EUROCarbDB: An open-access platform for glycoinformatics.

The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb.

Insights into the dynamics and molecular recognition features of glycopeptides by protein receptors: the 3D solution structure of hevein bound to the trisaccharide core of N-glycoproteins.

Protein-carbohydrate interactions are at the heart of a variety of essential molecular recognition events. Hevein, a model lectin related to the superantigen family, recognizes the trisaccharide core of N-glycoproteins (1). A combined approach of NMR spectroscopy and molecular modeling has permitted us to demonstrate that an Asn-linked Man(GlcNAc)(2) (2) is bound with even higher affinity than (GlcNAc)(3). The molecular recognition process entails conformational selection of only one of the possibilities existing for chitooligosaccharides. The deduced 3D structure of the hevein/2 complex permits the extension of polypeptide chains from the Asn moiety of 2, as well as glycosylation at Man O-3 and Man O-6 of the terminal sugar. Given the ubiquity of the Man(GlcNAc)(2) core in all mammalian N-glycoproteins, the basic recognition mode presented herein might be extended to a variety of systems with biomedical importance.

C-Mannosylation Enhances the Structural Stability of Human RNase 2.

C-Mannosylation is a relatively rare form of protein glycosylation involving the attachment of an α-mannopyranosyl residue to C-2 of the indole moiety of the amino acid tryptophan. This type of linkage was initially discovered in RNase 2 from human urine but later confirmed to be present in many other important proteins. Based on NMR experiments and extensive molecular dynamics simulations on the hundred microsecond timescale we demonstrate that, for isolated glycopeptides and denatured RNase 2, the C-linked mannopyranosyl residue exists as an ensemble of conformations, among which 1C4 is the most abundant. However, for native RNase 2, molecular dynamics and NMR studies revealed that the mannopyranosyl residue favors a specific conformation, which optimally stabilizes the protein fold through a network of hydrogen bonds and which leads to a significant reduction of the protein dynamics on the microsecond timescale. Our findings contribute to the understanding of the biological role of C-mannosylation.

Development of a 1H NMR structural-reporter-group concept for the primary structural characterisation of alpha-D-glucans.

An NMR study of proton chemical shift patterns of known linear alpha-D-glucopyranose di- and trisaccharide structures was carried out. Chemical shift patterns for (alpha1-->2)-, (alpha1-->3)-, (alpha1-->4)- and (alpha1-->6)-linked D-glucose residues were analysed and compared to literature data. Using these data, a 1H NMR structural-reporter-group concept was formulated to function as a tool in the structural analysis of alpha-D-glucans.

The European lipidomics initiative: enabling technologies.

Lipidomics is a new term to describe a scientific field that is a lot broader than lipidology, the science of lipids. Besides lipidology, lipidomics covers the lipid-metabolizing enzymes and lipid transporters, their genes and regulation; the quantitative determination of lipids in space and time, and the study of lipid function. Because lipidomics is concerned with all lipids and their enzymes and genes, it faces the formidable challenge to develop enabling technologies to comprehensively measure the expression, location, and regulation of lipids, enzymes, and genes in time, including high-throughput applications. The second challenge is to devise information technology that allows the construction of metabolic maps by browsing through connected databases containing the subsets of data in lipid structure, lipid metabolomics, proteomics, and genomics. In addition, to understand lipid function, on the one hand we need a broad range of imaging techniques to define where exactly the relevant events happen in the body, cells, and subcellular organelles; on the other hand, we need a thorough understanding of how lipids physically interact, especially with proteins. The final challenge is to apply this knowledge in the diagnosis, monitoring, and cure of lipid-related diseases.

Oxygenated metabolites of anandamide and 2-arachidonoylglycerol: conformational analysis and interaction with cannabinoid receptors, membrane transporter, and fatty acid amide hydrolase.

This study was aimed at finding structural requirements for the interaction of the acyl chain of endocannabinoids with cannabinoid receptors, membrane transporter protein, and fatty acid amide hydrolase (FAAH). To this end, the flexibility of the acyl chain was restricted by introduction of an 1-hydroxy-2Z,4E-pentadiene system in anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) at various positions using different lipoxygenases. This brought about selectivity and attenuated the binding potency of AEA and 2-AG. Although the displacement constants were modest, 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoyl-N-(2-hydroxyethyl)amine was found to bind selectively to the CB(1) receptor, whereas its 1-arachidonoyl-sn-glycerol analogue and 13(S)-hydroxy-octadeca-9Z,11E-dienoyl-N-(2-hydroxyethyl)amine could selectively bind to the CB(2) receptor. 11(S)-Hydroxy-eicosa-5Z,8Z,12E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did not bind to either receptor, whereas 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine did bind to both CB receptors with an affinity similar to that of AEA. All oxygenated anandamide derivatives were good inhibitors of FAAH (low micromolar K(i)) but were ineffective on the AEA transporter. 2-AG rapidly isomerizes into 1(3)-arachidonoyl-sn-glycerol. Both 1- and 3-arachidonoyl-sn-glycerol did not bind to either CB receptor and did not interfere with AEA transport. Thus, after it is isomerized, 2-AG is inactivated, thereby decreasing effective concentrations of 2-AG. Analysis of (1)H NMR spectra revealed that chloroform did not induce notably different conformations in the acyl chain of 15(S)-hydroxy-eicosa-5Z,8Z,11Z,13E-tetraenoic acid as compared with water. Molecular dynamics (MD) simulations of AEA and its analogues in the presence of explicit water molecules revealed that a tightly folded conformation of the acyl chain is not the only requirement for CB(1) binding. Structural details of the C(2)-C(15) loop, such as an sp(2) carbon at position 11, are necessary for receptor binding. The MD simulations may suggest that the average orientations of the pentyl tail of AEA and 12(S)-hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2-hydroxyethyl)amine are different from that of the low-affinity, inactive ligands.

The structural basis of the difference in sensitivity for PNGase F in the de-N-glycosylation of the native bovine pancreatic ribonucleases B and BS.

In glycoanalysis protocols, N-glycans from glycoproteins are most frequently released with peptide- N (4)-( N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F). As the enzyme is an amidase, it cleaves the NH-CO linkage between the Asn side chain and the Asn-bound GlcNAc residue. Usually, the enzyme has a low activity, or is not active at all, on native glycoproteins. A typical example is native bovine pancreatic ribonuclease B (RNase B) with oligomannose-type N-glycans at Asn-34. However, native RNase BS, generated by subtilisin digestion of native RNase B, which comprises amino acid residues 21-124 of RNase B, is sensitive to PNGase F digestion. The same holds for carboxymethylated RNase B (RNase B (cm)). In this study, NMR spectroscopy and molecular modeling have been used to explain the differences in PNGase F activity for native RNase B, native RNase BS, and RNase B (cm). NMR analysis combined with literature data clearly indicated that the N-glycan at Asn-34 is more mobile in RNase BS than in RNase B. MD simulations showed that the region around Asn-34 in RNase B is not very flexible, whereby the alpha-helix of the amino acid residues 1-20 has a stabilizing effect. In RNase BS, the alpha-helix formed by amino acid residues 23-32 is significantly more flexible. Using these data, the possibilities for complex formation of both RNase B and RNase BS with PNGase F were studied, and a model for the RNase BS-PNGase F complex is proposed.

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains--selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG.

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose-glycerol-methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8-15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

Characterization of the N-linked oligosaccharides from human chorionic gonadotropin expressed in the methylotrophic yeast Pichia pastoris.

Human chorionic gonadotropin (hCG) is a heterodimeric, placental glycoprotein hormone involved in the maintenance of the corpus luteum during the first trimester of pregnancy. Biologically active hCG has been successfully expressed in the yeast Pichia pastoris (phCG). In the context of structural studies and therapeutic applications of phCG, detailed information about its glycosylation pattern is a prerequisite. To this end N-glycans were released with peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated via anion-exchange chromatography (Resource Q) yielding both neutral (80%) and charged, phosphate-containing (20%) high-mannose-type structures. Subfractionations were carried out via normal phase (Lichrosorb-NH(2)) and high-pH anion-exchange (CarboPac PA-1) chromatography. Structural analyses of the released N-glycans were carried out by using HPLC profiling of fluorescent 2-aminobenzamide derivatives, MALDI-TOF mass spectrometry, and 500-MHz(1)H-NMR spectroscopy. Detailed neutral oligosaccharide structures, in the range of Man(8)GlcNAc(2) to Man(11)GlcNAc(2) including molecular isomers, could be established, and structures up to Man(15)GlcNAc(2) were indicated. Phosphate-containing oligosaccharides ranged from Man(9)PGlcNAc(2) to Man(13)PGlcNAc(2). Mannosyl O-glycans were not detected. Profiling studies carried out on different production batches showed that the oligosaccharide structures are similar, but their relative amounts varied with the culturing media.

Conformational studies on five octasaccharides isolated from chondroitin sulfate using NMR spectroscopy and molecular modeling.

Chondroitin sulfate proteoglycans (CS-PG) are involved in the regulation of the central nervous system in vertebrates due to their presence on cell surfaces and in the extracellular matrix of tissues. The CS moieties are built up from repeating -4)GlcA(beta1-3)GalNAc(beta1- disaccharide units, partly O-sulfated at different positions. The presence of the disulfated disaccharide D-unit, GlcA2S(beta1-3)GalNAc6S, in the CS moiety of the proteoglycan DSD-1-PG/phosphacan, correlates with neurite outgrowth promotion. The binding of monoclonal antibody (mAb) 473HD to DSD-1-PG, reducing neuronal stimulation, is inhibited by shark cartilage CS-D. CS-D is also recognized by two other mAbs, MO-225 and CS-56. Conformational studies were performed using NMR spectroscopy and molecular modeling on five octasaccharides isolated from shark cartilage CS-D. These octasaccharides present different binding properties toward the three mAbs. The combination of the experimental and theoretical approaches revealed that the sulfate group at position 2 of GlcA in disaccharide D and the presence of an exocyclic negative tail in disaccharides C [GlcA(beta1-3)GalNAc6S] and DeltaC [Delta4,5HexA(alpha1-3)GalNAc6S] are important for antibody recognition.

N-glycans of recombinant human acid alpha-glucosidase expressed in the milk of transgenic rabbits.

Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this disease are being treated by intravenous injection of recombinant forms of the enzyme. Focusing on recombinant approaches to produce the enzyme means that specific attention has to be paid to the generated glycosylation patterns. Here, human GAA was expressed in the mammary gland of transgenic rabbits. The N-linked glycans of recombinant human GAA (rhAGLU), isolated from the rabbit milk, were released by peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The N-glycan pool was fractionated and purified into individual components by a combination of anion-exchange, normal-phase, and Sambucus nigra agglutinin-affinity chromatography. The structures of the components were analyzed by 500 MHz one-dimensional and 600 MHz cryo two-dimensional (total correlation spectroscopy [TOCSY] nuclear Overhauser enhancement spectroscopy) (1)H nuclear magnetic resonance spectroscopy, combined with two-dimensional (31)P-filtered (1)H-(1)H TOCSY spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and high-performance liquid chromatography (HPLC)-profiling of 2-aminobenzamide-labeled glycans combined with exoglycosidase digestions. The recombinant rabbit glycoprotein contained a broad array of different N-glycans, comprising oligomannose-, hybrid-, and complex-type structures. Part of the oligomannose-type glycans showed the presence of phospho-diester-bridged N-acetylglucosamine. For the complex-type glycans (partially) (alpha2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the upper antenna (Lewis x). Using HPLC-mass spectrometry of glycopeptides, information was generated with respect to the site-specific location of the various glycans.

The Giardia intestinalis filamentous cyst wall contains a novel beta(1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study.

Assembly of a protective cyst wall by Giardia is essential for the survival of the parasite outside the host intestine and for transmission among susceptible hosts. The structure of the G. intestinalis filamentous cyst wall was studied by chemical methods, mass spectrometry, and (1)H nuclear magnetic resonance spectroscopy. Isolated cyst wall material contains carbohydrate and protein in a ratio of 3:2 (w/w), and the carbohydrate moiety is composed of a beta(1-3)-N-acetyl-D-galactopyranosamine homopolymer. Conformational analysis by molecular dynamics and persistence length calculations of GalNAc oligomers in solution demonstrated a flexible structure consisting of left- and right-handed helical elements. It is most likely that in the solid state, the polysaccharide forms ordered helices or possibly multiple helical structures having strong interchain interactions. The highly insoluble nature of the Giardia cyst wall must be due to these strong interchain interactions and, probably, a strong association between the carbohydrate and the protein moiety.