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PubMed1995

Strategy for the investigation of O-linked oligosaccharides from mucins based on the separation into neutral, sialic acid- and sulfate-containing species.

A method for the separation of O-linked oligosaccharides into neutral, sialic acid-containing and sulfated species was applied to oligosaccharides released by alkaline borohydride from mucin glycopeptides from porcine small intestine. The released mixture of reduced oligosaccharides was applied to an anion exchange column, and the neutral oligosaccharides were collected as the unretarded fraction. A mixture of dimethyl sulfoxide and iodomethane was passed through the column to convert the sialic acid-containing oligosaccharides into methyl esters that were eluted and converted to methyl amides by methyl amine. Finally the sulfated oligosaccharide fraction was eluted with salt. The neutral and the derivatized sialic acid-containing oligosaccharides were analysed by gas chromatography-mass spectrometry after permethylation and the sulfated oligosaccharide fraction was analysed by high performance anion exchange chromatography.

Animals↗
PubMed1990

Structural requirements for the binding of oligosaccharides to immobilized lectin of Erythrina variegata (Linn) var. orientalis.

The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins with Erythrina variegata agglutinin (EVA) were investigated by means of affinity chromatography on an EVA-Sepharose column. Some of the branched poly-N-acetyllactosamine-type oligosaccharides obtained from human erythrocyte band 3 glycoprotein were found to show high affinity to EVA-Sepharose, whereas complex-type oligosaccharides were shown to have low affinity. Hybrid type, oligomannose-type and unbranched poly-N-acetyllactosamine-type oligosaccharides bound very little or not at all to EVA-Sepharose. To further study the carbohydrate-binding specificity of this lectin, we investigated the interaction of immobilized EVA and oligosaccharide fragments obtained through partial hydrolysis from branched poly-N-acetyllactosamine-type oligosaccharides. Branched poly-N-acetyllactosamine-type oligosaccharides were subjected to limited hydrolysis with 0.1% trifluoroacetic acid at 100 degrees C for 40 min and then separated on an amino-bonded silica column. One of pentasaccharides thus prepared strongly bound to the EVA-Sepharose column. Structural analysis of this pentasaccharide showed that the Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal sugar sequence, which is an I-antigen determinant, was essential for the high affinity binding of the oligosaccharides to the EVA-Sepharose column.

Amino Acid Sequence↗
PubMed1982

Inhibition by nojirimycin and 1-deoxynojirimycin of microsomal glucosidases from calf liver acting on the glycoprotein oligosaccharides Glc1-3Man9GlcNAc2.

Particulate membrane fractions from calf liver catalyze the release of glucose from GlcNAc2-Man9-Glc1-3-oligosaccharides. Maximal oligosaccharide-glucosidase activity was obtained at pH 6.2 and a detergent concentration of 0.5% Triton X-100. This activity could be distinguished from non-specific alpha-glucosidase activity on the basis of different pH-dependence and lack of activation by detergent. The relative rates for the hydrolysis of the Glc3-, Glc2-, and Glc1-oligosaccharide, estimated from the initial velocity, was 1:12:3. There is no significant difference in the enzyme activity towards free, peptide-bound, or lipid-linked oligosaccharide. Nojirimycin and 1-deoxynojirimycin were strong inhibitors of microsomal oligosaccharide-glucosidases. Hydrolysis of Glc3-oligosaccharide was inhibited by 50% at concentrations of 0.16 mM and 2 microM, respectively. Hydrolysis of the Glc2- and Glc1-oligosaccharide was inhibited to a somewhat lower extent, suggesting the presence of at least two glucosidases, one acting on Glc3- and one acting on Glc1- and Glc2-oligosaccharide.

1-Deoxynojirimycin↗
PubMed2003

Enzymic production of a feruloylated oligosaccharide with antioxidant activity from wheat flour arabinoxylan.

BACKGROUND: Main cereals such as rice, wheat, barley, and corn belong to the family Gramineae and have similar cell-wall composition. Since cereal cell walls are a good source of dietary fibre, meeting one-half of the daily requirement of 30 g of dietary fibre can be achieved by the regular consumption of cereals. Many studies have dealt with the isolation of feruloylated oligosaccharides from Gramineae by treatment with polysaccharide hydrolysing enzymes. AIM OF THIS STUDY: Therefore, the purpose of this study was to investigate the production of feruloylated oligosaccharides from insoluble wheat flour arabinoxylan (WFAX) by treatment with a Thermoascus aurantiacus family 10 endoxylanase (XYLI) and the evaluation of their antioxidant activity. METHODS: The main feruloylated oligosaccharide was purified by anion-exchange and size-exclusion chromatography (SEC). Alkaline saponification and acid hydrolysis were used for product identification. Evaluation of antioxidant activity was performed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction assay and the inhibition of copper-mediated oxidation of low density lipoprotein (LDL). RESULTS: The optimal conditions for WFAX hydrolysis using the XYLI have been determined to be 100 U g(-1) of WFAX for 30 min at 50 degrees C. Saponification of the oligosaccharide released FA and oligosaccharide. The released oligosaccharide consisted of arabinose and xylose in a molar ratio of 1:3 and these results support the identity of the feruloylated oligosaccharide as feruloyl arabinoxylotrisaccharide (FAX(3)). FAX(3) showed profound antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) reduction assay exhibiting an antiradical efficiency of 0.035 (x 10(-3)) and inhibited the copper-mediated oxidation of human low density lipoprotein (LDL) in a dose-dependent manner with almost complete inhibition at 32 microM. CONCLUSION: A feruloylated oligosaccharide (FAX3) was isolated from WFAX after enzymatic treatment with XYLI. We verified antioxidant activity of FAX(3) which may be important in preventing or reducing the progression of atherosclerosis by inhibiting the peroxidation of lipoproteins.

Antioxidants↗
PubMed1988

High-performance anion-exchange chromatography of oligosaccharides using pellicular resins and pulsed amperometric detection.

High-performance liquid chromatography using pellicular quaternary amine-bonded resins was used to separate a variety of neutral, sialylated, and phosphorylated oligosaccharides. At pH 4.6, sialylated compounds were separated according to number of negative charges, sialic acid linkage [alpha(2,3) compared to alpha(2,6)], and position of sialic acid linkage along a linear saccharide chain. At pH 13, the neutral sugar portion of the sialylated chain had a significant effect on the separation, due to oxyanion formation. Specifically, sialylated tetrasaccharides containing the Gal beta(1,3)GlcNAc sequence were retained much more than their Gal beta(1,4)GlcNAc- or Gal-beta(1,4)GalNAc-sialylated counterparts. Linear phosphorylated oligosaccharides could be completely separated according to number of charges and net carbohydrate content. Partial separation of linear-chain positional isomers, differing in either location of Man-6-PO4 in the chain or linkage position of Man or Man-6-PO4, was accomplished. Branched-chain phosphorylated compounds could be completely separated according to which antennae contained the Man-6-PO4. The electrochemical current generated by oxidation of sialylated, phosphorylated, and neutral oligosaccharides was compared to that of a glucose. The relative molar response factors for neutral, sialylated, and phosphorylated oligosaccharides ranged from 0.2 to 3.2. Neutral oligosaccharides gave the following molar responses for each group of structurally related compounds: (1) mono- and disaccharide, 1-1.3; (2) linear tri- and tetrasaccharides, 1.5-2.0; and (3) branched pentasaccharide-nonasaccharides, 2.4-3.1. Response factors for the sialyated compounds were not as consistent and were affected by linkage position of sialic acid. For oligosaccharides of the same size, increasing phosphorylation resulted in a twofold decrease in response factor for each added phosphate group. Therefore, conversion of sialylated and phosphorylated oligosaccharides to their neutral counterparts, using alkaline phosphatase or neuraminidase, respectively, was required for quantitative analysis of oligosaccharide mixtures using electrochemical response. Using this approach, complete separation of the parent neutral structures was obtained, the relative proportions of the neutral species were quantified, and the amount of sialic acid released was easily determined in a neuraminidase digest.

Anion Exchange Resins↗
PubMed1990

Amino and hydrazino alkyl benzoates as derivatizing agents for the separation and mass spectrometric analysis of oligosaccharides from bacterial lipooligosaccharides.

In an attempt to develop more sensitive and versatile methods for the structure analysis of oligosaccharides derived from lipooligosaccharides (LOS) of gram-negative bacteria, amino and hydrazino alkyl benzoate derivatives were prepared. These oligosaccharide derivatives were separated by HPLC and then analyzed by liquid secondary ion mass spectrometry (LSIMS). Both the amino and hydrazino alkyl benzoates react with the free reducing termini of acid-treated LOS, increasing the hydrophobicity of the released oligosaccharides and allowing them to be separated by reverse-phase HPLC. In addition, these oligosaccharide derivatives now contain a sensitive uv chromophore for subsequent peak detection and improve the quality of the LSIMS spectra compared to underivatized oligosaccharides. However, the amino alkyl benzoates reacted poorly compared to the analogous hydrazino alkyl benzoates with 3-deoxy-manno-2-keto octulosonic acid (KDO), and oligosaccharides with KDO at the reducing terminus, especially when the oligosaccharide also contained phosphoethanolamine. Derivatization with the hydrazino compounds can be carried out quickly and under mild conditions using a minimal amount of reagent, and is therefore suitable for microscale analyses. The chromatographic and mass spectrometric characteristics of these derivatives make them excellent alternatives to permethylation and peracetylation techniques for the structural analysis of complex bacterial oligosaccharides derived from glycolipids.

Aminobenzoates↗
PubMed1984

Biosynthesis of glycoproteins in human placenta: differential labeling of mannose and heterogeneity of oligosaccharide lipid intermediates.

The biosynthesis of lipid-linked oligosaccharides has been studied in first trimester human placentas. Tissue was pulsed with [2-3H]mannose, [1-3H]glucosamine, and [1-3H]galactose (for [3H]glucose incorporation). The lipid-linked oligosaccharides (OSL) were purified on DEAE-cellulose. After cleaving the lipid from OSL the oligosaccharides were purified by paper chromatography and borate high-voltage electrophoresis. Four major oligosaccharides with the composition Glc(0-3)Man9GlcNAc2 thus obtained were characterized enzymatically by digestion with endo-beta-N-acetylglucosaminidase H and alpha-mannosidase, and chemically by methylation, acetolysis, and Smith degradation. While identical Glc(1-3)Man9GlcNAc2 oligosaccharides have been isolated from other in vivo systems, the presence of Man9Glc2 is novel for human placenta. Furthermore, Man9GlcNAc2 is present in appreciable amounts in placenta. Second, one mannose in Man9GlcNAc2 had a significantly higher specific radioactivity than the other mannosyl residues of the Glc(0-3)Man9GlcNAc2 oligosaccharides. Third, the differential labeling in Man9GlcNAc2 and our pulse-chase studies indicate that Man9GlcNAc2 is not a major precursor of Glc3Man9GlcNAc2. The data also suggest that the ninth mannose, which has the highest radioactivity, may be incorporated in a different subcellular compartment and may serve as a regulatory step in the biosynthesis of lipid-linked oligosaccharides. A model is proposed which outlines possible multiple pathways of lipid-linked oligosaccharide biosynthesis in human placenta.

Chemical Phenomena↗
PubMed1986

Characterization of cellular oligosaccharides from normal and cystic fibrotic fibroblasts using sequential endoglycosidase digestions.

A method was developed for obtaining detailed oligosaccharide profiles from [2-3H]mannose- or [6-3H]fucose-labeled cellular glycoproteins. The oligosaccharides were segregated first according to class, using endo-beta-N-acetylglucosaminidase H (Endo H) to release the high mannose species, and then with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F), which provided a complete array of complex oligosaccharide chains. The high mannose and complex oligosaccharides were fractionated subsequently according to net negative charge on QAE-Sephadex. High resolution gel filtration on TSK HW-40(S) resolved the neutral high mannose population into species of the type Man9-5 N-acetylglucosamine. Desialylation of the complex chains with neuraminidase allowed resolution of these oligosaccharides into their corresponding asialo bi-, tri-, and tetraantennary species. Fibroblasts from normal and cystic fibrosis cells were analyzed for differences in their glycosylation patterns using these techniques. Over 95% of the [2-3H]mannose-labeled glycoproteins were susceptible to the combined glycosidase digestions, but no difference in either the high mannose or complex oligosaccharides were observed. Nonetheless, the methodology developed in this study provides an important new approach for investigating oligosaccharides of different cell types and variants of the same type. Metabolic changes induced in cellular glycoproteins, as illustrated by use of the processing inhibitor swainsonine, demonstrated the versatility of this procedure for investigating questions relating to glycoprotein structure and enzyme specificity. Thus, by employing a variation of this method, it was possible to confirm the location of fucose in the core of PNGase F-released hybrid oligosaccharides by the subsequent release with Endo H of the disaccharide, fucosyl-N-acetylglucosamine.

Acetylglucosaminidase↗
PubMed1988

Detection and isolation of oligosaccharides with Lea and Leb blood group activities by affinity chromatography using monoclonal antibodies.

Affinity columns prepared by immobilizing monoclonal antibodies that specifically recognize the Lea or the Leb blood group antigens can be used for analytical or preparative isolation of oligosaccharides with the corresponding reactivities. The number of immobilized functional antibody combining sites on a column and the dissociation constants for standard oligosaccharides are determined by frontal analysis. By employing a simple approximation [K.-I. Kasai et al. (1986) J. Chromatogr. 376, 33-47] these parameters can be used to rationally design columns with properties appropriate for zonal affinity chromatography. The affinity for binding of the Lea-active oligosaccharide lacto-N-fucopentaose II (LNF II) by the anti-Lea antibody CO-514 doubles for each 8 degrees C downward shift in temperature between 37 and 4 degrees C. By zonal chromatography, Lea- or Leb-active oligosaccharides are recovered from a complex mixture of milk oligosaccharides containing more than a 20-fold molar excess of structurally similar but antigenically distinct oligosaccharides. The capacity for preparative isolation of an oligosaccharide increases in a linear fashion with the amount of antibody loaded on the solid support. The monoclonal antibodies used in these studies are products of hybridomas derived from mice immunized with human colorectal carcinoma cell lines [M. Blaszczyk et al. (1984) Arch. Biochem. Biophys. 233, 161-168]. The experiments establish that affinity chromatography applied to mixtures of oligosaccharides released by enzymatic or chemical cleavage of glycoconjugates may simplify the task of isolating and characterizing biologically interesting target antigens of monoclonal antibodies.

Animals↗
PubMed1992

Sulfated sialyl-oligosaccharides derived from tracheobronchial mucous glycoproteins of a patient suffering from cystic fibrosis.

Thirteen novel oligosaccharides, each possessing both a sulfate ester and a sialic acid residue, were isolated from tracheobronchial mucous glycoproteins from a patient with cystic fibrosis via cleavage by alkaline borohydride treatment, and by employing immobilized Limulus polyphemus lectin affinity chromatography, SynChroprep AX300 anion-exchange chromatography, Bio-Gel P-2 size-exclusion chromatography, and Hypersil 120A APS-2 high-performance liquid chromatography (HPLC). Proposed structures for the resulting purified sulfated sialyl-oligosaccharides were based on carbohydrate/permethylation analyses, periodate oxidation, complete sequential exoglycosidase digestion, analysis of desulfated products and, analysis by positive-ion fast-atom-bombardment mass spectrometry (FABMS). Sulfate esters on these sialyl-oligosaccharides resided on C-6 of a terminal or an internal D-galactose or 2-acetamido-2-deoxy-D-glucose residue or C-4 of a terminal D-galactose residue. The sialic acid residues were found to be either bound (2-->6)-alpha to 2-acetamido-2-deoxy-D-galactitol or (2-->3)-alpha or (2-->6)-alpha to a D-galactose residue occupying a nonreducing terminus. For this group of oligosaccharides, ranging in size from tri- to hepta-saccharides, it was also observed that a sialic acid residue and a sulfate ester did not residue on the same oligosaccharide branch when more than one branch existed. On linear unbranched sulfated sialyl-oligosaccharides, the sialic acid residue was bound to a D-galactose residue occupying a nonreducing terminus with the sulfate ester residing on an internal D-galactose or a 2-acetamido-2-deoxy-D-glucose residue. These results demonstrate that it is possible for sialic acid and a sulfate ester to exist on the same oligosaccharide and that this oligosaccharide can be as small as a trisaccharide.

Adolescent↗
PubMed1983

Diagnosis of GM1 gangliosidosis based on detection of urinary oligosaccharides with high performance liquid chromatography.

An improved, rapid, and sensitive method for the biochemical diagnosis of GM1 gangliosidosis based on the detection and quantification of urinary galactosyl-oligosaccharides with high performance liquid chromatography was developed. The oligosaccharides, in 50-100 microliters of urine, were converted to radioactively labeled oligosaccharide-alditols with NaB3H4 and fractionated on commercial silica-amine bonded, high performance liquid chromatography columns. Delineation between infantile, juvenile, and adult onset subtypes of GM1 gangliosidosis was possible by analysis of the levels of the excreted oligosaccharides and their characteristic elution profile. Infantile and juvenile patients contain identical numbers of oligosaccharide fractions (13 resolved components) but can be distinguished by 3-10-fold lower levels of oligosaccharides in juvenile patients and, in some cases by a disproportionately lower concentration of high molecular weight compounds. Adult onset patients were distinguished by substantially lower concentrations of urinary oligosaccharides, 130-180-fold below those in infantile patients, and the apparent absence of high molecular weight oligosaccharides.

Adult↗
PubMed1995

Separation of 8-aminonaphthalene-1,3,6-trisulfonic acid-labelled neutral and sialylated N-linked complex oligosaccharides by capillary electrophoresis.

Complex oligosaccharides, both neutral and sialylated, were derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and separated by capillary electrophoresis. The derivatization reaction was carried out in a total reaction volume of 2 microliters. The separated peaks were detected by laser-induced fluorescence detection using the 325-nm line of a He-Cd laser. Concentration and mass detection limits of 5 x 10(-8) M and 500 amol, respectively, could be achieved. The limiting step for higher sensitivity is not the detector performance, however, but the chemistry with a derivatization limit of 2.5 x 10(-6) M. Two labelling protocols were established, one with overnight reaction at 40 degrees C and the other with a 2.5-h derivatization time at 80 degrees C. Neutral oligosaccharides could be labelled with either protocol. However, sialylated oligosaccharides hydrolysed when labeled at 80 degrees C. Low nanomole to picomole amounts of oligomannose-type and complex-type oligosaccharide mixtures were derivatized and separated in less than 8 min with excellent resolution using a phosphate background electrolyte at pH 2.5. The linear relationship between the electrophoretic mobility and the charge-to-mass ratios of the ANTS conjugates was used for peak assignment. Further, the influence of the three-dimensional structure of the complex oligosaccharides on their migration behaviour is discussed. The suitability of the ANTS derivatization and the subsequent separation for the analysis of complex oligosaccharide patterns is demonstrated with oligosaccharide libraries derived from ovalbumin and bovine fetuin. For peak assignment the patterns are compared with those of the oligomannose and the complex-type oligosaccharide mixtures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1979

Abnormal lipid-linked oligosaccharides in class E Thy-1-negative mutant lymphomas.

The glycosylation defect of Thy-1-mutant lymphomas of the class E complementation group has been identified as a block in the synthesis of the lipid-linked oligosaccharide precursor of the asparagine-linked oligosaccharides of glycoproteins. Two major lipid-linked oligosaccharides were isolated from the mutant cells. Both oligosaccharides were smaller than the lipid-linkid oligosaccharides of wild-type lymphomas and, in contrast to the lipid-linked oligosaccharides isolated from wild-type cells, both were resistant to digestion with endoglycosidase H. The oligosaccharides of newly synthesized polypeptides in class E Thy-1-cells were also resistant to endoglycosidase H digestion, providing strong evidence that they are derived from the abnormal lipid-linked oligosaccharides.

Animals↗
PubMed1980

Biosynthetic processing of the oligosaccharide chains of cellular fibronectin.

We have examined the maturation or processing of the oligosaccharides of cellular fibronectin in cultured chick embryo fibroblasts. Fibronectin was pulse-labeled with [2-3H]mannose of [35S]methionine, and the turnover rates of carbohydrate and polypeptide portions of immunoprecipitated fibronectin were compared. The oligosaccharides on fibronectin were analyzed by gel electrophoresis for alterations in sensitivity to the enzyme endo-beta-N-acetylgluosaminidase H, which specifically cleaves the 'high-mannose' class of asparagine-linked oligosaccharide. Incorporated mannose was removed only at early time points, suggesting that the structure of fibronectin oligosaccharides was altered due to processing. This possibility was confirmed by the analysis of glycopeptides generated by exhaustive pronase digestion. Two major glycopeptide structures were detected; their properties correspond to a 'high-mannose' oligosaccharide precursor and a 'complex' carbohydrate product. The precursor-product relationship of these two forms of oligosaccharide chains was demonstrated by pulse-chase labeling experiments. The precursor glycopeptide had an apparent size (Mr 2100) comparable to (Man)9GlcNAc (Mr 2080), and was sensitive to endo-beta-N-acetylglucosaminidase H; nearly all of the labeled mannose incorporated in a 10 min pulse was released from fibronectin glycopeptides by this enzyme. During a 90 min chase period, the glycopeptides became larger and increasingly resistant to endo-beta-N-acetylglucosaminidase H cleavage. The final 'complex' or processed oligosaccharide structure contained approximately two-thirds less [3H]mannose, was insensitive to endo-beta-N-acetylglucosaminidase H and had an apparent Mr of 2300 as estimated by gel filtration. We conclude that the carbohydrate portion of fibronectin is synthesized as a 'high-mannose' intermediate and is subsequently processed to give the characteristic 'complex' oligosaccharide chains of fibronectin.

Acetylglucosaminidase↗
PubMed1987

Oligosaccharides of the Hazelhurst vesicular stomatitis virus glycoprotein are more extensively processed in Rous sarcoma virus-transformed baby hamster kidney cells.

Because of the extensive oligosaccharide heterogeneity of the membrane glycoprotein (G) from the Hazelhurst strain of vesicular stomatitis virus, this virus has been used as a specific intracellular probe of altered protein glycosylation in Rous sarcoma virus-transformed versus normal baby hamster kidney cells. Over 70% of G protein from virus released from the transformed cells had acidic-type oligosaccharides at both glycosylation sites, compared to less than 50% from the corresponding normal host cells. The remaining G protein contained an acidic-type oligosaccharide at one site and an endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide at the other. The major endoglycosidase-sensitive species were sialylated hybrid-type (NeuNAc-Gal-GlcNAc-Man5GlcNAc2-Asn) from the transformed and neutral-type (Man5-6GlcNAc2-Asn) from the normal host cells. The degree of branching of the acidic-type oligosaccharides was not increased in the transformed cells (approx. 80% biantennary for viral G protein from both cell types). At a reduced growth temperature (24 versus 37 degrees C), the G protein oligosaccharides were more extensively processed in both cell types (approximately 85-95% of G protein contained acidic-type structures at both sites), even though the level of viral protein synthesis and virus release was decreased. Essentially all of the minor, endoglycosidase-sensitive oligosaccharides on mature viral G protein were sialic acid-containing hybrid-type structures. At 24 degrees C the branching of the acidic-type oligosaccharides was increased in the virus released from the transformed cells versus normal cells.

Animals↗
PubMed1996

Preferential transfer to truncated oligosaccharides to the first sequon of yeast exoglucanase in Saccharomyces cerevisiae alg3 cells.

In addition to the exoglucanases (Exg) secreted into the culture medium by wild type cells, ExgIa and ExgIb, which have oligosaccharides attached to both potential N-glycosylation sites, Saccharomyces cerevisiae alg3 mutant secreted substantial amounts (35--44%) of underglycosylated and unglycosylated forms. Quantification of these forms indicated that no more than 78% of the available N-sites were occupied. About 50% of the transferred oligosaccharides were endo H sensitive, indicating that the lipid-linked precursor had completed its synthesis to Glc3-Man9-GlcNAc2. The other 50% remained endo H-resistant and, accordingly, it should be derived from the precursor oligosaccharide Man5-GlcNAc2 synthesized by this mutant. A closer analysis of forms that have received two oligosaccharides (ExgIb) showed that the first sequon was enriched in truncated residues, whereas the second one was enriched in regular counterparts. Similarly, analysis of the individual underglycosylated glycoforms indicated that 38% of the oligosaccharides attached to the second site were regular. This percentage dropped to 20% for glycoforms carrying the oligosaccharide in the first sequon. The preferential transfer of truncated oligosaccharides to the first glycosylation site seems to be a consequence of (1) the low percentage of truncated lipid linked oligosaccharides that receives the glucotriose unit, and (2) the effect of the glucotriose unit on the selection of N-sites to be glycosylated.

Binding Sites↗
PubMed2004

Profiling the morphological distribution of O-linked oligosaccharides.

The morphological distribution of oligosaccharides is determined in the egg jelly surrounding Xenopus laevis eggs. This biological system is used to illustrate a method for readily identifying and quantifying oligosaccharides in specific tissues. The extracellular matrix surrounding X. laevis eggs consists of a vitelline envelope and a jelly coat. The jelly coat contains three morphologically distinct layers designated J1, J2, and J3 from the innermost to the outermost and is composed of 9-11 distinct glycoproteins. Each jelly layer is known to have specific functions in the fertilization of the egg. We developed a rapid method to separate and identify the oligosaccharides from X. laevis egg jelly layers. Identification was based on the retention times in high-performance liquid chromatography (porous graphitized carbon column), exact masses, and tandem mass spectrometry. Over 40 neutral and 30 sulfated oligosaccharides were observed in the three jelly layers. Neutral oligosaccharide structures from different jelly layers were both unique and overlapping, while sulfated oligosaccharides were detected only in layers J1 and J2. Neutral oligosaccharides unique to jelly layer J3 and the combined layers J1+J2 had similar core structures and similar residues. However, differences between these two sets of unique oligosaccharides were also observed and were primarily due to the branching carbohydrate moieties rather than the core structures.

Animals↗
PubMed2006

Monosaccharide composition, chain length and linkage type influence the interactions of oligosaccharides with dry phosphatidylcholine membranes.

Sugars play an important role in the desiccation tolerance of most anhydrobiotic organisms and disaccharides have been extensively investigated for their ability to stabilize model membranes in the dry state. Much less is known about the ability of oligosaccharides to protect dry membranes. However, it has been shown that different structural families of oligosaccharides have different efficacies to interact with and protect membranes during drying. Here, we have compared three families of linear oligosaccharides (fructans, malto-oligosaccharides, manno-oligosaccharides) for their chain-length dependent lyoprotective effect on egg phosphatidylcholine liposomes. We found increased protection with chain length for the fructans, a moderate decrease in protection with chain length for malto-oligosaccharides, and a strong decrease for manno-oligosaccharides. Using Fourier-transform infrared spectroscopy and differential scanning calorimetry, we show that the degree of lyoprotection of the different sugars is closely related to their influence on the gel to liquid-crystalline phase behavior of the dry membranes and to the extent of H-bonding to different groups (C=O, P=O, choline) in the lipids. Possible structural characteristics of the different oligosaccharides that may determine the extent to which they are able to interact with and protect membranes are discussed.

Calorimetry, Differential Scanning↗