[Application of radioisotopes in the studies of glucoconjugates].
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Carbohydrates↗
Biomedical subjects
A Kobata
Publications and source records attributed to A Kobata.
Synthesis of a heptasaccharide hapten related to a biantennary glycan chain of human chorionic gonadotropin of a choriocarcinoma patient. A convergent approach.
Synthesis of the heptasaccharide hapten 8-methoxycarbonyloctyl O-beta-D-galactopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----2)-O-[beta-D-galactopyranosyl-(1----4)-O-(2-acetam ido-2- deoxy-beta-D-glucopyranosyl)-(1----4)]-O-alpha-D-mannopyranosyl-(1----3) -O- [alpha-D-mannopyranosyl-(1----6)]-beta-D-mannopyranoside is described, by use of the known, protected glycosyl acceptor 8-ethoxy-carbonyloctyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-(1----6)-2,4-di-O-benz yl-beta - D-mannopyranoside, and the key glycopentaosyl donors O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----4)-O-(2-acetami do-3,6- di-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-(1----2)-O-[(2,3,4,6-tetra-O- acetyl-beta-D-galactopyranosyl)-(1----4)-O-(2-acetamido-3,6-di-O-acetyl- 2- deoxy-beta-D-glucopyranosyl)-(1----4)]-3,6-di-O-benzyl-alpha-D-mannopyra nosyl trichloroacetimidate (5) and the corresponding fluoride 7, which, in turn, were prepared in 5 steps from allyl 3,6-di-O-benzyl-alpha-D-mannopyranoside in 35 and 22% overall yields, respectively. In model experiments, the key glycosyl donors 5 and 7 were also treated with the simple glycosyl acceptor 8-ethoxycarbonyloctanol, to give 8-methoxycarbonyloctyl O-beta-D-galactopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----2)-O-[beta-D-galactopyranosyl-(1----4)-O-(2-acetam ido-2- deoxy-beta-D-glucopyranosyl)-(1----4)]-alpha (and beta)-D-mannopyranoside.
Choriocarcinoma↗
Asparagine-linked sugar chains of fetuin: occurrence of tetrasialyl triantennary sugar chains containing the Gal beta 1----3GlcNAc sequence.
Asparagine-linked sugar chains were quantitatively released from fetuin by hydrazinolysis. Structural analysis of the sugar chains by sequential exoglycosidase digestion in combination with methylation analysis and Smith degradation revealed that most of them have typical biantennary (8%) and triantennary (74%) structures containing different amounts of N-acetylneuraminic acid residues. In addition, an unusual tetrasialyl triantennary sugar chain (17%) containing the Gal beta 1----3GlcNAc sequence in the outer chain moiety was detected, and its structure was elucidated as NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)-GlcNAc beta 1----4(NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2)Man alpha 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6)Man beta 1----4GlcNAc beta 1----4GlcNAc.
Acetylglucosamine↗
Synthesis of a pentasaccharide hapten related to a monoantennary glycan chain of human chorionic gonadotropin.
A stereocontrolled synthesis of a pentasaccharide hapten, namely, 8-ethoxy-carbonyloctyl O-beta-D-galactopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D-gluc opy ranosyl) -(1----2)-O-alpha-D-mannopyranosyl-(1----3)-O-[alpha-D-mannopyranosyl- (1----6)]-alpha-D-mannopyranoside, is described employing a trihexosyl glycosyl-donor, O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----4)-O-(2-acet amido-3,6- di-O-acetyl-2-deoxy-beta- D-glucopyranosyl)-(1----2)-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl trichloroacetimidate, and a mannobiosyl glycosyl-acceptor, 8-ethoxycarbonyl-octyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-(1----6)-2,4-di-O-benz yl-beta - D-mannopyranoside, as the key intermediates.
Carbohydrate Conformation↗
Synthesis of a heptasaccharide hapten related to an anomalous biantennary glycan-chain of human chorionic gonadotropin of a patient with choriocarcinoma. A stepwise approach.
A stereocontrolled synthesis of a heptasaccharide hapten, 8-methoxycarbonyloctyl 3-O-[2,4-di-O-(2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-beta-D- glucopyranosyl)-alpha-D-mannopyranosyl]-6-O-alpha-D-mannopyranosyl-beta- D-mannopyranoside (2), is described employing the lactosaminyl donor 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4, 6-tetra-O-acetyl-beta-D-galactopyranosyl)-beta-D-glucopyranosyl bromide and the mannotriosyl glycosyl acceptor 8-ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O-(3,6-di-O-benzyl-alpha-D-mannopyranosyl)-6-O-(2,3, 4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-beta-D-mannopyranoside, the reaction of which gave the biantennary structure 8-ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O-(3,6- di-O-benzyl-2,4-di-O-[3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3, 4,6-tetra-O-benzyl-alpha-D-mannopyranosyl)-beta-D-mannopyranoside (9), as well as a monoglycosylated product, 8-ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O-(3,6-di-O-benzyl-4-O-[3,6- di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6- tetra-O-benzyl-alpha-D-mannopyranosyl)-beta-D-mannopyranoside. The diglycosylated product 9 was transformed into 2, and the structure was confirmed by 1H-n.m.r. data.
Amino Acid Sequence↗
Structure identification of the complex-type, asparagine-linked sugar chains of beta-D-galactosyl-transferase purified from human milk.
The asparagine-linked sugar chains of human milk galactosyltransferase were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted into radioactive oligosaccharides by sodium borotritiate reduction after N-acetylation, and fractionated by paper electrophoresis and by Bio-Gel P-4 column chromatography after sialidase treatment. Structural studies of each oligosaccharides by sequential exoglycosidase digestion and methylation analysis indicated that the galactosyltransferase contains bi, tri-, and probably tetra-antennary, complex-type oligosaccharides having alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D- GlcpNac-(1----4)-alpha-L-[Fucp-(1----6)]-D-GlcNAc as their common core. Variation is produced by the different locations and numbers of the five different outer chains: beta-D-Galp-(1----4)-D-GlcNAc, alpha-L-Fucp-(1----3)-[beta-D-Galp-(1----4)]-D-GlcNAc, alpha-NeuAc-(2----6)-beta-D-Galp-(1----4)-D-GlcNAc, alpha-L-Fucp-(1----3)-[beta-D-Galp-(1----4)]-beta-D-GlcpNAc-(1---- 3)-beta- D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-GlcNAc, and alpha-NeuAc-(2----6)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D - Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcNAc.
Asparagine↗
The structures of the asparagine-linked sugar chains of bovine interphotoreceptor retinol-binding protein. Occurrence of fucosylated hybrid-type oligosaccharides.
The sugar chains of interphotoreceptor retinol-binding protein purified from the interphotoreceptor matrix of bovine eyes were liberated from the polypeptide portion by hydrazinolysis followed by N-acetylation and NaB[3H]4 reduction. The oligosaccharide fraction thus obtained was separated into four acidic fractions by paper electrophoresis. The four acidic fractions were confirmed to be mixtures of mono-, di-, tri-, and tetrasialyloligosaccharides. Both N-acetyl- and N-glycolylneuraminic acids were found as sialic acids of interphotoreceptor retinol-binding protein. The monosialylated oligosaccharide fraction, which accounted for 40 molar per cent of the total oligosaccharides liberated, was a mixture of the following hybrid-type oligosaccharides: (Formula: see text) This is the first time that fucosylated hybrid-type oligosaccharides have been found in any glycoprotein. The di-, tri-, and tetrasialyloligosaccharide fractions were composed of biantennary complex-type oligosaccharides, the outer chains of which are either Sia alpha 2----(3- or 6-linked)Gal beta 1----3(Sia alpha 2----6)GlcNac or Sia alpha 2----(3- or 6-linked)Gal beta 1----4GlcNAc.
Animals↗
Fractionation by lectin affinity chromatography indicates that the glycosylation of most ribonucleases in human viscera and body fluids is organ specific.
The behavior of ribonucleases extracted from various human viscera in five lectin-Sepharose columns shows that almost all contain carbohydrates and that there are organ-specific differences in the structure of these carbohydrates.
Body Fluids↗
Structures of sugar chains of human kidney gamma-glutamyltranspeptidase.
gamma-Glutamyltranspeptidase purified from human kidneys contains 4-5 asparagine-linked sugar chains in each molecule. The sugar chains were released from the polypeptide portion of the enzyme by hydrazinolysis as oligosaccharides and separated by paper electrophoresis into one neutral and two acidic fractions. By sequential exoglycosidase digestion and methylation analysis, the neutral fraction, which comprised 69% of total oligosaccharides, was shown to be a mixture of bisected bi- and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups in their outer chain moieties. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of bisected triantennary complex-type oligosaccharides with Gal beta 1----4GlcNAc and/or Gal beta 1----4(Fuc alpha 1----3)GlcNAc group in their outer chain moieties. Some of the outer chains of the acidic oligosaccharides were considered to be sialylated X-antigenic structures.
Carbohydrates↗
Structure of asparagine-linked sugar chains in the major polypeptide of hepatitis B surface antigen.
Asparagine-linked sugar chains were quantitatively released from hepatitis B surface antigen on hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The released oligosaccharides were composed of two major sialylated components and a trace of a neutral component. From the results of the combination of sequential exoglycosidase digestion and methylation analysis, the structures of the acidic and the neutral components were deduced to be NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3(+/- NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6)Man beta 1----4GlcNAc beta 1----4GlcNAcOT and Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6)Man beta 1----4GlcNAc beta 1----4GlcNAcOT, respectively.
Antigens, Surface↗
Purification and characterization of a novel alpha-mannosidase from Aspergillus saitoi.
An alpha-mannosidase differing from 1,2-alpha-mannosidase was found to occur in Aspergillus saitoi. By a series of column chromatographies the enzyme was purified up to 1,000-fold, and its properties were studied in detail. The enzyme preparation, which was practically free from other exoglycosidases, showed a pH optimum of 5.0. In contrast to 1,2-alpha-mannosidase, the enzyme was strongly activated by Ca2+ ions. p-Nitrophenyl alpha-mannopyranoside was not hydrolyzed by the enzyme. Accordingly, the substrate specificity of the new alpha-mannosidase was studied by using a variety of tritium-labeled oligosaccharides. Studies with linear oligosaccharides revealed that the enzyme cleaves the Man alpha 1----3Man linkage more than 10 times faster than the Man alpha 1----6Man and the Man alpha 1----2Man linkages. Furthermore, it cleaves the Man alpha 1----6Man linkage of the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT only after its Man alpha 1----3 residue is removed. Because of this specificity, the enzyme can be used as an effective reagent to discriminate R----Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT from its isomeric counterparts, Man alpha 1----6(R----Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, in which R represents sugars.
Aspergillus↗
[Glycoproteins produced by tumor cells].
Many glycoproteins which are produced by tumor cells and released into serum and urine, are useful for the diagnosis and prognosis of tumors. This review article describes the representative glycoproteins in this field and includes details about their carbohydrate moieties which have been clarified in recent years.
Acid Phosphatase↗
[Abnormal sugar chains found in tumor glycoproteins].
Comparative study of the asparagine-linked sugar chains of hCGs purified from urine of pregnant women and of choriocarcinoma patients revealed that extensive and structural changes occur common in the sugar chains of tumor hCGs. The changes can be ascribed to enhancement of an alpha-fucosyltransferase that forms the Fuc alpha 1----6GlcNAc group in the sugar chains and the appearance of N-acetylglucosaminyltransferase IV.
Asparagine↗
Carbohydrates of lysosomal enzymes secreted by Tetrahymena pyriformis.
The carbohydrate structures of acid phosphatase and alpha-glucosidase secreted into culture medium by Tetrahymena pyriformis strain W were studied. Their asparagine-linked sugar chains were quantitatively liberated as radioactive oligosaccharides from their polypeptide moieties by controlled hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The approximate amounts of total sugar chains liberated from 1 mol each of acid phosphatase and alpha-glucosidase were 6 and 4 mol, respectively. Paper electrophoresis revealed that only neutral oligosaccharides were obtained from both enzymes. The oligosaccharide fraction from acid phosphatase was separated into seven components by Bio-Gel P-4 column chromatography while that from alpha-glucosidase was resolved into three components. The structures of these oligosaccharides were determined by sequential glycosidase digestion in combination with methylation analysis. The sugar chains of the two enzymes can be primarily classified as high mannose-type oligosaccharides. However, they have the following characteristic features: 1) their common core is not the usual Man5 . GlcNAc2 structure, it is Man3 . GlcNAc2; 2) some of the sugar chains of acid phosphatase have 1 approximately 3 glucose residues linked to the nonreducing terminal Man alpha 1----2 residue. The structural characteristics of the sugar moieties of the two enzymes indicate that they might be produced by the so-called "alternate pathway," in which lipid-linked Glc3 . Man5 . GlcNAc2 functions as an oligosaccharide donor.
Acid Phosphatase↗
Structures of the sugar chains of rabbit immunoglobulin G: occurrence of asparagine-linked sugar chains in Fab fragment.
The asparagine-linked sugar chains of rabbit immunoglobulin G (IgG) and its Fc and Fab fragments were quantitatively liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Rabbit IgG was shown to contain 2.3 mol of asparagine-linked sugar chains per molecule distributed in both the Fc and Fab fragments. The sugar chains were of the biantennary complex type containing four cores: Man alpha 1----6(Man alpha 1----3)(+/- GlcNAc beta 1----4)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)-GlcNAc. A total of 16 distinct neutral oligosaccharide structures was found after sialidase treatment. The galactose residue in the monogalactosylated oligosaccharides was present on either the alpha 1----3 or alpha 1----6 side of the trimannosyl core. The Fab fragments contained neutral, monosialylated, and disialylated oligosaccharides, whereas the Fc fragment contained only neutral and monosialylated structures. The oligosaccharides isolated from the Fab fragments also contained more galactose and bisecting N-acetylglucosamine residues than those from the Fc fragments.
Animals↗
The structures of the carbohydrate moieties of mouse kidney gamma-glutamyltranspeptidase: occurrence of X-antigenic determinants and bisecting N-acetylglucosamine residues.
Paper electrophoresis and Bio-Gel P-4 column chromatography of the oligosaccharides released from mouse kidney gamma-glutamyltranspeptidase by hydrazinolysis gave fractionation patterns quite distinct from those of the bovine and rat kidney enzymes. Structural studies of the fractionated oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis showed that mouse kidney gamma-glutamyltranspeptidase contains a series of bisected complex-type asparagine-linked sugar chains with the following oligosaccharides as their outer chain moieties: GlcNAc beta 1----, Sia alpha 2----Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, and sialylated N-acetyllactosamine repeating sugar chains. Some of these sugar chains were found for the first time in glycoproteins.
Acetylglucosamine↗
Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin.
The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-Sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the alpha-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-Sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fuc alpha 1----2Gal beta 1----4GlcNAc and Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol II, which has a Gal beta 1----3(Fuc alpha 1----4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fuc alpha 1----2Gal beta 1----3GlcNAc and Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups showed almost no interaction with the matrix.
Acetylglucosamine↗
Enzymatic basis for the structural changes of asparagine-linked sugar chains of membrane glycoproteins of baby hamster kidney cells induced by polyoma transformation.
Previous studies indicated that enrichment of the GlcNAc beta 1----6Man alpha 1---- group with concomitant decrease of the GlcNAc beta 1----4Man alpha 1---- group occurs in the complex-type asparagine-linked sugar chains of the membrane glycoproteins of baby hamster kidney cells transformed by polyoma virus. The enzymatic basis of the chemical change is reported in this paper. By using oligosaccharides isolated from the urine of patients with a variety of exoglycosidase deficiencies, beta-N-acetylglucosaminyltransferases in the cell homogenate were successfully assayed separately. Both baby hamster kidney cells and their polyoma transformants contain beta-N-acetylglucosaminyltransferases I, II, IV, V, and VI, but not beta-N-acetylglucosaminyltransferase III. The beta-N-acetylglucosamine residue added by each beta-N-acetylglucosaminyltransferase (GnT) is shown below. (formula see text) Comparative studies of the specific activities of the five beta-N-acetylglucosaminyltransferases in the two cell lines revealed that the value of beta-N-acetylglucosaminyltransferase V in the polyoma transformant was twice of that in the normal cells, while those of the other four transferases in the two cell lines were not significantly different. Therefore the increase in beta-N-acetylglucosaminyltransferase V may be the direct cause of the changes found in the sugar chains of surface glycoproteins in baby hamster kidney cells transformed by polyoma virus.
Animals↗