Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (G... more 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 correlatio...
Glycosylation of proteins represents one of the most important post-(co-)translational events 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.
alpha 2HS-Glycoprotein, a normal human plasma protein, was recently shown to consist of two polyp... 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...
Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells wer... 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.
The cellulase complexes of two cellulolytic bacteria, Clostridium thermocellum and Bacteroides ce... 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...
We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in th... 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.
Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells wer... 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.
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 252, No. 2, February 1, pp. 574590,1987 The Structur... 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 ...
We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in th... 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.
An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous... 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...
The structure of the carbohydrate chains of mucous glycoproteins from the gastro-intestinal tract... 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...
alpha 2HS-Glycoprotein, a normal human plasma protein, was recently shown to consist of two polyp... 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...
Bollettino della Società italiana di biologia sperimentale, Jan 30, 1984
The nitrosamines have been extensively studied for their toxicity. Their formation in the foods c... 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.
In addition to the already knownonosaccharides fucose, xylose, mannose, galactose, glucose, N-ace... 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.
The separation of the enantiomers of lactic and glyceric acids can be achieved by capillary gas c... 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.
Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (G... more 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 correlatio...
Glycosylation of proteins represents one of the most important post-(co-)translational events 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.
alpha 2HS-Glycoprotein, a normal human plasma protein, was recently shown to consist of two polyp... 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...
Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells wer... 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.
The cellulase complexes of two cellulolytic bacteria, Clostridium thermocellum and Bacteroides ce... 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...
We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in th... 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.
Two variants (I and II) of tissue-type plasminogen activator (t-PA) from human melanoma cells wer... 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.
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 252, No. 2, February 1, pp. 574590,1987 The Structur... 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 ...
We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in th... 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.
An extracellular protein complex was isolated from the supernatant of a pectin-limited continuous... 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...
The structure of the carbohydrate chains of mucous glycoproteins from the gastro-intestinal tract... 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...
alpha 2HS-Glycoprotein, a normal human plasma protein, was recently shown to consist of two polyp... 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...
Bollettino della Società italiana di biologia sperimentale, Jan 30, 1984
The nitrosamines have been extensively studied for their toxicity. Their formation in the foods c... 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.
In addition to the already knownonosaccharides fucose, xylose, mannose, galactose, glucose, N-ace... 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.
The separation of the enantiomers of lactic and glyceric acids can be achieved by capillary gas c... 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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