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A Kobata

Publications and source records attributed to A Kobata.

At least 55 records · Page 3Linked to original sources
PubMed1993

Role of sugar chains in the in-vitro activity of recombinant human interleukin 5.

The structures of O-linked sugar chains of recombinant human interleukin 5 (rhIL-5) produced by Chinese hamster ovary cells were determined employing high-performance anion-exchange column chromatography under high pH conditions. The core sequence was elucidated to be Gal beta 1-->3-GalNAc by its susceptibility to endo-alpha-N-acetylgalactosaminidase and sialic acid linkages were determined using sialidases of different specificities. To investigate the role of sugar chains in the in-vitro activity of rhIL-5, it was digested with various glycosidases. While removal of N-linked sugar chains resulted in 2.8-fold increase of the activity, de-O-glycosylated rhIL-5 showed 10-fold higher activity than intact rhIL-5, suggesting that the presence of O-linked sugar chains suppresses the activity more effectively than that of N-linked chains. While incubation of de-N-glycosylated rhIL-5 at 70 degrees C for 30 min decreased the activity, intact and de-O-glycosylated rhIL-5 lost little activity, suggesting that N-linked sugar chains contributed to the thermostability of the molecule.

Borohydrides↗
PubMed1993

Altered glycosylation of beta 1 integrins associated with reduced adhesiveness to fibronectin and laminin.

The carbohydrate structures of the beta 1 integrins obtained from a mouse metastatic melanoma B16 F1 and its weakly metastatic wheat-germ agglutinin-resistant mutant Wa4-b1 were studied comparatively. The results indicated that the integrins from both cells contain high mannose-type and bi-, tri- and tetra-antennary complex-type sugar chains. No significant difference was found in the outer chain branching between both integrins, but sialylation of the sugar chains of the mutant's integrin was markedly decreased and almost all the outer chain moieties of tri- and tetra-antennary oligosaccharides of the mutant's integrin were fucosylated, resulting in the formation of X-antigenic determinants, Gal beta 1-->4 (Fuc alpha 1-->3) GlcNAc. In contrast, the integrin from parental cell contained no X-antigenic determinant. These structural differences found in the integrin are thought to account for the reduction in the metastatic potential of the mutant which also shows reduced adhesion to fibronectin and laminin as compared with the parental cell.

Animals↗
PubMed1993

Structures of mucin-type sugar chains on human erythropoietins purified from urine and the culture medium of recombinant Chinese hamster ovary cells.

Less is known about the mucin-type sugar chains attached to human erythropoietin as compared with N-linked sugar chains which structures and function have been well studied. In this study, we purified urinary human erythropoietin from three independent groups of aplastic anemia patients, and analyzed the structures of mucin-type sugar chains as well as that obtained from recombinant human erythropoietin produced by Chinese hamster ovary cells. Unlike the N-linked sugar chains, the mucin-type sugar chains are totally different between the urinary and the recombinant erythropoietins. All of the three independent samples of urinary erythropoietin contained oligosaccharides with the structures of +/- Neu5Ac alpha 2-->6GalNAc, while recombinant human erythropoietin contained those with the structures of Neu5Ac alpha 2-->3Gal beta 1-->3(Neu5Ac alpha 2-->6)GalNAc, Neu5Ac alpha 2-->3Gal beta 1-->3GalNAc, and Gal beta 1-->3(Neu5Ac alpha 2-->6)GalNAc.

Anemia, Aplastic↗
PubMed1993

Purification and characterization of an alpha-L-fucosidase from Pomacea canaliculata.

An alpha-L-fucosidase (EC 3.2.1.51) was isolated from the hepatopancreas of Pomacea canaliculata. The enzyme was purified 285-fold from the crude enzyme extract by procedures involving first heat treatment, ammonium sulfate fractionation, second heat treatment, and chromatography on DEAE-Sepharose, hydroxylapatite, and L-fucosylamine-CH-Sepharose. When assayed by using p-nitrophenyl glycosides as substrates, the final preparation was free from other glycosidase activities and gave a single protein band which corresponded to alpha-L-fucosidase activity on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be 260,000 by Sephacryl S-300 column chromatography. The enzyme has two optimum pH values, 2.5 and 5.0, and the apparent Km value and the maximum velocity for p-nitrophenyl alpha-L-fucoside at both pH were calculated to be 0.45 mM and 1.46 mumol/min/mg of protein, respectively. The enzyme was shown to hydrolyze the Fuc alpha 1-->2Gal, the Fuc alpha 1-->4GlcNAc, and the Fuc alpha 1-->6GlcNAc linkages, but hardly acts on the Fuc alpha 1-->3GlcNAc linkage in various oligosaccharides.

Ammonium Sulfate↗
PubMed1993

Structural study of the sugar chains of porcine factor VIII--tissue- and species-specific glycosylation of factor VIII.

The asparagine-linked sugar chains of blood coagulation factor VIII purified from porcine plasma were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and the sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns. Structural study of each oligosaccharide by sequential exoglycosidase digestion and by methylation analysis revealed that porcine factor VIII contains high mannose-type and bi-, tri-, and tetraantennary complex-type sugar chains. Sixty-seven percent of the complex-type sugar chains contained the Gal alpha 1-->3Gal group, and 23% of the biantennary complex-type sugar chains contained the bisecting N-acetylglucosamine residue. These structures were not detected in the sugar chains of human plasma factor VIII. An in vitro competition study of von Willebrand factor and anti-Gal antibody for binding to factor VIII revealed that von Willebrand factor prevented antibody binding to Gal alpha 1-->3Gal groups in porcine factor VIII sugar chains. This suggests that anti-Gal antibody present in human plasma may not interact with the sugar chains of therapeutic porcine factor VIII. Reverse-transcription polymerase chain reaction was used to identify porcine tissues producing FVIII mRNA. These studies revealed that the kidney is one of the major tissues expressing factor VIII which may contain the sugar chains with the bisecting N-acetylglucosamine residue.

Animals↗
PubMed1993

Kinetic study of human beta-1,4-galactosyltransferase expressed in E. coli.

Recombinant beta-1,4-galactosyltransferase which synthesizes the Gal beta 1-->4GlcNAc group of glycoprotein sugar chains was obtained as a soluble form from Escherichia coli by transfection of the human cDNA lacking the transmembrane segment. Kinetic study revealed that the soluble transferase has the same apparent Km values toward sugar nucleotide and sugar acceptors as those of mouse membrane-bound beta-1,4-galactosyltransferase previously characterized [Nakazawa et al. (1991) Eur. J. Biochem. 196, 363-368]. However, the Vmax value of this transferase was low when compared to that of the mammalian transferase, probably due to the instability of the transferase caused by the lack of protein glycosylation. The soluble transferase was purified from the E. coli lysates almost to homogeneity by chromatography on DEAE-Sepharose and alpha-lactalbumin-Sepharose columns. Using this purified transferase, the acceptor specificity of the transferase has been studied. The results showed that the transferase has apparent Km values of 170, 190, and 830 microM for agalacto-poly-N-acetyllactosamine, lacto-N-triose II, and lacto-N-triaosylceramide, respectively, but has apparently no activity toward glucosylceramide. These results suggest that the beta-1,4-galactosyltransferase may be involved in the synthesis of poly-N-acetyllactosamine, lacto-N-neotetraose, and probably lacto-N-neotetraosylceramide in addition to the formation of the Gal beta 1-->4GlcNAc group of glycoprotein sugar chains and lactose.

Carbohydrate Sequence↗
PubMed1993

Most bovine milk fat globule membrane glycoproteins contain asparagine-linked sugar chains with GalNAc beta 1-->4GlcNAc groups.

N-Acetylgalactosamine is usually not a constitutive monosaccharide of Asn-linked sugar chains. Our previous study showed that the Asn-linked sugar chains of bovine CD36 prepared from milk fat globule membranes (MFGM) contain this unique monosaccharide as the GalNAc beta 1-->4GlcNAc group [Nakata et al. (1993) Biochemistry 32, 4369-4383]. Western blot analysis of bovine MFGM glycoproteins with Wistaria floribunda agglutinin (WFA), which binds oligosaccharides terminating with either an alpha- or beta-N-acetylgalactosamine residue, showed that WFA binding is observed for most of the protein bands as detected with Coomassie Brilliant Blue staining. However, no WFA binding was observed for protein bands after treatment of MFGM glycoproteins with N-glycanase. Structural analyses of the sugar chains released by hydrazinolysis from the MFGM glycoproteins by sequential exoglycosidase digestion and by methylation analysis revealed that oligosaccharides, which bound to a WFA-agarose column, are bi-, tri-, and tetraantennary complex-type and hybrid-type sugar chains with the GalNAc beta 1-->4GlcNAc group in their outer chain moieties, while oligosaccharides, which passed through the column, were of high-mannose-type, hybrid-type, and complex-type, of which the latter two groups contained the Gal beta 1-->4GlcNAc groups. These results indicated that many bovine MFGM glycoproteins contain Asn-linked sugar chains with the GalNAc beta 1-->4GlcNAc group.

Acetylgalactosamine↗
PubMed1992

Structures and functions of the sugar chains of glycoproteins.

Most proteins within living organisms contain sugar chains. Recent advancements in cell biology have revealed that many of these sugar chains play important roles as signals for cell-surface recognition phenomena in multi-cellular organisms. In order to elucidate the biological information included in the sugar chains and link them with biology, a novel scientific field called 'glycobiology' has been established. This review will give an outline of the analytical techniques for the structural study of the sugar chains of glycoproteins, the structural characteristics of the sugar chains and the biosynthetic mechanism to produce such characteristics. Based on this knowledge, functional aspects of the sugar chains of glycohormones and of those in the immune system will be described to help others understand this new scientific field.

Animals↗
PubMed1992

Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.

Animals↗
PubMed1992

Immobilized lectin columns: useful tools for the fractionation and structural analysis of oligosaccharides.

Elucidation of the binding specificity of a concanavalin A-Sepharose column led to the possibility of the affinity chromatography of oligosaccharides and glycopeptides with the use of immobilized lectin columns. Subsequent addition of immobilized erythroagglutinating phytohaemagglutinin, Aleuria aurantia lectin, Datura stramonium agglutinin, Ricinus communis agglutinin and Allomyrina dichotoma agglutinin to the range of well characterized lectin columns has afforded a way to fractionate a mixture of N-linked oligosaccharides even to a single component.

Animals↗
PubMed1992

Carbohydrate binding specificity of immobilized Psathyrella velutina lectin.

The carbohydrate binding specificity of Psathyrella velutina lectin (PVL) was thoroughly investigated by analyzing the behavior of various complex-type oligosaccharides and human milk oligosaccharides on a PVL-Affi-Gel 10 column. Basically, the lectin interacts with the nonreducing terminal beta-N-acetylglucosamine residue, but does not show any affinity for the nonreducing terminal N-acetylgalactosamine or N-acetylneuraminic acid residue. Substitution of the terminal N-acetylglucosamine residues of oligosaccharides by galactose completely abolishes their affinity to the column. GlcNAc beta 1----3Gal beta 1----4sorbitol binds to the column, but GlcNAc beta 1----6Gal beta 1----4sorbitol is only retarded in the column. The behavior of degalactosylated N-linked oligosaccharides is quite interesting. Although all degalactosylated monoantennary sugar chain isomers are retarded in the column, those with the GlcNAc beta 1----2Man group interact more strongly with the column than those with the GlcNAc beta 1----4Man group or the GlcNAc beta 1----6Man group. The degalactosylated bi- and triantennary sugar chains bind to the column, but the tetraantennary ones are only retarded in the column. These results indicated that the binding affinity is not simply determined by the number of terminal N-acetylglucosamine residues. Addition of the bisecting N-acetylglucosamine residue reduces the affinity of oligosaccharides to the column, but addition of an alpha-fucosyl residue at the C-6 position of the proximal N-acetylglucosamine residue does not affect the behavior of oligosaccharides in the column. These results indicated that the binding specificity of PVL is quite different from those of other N-acetylglucosamine-binding lectins from higher plants, which interact preferentially with the GlcNAc beta 1----4 residue.

Basidiomycota↗
PubMed1992

Protein glycosylation.

Glycan structures can modulate the biological properties and functions of glycoproteins. This has been shown by investigation of the biological activities and glycan structures of several recombinant glycoproteins. Glycan structures of glycoproteins differ according to the species and tissue producing them, and selection of an appropriate host-cell type can generate recombinant glycoproteins with new characteristics.

Animals↗
PubMed1992

Characterization of recombinant murine interleukin 5 expressed in Chinese hamster ovary cells.

We have purified recombinant murine interleukin 5 (rmIL-5) from the supernatant of Chinese hamster ovary cells. Each peptide fragment of the purified rmIL-5 generated by Achromobacter protease I digestion was characterized and glycosylation sites were determined. Although rmIL-5 contains three potential sites of N-linked glycosylation (Asn-26, Asn-55 and Asn-69), Asn-69 is not glycosylated. The oligosaccharides released from the protein by hydrazinolysis were fractionated by paper electrophoresis, lectin column chromatography and gel permeation chromatography, and their structures were analysed by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bi-, tri- and tetraantennary complex-type sugar chains with and without a fucose at the C-6 position of the proximal N-acetylglucosamine residue and high-mannose-type sugar chains. Although > 80% of the sugar chains are neutral oligosaccharides similar to recombinant human IL-5 (rhIL-5; Kodama, S., Endo, T., Tsuroka, N., Tsujimoto, M. and Kobata, A. (1991) J. Biochem., 110, 693-701), rmIL-5 has more tetraantennary oligosaccharides than rhIL-5. A site differential study revealed that Asn-55 has more tetraantennary oligosaccharides than Asn-26.

Amino Acid Sequence↗
PubMed1992

Altered glycosylation of membrane glycoproteins associated with human mammary carcinoma.

N-Linked sugar chains of normal mammary gland, mammary carcinomas (primary lesion), and axillary lymph node metastases of mammary carcinomas were released from their membrane preparations by hydrazinolysis and their structures were analyzed. Fractionation using a Datura stramonium agglutinin (DSA)-Sepharose column revealed that the metastasized carcinomas contain more than twice as much DSA-binding oligosaccharides as the normal gland, and the primary carcinomas contain an intermediate amount. These oligosaccharides were elucidated to have tri- and tetraantennary structures containing the GlcNAc beta 1-->6(GlcNAc beta 1-->2)Man group with and without N-acetyllactosamine repeating units. Lectin blot analysis of membrane glycoproteins and histochemical staining of tissues using biotinylated DSA indicated that these glycosylation changes predominantly occur in a limited number of glycoproteins with apparent molecular weights of 90, 160, and 210 kilodaltons, and mammary carcinomas are distinguishable from normal gland by their intense intracytoplasmic staining.

Acrylic Resins↗
PubMed1992

Carbohydrate structures of beta-core fragment of human chorionic gonadotropin isolated from a pregnant individual.

Highly purified beta-core fragment was obtained from urine of a pregnant woman with use of an immunoaffinity column. The amino acid sequence of beta-core fragment indicated that it is composed of two polypeptides linked by a disulfide bond. The two polypeptides correspond to the 6-40 and 55-92 portions of hCG beta-subunit. Both Asn13 and Asn30 residues were glycosylated. The N-linked sugar chains of beta-core fragment were quantitatively released as radioactive oligosaccharides by hydrazinolysis, followed by N-acetylation and NaB3H4 reduction. The radioactive oligosaccharides were fractionated by serial lectin column chromatography and Bio-Gel P-4 column chromatography, and their structures were investigated by sequential exoglycosidase digestion and periodate oxidation. The results indicated that they were a mixture of the four oligosaccharides: Man alpha 1----6(+/- Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The structural characteristics of the sugar chains of beta-core fragment are quite different from those of the beta-subunit of hCG whose structures were typical biantennary sugar chains containing the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----2 group as their outer chains.

Amino Acid Sequence↗