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Glycans
Kinds of Glycans Terms modified by Glycans Selected AbstractsO -Glycosyl Amino Acids by 2-Nitrogalactal Concatenation , Synthesis of a Mucin-Type O -GlycanEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 6 2003Gottfried A. Winterfeld Abstract Base-promoted Michael-type addition of N -Boc- and N -Fmoc-protected serine and threonine esters to 2-nitrogalactal derivatives 2 and 26 led highly selectively to ,-glycosides 4a,d and 27a,c, respectively. Ensuing transformation of threonine derivative 4d and serine derivatives 4a,b resulted in compounds useful as lysine and dipeptide mimetics. 6- O -Desilylation of 27a,c, then 6- O -sialylation, and transformation of the nitro group of the galactose moiety into a 2-acetamido functionality, afforded N -Boc-protected serine and threonine tert -butyl esters 31a,c carrying the O -protected STN -antigen at the hydroxy group. The threonine derivative 31c was then transformed into the N -Fmoc-protected amino acid building block 33, which was employed for the synthesis of mucin repeating unit partial structure Ac-GS(STN)-TAPPAHG-NH2 (1). (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003) [source] ChemInform Abstract: The Controlled Glycosylation of a Protein with a Bivalent Glycan: Towards a New Class of Glycoconjugates, Glycodendriproteins.CHEMINFORM, Issue 22 2001Benjamin G. Davis Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a "Full Text" option. The original article is trackable via the "References" option. [source] Improved sample preparation method for glycan analysis of glycoproteins by CE-LIF and CE-MSELECTROPHORESIS, Issue 8 2010Zoltan Szabo Abstract CE is a high-resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37°C, using ,100 fold excess of fluorophore reagent, 8-aminopyrene-1,3,6-trisulfonic-acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:,100 ratio) maintained the >95% derivatization yield at 55°C with only 50,min reaction time. Terminal sialic acid loss was negligible at 55°C during the derivatization process, and indicating that the kinetics of labeling at 55°C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8-aminopyrene-1,3,6-trisulfonic-acid simplified the removal of excess reagent, important in both CE-LIF (electrokinetic injection bias) and CE-MS (ion suppression). Coupling CE- ESI-MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values. [source] Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis , electrospray , time-of-flight mass spectrometryELECTROPHORESIS, Issue 13 2006Elvira Balaguer Abstract Glycosylation of recombinant human erythropoietin (rHuEPO) is a post-translational process that alters biological activity, solubility and lifetime of the glycoprotein in blood, and strongly depends on the type of cell and the cell culture conditions. A fast and simple method providing extensive carbohydrate information about the glycans present in rHuEPO and other glycoproteins is needed in order to improve current methods in drug development or product quality control. Here, an improved method for intact rHuEPO glycoform characterization by CZE-ESI-TOF MS has been developed using a novel capillary coating and compared to a previous study. Both methods allow a fast separation in combination with accurate mass characterization of the single protein isoforms. The novel dynamic coating provides a separation at an EOF close to zero, enabling better separation. This results in an improved mass spectrometric resolution and the detection of minor isoforms. In order to assign an unequivocal carbohydrate composition to every intact glycoform, a CZE-ESI-MS separation method for enzymatically released underivatized N -glycans has been developed. The TOF,MS allows the correct identification of the glycans due to its high mass accuracy and resolution. Therefore, glycan modifications such as acetylation, oxidation, sulfation and even the exchange of OH by NH2 are successfully characterized. Information of the protein-backbone molecular mass has been combined with results from peptide analysis (revealing information about O -glycosylation) and from the glycan analysis, including the detection of as yet undescribed glycans containing four antennae and five sialic acids. This allows an unequivocal assignment of an overall glycosylation composition to the molecular masses obtained for the intact rHuEPO glycoforms. [source] Metagenomic approach studying the taxonomic and functional diversity of the bacterial community in a mesotrophic lake (Lac du Bourget , France)ENVIRONMENTAL MICROBIOLOGY, Issue 9 2009Didier Debroas Summary The main goals of this work were to identify the metabolic pathways of the bacterial community in a lacustrine ecosystem and to establish links between taxonomic composition and the relative abundances of these metabolic pathways. For this purpose, we analysed a 16S rRNA gene library obtained by gene amplification together with a sequence library of both insert ends on c. 7700 fosmids. Whatever the library used, Actinobacteria was the most abundant bacterial group, followed by Proteobacteria and Bacteroidetes. Specific aquatic clades such as acI and acIV (Actinobacteria) or LD12 and GOBB-C201 (Alphaproteobacteria) were found in both libraries. From comparative analysis of metagenomic libraries, the metagenome of this lake was characterized by overrepresentation of genes involved in the degradation of xenobiotics mainly associated with Alphaproteobacteria. Actinobacteria were mainly related to metabolic pathways involved in nucleotide metabolism, cofactors, vitamins, energy, replication and repair. Betaproteobacteria appeared to be characterized by the presence of numerous genes implicated in environmental information processing (membrane transport and signal transduction) whereas glycan and carbohydrate metabolism pathways were overrepresented in Bacteroidetes. These results prompted us to propose hypotheses on the ecological role of these bacterial classes in lacustrine ecosystems. [source] Chemoselective Reagents for Covalent Capture and Display of Glycans in Microarrays,EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 3 2010Emiliano Cló Abstract Glycobiology has made very significant progress in the past decades. However, further progress will significantly depend on the establishment of novel methods for miniaturized, high-throughput analysis of glycan,protein interactions. Robust solid-phase chemical tools and new, chemoselective reagents for biologically meaningful display of surface-immobilized glycans are likely to play a key role. Here we present four new bifunctional linkers that allow highly chemoselective capture of unprotected glycans in solution to form glycan-linker conjugates for direct construction of glycan microarrays (glycochips). The bifunctional linkers carry O -linked aminooxy moieties, some with N -substituents at one end and an amino group at the other. In addition, they contain a substituted benzene ring for UV traceability and improved purification of glycan-linker conjugates. NMR spectroscopic studies in solution proved that N -substituted aminooxy linkers provided model glycan-linker conjugates with the ,-glucopyranoside configuration, i.e. the ring-closed form required for biological recognition. Then an ensemble of glycan-linker conjugates were assembled from mannobiose, lactose, and N -acetyl-lactosamine and used for covalent printing of glycan microarrays. The stability of oximes were studied both in solution and on-chip. In solution, two of the linkers provided glycan-linker conjugates with a remarkable stability at pH 4 or higher, on-chip this relative stability was upheld. Two of the linkers, with different properties, are recommended for the glycobiology toolbox for the construction of glycan microarrays from unprotected glycans. [source] Engineering of a monomeric and low-glycosylated form of human butyrylcholinesteraseFEBS JOURNAL, Issue 2 2002Expression, characterization, crystallization, purification Human butyrylcholinesterase (BChE; EC 3.1.1.8) is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0 Å resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 Å, c = 124.9 Å, giving a Vm of 2.73 Å3 per Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general. [source] A novel type of carbohydrate,protein linkage region in the tyrosine-bound S-layer glycan of Thermoanaerobacterium thermosaccharolyticum D120-70FEBS JOURNAL, Issue 17 2000Christina Schäffer The surface-layer (S-layer) protein of Thermoanaerobacterium thermosaccharolyticum D120-70 contains glycosidically linked glycan chains with the repeating unit structure ,4)[,- d -Galp -(1,2)]-,- l -Rhap -(1,3)[,- d -Glcp -(1,6)]-,- d -Manp -(1,4)-,- l -Rhap -(1,3)-,- d -Glcp -(1,. After proteolytic degradation of the S-layer glycoprotein, three glycopeptide pools were isolated, which were analyzed for their carbohydrate and amino-acid compositions. In all three pools, tyrosine was identified as the amino-acid constituent, and the carbohydrate compositions corresponded to the above structure. Native polysaccharide PAGE showed the specific heterogeneity of each pool. For examination of the carbohydrate,protein linkage region, the S-layer glycan chain was partially hydrolyzed with trifluoroacetic acid. 1D and 2D NMR spectroscopy, including a novel diffusion-edited difference experiment, showed the O-glycosidic linkage region ,- d -glucopyranose,O -tyrosine. No evidence was found of additional sugars originating from a putative core region between the glycan repeating units and the S-layer polypeptide. For the determination of chain-length variability in the S-layer glycan, the different glycopeptide pools were investigated by matrix-assisted laser desorption ionization-time of flight mass spectrometry, revealing that the degree of polymerization of the S-layer glycan repeats varied between three and 10. All masses were assigned to multiples of the repeating units plus the peptide portion. This result implies that no core structure is present and thus supports the data from the NMR spectroscopy analyses. This is the first observation of a bacterial S-layer glycan without a core region connecting the carbohydrate moiety with the polypeptide portion. [source] Glycosylation status of haptoglobin in sera of patients with prostate cancer vs. benign prostate disease or normal subjectsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2008Tsutomu Fujimura Abstract We studied chemical level and glycosylation status of haptoglobin in sera of patients with prostate cancer, as compared to benign prostate disease and normal subjects, with the following results. (i) Haptoglobin level was enhanced significantly in sera of prostate cancer. (ii) Sialylated bi-antennary glycans were the dominant structures in haptoglobins from all 3 sources, regardless of different site of N-linked glycan. The N-linked glycans at N184 were exclusively bi-antennary, and showed no difference between prostate cancer vs. benign prostate disease. (iii) Tri-antennary, N-linked, fucosylated glycans, carrying at least 1 sialyl-Lewisx/a antenna, were predominantly located on N207 or N211 within the amino acid 203-215 sequence of the ,-chain of prostate cancer, and were minimal in benign prostate disease. Fucosylated glycans were not observed in normal subjects. A minor tri-antennary N-linked glycan was observed at N241 of the ,-chain in prostate cancer, which was absent in benign prostate disease. (iv) None of these N-linked structures showed the expected presence of disialylated antennae with GalNAc,4(NeuAc,3)Gal,3(NeuAc,6)GlcNAc,Gal, or its analogue, despite cross-reactivity of prostate cancer haptoglobin with monoclonal antibody RM2. (v) Minor levels of O -glycosylation were identified in prostate cancer haptoglobin for the first time. Mono- and disialyl core Type 1 O-linked structures were identified after reductive ,-elimination followed by methylation and mass spectrometric analysis. No evidence was found for the presence of specific RM2 or other tumor-associated glycosyl epitopes linked to this O -glycan core. In summary, levels of haptoglobin are enhanced in sera of prostate cancer patients, and the N -glycans attached to a defined peptide region of its ,-chain are characterized by enhanced branching as well as antenna fucosylation. © 2007 Wiley-Liss, Inc. [source] Evaluation of glycosylation and malonylation patterns in flavonoid glycosides during LC/MS/MS metabolite profilingJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2008P. Kachlicki Abstract Flavonoid conjugates constitute several classes of plant phenolic secondary metabolites including many isomeric compounds differing in the hydroxylation pattern and substitution of their rings with different groups such as alkyls, acyls or sugars. These compounds occur in plant tissues mainly as glycosides and in many cases it is necessary to have reliable and detailed information concerning the structure of these natural products. Our results were obtained using leaf extracts of Arabidopsis thaliana and Lupinus angustifolius in which different glycosides of flavones, flavonols and isoflavones are present. Analysis of collision-induced dissociation (CID)/MS/MS spectra of protonated [M + H]+, sodiated [M + Na]+ or deprotonated [M , H], molecules recorded during HPLC runs may bring needed information in this respect. However, registration of mass spectra of [M + Na]+ ions with a good efficiency is possible only after post-column addition of a sodium acetate solution to the LC column eluate. The retention of sodium cation on the saccharidic parts of the molecule is observed after the CID fragmentation. In many cases, the location of this cation on the glycan attached to C-3 hydroxyl group of flavonol led to assignment of its structure. Additionally, the determination of the structure of the aglycone and of the sequence of the glycan part was made possible through the CID data obtained from the [M + H]+ and [M , H], ions. CID spectra show a different order of sugar elimination from hydroxyl groups at C-3 and C-7 in flavonol glycosides isolated from A. thaliana leaves and give sufficient information to discriminate flavonoid O-diglycosides from flavonoid di-O-glycosides. Copyright © 2007 John Wiley & Sons, Ltd. [source] Development of fully functional proteins with novel glycosylation via enzymatic glycan trimmingJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 8 2009Melinda L. Toumi Abstract Recombinant glycoproteins present unique challenges to biopharmaceutical development, especially when efficacy is affected by glycosylation. In these cases, optimizing the protein's glycosylation is necessary, but difficult, since the glycan structures cannot be genetically encoded, and glycosylation in nonhuman cell lines can be very different from human glycosylation profiles. We are exploring a potential solution to this problem by designing enzymatic glycan optimization methods to produce proteins with useful glycan compositions. To demonstrate viability of this new approach to generating glycoprotein-based pharmaceuticals, the N -linked glycans of a model glycoprotein, ribonuclease B (RNase B), were modified using an ,-mannosidase to produce a new glycoprotein with different glycan structures. The secondary structure of the native and modified glycoproteins was retained, as monitored using circular dichroism. An assay was also developed using an RNA substrate to verify that RNase B had indeed retained its function after being subjected to the necessary glycan modification conditions. This is the first study that verifies both activity and secondary structure of a glycoprotein after enzymatic glycan trimming for use in biopharmaceutical development methods. The evidence of preserved structure and function for a modified glycoprotein indicates that extracellular enzymatic modification methods could be implemented in producing designer glycoproteins. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:2581,2591, 2009 [source] JET-SUSPENDED, CALCITE-BALLASTED CYANOBACTERIAL WATERWARTS IN A DESERT SPRING1JOURNAL OF PHYCOLOGY, Issue 3 2002Ferran Garcia-Pichel We describe a population of colonial cyanobacteria (waterwarts) that develops as the dominant primary producer in a bottom-fed, O2 -poor, warm spring in the Cuatro Ciénegas karstic region of the Mexican Chihuahuan Desert. The centimeter-sized waterwarts were suspended within a central, conically shaped, 6-m deep well by upwelling waters. Waterwarts were built by an Aphanothece -like unicellular cyanobacterium and supported a community of epiphytic filamentous cyanobacteria and diatoms but were free of heterotrophic bacteria inside. Sequence analysis of 16S rRNA genes revealed that this cyanobacterium is only distantly related to several strains of other unicellular cyanobacteria (Merismopedia, Cyanothece, Microcystis). Waterwarts contained orderly arrangements of mineral crystallites, made up of microcrystalline low-magnesium calcite with high levels of strontium and sulfur. Waterwarts were 95.9% (v/v) glycan, 2.8% cells, and 1.3% mineral grains and had a buoyant density of 1.034 kg·L,1. An analysis of the hydrological properties of the spring well and the waterwarts demonstrated that both large colony size and the presence of controlled amounts of mineral ballast are required to prevent the population from being washed out of the well. The unique hydrological characteristics of the spring have likely selected for both traits. The mechanisms by which controlled nucleation of extracellular calcite is achieved remain to be explored. [source] The plasma von Willebrand factor O -glycome comprises a surprising variety of structures including ABH antigens and disialosyl motifsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2010K. CANIS Summary.,Background: von Willebrand factor (VWF) is a key component for maintenance of normal hemostasis. Its glycan moieties, accounting for about 20% of its molecular weight, have been shown to affect many of its properties. Previous studies reported correlations between VWF secretion, half-life and the nature or presence of its N -glycans, and more importantly between VWF plasma level and the type of N -linked ABH antigens. Despite the presence of 10 predicted O -glycosylation sites, the O -glycome remains poorly characterized, impairing the complete elucidation of its influence on VWF functions. So far only a single glycan structure, a disialyl core 1 glycan, has been identified. Objectives: To define an exhaustive profile of the VWF O -glycan structures to help the understanding of their role in VWF regulation and properties. Methods: Plasma-derived VWF O -linked sugars were isolated and analyzed using state-of-the-art mass spectrometry methodologies. Results and conclusions: We provide here a detailed analysis of the human plasma-derived VWF O -glycome. Eighteen O -glycan structures including both core 1 and core 2 structures are now demonstrated to be present on VWF. Amongst the newly determined structures are unusual tetra-sialylated core 1 O -glycans and ABH antigen-containing core 2 O -glycans. In conjunction with current models explaining VWF activity, knowledge of the complete O -glycome will facilitate research aimed at providing a better understanding of the influence of glycosylation on VWF functions. [source] Protein N-glycosylation determines functionality of the Saccharomyces cerevisiae cell wall integrity sensor Mid2pMOLECULAR MICROBIOLOGY, Issue 6 2008Franziska Hutzler Summary The fungal cell wall is a highly dynamic structure that is essential to maintain cell shape and stability. Hence in yeasts and fungi cell wall integrity is tightly controlled. The Saccharomyces cerevisiae plasma membrane protein Mid2p is a putative mechanosensor that responds to cell wall stresses and morphological changes during pheromone induction. The extracellular domain of Mid2p, which is crucial to sensing, is highly O- and N-glycosylated. We showed that O-mannosylation is determining stability of Mid2p. If and how N-glycosylation is linked to Mid2p function was unknown. Here we demonstrate that Mid2p contains a single high mannose N-linked glycan at position Asn-35. The N -glycan is located close to the N-terminus and is exposed from the plasma membrane towards the cell wall through a highly O-mannosylated domain that is predicted to adopt a rod-like conformation. In contrast to O-mannosylation, lack of the N-linked glycan affects neither, stability of Mid2p nor distribution at the plasma membrane during vegetative and sexual growth. However, non-N-glycosylated Mid2p fails to perceive cell wall challenges. Our data further demonstrate that both the extent of the N-linked glycan and its distance from the plasma membrane affect Mid2p function, suggesting the N -glycan to be directly involved in Mid2p sensing. [source] Core glycan in the yeast multicopper ferroxidase, Fet3p: A case study of N-linked glycosylation, protein maturation, and stabilityPROTEIN SCIENCE, Issue 9 2010Lynn Ziegler Abstract Glycosylation is essential to the maintenance of protein quality in the vesicular protein trafficking pathway in eukaryotic cells. Using the yeast multicopper oxidase, Fet3p, the hypothesis is tested that core glycosylation suppresses Fet3p nascent chain aggregation during synthesis into the endoplasmic reticulum (ER). Fet3p has 11 crystallographically mapped N-linked core glycan units. Assembly of four of these units is specifically required for localization of Fet3p to the plasma membrane (PM). Fet3 protein lacking any one of these glycan units is found in an intracellular high-molecular mass species resolvable by blue native gel electrophoresis. Individually, the remaining glycan moieties are not required for ER exit; however, serial deletion of these by N , A substitution correlates with these desglycan species failure to exit the ER. Desglycan Fet3 proteins that localize to the PM are wild type in function indicating that the missing carbohydrate is not required for native structure and biologic activity. This native function includes the interaction with the iron permease, Ftr1p, and wild type high-affinity iron uptake activity. The four essential sequons are found within relatively nonpolar regions located in surface recesses and are strongly conserved among fungal Fet3 proteins. The remaining N-linked sites are found in more surface exposed, less nonpolar environments, and their conservation is weak or absent. The data indicate that in Fet3p the N-linked glycan has little effect on the enzyme's molecular activity but is critical to its cellular activity by maximizing the protein's exit from the ER and assembly into a functional iron uptake complex. [source] Determination of the site-specific and isoform-specific glycosylation in human plasma-derived antithrombin by IEF and capillary HPLC-ESI-MS/MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 15 2005Alexander Plematl Abstract The glycan structures of the major and more than ten minor populated isoforms of antithrombin (AT) were determined after separation of the isoforms by IEF using IPG strips. The bands excised from the gel were reduced, derivatized by iodoacetamide and submitted to tryptic digestion. The digest was analyzed by RP-HPLC-ESI-MS equipped with a quadrupole ion-trap mass analyzer. MS/MS experiments allowed establishing the monosaccharide compositions in the glycopeptides. For the major isoform of ,-AT four identical biantennary glycans with two terminal sialic acids (SA) each, a total of eight SA, were found in full agreement with the literature. In the IEF-band containing this major isoform (pI 5.18) a further, much less abundant, isoform was detected showing a fucosylation on the glycan attached to Asn155 but being of otherwise identical structure as described above. The isoforms with pI 5.10 were found to include one triantennary glycan, all antennas carrying terminal SA. The occurrence of triantennary structure is site specific, involving the peptides with Asn135 and Asn155, alternately. At pI 5.24 we found those four isoforms that carry the glycans like the main-isoform of ,-AT but missing one terminal SA. There was no site specificity found for the mono-sialo structure. The isoform at pI 5.31 is the major isoform of ,-AT containing three identical biantennary structures being fully sialylated. No isoforms (above 0.5% abundance) with two glycans only or three glycans other than ,-AT were detected. Fucosylation was found in the main isoform with an abundance of about 5%, and as expected with all the other isoforms with a comparable abundance. [source] Software utilities for the interpretation of mass spectrometric data of glycoconjugates: application to glycosphingolipids of human serumRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 7 2010Jamal Souady Glycosphingolipids (GSLs) are major components of the outer leaflet of the cell membrane. These lipids are involved in many cell surface events and show disease-related expression changes. GSLs could thus serve as useful targets for biomarker discovery. The GSL structure is characterized by two entities: a hydrophilic glycan and a hydrophobic ceramide moiety. Both components exhibit numerous structural variations, the combination of which results in a large diversity of GSL structures that can potentially exist. Mass spectrometry (MS) is a powerful tool for high-throughput analysis of GSL expression analysis and structural elucidation. Yet, the assignment of GSL structures using MS data is tedious and demands highly specialized expertise. SysBioWare, a software platform developed for MS data evaluation in glycomics, was here applied for the MS analysis of human serum GSLs. The program was tuned to provide automated compositional assignment, supporting a variety of glycan and ceramide structures. Upon in silico fragmentation, the masses of predicted ions arising from cleavages in the glycan as well as the ceramide moiety were calculated, thus enabling structural characterization of both entities. Validation of proposed structures was achieved by matching in silico calculated fragment ions with those of experimental MS/MS data. These results indicate that SysBioWare can facilitate data interpretation and, furthermore, help the user to deal with large sets of data by supporting management of MS and non-MS data. SysBioWare has the potential to be a powerful tool for high-throughput glycosphingolipidomics in clinical applications. Copyright © 2010 John Wiley & Sons, Ltd. [source] Application of the StrOligo algorithm for the automated structure assignment of complex N-linked glycans from glycoproteins using tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2003Martin Ethier Oligosaccharides associated with proteins are known to give these molecules specific conformations and functions. Analysis of proteins would not be complete without studying the glycans. However, high-throughput techniques in proteomics will soon overwhelm the current capacity of methods if no automation is incorporated into glycomics. New capabilities of the StrOligo algorithm introduced for this purpose (Ethier et al., Rapid Commun. Mass Spectrom., 2002; 16: 1743) will be discussed here. Experimental tandem mass spectra were acquired to test the algorithm using a hybrid quadrupole-time-of-flight (QqTOF) instrument with a matrix-assisted laser desorption/ionization (MALDI) source. The samples were N-linked oligosaccharides from monoclonal antibody IgG, beta interferon and fetuin, detached by enzymatic deglycosylation and labeled at the reducing end. Improvements to the program were made in order to reduce the need for user intervention. StrOligo strips the spectra down to monoisotopic peaks only. The algorithm first builds a relationship tree, accounting for each observed loss of a monosaccharide moiety, and then analyzes the tree and proposes possible structures from combinations of adducts and fragment ion types. A score, which reflects agreement with experimental results, is then given to each proposed structure. The program then decides which combination is the best one and labels relevant peaks in the experimental mass spectrum using a modified nomenclature. The usefulness of the algorithm has been demonstrated by assigning structures to several glycans released from glycoproteins. The analysis was completed in less than 2 minutes for any glycan, which is a substantial improvement over manual interpretation. Copyright © 2003 John Wiley & Sons, Ltd. [source] Defining the glycophenotype of squamous epithelia using plant and mammalian lectins.APMIS, Issue 12 20023-linked N-acetylneuraminic acid in squamous epithelia, Differentiation-dependent expression of, carcinomas, its differential effect on binding of the endogenous lectins galectins- A thorough characterization of the properties of squamous epithelial cells is necessary in order to improve our understanding of the functional aspects of normal development and malignant aberrations. Up to now, studies have focused almost exclusively on monitoring distinct protein markers. With our growing awareness of the coding function of glycan chains of cellular glycoconjugates and their interaction with receptors (lectins) in situ, defining the glycophenotype of these cells has become an important issue. Whereas the commonly applied plant lectins are tools used to map the presence and localization of biochemically defined saccharide epitopes, the introduction of endogenous (mammalian) lectins to this analysis enables us to take the step from monitoring the presence of glycan to understanding the functional implications by revealing ligand properties of the detected epitope for tissue lectin. Thus, in this study we investigated a distinct aspect of glycosylation using plant and mammalian lectins, i.e. the linkage type of sialylation. We first mapped the expression profile of the type of sialylation (,2,3- or ,2,6-linked) by plant lectins. Based on the hypothesis that this factor regulates accessibility of ligands for endogenous lectins we introduced two labeled galectins to this study. Galectin-3 (but not galectin-1) binding was related to cell differentiation in normal adult and developing epithelia, cultured epidermal cells, and carcinomas derived from these epithelia. The presented data suggest that ,2,6-linked N-acetyl- D -neuraminic acid moieties could serve to mask galectin-3-reactive glycoepitopes. As a consequence, monitoring of the linkage type of sialic acid in glycans by plant lectins therefore has implications for the extent of glycan reactivity with endogenous lectins, pointing to a potential function of changes in sialylation type beyond these cell and lectin systems. [source] Detection of orientation-specific anti-gp120 antibodies by a new N-glycanase protection assayAPMIS, Issue 2 2002G. J. Gram Several functions have been assigned to the extensive glycosylation of HIV-1 envelope glycoprotein gp120, especially immune escape mechanisms, but the intramolecular interactions between gp120 and its carbohydrate complement are not well understood. To analyse this phenomenon we established a new microwell deglycosylation assay for determining N-linked glycan accessibility after binding of gp120-specific agents. Orientation-specific exposition of gp120 in ELISA microplates was achieved by catching with either anti-C5 antibody D7324 or anti-V3 antibody NEA-9205. We found that soluble CD4 inhibited the deglycosylation of gp120 only when gp120 was caught by D7324 and not by NEA9205. In contrast, antibodies from HIV-infected individuals inhibited the deglycosylation best when gp120 was caught by NEA9205. These results demonstrated that both the CD4-binding site and the epitopes recognised by antibodies from HIV-infected individuals have N-glycans in the close vicinity. However, the difference in gp120 orientation indicates that antibodies in HIV-infected individuals, at least partly, bind to epitopes different from the CD4-binding site. Finally, we determined the structural class of the glycan of one V1 glycosylation site of prototype HIV-1 LAI gp120, which remained unsolved from previous studies, and found that it belonged to the complex type of glycans. [source] Aberrant IgG galactosylation precedes disease onset, correlates with disease activity, and is prevalent in autoantibodies in rheumatoid arthritis,ARTHRITIS & RHEUMATISM, Issue 8 2010Altan Ercan Objective To examine the association between aberrant IgG galactosylation and disease parameters in rheumatoid arthritis (RA). Methods Analysis of N -glycan in serum samples from multiple cohorts was performed. The IgG N -glycan content and the timing of N -glycan aberrancy relative to disease onset were compared in healthy subjects and in patients with RA. Correlations between aberrant galactosylation and disease activity were assessed in the RA cohorts. The impact of disease activity, sex, age, anti,cyclic citrullinated peptide (anti-CCP) antibody titer, disease duration, and C-reactive protein level on aberrant galactosylation was determined using multivariate analysis. The N -glycan content was also compared between epitope affinity,purified autoantibodies and the remaining IgG repertoire in RA patients. Results Our results confirm the aberrant galactosylation of IgG in RA patients as compared with healthy controls (mean ± SD 1.36 ± 0.43 versus 1.01 ± 0.23; P < 0.0001). We observed a significant correlation between levels of aberrant IgG galactosylation and disease activity (Spearman's , = 0.37, P < 0.0001). This correlation was higher in women (Spearman's , = 0.60, P < 0.0001) than in men (Spearman's , = 0.16, P = 0.10). Further, aberrant IgG galactosylation substantially predated the onset of arthritis and the diagnosis of RA (3.5 years) and resided selectively in the anticitrullinated antigen fraction. Conclusion Our findings identify aberrant IgG galactosylation as a dysregulated component of the humoral immune response in RA that begins prior to disease onset, associates with disease activity in a sex-specific manner, and resides preferentially in autoantibodies. [source] Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N -glycan by blocking core oligosaccharide assemblyBIOTECHNOLOGY JOURNAL, Issue 5 2008Doo-Byoung Oh Abstract The initial lipid-linked oligosaccharide Glc3Man9GlcNAc2 -dolichyl pyrophosphate (Dol-PP) for N -glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog (HpALG3) coding for a dolichyl-phosphate-mannose dependent ,-1,3-mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage-specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc2 -Dol-PP to Man6GlcNAc2 -Dol-PP, the first step to attach a mannose to the lipid-linked oligosaccharide in the ER. The N -glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast-specific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi , -1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N -glycan (Man3GlcNAc2), which is the common core backbone of various human-type N -glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans. [source] Immune biasing by helminth glycansCELLULAR MICROBIOLOGY, Issue 1 2004Paul G. Thomas Summary The ability of helminth parasites to drive polarized Th2 responses has been known for some time. Interestingly, many recent studies have shown that helminth-expressed glycan activation of host immune cells accounts for much of the anti-inflammatory and Th2-biasing observed. This microreview attempts to cover the biology of expression of immunomodulatory glycans in various helminth parasites, the immune cells they interact with including the production of cytokines, chemokines and antibodies. We also discuss the potential,, cell,, surface,, receptors,, which,, are,, capable of binding certain glycans and the known mechanisms which ultimately lead to production of anti-inflammatory,, mediators,, as,, well,, as,, polarizing,, CD4+ T-cell responses to Th2-type in the host. Lastly, we discuss a novel mechanism for activation of antigen-presenting cells by a specific helminth glycan that leads to maturation of Type 2 dendritic cells. [source] Chemoselective Capture of Glycans for Analysis on Gold Nanoparticles: Carbohydrate Oxime Tautomers Provide Functional Recognition by ProteinsCHEMISTRY - A EUROPEAN JOURNAL, Issue 7 2009Mikkel Abstract Open or closed: N -Glycosyl oxyamine versus open-chain glycosyl oxime is the key to protein recognition on glyconanoparticles. Unprotected glycans are captured by oxime formation with a novel bifunctional reagent and the resulting glycan-linker conjugates are anchored to gold nanoparticles (AuNPs). These glyconanoparticles maintain the structural integrity of glycans in the study of protein,carbohydrate interactions (see figure). Nanoparticles functionalized with glycans are emerging as powerful solid-phase chemical tools for the study of protein,carbohydrate interactions using nanoscale properties for detection of binding events. Methods or reagents that enable the assembly of glyconanoparticles from unprotected glycans in two consecutive chemoselective steps with meaningful display of the glycan are highly desirable. Here, we describe a novel bifunctional reagent that 1),couples to glycans by oxime formation in solution, 2),aids in purification through a lipophilic trityl tag, and 3),after deprotection then couples to gold nanoparticles through a thiol. NMR studies revealed that these oximes exist as both the open-chain and N -glycosyl oxy-amine tautomers. Glycan-linker conjugates were coupled through displacement of ligands from preformed, citrate-stabilized gold nanoparticles. Recognition of these glycans by proteins was studied with a lectin, concanavalin A (ConA), in an aggregation assay and with a processing enzyme and glucoamylase (GA). We demonstrate that the presence of the N -glycosyl oxy-amines clearly enables functional recognition in sharp contrast to the corresponding reduced oxy-amines. This concept is then realized in a novel reagent, which should facilitate nanoglycobiology by enabling the operationally simple capture of glycans and their biologically meaningful display. [source] Assembly of a Series of Malarial Glycosylphosphatidylinositol Anchor OligosaccharidesCHEMISTRY - A EUROPEAN JOURNAL, Issue 8 2005Yong-Uk Kwon Dr. Abstract We report an efficient and convergent synthesis of a series of oligosaccharides comprised of the malaria GPI glycan (2,a), a promising anti-malaria vaccine candidate currently in preclinical trials and several related oligosaccharide sequences (3,8) that are possible biosynthetic precursors of the malarial GPI. A flexible synthetic strategy is disclosed that relies on a late-stage coupling between oligomannosides of varying length and pseudo-disaccharide glycosyl acceptor 11 to readily access various malarial GPI structures. Phosphorylation was accomplished by mild and efficient H-phosphonate chemistry before the final deprotection was carried out by using sodium in ammonia. The direct connection of a thiol group via a phosphate diester linkage to the inositol moiety provides a handle for easy conjugation of the GPI glycan to carrier proteins, immobilization on carbohydrate microarrays and photo-affinity labels identification. These synthetic oligosaccharides will serve as molecular probes. [source] Characterization of peach thaumatin-like proteins and their identification as major peach allergensCLINICAL & EXPERIMENTAL ALLERGY, Issue 9 2010A. Palacín Summary Background Peach is the most important fruit related to food allergy in the Mediterranean area. Pru p 3, its lipid transfer protein, has been described as the principal allergen responsible for cross-reactivities with other foods and pollen and the severity of clinical symptoms. However, the involvement of other allergenic families cannot be ruled out. Thaumatin-like proteins (TLPs) have been described as food allergen in several fruits, such as apple, cherry, kiwi and banana, and pollen. Objective To identify members of the TLP family in peach fruit and to characterize putative allergens. Methods Through two-dimensional (2D) electrophoresis of peach extract and immunodetections with a pool of peach-allergic patients, IgE-binding spots were identified and the corresponding proteins purified and characterized as allergens by in vitro and in vivo assays. Three isoforms, belonging to the TLP family, were purified by different chromatographic systems and characterized by N -terminal amino acid sequences, molecular weight determination (MALDI) and enzymatic activity analysis (,-1,3-gluconase test and inhibition growth of fungi). In the same way, their IgE-binding capacity and allergenic activity were tested by ELISA assays, basophil activation tests and skin prick tests (SPT). Results Two peach-TLPs, Pru p 2.0101 and Pru p 2.0201, were identified as IgE-binding spots by 2D electrophoresis. Another peach-TLP, Pru p 2.0301, was cloned and produced as recombinant protein in a yeast system. The three isoforms were purified and characterized as TLPs by immunoblotting with anti-chestnut TLP antibodies and anti-plant N -asparagine complex glycan (anti-cross-reactive carbohydrate determinant). All of them showed ,-1,3-glucanase activity and inhibition of fungal growth. The three TLPs were recognized by around 50% of the sera from 31 patients analysed in ELISA experiments. All three gave a positive response to an SPT and/or in basophil activation experiments. Conclusion Three isoforms, belonging to the TLP family, were identified in peach as principal allergens. Their prevalence, observed in in vitro, ex vivo and in vivo analyses, suggests that they are important allergens and should therefore be included in the routine diagnosis of peach allergy, at least in the Mediterranean area. Cite this as: A. Palacín, L. Tordesillas, P. Gamboa, R. Sanchez-Monge, J. Cuesta-Herranz, M. L. Sanz, D. Barber, G. Salcedo and A. Díaz-Perales, Clinical & Experimental Allergy, 2010 (40) 1422,1430. [source] Presynaptic secretion of mind-the-gap organizes the synaptic extracellular matrix-integrin interface and postsynaptic environmentsDEVELOPMENTAL DYNAMICS, Issue 3 2009Emma Rushton Abstract Mind-the-Gap (MTG) is required during synaptogenesis of the Drosophila glutamatergic neuromuscular junction (NMJ) to organize the postsynaptic domain. Here, we generate MTG::GFP transgenic animals to demonstrate MTG is synaptically targeted, secreted, and localized to punctate domains in the synaptic extracellular matrix (ECM). Drosophila NMJs form specialized ECM carbohydrate domains, with carbohydrate moieties and integrin ECM receptors occupying overlapping territories. Presynaptically secreted MTG recruits and reorganizes secreted carbohydrates, and acts to recruit synaptic integrins and ECM glycans. Transgenic MTG::GFP expression rescues hatching, movement, and synaptogenic defects in embryonic-lethal mtg null mutants. Targeted neuronal MTG expression rescues mutant synaptogenesis defects, and increases rescue of adult viability, supporting an essential neuronal function. These results indicate that presynaptically secreted MTG regulates the ECM-integrin interface, and drives an inductive mechanism for the functional differentiation of the postsynaptic domain of glutamatergic synapses. We suggest that MTG pioneers a novel protein family involved in ECM-dependent synaptic differentiation. Developmental Dynamics 238:554,571, 2009. © 2009 Wiley-Liss, Inc. [source] Improved sample preparation method for glycan analysis of glycoproteins by CE-LIF and CE-MSELECTROPHORESIS, Issue 8 2010Zoltan Szabo Abstract CE is a high-resolution separation technique broadly used in the biotechnology industry for carbohydrate analysis. The standard sample preparation protocol for CE analysis of glycans released from glycoproteins generally requires derivatization times of overnight at 37°C, using ,100 fold excess of fluorophore reagent, 8-aminopyrene-1,3,6-trisulfonic-acid, if the sample is unknown, or it is a regulated biotherapeutic product, possibly containing terminal sialic acid(s). In this paper, we report on significant improvements for the standard CE sample preparation method of glycan analysis. By replacing the conventionally used acetic acid catalyst with citric acid, as low as 1:10 glycan to fluorophore molar ratio (versus the typical 1:,100 ratio) maintained the >95% derivatization yield at 55°C with only 50,min reaction time. Terminal sialic acid loss was negligible at 55°C during the derivatization process, and indicating that the kinetics of labeling at 55°C was faster than the loss of sialic acid from the glycan. The reduced relative level of 8-aminopyrene-1,3,6-trisulfonic-acid simplified the removal of excess reagent, important in both CE-LIF (electrokinetic injection bias) and CE-MS (ion suppression). Coupling CE- ESI-MS confirmed that the individual peaks separated by CE corresponded to single glycans and increased the confidence of structural assignment based on glucose unit values. [source] Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis , electrospray , time-of-flight mass spectrometryELECTROPHORESIS, Issue 13 2006Elvira Balaguer Abstract Glycosylation of recombinant human erythropoietin (rHuEPO) is a post-translational process that alters biological activity, solubility and lifetime of the glycoprotein in blood, and strongly depends on the type of cell and the cell culture conditions. A fast and simple method providing extensive carbohydrate information about the glycans present in rHuEPO and other glycoproteins is needed in order to improve current methods in drug development or product quality control. Here, an improved method for intact rHuEPO glycoform characterization by CZE-ESI-TOF MS has been developed using a novel capillary coating and compared to a previous study. Both methods allow a fast separation in combination with accurate mass characterization of the single protein isoforms. The novel dynamic coating provides a separation at an EOF close to zero, enabling better separation. This results in an improved mass spectrometric resolution and the detection of minor isoforms. In order to assign an unequivocal carbohydrate composition to every intact glycoform, a CZE-ESI-MS separation method for enzymatically released underivatized N -glycans has been developed. The TOF,MS allows the correct identification of the glycans due to its high mass accuracy and resolution. Therefore, glycan modifications such as acetylation, oxidation, sulfation and even the exchange of OH by NH2 are successfully characterized. Information of the protein-backbone molecular mass has been combined with results from peptide analysis (revealing information about O -glycosylation) and from the glycan analysis, including the detection of as yet undescribed glycans containing four antennae and five sialic acids. This allows an unequivocal assignment of an overall glycosylation composition to the molecular masses obtained for the intact rHuEPO glycoforms. [source] Analysis of chicken and turkey ovalbumins by microchip electrophoresis combined with exoglycosidase digestionELECTROPHORESIS, Issue 18 2003Xiuli Mao Abstract The polypeptide and carbohydrate patterns of two glycoproteins, chicken ovalbumin (CO) and turkey ovalbumin (TO), were analyzed by microchip electrophoresis (ME), following digestion with proteases and exoglycosidases. Glycopeptides derived from ovalbumin were obtained by digestion with Pronase, followed by dialysis, and then separated by ME. Using CO as model, the method was developed to deduce the structure of glycans from glycoproteins by comparing the electropherograms of glycopeptides with and without digestion of exolycosidases. Applying the same approach, the structure of oligosaccharides linked to TO was determined. TO was found to contain high-mannose type oligosaccharides and oligosaccharides with terminal N -acetylglucosamine residues. The complete primary analysis of CO and TO by ME described in this paper provides a basis for an analysis of glycoproteins with an integrated microfluidic chip. [source] | |||||||||||||||||||||||||||||||||||||