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Glyco Resources, Labs, and Companies

This sub-forum is for learning about and discussing Glycobiology resources both online and in the physical world
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Consortium for Functional Glycomics

by R Bishop
May 28, 2008, 10:27 AM


This sub-forum is for discussing all things related to Glycobiology including removal of glycans, glycan structure, O-GlcNAc, N-glycans, O-glycans, and proteoglycans
28 70 33466

glycan analysis

by Pippuri
Oct 04, 2013, 18:41 PM

Society Overview

The Society for Glycobiology is a non-profit scholarly society devoted to the pursuit of knowledge of glycan structures and functions, and to the sharing of that knowledge among scientists worldwide.  For more information, please visit The Society for Glycobiology homepage.

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N-linked glycosylation is an enzymatic reaction in which an oligosaccharide is transferred en bloc onto an asparagine residue of an acceptor polypeptide, catalyzed by oligosaccharyltransferase (OST). Despite the available crystal structures, the role of the external loop EL5, which is critical for the catalytic cycle, is enigmatic as EL5 in the crystal structures is partially absent or blocks a pathway of lipid-linked oligosaccharide to the active site. Here we report the molecular origin of EL5 conformational changes through a series of molecular dynamics simulations of a bacterial OST, Campylobacter lari PglB. The simulations reveal that the isoprenoid moiety of lipid-linked oligosaccharide favorably binds to a hydrophobic groove of the PglB transmembrane domain. This binding triggers the conformational changes of the transmembrane domain and subsequently impairs the structural stability of EL5, leading to disordered EL5 with open conformations that are required for correct placement of the oligosaccharide in the active site.
Bacterial levansucrases produce β(2,6)-linked levan-type polysaccharides using sucrose or sucrose analogs as donor/acceptor substrates. However, the dominant reaction of Bacillus megaterium levansucrase (Bm-LS) is hydrolysis. Single domain levansucrases from Gram-positive bacteria display a wide substrate-binding pocket with open access to water, challenging engineering for transfructosylation-efficient enzymes. We pursued a shift in reaction specificity by either modifying the water distribution in the active site or the coordination of the catalytic acid/base (E352) and the nucleophile (D95), thus affecting the fructosyl-transfer rate and allowing acceptors other than water to occupy the active site. Two serine (173/422) and two water-binding tyrosine (421/439) residues located in the first shell of the catalytic pocket were modified. Library variants of S173, Y421 and S422, which coordinate the position of D95 and E352, show increased transfructosylation (30–200%) and modified product spectra. Substitutions at position 422 have a higher impact on sucrose affinity, while changes at position 173 and 421 have a strong effect on the overall catalytic rate. As most retaining glycoside hydrolases (GHs) Bm-LS catalyzes hydrolysis and transglycosylation via a double displacement reaction involving two-transition states (TS1 and TS2). Hydrogen bonds of D95 with the side chains of S173 and S422 contribute a total of 2.4 kcal mol−1 to TS1 stabilization, while hydrogen bonds between invariant Y421, E352 and the glucosyl C-2 hydroxyl-group of sucrose contribute 2.15 kcal mol−1 stabilization. Changes at Y439 render predominantly hydrolytic variants synthesizing shorter oligosaccharides.
Wisteria floribunda agglutinin (WFA) is a useful probe for distinguishing glycan structural alterations in diseases such as intrahepatic bile duct carcinoma and hepatic fibrosis; however, the gene encoding WFA has not been identified. Here, we identified the gene encoding WFA, and recombinant WFA (rWFA) was expressed in Escherichia coli and purified. The natural complementary DNA sequence obtained from wisteria seeds contained an open reading frame of 861 nucleotides encoding a WFA precursor, which included a hydrophobic signal peptide at the N-terminus, a propeptide at the C-terminus and a single cysteine (Cys) residue for dimer formation. We characterized the natural and rWFA by the glycoconjugate microarray and frontal affinity chromatography. rWFA exhibited glycan binding specificity similar to that of natural WFA: both bound to Gal- and N-acetylgalactosamine (GalNAc)-terminated glycans. Moreover, the engineered WFA with an amino acid substitution in Cys-272 yielded a recombinant monomeric lectin with limited binding specificity but wild-type affinity for GalNAc-terminated glycans, specifically GalNAcβ1,4GlcNAc. Thus, this engineered lectin may be applied to highly sensitive biomarker detection.
N-acetylglucosaminyltransferase V (GnT-V), an enzyme that catalyzes the formation of the N-linked β-1-6 branching of oligosaccharides, is related to the radiosensitivity of nasopharyngeal carcinoma (NPC). Cetuximab (C225) is an epidermal growth factor receptor (EGFR) inhibitor used as a radiosensitizer in the treatment of NPC. In this study, we used GnT-V as a molecular target to further sensitize cetuximab-treated NPC cells to radiation. The results from two NPC cell lines (CNE1 and CNE2) revealed that the silencing of GnT-V enhanced cetuximab-induced radiosensitivity by decreasing the β-1-6 branching of oligosaccharides on the EGFR. GnT-V down-regulation combined with cetuximab decreased the survival fraction, healing rate and cell viability and increased the apoptosis rate. Concomitantly, the combination of cetuximab and irradiation did not change the EGFR mRNA and protein levels and decreased the β-1-6 branching on the EGFR. Subsequently, we further explored the signaling downstream of EGF, particularly the PI3K/Akt signaling pathway, and discovered that treatment consisting of GnT-V down-regulation, irradiation and cetuximab was negatively correlated with phospho-Akt and phspho-PI3K. Finally, an in vivo experiment with radiotherapy revealed that the combination of GnT-V down-regulation and cetuximab decelerated tumor growth. In summary, our study demonstrated that the combination of decreased GnT-V activity and cetuximab enhanced NPC radiosensitivity, and the possible mechanism underlying this effect might involve the N-linked β1-6 branching of the EGFR.
Retinopathy and nephropathy are life-threatening diabetic complications that decrease patient quality of life. Although the mechanisms underlying these conditions have been extensively studied, they remain unknown. Recent reports have demonstrated the presence of sodium glucose cotransporter 2 (SGLT2) in retinal pericytes and mesangial cells. Hyperglycemia results in functional and morphological changes in these cells, but these effects are attenuated by phlorizin, a nonselective SGLT inhibitor. Based on these findings, we hypothesized that SGLT2 plays a pivotal role in the development of diabetic nephropathy and retinopathy and that SGLT2 inhibitors may directly protect against these complications.
A symbiosis-related lectin, SLL-2, from the octocoral Sinularia lochmodes, distributes densely on the cell surface of microalgae, Symbiodinium sp., an endosymbiotic dinoflagellate of the coral, and is also shown to be a chemical cue that transforms dinoflagellates into a nonmotile (coccoid) symbiotic state. SLL-2 binds to the sugar chain of the molecule similar to Forssman antigen pentasaccharide (GalNAcα1-3GalNAcβ1-3 Galα1-4 Galβ1-4Glc) on the surface of microalgae with high affinity. Here we report the crystal structure of the complex between SLL-2 and Forssman antigen tetrasaccharide (GalNAcα1-3GalNAcβ1-3 Galα1-4 Galβ) at 3.4 Å resolution. In an asymmetric unit of the crystal, there are two hexameric molecules with totally 12 sugar recognition sites. At 9 in 12 sites, the first and second saccharides of the Forssman antigen tetrasaccharide bind directly to galactopyranoside binding site of SLL-2, whereas the third and fourth saccharides have no interaction with the SLL-2 hexameric molecule that binds the first saccharide. The sugar chain bends at α-1,4-glycosidic linkage between the third and fourth saccharides toward the position that we defined as a pyranoside binding site in the crystal structure of the complex between SLL-2 and GalNAc. The structure allowed us to suggest a possible binding mode of the Forssman antigen pentasaccharide to SLL-2. These observations support our hypothesis that the binding of SLL-2 to the cell surface sugars of zooxanthella in a unique manner might trigger some physiological changes of the cell to adapt symbiosis with the host coral.
Pyrobaculum calidifontis is a hyperthermophilic archaeon that belongs to the phylum Crenarchaeota. In contrast to the phylum Euryarchaeota, only the N-glycan structure of the genus Sulfolobus is known in Crenarchaeota. Here, we enriched glycoproteins from cultured P. calidifontis cells, by ConA lectin chromatography. The MASCOT search identified proteins with at least one potential N-glycosylation site. The tandem mass spectrometry (MS/MS) analysis of 12 small tryptic glycopeptides confirmed the canonical N-glycosylation consensus in P. calidifontis. We determined the N-linked oligosaccharide structure produced by an in vitro enzymatic oligosaccharyl transfer reaction. Pyrobaculum calidifontis cells were cultured in rich medium supplemented with 13C-glucose, for the metabolic labeling of N-oligosaccharide donors. An incubation with a synthetic peptide substrate produced glycopeptides with isotopically labeled oligosaccharide moieties. The MS and nuclear magnetic resonance analyses revealed that the P. calidifontisN-glycan has a biantennary, high-mannose-type structure consisting of up to 11 monosaccharide residues. The base portion of the P. calidifontisN-glycan strongly resembles the eukaryotic core structure, α-Man-(1-3)-(α-Man-(1-6)-)β-Man-(1-4)-β-GlcNAc-(1-4)-β-GlcNAc-Asn. Structural differences exist in the anomeric configuration between Man and GlcNAc, and the chitobiose structure is chemically modified: one GlcNAc residue is oxidized to glucoronate, and the GlcNAc residues are both modified with an additional acetamido group at the C-3 position. As a result, the core structure of the P. calidifontisN-glycan is α-Man-(1-3)-(α-Man-(1-6)-)α-Man-(1-4)-β-GlcANAc3NAc-(1-4)-β-GlcNAc3NAc-Asn, in which the unique features of the P. calidifontisN-glycan are underlined. In spite of these differences, the structure of the P. calidifontisN-glycan is the most similar to the eukaryotic counterparts, among all archaeal N-glycans reported to date.
The biosynthesis of eukaryotic lipid-linked oligosaccharides (LLOs) that act as donor substrates in eukaryotic protein N-glycosylation starts on the cytoplasmic side of the endoplasmic reticulum and includes the sequential addition of five mannose units to dolichol-pyrophosphate-GlcNAc2. These reactions are catalyzed by the Alg1, Alg2 and Alg11 gene products and yield Dol-PP-GlcNAc2Man5, an LLO intermediate that is subsequently flipped to the lumen of the endoplasmic reticulum. While the purification of active Alg1 has previously been described, Alg11 and Alg2 have been mostly studied in vivo. We here describe the expression and purification of functional, full length Alg2 protein. Along with the purified soluble domains Alg1 and Alg11, we used Alg2 to chemo-enzymatically generate Dol-PP-GlcNAc2Man5 analogs starting from synthetic LLOs containing a chitobiose moiety coupled to oligoprenyl carriers of distinct lengths (C10, C15, C20 and C25). We found that while the addition of the first mannose unit by Alg1 was successful with all of the LLO molecules, the Alg2-catalyzed reaction was only efficient if the acceptor LLOs contained a sufficiently long lipid tail of four or five isoprenyl units (C20 and C25). Following conversion with Alg11, the resulting C20 or C25 -containing GlcNAc2Man5 LLO analogs were successfully used as donor substrates of purified single-subunit oligosaccharyltransferase STT3A from Trypanosoma brucei. Our results provide a chemo-enzymatic method for the generation of eukaryotic LLO analogs and are the basis of subsequent mechanistic studies of the enigmatic Alg2 reaction mechanism.
Glycosyltransferases are essential tools for in vitro glycoengineering. Bacteria harbor an unexplored variety of protein glycosyltransferases. Here, we describe a peptide glycosyltransferase (EntS) encoded by ORF0417 of Enterococcus faecalis TX0104. EntS di-glycosylates linear peptide of enterocin 96 – a known antibacterial, in vitro. It is capable of transferring as well as extending the glycan onto the peptide in an iterative sequential dissociative manner. It can catalyze multiple linkages: Glc/Gal(-O)Ser/Thr, Glc/Gal(-S)Cys and Glc/Gal(β)Glc/Gal(-O/S)Ser/Thr/Cys, in one pot. Using EntS generated glycovariants of enterocin 96 peptide, size and identity of the glycan are found to influence bioactivity of the peptide. The study identifies EntS as an enzyme worth pursuing, for in vitro peptide glycoengineering.
Protein O-fucosyltransferase-1 (POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signaling are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1−/− background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.
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