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At least 919 records · Page 51Linked to original sources
PubMed1991

gp160 of HIV-I synthesized by persistently infected Molt-3 cells is terminally glycosylated: evidence that cleavage of gp160 occurs subsequent to oligosaccharide processing.

The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.

Asparagine↗
PubMed1992

Lec15 cells transfer glucosylated oligosaccharides to protein.

B4-2-1 cells (Lec15 cells) are Chinese hamster ovary cells deficient in mannosylphosphoryldolichol synthase activity. They synthesize the truncated lipid intermediate Man5GlcNAc2-P-P-dolichol rather than the Glc3Man9GlcNAc2-P-P-dolichol synthesized by wild-type cells. In this report we present evidence that these cells did synthesize glucosylated Man5GlcNAc2-P-P-dolichol, but this species represented only a minor fraction of the labeled oligosaccharide-lipid. On the other hand, glucosylated oligosaccharides were a major species transferred to protein in these cells, showing that in vivo, glucosylated oligosaccharides are preferentially transferred to protein. The truncated oligosaccharides found in B4-2-1 cells were removed from the protein by N-glycanase treatment, since they were resistant to both endo-beta-N-acetylglucosaminidase H and F activity. B4-2-1 cells processed the glucosylated, truncated oligosaccharides transferred to G protein of vesicular stomatitis virus, leading to infectious virus.

Animals↗
PubMed1994

Behaviour of complex oligosaccharides at a bilayer membrane surface: probed by 2H-NMR.

Deuterium wideline NMR was used in an attempt to directly assess oligosaccharide arrangement and motional characteristics of complex glycosphingolipids dispersed as minor components in phospholipid membranes. A convenient, general synthetic approach was developed which involved replacement of the acetate group of amido sugars with deuteroacetate (-COCD3). This provided excellent signal-to-noise when applied to the terminal GalNAc residue of globoside, and the terminal NANA residue of GM1. Simultaneously, globoside and GM1 fatty acids were replaced with stearic acid deuterated at C-2- a probe location sensitive to glycolipid hydrophobic backbone orientation and rigid body motion. Deuterated GM1 and globoside were studied by 2H-NMR in bilayers of 1-palmitoyl-2-oleoyl phosphatidylcholine, in the presence and absence of physiological quantities of cholesterol. The monoglycosyl glycosphingolipid, glucosyl ceramide, which is the common skeleton of many complex glycosphingolipids including those studied here, was also deuterated at fatty acid C-2 for comparative study in the same matrices. Correlation with spectra of the complex glycolipids demonstrated that, for a given temperature and membrane composition, ceramide backbone conformation was very similar amongst the species studied. Spectral features of GM1 deuterated on terminal NANA and assembled at a membrane surface, were found to be highly consistent with the oligosaccharide conformation determined in studies of GM1 in solution. In contrast, globoside deuterated in the terminal GalNAc residue gave spectra very different from those predicted on the basis of the conformation considered to exist in solution. It seems likely that this result reflects a combination of greater oligosaccharide chain flexibility relative to GM1, and the presence of the membrane environment. Interestingly, although there was highly significant spatial geometry associated with the complex oligosaccharide chains, and although temperature and the presence of cholesterol exert measurable effects on the membrane-inserted portion, these factors had very little impact on the measured spectral parameters associated with the NANA residue of GM1 or the terminal GalNAc residue of globoside. This seems to indicate lack of sensitivity of the complex oligosaccharide chains to conformation and internal motions of the hydrophobic chain segments in these fluid and semi-fluid membranes; and has important implications for mechanisms of crypticity.

Carbohydrate Sequence↗
PubMed1987

A phospho-oligosaccharide mimics insulin action on glycogen phosphorylase and pyruvate kinase activities in isolated rat hepatocytes.

A phospho-oligosaccharide which is the polar head group of a novel insulin-sensitive glycophospholipid has recently been involved in insulin action. We have investigated the insulin-like effects of this phospho-oligosaccharide on both glycogen phosphorylase a and pyruvate kinase activities of hepatocytes incubated in the presence of glucagon (0.1 nM). Similarly to insulin, the phospho-oligosaccharide antagonized glucagon-dependent activation of glycogen phosphorylase, as well as the inactivation of pyruvate kinase caused by this hormone. The antagonistic action of the phospho-oligosaccharide on glucagon effects was dose-dependent. Furthermore, it partially antagonized glucagon-stimulated cyclic AMP levels. These results support the hypothesis that this phospho-oligosaccharide mediates at least some insulin actions in hepatocytes.

Animals↗
PubMed1989

Liberation of oligosaccharides from glycosphingolipids on PC12 cell surface with endoglycoceramidase.

Endoglycoceramidase (EGCase) cleaves the linkage between the oligosaccharide and ceramide of glycosphingolipids (Ito, M., and Yamagata, T., J. Biol. Chem. 261, 14278-14282, ibid, 264, in press). Intact cells of rat pheochromocytoma line PC12 were treated with the highly purified EGCase I and the oligosaccharides released were analyzed by HPLC. Cleavage of the oligosaccharides with the enzyme reached a plateau as the amount of the enzyme was increased. At maximum, 42% of the oligosaccharides from globoside, 40% from GalGb3Cer, and 60% from Gb3Cer were liberated during 1h of incubation without impairing the viability of cells. The only partial liberation indicates that not all oligosaccharides of cell surface glycosphingolipids are accessible to the enzyme. The use of EGCase offers an important new method to access the functions of glycosphingolipids on cell surface in situ.

Adrenal Gland Neoplasms↗
PubMed1991

Characterization of mucin-type oligosaccharides with the sialyl-Le(a) structure from human colorectal adenocarcinoma cells.

Oligosaccharides with the sialyl-Le(a) structure have been isolated on an affinity column of a monoclonal antibody, MSW 113, from mucin-type glycoproteins derived from the surfaces of SW 1116 and LS 180 cells, and their secretions. The oligosaccharides were polydisperse with respect to molecular size, the oligosaccharides derived from glycoproteins in culture media being larger than those in cell lysates, as assessed by gel filtration. Some of the oligosaccharides were susceptible to degradation by endo-beta-galactosidase (E. freundii), as judged from the change in the gel filtration pattern. These results indicate that oligosaccharides with the sialyl-Le(a) structure derived from mucin-type glycoproteins produced by human colonic cancer cells are extremely large in size and complex in structure, and that some of them contain the poly-N-acetyllactosamine structure.

Adenocarcinoma↗
PubMed1992

Hepatocyte growth factor is linked by O-glycosylated oligosaccharide on the alpha chain.

The glycosylation site and the structure of O-glycosylated oligosaccharide of recombinant human HGF were investigated. N-acetylgalactosamine (GalNAc) in the alpha chain suggested the presence of O-glycosylated oligosaccharide. Sugar analysis and amino acid sequence analysis of peptide fragments produced by limited degradation revealed that O-glycosylated oligosaccharide linked to Thr445 of the alpha chain. The molecular weight of the oligosaccharide was determined with ion spray mass spectrometry. From these studies, the structure of the O-glycosylated oligosaccharide on the alpha chain of HGF was concluded as [formula: see text].

Amino Acid Sequence↗
PubMed1988

A micromethod for the estimation of oligosaccharides containing glycosidically linked sialic acid or hexoses, or both, in glycoproteins.

The peeling reaction, the process by which oligosaccharides are degraded in alkali, was used as the basis for an assay to provide structural information about glycosidically linked oligosaccharides in glycoproteins. Glycoproteins were treated with 0.05 M NaOH at 50 degrees to induce release, and subsequent degradation ("peeling"), of glycosidically linked, but not of N-glycosydically linked, oligosaccharides. Among the degradation products generated from O-linked chains were three 3-deoxy sugar acids whose formation was correlated with certain structural features of the oligosaccharides. N-Acetylneuraminic acid was released from terminal positions in the oligosaccharides, and iso- and meta-saccharinic acids were derived from the degradation of 4-O- and 3-O-substituted hexoses, respectively. All of these sugar acids were detected colorimetrically by periodate oxidation and reaction of the product with 2-thiobarbituric acid. The ability of the method to generate 3-deoxy sugar acids was tested in 8 alkali-treated glycoproteins. 3-Deoxy sugar acids were detected only in those glycoproteins whose glycosidically linked carbohydrates contained N-acetylneuraminic acid, or 3-O- or 4-O-substituted hexoses, or both. As little as 0.12 microgram of 3-deoxy sugar acid produced from 5 micrograms of human chorionic gonadotropin was sufficient for detection. This method is novel in its ability to distinguish sialylation of glycosidically linked carbohydrates. Furthermore, it combines the specificity of beta-elimination with the sensitivity of the 2-thiobarbituric acid assay in targeting degradation products of the peeling reaction as candidates for an assay method.

Glycoproteins↗
PubMed1989

Liquid ion-exchange chromatography under pressure of milk oligosaccharides using a pulsed amperometric detector.

Several oligosaccharides from human milk were separated completely on a Dionex AS6 ion-exchange column under mild alkaline conditions, such that no degradation of oligosaccharides was detectable after incubation for 6 h at room temperature in the eluent buffer. In general, both the presence of fucosyl groups and branching within oligosaccharide chains tend to reduce retention times for oligosaccharides in this system. Thus, both lacto-N-fucopentaose II [beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1--- -3)- beta-D-Galp-(1----4)-D-Glc] and lacto-N-fucopentaose III [beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1--- -3)- beta-D-Galp-(1----4)-D-Glc] were eluted before lacto-N-fucopentaose I [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3 )-beta-D- Galp-(1----4)-D-Glc], and all three fucopentaoses were eluted earlier than lacto-N-tetraose [beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc ]. 2'-Fucosyllactose [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-D-Glc] and 3-fucosyllactose [alpha-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-Glc] were separated completely under a wide range of conditions. Introduction of substituents at either the O-3 or O-4 of 2-acetamido-2-deoxy-D-glucose produced large differences in retention: lacto-N-tetraose and lacto-N-neotetraose [beta-D-Gal-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc] could be separated by more than 8 min. The l.c. ion-exchange chromatographic system described represents a useful addition to existing methods for separating oligosaccharides derived from glycoproteins and glycolipids.

Asialoglycoproteins↗
PubMed1990

High-sensitivity structural analyses of oligosaccharide probes (neoglycolipids) by liquid-secondary-ion mass spectrometry.

The sensitivity of detection and extent of information on structure obtainable by liquid-secondary-ion mass spectrometry (l.s.i.-m.s.) of neoglycolipids on the conventional target probe and directly from the surface of silica plates following t.l.c. has been assessed. Neoglycolipids were derived from malto-oligosaccharides, chitin oligosaccharides, and a range of deoxyhexose-, hexose-, 2-acetamido-2-deoxyhexose-, and sialic acid-containing mammalian oligosaccharides by reductive amination using phosphatidylethanolamine dipalmitoate (PPEADP). Sub-pmol amounts of the maltopentaose-PPEADP derivative applied directly to the target probe provided information on molecular weight, whereas approximately 1 pmol was required when analysed on the silica gel t.l.c. plate. With a biantennary octasaccharide derivative, the sensitivity of detection was 20-50 times lower and the other oligosaccharides had intermediate sensitivities. Information on composition and sequence was obtained readily from fragment ions, using 5 pmol of the maltopentaose derivative and 50 pmol of the octasaccharide derivative on the target probe, and 50 and 200 pmol, respectively, on the silica gel chromatogram. The optimised conditions formed the basis for characterising the structures of the components of mixtures of oligosaccharides generated from glycoproteins.

Carbohydrate Conformation↗
PubMed1991

Analysis of sialic acid-containing mucin oligosaccharides from porcine small intestine by high-temperature gas chromatography-mass spectrometry of their dimethylamides.

Acetylation of sialic acid-containing oligosaccharides lactonises the sialic acid residue quantitatively for all oligosaccharides studied except for 6'-sialyl-lactose. The modified, unsulphated, sialylated and sulphated oligosaccharides can then be fractionated by anion-exchange chromatography. Ammonolysis of the lactones followed by methylation yielded the dimethylamides, which are amenable to g.l.c.-m.s. and give intense and informative mass spectra. This approach has been used to characterise the sialic acid-containing O-linked oligosaccharides obtained from the mucin glycopeptides of the small intestine of the pig. At least 28 structures were found, having NeuAc or NeuGc 6-linked to the HexNAc attached to the peptide core or to a Hex 3-linked to HexNAc. Four different disialylated oligosaccharides were found having NeuAc or NeuGc on the Hex residue 3-linked to HexNAc and 6-linked to HexNAc.

Animals↗
PubMed1992

Separation of mucin oligosaccharide-alditols by high performance liquid chromatography on alkylamine-bonded silica columns. Effects of structural parameters.

Forty five oligosaccharide-alditols, purified after reductive beta-elimination of human bronchial mucins, were analyzed by HPLC on alkylamine-bonded silica column (Lichrosorb-NH2). The comparison of their structural features and retention times permitted the extension of some previous findings. The chromatographic behavior of the oligosaccharide depends more on the accessibility of oligosaccharide hydroxyl groups than on the sugar composition. The use of this type of fractionation is very efficient for low-molecular-mass oligosaccharide-alditols but needs to be completed by a second chromatographic step for higher molecular-mass oligosaccharide-alditols.

Alkylation↗
PubMed1995

Structure determination of galacto-oligosaccharides by pyridylamination and NMR spectroscopy.

Galacto-oligosaccharides formed from lactose by the action of some beta-galactosidases were subjected to gel chromatography on Bio-Gel P-2, and the resulting oligosaccharide fractions were converted into pyridylamino (PA) derivatives. Each PA-oligosaccharide fraction, which consisted of several isomers in a given size-class, was then subjected to HPLC on an ODS column. Twenty-one individual galacto-oligosaccharide components were isolated in this way. The structures of most of these compounds, namely six disaccharides, five trisaccharides, two tetrasaccharides, and a pentasaccharide, were determined by 13C-NMR spectroscopy. The results obtained will be useful for the study of the activity of various galacto-oligosaccharides on the growth of Bifidobacterium species.

Aminopyridines↗
PubMed1994

Degradation of feruloylated oligosaccharides from sugar-beet pulp and wheat bran by ferulic acid esterases from Aspergillus niger.

The activity of two forms of ferulic acid esterase (FAE) from Aspergillus niger on a synthetic feruloylated substrate (methyl ferulate) and on 11 different feruloylated oligosaccharides from sugar-beet pulp and wheat bran was determined. The enzymes exhibited different specificities for the various feruloylated substrates and were more active on certain substrates of cell-wall origin than on methyl ferulate. Both enzymes preferred the arabinose residue to which ferulic acid is attached in the furanose form. FAE-I had no clear preference for the type of linkage involved between the ferulic acid units and the oligosaccharide chain. In contrast, FAE-III had a clear requirement for ferulic acid to be attached to O-5 of the Ara f ring while no catalysis was observed when ferulic acid was attached to O-2. Both enzymes showed maximum activity on feruloylated trisaccharides. An increase in the length of the oligosaccharide chain did not preclude catalysis, but feruloylated oligosaccharides of a dp > 3 were hydrolysed at a reduced rate. Our results support the hypothesis that different kinds of ferulic acid esterases exist with different specificities for the oligosaccharide chain of the feruloylated substrates.

Aspergillus niger↗
PubMed1994

Induced circular dichroism study of the aqueous solution complexation of cello-oligosaccharides and related polysaccharides with aromatic dyes.

Acetobacter xylinum, grown in the presence of low levels of the water-soluble dye Calcofluor White ST produces a pellicle of cellulose that has no detectable crystallinity. Biological factors of this sort are probably more important than physical factors in controlling the higher order structures of celluloses. Circular dichroism (CD) is induced by complexes that are formed by specific interactions between chiral oligosaccharides and dye molecules. Using CD, equilibrium constants were measured for the association reactions between various dyes with a series of cello-oligosaccharides (n = 2-6), methylcellulose, hydroxypropylcellulose (HPC), amylose, cyclomalto-oligosaccharides (cyclodextrins), and the linear malto-oligosaccharides (n = 3-7). Possible structural features of the complexes are discussed. Dyes that are capable of binding to the higher cello-oligomers in aqueous solutions are the same dyes that modify the solid structure of bacterial cellulose. An analogy between the binding of water-soluble dyes to cello-oligosaccharides and the binding of the cellulose-degrading enzyme, cellobiohydrolase I, to cellulose is discussed.

Acetobacter↗
PubMed1995

Structural studies of the O-antigen oligosaccharides from two strains of Moraxella catarrhalis serotype C.

The oligosaccharide parts from Moraxella (Branhamella) catarrhalis serotype C lipooligosaccharides were isolated by mild acid hydrolysis followed by gel permeation chromatography. Four different oligosaccharides could be identified from strain RS26 and two from strain RS10. The structures of the O-oligosaccharides were established by methylation analyses, mass spectrometry, and NMR spectroscopy. It is concluded that the oligosaccharide O-antigens from RS26 are a mixture of octa-, deca-, and undeca-saccharides, and most likely a heptasaccharide. Strain RS10 contains the deca- and the undeca-saccharide only. The structures for the oligosaccharides are shown below. [formula: see text] OS(7) [formula: see text] OS(8) [formula: see text] OS(10) [formula: see text] OS(11) Methylation analysis of the intact lipooligosaccharides showed that two Kdo residues were present, one terminal and one 4,5-substituted residue. It also showed that they consisted of a lipid A portion with 6-substituted glucosamine residues.

Carbohydrate Sequence↗
PubMed1994

A detailed structural characterization of ribonuclease B oligosaccharides by 1H NMR spectroscopy and mass spectrometry.

The structures of ribonuclease B oligosaccharides have previously been shown to be high mannose type by methylation analyses and sequential exoglycosidase digestion. Due to the unique nature of these oligosaccharides, in that all mannosyl residues are attached by alpha-(1-->2)-linkages beyond the branch points, methylation analysis fails to solve the exact structures beyond Man5. Therefore, we have undertaken this study using 1H nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. In this study, bovine pancreatic ribonuclease B was first reduced and carboxymethylated, and was then deglycosylated by peptide/N-glycosidase F (PNGase F). The released oligosaccharides were fractionated by Bio-Gel P-4 chromatography to give five pools, Man5 through Man9. The structures of the oligosaccharide pools were then studied by laser desorption time of flight mass spectrometry and 1H NMR spectroscopy at 300 MHz. For Man5, Man-A and Man-B are attached in alpha-(1-->3)- and alpha-(1-->6)-linkages to the alpha-(1-->6)-linked Man-4' of the pentasaccharide core structure. For Man6, Man-C is linked alpha-(1-->2) to the alpha-(1-->3)-linked Man-4. Man7 exists as three structural isomers, and has the additional mannosyl residue (Man-D) linked alpha-(1-->2) to Man-A, Man-B, and Man-C is linked alpha-(1-->2) to the alpha-(1-->3)-linked Man-4. Man-7 exists as three structural isomers, with the additional two mannosyl residues linked alpha-(1-->2) to Man-A, Man-B, and Man-C. For each position, Man-A, Man-B, and Man-C, the extent of occupancy by one of the additional alpha-(-->)-linked mannosyl residues was 15, 94, and 91%, respectively. Man9 is a single component, with the three additional mannosyl residues linked alpha-(1-->2) to Man-A, Man-B, and Man-C, respectively. The relative molar proportions of Man5 to Man9 are 57, 31, 4, 7, and 1%, respectively. This report presents for the first time the complete structural characterization of the oligosaccharides from ribonuclease B.

Amidohydrolases↗
PubMed1991

Immunosuppressive activity of Tamm-Horsfall glycoprotein oligosaccharides: effect of removal of outer sugars and conjugation with a protein carrier.

Tamm-Horsfall (TH) glycoprotein, the major protein of human urine, is, in vitro, a powerful immunosuppressive agent and the activity resides in its oligosaccharide chains. In this study we investigated structural features required for the inhibitory activity of TH glycoprotein oligosaccharides in the one-way mixed lymphocyte reaction (MLR). We found that both high-mannose and complex-type TH glycopeptides, fractionated from Pronase-digested TH glycoprotein, behaved as inhibitors. Sequential exoglycosidase digestion of complex-type TH glycopeptide results in a slight increase of the inhibitory activity, with a maximum after desialylation and beta-galactosidase treatment. These results suggest that the immunosuppressive activity resides in the central portion of TH glycoprotein N-linked oligosaccharides. The conjugation of complex-type TH glycopeptides to a protein carrier, such as bovine serum albumin, greatly enhanced the inhibitory activity. This effect occurred if the TH-glycopeptide conjugate was added to MLR within the first 24 hr. These results indicate that (i) the immunosuppressive activity is strongly dependent on a multivalent interaction between TH oligosaccharides and ligand(s) at the lymphocyte surface; (ii) an early step of cell-cell recognition is the target of the immunosuppressive conjugate; (iii) TH oligosaccharides compete with a carbohydrate recognition system between effector and stimulator cells which contributes to the MLR-induced blastogenesis.

Carbohydrate Sequence↗