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PubMed1989

Characterization and charge distribution of the asparagine-linked oligosaccharides on secreted mouse thyrotropin and free alpha-subunits.

Mouse hemipituitaries in vitro secrete TSH, composed of an alpha-beta heterodimer, as well as excess (free) alpha-subunits. By dual metabolic labeling with [35S]sulfate and [3H]mannose, we have characterized oligosaccharides from secreted TSH alpha, TSH beta, and free alpha-subunits released from the apoprotein by enzymatic deglycosylation. Oligosaccharides from each subunit displayed a distinct anion exchange HPLC profile due to a specific pattern of sialylation and sulfation. Six species were obtained from TSH alpha (with two glycosylation sites), including neutral oligosaccharides as well as those with one or two negative charges. For TSH beta (with one glycosylation site) at least eight oligosaccharide species were noted, representing nearly every permutation of sialylation and sulfation; approximately 30% contained three or more negative charges. Analysis of [3H]mannose-labeled oligosaccharides on Concanavalin-A-agarose showed 85% binding for those from TSH alpha, 70% for free alpha, and 50% for those from TSH beta. These data demonstrate that oligosaccharides from secreted TSH beta were more sialylated and sulfated, consistent with a more complex branching pattern, than those from TSH alpha. Oligosaccharides from free alpha-subunit were more sialylated than those from TSH alpha, and the net negative charge was intermediate between those of TSH alpha and TSH beta. Although great microheterogeneity is present even at the single glycosylation site on the beta-subunit of secreted TSH, a pattern of sialylation and sulfation could be discerned. If one assigns probabilities of sialylation [p(N)] and sulfation [p(S)] based on the observed distribution within monoacidic (charge -1) species, the proportion of diacidic (charge -2) oligosaccharides could be predicted for each subunit by [p(N)]2, 2[p(N)] [p(S)], [p(S)]2, corresponding to species containing two sialic acid, one sialic acid and one sulfate, and two sulfate residues, respectively. This suggests that the probability of sialylation or sulfation at a second site on these oligosaccharides is similar to that at the first and that anionic oligosaccharides in secreted TSH and free alpha are distributed binomially with regard to sialic acid and sulfate residues.

Animals↗
PubMed1996

Oligosaccharide mapping reveals hormone-specific glycosylation patterns on equine gonadotropin alpha-subunit Asn56.

Equine gonadotropin alpha-subunit glycosylation was examined by releasing oligosaccharides using a sequential enzymatic deglycosylation protocol and comparing the released oligosaccharide populations using a high resolution oligosaccharide mapping technique. Digestion of native alpha-subunit preparations with peptide-N-glycosidase altered their mobilities during SDS-PAGE under reducing conditions to positions intermediate between the corresponding native alpha-subunit and completely deglycosylated alpha-subunit bands. Complete alpha-subunit deglycosylation required reduction of disulfide bonds. Results of solid-phase Edman degradation demonstrated that partial deglycosylation occurred exclusively at Asn56. Oligosaccharide mapping of total oligosaccharides obtained by enzymatic deglycosylation of reduced, carboxymethylated alpha-subunit preparations revealed hormone-specific patterns of glycosylation in eLH alpha and eCG alpha. Oligosaccharide mapping of individual glycosylation sites revealed that hormone-specific glycosylation was primarily restricted to Asn56 of both subunit preparations and revealed a hormone-specific pattern of Asn56 glycosylation in eFSH alpha that was obscured in the total oligosaccharide map. eLH alpha Asn56 oligosaccharides appeared to be primarily seven variants of a monoantennary structure. eCG alpha Asn56 oligosaccharides consisted of one of two forms, either a sialylated biantennary oligosaccharide that appeared identical to a commercial carbohydrate standard or a lactosamine variant of that structure.

Amidohydrolases↗
PubMed1991

Developmental regulation of asparagine-linked oligosaccharide synthesis in Dictyostelium discoideum.

In the preceding report we demonstrated that the expression of two developmentally regulated alpha-mannosidase activities is induced in Dictyostelium discoideum during its differentiation from single-cell amoebae to multicellular organism (Sharkey, D. J., and Kornfeld, R. (1991) J. Biol. Chem. 266, 18477-18484). These activities, designated membrane alpha-mannosidase I (MI) and membrane alpha-mannosidase II (MII), were shown to have several properties in common with rat liver Golgi alpha-mannosidases I and II, respectively, suggesting that MI and MII may play a role in the processing of asparagine-linked oligosaccharides in developing D. discoideum. In this study we analyzed the structures of the asparagine-linked oligosaccharides synthesized by D. discoideum at various stages of development to determine the timing and extent of asparagine-linked oligosaccharide processing. Cells were labeled with [2-3H] mannose, and then total cellular glycoproteins were digested with Pronase to generate glycopeptides that were fractionated on concanavalin A-Sepharose. Glycopeptides from each fraction were digested with endoglycosidase H, both before and after desulfation by solvolysis, and the released, neutral oligosaccharides were sized by high pressure liquid chromatography. At early stages of development, D. discoideum contain predominantly large high mannose-type oligosaccharides (Man9GlcNAc and Man8GlcNAc). Some of these are modified by GlcNAc residues attached beta 1-4 to the mannose-linked alpha 1-6 to the beta-linked core mannose (the "intersecting" position), as well as by fucose, sulfate, and phosphate. In contrast, the oligosaccharides found at late stages of development (18-24 h) have an array of sizes from Man9GlcNAc to Man3GlcNAc. These are still modified by GlcNAc, fucose, sulfate, and phosphate, but the percent of larger high mannose oligosaccharides that are modified with GlcNAc in the intersecting position decreases after 6 h of development, in parallel with the decrease in the intersecting GlcNAc transferase activity. Similarly, the changes in the size of asparagine-linked oligosaccharides synthesized during development correlate well with the appearance of MI and MII activities and suggest that these developmentally regulated alpha-mannosidase activities function in the processing of these oligosaccharides. This is supported further by the observation that oligosaccharide processing was inhibited in late stage cells labeled in the presence of either deoxymannojirimycin, an inhibitor of MI, or swainsonine, an inhibitor of MII.

Animals↗
PubMed1987

Structural determination of the oligosaccharide side chains from a glycoprotein isolated from the mucus of the coral Acropora formosa.

An extracellular mucous glycoprotein has been isolated from the hard coral Acropora formosa. The glycoprotein contains sulfated oligosaccharide side chains attached through O-glycosidic linkages to serine and threonine, the principal amino acids (77%) in the polypeptide. The oligosaccharide side chains consist of D-arabinose, D-mannose, and N-acetyl-D-glucosamine with smaller amounts of D-galactose, L-fucose, and N-acetyl-D-galactosamine, but no sialic or uronic acids. Alkaline borohydride reductive cleavage resulted in a mixture of oligosaccharide alditols. Six oligosaccharides were purified by high performance liquid chromatography. The structures of these oligosaccharides, which do not resemble those of any other glycoprotein so far examined, were determined by a combination of gas chromatography/mass spectrometry analysis of methylation products and NMR spectroscopy. All oligosaccharides contain a reducing terminal mannitol residue with N-acetylglucosamine linked to carbon 2, 4, or 6 of the mannitol. There is no evidence for linkage of N-acetylglucosamine to any other glycoses in the glycoprotein. Galactose was detected in two oligosaccharides linked to the 4-position of mannitol. Arabinose (Ara) was found in only one oligosaccharide. This was probably due to hydrolysis of the labile arabino-furanoside linkages. Evidence is presented which indicates the arabinose occurs primarily at the terminal position of oligosaccharide side chains. The structures of the oligosaccharides isolated from the glycoprotein were: (Formula: see text).

Animals↗
PubMed1986

Oligosaccharide processing at individual glycosylation sites on MOPC 104E immunoglobulin M. Differences in alpha 1,2-linked mannose processing.

Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.

Acetylglucosaminidase↗
PubMed1983

Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices.

Incubations of thyroid microsomes with radiolabeled dolichyl pyrophosphoryl oligosaccharide (Glc3Man9-GlcNAc2) under conditions optimal for the N-glycosylation of protein resulted in the release, by apparently independent enzymatic reactions, of two types of neutral glucosylated polymannose oligosaccharides which differed from each other by terminating either in an N-acetylglucosamine residue (Glc3Man9GlcNAc1) or a di-N-acetylchitobiose moiety (Glc3Man9GlcNAc2). The first mentioned oligosaccharide, which was released in a steady and slow process unaffected by the addition of EDTA, appeared to be primarily the product of endo-beta-N-acetylglucosaminidase action on newly synthesized glycoprotein and such an enzyme with a neutral pH optimum capable of hydrolyzing exogenous glycopeptides and oligosaccharides (Km = 18 microM) was found in the thyroid microsomal fraction. The Glc3Man9GlcNAc2 oligosaccharide, in contrast, appeared to originate from the oligosaccharide-lipid by a rapid hydrolysis reaction which closely paralleled the N-glycosylation step, progressing as long as oligosaccharide transfer to protein occurred and terminating when carbohydrate attachment ceased either due to limitation of lipid-saccharide donor or addition of EDTA. There was a striking similarity between oligosaccharide release and transfer to protein with lipid-linked Glc3Man9GlcNAc2 serving as a 10-fold better substrate for both reactions than lipid-linked Man9-8GlcNAc2. The coincidence of transferase and hydrolase activities suggest the possibility of the existence of one enzyme with both functions. The physiological relevance of oligosaccharide release was indicated by the formation of such molecules in thyroid slices radiolabeled with [2-3H]mannose. Large oligosaccharides predominated (12 nmol/g) and consisted of two families of components; one group terminating in N-acetylglucosamine, ranged from Glc1Man9GlcNAc1 to Man5GlcNAc1 while the other contained the di-N-acetylchitobiose sequence and included Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, and Man9GlcNAc2.

Acetylglucosaminidase↗
PubMed1983

Structures of the oligosaccharides present at the three asparagine-linked glycosylation sites of human IgD.

The complete amino acid sequence of the human myeloma IgD:WAH has been determined and the sites of asparagine glycosylation identified as residues 354, 445, and 496 (Takahashi, N., Tetaert, D., Debuiere, B., Lin, L.-C., and Putnam, F. W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2850-2854). We have determined the structures of the oligosaccharides at each of these positions. Asn 354 bears oligosaccharides exclusively of the high mannose type containing from 5 to 9 mannose residues. Twenty per cent of the oligosaccharides at this site contain 1 glucose residue at the terminus of the branch emanating from the alpha 1 leads to 3-linked core mannose which is believed to reflect incomplete processing of the triglucosyl-high mannose oligosaccharide intermediate following transfer from dolichol to nascent peptide. Asn 445 and Asn 496 bear exclusively dibranched complex oligosaccharide structures; 30-40% of these molecules contain a bisecting GlcNAc-linked beta 1 leads to 4 to the innermost core mannose residue. At Asn 445, 40% of both the bisected and nonbisected oligosaccharides contain 1 residue of fucose on the Asn-linked GlcNAc and 50% bear a single N-acetylneuraminic acid residue. The oligosaccharides at Asn 496 are devoid of sialic acid and fucose. Thus, IgD:WAH is notable for the presence of virtually unprocessed oligosaccharide structures (glucosylated high mannose) on the same peptide backbone as extensively processed complex type molecules. The finding that each of the 3 Asn glycosylation sites of IgD:WAH bears either exclusively a complex or a high mannose type oligosaccharide indicates that there is considerable specificity in the glycosylation process. These oligosaccharides, nonetheless, display extensive microheterogeneity at each location.

Amino Acid Sequence↗
PubMed1982

Adsorptive pinocytosis of phosphorylated oligosaccharides by human fibroblasts.

Adsorptive pinocytosis of lysosomal enzymes by human fibroblasts depends on phosphomannosyl recognition markers on the enzymes and on high affinity receptors on the cell surface. To define the role of phosphorylated oligosaccharides in enzyme recognition, we studied the pinocytosis of [2-3H]mannose-labeled oligosaccharides purified from glycoproteins secreted by fibroblasts. Uptake of the oligosaccharides was inhibited 97% by 2 mM mannose-6-phosphate, 33% by 2 mM glucose 6-phosphate, and 5% or less by 2 mM alpha-methylmannoside, mannose, galactose, or L-fucose. The oligosaccharides were separated into neutral and five anionic species by chromatography on quaternary aminoethyl-Sephadex, characterized, and compared for susceptibility to pinocytosis. Treatment of the phosphorylated oligosaccharides with alkaline phosphatase before or after mild acid hydrolysis demonstrated that they contained one or two phosphates in phosphodiester linkage (covered) or phosphomonoester linkage (uncovered), or two phosphates, one in monoester linkage and one in diester linkage. Neutral oligosaccharides and those with one covered phosphate were not taken up by fibroblasts. Species with one uncovered phosphate or two covered phosphates showed low but detectable uptake. Oligosaccharides isolated as species with two uncovered phosphates, or those converted to this form by mild acid hydrolysis, were taken up 30-fold greater than the lower uptake forms during a 12-h incubation. Thus, oligosaccharides with two uncovered phosphates were far better ligands for the phosphomannosyl receptor than other oligosaccharides on acid hydrolases secreted by fibroblasts and initial rates of uptake of these oligosaccharides were comparable to those reported for several "high uptake" lysosomal enzymes.

Cells, Cultured↗
PubMed1977

Quantitation of some urinary oligosaccharides during pregnancy and lactation.

The excretion rates of different oligosaccharides in the urine of two individuals, a blood group O, nonsecretor woman, and a blood group A, secretor woman, were studied at different times during pregnancy and lactation. Quantitative determinations of di-, tri-, and tetrasaccharides were performed by combined gas-liquid chromatography-mass spectrometry. Larger oligosaccharides were quantitated by their content of total hexose or 6-deoxyhexose (fucose). The urinary oligosaccharide excretion during pregnancy is detectable by the 13th week. All oligosaccharides studied increased and reached the highest levels during the last trimester. During lactation more complex excretion patterns were observed. Lactose and oligosaccharides with a lactose backbone were the most predominant compounds. Oligosaccharides with a lacto-K-tetraose or a lacto-N-neotetraose backbone were present both during pregnancy and lactation, but the former type showed a much more pronounced increase of excretion during lactation. Four oligosaccharides containing only D-glucose were found in largest amounts during pregnancy. One of these oligosaccharides, a glucose tetrasaccharide, was quantitatively determined in the urine of 13 individuals and the excretion was found to decrease 2- to 5-fold in the first week post partum relative to the last week of pregnancy. Urine from a woman whose fetus died unexpectedly in the 24th week of pregnancy was also studied. No oligosaccharides characteristic of a normal pregnancy could be detected, suggesting that at least some oligosaccharides appearing specifically during pregnancy might reflect the function of the placenta or the fetus, or both.

ABO Blood-Group System↗
PubMed1993

Structural elucidation of a variety of GalNAc-containing N-linked oligosaccharides from human urinary kallidinogenase.

Fifteen different structures of terminal GalNAc-containing N-linked oligosaccharides from human urinary kallidinogenase have been identified. These N-linked oligosaccharides were mostly neutral, because sialic acid content was lower than 0.13 mol of sialic acid/mol of sugar chain, and sulfate was not detected. The oligosaccharides were released from pepsin-digested protein by glycoamidase A (from almond) digestion. The reducing ends of the oligosaccharide chains were aminated with a fluorescent reagent, 2-aminopyridine. The resulting mixture of pyridylamino derivatives of the oligosaccharides were separated by high performance liquid chromatography on an ODS-silica column, and 15 oligosaccharides were isolated. The structure of each oligosaccharide fraction was analyzed by two-dimensional sugar mapping, component sugar analysis, high resolution proton nuclear magnetic resonance and methylation analysis. It was found that each N-linked oligosaccharide associated with human urinary kallidinogenase contains unsubstituted GalNAc residues at the nonreducing terminal. These 15 oligosaccharides include 5 biantennary, 7 triantennary, and 3 tetraantennary oligosaccharides.

Acetylgalactosamine↗
PubMed1976

Lipid-saccharide intermediates in glycoprotein biosynthesis. II. Studies on the structure of an oligosaccharide-lipid from thyroid.

Structural studies have been performed on an oligosaccharide-lipid from thyroid believed to be an intermediate in glycoprotein synthesis. For these investigations the compound was isolated from the gland in unlabeled form as well as differentially radiolabeled in its saccharide, lipid, and phosphate portions by incubation of slices with [14C]- or [3H]glucose, [3H]mevalonic acid and [32P]phosphate, respectively. The unlabeled oligosaccharide-lipid was obtained in a chloroform/methanol/water (10/10/3) extract in a yield of about 1 nmol/g of thyroid and was purified therefrom by DEAE-cellulose chromatography. The saccharide moiety released from the glycolipid by mild acid hydrolysis was isolated by gel filtration and contained 11 mannose, 1 to 2 glucose, and 2 N-acetylglucosamine residues. The reducing terminal position of the oligosaccharide was occupied by 1 of the glucosamine residues and from these analyses a molecular weight of 2,415 was calculated. That glucose is an integral part of the molecule was further demonstrated by the finding that during Dowex 50 chromatography it remained as a constituent of the positively charged oligosaccharide produced by deacetylation with alkaline borohydride at 80 degrees. The phosphorus content of the purified unlabeled oligosaccharide-lipid was determined to be 2 residues per molecule, suggesting the presence of a pyrophosphate bridge between its carbohydrate and lipid portions. Further evidence for such a linkage region was provided by characterization of the products from mild acid and alkaline hydrolysis of the differentially radiolabeled glycolipid. These included dolichyl mono- and pyrophosphate, oligosaccharide phosphate, and free oligosaccharide. Digestion with alpha-mannosidase of the radiolabeled glycolipid led to the release of 39% of its mannose while from the free oligosaccharide 53% of this sugar was removed. Acetolysis of the [14C]oligosaccharide yielded a mannobiose and mannotriose as well as larger fragments consisting of mannose, glucose, and glucosamine. Smith periodate degradation gave rise to a small core segment (6 glycose residues) made up only of mannose and glucosamine from which half of the mannose residues could be released by alpha-mannosidase digestion. From these studies a tentative structure for the carbohydrate moiety of the oligosaccharide-lipid has been proposed. In this formulation an inner core (periodate-resistant) made up of 4 mannose and 2 N-acetylglucosamine residues is attached to the pyrophosphate group by the most internal glucosamine. This core, as well as an additional mannose and 1 to 2 glucose residues, constitutes the alpha-mannosidase-resistant fragment. More peripherally are found other mannose residues, all in alpha-linkage. In this structural scheme the glucose is located so as to prevent the enzymatic release of more internally situated alpha-linked mannose residues.

Acetylglucosamine↗
PubMed2005

Chitosan oligosaccharide inhibits 203HgCl2-induced genotoxicity in mice: micronuclei occurrence and chromosomal aberration.

The purpose of this study was to investigate the safety of chitosan oligosaccharide and the effects of chitosan oligosaccharide on mercury induced genotoxicity in mice using the micronuclei and chromosome aberration. The micronuclei test was performed by microscopic examination (x1,000, stained using a May-Grunwald solution) after administering 0.01, 0.1, and 1% (10 mg/mL) chitosan oligosaccharide for 7, 60, and 180 days ad libitum in mice. Total micronuclei of 1,000 polychromatic erythrocytes were recorded for each group. There was no difference between the untreated and experimental groups. The intake periods and concentrations of chitosan oligosaccharide did not affect the occurrence of micronuclei in bone marrow cells (P>0.05). The chromosomal aberration test was performed by microscopic examination (x1,000, stained using a 4% Giemsa solution) after administering the same concentration of chitosan oligosaccharide to mice, in F1, F2, F3 generations and parents. The frequency of chromosomal aberrations was defined as [Ydr = (D+R)/total number of counted lymphocytes]. Similar to the micronuclei test, there was no difference between the untreated and treated groups. These results showed that the intake periods and concentrations of chitosan oligosaccharide did not affect chromosomal aberrations in bone marrow cells (P>0.05). To investigate the effect of chitosan oligosaccharide on mercury-induced chromosome aberration, mice in each condition were supplied with 203HgCl2 and chitosan oligosaccharide ad libitum. Chitosan oligosaccharide significantly inhibited 203HgCl2-induced chromosome aberration in mice. Based on the results of this study, it may be concluded that the chitosan oligosaccharide is a nontoxic material that could be used as a suppressor of heavy metal-induced genotoxicity.

Animals↗
PubMed1990

Xyloglucan oligosaccharides promote growth and activate cellulase: evidence for a role of cellulase in cell expansion.

Oligosaccharides produced by the action of fungal cellulase on xyloglucans promoted the elongation of etiolated pea (Pisum sativum L.) stem segments in a straight-growth bioassay designed for the determination of auxins. The oligosaccharides were most active at about 1 micromolar. We tested the relative growth-promoting activities of four HPLC-purified oligosaccharides which shared a common glucose(4). xylose(3) (XG7) core. The substituted oligosaccharides XG8 (glucose(4). xylose(3). galactose) and XG9n (glucose(4). xylose(3). galactose(2)) were more effective than XG7 itself and XG9 (glucose(4). xylose(3). galactose. fucose). The same oligosaccharides also promoted the degradation, assayed viscometrically, of xyloglucan by an acidic cellulase from bean (Phaseolus vulgaris L.) leaves. The oligosaccharides were highly active at 10(-4) molar, causing up to a fourfold increase in activity, but the effect was still detectable at 1 micromolar. Those oligosaccharides (XG8 and XG9n) which best promoted growth, stimulated cellulase activity to the greatest extent. The oligosaccharides did not stimulate the action of the cellulase in an assay based on the conversion of [(3)H]xyloglucan to ethanol-soluble fragments. This suggest that the oligosaccharides enhanced the midchain hydrolysis of xyloglucan molecules (which would rapidly reduce the viscosity of the solution), at the expense of cleavage near the termini (which would yield ethanol-soluble products). We suggest that the promotion of midchain xyloglucan cleavage, by loosening the primary cell wall matrix, explains the promotion of growth by the oligosaccharides.

Journal Article↗
PubMed2001

Cyanovirin-N defines a new class of antiviral agent targeting N-linked, high-mannose glycans in an oligosaccharide-specific manner.

Herein we report that the novel HIV-inactivating protein cyanovirin-N (CV-N) targets specific, N-linked high-mannose oligosaccharides found on the viral envelope of HIV-1. First, we released the oligosaccharides by PnGase-treatment of HIV-gp120 (containing high-mannose, hybrid-type and complex-type oligosaccharides) or HSV-1 gC (containing only complex-type). Then, in an affinity chromatographic system, we found that CV-N bound to the free oligosaccharides from gp120 but not from gC-1, suggesting that high-mannose oligosaccharides constitute a target structure for CV-N. This was supported by the affinity of CV-N for high-mannose glycans released from gp120 by endo-H as well as high-mannose glycans released from castanospermine-treated HSV-1 gC. Furthermore, free Man-8 or Man-9 oligosaccharides partially inhibited the binding of CV-N to gp120, although neither oligosaccharides smaller than Man-7 nor monosaccharides interfered with CV-N/gp120 interaction, thereby establishing the oligosaccharide-specific affinity of CV-N to high-mannose glycans. This affinity for high-mannose oligosaccharides may explain the broad antiviral activity of CV-N against human and primate immunodeficiency retroviruses as well as certain other viruses that carry these oligosaccharides.

Antiviral Agents↗
PubMed1984

Cleavage of dolichyl pyrophosphoryl oligosaccharides by endo-beta-N-acetylglucosaminidase H: comparison of enzymatic and acid hydrolysis techniques for saccharide release.

Endo-beta-N-acetylglucosaminidase H (endo H) was found to bring about the complete hydrolysis of dolichyl pyrophosphoryl oligosaccharides. Both glycosylated and unglucosylated polymannose oligosaccharides were released by the enzyme through cleavage of the di-N-acetylchitobiose sequence. The action of the endo H on the oligosaccharide-lipids was facilitated by the inclusion of Triton X-100 (maximal stimulation at concentrations greater than 0.03%) or small amounts of a variety of other detergents; however, sodium dodecyl sulfate (0.1%) was strongly inhibitory. Although incubations were routinely carried out at pH 5.2, the enzyme was noted to be equally effective at pH 6.5 and to retain 75% of its activity toward oligosaccharide-lipid at pH 7.4. While these results broaden the known specificity of the endo H for the aglycon moiety, it was observed that even under optimal conditions the rate of hydrolysis of lipid-linked Glc3Man9GlcNAc2 was substantially slower than that of the same oligosaccharide attached to asparagine in a peptide sequence. The use of endo H, an enzyme which can be obtained free of exoglycosidases, appears to have a number of advantages over mild acid hydrolysis as a tool for cleaving oligosaccharide-lipids. It was found that the latter procedure causes a small but detectable degradation of the sugar chains and, when carried out in the presence of methanol, leads to the release of about 10% of the oligosaccharide as its beta-methyl glycoside. Furthermore, the oligosaccharides released by the endo H can be directly compared to those liberated by this enzyme from glycoproteins; this may prove to be useful in metabolic studies dealing with oligosaccharide-lipid assembly and their involvement in the N-glycosylation of proteins.

Acetylglucosaminidase↗
PubMed2001

Helix pomatia agglutinin lectin-binding oligosaccharides of aggressive breast cancer.

Predicting long-term outcome after breast-cancer diagnosis remains problematic, particularly for patients with clinically small, axillary lymph node- negative tumours. Evidence suggests that the lectin Helix pomatia agglutinin (HPA) identifies oligosaccharides associated with poor-prognosis cancer. Our aim was to identify oligosaccharides that bind HPA in aggressive breast cancers. Breast-cancer cell lines (MCF-7, BT-549 and BT-20) and a cell line from human milk (HBL-100), which showed a range of HPA-binding intensities, were used to extract HPA-binding glycoproteins. Oligosaccharides were released using anhydrous hydrazine and separated on a range of HPLC matrices. We investigated whether HPA-binding oligosaccharides from cell lines were present in human breast-cancer tissues, using 69 breast-cancer specimens from patients with between 5 and 10 years' follow-up. A monosialylated oligosaccharide was over-expressed in the cell line that bound HPA strongly. Further analysis by normal-phase HPLC showed that the 2-aminobenzamide-conjugated oligosaccharide had a hydrodynamic volume of 4.58 glucose units (HPAgly1). Increased expression of HPAgly1 was associated with HPA staining of breast-cancer specimens (Student's t-test p = 0.025). Analysis of oligosaccharide levels and disease-free survival after treatment for breast cancer indicated a shorter disease-free interval for patients with elevated levels of HPAgly1. This is the first time that histochemical lectin staining has been correlated with biochemical mapping of oligosaccharides. Using this approach, we have identified a monosialylated HPA lectin-binding oligosaccharide present in breast-cancer cells grown in vitro which is elevated in breast-cancer specimens that bind the lectin.

Adult↗
PubMed1989

Heparin-derived oligosaccharides: affinity for acidic fibroblast growth factor and effect on its growth-promoting activity for human endothelial cells.

The minimal structural requirements for the interaction of heparin with acidic fibroblast growth factor (aFGF) were investigated. Oligosaccharides (tetra- to decasaccharides) obtained by nitrous acid depolymerisation of standard heparin were separated by affinity chromatography on Sepharose-immobilised aFGF. The shortest fragment retained by the affinity column at 0.2 M NaCl and eluted at 1 M NaCl was a "regular" hexasaccharide, a trimer of the most abundant disaccharide sequence in heparin. More complex octa- and decasaccharides were also retained by the column. The oligosaccharides eluted by 1 M NaCl from the affinity column ("high-affinity" oligosaccharides) and those washed from the column at 0.2 M NaCl ("low-affinity" oligosaccharides) were compared for their capacity to protect aFGF from proteolysis and to potentiate its mitogenic activity. At a low ionic strength, all oligosaccharides tested, except the "regular" disaccharide, protected aFGF against trypsin and collagenase digestion. At higher ionic strength (greater than 0.2 M NaCl), only high-affinity oligosaccharides showed a protective effect. The high-affinity oligosaccharides (hexa- to decasaccharides) potentiated the mitogenic activity of aFGF, as measured by [3H]thymidine incorporation into DNA of human fibroblasts. The effect of the oligosaccharides on human endothelial cell proliferation was more complex: inhibition of proliferation was observed in the presence of serum and low concentrations of aFGF (1-5 ng/ml) and potentiation in the presence of higher concentrations of aFGF. The potentiating effect increased as a function of molecular size of the heparin fragments and, for a given size, as a function of the anionic charge of the oligosaccharide. Our results suggest that inhibition of cell proliferation by heparin may result from interference with an autocrine basic FGF-like activity.

Cell Division↗
PubMed1993

Endo beta-N-acetylglucosaminidase F cleavage specificity with peptide free oligosaccharides.

Endo beta-N-acetylglucosaminidase activities were determined based on conversion of oligosaccharides containing two N-acetylglucosamines to the oligosaccharides with a single N-acetylglucosamine at the reducing terminal and following their separation on a carbohydrate analyzer. The oligosaccharides eluted from the high performance anion exchange column in the order of fucosyl-N,N'-diacetylchitobiose, N,N'-diacetylchitobiose and N-acetylglucosamine containing reducing terminals. Using this assay, differences in cleavage specificity of the endo beta-N-acetylglucosaminidase F (Endo F) activity on various free oligosaccharides obtained from the standard glycoproteins was determined. The commercial Endo F-peptide N-glycosidase/glycanyl amidase (PNGase) mixture readily cleaved high mannose and complex oligosaccharides (neutral and sialyated) with common core alpha 1-6 linked fucose found in porcine thyroglobulin including the trimannosyl-chitobiose core structure. However, the same Endo F mixture did not cleave the non-fucosylated complex oligosaccharides found in human transferrin and also the common core structure. Glycopeptide counterparts with and without fucose were good substrates for the endoglycosidases. These results show that the specificity of these enzymes is such that they can recognize the conformational differences between free oligosaccharides and glycopeptides with and without the common core alpha 1-6 linked fucose. In contrast, highly purified Endo F cleaved only the high mannose type oligosaccharides and was unable to cleave ovalbumin hybrid type oligosaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)

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