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Biomedical subjects

A Kobata

Publications and source records attributed to A Kobata.

At least 19 recordsLinked to original sources
PubMed2001

The history of glycobiology in Japan.

This mini review surveys the major accomplishments in the field of glycoconjugates research in Japan, which were made after World War II. It describes early movements in the field of glycoconjugate research in Japan, development of the new techniques to investigate structures of the sugar chains of glycoconjugates, studies of the functions of the sugar chain moieties, and the political movement in Japan to support the basic research necessary for the development of glycotechnology. As introduced in this short article, important groundwork for glycobiology was made by Japanese researchers.

Biotechnology↗
PubMed1999

Glycosylation differences between the normal and pathogenic prion protein isoforms.

Prion protein consists of an ensemble of glycosylated variants or glycoforms. The enzymes that direct oligosaccharide processing, and hence control the glycan profile for any given glycoprotein, are often exquisitely sensitive to other events taking place within the cell in which the glycoprotein is expressed. Alterations in the populations of sugars attached to proteins can reflect changes caused, for example, by developmental processes or by disease. Here we report that normal (PrP(C)) and pathogenic (PrP(Sc)) prion proteins (PrP) from Syrian hamsters contain the same set of at least 52 bi-, tri-, and tetraantennary N-linked oligosaccharides, although the relative proportions of individual glycans differ. This conservation of structure suggests that the conversion of PrP(C) into PrP(Sc) is not confined to a subset of PrPs that contain specific sugars. Compared with PrP(C), PrP(Sc) contains decreased levels of glycans with bisecting GlcNAc residues and increased levels of tri- and tetraantennary sugars. This change is consistent with a decrease in the activity of N-acetylglucosaminyltransferase III (GnTIII) toward PrP(C) in cells where PrP(Sc) is formed and argues that, in at least some cells forming PrP(Sc), the glycosylation machinery has been perturbed. The reduction in GnTIII activity is intriguing both with respect to the pathogenesis of the prion disease and the replication pathway for prions.

Animals↗
PubMed1999

Study of the sugar chains of recombinant human amyloid precursor protein produced by Chinese hamster ovary cells.

The N- and O-glycans of recombinant amyloid precursor protein (APP), purified from Chinese hamster ovary cells transfected with the human 695-amino acid form of APP, were separately released by hydrazinolysis under different conditions. The reducing ends of the released N- and O-glycans were reduced with NaB3H4 and derivatized with 2-aminobenzamide (2AB), respectively. After acidic N-glycans were obtained by anion-exchange column chromatography, these were converted to neutral oligosaccharides by sialidase digestion, demonstrating that their acidic nature was entirely due to sialylation. The sialidase-treated N-glycans were then fractionated by lectin column chromatography and their structures were determined by linkage-specific sequential exoglycosidase digestion. These results demonstrated that recombinant APP has bi- and triantennary complex type N-glycans with fucosylated and nonfucosylated trimannosyl cores. In a similar fashion, the 2AB-labeled O-glycans derived from APP were determined to be mono- and disialylated core type 1 structures. Taken together, these results indicate that recombinant APP has sialylated bi- and triantennary N-glycans with fucosylated and nonfucosylated cores and sialylated O-glycans with core type 1 structures.

Amyloid beta-Protein Precursor↗
PubMed1999

Structure, pathology and function of the N-linked sugar chains of human chorionic gonadotropin.

Human chorionic gonadotropin (hCG) contains five acidic N-linked sugar chains, which are derived from three neutral oligosaccharides by sialylation. Each of the two subunits (hCGalpha and hCGbeta) of hCG contain two glycosylated Asn residues. Glycopeptides, each containing a single glycosylated Asn, were obtained by digestion of hCGalpha with trypsin, and of hCGbeta with chymotrypsin and lysyl endopeptidase. Comparative study of the sugar chains of the four glycopeptides revealed the occurrence of site-directed glycosylation. Studies of the sugar chains of hCGs, purified from urine of patients with various trophoblastic diseases, revealed that choriocarcinoma hCGs contain sialylated or non-sialylated forms of eight neutral oligosaccharides. In contrast, hCGs from invasive mole patients contain sialyl derivatives of five neutral oligosaccharides. The structural characteristics of the five neutral oligosaccharides, detected in choriocarcinoma hCGs but not in normal placental hCGs, indicate that N-acetylglucosaminyltransferase IV (GnT-IV) is abnormally expressed in the malignant cells. This supposition was confirmed by molecular biological study of GnT-IV in placenta and choriocarcinoma cell lines. The appearance of tumor-specific sugar chains in hCG has been used to develop a diagnostic method of searching for malignant trophoblastic diseases. In addition, a summary of the current knowledge concerning the functional role of N-linked sugar chains in the expression of the hormonal activity of hCG has been presented.

Animals↗
PubMed1999

Unusually high expression of N-acetylglucosaminyltransferase-IVa in human choriocarcinoma cell lines: a possible enzymatic basis of the formation of abnormal biantennary sugar chain.

Structural analysis of the sugar chains of human chorionic gonadotropin (hCG) has revealed that abnormal biantennary structures appear specifically on hCG in the urine of choriocarcinoma patients. However, the enzymatic and molecular mechanisms of the biosynthesis of abnormal biantennary sugar chains have not yet been elucidated. In this report, the enzyme activities and the expression levels of mRNAs of N-acetylglucosaminyltransferases (GnT)-I to -V, beta-1,4-galactosyltransferase, and alpha-mannosidase II in normal human placentae and three human choriocarcinoma cell lines were investigated. GnT-IV activities in choriocarcinoma cell lines were increased from 16- to 66-fold and GnT-III activity was increased from 15- to 25-fold as compared with those in human placentae, whereas other enzyme activities were not increased significantly. The mRNA expression levels generally correlated with their enzyme activities. Among the two GnT-IV genes found in human tissues only GnT-IVa gene was strongly expressed in the cancer cells: from three to seven times as much as in the normal tissue, whereas that of GnT-IVb remained constant. On the basis of these results, we proposed that ectopic expression of GnT-IVa gene should occur along with the malignancy of trophoblastic tissues, and that the increased GnT-IV activity should be the main cause of the formation of abnormal biantennary sugar chains in choriocarcinoma. A possible enzymatic basis of the biosynthesis of abnormal biantennary sugar chains is discussed.

Carbohydrate Metabolism↗
PubMed1999

Detection of novel carbohydrate binding activity of interleukin-1.

Tamm-Horsfall glycoprotein (THGP) and the oligosaccharide fraction liberated from THGP by hydrazinolysis inhibited tetanus toxoid-induced T cell proliferation. Intact THGP showed approximately 100-fold more inhibitory activity than the free oligosaccharides. After fractionating the oligosaccharides by anion-exchange column chromatography, the inhibitory activity could be detected in a sialidase-resistant acidic oligosaccharide fraction (fraction AR). The inhibitory activity of fraction AR was not observed when the fraction was added to the T cell culture medium 24 h after the addition of tetanus toxoid. Increased concentration of interleukin (IL) 1beta and decreased concentration of IL-2 were observed in the T cell culture medium after the addition of fraction AR. The oligosaccharides in fraction AR also inhibited the growth of an IL-1-dependent cell line, D10-G4. These results strongly suggested that the oligosaccharides in fraction AR bind to IL-1beta and suppress its cytokine activity. IL-1beta actually bound to the fraction AR immobilized on an amino-bonded thin layer plate. Fractionation of the oligosaccharides indicated that only oligosaccharides containing an N-acetylgalactosamine residue and a sulfate residue bound specifically to IL-1beta. Removal of either the sulfate residue or the N-acetylgalactosamine residue from the oligosaccharides abolished both the proliferation-inhibition and IL-1beta binding activities. Since IL-1beta did not bind to thyroid-stimulating hormone, which has the sulfate group at C-4 of the N-acetylgalactosamine residue in its N-linked sugar chains, the binding of IL-1beta toward oligosaccharides in fraction AR was considered to be highly specific.

Acetylation↗
PubMed1999

Evidence for the presence of major peripheral myelin glycoprotein P0 in mammalian spinal cord and a change of its glycosylation state during aging.

Glycoproteins, which react with Lens culinaris agglutinin, in the membrane preparation of various portions of brains and spinal cords, obtained from 9-week-old rats and 29-month-old rats, were comparatively analyzed by SDS-polyacrylamide gel electrophoresis. In contrast to the samples from brain, which showed similar staining patterns in the two different age groups, the glycoprotein patterns of spinal cords showed marked differences by the age of donors. The most prominent evidence is that a glycoprotein with an apparent molecular weight of 30 kDa (gp30) was detected in the aged rats, but not in the young adult rats. Based on the amino acid sequence data around the glycosylation site, the gp30 was identified as P0, which is a member of immunoglobulin superfamily and a major structural component of mammalian peripheral nerve myelin. This is the first report indicating that P0, which has been considered as a peripheral nerve-specific glycoprotein, occurs also in the spinal cord of mammals. In addition, nonglycosylated P0 molecule could be detected in the spinal cord of young adult rats by anti-P0 polyclonal antibody. These results indicate that the glycosylation state of the P0 molecule in the spinal cord changes during aging.

Aging↗
PubMed1998

Detection of O-mannosyl glycans in rabbit skeletal muscle alpha-dystroglycan.

alpha-Dystroglycan, which is a cell surface component of dystroglycan complex, is known to bind laminin in basal lamina of muscle cells and Schwann cells. We found previously that a novel O-glycan, Siaalpha2-3Galbeta1-4GlcNAcbeta1-2Man, is the major oligosaccharide in bovine peripheral nerve alpha-dystroglycan, and that this structure might mediate the binding of laminin. In order to determine whether this structure is specific for peripheral nerve alpha-dystroglycan or present on different forms of alpha-dystroglycan, we analyzed the structures of the sialylated O-glycans of rabbit skeletal muscle alpha-dystroglycan. Their structures were elucidated to be a mixture of a core 1 O-glycan and the same O-mannosyl glycan that we found in bovine peripheral nerve. These results indicate that alpha-dystroglycan in different species and tissues share a common structure of its major O-linked acidic carbohydrate, suggesting its relevance to the basic functional role of alpha-dystroglycan.

Animals↗
PubMed1998

Structural study of the O-linked sugar chains of human leukocyte tyrosine phosphatase CD45.

The O-linked sugar chains of the human leukocyte cell surface glycoprotein CD45 were released as tritium-labeled oligosaccharides by beta-elimination in the presence of NaB3H4. Mono Q column chromatography revealed that they comprise neutral (64%) and acidic (36%) oligosaccharides, the latter of which were converted to neutral ones by Arthrobacter ureafaciens sialidase treatment. Structural studies of each oligosaccharide fractionated on a Bio-Gel P-4 column by sequential exoglycosidase digestion and by methylation analysis revealed that human leukocyte CD45 contains mainly core 1 and core 2 oligosaccharides, 15% of which are modified with poly (N-acetyllactosamine) chains in different extensions. CD45 consists of several isoforms which were isolated after cell surface sialic acid residues were labeled by periodate/NaB3H4 treatment. Bio-Gel P-6 column chromatography of a mixture of the tritium-labeled glycopeptide/oligosaccharides obtained by pronase-digestion followed by mild alkaline borohydride treatment showed that distribution of the sialylated core 2 oligosaccharides is different among CD45 isoforms.

Carbohydrate Sequence↗
PubMed1997

A role of dystroglycan in schwannoma cell adhesion to laminin.

Dystroglycan is encoded by a single gene and cleaved into two proteins alpha- and beta-dystroglycan by posttranslational processing. Recently, alpha-dystroglycan was demonstrated to be an extracellular laminin-binding protein anchored to the cell membrane by a transmembrane protein beta-dystroglycan in striated muscle and Schwann cells. However, the biological functions of the dystroglycan-laminin interaction remain obscure, and in particular, it is still unclear if dystroglycan plays a role in cell adhesion. In the present study, we characterized the role of dystroglycan in the adhesion of schwannoma cells to laminin-1. Immunochemical analysis demonstrated that the dystroglycan complex, comprised of alpha- and beta-dystroglycan, was a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4. It also demonstrated the presence of alpha-dystroglycan, but not beta-dystroglycan, in the culture medium, suggesting secretion of alpha-dystroglycan by RT4 cells. RT4 cells cultured on dishes coated with laminin-1 became spindle in shape and adhered to the bottom surface tightly. Monoclonal antibody IIH6 against alpha-dystroglycan was shown previously to inhibit the binding of laminin-1 to alpha-dystroglycan. In the presence of IIH6, but not several other control antibodies in the culture medium, RT4 cells remained round in shape and did not adhere to the bottom surface. The adhesion of RT4 cells to dishes coated with fibronectin was not affected by IIH6. The known inhibitors of the interaction of alpha-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan, dextran sulfate, heparin, and sialic acid, also perturbed the adhesion of RT4 cells to laminin-1, whereas the reagents which do not inhibit the interaction, including dextran, chondroitin sulfate, dermatan sulfate, and GlcNAc, did not. Altogether, these results support a role for dystroglycan as a major cell adhesion molecule in the surface membrane of RT4 cells.

Animals↗
PubMed1997

Increased expression of highly branched N-glycans at cell surface is correlated with the malignant phenotypes of mouse tumor cells.

Three NIH3T3 transformants, MTAg, MTPy, and MT1, which grow similarly in soft agar media, showed remarkable differences in athymic mice: MTAg grew more rapidly than MTPy, whereas MT1 and NIH3T3 did not, and only MTAg metastasized in lung. Structural analysis of N-glycans from plasma membrane glycoproteins revealed that each sample contains similar amounts of N-glycans, but the relative amounts of 2,6-branched tri- and tetra-antennary oligosaccharides prominently increase and the relative amounts of biantennary oligosaccharides prominently decrease in the order of NIH3T3, MT1, MTPy, and MTAg, whereas those of others remained constant. Western blot analysis revealed that binding of Datura stramonium agglutinin, which interacts with 2,6-branched tri- and tetra-antennary oligosaccharides, is significantly increased in several bands from MTAg compared with NIH3T3, two of which are tentatively identified as lysosome-associated membrane protein-1 and fibronectin (FN)-receptor. It was also shown that the spreading of MTAg on FN-coated plates is dramatically inhibited with the anti-FN-receptor antiserum when compared with NIH3T3. These results indicate that the increased expression of highly branched N-glycans at cell surface is correlated with the rapidness of tumor formation and altered adhesive properties of tumor cells in vivo.

3T3 Cells↗
PubMed1997

Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan. The role of a novel O-mannosyl-type oligosaccharide in the binding of alpha-dystroglycan with laminin.

alpha-Dystroglycan is a heavily glycosylated protein, which is localized on the Schwann cell membrane as well as the sarcolemma, and links the transmembrane protein beta-dystroglycan to laminin in the extracellular matrix. We have shown previously that sialidase treatment, but not N-glycanase treatment, of bovine peripheral nerve alpha-dystroglycan greatly reduces its binding activity to laminin, suggesting that the sialic acid of O-glycosidically-linked oligosaccharides may be essential for this binding. In this report, we analyzed the structures of the sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan by two methods. O-Glycosidically-linked oligosaccharides were liberated by alkaline-borotritide treatment or by mild hydrazinolysis followed by 2-aminobenzamide-derivatization. Acidic fractions obtained by anion exchange column chromatography that eluted at a position corresponding to monosialylated oligosaccharides were converted to neutral oligosaccharides by exhaustive sialidase digestion. The sialidases from Arthrobacter ureafaciens and from Newcastle disease virus resulted in the same degree of hydrolysis. The neutral oligosaccharide fraction, thus obtained, gave a major peak with a mobility of 3.8-3.9 glucose units upon gel filtration, and its reducing terminus was identified as a mannose derivative. Based on the results of sequential exoglycosidase digestion, lectin column chromatography, and reversed-phase high-performance liquid chromatography, we concluded that the major sialylated O-glycosidically-linked oligosaccharide of the alpha-dystroglycan was a novel O-mannosyl-type oligosaccharide, the structure of which was Siaalpha2-3Galbeta1-4GlcNAcbeta1-2Man-Ser/Thr (where Sia is sialic acid). This oligosaccharide constituted at least 66% of the sialylated O-linked sugar chains. Furthermore, a laminin binding inhibition study suggested that the sialyl N-acetyllactosamine moiety of this sugar chain was involved in the interaction of the alpha-dystroglycan with laminin.

Animals↗
PubMed1997

A novel monoantennary complex-type sugar chain found in octopus rhodopsin: occurrence of the Gal beta1-->4Fuc group linked to the proximal N-acetylglucosamine residue of the trimannosyl core.

The N-linked sugar chains were liberated as oligosaccharides from octopus rhodopsin by hydrazinolysis. Most of the oligosaccharides were neutral, and separated into two major components by column chromatography using immobilized lectins and Bio-Gel P-4. Structural analysis of the one major component by sequential exoglycosidase digestion, chemical fragmentation in combination with methylation analysis revealed that it is a nonasaccharide; Man alpha1-->6(Gal beta1-->3GlcNAc beta1-->2Man alpha1-->3)Man beta1--> 4GlcNAc beta1-->4(Gal beta1-->4Fuc alpha1-->6)GlcNAc. This structure is quite unique in that a novel galactosylated fucose residue is attached to the reducing terminal N-acetylglucosamine residue.

Acetylglucosamine↗
PubMed1996

Transglycosylation of intact sialo complex-type oligosaccharides to the N-acetylglucosamine moieties of glycopeptides by Mucor hiemalis endo-beta-N-acetylglucosaminidase.

The endo-beta-N-acetylglucosaminidase (endo-beta-GlcNAc-ase) of Mucor hiemalis, endo-M, was found to transfer the sialo complex-type oligosaccharides from transferrin glycopeptide to the N-acetylglucosamine (GlcNAc) moieties of peptidyl-GlcNAc. Disialo complex-type oligosaccharide of transferrin glycopeptide was transferred to 9-fluorenylmethyloxycarbonyl (Fmoc)-asparaginyl-N-acetylglucosaminide (Fmoc-Asn-GlcNAc) by endo-M in a high yield. The structure of the reaction product was confirmed to be Fmoc-Asn-(GlcNAc)2-Man-(Man-GlcNac-Gal-NeuAc)2 by mass spectrometry. Endo-M also transferred disialo complex-type oligosaccharide to the GlcNAc residue of chemically synthesized H-Ile-Asn(GlcNAc)-Ala-Thr-Leu-OH. Asn-linked asialo complex-type oligosaccharide and Asn-linked high-mannose type oligosaccharide were also effective as oligosaccharide donors. Transfer of disialo complex-type oligosaccharide to the GlcNAc-peptide was the most effective among the three types of oligosaccharides, although the disialo complex-type oligosaccharide attached to the peptide was the poorest substrate for the hydrolytic activity of endo-M.

Acetylglucosamine↗
PubMed1996

Comparative study of the sugar chains of alkaline phosphatases purified from rat liver and rat AH-130 hepatoma cells. Occurrence of fucosylated high-mannose-type and hybrid-type sugar chains.

The N-linked sugar chains of alkaline phosphatases, purified from rat AH-130 hepatoma and from normal rat liver, were released quantitatively as oligosaccharides by hydrazinolysis and were labeled by reduction with NaB3H4. A comparative study of their structures revealed that following structural differences are induced by hepatocyte carcinogenesis: complex-type tetraantennary sugar chains and hybrid-type sugar chains appear; outer-chain moieties of the sugar chains of the hepatoma enzyme contain exclusively the Gal(Beta 1-4)GlcNAc groups (type 2 chains) but those of the normal enzyme contain other Gal(Beta 1-)GlcNAc groups and type 2 chains; and novel fucosylated high-mannose-type sugar chains are found in the oligosaccharides of the hepatoma enzyme.

Alkaline Phosphatase↗
PubMed1996

Analysis of the oligosaccharide chains of rhodopsin from normal rats and those with hereditary retinal dystrophy.

The nature of the oligosaccharides of rhodopsin from normal rats and from the Royal College of Surgeons (RCS) rats was examined by chemical, enzymatic and chromatographic procedures. This report is the first description of the structures of oligosaccharides of rat rhodopsin. The major oligosaccharide isomer of rat rhodopsin was shown to have the same structure as that from cow, human and frog. No neutral galactosylated species were detected. Although the site of the dystrophy in the RCS rat has been shown to be located at the retinal pigment epithelium, the possibility was examined that alterations in the glycosylation of rhodopsin might also be present. No differences were observed in either the amounts or structures of the rhodopsin oligosaccharide chains from young or adult control rats and RCS rats, although some differences were observed in the relative distribution of some oligosaccharide isomers between the RCS and controls. In addition, no differences in the amino acid content or SDS-PAGE patterns were detected.

Amino Acid Sequence↗
PubMed1996

Characterization of dp6troglycan-laminin interaction in peripheral nerve.

Dystoroglycan is encoded by a single gene and cleaved into two proteins, alpha and beta-dystroglycan, by posttranslational processing. The 120kDa peripheral nerve isoform of alpha-dystroglycan binds laminin-2 comprised of the alpha 2, beta 1, and gamma 1 chains. In congenital muscular dystrophy and dy mice deficient in laminin alpha 2 chain, peripheral myelination is disturbed, suggesting a role for the dystroglycan- laminin interaction in peripheral myelinogenesis. To begin to test this hypothesis, we have characterized the dystroglycan-laminin interaction in peripheral nerve. We demonstrate that (1) alpha-dystroglycan is an extracellular peripheral membrane glycoprotein that links beta-dystroglycan in the Schwann cell outer membrane with laminin-2 in the endoneurial basal lamina, and (2) dystrophin homologues Dp116 and utrophin are cytoskeletal proteins of the Schwann cell cytoplasm. We also present data that suggest a role for glycosylation of alpha-dystroglycan in the interaction with laminin.

Animals↗