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At least 757 records · Page 42Linked to original sources
PubMed1999

Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells.

A CHO mutant MI8-5 was found to synthesize Man9-GlcNAc2-P-P-dolichol rather than Glc3Man9GlcNAc2-P-P-dolichol as the oligosaccharide-lipid intermediate in N-glycosylation of proteins. MI8-5 cells were incubated with labeled mevalonate, and the prenol was found to be dolichol. The mannose-labeled oligosaccharide released from oligosaccharide-lipid of MI8-5 cells was analyzed by HPLC and alpha-mannosidase treatment, and the data were consistent with a structure of Man9GlcNAc2. In addition, MI8-5 cells did not incorporate radioactivity into oligosaccharide-lipid during an incubation with tritiated galactose, again consistent with MI8-5 cells synthesizing an unglucosylated oligosaccharide-lipid. MI8-5 cells had parental levels of glucosylphosphoryldolichol synthase activity. However, in two different assays, MI8-5 cells lacked dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase activity. MI8-5 cells were found to synthesize glucosylated oligosaccharide after they were transfected with Saccharomyces cerevisiae ALG 6, the gene for dolichol-P-Glc:Man9GlcNAc2-P-P-dolichol glucosyltransferase. MI8-5 cells were found to incorporate mannose into protein 2-fold slower than parental cells and to approximately a 2-fold lesser extent.

Animals↗
PubMed2003

Beta-galactofuranose-containing O-linked oligosaccharides present in the cell wall peptidogalactomannan of Aspergillus fumigatus contain immunodominant epitopes.

O-linked oligosaccharide groups ranging from di- to hexasaccharide were beta-eliminated by mild alkaline treatment under reducting conditions from the peptidogalactomannan of Aspergillus fumigatus mycelial cell wall. The resulting reduced oligosaccharides, which were the minor components of the peptidogalactomannan fraction, were fractionated to homogeneity by successive gel filtration and high-performance liquid chromatography. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-QTOF-MS, 1H COSY and TOCSY, and 1H-13C HMQC NMR spectroscopy and methylation analysis, to be: alpha-Glcp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 5)-beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol and beta-Galf-(1 --> 5)-[beta-Galf-(1 --> 5]3-beta-Galf-(1 --> 6)-Man-ol. The beta-Galf containing oligosaccharides have not been previously described as fungal O-linked oligosaccharides. The peptidogalactomannan is antigenic and was recognized by human sera of patients with aspergillosis when probed by ELISA, but de-O-glycosylation rendered a 50% decrease in its reactivity. Furthermore, when tested in a hapten inhibition test, the isolated oligosaccharide alditols were able to block, on a dose-response basis, recognition between human sera and the intact peptidogalactomannan. The immunodominant epitopes were present in the tetra- and hexasaccharide, which contain a beta-Galf-(1 --> 5)-beta-Galf terminal group. These results suggest that the O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the A. fumigatus peptidogalactomannan, may account for a significant part of its antigenicity, other than the known activity associated with the galactomannan component.

Aspergillus fumigatus↗
PubMed2005

Mild acid hydrolysis of sulfated fucans: a selective 2-desulfation reaction and an alternative approach for preparing tailored sulfated oligosaccharides.

Sulfated fucans from marine invertebrates have simple, linear structures, composed of repeating units of oligosaccharides. Most of these polysaccharides contain 3-linked fucosyl units, but each species of invertebrate has a specific pattern of sulfation. No specific enzyme able to cleave or to desulfate these polysaccharides has been described yet. Therefore, we employed an alternative approach, based on mild acid hydrolysis, in an attempt to obtain low molecular-weight derivatives from sulfated fucans. Surprisingly, we observed that sulfated fucans from Lytechinus variegatus and Strongylocentrotus pallidus (but not the sulfated fucans from other species) yield by mild acid hydrolysis oligosaccharides with well-defined molecular size as shown by narrow bands in polyacrylamide gel electrophoresis (PAGE). The sulfated oligosaccharides obtained by mild acid hydrolysis were purified by gel-filtration chromatography, and their structures were identified by (1)H-nuclear magnetic resonance (NMR) spectroscopy, revealing an identical chemical composition for all oligosaccharides. When we followed the acid hydrolysis by (1)H-NMR spectroscopy, we found that a selective 2-desulfation occurs in the fucans from S. pallidus and from L. variegatus. The reaction has two stages. Initially, 2-sulfate esters at specific sites are removed. Then the desulfated units are cleaved, yielding oligosaccharides with well-defined molecular size. The apparent requirement for the selective 2-desulfation is the occurrence of an exclusively 2-sulfated fucosyl unit linked to or preceded by a 4-sulfated residue. Thus, a homofucan from Strongylocentrotus franciscanus resists desulfation by mild acid hydrolysis, because it lacks the neighboring 4-sulfated unit. Overall, our results show a new approach for desulfating sulfated fucans at specific sites and obtaining tailored sulfated oligosaccharides.

Animals↗
PubMed2006

Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types.

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an alpha-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgammaRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgammaRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.

Animals↗
PubMed2001

Is there any physiological role for gonadotrophin oligosaccharide heterogeneity in humans? II. A biochemical point of view.

Heterogeneity of gonadotrophin oligosaccharides caused either by pharmacological intervention or which occurs naturally during normal physiological changes is well documented. Recent advances in structure determination of oligosaccharides has to some extent led to a better appreciation of how oligosaccharide heterogeneity may affect protein folding, stability, measurement and modulation of receptor binding. Here it is discussed how carbohydrate structure can impact upon gonadotrophin structure and function. It is well documented that oligosaccharides can serve as a cognate site for protein binding. One functional aspect of gonadotrophin glycosylation heterogeneity is the modulation of receptor binding affinity, yielding partially agonistic glycoforms. Carbohydrate heterogeneity is problematic for a clinical chemist if immunochemical assays are sensitive to heterogeneity. However, even measurements made without such interference may not accurately reflect the biological activity that is a collective result of all isoforms in the circulation, and perhaps of the genotype of each individual. Moreover, oligosaccharide heterogeneity may affect heterodimer stability, therefore, biological activity and immunochemical activity, not to mention clearance. It seems reasonable to conclude that from a biochemical point of view, oligosaccharide heterogeneity is of considerable importance. However, accurate measurement of isoforms in blood, and appropriate in-vitro bioassays that are insensitive to matrix effects are needed to define the physiological significance of each glycoform, and thereby better define target therapeutics and interpret diagnostic results.

Binding Sites↗
PubMed1983

Utilization of intravenously infused glucose-oligosaccharides in fasted and fed pigs.

The ability of fed or fasted 30-day-old pigs to utilize intravenously administered glucose-oligosaccharides (supplied at 20 g/day) was compared in a randomized crossover design. Six pigs were fed a stock diet from days 10 to 30 of life. A central venous catheter was placed on day 30. From days 30 to 39 of life, either a balanced electrolyte solution or a glucose-oligosaccharide solution was infused through the intravenous (i.v.) catheter. On the first i.v. feeding day, all animals were infused with an isotonic, balanced electrolyte solution and were allowed food and water ad libitum per os. On days 2 through 5 of the i.v. period, 3 animals received glucose-oligosaccharides intravenously (20 g/day), with all other needed nutrients, including energy, supplied enterally. On days 6-9 of the i.v. infusion period these animals continued to receive oligosaccharides intravenously, but were denied food. The other three animals were infused with oligosaccharides in the fed or fasting state in the reverse order. Based on urinary carbohydrate excretion, mean (+/- SD) glucose-oligosaccharide utilization was 92% whether the animals were fed or fasted. These results differ from those observed in fasted human subjects.

Animals↗
PubMed1997

Selected indigestible oligosaccharides affect large bowel mass, cecal and fecal short-chain fatty acids, pH and microflora in rats.

Certain indigestible oligosaccharides may benefit gastrointestinal tract health via fermentation and proliferation of desirable bacterial species. The purpose of this study was to elucidate effects of selected oligosaccharides on cecal and fecal short-chain fatty acid (SCFA) concentration, pH, total large bowel wet weight and wall weight, and gut microbiota levels in rats. Fifty male Sprague-Dawley rats were randomly assigned to one of five treatments: 1) control diet; 2) control diet + 5% microcrystalline cellulose (5% CC); 3) control diet + 5% CC + 6% fructooligosaccharides; 4) control diet + 5% CC + 6% oligofructose; or 5) control diet + 5% CC + 6% xylooligosaccharides. The control diet consisted of (dry matter basis) 20% protein, 65% carbohydrate, 10.5% fat, vitamin and mineral mixes. The duration of the study was 14 d. The oligofructose- and fructooligosaccharide-containing diets resulted in higher cecal butyrate concentrations compared with the control, cellulose and xylooligosaccharide diets. Generally, total cecal SCFA pools were higher while pH was lower from ingesting oligosaccharide-containing diets compared with control or cellulose diets. Cecal total weight and wall weight were higher from oligosaccharide consumption, whereas colonic total wet weight was higher for rats consuming xylooligosaccharides compared with other treatments; colon wall weight was unaffected by treatments. Cecal bifidobacteria and total anaerobes were higher whereas total aerobes were lower in rats fed oligosaccharide diets compared with those fed the control diet. Cecal lactobacilli levels were unaffected by treatment. Dietary incorporation of fermentable, indigestible oligosaccharides, by providing SCFA, lowering pH, and increasing bifidobacteria, may be beneficial in improving gastrointestinal health.

Animals↗
PubMed1997

Molecular dynamics simulations of the N-linked oligosaccharide of the lectin from Erythrina corallodendron.

Molecular dynamics simulations have been used to model the flexibility of the seven-sugar oligosaccharide of the lectin from Erythrina corallodendron in three separate simulations: one of the isolated oligosaccharide in vacuo, one of the oligosaccharide in solution and one of the oligosaccharide linked to the protein in solution. Adiabatic conformational energy maps were prepared for each of the disaccharide linkages as a means of interpreting the observed dynamics and conformational averages in terms of intramolecular energy. The inclusion of aqueous solvent molecules appears to be necessary to reproduce the experimental conformational behavior, which also cannot be predicted well from conformational energy maps for the disaccharide linkages alone. The crystallographically determined conformation does not appear to be induced by the crystal dimerization, but is rather stable in solution. The build-up of fluctuations along the successive linkages of the oligosaccharide is significant and would be sufficient to prevent branch residues from being located in most crystal structure determinations. Good general agreement between the calculated solution structure and the average structure determined by NMR was found for most of the oligosaccharide linkages.

Carbohydrate Conformation↗
PubMed2003

Effect of enzyme addition on the digestibilities of cell wall polysaccharides and oligosaccharides from whole, dehulled, and ethanol-extracted white lupins in chickens.

Three experiments were conducted to examine the effects of a commercial enzyme preparation on chicks performance and digestibilities of nonstarch polysaccharides (NSP), raffinose (R), stachyose (S), and total oligosaccharides (O) in diets containing whole and dehulled lupin and ethanol-extracted dehulled lupin meal. Ethanol extract was also used to produce a rich oligosaccharide fraction. In the first experiment, the dehulling treatment and the addition of enzyme to the diet improved (P < 0.05) weight gain (24 and 15%), feed-to-gain ratio (13 and 9%), dry matter retention (32 and 8%), apparent protein digestibility (6 and 3%) and ileal digestibilities of raffinose (19 and 119%), stachyose (85 and 204%), and total oligosaccharides (68 and 178%), respectively. In addition, enzyme treatment improved (P < 0.05) excreta digestibility values for NSP (from -1.7 to 5.5%), R (from 64.6 to 92.6%), S (from 48.8 to 82.4%), and O (from 55.8 to 83.5%). In the second experiment, extraction of the ethanol soluble components from dehulled lupin decreased (P < 0.0001) weight gain (51%), and increased (P < 0.0001) feed consumption (34%), feed-to-gain ratio (32%), relative gizzard weight (14%), and the relative ceca length (20%). The addition of the isolated fraction of oligosacharides to the corn-extracted lupin diet (7.5 and 15%) yielded performance values similar to those obtained with the unextracted dehulled lupin. The addition of enzymes to the diets significantly improved the weight gain (11%) and feed to gain ratio (6%), and decreased relative gizzard weight (12%) and relative ceca length (7%). Ileal digestibilities of R, S, and O were considerably lower than corresponding excreta digestibilities. The excreta digestibility of NSP and excreta and ileal digestibilities of R, S, and O were lower (P < 0.05) in chicks fed the lupin diet containing the high concentration of extract compared with those fed the same diet containing the lower concentration of extract. Enzymes, when added to the diets, increased (P < 0.05) the digestibilities of R, S, and O with the relative effects being much greater for ileal than excreta samples. In the third experiment, the ethanol extract (7.5 and 15%) added to a corn-soybean diet improved weight gain, feed consumption, and feed-to-gain ratio by 19, 13, and 6%, respectively. Ileal digestibilities of R, S, and O were low (< 45%), especially in chicks fed the diets containing the high amount of added oligosaccharide fraction. There was no effect of enzyme addition on the performance parameters. However, supplemental dietary enzymes improved (P < 0.05) ileal and excreta digestibilities of oligosaccharides from 15.6 and 68.6% to 52.3 and 84.8%, respectively. In summary, dehulling greatly improved the nutritional value of lupin, while removal of the ethanol soluble materials, including raffinose and stachyose considerably decreased its value. The oligosaccharide fraction does not seem to have an antinutritive effect and the addition of enzymes increased the digestibilities of NSP, raffinose, and stachyose but did not significantly improve chick performance.

Animal Feed↗
PubMed1990

Structure of asparagine-linked oligosaccharides on human and rabbit testosterone-binding globulin.

We have previously demonstrated, using two-dimensional polyacrylamide gel electrophoresis, that much of the microheterogeneity of human (h) and rabbit (rb) testosterone-binding globulin (TeBG) is due to differential glycosylation of a single protomer. Since glycosylation has been shown to be a physiologically important modification of proteins, we have examined the structure of the oligosaccharide chains attached to hTeBG and rbTeBG to facilitate future studies on the mechanisms of action of the proteins. The structures of the oligosaccharides attached to TeBG were determined by using serial lectin chromatography. About 10% of the TeBG from castrated male rabbits and about 20% of the TeBG from pregnant rabbits and from a human sample were not retained on a column of immobilized concanavalin-A (Con-A). This fraction would consist of TeBG with attached asparagine (Asn)-linked tri- and tetraantennary complex and serine/threonine (O)-linked oligosaccharides as well as non-glycosylated forms. None of the lectins used to subfractionate these species was effective. Forty to 50% of the TeBG applied to Con-A possessed biantennary complex oligosaccharides as indicated by the fact that it could be eluted with 10 mM 1-O-methyl-alpha-D-glucopyranoside and by its retention on wheat germ agglutinin (WGA). About 8% of the biantennary complex oligosaccharides on hTeBG and none of those on rbTeBG were fucosylated on the chitobiose core, as determined by chromatography on Lens culinaris lectin (LcH). Galactosylated oligosaccharides were also present on the TeBG in this fraction as indicated by its interaction with Ricinus communis-I (RCA-I). Thirty to 40% of the TeBG applied to Con-A was retained and could be eluted with 0.5 M methyl-alpha-D-mannopyranoside. This fraction contains TeBG possessing high mannose-type, hybrid-type, and complex galactosylated glycans as determined by chromatography on Con-A, WGA, and RCA-I. Evidence based on the binding of mannoside-eluted TeBG to Con-A, WGA, and RCA-I indicated that at least the TeBG in this fraction contained two glycosylation sites and that the sites were differentially glycosylated.

Animals↗
PubMed1991

Glycosidase inhibitors: inhibitors of N-linked oligosaccharide processing.

The biosynthesis of the various types of N-linked oligosaccharide structures involves two series of reactions: 1) the formation of the lipid-linked saccharide precursor, Glc3Man9(GlcNAc)2-pyrophosphoryl-dolichol, by the stepwise addition of GlcNAc, mannose and glucose to dolichyl-P, and 2) the removal of glucose and mannose by membrane-bound glycosidases and the addition of GlcNAc, galactose, sialic acid, and fucose by Golgi-localized glycosyltransferases to produce different complex oligosaccharide structures. For most glycoproteins, the precise role of the carbohydrate is still not known, but specific N-linked oligosaccharide structures are key players in targeting of lysosomal hydrolases to the lysosomes, in the clearance of asialoglycoproteins from the serum, and in some cases of cell:cell adhesion. Furthermore, many glycoproteins have more than one N-linked oligosaccharide, and these oligosaccharides on the same protein frequently have different structures. Thus, one oligosaccharide may be of the high-mannose type whereas another may be a complex chain. One approach to determining the role of specific structures in glycoprotein function is to use inhibitors that block the modification reactions at different steps, causing the cell to produce glycoproteins with altered carbohydrate structures. The function of these glycoproteins can then be assessed. A number of alkaloid-like compounds have been identified that are specific inhibitors of the glucosidases and mannosidases involved in glycoprotein processing. These compounds cause the formation of glycoproteins with glucose-containing high mannose structures, or various high-mannose or hybrid chains, depending on the site of inhibition. These inhibitors have also been useful for studying the processing pathway and for comparing processing enzymes from different organisms.

Alkaloids↗
PubMed1990

Variations in oligosaccharides and lactose in human milk during the first week of lactation.

Variations in oligosaccharides and lactose in human milk were studied in 15 mothers during the first week of lactation. The neuraminyloligosaccharides and heavy neutral oligosaccharides increased slightly from days 2 to 5 postpartum and appeared to decrease until day 7. The lacto-N-difucohexaoses, lacto-N-fucopentaoses, and lacto-N-tetraose increased until day 5 and then decreased. Lactodifucotetraose and the fucosidolactoses decreased substantially until day 5 (p less than 0.05) and appeared to stablize in the following days. Lactose increased until day 5 (p less than 0.05) and continued to increase thereafter. Lactose was negatively correlated with total oligosaccharides (p less than 0.10). The fluctuations observed in total oligosaccharides from days 2 to 5 postpartum and their subsequent stabilization and regular decrease during lactation confirm the hypothesis of Kulski and Hartmann that mammary secretion occurs in three periods: colostrum for the first 36 h postpartum, transitional milk from days 2 to 5 postpartum, and mature milk after day 5. The oligosaccharide variations we found corresponded to those of other milk constituents observed by other authors. The significance of the oligosaccharide variations is discussed.

Adult↗
PubMed2004

Prebiotic oligosaccharides in dietetic products for infants: a commentary by the ESPGHAN Committee on Nutrition.

This article by the ESPGHAN Committee on Nutrition summarizes available information on the effects of adding prebiotic oligosaccharides to infant and follow-on formulae. Currently there are only limited studies evaluating prebiotic substances in dietetic products for infants. Although administration of prebiotic oligosaccharides has the potential to increase the total number of bifidobacteria in feces and may also soften stools, there is no published evidence of clinical benefits of adding prebiotic oligosaccharides to dietetic products for infants. Data on oligosaccharide mixtures in infant formulae do not demonstrate adverse effects, but further evaluation is recommended. Combinations and dosages in addition to those so far studied need to be fully evaluated with respect to both safety and efficacy before their use in commercial infant food products. Well-designed and carefully conducted randomized controlled trials with relevant inclusion/exclusion criteria, adequate sample sizes and validated clinical outcome measures are needed both in preterm and term infants. Future trials should define optimal quantity and types of oligosaccharides with prebiotic function, optimal dosages and duration of intake, short and long term benefits and safety. At the present time, therefore, the Committee takes the view that no general recommendation on the use of oligosaccharide supplementation in infancy as a prophylactic or therapeutic measure can be made.

Bifidobacterium↗
PubMed1988

Comparative study on O-linked oligosaccharides of glycoprotein D of herpes simplex virus types 1 and 2.

Glycoproteins D1 (gD1) and D2 (gD2) of herpes simplex virus type 1 and type 2, respectively, were purified from infected HEp-2 cells labelled with [3H]glucosamine for 14 h followed by a 3 h chase using HD1 monoclonal antibody linked to Sepharose. O-linked oligosaccharides were found to be present in both glycoproteins. The identification of N-acetyl [3H]galactosaminitol as the major labelled component in the oligosaccharides generated by mild alkaline borohydride treatment demonstrated that these chains have N-acetylgalactosamine at the reducing end. These oligosaccharides consist of mono- and disialylated species with a predominance of the latter species in gD1. Size analysis and radioactive amino sugar composition strongly suggest a structure in which the galactosyl-N-acetylgalactosamine core is substituted with one or two sialic acid residues. In terms of [3H]glucosamine-derived radioactivity, O-linked oligosaccharides are less represented than N-linked oligosaccharides. The O-linked oligosaccharide number determination showed that gD1 and gD2 carry two and three chains, respectively.

Acetylgalactosamine↗
PubMed2002

Effect of addition of new oligosaccharide chains to the globular head of influenza A/H2N2 virus haemagglutinin on the intracellular transport and biological activities of the molecule.

The haemagglutinin (HA) of influenza A/H2N2 virus possesses six antigenic sites (I-A to I-D, II-A and II-B), and sites I-A, I-B and I-C are located in the regions corresponding to sites A, B and D on the H3 HA. We demonstrated previously that most escape mutants selected by mAbs to site I-A, I-B or I-C had acquired a new oligosaccharide at position 160, 187 or 131, respectively, but this has never occurred during circulation of A/H2N2 virus in humans. Here, to examine whether the H2 HA has the potential to gain two new oligosaccharides on its tip, 31 double escape mutants were isolated by using a single escape mutant with an oligosaccharide at position 160, 187 or 131 as a parental virus and a mAb to an antigenic site different from that to which the mAb used for selection of the parental virus was directed as a selecting antibody, but there were no mutants with two new oligosaccharides. Glycosylation-site HA mutants containing one to three oligosaccharides at positions 160, 187 and 131 were also constructed and their intracellular transport and biological activities were analysed. The results showed that all of the mutant HAs were transported to the cell surface but exhibited a decrease in both receptor-binding and cell-fusing activities. Thus, influenza A/H2N2 virus may have failed to increase the number of oligosaccharides on the HA because, if this happens, the biological activities of the HA are reduced, decreasing the ability of the virus to replicate in humans.

Animals↗
PubMed1980

Fluoroglucose-inhibition of protein glycosylation in vivo. Inhibition of mannose and glucose incorporation into lipid-linked oligosaccharides.

The effects of the glycosylation inhibitor 2-deoxy-2-fluoro-D-glucose on the formation of the lipid-linked oligosaccharides and monosaccharides that are involved in protein glycosylation were investigated. In chick embryo cells treated with fluoroglucose the formation of lipid-linked oligosaccharides cannot go to completion and oligosaccharides with decreased amounts of glucose and mannose can be detected. These oligosaccharides are probably biosynthetic intermediates and serve as acceptors of sugar residues while reversing fluoroglucose-inhibition by the addition of mannose and glucose to the culture medium. In contrast to deoxyglucose, fluoroglucose was not incorporated into lipid-linked oligosaccharides. Fluoroglucose inhibits the formation in vivo of dolichyl phosphate glucose and dolichyl phosphate mannose, but not the transfer of those sugar residues from the lipid monophosphate derivative to the lipid-linked oligosaccharides. The pool size of UDP-glucose, but not of GDP-mannose and UDP-N-acetylglucosamine, was decreased. Also, the formation of lipid-linked N-acetylglucosamine was not affected by fluoroglucose. Fluoroglucose was applied to deplete cellular membranes of endogenous lipid-linked mannose and glucose, and can possibly be used to discern different pathways of glycosylation.

Animals↗
PubMed1981

Dolichol pathway in lymphocytes from rat spleen. Influence of the glucosylation on the cleavage of dolichyl diphosphate oligosaccharides into phosphooligosaccharides.

Incubation of rat-spleen lymphocytes with UDP-glucose together with GDP-mannose and UDP-N-acetylglucosamine leads to the formation of glucosylated lipid intermediates characterized as dolichyl phosphate glucose and dolichyl diphosphate oligosaccharides. This latter can be either transferred onto endogenous protein acceptors or cleaved into phosphooligosaccharides. The striking fact is that phosphooligosaccharide populations contain far less glucosylated products than the dolichyl diphosphate oligosaccharide ones from which they are derived. Two hypotheses have been investigated: either a rapid action of glucosidases on the liberated phosphooligosaccharides or a preferential splitting of the non-glucosylated population of dolichyl diphosphate oligosaccharides. Addition of p-nitrophenyl-alpha-D-glucoside inhibits glucosidase activities and allows the production of a major population of dolichyl diphosphate oligosaccharides containing three glucose residues. Using these conditions, it is shown that the amount of phosphooligosaccharides generated from the splitting of dolichyl diphosphate oligosaccharides is greatly decreased and that the major part of these remaining phosphooligosaccharides do not contain glucose. These results show that the presence of glucosyl units prevent dolichyl diphosphate oligosaccharides from further degradation into phosphooligosaccharides.

Animals↗
PubMed1986

Carbohydrate structures of acetylcholine receptor from Torpedo californica and distribution of oligosaccharides among the subunits.

The structure of carbohydrates in acetylcholine receptor (AChR) from Torpedo californica is reported. Oligosaccharides released quantitatively from the whole molecule by N-oligosaccharide glycopeptidase digestion were fractionated by thin-layer chromatography and further purified by high-performance liquid chromatography. We show that more than 70% of the total oligosaccharide chains in Torpedo AChR are of the high-mannose type with the structures (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2. The structure of these oligosaccharides were determined by proton nuclear magnetic resonance spectroscopy. These two types of oligosaccharides were shown to be distributed different proportions in all subunits of Torpedo AChR. We also show that several kinds of complex-type oligosaccharides comprising the rest of the carbohydrate in the protein exist mainly in the gamma and delta subunits. The structure of the carbohydrate moiety that is distributed on the four subunits of AChR was also examined by susceptibility to endo-beta-N-acetylglucosaminidase and sialidase and by binding affinity to lectins, e.g. concanavalin A, leucoagglutinating phytohemagglutinin, and wheat germ agglutinin.

Animals↗