PubMed Health⌕ Search

Biomedical subjects

A Kobata

Publications and source records attributed to A Kobata.

At least 37 records · Page 2Linked to original sources
PubMed1995

Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin.

To elucidate the role of the branched structure of sugar chains of human erythropoietin (EPO) in the expression of in vivo activity, the pharmacokinetic profile of a less active recombinant human EPO sample (EPO-bi) enriched with biantennary sugar chains was compared with that of a highly active control EPO sample enriched with tetraantennary sugar chains. After an intravenous injection in rats, 125I-EPO-bi disappeared from the plasma with 3.2 times greater total body clearance (Cltot) than control 125I-EPO. Whole-body autoradiography after 20 minutes of administration indicated that the overall distribution of radioactivity is similar, but 125I-EPO-bi showed a higher level of radioactivity in the kidneys than control 125I-EPO. Quantitative determination of radioactivity in the tissues also indicated that radioactivity of 125I-EPO-bi in the kidneys was two times higher than that of control 125I-EPO. The difference in plasma disappearance between 125I-EPO-bi and control 125I-EPO was not observed in bilaterally nephrectomized rats. The distribution of 125I-EPO-bi to bone marrow and spleen was similarly inhibited by simultaneous injection of excess amounts of either the nonlabeled EPO-bi or control EPO. These results indicate that the low in vivo biologic activity of EPO-bi results from rapid clearance from the systemic circulation by renal handling. Thus, the well-branched structure of the N-linked sugar chain of EPO is suggested to play an important role in maintaining its higher plasma level, which guarantees an effective transfer to target organs and stimulation of erythroid progenitor cells.

Animals↗
PubMed1995

Characterization of glycosylphosphatidylinositol (GPI)-anchored NCAM on mouse skeletal muscle cell line C2C12: the structure of the GPI glycan and release during myogenesis.

The mouse myoblast cell line C2C12 constitutively expressed 160-kDa transmembrane NCAM isoform and 135-kDa GPI-anchored isoform before differentiation. During differentiation into multinucleated myotubes, the cells newly expressed 150-kDa GPI-anchored isoform and the level of 135-kDa GPI-anchored isoform increased. Structural analysis of the GPI glycan of NCAM, which was purified from C2C12 myotubes after metabolic labeling with [3H]inositol, was performed by sequential exoglycosidase digestion and Wistaria floribunda agglutinin-agarose column chromatography. The core GPI glycan structure, Man alpha 1-2Man alpha-Man alpha-GlcNH2-myoInositol, was conserved and variations were observed in additional mannose and N-acetylgalactosamine residues. Structural analysis of the GPI glycans of the two GPI-anchored isoforms, GPI-NCAM 135 and GPI-NCAM 150, showed the enhanced attachment of the N-acetylgalactosamine residue to the GPI glycan core of GPI-NCAM 150. These GPI-anchored NCAM isoforms were released from C2C12 cells during the myoblast differentiation. Release of GPI-anchored NCAMs was observed when C2C12 cells were cultured in a serum-free medium, and inositol but not inositol phosphate was detected after nitrous acid deamination of the released NCAM. These results suggest that the GPI-anchored NCAM was released from the cell surface by the action of an endogeneous phospholipase D.

Animals↗
PubMed1995

Glycosylation of the variable region of immunoglobulin G--site specific maturation of the sugar chains.

The structure of the N-linked sugar chains attached to three IgG antibodies, identical in amino acid sequence except for the changes required to introduce the carbohydrate addition sites, has been determined. All three antibodies are specific for dextran but differ in their ability to bind antigen. The heavy chains with a murine variable region (V region) attached to the human gamma 4 constant region were expressed in a murine hybridoma synthesizing the specific light chain. In addition to the glycosylation site in the Fc portion, each antibody has a different glycosylation site in the second complementarity determining region (CDR2) of the heavy chain (Asn54, Asn58, or Asn60). The sugar chains were released from purified Fab and Fc fragments by hydrazinolysis and converted to radioactive oligosaccharides by reduction with sodium borotritide. The structures of these radioactive oligosaccharides were determined by a combination of sequential exoglycosidase digestion and Bio-Gel P-4 and lectin column chromatography. For all three antibodies, the carbohydrate attached to the Fc portion was a mixture of complex-type biantennary sugar chains. The variable region carbohydrate structures attached at Asn54 and Asn58 were also complex-type but more highly sialylated than were the Fc-associated sugars. Moreover, unlike the Fc-associated sugars, a significant population of Fab-associated sugars contained a Gal alpha 1-->3 residue as a non-reducing terminus. In contrast, the carbohydrate attached at Asn60 was a high mannose structure. These results demonstrate that slight changes in the position of carbohydrate attachment within CDR2 of the variable region of the heavy chain can substantially alter carbohydrate processing and that complex-type carbohydrates contained within the same polypeptide chain can have different structures.

Amino Acid Sequence↗
PubMed1995

Carbohydrate structures of a normal counterpart of the carcinoembryonic antigen produced by colon epithelial cells of normal adults.

Normal faecal antigen-2 (NFA-2) and non-specific cross-reacting antigen-2 (NCA-2), cross-reacting with anticarcinoembryonic antigen (CEA) antibodies, were found in normal human faeces and meconium, respectively. Because NFA-2, NCA-2 and CEA are considered as the same gene products, NFA-2 and NCA-2 should be normal counterparts of CEA produced by colon epithelial cells of normal adults and fetuses, respectively. Comparison of sugar chain structures of these three antigens is indispensable in order to unravel the structural alteration induced by malignant transformation and development of colon epithelial cells. The sugar chain structures of CEA (Yamashita, K. et al., Cancer Res., 47, 3451-3459, 1987) and NCA-2 (Yamashita, K. et al., J. Biol. Chem., 264, 17873-17881, 1989) were previously reported. In this paper, the structures of the oligosaccharides released from four NFA-2 samples by hydrazinolysis were studied by means of lectin-affinity column chromatography, endo- and exo-glycosidase digestion, methylation analysis, hydrazinolysis-nitrous acid deamination and electrospray ionization mass spectrometry. NFA-2 contains 24-27 mol of N-linked sugar chains/molecule, which is similar to NCA-2 (27 mol) and CEA (24-27 mol). In contrast to CEA, which contains approximately 8% high-mannose-type sugar chains, all sugar chains of NFA-2 are mono- to tetra-antennary complex-type chains having four types of tri-mannosyl cores, with or without bisecting N-acetylglucosamine and fucose residues. The structures of their outer chain moieties comprise Gal beta 1-->3(HSO(3-)-->6)GlcNAc, Neu5Ac alpha 2-->3Gal beta 1-->3GlcNAc, Type 1, repeating chain, Type 2, Type 2H, Type 1H, Lex, Lea and Leb antigenic determinants. Approximately 50% of the outer chain moieties of the oligosaccharides of NFA-2 contain Type 1 chain, in contrast to those of CEAs produced by the liver metastases of colon tumours in which only a trace amount of Type 1 chain was detected.

Adult↗
PubMed1995

Site-specific glycosylation of bovine butyrophilin.

Our previous studies showed that the N-linked sugar chains of most bovine glycoproteins from milk fat globule membranes (MFGM) contain the GalNAc beta 1-->4GlcNAc group [Sato et al. (1993) J. Biochem. 114, 890-900]. Since expression of the disaccharide structure is influenced by peptide sequences near the glycosylation sites [Smith and Baenziger (1992) Proc. Natl. Acad. Sci. USA 89, 329-333], the site-specificity of the N-acetylgalactosaminylated sugar chains was investigated using bovine butyrophilin, a major MFGM glycoprotein with known primary structure. Two glycopeptide fragments which contained the N-linked sugar chains linked to either Asn-55 or Asn-215 residue were obtained by digestion of the protein with Achromobacter protease I. The sugar chains released from each glycopeptide by hydrazinolysis were reduced with NaB3H4. Structural analyses of the oligosaccharides by sequential exoglycosidase digestion and methylation analysis revealed that only complex-type sugar chains with the GalNAc beta 1-->4GlcNAc structure are included in Asn-55-linked oligosaccharides, while only novel hybrid-type sugar chains detected previously in bovine MFGM glycoproteins are included in Asn-215-linked oligosaccharides. The results show that the glycosylation of butyrophilin occurs in a site-specific manner.

Amino Acid Sequence↗
PubMed1994

Structural study on the glycosyl-phosphatidylinositol anchor and the asparagine-linked sugar chain of a soluble form of CD59 in human urine.

CD59 is an 18-kDa glycoprotein widely expressed on human cells. An important structural feature of CD59 is its attachment to the cell surface via a glycosyl-phosphatidylinositol (GPI) anchor. CD59, like many GPI-anchored proteins, has been found in urine, serum, and other body fluids. The structures of the GPI anchor and the asparagine-linked sugar chain of a soluble form of CD59 in urine, U-CD59, were determined. Purified U-CD59 released 1 mol of inositol per mole of protein by nitrous acid deamination, which cleaved between glucosamine and inositol present commonly in the GPI anchor. This indicates that a GPI anchor, which ended with inositol, is linked at the carboxy terminus of U-CD59. The peptide containing an asparagine-linked sugar chain and the peptide containing a glycan portion of the GPI anchor were isolated after trypsin digestion of U-CD59. The asparagine-linked sugar chains and the glycan portion of the GPI anchor were isolated from these peptides following hydrazinolysis or deamination and dephosphorylation, respectively. Their structures were analyzed by sequential exoglycosidase digestion and methylation analyses. The structures of the asparagine-linked sugar chains of U-CD59 were biantennary complex type, only 4.2% of which are monosialylated. The backbone structure of the GPI anchor was similar to that of Try-panosoma brucei variant surface glycoprotein, but showed significant variations in its side-chain moieties. This is the first detailed structural analysis of the human GPI anchor and the first detailed analysis of the carboxyl-terminal structure of the soluble-form GPI-anchored protein. The results indicate that the backbone structure of the GPI anchor is conserved from parasites to human and that at least a part of the soluble-form GPI-anchored protein has the structure produced by the action of glycan-phosphatidylinositol-specific phospholipase D.

Antigens, CD↗
PubMed1994

Distribution of glycosylation abnormality among serum IgG subclasses from patients with rheumatoid arthritis.

IgG from patients with rheumatoid arthritis possess significantly fewer galactose residues in its sugar chains. We made an attempt to analyze the distribution of a galactosylation defect among IgG subclasses by immunoassay using PVL, a recently described lectin specific for N-acetylglucosamine (GlcNAc). A distinct difference between RA and normal controls was present in IgG2 (P = 0.0007). A less remarkable but significant difference was seen in IgG1 (P = 0.0118) and in IgG4 (P = 0.022). A significant difference was not observed in IgG3 (P = 0.169). The possible relationship between such a glycosylation abnormality and RF production was discussed.

Adult↗
PubMed1994

Carbohydrate heterogeneity of human myeloma proteins of the IgA1 and IgA2 subclasses.

Comparative studies of the N-linked carbohydrate chains of human myeloma proteins of the IgA1 and IgA2 subclasses were performed. The N-linked carbohydrate chains were released by hydrazinolysis from the polypeptide backbone, converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation and separated into one neutral and two acidic fractions by paper electrophoresis. The acidic oligosaccharides were completely converted to neutral oligosaccharides by sialidase treatment, indicating that they were sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that human myeloma IgA proteins contained significant amounts of biantennary complex-type carbohydrate chains in addition to a small amount of the high mannose-type. The results indicated that the oligosaccharide structures of human IgA1 and IgA2 display a high degree of heterogeneity not only in the number of carbohydrate chains, but also in their composition.

Anions↗
PubMed1994

Structural studies of the N-linked sugar chains of human rhodopsin.

Human rhodopsin is a glycoprotein containing two N-linked sugar chains. After the isolation and purification of rhodopsins from human retinas, structural studies of their N-linked sugar chains were performed. The sugar moieties, quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis, were converted to radioactive oligosaccharides by reduction with NaB3H4 after N-acetylation. As indicated by high-voltage paper electrophoresis, > 96% of the sugar chains were free of sialic acid and the remaining were sialylated derivatives. Structural studies of each oligosaccharide by lectin affinity column chromatography, and sequential exoglycosidase digestion in combination with methylation analysis, revealed that almost all of the oligosaccharides were hybrid-type sugar chains. While the major oligosaccharide species of bovine and human rhodopsin are identical, in contrast to the sugar chains of bovine rhodopsin, human rhodopsin also contains sialylated isomers and a high concentration of a galactosylated isomer. These results suggest that species-specific processing of the sugar chains of rhodopsin occurs.

Amino Acids↗
PubMed1993

Structural study of the sugar chains of human leukocyte common antigen CD45.

The leukocyte cell surface glycoprotein CD45 is a protein tyrosine phosphatase and is involved in signal transduction mediated by the T cell antigen receptor. The asparagine-linked sugar chains were released as oligosaccharides from purified CD45 by hydrazinolysis. Approximately 6 mol of sugar chains was released from 1 mol of CD45. These sugar chains were converted to radioactive oligosaccharides by reduction with NaB3H4 and separated into neutral and acidic fractions by paper electrophoresis. All of the acidic oligosaccharides were converted to neutral ones by digestion with sialidase, indicating that they are sialyl derivatives. Binding of the sialylated oligosaccharides to an SNA-agarose column as well as methylation analysis revealed that the oligosaccharides have only alpha-2,6-linked sialic acid residues. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial lectin column chromatography followed by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that CD45 contains mainly bi-, tri-, and tetraantennary complex-type sugar chains. About 46% of the tetraantennary complex-type sugar chains had the poly(N-acetyllactosamine) groups and 18% of the 2,4-branched triantennary complex-type sugar chains had the fucosyl N-acetyllactosamine group. A portion of the bi- and 2,4-branched triantennary complex-type sugar chains were bisected. In addition to these sugar chains, a small amount of high mannose-type and hybrid-type sugar chains were detected.

Asparagine↗
PubMed1993

Alterations in the carbohydrate structures of an abnormal protein from sera of patients with rheumatoid arthritis.

G4-11, an abnormal immunoglobulin G4 (IgG4) glycoprotein, highly purified from the sera of patients with rheumatoid arthritis and from healthy individuals, contains two asparagine-linked sugar chains in one molecule. Comparative studies of the sugar chains released by hydrazinolysis revealed that the structures of the sugar chains of rheumatoid arthritis G4-11 are quite different from those of normal individuals. Although all G4-11 contained biantennary complex-type oligosaccharides like in the case of serum IgGs, samples from patients with rheumatoid arthritis had less galactosylated forms than those from normal individuals. These results indicated that this galactose deficiency of the sugar chains occurs in the abnormal IgG4 molecule of rheumatoid arthritis patients, just as was found in whole serum IgG.

Arthritis, Rheumatoid↗
PubMed1993

The structure of N-linked oligosaccharides of human pancreatic bile-salt-dependent lipase.

This study describes the structure of the N-linked oligosaccharide chains of bile-salt-dependent lipase isolated from the pancreatic juice of a normal donor. After hydrazinolysis, neutral sugar chains were separated from acidic chains by paper electrophoresis and were fractionated using serial column chromatography with immobilized lectins and Bio-Gel P-4 filtration. Structural analysis was performed by means of sequential glycosidase digestion and revealed that the neutral sugar chains are mainly of the biantennary complex type. Fucose residues were identified for some trimannosyl core structures and were alpha(1-6) or alpha(1-2) linked to the innermost GlcNAc residue and a terminal Gal residue, respectively. Sialyl residues were also involved in the oligosaccharide structures. Most of these structures have no linear N-acetyllactosamine repeats. Evidence from several approaches suggests that the sugar chains of the human pancreatic bile-salt-dependent lipase possess a blood-group-related antigenic determinant.

Bile Acids and Salts↗
PubMed1993

O-linked neutral sugar chains of porcine zona pellucida glycoproteins.

The O-linked sugar chains were liberated from porcine zona pellucida glycoproteins by alkaline-borohydride treatment and the structures of neutral oligosaccharides were determined. The major oligosaccharides had the following structures with type-1 cores, (Gal beta 1-->4GlcNAc beta 1-->3)1 3-Gal bdta 1-->3N-acetylgalactosaminitol (54%) and GlcNAc beta 1-->3(Gal beta 1-->4GlcNAc beta 1-->3)0-1Gal beta 1-->3N- acetylgalactosaminitol (16%). Approximately 6% of the oligosaccharides had the structure with a type-3 core, Gal beta 1-->4GlcNAc beta 1-->3N-acetylgalactosaminitol. Oligosaccharides having alpha-galactose (2%) and beta-GalNAc (4%) at their non-reducing termini were also found as minor components.

Animals↗
PubMed1993

Structural changes in the N-linked sugar chains of serum immunoglobulin G of HTLV-I transgenic mice.

IgGs were purified from the sera of HTLV-I transgenic and nontransgenic mice. Comparative studies of the N-linked sugar chains released by hydrazinolysis revealed that their structures of transgenic IgG are quite different from those of nontransgenic IgG. Although both IgGs contained biantennary complex-type oligosaccharides, transgenic IgG had more agalactosylated forms (45%) than those from nontransgenic IgG (28%), just as was found in patients with rheumatoid arthritis (RA). Since these transgenic mice express arthritis similar to RA, it will be a useful model to investigate the relationship between the galactosylation of IgG and the development of RA.

Animals↗
PubMed1993

Structural study of the sugar chains of CD36 purified from bovine mammary epithelial cells: occurrence of novel hybrid-type sugar chains containing the Neu5Ac alpha 2-->6GalNAc beta 1-->4GlcNAc and the Man alpha 1-->2Man alpha 1-->3Man alpha 1-->6Man groups.

CD36 is a glycoprotein included in the bovine milk fat globule membrane derived from mammary secretory epithelial cells during lactation. Asparagine-linked sugar chains were quantitatively released from CD36 as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with NaB3H4 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 Bio-Gel P-4 column chromatography in combination with serial chromatography on immobilized lectin columns including a Wistaria floribunda agglutinin (WFA)-agarose column. WFA is known to bind oligosaccharides terminating with either an alpha- or beta-N-acetylgalactosamine residue. Structural studies of oligosaccharides in each fraction by sequential exoglycosidase digestion as well as methylation analysis revealed that CD36 contains high mannose-type, hybrid-type, and bi, tri-, and tetraantennary complex-type sugar chains. A portion of the hybrid-type and the complex-type sugar chains which bound to a WFA-agarose column (28% of all oligosaccharides) contained the GalNAc beta 1-->4GlcNAc group(s) instead of the Gal beta 1-->4GlcNAc group(s) in their outer chain moieties. Like oligosaccharides found in human luteinizing hormone [Weisshaar, G., Hiyama, J., Renwick, A. G., & Nimtz, M. (1991) Eur. J. Biochem. 195, 257-268], some of the GalNAc beta 1-->4GlcNAc groups found in the CD36 oligosaccharides were sialylated as the Neu5Ac alpha 2-->6GalNAc group. Furthermore, most of the hybrid-type sugar chains of CD36 with the Gal/GalNAc beta 1-->4GlcNAc beta 1-->2 outer chain on their Man alpha 1-->3 arm contained an unusual Man alpha 1-->2Man alpha 1-->3 group on their Man alpha 1-->6 arm.

Animals↗