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Biomedical subjects

M N Fukuda

Publications and source records attributed to M N Fukuda.

At least 73 records · Page 4Linked to original sources
PubMed1986

Structures of glycosphingolipids isolated from human embryonal carcinoma cells. The presence of mono- and disialosyl glycolipids with blood group type 1 sequence.

Structures of glycolipids present in the human embryonal carcinoma cell PA1, were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase digestion. PA1 cells contain globotriaosylceramide, sialosylgangliotriaosylceramide, sialylated and nonsialylated lacto-N-neotetraosylceramide, and the following glycolipids with a blood group type 1 sequence: (formula; see text) The two former glycolipids, lacto-N-tetraosylceramide and sialosyllacto-N-tetraosylceramide, reacted with monoclonal antibodies, K21 and K4, respectively. K21 and K4 antigens are present in many of the human embryonal carcinoma cells but not in a variety of other cell lines, suggesting that sialylated but not fucosylated blood group type 1 sequences are characteristic markers for human embryonal carcinoma cells and malignant teratocarcinomas.

Antigens↗
PubMed1986

Structure of a novel sialylated fucosyl lacto-N-norhexaosylceramide isolated from chronic myelogenous leukemia cells.

A novel sialylated fucosyl glycolipid, which is present at an elevated level in chronic myelogenous leukemia cells, was isolated. The structure of this fucoganglioside was elucidated by methylation analysis, fast atom bombardment-mass spectrometry, and enzymatic degradation, followed by reaction with anti-Lex, Gal beta 1----4 (Fuc alpha 1----3) GlcNAc beta 1----, monoclonal antibody. The structure of this ganglioside was found to be: (Formula: see text). This structure is unique in that a fucose is attached to the internal N-acetylglucosamine but not to the subterminal N-acetylglucosamine. Since this glycolipid is apparently absent in normal granulocytes or acute myelogenous leukemia cells, it can be a specific marker for chronic myelogenous leukemia cells. Based on the structures of this fucoganglioside and normal granulocyte glycolipids, a biosynthetic pathway of extension, sialylation, followed by fucosylation is proposed.

Antibodies, Monoclonal↗
PubMed1986

Trophoblastic differentiation of human teratocarcinoma cell line HT-H1.

In culture the human teratocarcinoma cell line HT-H generates both adherent monolayer and free-floating aggregates. Some populations of aggregated cells develop further to form cystic bodies. A previous study showed the morphological resemblance of the cystic bodies to cells of blastocyst of preimplantation embryo. In this study, HT-H adherent cells were further separated into two subpopulations, fast adhering and slow adhering cells. Fast adhering cells produce fibronectin, spread well onto substratum, and do not proliferate. In contrast, slow adhering cells do not produce fibronectin. Trophoblastic markers were examined in each morphological stage of HT-H cells and the following results were obtained. Only fast adhering cells produce progesterone. Human chorionic gonadotropin was secreted preferentially by fast adhering cells, about six times less by slow adhering cells, and was not secreted by aggregates or cystic bodies. All stages of HT-H cells express c-fos but only fast adhering cells express c-fms oncogene. Cytokeratin 18 was expressed in all stages of HT-H cells. The level of cytokeratin 18 is modestly decreased from adherent to aggregates further into cystic bodies. These results indicate that HT-H cells share properties with cells in trophoblast, placenta, and extraembryonic endoderm. Spontaneous differentiation of HT-H cultures results in the appearance of fast adhering cells which exhibit biochemical properties expected for syncytiotrophoblast.

Cell Adhesion↗
PubMed1986

Anomalous clustering of underglycosylated band 3 in erythrocytes and their precursor cells in congenital dyserythropoietic anemia type II.

Congenital dyserythropoietic anemia type II (CDA II or HEMPAS) is a genetic anemia caused by membrane abnormality. Our previous studies indicated that in HEMPAS, erythrocytes band 3 and band 4.5 are not glycosylated by polylactosaminoglycans. The present study was aimed at determining how such underglycosylated band 3 behaves in erythrocyte membranes. By using anti-band 3 antibodies, immunogold electron microscopy revealed that band 3s are clustered in HEMPAS erythrocyte membranes. By freeze-fracture electron microscopy, band 3s were also seen as lightly clumped intramembrane particles on a protoplasmic fracture face. Erythrocyte precursor cells stained by anti-band 3 antibodies showed that band 3s are present in the cytoplasmic area of the reticulocytes as scattered single particles. However, in young erythrocytes in which intracellular membranes are almost degenerated, band 3s were clustered in the cytoplasmic area of the cell. These observations suggest that band 3s cluster before they are incorporated into the plasma membranes of HEMPAS erythrocytes. In contrast to band 3, glycophorin A detected by anti-glycophorin A antibodies did not show a noticeable difference between normal and HEMPAS. Such a clustering of band 3 may cause abnormal localization of band 3-associated proteins and may thus result in the macroscopic membrane abnormality seen in HEMPAS erythrocytes.

Anemia, Dyserythropoietic, Congenital↗
PubMed1985

Embryonal lactosaminoglycan. The structure of branched lactosaminoglycans with novel disialosyl (sialyl alpha 2----9 sialyl) terminals isolated from PA1 human embryonal carcinoma cells.

Lactosaminoglycan glycopeptides were isolated from human PA1 embryonal carcinoma cells and their structures were elucidated. The glycopeptides were digested by Escherichia freundii endo-beta-galactosidase before and after the modifications by exoglycosidases. The core glycopeptides and oligosaccharides thus obtained and the intact glycopeptides were analyzed by methylation, fast atom bombardment-mass spectrometry, and high-performance liquid chromatography. Based on these experiments, the structures of PA1 lactosaminoglycans were found to have the following unique features. 1) Three lactosaminoglycan fractions of different molecular weights were isolated by Sephadex G-50 gel filtration. Lactosaminoglycans of the highest molecular weight (GpI) have tetra-antennary cores, those of intermediate molecular weight (GpII) have triantennary cores and those of low molecular weight (GpIII) have triantennary and tetra-antennary cores. 2) GpI is composed of 22-26 lactosaminyl units and 7-9 branched galactose residues, GpII is composed of 16-22 lactosaminyl units and 5-7 branched galactose residues, and GpIII is composed of 12-16 lactosaminyl units and 3-4 branched galactose residues. 3) Each branch is short and is composed of the Gal beta 1----4GlcNAc beta 1----6 structure. 4) Sialic acid is preferentially linked to nonreducing terminal regions and a significant amount of the novel disialosyl structure, NeuNAc alpha 2----9NeuNAc alpha 2----3/6Gal, is present at the terminals of the longer polylactosaminyl side chains. 5) These lactosaminoglycans are carried by cell surface glycoproteins of Mr = 80,000 approximately 120,000, as evidenced by lectin-agarose chromatography.

Amino Sugars↗
PubMed1985

Isolation and characterization of a new endo-beta-galactosidase from Diplococcus pneumoniae.

An endo-beta-galactosidase, which hydrolyzes the internal beta-galactosidic linkages of R----GlcNAc (or GalNAc) beta 1----3Gal beta 1----4GlcNAc (or Glc), was isolated from the culture supernatant of Diplococcus pneumoniae. The enzyme, named endo-beta-galactosidase DII, hydrolyzed linear N-acetyllactosamine repeating structures in glycolipids and glycopeptides to release oligosaccharides. The specificity of endo-beta-galactosidase DII is the same as that of Escherichia freundii endo-beta-galactosidase as far as described above, but the following differences between these two enzymes were found: Branched lactosaminyl glycolipids and H-antigenic glycolipids were resistant to endo-beta-galactosidase DII, even when linear structure was present at the inner part. Throughout the enzymic hydrolysis, endo-beta-galactosidase DII released mostly small oligosaccharides (tetra-, tri-, and disaccharides) from substrates, suggesting that the enzyme split off the oligosaccharides stepwise from the nonreducing terminal. Lactosaminoglycans were partially hydrolyzed by endo-beta-galactosidase DII to produce small oligosaccharides as the major product and residual glycopeptides. The residual glycopeptides were readily hydrolyzed by E. freundii endo-beta-galactosidase to produce various sizes of oligosaccharides. Keratan sulfate was not degraded by endo-beta-galactosidase DII. These properties of endo-beta-galactosidase DII characterize it as a new endo-beta-galactosidase with a unique specificity.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1985

Structures of glycosphingolipids isolated from human granulocytes. The presence of a series of linear poly-N-acetyllactosaminylceramide and its significance in glycolipids of whole blood cells.

Structures of glycolipids isolated from human granulocytes were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase treatment. All neutral glycolipids, with saccharide residues ranging from 2 to 10, were found to have linear N-acetyllactosaminyl backbones. The majority of neutral glycolipids contain one or two fucosyl residues attached to N-acetylglucosamine residues through the Fuc alpha 1----3 linkage and were reactive with the monoclonal antibody specific to Gal beta 1----4(Fuc alpha 1----3)GlcNAc, the Lex structure. Their general structure can be expressed as follows: (formula; see text) where n = 0-3. Glycolipids containing sialic acid (gangliosides) were also found to have linear N-acetyllactosaminyl backbones with sialic acid joined to this backbone by either alpha 2----3 or alpha 2----6 linkage. The gangliosides have the following general structure: (formula; see text) where n = 0-3. The ceramide was composed of sphingosine with d18:1 as the long-chain base and C16:0 (as a major component) or C24:1 (as a minor component) fatty acid. Analysis of glycolipids isolated from granulocytes, erythrocytes, and whole blood cells revealed that, among the glycolipids prepared from the whole blood cells, dihexaosylceramide, lactoneotetraosylceramide, and the above described linear lactoneo series neutral glycolipids are present in granulocytes but barely present in erythrocytes.

Antibodies, Monoclonal↗
PubMed1984

Structure of branched lactosaminoglycan, the carbohydrate moiety of band 3 isolated from adult human erythrocytes.

The structure of branched lactosaminoglycan isolated from Band 3 glycoprotein of adult human erythrocytes was elucidated. The glycopeptides were digested by endo-beta-galactosidase under various conditions and oligosaccharides and core glycopeptides thus obtained and intact glycopeptides were analyzed by methylation, exoglycosidase digestion, and fast atom bombardment mass spectrometry. Based on these experiments, the structure of adult lactosaminoglycan was found to have the following unique features: 1) two (as major components) or three (as minor components) polylactosaminyl side chains composed of (Gal beta 1----4 GlcNAc beta 1----3) repeating units are attached to the core portion, the structure of which is Man alpha 1----6(Man alpha 1----3) [+/- GlcNAc beta 1----4]Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc----Asn, 2) the polylactosaminyl side chain arising from the mannose C-6 side is composed of 10-12 N-acetyllactosaminyl units and three branches; the lactosaminyl side chain arising from the mannose C-3 side is composed of 5-6 lactosaminyl units and contains one or two branches, 3) each branch is short and is composed of the Gal beta 1----4GlcNAc beta 1----6 structure, 4) fucose and sialic acid are preferentially linked to nonreducing terminal regions but not on the branches in the internal portion of the polylactosaminyl chain.

Adult↗
PubMed1984

Structure of fetal lactosaminoglycan. The carbohydrate moiety of Band 3 isolated from human umbilical cord erythrocytes.

The structure of lactosaminoglycan prepared from Band 3 glycoprotein of umbilical cord blood erythrocytes was elucidated. The glycopeptides were digested by endo-beta-galactosidase and oligosaccharides, core glycopeptides, and intact glycopeptides were analyzed by permethylation, exoglycosidase digestion, and fast atom bombardment mass spectrometry. The structure of one of the major components was found to be: sequence in text This structure is unique in that 1) two linear polylactosaminyl chains are attached to the core portion, 2) the polylactosaminyl side chain is much longer on the Man alpha 1----6 side than on the Man alpha 1----3 side, 3) alpha 2----3-linked sialic acid is present at the terminal of the long polylactosaminyl side chain, whereas alpha 2----6-linked sialic acid is present in the other short side chain, and 4) fucose is linked only to the external position of the lactosaminyl side chain and fucose, alpha 1----6-linked to the innermost N-acetylglucosamine, is absent.

Amino Sugars↗
PubMed1984

Defect in glycosylation of erythrocyte membrane proteins in congenital dyserythropoietic anaemia type II (HEMPAS).

Congenital dyserythropoietic anaemia type II (HEMPAS) is a hereditary disease believed to be caused by a membrane abnormality of erythroid cells. Since the molecular basis of this membrane abnormality has not yet been defined, membrane glycoproteins of HEMPAS erythrocytes were analysed by cell surface labelling and endo-beta-galactosidase digestion in this study. HEMPAS erythrocytes showed an abnormal glycoprotein profile when cells were labelled by the galactose oxidase/NaB[3H]4 method; Band 3 and Band 4.5 glycoproteins in HEMPAS are labelled but with less intensity although normally these proteins are the major components revealed by the same method. Instead, in HEMPAS, labelled lactosaminoglycans were found as a lower molecular weight glycoconjugate (HEMPAS glycan). HEMPAS glycan was characterized by micelle formation, a monomer molecular weight of 4000, susceptibility to endo-beta-galactosidase and resistance to protease. These characteristics suggest that HEMPAS glycan has the nature of macroglycolipid. Proteins of Band 3 and the glucose transport protein (a component of Band 4.5), which were detected by antibodies showed a slightly decreased molecular weight in HEMPAS erythrocytes compared to those from normal erythrocytes, which was consistent with the decreased glycosylation of these proteins. The results indicate that anomalies in glycosylation occurred specifically in lactosaminoglycan glycoproteins of HEMPAS erythrocytes.

Anemia, Dyserythropoietic, Congenital↗
PubMed1983

Glycolipids of fetal, newborn, and adult erythrocytes: glycolipid pattern and structural study of H3-glycolipid from newborn erythrocytes.

The glycolipids of blood group type O adult, newborn, and fetal erythrocytes were compared. The total amount of glycolipids was indistinguishable between adult and newborn erythrocytes. However, glycolipids with long and neutral carbohydrates and the H determinant were greatly reduced in newborn cells. On the other hand, the amount of sialylated glycolipids (gangliosides) was significantly higher in newborn cells, suggesting that during erythropoiesis sialyltransferases are more active in fetuses than in adults. The amount of each core structure, lacto-N-tetraosyl, linear lacto-N-hexaosyl, and branched lacto-N-octaosyl, was compared between adult and newborn erythrocytes. It was found that branched lacto-series glycolipids were reduced in newborn cells compared with adult cells. Thus, development from fetal to adult human erythrocytes is associated with an increase of branching and a decrease of sialylation of N-acetyllactosaminyl carbohydrate chains. The study indicates that glycolipids are quantitatively different between adult and newborn or fetus.

ABO Blood-Group System↗
PubMed1983

Isolation and characterization of an endo-beta-galactosidase from Bacteroides fragilis.

Six strains of Bacteroides fragilis were examined and all found to produce endo-beta-galactosidase, an enzyme that hydrolyses internal beta-galactosidic linkages of oligosaccharides belonging to the poly-N-acetyl-lactosamine series, with the common structure GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc/Glc. The enzyme was produced without the addition of an inducer such as keratan sulphate. It was purified 7000-fold from the culture supernatant and obtained with a yield 4-10-fold greater than from sources described previously. The specificity of the enzyme towards bovine corneal keratan sulphate, milk oligosaccharides and the glycolipids lacto-N-neotetraosylceramide and lacto-N-tetraosylceramide closely resembled that of the endo-beta-galactosidase isolated from Escherichia freundii. A novel observation was that both enzymes hydrolysed the type 2 sequence, Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc, at about twice the rate of the type 1 isomer, Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc. Because of the ease of purification of the enzyme and high yield in the absence of contaminating glycosidases and proteinases, Bacteroides fragilis is a valuable source of endo-beta-galactosidase for the structural analysis of carbohydrate chains.

Bacteroides fragilis↗
PubMed1982

A new glycolipid antigen isolated from human erythrocyte membranes reacting with antibodies directed to globo-N-tetraosylceramide (globoside).

Two glycolipid fractions were separated by high performance liquid chromatography from human erythrocyte membranes and which were reactive with antibodies to globo-N-tetraosylceramide (globoside). The reactivity to the antibody was abolished by treatment with endo-beta-galactosidase of Escherichia freundii which specifically hydrolyzes lacto-N-glycosyl series glycolipids, but does not hydrolyze globo series glycolipids. One of the fractions was isolated by repeated high performance liquid chromatography, and its structure was determined by direct probe mass spectrometry, methylation analysis, and by degradation with exo- and endoglycosidases as follows: GalNAc beta 1 leads to 3Gal beta 1 leads to 4GLcNAc beta 1 leads to 3 Gal beta 1 leads to 4Glc beta 1 leads to 1ceramide. The carbohydrate structure was identical with the asialo core of "G3-ganglioside" (IV3NeuAc2 leads to 3GalNAcLcnOs4Cer) (Watanabe, K., and Hakomori, S. (1979) Biochemistry 14,5502-5504), but the ceramide moiety of this glycolipid was characterized by having C22 and C24 fatty acids in a striking contrast to that G3-ganglioside was characterized by the predominance of C14 fatty acid. Since globoside was previously identified as blood group P antigen (Marcus, D. M., Kundu, S. K., and Suzuki, A. (1981) Semin. Hematol. 18, 63-71), and both globoside and this glycolipid possess the common terminal structure GalNAc beta 1 leads to 3Gal, this glycolipid may represent the second blood group P antigen belonging to the lacto series.

Antibodies↗
PubMed1982

Structures of branched blood group A-active glycosphingolipids in human erythrocytes and polymorphism of A- and H-glycolipids in A1 and A2 subgroups.

The structures of two branched variants of blood group A-active glycolipid, Ac and Ad (Hakomori, S., Stellner, K., and Watanabe, K. (1972) Biochem. Biophys. Res. Commun. 49, 1061-1068), isolated from human type A-erythrocytes, were elucidated. The intact glycolipids and fragments produced by treatment with endo-beta-galactosidase from Escherichia freundii (Fukuda, M. N., and Matsumura, G. (1976) J. Biol. Chem. 251, 6218-6225) were analyzed by methylation. In addition, a specific limited enzyme digestion of Ad-glycolipid was made by incorporating the glycolipid into liposomes before endo-beta-galactosidase digestion. The method preferentially hydrolyzed one of the side chains carrying an A-determinant leaving a core which was recovered from the liposomes. The Ac- and Ad- glycolipids have two branched carbohydrate chains with 12 and 14 sugar residues, respectively, and both have two A-determinants as shown below. (formula see text). The structures of A- and H-active variants isolated from subgroups A1 and A2 have been compared. The results indicate that A variants from A1 or A2 erythrocytes have identical structures, and that H-variants isolated from A2 erythrocytes are identical to those isolated from O erythrocytes. Thus, there is no qualitative structural difference in A-active glycolipids between A1 and A2 erythrocytes.

ABO Blood-Group System↗
PubMed1981

Purification and characterization of endo-beta-galactosidase from Escherichia freundii induced by hog gastric mucin.

A new procedure for inducing and purifying endo-beta-galactosidase from Escherichia freundii was described. The enzyme was found to be induced with high efficiency in culture medium containing Smith-degraded hog gastric mucin, which was prepared from a commercially available starting material. Endo-beta-galactosidase was then purified by ammonium sulfate fractionation, DEAE-Sephadex chromatography, and affinity chromatography on Sepharose conjugated with the Smith-degraded mucin. The enzyme thus purified by only three steps showed no other glycosidase or protease activities and had higher specific activity compared to the previous method. This new method has a great advantage since the gastric mucin is abundantly available and the efficiency of enzyme production was high without significant induction of exoglycosidase. The hydrolysis of oligosaccharides, glycosphingolipid, and keratansulfate was studied by using this newly purified enzyme. Kinetic data indicate that hydrolyzability of these substrates is largely affected by substrate concentration, enzyme concentration and the structure of substrates. Based on these results, the specificity of E. freundii endo-beta-galactosidase was discussed.

Animals↗
PubMed1981

Isolation and structural characterization of human lymphocyte and neutrophil gangliosides.

Gangliosides were isolated from purified preparations of human peripheral blood lymphocytes and neutrophils. Structural analyses and comparisons were performed by direct probe mass spectrometry and by degradation studies with the following enzymes: Escherichia freundii endo-beta-galactosidase; Clostridium perfringens and Arthrobacter ureafaciens neuraminidase; and jack bean beta-N-acetylhexosaminidase and beta-galactosidase. This combination of techniques allowed us to obtain carbohydrate composition and sequence information without the aid of methylation or carbohydrate compositional analyses using only 1-2 mg of purified gangliosides. On the basis of these studies we propose that human lymphocytes and neutrophils have gangliosides with the following structures. NeuAc alpha 2 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Structure A NeuAc alpha 2 leads to ? GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Structure B NeuAc alpha 2 leads to ? Gal beta 1 leads to 3,4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1Cer Structure C All three compounds were isolated from both cell types with structure A being the major lymphocyte ganglioside and structure C the major neutrophil ganglioside. Structure B is a novel ganglioside and may represent a leukocyte-specific glycosphingolipid. Neuraminidase degradation studies demonstrated that only one ganglioside species of each cell type contains an internally linked sialic acid residue, and on the basis of thin layer chromatographic analysis this component is the same as the major brain ganglioside, GM1 (II3-N-acetylneuraminosyl-gangliotetraosylceramide). In addition, large gangliosides with the general structure NeuAc alpha 2 leads to ?(Gal beta 1 leads to 3,4GlcNAc beta 1 leads to 3)n Gal beta 1 leads to 4Glc beta 1 leads to 1Cer were isolated. These results are discussed as they relate to blood group antigens and specific cell surface markers in human leukocytes.

Carbohydrate Conformation↗