G.J. Gerwig
Utrecht University, Departement of Natural Sciences, Faculty Member
- life-science/biochemistedit
Research Interests:
Research Interests:
Glycosylation of proteins represents one of the most important post-(co-)translational events in view of the ubiquity of the phenomenon. In most cases, the covalently linked glycans are involved in the functioning of these biomolecules in... more
Glycosylation of proteins represents one of the most important post-(co-)translational events in view of the ubiquity of the phenomenon. In most cases, the covalently linked glycans are involved in the functioning of these biomolecules in biological systems. Detailed information on the carbohydrate moieties including monosaccharide composition, anomeric configurations, type of glycosidic linkages and attachment sites at the protein is indispensable in describing the ultimate structure of a specific glycoprotein. This chapter presents a general strategy for the structural characterization of glycoproteins/glycopeptides focussed on the glycan part. Some of the techniques commonly used, like enzyme treatments, separation methods, chemical analyses, mass spectrometry and nuclear magnetic resonance spectroscopy are briefly reviewed.
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alpha 2HS-Glycoprotein, a normal human plasma protein, was recently shown to consist of two polypeptide chains. In the present study, we have separated these two chains from one another and have elucidated the complete primary structure... more
alpha 2HS-Glycoprotein, a normal human plasma protein, was recently shown to consist of two polypeptide chains. In the present study, we have separated these two chains from one another and have elucidated the complete primary structure of the B-chain. Employing automated Edman degradation, the polypeptide moiety of this chain was shown to consist of 27 amino acid residues with an unequal distribution of the neutral and charged amino acid residues. The first 20 residues are uncharged, whereas the carboxyl-terminal heptapeptide contains all charged residues. Utilizing 500-MHz 1H-NMR spectroscopy, the carbohydrate unit proved to be a trisaccharide consisting of sialic acid, galactose, and N-acetylgalactosamine O-glycosidically linked to serine (residue 6). The structure of the B-chain was found to be as follows. (formula; see text) Thus, the molecular weight of the B-chain is 3386. Evaluation of the polypeptide chain by the procedure of Chou and Fasman (Chou, P.Y., and Fasman, G.D. (1...
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Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells were separated by Lysine Sepharose chromatography. The carbohydrate compositions of the forms were determined by gas-liquid chromatography.... more
Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells were separated by Lysine Sepharose chromatography. The carbohydrate compositions of the forms were determined by gas-liquid chromatography. Variant I contained 12.8 g and variant II 7.1 g of carbohydrate per 100 g protein. Both variants contained N-acetylgalactosamine, suggesting O-glycosylation in addition to N-glycosylation. The possible role of N-linked oligosaccharides for the biological activity of t-PA was studied using t-PA secreted by melanoma cells in the presence of tunicamycin, an inhibitor of N-glycosylation. The latter t-PA showed the same plasminogen activating and fibrin binding properties as normally glycosylated t-PA, indicating that N-linked carbohydrate is not involved in the fibrinolytic activity of t-PA.
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The cellulase complexes of two cellulolytic bacteria, Clostridium thermocellum and Bacteroides cellulosolvens, were subjected to extensive Pronase digestion. Glycopeptide fractions were isolated by gel permeation and fast protein liquid... more
The cellulase complexes of two cellulolytic bacteria, Clostridium thermocellum and Bacteroides cellulosolvens, were subjected to extensive Pronase digestion. Glycopeptide fractions were isolated by gel permeation and fast protein liquid chromatography and analyzed by monosaccharide analysis, amino acid analysis, methylation analysis, and 1H NMR spectroscopy. Alkaline borohydride-induced deglycosylation/amino acid conversion and periodate oxidation studies on the glycopeptide fraction of the C. thermocellum cellulosome demonstrated that the earlier established collection of carbohydrate moieties with 3-O-Me-D-GlcpNAc-alpha (1-->2)-[D-Galp-alpha (1-->3)]-D-Galf-alpha (1-->2)-D-Gal (where 3-O-Me-D-GlcpNAc is 3-O-methyl-N-acetylglucopyranosamine, Galp is galactopyranose, and Galf is galactofuranose) as the major component, is O-linked to threonine via galactopyranose. Using the same approach for the glycopeptide fraction of the cellulase complex of B. cellulosolvens, it was fou...
Research Interests: Chemistry, NMR Spectroscopy, Biological Chemistry, Magnetic Resonance Spectroscopy, Medicine, and 14 moreCellulase, Biological Sciences, P-glycoprotein, Peptides, Glycoproteins, CHEMICAL SCIENCES, Amino Acid Profile, CARBOHYDRATES, Amino Acid Sequence, Bacteroides Sp, Clostridium, Carbohydrate Sequence, Molecular Sequence Data, and Medical and Health Sciences
We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and... more
We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.
Research Interests:
Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells were separated by Lysine Sepharose chromatography. The carbohydrate compositions of the forms were determined by gas-liquid chromatography.... more
Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells were separated by Lysine Sepharose chromatography. The carbohydrate compositions of the forms were determined by gas-liquid chromatography. Variant I contained 12.8 g and variant II 7.1 g of carbohydrate per 100 g protein. Both variants contained N-acetylgalactosamine, suggesting O-glycosylation in addition to N-glycosylation. The possible role of N-linked oligosaccharides for the biological activity of t-PA was studied using t-PA secreted by melanoma cells in the presence of tunicamycin, an inhibitor of N-glycosylation. The latter t-PA showed the same plasminogen activating and fibrin binding properties as normally glycosylated t-PA, indicating that N-linked carbohydrate is not involved in the fibrinolytic activity of t-PA.
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Research Interests: Chemistry, Magnetic Resonance Spectroscopy, Medicine, Methylation, Hydrofluoric acid, and 11 moreEuropean, Gas Chromatography/mass Spectrometry, CARBOHYDRATES, Hydrolysis, Oxidation-Reduction, Biochemistry and cell biology, Serotyping, N-dealkylation, Chemical Phenomena, Neisseria meningitidis, and Medical biochemistry and metabolomics
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 252, No. 2, February 1, pp. 574590,1987 The Structures of N and 0Glycosidic Carbohydrate Chains of a Chondroitin Sulfate Proteoglycan Isolated from the Media of the Human Aorta1 FUMIKO... more
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 252, No. 2, February 1, pp. 574590,1987 The Structures of N and 0Glycosidic Carbohydrate Chains of a Chondroitin Sulfate Proteoglycan Isolated from the Media of the Human Aorta1 FUMIKO AKIYAMA,*2 ...
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We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and... more
We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.
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An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture of Clostridium thermosaccharolyticum Haren. The complex possessed both pectin methylesterase (EC 3.1.1.11) and... more
An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous culture of Clostridium thermosaccharolyticum Haren. The complex possessed both pectin methylesterase (EC 3.1.1.11) and exo-poly-alpha-galacturonate hydrolase (EC 3.2.1.82) activity and produced digalacturonate from the nonreducing end of the pectin chain. The protein consisted of 230- and 25-kDa subunits. The large subunit contained 10% (wt/wt) sugars (N-acetylgalactosamine and galactose). Under physiological conditions both activities acted in a coordinated manner: the ratio between methanol and digalacturonate released during degradation was constant and equal to the degree of esterification of the pectin used. Prolonged incubation of the enzyme with pectin led to a nondialyzable fraction that was enriched in neutral sugars, such as arabinose, rhamnose, and galactose; the high rhamnose/galacturonic acid ratio was indicative of hairy region-like structures. The smallest substrate utili...
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The structure of the carbohydrate chains of mucous glycoproteins from the gastro-intestinal tract was examined for species- and tissue-specificity. To this purpose, oligosaccharides were released from purified glycoprotein preparations of... more
The structure of the carbohydrate chains of mucous glycoproteins from the gastro-intestinal tract was examined for species- and tissue-specificity. To this purpose, oligosaccharides were released from purified glycoprotein preparations of rat and pig gastric, duodenal-gland and small-intestinal mucus, by alkaline borohydride reductive cleavage. Based on the results of 500-MHz 1H-NMR spectroscopy and of sugar analysis of the total oligosaccharide fractions, terminal GlcNAc, alpha (1 leads to 4)-linked to galactose, appears to be a characteristic constituent of duodenal-gland oligosaccharides. Similarly, NeuAc in alpha (2 leads to 3)-linkage to galactose turns out to be a typical constituent of small-intestinal mucous glycoproteins. In general, glycoproteins from gastric mucus possess larger and more-branched carbohydrate chains than those from duodenal-gland and small-intestinal mucus. Comparing rat and pig, oligosaccharide structures for corresponding tissues are less complex for th...
Research Interests: Magnetic Resonance Spectroscopy, Biological Sciences, Intestinal Mucosa, Animals, Male, and 12 morePhysical sciences, Glycoproteins, Rats, Glucosamine, Swine, Species Specificity, Structure activity Relationship, Oligosaccharides, Duodenum, N-acetylglucosamine, Carbohydrate Sequence, and Gastric mucosa
alpha 2HS-Glycoprotein, a normal human plasma protein, was recently shown to consist of two polypeptide chains. In the present study, we have separated these two chains from one another and have elucidated the complete primary structure... more
alpha 2HS-Glycoprotein, a normal human plasma protein, was recently shown to consist of two polypeptide chains. In the present study, we have separated these two chains from one another and have elucidated the complete primary structure of the B-chain. Employing automated Edman degradation, the polypeptide moiety of this chain was shown to consist of 27 amino acid residues with an unequal distribution of the neutral and charged amino acid residues. The first 20 residues are uncharged, whereas the carboxyl-terminal heptapeptide contains all charged residues. Utilizing 500-MHz 1H-NMR spectroscopy, the carbohydrate unit proved to be a trisaccharide consisting of sialic acid, galactose, and N-acetylgalactosamine O-glycosidically linked to serine (residue 6). The structure of the B-chain was found to be as follows. (formula; see text) Thus, the molecular weight of the B-chain is 3386. Evaluation of the polypeptide chain by the procedure of Chou and Fasman (Chou, P.Y., and Fasman, G.D. (1...
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The nitrosamines have been extensively studied for their toxicity. Their formation in the foods containing nitrous and nitric derivatives is favoured by the presence of suitable substrata. In the present work have been used column and... more
The nitrosamines have been extensively studied for their toxicity. Their formation in the foods containing nitrous and nitric derivatives is favoured by the presence of suitable substrata. In the present work have been used column and capillari GL Chromatography for the determination of some nitrosamines in altered foods. The results obtained by applying the suggested procedure to a number of altered samples have been very encouraging.
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In addition to the already knownonosaccharides fucose, xylose, mannose, galactose, glucose, N-acetylgalactosamine and N-acetylglucosamine, the carbohydrate part of the haemocyanin from Helix pomatia (Roman snail) contains... more
In addition to the already knownonosaccharides fucose, xylose, mannose, galactose, glucose, N-acetylgalactosamine and N-acetylglucosamine, the carbohydrate part of the haemocyanin from Helix pomatia (Roman snail) contains 3-O-methylgalactose, and that from Lymnaea stagnalis (a freshwater snail) 3-O-methylgalactose and 3-O-methylmannose. The 3-O-methyl sugars were identified by g.l.c.-mas spectrometry of the corresponding trimethylsilyl methyl glycosides and the alditol acetates, and by co-chromatography with the synthetic reference substances.
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The separation of the enantiomers of lactic and glyceric acids can be achieved by capillary gas chromatography on SP-1000 using the corresponding O-acetylated methyl esters. The structures of the derivatives were proved by proton magnetic... more
The separation of the enantiomers of lactic and glyceric acids can be achieved by capillary gas chromatography on SP-1000 using the corresponding O-acetylated methyl esters. The structures of the derivatives were proved by proton magnetic resonance spectroscopy and mass spectrometry. The method has been used for the determination of the absolute configuration of lactic and glyceric acids isolated from serum and urine from different patients.
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Aqueous extracts of sheep pineal bodies were separated on Sephadex G-25. Two low molecular weight Sephadex G-25 fractions, F2 and F3, were ultrafiltrated through the Amicon membrane UM-2. The UM-2 filtrate was subsequently filtrated... more
Aqueous extracts of sheep pineal bodies were separated on Sephadex G-25. Two low molecular weight Sephadex G-25 fractions, F2 and F3, were ultrafiltrated through the Amicon membrane UM-2. The UM-2 filtrate was subsequently filtrated through the ultramembrane UM-05 and the UM-05 filtrate was separated on Sephadex G-10 columns. After paper electrophoresis, preparative paper chromatography was carried out. The fluorescent band showing a Rf value identical with synthetic 6-biopterin was eluted; gas liquid chromatography and mass spectrometry of the isolated compound were carried out. The mass spectra of the isolated compound were shown to be identical with synthetic 6-biopterin. The results of the Crithidia fasciculata test and thinlayer chromatography study revealed that the isolated compound is identical with 6-L-erythro-biopterin. The activities of the isolated compound and of synthetic biopterin in in vitro and in vivo bioassays are demonstrated.
Research Interests: Mass Spectrometry, Fluorescence, Melatonin, Methods, Cerebral Cortex, and 15 moreMice, Hypothalamus, Female, Animals, Male, GAS LIQUID CHROMATOGRAPHY, Neural, Pineal Gland, Biological Assay, Luteinizing Hormone, Aqueous Extract, Gonadotropins, Neurosciences, Low molecular weight, and Anterior Pituitary
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The absolute configurations of urinary 2-hydroxybutyrate and 3-hydroxybutyrate were determined in patients with lactic acidemia and ketosis by capillary gas-liquid chromatography of their O-acetylated (--)-menthyl ester derivatives.... more
The absolute configurations of urinary 2-hydroxybutyrate and 3-hydroxybutyrate were determined in patients with lactic acidemia and ketosis by capillary gas-liquid chromatography of their O-acetylated (--)-menthyl ester derivatives. 2-Hydroxybutyrate had the L-configuration, whereas 3-hydroxybutyrate was in the D-configuration.
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Three siblings suffered from an unusual disorder of cyclic vomiting and congenital hepatic fibrosis. Serum transferrin isoelectric focusing showed increased asialo- and disialotransferrin isoforms as seen in the carbohydrate-deficient... more
Three siblings suffered from an unusual disorder of cyclic vomiting and congenital hepatic fibrosis. Serum transferrin isoelectric focusing showed increased asialo- and disialotransferrin isoforms as seen in the carbohydrate-deficient glycoprotein (CDG) syndrome type I. Phosphomannomutase, which is deficient in most patients with type I CDG syndrome, was found to be normal in all three patients. Structural analysis of serum transferrin revealed nonglycosylated, hypoglycosylated, and normoglycosylated transferrin molecules. These findings suggested a defect in the early glycosylation pathway. Phosphomannose isomerase was found to be deficient and the defect was present in leucocytes, fibroblasts, and liver tissue. Phosphomannose isomerase deficiency appears to be a novel glycosylation disorder, which is biochemically indistinguishable from CDG syndrome type I. However, the clinical presentation is entirely different.
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500-MHz H-NMR spectroscopy of the oligosaccharides derived from gamma-seminoprotein, a human seminal plasma glycoprotein, revealed considerable microheterogeneity both with respect to the degree of branching and with regard to the... more
500-MHz H-NMR spectroscopy of the oligosaccharides derived from gamma-seminoprotein, a human seminal plasma glycoprotein, revealed considerable microheterogeneity both with respect to the degree of branching and with regard to the peripheral sugars. Although the protein possesses only one N-glycosylation site, di-, tri- and tetra-antennary glycans in a ratio of 40:15:45 were found to be present. Moreover, certain branches of the tri- and tetra-antennas contain one or two Fuc residues which form part of the SSEA-1 and Y determinants. It should be noted that this is the first report that describes the occurrence and localization of the Y determinant, i.e. Fuc alpha (1----2)Gal beta (1----4) [Fuc alpha (1----3)]GlcNAc beta (1----.), in an N-glycan of a glycoprotein.
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Research Interests: Mass Spectrometry, Polysaccharides, Protein Engineering, Erythropoietin, Cell line, and 15 moreHumans, Animals, Goats, Epidermal Growth Factor, Epithelial cells, Mammary gland, Protein Expression, Primary Culture, Growth Factor, Culture Media, Recombinant Proteins, Capra Hircus, Biochemistry and cell biology, Doubling Time, and Chinese hamster ovary
The quantitative analysis by gas chromatography of monosaccharides present in glycoproteins and glycopeptides using methanolysis, followed by re-N-acetylation and trimethylsilylation, gives rise to several peaks for each monosaccharide.... more
The quantitative analysis by gas chromatography of monosaccharides present in glycoproteins and glycopeptides using methanolysis, followed by re-N-acetylation and trimethylsilylation, gives rise to several peaks for each monosaccharide. The identity of these peaks for xylose, fucose, mannose, galactose, glucose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid was established for alpha- and beta-methyl pyranosides and furanosides by combined g.l.c.-mass spectrometry and proton-magnetic-resonance spectroscopy. These data provide for the unambiguous interpretation of the gas chromatograms obtained in the application of this g.l.c. method, and supply basic information for the further application of mass spectrometry in this field.
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The primary structure of the carbohydrate chains of hemocyanin from the crayfish Astacus leptodactylus were investigated. The carbohydrate content is 0.2% (w/w) as referred to total hemocyanin content, resp. 1.8% as referred only to the... more
The primary structure of the carbohydrate chains of hemocyanin from the crayfish Astacus leptodactylus were investigated. The carbohydrate content is 0.2% (w/w) as referred to total hemocyanin content, resp. 1.8% as referred only to the one subunit which is glycosylated. Mannose and M-acetylglucosamine are present in a molar ratio of 6:2. The carbohydrate chains are N-glycosidically linked as revealed by dot blot analysis using various lectins and enzyma-tic deglycosylation. Furthermore, they are part of only one hemocyanin subunit of A. leptodactylus. After enzymatic deglycosylation with PNGase F, the oligosaccharide pool was separated by FPLC on Mono Q and subsequent HPLC on Lichrosorb-NH 2 , the subfractions were characterized by 1 H NMR spectroscopy. A total of six oligosaccharides, ranging from Man 4 GlcNAc 2 to Man 9 GlcNAc 2 is present, Man e GlcNAc 2 representing the most abundant one with 57% of all oligosaccharides.
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Many viruses achieve reversible attachment to sialic acid (Sia) by encoding envelope glycoproteins with receptor-binding and receptor-destroying activities. Toroviruses and group 2 coronaviruses bind to O-acetylated Sias, presumably via... more
Many viruses achieve reversible attachment to sialic acid (Sia) by encoding envelope glycoproteins with receptor-binding and receptor-destroying activities. Toroviruses and group 2 coronaviruses bind to O-acetylated Sias, presumably via their spike proteins (S), whereas other glycoproteins, the hemagglutinin-esterases (HE), destroy Sia receptors by de-O-acetylation. Here, we present a comprehensive study of these enzymes. Sialate-9-O-acetylesterases specific for 5-N-acetyl-9-O-acetylneuraminic acid, described for bovine and human coronaviruses, also occur in equine coronaviruses and in porcine toroviruses. Bovine toroviruses, however, express novel sialate-9-O-acetylesterases, which prefer the di-O-acetylated substrate 5-N-acetyl-7(8),9-di-O-acetylneuraminic acid. Whereas most rodent coronaviruses express sialate-4-O-acetylesterases, the HE of murine coronavirus DVIM cleaves 9-O-acetylated Sias. Under the premise that HE specificity reflects receptor usage, we propose that two types...
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Toxoplasma gondii is a ubiquitous parasitic protozoan causing congenital infection and severe encephalitis in the course of the acquired immunodeficiency syndrome. Glycosyl-phosphatidylinositols of T. gondii have been shown to be... more
Toxoplasma gondii is a ubiquitous parasitic protozoan causing congenital infection and severe encephalitis in the course of the acquired immunodeficiency syndrome. Glycosyl-phosphatidylinositols of T. gondii have been shown to be identical with the low molecular weight antigen which elicits an early immunoglobulin M immune response in humans. A detailed study of the structures of these glycolipid antigens was performed. Radiolabelled glycolipids were extensively analysed by chemical and exoglycosidase treatments in combination with high pH anion-exchange chromatography, gel-filtration and lectin affinity chromatography. In addition, carbohydrate fragments prepared and purified from bulk preparations of unlabelled glycolipids by high performance liquid chromatography were subjected to two-dimensional 1H nuclear magnetic resonance spectroscopy, fast-atom bombardment-mass spectrometry, and methylation linkage analysis in order to elucidate the structure of T. gondii GPIs. The following structures were identified: (ethanolamine-PO4)-Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha-inositol-PO4-lipid and the novel structure (ethanolamine-PO4)-Man alpha 1-2Man alpha 1-6(Glc alpha 1-4GalNAc beta 1-4)Man alpha 1-4 GlcN alpha-inositol-PO4-lipid both with and without terminal ethanolamine phosphate. Evidence is provided, that only T. gondii GPIs bearing the unique glucose-N-acetylgalactosamine side branch are immunogenic in humans and that this structure is widely distributed among T. gondii isolates. Monoclonal antibodies have been characterized to recognize structures with different degrees of side-chain modification. We suggest that these reagents in combination with recently devised techniques for insertional mutagenesis in T. gondii should greatly facilitate the cloning of genes essential for GPI side-chain modification.
Research Interests: Molecular Biology, Toxoplasma, Polysaccharides, Magnetic Resonance Spectroscopy, Toxoplasmosis, and 14 moreToxoplasma gondii, Western blotting, Humans, Methylation, Animals, Monoclonal Antibodies, Affinity chromatography, High Pressure Liquid Chromatography, Ethanolamine, Epitopes, Low molecular weight, Biochemistry and cell biology, Molecular Structure, and Oligosaccharides
Toxoplasma gondii is a ubiquitous parasitic protozoan causing congenital infection and severe encephalitis in the course of the acquired immunodeficiency syndrome. Glycosyl-phosphatidylinositols of T. gondii have been shown to be... more
Toxoplasma gondii is a ubiquitous parasitic protozoan causing congenital infection and severe encephalitis in the course of the acquired immunodeficiency syndrome. Glycosyl-phosphatidylinositols of T. gondii have been shown to be identical with the low molecular weight antigen which elicits an early immunoglobulin M immune response in humans. A detailed study of the structures of these glycolipid antigens was performed. Radiolabelled glycolipids were extensively analysed by chemical and exoglycosidase treatments in combination with high pH anion-exchange chromatography, gel-filtration and lectin affinity chromatography. In addition, carbohydrate fragments prepared and purified from bulk preparations of unlabelled glycolipids by high performance liquid chromatography were subjected to two-dimensional 1H nuclear magnetic resonance spectroscopy, fast-atom bombardment-mass spectrometry, and methylation linkage analysis in order to elucidate the structure of T. gondii GPIs. The following...
Research Interests: Molecular Biology, Toxoplasma, Polysaccharides, Magnetic Resonance Spectroscopy, Toxoplasmosis, and 14 moreToxoplasma gondii, Western blotting, Humans, Methylation, Animals, Monoclonal Antibodies, Affinity chromatography, High Pressure Liquid Chromatography, Ethanolamine, Epitopes, Low molecular weight, Biochemistry and cell biology, Molecular Structure, and Oligosaccharides
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Different fungi, including the genera Neosartorya, Byssochlamys and Talaromyces, produce (asco)spores that survive pasteurization treatments and are regarded as the most stress-resistant eukaryotic cells. Here, the NMR analysis of a... more
Different fungi, including the genera Neosartorya, Byssochlamys and Talaromyces, produce (asco)spores that survive pasteurization treatments and are regarded as the most stress-resistant eukaryotic cells. Here, the NMR analysis of a series of trehalose-based oligosaccharides, being compatible solutes that are accumulated to high levels in ascospores of the fungus Neosartorya fischeri, is presented. These oligosaccharides consist of an α,α-trehalose backbone, extended with one [α-d-Glcp-(1→6)-α-d-Glcp-(1↔1)-α-d-Glcp; isobemisiose], two [α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1↔1)-α-d-Glcp] or three [α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1→6)-α-d-Glcp-(1↔1)-α-d-Glcp] glucose units. The tetra- and pentasaccharide, dubbed neosartose and fischerose, respectively, have not been reported before to occur in nature.
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Decoration of prebiotic galacto-oligosaccharides (GOS) with sialic acid yields mixtures of GOS and sialylated GOS (Sia-GOS), novel products that are expected to have both prebiotic and anti-adhesive functionalities. The recombinantly... more
Decoration of prebiotic galacto-oligosaccharides (GOS) with sialic acid yields mixtures of GOS and sialylated GOS (Sia-GOS), novel products that are expected to have both prebiotic and anti-adhesive functionalities. The recombinantly produced trans-sialidase enzyme from Trypanosoma cruzi (TcTS), an enzyme with the ability to transfer (α2-3)-linked sialic acid from sialogalactoglycans to asialogalactoglycans, was employed to catalyze this sialylation. As sialic acid acceptor substrates, Vivinal® GOS and derived fractions of specific degree of polymerization, were taken. As sialic acid donor substrates, bovine κ-casein-derived glycomacropeptide [> 99% N-acetylneuraminic acid (Neu5Ac); < 1% N-glycolylneuraminic acid (Neu5Gc)] and bovine blood plasma glycoprotein mixture (45% Neu5Ac; 55% Neu5Gc) were selected, yielding potential food and feed products, respectively. High-pH anion-exchange chromatography, matrix-assisted laser-desorption ionization time-of-flight mass spectrometry,...
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Glucansucrases are exclusively found in lactic acid bacteria and synthesize a variety of α-glucans from sucrose. They are large multidomain enzymes belonging to the CAZy family 70 of glycoside hydrolase enzymes (GH70). The crystal... more
Glucansucrases are exclusively found in lactic acid bacteria and synthesize a variety of α-glucans from sucrose. They are large multidomain enzymes belonging to the CAZy family 70 of glycoside hydrolase enzymes (GH70). The crystal structure of the N-terminal truncated GTF180 of Lactobacillus reuteri 180 (GTF180-ΔN) revealed that the polypeptide chain follows a U shape course to form five domains, including domains A, B, and C, which resemble those of family GH13 enzymes, and two extra and novel domains (domains IV and V), which are attached to the catalytic core. To elucidate the functional roles of domain V, we have deleted the domain V fragments from both the N- and C-terminal ends (GTF180-ΔNΔV). Truncation of domain V of GTF180-ΔN yielded a catalytically fully active enzyme but with heavily impaired polysaccharide synthesis ability. Instead, GTF180-ΔNΔV produced a large amount of oligosaccharides. Domain V is not involved in determining the linkage specificity, and the size of po...
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The primary structure of the carbohydrate chains of hemocyanin from the crayfish Astacus leptodactylus were investigated. The carbohydrate content is 0.2% (w/w) as referred to total hemocyanin content, resp. 1.8% as referred only to the... more
The primary structure of the carbohydrate chains of hemocyanin from the crayfish Astacus leptodactylus were investigated. The carbohydrate content is 0.2% (w/w) as referred to total hemocyanin content, resp. 1.8% as referred only to the one subunit which is glycosylated. Mannose and N-acetylglucosamine are present in a molar ratio of 6:2. The carbohydrate chains are N-glycosidically linked as revealed by dot blot analysis using various lectins and enzymatic deglycosylation. Furthermore, they are part of only one hemocyanin subunit of A. leptodactylus. After enzymatic deglycosylation with PNGase F, the oligosaccharide pool was separated by FPLC on Mono Q and subsequent HPLC on Lichrosorb-NH2, the subfractions were characterized by 1H NMR spectroscopy. A total of six oligosaccharides, ranging from Man4GlcNAc2 to Man9GlcNAc2 is present, Man6GlcNAc2 representing the most abundant one with 57% of all oligosaccharides.