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PubMed1986

Isolation and characterization of sulphated oligosaccharides released from bovine corneal keratan sulphate by the action of endo-beta-galactosidase.

A series of oligosaccharides has been isolated from the keratan sulphate peptidoglycan (3 M NaCl fraction) of bovine cornea after digestion with the endo-beta-galactosidase of Bacteroides fragilis. Structural information on the major oligosaccharides was obtained from (a) their susceptibilities to endo-beta-galactosidase before and after desulphation, (b) their elution positions on a column of Bio-Gel P-4 and retention times on a high-performance anion-exchange column and (c) negative-ion fast-atom-bombardment mass spectrometry. More than 75% of the oligosaccharides were sulphated unbranched poly(N-acetyllactosamine) sequences, (-3/4GlcNAc beta 1-3Gal beta 1-)n, and approximately 3% was the neutral disaccharide, GlcNAc beta 1-3Gal. The sulphated disaccharide, GlcNAc-SO-3 beta 1-3Gal, accounted for almost 35% of the oligosaccharide material while 40% consisted of four oligosaccharides, unbranched tetra-, hexa-, octa- and decasaccharides of poly(N-acetyllactosamine) type, having 3, 5, 7 and 9 sulphate residues respectively. Proton nuclear magnetic resonance studies at 500 MHz (Hounsell, E. F., et al. following paper in this journal) have shown that a sulphate residue is attached to the C-6 position of each N-acetylglucosamine and each internal galactose residue of these four oligosaccharides which express to varying degrees the antigenic determinants recognised by three monoclonal antibodies to keratan sulphate (Mehmet, H. et al., paper which follows the next paper in this journal).

Animals↗
PubMed1988

Assembly of asparagine-linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases.

We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases.

Alkaloids↗
PubMed1989

Catabolic pathway of oligosaccharide-diphospho-dolichol. Subcellular sites of the degradation of the oligomannoside moiety.

The degradation of oligosaccharide-diphospho-dolichol leads to the release of oligosaccharide material ranging from (Glc)3(Man)9(GlcNAc)2-P to (Man)3 species and further smaller species. The subcellular location of the glucosidases and mannosidases involved in this catabolic process has been investigated on the basis of their differential sensitivity towards specific inhibitors (castanospermine, deoxymannojirimycin and swainsonine). The results indicate that the first steps of degradation down to the (Man)6 species occurs in the rough endoplasmic reticulum. This result is supported by the fact that the (Man)6 species is the end product when lipid-intermediate-derived glucosylated oligosaccharides are incubated with purified rough endoplasmic reticulum membranes. Swainsonine and lysosomotropic agents (chloroquine and ammonium chloride) do not affect the degradation process, thus indicating that neither Golgi apparatus nor lysosomes are involved in this catabolism. The observation of the same degradation pattern of the released oligosaccharide material in mannosidosis fibroblasts, lacking lysosomal mannosidases, confirms these results. Finally, the subcellular distribution of the released oligosaccharide material indicates that the oligomannosides larger than (Man)6 species are sequestered in the particulate fraction whereas, in contrast, oligomannosides smaller than (Man)6 species are found predominantly in the cytosol. Taken altogether, the experiments demonstrate that the first steps of the degradation of oligosaccharide-diphospho-dolichol occurs in the rough endoplasmic reticulum producing oligomannosides of the (Man)6 species which are then translocated to the cytoplasm to be further degraded.

1-Deoxynojirimycin↗
PubMed1993

Comparative study of high-mannose-type oligosaccharides in membrane glycoproteins of rat hepatocytes and different rat hepatoma cell lines.

A comparative study was undertaken to characterize the oligosaccharides released by endo-beta-N-acetylglucosaminidase H (endo H) from the membrane glycoproteins of rat hepatocytes and three different Morris hepatoma cell lines (NA-MH 7777, HTC and MH1C1). It is shown that the membrane glycoproteins of hepatocytes and hepatoma cells contain markedly different quantities and forms of high-mannose-type carbohydrate chains. After radiolabelling of the cells with D-[2-3H]mannose, in the absence and presence of 1 mM 1,5-dideoxy-1,5-imino-D-mannitol (1-deoxymannojirimycin), high-mannose-type oligosaccharides were released from delipidated membrane glycoproteins by enzymic digestion with endo H. The carbohydrate chains were converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride, then further analysed by HPLC using an APS-2 Hypersil column. In the absence of 1-deoxymannojirimycin, up to 10% of the radiolabelled oligosaccharides were released by endo H-treatment of the membrane glycoprotein fraction from rat hepatocytes. In contrast, the quantity of radiolabelled high-mannose-type carbohydrate chains released by endo H-treatment from tumour-cell membrane glycoproteins of hepatoma cell lines NA-MH 7777 (31.5%). MH1C1-MH 7795 (37.2%) and HTC-MH 7288c (48%) was increased up to fivefold. The formation of higher-mannosylated structures after oligosaccharide analysis was observed in all hepatoma cell lines, with Man8GlcNAcOH as the major component, whereas in hepatocytes Man5GlcNAcOH was the predominant high-mannose-type structure. In contrast, in the presence of the Golgi alpha-D-mannosidase I inhibitor, 1-deoxymannojirimycin, no significant differences were observed between the distribution of high-mannose-type oligosaccharides in the membrane glycoproteins of hepatocytes and hepatoma cells. However, in the presence of this inhibitor, the proportion of radiolabelled glycans sensitive to deglycosylation by endo H was greatly increased (> 85%) in all the cell lines investigated, the predominant structures being Man8-9-GlcNAcOH. This study shows that an increased content of high-mannose-type sugar chains is a general characteristic of membrane-bound glycoproteins for malignant transformed hepatocytes.

1-Deoxynojirimycin↗
PubMed1993

Structures of the N-linked oligosaccharides on porcine plasma vitronectin.

The structures of N-linked oligosaccharides, especially the distribution of sialic acid species, present on porcine plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N-glycosidase F digestion and tagged with 2-aminopyridine, and the pyridylamino-oligosaccharides were fractionated by anion-exchange and reverse-phase HPLC. Nine major pyridyl-amino-oligosaccharides were isolated. The linkages and locations of sialic acids were determined by a novel approach involving desialylation with Salmonella sialidase in combination with acid desialylation. After desialylation, the asialo-forms were analyzed by two-dimensional sugar mapping, component sugar analysis and 400-MHz 1H-NMR spectroscopy. The major oligosaccharides of porcine vitronectin were of the fucosylated biantennary type, with a small amount of the triantennary N-acetyllactosamine type, to which 1-3 mol sialic acids was linked. Sialic acids were linked predominantly through alpha 2-6 linkages, although alpha 2-3 linkages were also present, and fucose was linked to the innermost N-acetylglucosamine through an alpha 1-6 linkage. It was found that every pyridylamino-oligosaccharide population contained N-glycolylneuraminic acid and N-acetylneuraminic acid in a molar ratio of 1:2-9, and that N-glycolylneuraminic acids were located predominantly on the Man alpha 1-6 arm.

Aminopyridines↗
PubMed1995

Structures of the N-linked oligosaccharides on human plasma vitronectin.

The structures of N-linked oligosaccharides present on human plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N-glycosidase F digestion and tagged with 2-aminopyridine; the pyridylamino-oligosaccharides were then fractionated by anion-exchange and reverse-phase HPLC. Ten major pyridylamino-oligosaccharides were isolated. The linkages and locations of sialic acid residues were determined by desialylation with Salmonella sialidase in combination with acid. The asialo forms were then analyzed by two-dimensional sugar mapping, component sugar analysis and 400-MHz 1H-NMR spectroscopy. The major oligosaccharides of human vitronectin were of the diantennary N-acetyllactosamine type, with a lesser amount of the tri- and a small amount of the mono-antennary type, to which 1-3 mol sialic acid residues were linked, mostly through alpha 2-6 linkages, although alpha 2-3 linkages were also present. The possibility that several binding activities of vitronectin can be ascribed to its glycan moiety was discussed, based on the specific features of the N-linked oligosaccharides on human vitronectin revealed here.

Carbohydrate Sequence↗
PubMed1983

Structure of the mannose-rich oligosaccharide chains of concanavalin A-binding glycopeptides derived from beef brain glycoproteins.

A neutral, mannose-rich, concanavalin A (Con A)-binding glycopeptide fraction was obtained by proteolytic digestion of defatted beef brain tissue. Hydrazinolysis followed by gel filtration of the reaction products provided three oligosaccharides. A portion of each oligosaccharide was treated by exhaustive digestion with alpha-mannosidase. Another portion was subjected to selective acetolysis of Man alpha 1 leads to 6Man linkages, providing two fragments that were recovered by gel filtration. The structure of the intact oligosaccharides, as well as the fragments obtained by selective acetolysis and enzymatic treatment, were resolved by gas-liquid chromatographic-mass spectrometric analysis. The structures of the three oligosaccharides were: (a) Man alpha 1 leads to 2Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4-N-acetylglucosamine (GlcNAc)beta 1 leads to 4N-acetylglucosaminitol (GlcOLNAc); (b) Man alpha 1 leads to 2Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man alpha 1 leads to 6(Man alpha 1 leads to 2Man alpha 1 leads to 3)-Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcOLNAc; and (c) Man alpha 1 leads to 6(Man alpha 1 leads to 3) Man alpha 1 leads to 6(Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc-beta 1 leads to 4GlcOLNAc. These structures account for 15-20% of the glycoprotein-carbohydrate of whole beef brain and most of the oligosaccharides that demonstrate a high affinity for Con A. In view of the large number of Con A-binding glycoproteins in brain tissue, it appears that many of these different glycoproteins must contain structurally identical oligosaccharides.

Animals↗
PubMed1987

Characterization of fucosyl oligosaccharides associated with synaptic membrane and synaptic junctional glycoproteins.

Rats were administered [3H]fucose by intracranial injection and synaptic membranes (SMs) isolated 18 h later. Oligosaccharides associated with SM glycoproteins were prepared by hydrazinolysis and analyzed by a combination of affinity chromatography on concanavalin A (Con A)-agarose, ion-exchange chromatography on DEAE-cellulose, and gel permeation chromatography. Most (94%) of the [3H]fucose-labelled oligosaccharides were present in the fraction that did not bind to Con A. Of these 41% did not bind to DEAE-cellulose, indicating the absence of negatively charged groups and the remainder were resolved into four fractions of increasing acidity. Gel permeation chromatography of the fractions from the DEAE-cellulose column suggested that the major oligosaccharides corresponded to fucosylated triantennary structures containing varying amounts of sialic acid although more highly branched structures containing peripheral branches lacking one or more sugars may also have been present. Comparison of fucosyl oligosaccharides associated with SMs prepared from 10- and 28-day-old animals indicated that although the general oligosaccharide content was similar at both ages, membranes from younger animals were characterized by an increase in the proportion of highly acidic structures. Fucosylated glycans derived from synaptic junctional (SJ) glycoproteins were also characterized by a greater percentage of highly acidic structures than SMs. The results indicate that SMs and SJs are characterized by specific complements of fucosylated glycoprotein oligosaccharides.

Animals↗
PubMed1989

Oligosaccharide composition, localization, and developmental changes of a CNS-specific (F3-87-8) glycoprotein.

The F3-87-8 glycoprotein was isolated from rat brain by immunoaffinity chromatography after biosynthetic labeling by intracerebral administration of [3H]glucosamine, and the oligosaccharide composition of pronase-derived glycopeptides was determined by sequential lectin affinity chromatography and alkali treatment. Triantennary complex oligosaccharides (65%) and O-glycosidic oligosaccharides (18%) were the predominant types present, accompanied by 7-10% each of biantennary and high-mannose oligosaccharides. Twenty-two percent of the complex oligosaccharides had a fucose residue linked to the proximal N-acetylglucosamine of the chitobiose units. No poly(N-acetyllactosaminyl) or hybrid oligosaccharides were detected. Immunocytochemical studies on the localization of this glycoprotein in developing rat brain demonstrated that in 1-week postnatal cerebellum, there is light staining of the internal granule cell layer and surrounding the Purkinje cells. By 2 weeks, an intense staining of myelinating fiber tracts appears, accompanied by much lighter staining in the granule cell layer and at the base of the molecular layer. Staining of the white matter remains strong at 3 weeks postnatal, together with significant staining throughout the molecular layer, and then decreases in both areas by 1 month. In adult brain there is relatively uniform staining of approximately equal intensity in the white matter, granule cell layer, and molecular layer, whereas the Purkinje cell bodies appear unstained throughout development. In agreement with a previously reported immunochemical analysis, no staining was seen in other tissues, confirming the CNS-specific localization of this glycoprotein.

Aging↗
PubMed1990

The epitope(s) recognized by HNK-1 antibody and IgM paraprotein in neuropathy is present on several N-linked oligosaccharide structures on human P0 and myelin-associated glycoprotein.

The mouse monoclonal antibody HNK-1 and the human monoclonal IgM antibody present in patients with polyneuropathy both recognize carbohydrate epitope(s) on human myelin-associated glycoprotein and P0. In the present study, the oligosaccharide structures that bear the antibody epitope(s) were investigated. The extracellular derivative of myelin-associated glycoprotein (dMAG) was purified by immunoaffinity chromatography. P0 was electroeluted from gel slices. Western blot analysis of whole glycoproteins demonstrated that the epitopes for HNK-1 and the human monoclonal IgM antibody were different. The glycopeptides obtained by proteolysis of purified dMAG and P0 were separated and characterized by affinity chromatography on concanavalin A-Sepharose. Both dMAG and P0 displayed heterogeneity in their oligosaccharide structures, i.e., they both contained mainly tri- and tetraantennary oligosaccharides (approximately 80%), although biantennary (10%) and high-mannose and/or hybrid (10%) oligosaccharides were present. The human monoclonal IgM antibody epitope was present on all types of isolated oligosaccharide structures from either dMAG and P0. The HNK-1 epitope was present on all types of oligosaccharide structures of dMAG, whereas it was present only on tri- and tetraantennary structures of P0.

Antibodies, Monoclonal↗
PubMed1990

Regional central nervous system oligosaccharide storage in caprine beta-mannosidosis.

Goats affected with beta-mannosidosis, an autosomal recessive disease of glycoprotein metabolism, have deficient activity of the lysosomal enzyme beta-mannosidase along with tissue storage of oligosaccharides, including a trisaccharide [Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc] and a disaccharide [Man(beta 1-4)GlcNAc]. CNS myelin deficiency, with regional variation in severity, is a major pathological characteristic of affected goats. This study was designed to investigate regional CNS differences in oligosaccharide accumulation to assess the extent of correlation between oligosaccharide accumulation and severity of myelin deficits. The concentrations of accumulated disaccharide and trisaccharide and the activity of beta-mannosidase were determined in cerebral hemisphere gray and white matter and in spinal cord from three affected and two control neonatal goats. In affected goats, the content of trisaccharide and disaccharide in spinal cord (moderate myelin deficiency) was similar to or greater than that in cerebral hemispheres (severe myelin deficiency). Thus, greater oligosaccharide accumulation was not associated with more severe myelin deficiency. Regional beta-mannosidase activity levels in control goats were consistent with the affected goat oligosaccharide accumulation pattern. The similarity of trisaccharide and disaccharide content in cerebral hemisphere gray and white matter suggested that lysosomal storage vacuoles, more numerous in gray matter, may not be the only location of stored CNS oligosaccharides.

Animals↗
PubMed1996

Urinary excretion of lactose and oligosaccharides in preterm infants fed human milk or infant formula.

At present, not much is known about the absorption and metabolism of human milk (HM) oligosaccharides in term and preterm infants. We investigated the renal excretion of lactose and complex oligosaccharides in preterm infants fed HM (n = 9, mean actual body weight 2290 g) or a cow's milk-based infant formula (n = 9, mean actual body weight 2470 g). We found that the renal excretion of lactose in HM-fed infants was slightly lower than in formula-fed infants (14.0 +/- 7.4 versus 20.4 +/- 8.7 mg kg-1 day-1, mean +/- SD). The excretion of neutral sugars deriving from oligosaccharides was similar in HM-fed and formula-fed infants (3.8 +/- 2.1 versus 2.9 +/- 0.9 mg kg-1 day-1); the difference between means was not statistically significant. The separation and characterization of oligosaccharides by high-pH anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) and subsequent analysis by fast atom bombardment-mass spectrometry (FAB-MS) revealed a more complex pattern in HM-fed infants compared to the formula-fed group. Lactose-derived oligosaccharides characteristic for HM (e.g. lacto-N-tetraose, and lacto-N-fucopentaoses I and II) were excreted in HM-fed but not in formula-fed infants. These results indicate that nutrition has a significant impact on the oligosaccharide composition in urine of preterm infants.

Chromatography, High Pressure Liquid↗
PubMed1994

Alteration of oligosaccharide biosynthesis by genetic manipulation of glycosyltransferases.

The alteration of oligosaccharide structures through genetic manipulation of glycosyltransferase activities is now a reality. It is apparent that this technique has greater consequences on oligosaccharide structure when an exogenous enzyme is introduced into cells, and in particular when this enzyme is responsible for a terminal glycosylation step. By contrast, only one study has examined the effects of overexpressing an endogenous glycosyltransferase, in which there was no detectable effect on glycosylation. However, there are still other key regulatory biosynthetic enzymes, such as GlcNAc transferase V and beta 1,3 GlcNAc transferase, whose overexpression may alter glycosylation. Both of these enzymes are required for the biosynthesis of polylactosaminoglycans (polymers of N-acetyllactosamine disaccharides), and their elevation in tumor cells correlates with increased expression of polylactosaminoglycans. Recently, the gene encoding GlcNAc transferase V has been isolated, but its transfection into cells and characterization of the resulting oligosaccharides awaits further study. Alternate strategies for modifying oligosaccharide structures could involve the introduction of more than one glycosyltransferase into cells to ensure the availability of biosynthetic intermediates. Alternatively, the disruption of specific glycosyltransferase genes by homologous recombination could be used to eliminate competing glycosyltransferases that act on a common substrate. Although oligosaccharide biosynthesis is directly dependent upon the presence or absence of specific glycosyltransferases, other factors also contribute to glycosylation. For example, the transport rate of a glycoprotein through the endoplasmic reticulum and Golgi complex, the levels of processing glycosidases, the availability of substrates, the host cell, and ultimately, the peptide backbone of the particular glycoprotein of interest are important contributors to the final outcome of oligosaccharide structure. Despite these complications, further study into the manipulation of glycosyltransferase genes may ultimately allow the controlled and predictable biosynthesis of glycoprotein sugar chains.

Animals↗
PubMed2001

Synthesis and fermentation properties of novel galacto-oligosaccharides by beta-galactosidases from Bifidobacterium species.

beta-Galactosidase enzymes were extracted from pure cultures of Bifidobacterium angulatum, B. bifidum BB-12, B. adolescentis ANB-7, B. infantis DSM-20088, and B. pseudolongum DSM-20099 and used in glycosyl transfer reactions to synthesize oligosaccharides from lactose. At a lactose concentration of 30% (wt/wt) oligosaccharide yields of 24.7 to 47.6% occurred within 7 h. Examination of the products by thin-layer chromatography and methylation analysis revealed distinct product derived spectra from each enzyme. These were found to be different to that of Oligomate 55, a commercial prebiotic galacto-oligosaccharide. Fermentation testing of the oligosaccharides showed an increase in growth rate, compared to Oligomate 55, with products derived from B. angulatum, B. bifidum, B. infantis, and B. pseudolongum. However B. adolescentis had a lower growth rates on its oligosaccharide compared with Oligomate 55. Mixed culture testing of the B. bifidum BS-4 oligosaccharide showed that the overall prebiotic effect was equivalent to that of Oligomate 55.

Bifidobacterium↗
PubMed2005

In vitro determination of prebiotic properties of oligosaccharides derived from an orange juice manufacturing by-product stream.

Fermentation properties of oligosaccharides derived from orange peel pectin were assessed in mixed fecal bacterial culture. The orange peel oligosaccharide fraction contained glucose in addition to rhamnogalacturonan and xylogalacturonan pectic oligosaccharides. Twenty-four-hour, temperature- and pH-controlled, stirred anaerobic fecal batch cultures were used to determine the effects that oligosaccharides derived from orange products had on the composition of the fecal microbiota. The effects were measured through fluorescent in situ hybridization to determine changes in bacterial populations, fermentation end products were analyzed by high-performance liquid chromatography to assess short-chain fatty acid concentrations, and subsequently, a prebiotic index (PI) was determined. Pectic oligosaccharides (POS) were able to increase the bifidobacterial and Eubacterium rectale numbers, albeit resulting in a lower prebiotic index than that from fructo-oligosaccharide metabolism. Orange albedo maintained the growth of most bacterial populations and gave a PI similar to that of soluble starch. Fermentation of POS resulted in an increase in the Eubacterium rectale numbers and concomitantly increased butyrate production. In conclusion, this study has shown that POS can have a beneficial effect on the fecal microflora; however, a classical prebiotic effect was not found. An increase in the Eubacterium rectale population was found, and butyrate levels increased, which is of potential benefit to the host.

Beverages↗
PubMed1985

Composition and antigenic activity of the oligosaccharide moiety of Haemophilus influenzae type b lipooligosaccharide.

The oligosaccharide moiety of the lipooligosaccharide of Haemophilus influenzae type b strain Eag was isolated from the lipid component by mild acid hydrolysis and purified by gel filtration. Fast atom bombardment-mass spectrometry indicated that the lipid-free oligosaccharide had a basic molecular weight of 1,768; polysaccharides comparable to high-molecular-weight O side chains were not found. Glucose, galactose, galactosamine, heptose, 3-deoxy-D-manno-2-octulosonic acid (KDO), ethanolamine, and phosphate were identified in the lipid-free oligosaccharide by colorimetric assays, gas chromatography-mass spectrometry, or an amino acid analyzer. The presence of KDO was not clearly established by a thiobarbituric acid assay or by growth inhibition by a diazaborine derivative thought to block KDO synthesis. However, the semicarbizide assay and gas chromatography-mass spectrometry confirmed the presence of KDO. Lectin precipitation by Eag lipooligosaccharide in gels indicated that beta-D-galactose was present and that some of this monosaccharide was a terminal, nonreducing residue linked to N-acetyl-D-galactosamine. The lipid-free oligosaccharide was antigenic and completely inhibited lipooligosaccharide antibody (predominantly immunoglobulin G [IgG] and IgM) in an enzyme-linked immunosorbent assay, whereas the solubilized lipid A moiety did not. H. influenzae type b lipid-free oligosaccharide differed from core oligosaccharide of Salmonella lipooligosaccharide by the presence of galactosamine and a smaller percentage of heptose and KDO.

Ethanolamines↗
PubMed1981

Structures of the oligosaccharides of the glycoprotein coded by early region E3 of adenovirus 2.

Early region E3 of adenovirus 2 encodes a glycoprotein, E3-gp25K, that is a good model with which to study structure-function relationships in transmembrane glycoproteins. We have determined the structures of the oligosaccharides linked to E3-gp25K. The oligosaccharides were labeled with [2-(3)H]mannose in adenovirus 2-early infected KB cells for 5.5h (pulse) or for 5.5 h followed by a 3-h chase (pulse-chase). E3-gp25K was extracted and purified by chromatography on DEAE-Sephacel in 7 M urea, followed by gel filtration on a column of Bio-Gel A-1.5m in 6 M guanidine hydrochloride. An analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that it was >95% pure. The oligosaccharides were isolated by pronase digestion followed by gel filtration on a column of Bio-Gel P-6, then by digestion with endo-beta-N-acetylglucosaminidase H, followed by gel filtration on Bio-Gel P-6, and finally by paper chromatography. The pulse sample contained equal amounts of Man(9)GlcNAc and Man(8)GlcNAc and small amounts of Man(7)GlcNAc and Man(6)GlcNAc. The pulse-chase sample had predominantly Man(8)GlcNAc and much less Man(9)GlcNAc, indicating that processing of the Man(9)GlcNAc to Man(8)GlcNAc had occurred during the chase period. Thus, Man(8)GlcNAc is the major oligosaccharide on mature E3-gp25K. The structures of these oligosaccharides were established by digestion with alpha-mannosidase, methylation analysis, and acetolysis. The oligosaccharides found had typical high-mannose structures that have been observed in other membrane and soluble glycoproteins, and the branching patterns and linkages of the mannose residues of Man(9)GlcNAc were identical to those of the lipid-linked Glc(3)Man(9)GlcNAc(2) donor. Thus, adenovirus 2 infection (early stages) apparently does not affect the usual cellular high-mannose glycosylation pathways, and despite being virus coded, E3-gp25K is glycosylated in the same manner as a typical mammalian cell-coded glycoprotein.

Adenoviruses, Human↗
PubMed1998

Increased sialylation of oligosaccharides on IgG paraproteins--a potential new tumour marker in multiple myeloma.

AIMS: To investigate whether changes in carbohydrate structure of IgG are related to malignancy and stage of disease in myeloma and monoclonal gammopathy of uncertain significance (MGUS). METHODS: 61 patients were studied at diagnosis: 14 with MGUS, nine with stage I multiple myeloma, 11 with stage II, 21 with stage III, and five with solitary plasmacytoma. IgG was extracted from serum by protein G affinity chromatography. Oligosaccharides were cleaved from the protein backbone enzymatically by N-glycosidase F. Oligosaccharide analysis was performed by high pressure anion exchange chromatography with pulsed electrochemical detection (HPAE-PED). RESULTS: Up to 15 oligosaccharide peaks were identified in three major fractions: neutral, monosialylated, and disialylated. Patients with myeloma showed an increase in the proportion of sialylated oligosaccharides in comparison with patients with MGUS. The ratio of neutral to sialylated oligosaccharides (N:S) was reduced at all stages of myeloma compared with MGUS: MGUS, 11.35; myeloma stage I, 7.6 (p = 0.047); stage II, 5.20 (p = 0.035); stage III, 3.60 (p = 0.0002); plasmacytoma, 7.5 (p = 0.046). The N:S ratio was independent of paraprotein concentration (r = 0.05). CONCLUSIONS: The ratio of neutral to sialylated oligosaccharides may act as a new marker of malignancy in IgG paraproteinaemia and warrants further investigation.

Aged↗