-Gal epitope (Gal(α1–3)Gal(β1–4)Glc(NAc)) and Lewis x (Gal(β1–4)[Fuc(α1–3)]GlcNAc) structures within oligosaccharides. By comparison to American black bear milk, cheetah milk had a much smaller array of oligosaccharides. Two cheetah milks contained Gal(α1–3)Gal(β1–4)Glc (isoglobotriose), while another cheetah milk did not, but contained Gal(β1–6)Gal(β1–4)Glc (6′-galactosyllactose) and Gal(β1–3)Gal(β1–4)Glc (3′-galactosyllactose). Two cheetah milks contained Gal(β1–4)GlcNAc(β1–3)[Gal(β1–4)GlcNAc(β1–6)]Gal(β1–4)Glc (lacto-N-neohexaose), and one cheetah milk contained Gal(β1–4)Glc-3’-O-sulfate. Neu5Ac(α2–8)Neu5Ac(α2–3)Gal(β1–4)Glc (disialyllactose) was the only sialyl oligosaccharide identified in cheetah milk. The heterogeneity of milk oligosaccharides was found between both species with respect of the presence/absence of B-antigen and Lewis x. The variety of milk oligosaccharides was much greater in the American black bear than in the cheetah. The ratio of milk oligosaccharides-to-lactose was lower in cheetah (1:1–1:2) than American black bear (21:1) which is likely a reflection of the requirement for a dietary supply of N-acetyl neuraminic acid (sialic acid), in altricial ursids compared to more precocial felids, given the role of these oligosaccharides in the synthesis of brain gangliosides and the polysialic chains on neural cell adhesion.

Functional analysis of glycosylation using Drosophila melanogaster Abstract The glycosylation of proteins and lipids has various essential roles in a diverse range of biological processes, including embryogenesis, organ development, neurogenesis, maintenance of homeostasis, immune response, and tumorigenesis. Drosophila melanogaster is one of the representative multicellular model organisms, which have many useful genetic manipulation tools; it is used in developmental biology as well as classical and molecular genetics. Glycobiology is not an exception and many studies using Drosophila have been performed in this field to clarify novel functions of glycans. Recently, genome-wide screening and functional analyses were performed in whole body, wings, eyes, neuromuscular junctions, and immune organs. Furthermore, detailed studies with Drosophila mutants of glycosyltransferases, nucleotide sugar transporters, and glycosidases revealed novel functions of N-linked glycans, glycosaminoglycans, glycolipids, and O-linked glycans including mucin type O-glycan, O-Fuc, O-Man, and O-GlcNAc. As many of these functions are common between Drosophila and humans, these mutants represent good models for human disease. In this review, recent studies of glycan functions using Drosophila are summarized.

Glycan-binding profile of DC-like cells Abstract Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-“vector” it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling. It would be more convenient to employ long-term cell cultures or even primary DCs from murine blood. We therefore examined whether THP-1 (human monocyte cell line) and DC2.4 (immature murine DC-like cell line) could serve as a model for human DCs. These cells were probed with a set of glycans previously identified as binding to circulating human CD14low/-CD16+CD83+ DCs. In addition, we tested a subpopulation of murine CD14low/-CD80+СD11c+CD16+ cells reported as relating to the human CD14low/-CD16+CD83+ cells. Manα1–3(Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ bound to both the cell lines and the murine CD14low/-CD80+СD11c+CD16+ cells. Primary cells, but not the cell cultures, were capable of binding GalNAcα1–3Galβ (Adi), the most potent ligand for binding to human circulating DCs. In conclusion, not one of the studied cell lines proved an adequate model for DCs processes involving lectin binding. Although the glycan-binding profile of BYRB-Rb (8.17)1Iem mouse DCs could prove useful for assessing human DCs, important glycan interactions were missing, a situation which was aggravated when employing cells from the BALB/c strain. Accordingly, one must treat results from murine work with caution when seeking vaccine targeting of human DCs, and certainly should avoid cell lines such as THP-1 and DC2.4 cells.

Synthesis and biological roles of O-glycans in insects Abstract Protein O-glycosylation is the attachment of carbohydrate structures to the oxygen atom in the hydroxyl group of Serine and Threonine residues. This post-translational modification is commonly found on the majority of proteins trafficking through the secretory pathway and is reported to influence protein characteristics such as folding, secretion, stability, solubility, oligomerization and intracellular localization. In addition, O-glycosylation is essential for cell-cell interactions, protein-protein interactions and many biological processes, such as stress response, immunization, phosphorylation, ubiquitination, cell division, metabolism and cell signaling. The availability of sequenced genomes and genetic tools to create mutants with clear phenotypes makes insects an interesting model system to study O-glycosylation. In this review, we provide an overview of the current knowledge of O-glycosylation, mainly obtained from the model organism Drosophila melanogaster, with a focus on the synthesis and biological roles of the common O-glycans in insects.

Anionic and zwitterionic moieties as widespread glycan modifications in non-vertebrates Abstract Glycan structures in non-vertebrates are highly variable; it can be assumed that this is a product of evolution and speciation, not that it is just a random event. However, in animals and protists, there is a relatively limited repertoire of around ten monosaccharide building blocks, most of which are neutral in terms of charge. While two monosaccharide types in eukaryotes (hexuronic and sialic acids) are anionic, there are a number of organic or inorganic modifications of glycans such as sulphate, pyruvate, phosphate, phosphorylcholine, phosphoethanolamine and aminoethylphosphonate that also confer a ‘charged’ nature (either anionic or zwitterionic) to glycoconjugate structures. These alter the physicochemical properties of the glycans to which they are attached, change their ionisation when analysing them by mass spectrometry and result in different interactions with protein receptors. Here, we focus on N-glycans carrying anionic and zwitterionic modifications in protists and invertebrates, but make some reference to O-glycans, glycolipids and glycosaminoglycans which also contain such moieties. The conclusion is that ‘charged’ glycoconjugates are a widespread, but easily overlooked, feature of ‘lower’ organisms.

UDP- N -acetyl-α-D-galactosamine:polypeptide N -acetylgalactosaminyltransferase from the snail Biomphalaria glabrata – structural reflections Abstract UDP-GalNAc:polypeptide GalNAc transferase (ppGalNAcT; EC 2.4.1.41) is the initiating enzyme for mucin-type O-glycosylation in animals. Members of this highly conserved glycosyltransferase family catalyse a single glycosidic linkage. They transfer an N-acetylgalactosamine (GalNAc) residue from an activated donor (UDP-GalNAc) to a serine or threonine of an acceptor polypeptide chain. A ppGalNAcT from the freshwater snail Biomphalaria glabrata is the only characterised member of this enzyme family from mollusc origin. In this work, we interpret previously published experimental characterization of this enzyme in the context of in silico models of the enzyme and its acceptor substrates. A homology model of the mollusc ppGalNAcT is created and various substrate peptides are modelled into the active site. We hypothesize about possible molecular interpretations of the available experimental data and offer potential explanations for observed substrate and cofactor specificity. Here, we review and synthesise the current knowledge of Bge-ppGalNAcT, supported by a molecular interpretation of the available data.