Isolation and fractionation of glycoprotein glycans in small amounts.
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Animals↗
Biomedical subjects
R Geyer
Publications and source records attributed to R Geyer.
Biosynthesis of sulfated glycoprotein-N-glycans present in recombinant human tissue plasminogen activator.
Recombinant human tissue plasminogen activator expressed in murine epithelial cells carries, in part, sulfated N-glycans, which are characterized by the presence of a NeuAc alpha 3[SO4-6]Gal unit. In order to study the biosynthesis of this novel structural element, corresponding sulfated asialooligosaccharide alditols were resialylated in vitro using a crude sialyltransferase preparation from murine liver which was shown to contain Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase activity. Products were analyzed for transfer of sialic acid residues by anion-exchange HPLC. The results demonstrated that resialylation of SO4-6Gal-residues did not occur. Therefore, it may be concluded that transfer of the sulfate group is the final step in the biosynthesis of this structural epitope.
Animals↗
Effect of substrate structure on the activity of Man9-mannosidase from pig liver involved in N-linked oligosaccharide processing.
Man9-mannosidase, an alpha 1,2-specific enzyme located in the endoplasmic reticulum and involved in N-linked-oligosaccharide processing, has been isolated from crude pig-liver microsomes and its substrate specificity studied using a variety of free and peptide-bound high-mannose oligosaccharide derivatives. The purified enzyme displays no activity towards synthetic alpha-mannosides, but removes three alpha 1,2-mannose residues from the natural Man9-(GlcNAc)2 substrate (M9). The alpha 1,2-mannosidic linkage remaining in the M6 intermediate is cleaved about 40-fold more slowly. Similar kinetics of hydrolysis were determined with Man9-(GlcNAc)2 N-glycosidically attached to the hexapeptide Tyr-Asn-Lys-Thr-Ser-Val (GP-M9), indicating that the specificity of the enzyme is not influenced by the peptide moiety of the substrate. The alpha 1,2-mannose residue which is largely resistant to hydrolysis, was found to be attached in both the M6 and GP-M6 intermediate to the alpha 1,3-mannose of the peripheral alpha 1,3/alpha 1,6-branch of the glycan chain. Studies with glycopeptides varying in the size and branching pattern of the sugar chains, revealed that the relative rates at which the various alpha 1,2-mannosidic linkages were cleaved, differed depending on their structural complexity. This suggests that distinct sugar residues in the aglycon moiety may be functional in substrate recognition and binding. Reduction or removal of the terminal GlcNAc residue of the chitobiose unit in M9 increased the hydrolytic susceptibility of the fourth (previously resistant) alpha 1,2-mannosidic linkage significantly. We conclude from this observation that, in addition to peripheral mannose residues, the intact chitobiose core represents a structural element affecting Man9-mannosidase specificity. A possible biological role of the enzyme during N-linked-oligosaccharide processing is discussed.
Amino Acid Sequence↗
Glycosphingolipids in cestodes. Chemical structures of ceramide monosaccharide, disaccharide, trisaccharide and tetrasaccharide from metacestodes of the fox tapeworm, Taenia crassiceps (Cestoda: Cyclophyllidea).
The presence of glycosphingolipids in the metacestodes of the fox tapeworm, Taenia crassiceps, has been established. The normal-phase TLC pattern of the neutral-fraction glycolipids revealed groups of bands corresponding to homologous components of increasing sugar chain length. The three simplest glycolipid components have been isolated and their chemical constitution determined as being of the neogala series: Gal beta 1Cer, Gal beta 6Gal beta 1Cer and Gal beta 6Gal beta 6Gal beta 1Cer. The ceramide tetrasaccharide fraction has been found to consist of a mixture of neogalatetraosylceramide, as an elongation of the neogala series, Gal beta 6Gal beta 6Gal beta 6Gal beta 1Cer and the component Gal alpha 4Gal beta 6Gal beta 6Gal beta 1Cer (both occurring in approximately equimolar proportions). The long-chain bases of the ceramide monogalactoside, digalactoside, trigalactoside and tetragalactosides contain, as well as small amounts of sphingosine, predominantly dihydrosphingosine/phytosphingosine in the approximate ratios 1.7:1, 1.4:1, 1:1 and 2.3:1, respectively. The major ceramide fatty acids have particularly long chains, with hexacosanoic and octacosanoic acids predominating. Upon reverse-phase TCL, the glycolipid components ceramide monogalactoside, digalactoside and trigalactoside were each separable into five component bands. Parent glycolipid components therefore show component band distributions comparable to one another in being governed by similar ceramide constitutions.
Animals↗
Carbohydrate structure of a thrombin-like serine protease from Agkistrodon rhodostoma. Structure elucidation of oligosaccharides by methylation analysis, liquid secondary-ion mass spectrometry and proton magnetic resonance.
The carbohydrate side chains of the thrombin-like serine protease ancrod from the venom of the Malayan pit viper Agkistrodon rhodostoma were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and fractionated by high-performance liquid chromatography. Glycans obtained were characterized by digestion with exoglycosidases, methylation analysis and, in part, by liquid secondary-ion mass spectrometry and 1H-NMR spectroscopy. The results reveal that this snake venom glycoprotein contains partially truncated di-, tri- and tetraantennary complex type N-glycans carrying Fuc(alpha 1-6) residues at the innermost N-acetylglucosamine and solely (alpha 2-3)-linked sialic acid substituents. As a characteristic feature, ancrod oligosaccharides comprise mainly sialylated Gal beta 3GlcNAc beta lactosamine antennae. Furthermore, a small proportion of the sugar chains were found to carry a NeuAc alpha 3GalNAc beta 4GlcNAc beta antenna exclusively linked to C-2 of Man(alpha 1-3) residues of the pentasaccharide core. Thus, many of the glycans found represent novel glycoprotein-N-glycan structures.
Carbohydrate Sequence↗
Structure of the N-linked oligosaccharides of the human transferrin receptor.
Human transferrin receptor was isolated from placenta and from the hepatocarcinoma cell line Hep G2. Asparagine-linked oligosaccharides were released by treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H or peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Oligosaccharide alditols were fractionated by anion-exchange high-performance liquid chromatography and by high-pH anion-exchange chromatography. Glycans from placental transferrin receptor were further characterized, after desialylation, by methylation analysis and, in part, by liquid secondary-ion mass spectrometry. Sialylation of placental transferrin receptor was examined by lectin affinity blotting with Sambucus nigra agglutinin and Maackia amurensis agglutinin. In order to trace possible inter-individual differences in N-glycosylation of the receptor, two preparations of placental transferrin receptor purified from two donors were compared. The results demonstrate that human transferrin receptor from placenta predominantly carries diantennary and triantennary N-acetyllactosaminic glycans as well as hybrid-type species, the galactose residues of which being almost completely substituted with (alpha 2-3)-linked sialic acid residues. Distinct differences were noted in the glycosylation pattern of the receptor from different individuals. Transferrin receptor from donor A carried predominantly diantennary and triantennary complex-type glycans, in part fucosylated at the innermost N-acetylglucosamine residue, in addition to small amounts of bisected and of incomplete diantennary species. Placental transferrin receptor from donor B predominantly carried triantennary N-acetyllactosaminic glycans without fucose and hybrid-type oligosaccharides with four or five mannose residues. Distinct from placental transferrin receptor, the receptor from Hep G2 cells contained larger amounts of oligomannosidic glycans with six to nine mannose residues and tetrasialylated complex-type oligosaccharides apart from mono-, di- and trisialylated species.
Agglutinins↗
The prognostic significance of postoperative residual contrast enhancement on CT scan in pediatric patients with medulloblastoma.
The clinical and therapeutic features of 20 patients with medulloblastoma treated at Children's Hospital and Medical Center, Seattle, from 1980 to 1987, were retrospectively analyzed with regard to prognosis. The overall actuarial 5-year survival rate was 63%, with 57% of patients free from recurrence at 5 years. Residual contrast enhancement on CT scans taken immediately postoperatively was associated with a significantly decreased 5-year recurrence-free survival rate; the 5-year recurrence-free survival rate was 100% for those patients without post-operative residual enhancement compared to 41% for those patients with residual enhancement. A high risk group of patients with residual contrast enhancement persisting one year following diagnosis was identified. No patient in this group survived without disease progression. Other factors, including sex, age at diagnosis, evidence of tumor dissemination, or extent of surgical resection as reported by the neurosurgeon, did not significantly influence prognosis.
Adolescent↗
Comparison of the carbohydrate moieties of recombinant soluble Fc epsilon receptor (sFc epsilon RII/sCD23) expressed in Saccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells.
Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFc epsilon RII/sCD23) was produced in Saccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFc epsilon RII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(alpha 2-3)Gal(beta 1-3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only alpha-mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.
Amino Acid Sequence↗
Carbohydrate structure of Marburg virus glycoprotein.
Marburg virus was propagated in E6 cells, a cloned cell line of Vero cells, in the presence of [6-3H]glucosamine. Radiolabelled viral glycoprotein was digested with trypsin, and oligosaccharides were liberated by sequential treatment with endo-beta-N-acetylglucosaminidase H, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and O-glycosidase, by beta-elimination, and by alkaline hydrolysis. After fractionation by HPLC and gel filtration, glycans were characterized chromatographically, by digestion with exoglycosidases and, in part, by methylation analysis and liquid secondary ion mass spectrometry. The oligosaccharide structures thus established include oligomannosidic and hybrid-type N-glycans, as well as neutral fucosylated bi-, tri- and tetraantennary species, most of which carry an additional bisecting N-acetylglucosamine. In addition, high amounts of neutral mucin-type O-glycans with type-1 and type-2 core structures were detected. None of the glycans present in this viral glycoprotein carried sialic acid residues.
Amidohydrolases↗
Structure elucidation of sulphated oligosaccharides from recombinant human tissue plasminogen activator expressed in mouse epithelial cells.
Sulphated N-linked carbohydrate chains isolated from recombinant human tissue plasminogen activator expressed in mouse epithelial (C127) cells were analysed as oligosaccharide alditols by methylation analysis, liquid secondary ion mass spectrometry, and one- and two-dimensional 1H-NMR spectroscopy. The results demonstrate that the major component has the following novel structure: NeuAc-alpha 2-6Gal beta 1-4GlcNAc beta 1-2[NeuAc alpha 2-3Gal beta 1- 4GlcNAc beta 1-4]-Man alpha 1-3[NeuAc alpha 2-3(SO4-6)Gal beta 1- 4-GlcNAc beta 1-2Man alpha 1-6]-Man beta 1-4GlcNAc beta 1- 4[Fuc alpha 1-6]GlcNAc-o1.
Animals↗
Carbohydrate structure of human pancreatic elastase 1.
Human pancreatic elastase 1 (E1) is a glycoprotein containing two potential N-glycosylation sites, one of which carries a carbohydrate moiety [Wendorf, Geyer, Sziegoleit & Linder (1989) FEBS Lett. 249, 275-278]. In order to study its glycosylation, glycoprotein isolated from post-mortem pancreas tissue of 75 donors was digested with trypsin. Oligosaccharides were liberated from resulting glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glycosaminyl)-asparagine amidase F, radiolabelled by reduction with KB3H4 and separated by h.p.l.c. and gel filtration. Major oligosaccharide alditol fractions, representing 67.8 mol% of total glycans, were characterized by methylation analysis and sequential degradation with exoglycosidases. The results revealed that about two-fifths of the partially truncated, mainly biantennary, complex-type glycans found comprised blood group A, B, Lea (or X), difucosyl A or difucosyl B determinants, which could be assigned to lactosamine antennae linked to Man(alpha 1-3)- residues of the sugar chains.
Carbohydrate Conformation↗
Processing of influenza virus hemagglutinin in insect cells.
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Animals↗
Infants with medulloblastoma: a single institution review of survival.
Although the prognoses of children with medulloblastoma have improved dramatically over the last several decades, the reported survival for very young children with this tumor remains poor. We undertook a retrospective review of patients less than 36 months of age at the time of the diagnosis of a medulloblastoma who were treated at our institution during a 36-year period. Of 28 such patients, 10 patients are presently surviving without disease at a median of 150 months from diagnosis. The presence of a metastatic tumor at the time of diagnosis was identified as a significant indicator of a poor prognosis, and all completely staged patients without metastasis are surviving without a recurrence of disease. This report suggests that the survival of very young children with a medulloblastoma may not be as poor as has been previously reported, particularly if a disseminated tumor is not present at the time of diagnosis.
Cerebellar Neoplasms↗
Glycosylation of the envelope glycoprotein from a polytropic murine retrovirus in two different host cells.
A polytropic recombinant retrovirus containing the envelope gene of Friend mink cell focus-inducing virus plus the remainder of the genome of an amphoropic murine leukemia virus was propagated on mouse embryo fibroblasts and mink lung cells. Virus particles, metabolically labeled with [2-3H]mannose, were harvested from the culture supernatants and lysed with detergents. The viral envelope glycoprotein was isolated from the lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Oligosaccharides were liberated by sequential treatment of tryptic glycopeptides with endo-beta-N-acetylglucosaminidase H and peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F and fractionated by high-performance liquid chromatography. Individual glycans were characterized chromatographically, by methylation analyses and in part, by enzymic microsequencing. The results demonstrated that viral glycoproteins, synthesized in mouse embryo fibroblasts, carried as major constituents partially fucosylated diantennary, 2,4- and 2,6-branched triantennary and tetraantennary complex type N-glycans with 0-4 sialic acid residues and only small amounts of high-mannose type species with 5-9 mannose residues. As a characteristic feature, part of the complex type glycans contained additional Gal(alpha 1-3) substituents. Glycoprotein obtained from virions propagated on mink lung cells, contained partially fucosylated diantennary and 2,4-branched triantennary oligosaccharides with 1-3 sialic acid residues, in addition to trace amounts of high-mannose type species with 8 or 9 mannose residues. Thus, the results reveal that predominantly, the complex type N-glycans of the retroviral envelope glycoprotein display cell-specific variations including differences in oligosaccharide branching, sialylation and substitution by additional Gal(alpha 1-3) residues.
Acetylglucosaminidase↗
Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus.
Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N-glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo-beta-N-acetylglucosaminidase H, or by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, were fractionated and subjected to structural analysis by one- and two-dimensional 1H NMR, as well as by methylation/gas-liquid-chromatography/mass-fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N-glycans: oligomannosidic oligosaccharides, mainly Man alpha 1----6(Man alpha 1----3)Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Man alpha 1----6(Man alpha 1----3)Man alpha 1----6 and Man alpha 1----3Man alpha 1----6 and Man alpha 1----3Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were found at Asn12, as previously published [Schlüter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. & Stirm, S. (1984) FEBS Lett. 169, 194-198] and at Asn334/341. N-Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAc alpha 2----3 or Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(NeuAc alpha 2----6 or NeuAc alpha 2----3Gal-beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNac beta 1----4GlcNAc beta 1---- with some bifurcation at ----6Man alpha 1----6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O-glycosidically substituted by Gal beta 1----3GalNAc alpha 1----, monosialylated or desialylated at position 3 of Gal or/and position 6 of GalNAc.
Amino Acid Sequence↗
Separation of glycoprotein-N-glycans by high-pH anion-exchange chromatography.
A series of high-mannose and complex type glycoprotein-N-glycans was subjected to high-pH anion-exchange chromatography. The results revealed that this method represents a useful tool for analytical characterization of single oligosaccharides as well as preparative separation of complex mixtures of carbohydrate side-chains. On the other hand, it became evident that in several cases a combination of different chromatographic techniques is required for efficient separation of individual oligosaccharide species.
Chromatography, Ion Exchange↗
The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector.
The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (SF) cell cultures using a baculovirus vector. To elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase H and glycopeptidase F. Sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures Man5-9GlcNAc2, and the truncated oligosaccharide cores Man3GlcNAc2 and Man3[Fuc]GlcNAc2. Polyacrylamide gel electrophoresis of endoglycosidase-treated hemagglutinin showed that most side chains of the HA1 subunit are truncated, whereas the HA2 subunit has one oligomannosidic and one truncated oligosaccharide. Comparison of these results with the glycosylation pattern of hemagglutinin obtained from vertebrate cells allowed a tentative allocation of the oligosaccharides to individual glycosylation sites. The results indicate that SF cells have the capacity to trim N-glycans to trimannosyl cores and to further process these by the addition of fucose. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains.
Animals↗
Rapid intramolecular turnover of N-linked glycans in plasma membrane glycoproteins. Extension of intramolecular turnover to the core sugars in plasma membrane glycoproteins of hepatoma.
Plasma membrane glycoproteins of rat hepatocytes undergo a rapid terminal deglycosylation in that the terminal sugars of the oligosaccharide side chains are rapidly removed from the otherwise intact glycoproteins [Tauber, R., Park, C.S. & Reutter, W. (1983) Proc. Natl Acad. Sci. USA 80, 4026-4029]. The present paper demonstrates that this rapid intramolecular turnover of plasma membrane glycoproteins is not restricted to peripheral sugars but, in contrast to liver, in hepatoma the core sugars of the oligosaccharide chains are also involved. Intramolecular turnover was measured in Morris hepatoma 7777 in five plasma membrane glycoproteins with Mr of 85,000 (hgp85), 105,000 (hgp105), 115,000 (hgp115), 125,000 (hgp125), 175,000 (hgp175) (hgp = hepatoma glycoprotein) that were isolated and purified to homogeneity by concanavalin-A--Sepharose affinity chromatography and semipreparative SDS gel electrophoresis. Analysis of the carbohydrates of hgp85, hgp105, hgp115 and hgp125 revealed the presence of N-linked oligosaccharides containing L-fucose, D-galactose, D-mannose and N-acetyl-D-glucosamine, but only of trace amounts of N-acetyl-D-galactosamine; hgp175 additionally contained significant amounts of N-acetyl-D-galactosamine, indicating the presence of both N- and O-linked oligosaccharides. As shown by digestion with endoglucosaminidase H, the N-linked oligosaccharides of hgp105, hgp115, hgp125 and hgp175 were of the complex type, whereas hgp85 also contained oligosaccharides of the high-mannose type. Half-lives of the turnover of the oligosacharide chains and of the protein backbone of the five glycoproteins were measured in the plasma membrane in pulse-chase experiments in vivo, using L-[3H]fucose as a marker of terminal sugars, D-[3H]mannose as marker of a core sugar and L-[3H]leucine for labelling the protein backbone. Protein backbones of the five glycoproteins were degraded with individual half-lives ranging over 41-90 h with a mean of 66 h. Compared to the degradation of the polypeptide backbone, both the terminal sugar L-fucose and the core sugar D-mannose turned over with much shorter half-lives averaging about 20 h in the five glycoproteins. The data show that, conversely to liver, within plasma membrane glycoproteins of hepatoma not only peripheral sugars but also core sugars of the oligosaccharides are split off during the life-span of the protein backbone. It may therefore be assumed that this reprocessing of plasma membrane glycoproteins is sensitive to malignant transformation.
Animals↗