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A Kobata

Publications and source records attributed to A Kobata.

At least 91 records · Page 5Linked to original sources
PubMed1990

Role of sugar chains in the in vitro biological activity of human erythropoietin produced in recombinant Chinese hamster ovary cells.

Human erythropoietin contains three Asn-type and one mucin-type sugar chains. That the branching structure of the outer portion of Asn-type sugar chains is correlated to its biological activity in vivo has been reported recently (Takeuchi, M., Inoue, N., Strickland, T. W., Kubota, M., Wada, M., Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S., and Kobata, A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7819-7822). In this study, the effect of trimming of sugar chains on the biological activity in vitro of this hormone was examined by using several glycosidases. Human erythropoietin produced by recombinant Chinese hamster ovary cells showed three times higher activity after desialylation. The activity was not changed significantly by further removal of the mucin-type sugar chain from the hormone, indicating no contribution of this type of sugar chain to the activity of erythropoietin in vitro. Sequential removal of galactose and N-acetylglucosamine from the outer chain moieties of the desialylated Asn-type sugar chains raised the activity of the hormone up to four and five times the intact erythropoietin, respectively. The activation effect was diminished slightly by further removing alpha-mannosyl residues and to a great extent by removing beta-mannosyl residues from the core portions of the Asn-type sugar chains. N-Glycanase digestion of intact erythropoietin resulted in almost complete loss of the activity in vitro. These results indicate that the core portion of the Asn-type sugar chains is necessary for erythropoietin to express its full biological activity in vitro and suggest that removal of the core portion of the sugar chains destroys the active conformation of erythropoietin.

Animals↗
PubMed1990

Structures of the asparagine-linked oligosaccharides of an alkaline phosphatase, kasahara isozyme, purified from FL amnion cells.

Asparagine-linked oligosaccharides were quantitatively released by hydrazinolysis from an alkaline phosphatase, Kasahara isozyme, which was purified from FL amnion cells. Almost all of the oligosaccharides (98%) were acidic components, all of which can be converted to neutral oligosaccharides upon sialidase digestion. Structural analysis of the oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis revealed that the alkaline phosphatase of FL cells contains sialylated mono-, bi-, tri-, and tetraantennary complex type sugar chains with the Gal beta 1----4GlcNAc beta 1---- outer chains. Some of the tetraantennary sugar chains contain a single Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1---- outer chain on their Man alpha 1----6 arm. Both fucosylated and nonfucosylated trimannosyl cores were found in the sugar chains. However, it is of interest that the core portion of monoantennary oligosaccharide was not fucosylated and that of the tetraantennary oligosaccharide with a tetrasaccharide outer chain was completely fucosylated.

Alkaline Phosphatase↗
PubMed1990

The structure of the asparagine-linked sugar chains of bovine brain ribonuclease.

The asparagine-linked sugar chains of bovine brain ribonuclease were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. After N-acetylation, they were converted into radioactively-labeled oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharide mixture was fractionated by ion-exchange chromatography, and the acidic oligosaccharides were converted into neutral oligosaccharides by sialidase digestion. The neutral oligosaccharides were then fractionated by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exoglycosidase digestion in combination with methylation analysis revealed that bovine brain ribonuclease showed extensive heterogeneity. It contains bi- and tri-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. Four different outside oligosaccharide chains, i.e., beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----, alpha-Neu5Ac-(2----6)-beta-D- Galp-(1----4)-beta-D-GlcpNAc-(1----, alpha-Neu5Ac-(2----3)-beta-D-Galp-(1----4)- beta-D-GlcpNAc-(1----, and alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----, were found. The preferential distribution of the alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc group on the alpha-D-Manp-(1----6) arm is a characteristic feature of the sugar chains of this enzyme.

Animals↗
PubMed1990

Transfection with fragments of the adenovirus 12 gene induces tumorigenicity-associated alteration of N-linked sugar chains in rat cells.

N-linked sugar chains of rat 3Y1 cells and their poorly tumorigenic (E1Y cells) and highly tumorigenic (CY1 cells) transformants carrying various adenovirus 12 gene fragments were quantitatively released as oligosaccharides from their membrane preparations by hydrazinolysis. After being fractionated by a series of immobilized lectin column chromatography, structures of oligosaccharides in each fraction were studied by sequential glycosidase digestion in combination with methylation analysis. All cells contain bi-, tri-, and tetraantennary complex-type oligosaccharides as well as high mannose-type oligosaccharides in different molar ratio. Expression of 2,4-branched triantennary oligosaccharides increased in both transformed cells. However, their contents were rather higher in poorly tumorigenic E1Y cells than in highly tumorigenic CY1 cells. In contrast, the increase of 2,6-branched triantennary and tetraantennary oligosaccharides was positively correlated to the tumorigenic potential of the transformed cells. The data indicate that glycosylation of cellular proteins is differently affected by the expression of specific regions of the adenovirus genome, and the combined action of E1 and E4 gene products is important for the expression of the GlcNAc beta 1----6Man group associated with tumorigenicity of the cells.

Adenoviridae↗
PubMed1990

Altered glycosylation of human chorionic gonadotropin decreases its hormonal activity as determined by cyclic-adenosine 3',5'-monophosphate production in MA-10 cells.

Human chorionic gonadotropin (hCG) purified from pooled urine of normal pregnant women contains four asparagine-linked sugar chains and four mucin-type sugar chains. The structures of asparagine-linked sugar chains of this hormone are constant and site-specific. hCGs obtained from the urine of patients with invasive mole or choriocarcinoma have quite different sets of oligosaccharides although the primary structures of the polypeptides and the numbers of the sugar chains are the same as those of normal hCG. In order to examine the biological activities of these hCGs with altered glycosylation, we measured the amount of cAMP produced in a murine Leydig tumour cell line, MA-10, after incubation with the hCG samples. The extent of sialylation of oligosaccharides in the three hCG samples used in this study were 88% in normal hCG, 82% in invasive mole hCG and 63% in choriocarcinoma hCG. The hormonal activity of invasive mole hCG was slightly lower while that of choriocarcinoma hCG was significantly (P less than 0.01) lower than that of normal hCG. Complete desialylation induced remarkable loss of full activities in all the samples. However, the hormonal activities of the three samples were different even after desialylation. The full activities of the desialylated samples of invasive mole hCG and choriocarcinoma hCG were 78 and 65% of that of desialylated normal hCG. These results indicated that the structures of the neutral oligosaccharide portion are also important for the expression of full hormonal activity.

Animals↗
PubMed1990

Kinetic study of a galactosyltransferase in the B cells of patients with rheumatoid arthritis.

The sugar chains of IgG samples purified from sera of patients with rheumatoid arthritis (RA) contain many fewer galactose residues than those from sera of healthy individuals. Enzymatic studies revealed that the low galactose content in the IgGs of RA patients results from the reduced activity in the B cells of a galactosyltransferase (EC 2.4.1.90), which preferentially transfers galactose to asialo-agalacto-IgG. Asialo-agalacto-transferrin and asialo-ovine submaxillary mucin were also galactosylated by detergent-activated human B cell homogenates. However, no difference in the enzymatic activities toward these two acceptors was detected between the B cells from RA patients and from non-RA patients and healthy individuals. Enzyme kinetic studies revealed that an affinity of the galactosyltransferase in the B cells from RA patients was lowered for UDP-Gal but not for asialo-agalacto-IgG, while the affinities for UDP-Gal and asialo-agalacto-transferrin of the galactosyltransferase were not changed between the B cells from RA patients and from non-RA patients and healthy individuals in accordance with their enzyme activities. The results indicated that the reduced galactosyltransferase activity toward asialo-agalacto-IgG in the B cells from RA patients can be ascribed to the lowered affinity for UDP-Gal.

Arthritis, Rheumatoid↗
PubMed1990

Primary structure of a fucose-specific lectin obtained from a mushroom, Aleuria aurantia.

Aleuria aurantia lectin (AAL) is a protein composed of two identical subunits having no carbohydrate chain and shows sugar-binding specificity for L-fucose. Full-length cDNA encoding for the lectin has been isolated from a lambda gt11 library, screened with an antiserum directed against AAL. The cDNA clone contained 1,370 nucleotides and an open reading frame of 939 nucleotides encoding 313 amino acids. The amino-terminal sequence (residues 1-30) of the lectin isolated from the mushroom coincided with the deduced amino acid sequence starting from proline at the 2nd residue, indicating that the mature AAL consists of 312 amino acids. Its molecular weight is calculated to be 33,398. The deduced amino acid sequence shows that AAL includes six internal homologous regions, and has considerable homology with a hemagglutinin from a Gram-negative bacterium, Myxococcus xanthus, which forms a fruiting body. No significant homology was observed with higher plant or animal lectins. The recombinant AAL produced by Escherichia coli JM109 carrying the AAL expression plasmid pKA-1 [Fukumori, F. et al. (1989) FEBS Lett. 250, 153-156] was purified from the cell lysate by affinity chromatography using a fucose-starch column, and hundreds of milligrams of the lectin was obtained. The recombinant lectin showed the same biochemical characteristics and sugar binding specificity as did the natural AAL.

Amino Acid Sequence↗
PubMed1990

Comparative study of the N-linked oligosaccharides released from normal human esophageal epithelium and esophageal squamous carcinoma.

N-Linked sugar chains of normal human esophageal epithelium and esophageal squamous carcinoma were quantitatively released as oligosaccharides from their membrane preparations by hydrazinolysis. After being fractionated by serial lectin column chromatography using concanavalin A-Sepharose and Datura stramonium agglutinin-Sepharose, their structures were elucidated by exoglycosidase digestion in combination with methylation analysis. Both normal epithelium and esophageal carcinoma contained bi-, tri- and tetraantennary oligosaccharides as well as high mannose-type oligosaccharides. Interestingly, carcinoma had about 1.6 times larger amounts of tri- and tetraantennary oligosaccharides with the GlcNAc beta 1----4Man alpha 1----and/or the GlcNAc beta 1----6Man alpha 1----linkages than normal epithelium. Tri- and tetraantennary oligosaccharides with N-acetyllactosamine repeating units (the Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc group) were also increased in carcinoma. These data indicated that the altered glycosylation of proteins previously found in transformed rodent cells also occurs widely in human esophageal carcinoma.

Acrylic Resins↗
PubMed1990

Secretory immunoglobulin A carries oligosaccharide receptors for Escherichia coli type 1 fimbrial lectin.

Type 1 fimbriae with mannose-specific lectins are widely distributed among members of the family Enterobacteriaceae and confer the ability to attach to a range of host cells, including colonic epithelial cells. The mucosal surfaces are protected by secretory immunoglobulin A (IgA), which agglutinates microorganisms and prevents their attachment to host epithelial cells. This action has been attributed to a specificity of the antigen-combining site of mucosal immunoglobulins for bacterial and viral surface components. Here, we report a novel mechanism for the antibacterial effect of secretory IgA. Secretory IgA and IgA myeloma proteins, especially those of the IgA2 subclass, were shown to possess carbohydrate receptors for the mannose-specific lectin of type 1-fimbriated Escherichia coli. The presence of the high-mannose oligosaccharide chain Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc correlated with binding activity. The interaction between bacterial mannose-specific lectins and IgA receptor oligosaccharide resulted in agglutination of the bacteria and in inhibition of bacterial attachment to colonic epithelial cells. Thus, this interaction could form the basis for a broad antibacterial function of secretory IgA against enterobacteria regardless of the specificity of antibody molecules.

Agglutination↗
PubMed1990

Recognition of N-glycosidic carbohydrates on esophageal carcinoma cells by macrophage cell line THP-1.

Cell-to-cell contact between macrophages and tumor cells is an important initial reaction in a host defense mechanism against tumor cells. The authors have studied cell surface components of human esophageal carcinoma cells recognized by macrophages. Superoxide release from THP-1 cells, a human macrophage cell line, was analyzed in their interaction with a battery of human squamous cell carcinoma cell lines (TE) originated from esophageal cancer patients. The macrophage-triggering ability of TE 1 cell line, a high stimulant, was reduced after treatment with trypsin or tunicamycin, an inhibitor of N-glycosidic glycosylation. Addition of monosaccharides was efficient in competitive inhibition of these cellular interaction. Moreover, con-A-resistant mutation of TE 1 cells was found to reduce their macrophage-triggering ability, associated with increase of L-PHA-binding capacity, suggesting substitution to the GlcNAc beta(1----6)-linked lactosamine antenna in N-glycosidic carbohydrates. These findings suggest that terminal residues of N-glycosidic carbohydrates on some esophageal carcinoma cells may contribute to the recognition sites of macrophages.

Carcinoma, Squamous Cell↗
PubMed1989

Novel methods to determine the epitopes on the asparagine-linked oligosaccharides of glycoproteins.

Oligosaccharides obtained from glycoproteins by hydrazinolysis followed by N-acetylation were covalently coupled to an amino-bonded thin-layer plate or to a poly-L-lysine coating on the microtiter plate. Bound oligosaccharides can be directly detected by immunostaining or by enzyme-linked immunosorbent assay (ELISA) using a mouse monoclonal antibody. These methods are simple and require small amounts of oligosaccharides and antibodies. The methods were successfully applied to determine the epitope of F48-60, a monoclonal antibody which reacts with the N-linked sugar chains of nonspecific cross-reacting antigen 2, but not with those of carcinoembryonic antigen. The results revealed that the antibody recognizes the Gal beta 1----3GlcNAc beta 1----group.

Animals↗
PubMed1989

Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains.

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.

Antigen-Antibody Complex↗
PubMed1989

Diversity of oligosaccharide structures linked to asparagines of the scrapie prion protein.

Prion proteins from humans and rodents contain two consensus sites for asparagine-linked glycosylation near their C-termini. The asparagine-linked oligosaccharides of the scrapie isoform of the hamster prion protein (PrP 27-30) were released quantitatively from the purified molecule by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The radioactive oligosaccharides were fractionated into one neutral and three acidic oligosaccharide fractions by anion-exchange column chromatography. All oligosaccharides in the acidic fractions could be converted to neutral oligosaccharides by sialidase digestion. Structural studies on these oligosaccharides including sequential exoglycosidase digestion in combination with methylation analysis revealed that PrP 27-30 contains a mixture of bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6(GlcNAc beta 1----4)(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4-(Fuc alpha 1----6)GlcNAc as their core. Variation is produced by the different combination of the oligosaccharides Gal beta 1----4GlcNAc beta 1----, Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----, GlcNAc beta 1----, Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----, and Sia alpha 2----6Gal beta 1----4GlcNAc beta 1---- in their outer chain moieties. When both asparagine-linked consensus sites are glycosylated, the diversity of oligosaccharide structures yields over 400 different forms of the scrapie prion protein. Whether these diverse asparagine-linked oligosaccharides participate in scrapie prion infectivity or modify the function of the cellular prion protein remains to be established.

Acetylation↗
PubMed1989

Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia.

Aleuria aurantia lectin (AAL) shows sugar-binding specificity for L-fucose. A lambda gt11 expression library was constructed from A. aurantia poly(A) RNA and screened with a polyclonal antiserum directed against AAL. An immunopositive clone carrying 1.3-kb EcoRI fragment was obtained. The fragment encoded AAL, but lacked a nucleotide sequence corresponding to the two amino-terminal amino acids. The 5'-terminal part of the fragment was replaced with a chemically synthesized DNA fragment and inserted into an expression vector to yield a plasmid pKA-1. Escherichia coli carrying pKA-1 expressed functional AAL and the recombinant AAL showed the same immunological properties as those of natural AAL.

Amino Acid Sequence↗
PubMed1989

4-O-acetyl-N-acetylneuraminic acid in the N-linked carbohydrate structures of equine and guinea pig alpha 2-macroglobulins, potent inhibitors of influenza virus infection.

To investigate the molecular basis of the differential ability of human, equine, and guinea pig alpha 2-macroglobulins to inhibit hemagglutination and infectivity of a human influenza virus, A/Memphis/102/72 (H3N2), the structures of oligosaccharides released from the three glycoproteins by hydrazinolysis were analyzed comparatively. Approximately seven to eight sugar chains were released from each subunit of two potent inhibitors (equine and guinea pig alpha 2-macroglobulins) and a weak inhibitor (human alpha 2-macroglobulin). More than 70% of the oligosaccharides contained sialic acids in all three cases. Structural analysis of these sialo-oligosaccharides revealed that all of the three glycoproteins contain biantennary oligosaccharides with one and two sialic acids as major sugar chains (70-80% of total sugar chains). Four percent of the biantennary oligosaccharides from equine sample, 10% of those from guinea pig, and 24% of those from human contain a fucosylated trimannosyl core. No triantennary oligosaccharide was detected in equine alpha 2-macroglobulin. However, human and guinea pig alpha 2-macroglobulins contain both fucosylated and nonfucosylated triantennary oligosaccharides. All sialic acid residues occur as the Sia alpha 2----6Gal group. The one unique feature of the carbohydrate groups of equine and guinea pig alpha 2-macroglobulins was the presence of 4-O-Ac-Neu5Ac as 30-50% of the total sialic acids, while human alpha 2-macroglobulin contained only Neu 5Ac. However, 4-O-Ac-Neu5Ac is not responsible for the potent inhibition of influenza virus infection and hemagglutination as will be described in the accompanying paper.

Animals↗
PubMed1989

Altered glycosylation of serum transferrin of patients with hepatocellular carcinoma.

The sugar chains of transferrin samples, purified from sera of patients with hepatocellular carcinoma and of healthy individuals, were released quantitatively as radioactive oligosaccharides by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Comparative study of their structure by serial lectin column chromatography, by Bio-Gel P-4 column chromatography, and by sequential exoglycosidase digestion revealed that prominently altered glycosylation is commonly found in the hepatoma transferrins, although they all contain two complex-type asparagine-linked sugar chains in one molecule like in the case of normal transferrins. The alteration is quite various, including the increase of highly branched sugar chains, of those with the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----and the Neu5Ac alpha 2----3Gal beta 1----4GlcNAc beta 1----groups in their outer chain moieties and of those with a fucosylated trimannosyl core. Many but not all of the hepatoma transferrin samples contained a small amount of a bisected biantennary sugar chain, which was not detected in the normal transferrin samples.

Biomarkers, Tumor↗
PubMed1989

Function and pathology of the sugar chains of human immunoglobulin G.

Human immunoglobulin G (IgG) is unique among serum glycoproteins because it contains more than 30 different biantennary complex-type asparagine-linked oligosaccharides. This extremely high microheterogeneity is probably produced because human individuals have a series of B cell clones equipped with different sets of glycosyltransferases. Despite this complex composition, IgG samples purified from whole human sera have the same mole ratios of oligosaccharides, indicating that the ratio of B cell clones synthesizing IgGs with different sugar chains is constant in healthy individuals. We found that the glycosylation patterns of whole serum IgGs obtained from patients with rheumatoid arthritis (RA) are quite different from those of whole serum from healthy individuals. Structural studies of the oligosaccharides revealed that the sugar chains of the IgGs obtained from patients with RA are depleted of the beta-galactose residues. The sugar chains of transferrin from patients with RA are fully galactosylated. Therefore the galactose deletion from IgG is probably brought about by a decrease in galactosyltransferase activity in B cells rather than by degradation by galactosidase during circulation. Enzymic study revealed that human B cells contain various beta-galactosyltransferases which form the Gal(beta 1-4)GlcNAc groups in the sugar chains of different glycoproteins. Among these enzymes, abnormality in patients with RA was found only in the one that transfers beta-galactose residues specifically to degalactosylated IgG. This enzyme showed lower affinity toward UDP-Gal in B cells of patients with RA than that in healthy individuals.

Arthritis, Rheumatoid↗