Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “OLIGOSACCHARIDES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 739 records · Page 41Linked to original sources
PubMed2002

Core oligosaccharides of Plesiomonas shigelloides O54:H2 (strain CNCTC 113/92): structural and serological analysis of the lipopolysaccharide core region, the O-antigen biological repeating unit, and the linkage between them.

The structure of the core oligosaccharide moiety of the lipopolysaccharide (LPS) of Plesiomonas shigelloides O54 (strain CNCTC 113/92) has been investigated by (1)H and (13)C NMR, fast atom bombardment mass spectrometry (MS)/MS, matrix-assisted laser-desorption/ionization time-of-flight MS, monosaccharide and methylation analysis, and immunological methods. It was concluded that the main core oligosaccharide of this strain is composed of a decasaccharide with the following structure: (see text) in which l-alpha-D-Hepp is l-glycero-alpha-D-manno-heptopyranose. The nonasaccharide variant of the core oligosaccharide ( approximately 10%), devoid of beta-D-Glcp substituting the alpha-D-GlcpN at C-6, was also identified. The core oligosaccharide substituted at C-4 of the outer core beta-D-Glcp residue with the single O-polysaccharide repeating unit was also isolated yielding a hexadecasaccharide structure. The determination of the monosaccharides involved in the linkage between the O-specific polysaccharide part and the core, as well as the presence of -->3)-D-beta-D-Hepp-(1--> instead of -->3,4)-D-beta-D-Hepp-(1--> in the repeating unit, revealed the structure of the biological repeating unit of the O-antigen. The core oligosaccharides are not substituted by phosphate residues and represent novel core type of bacterial LPS that is characteristic for the Plesiomonas shigelloides serotype O54. Serological screening of 69 different O-serotypes of P. shigelloides suggests that epitopes similar to the core oligosaccharide of serotype O54 (strain CNCTC 113/92) might also be present in the core region of the serotypes O24 (strain CNCTC 92/89), O37 (strain CNCTC 39/89) and O96 (strain CNCTC 5133) LPS.

Acetylation↗
PubMed2002

Biosynthetic oligosaccharide libraries for identification of protein-binding heparan sulfate motifs. Exploring the structural diversity by screening for fibroblast growth factor (FGF)1 and FGF2 binding.

Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, end-group (3)H-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2-O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-O-sulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries.

Binding Sites↗
PubMed2003

Structural analysis of oligosaccharides from lipopolysaccharide (LPS) of Escherichia coli K12 strain W3100 reveals a link between inner and outer core LPS biosynthesis.

Lipopolysaccharide (LPS) from Escherichia coli K12 W3100 is known to contain several glycoforms, and the basic structure has been investigated previously by methylation analyses (Holst, O. (1999) in Endotoxin in Health and Disease (Brade, H., Opal, S. M., Vogel, S. N., and Morrison, D., eds) pp. 115-154; Marcel Dekker, Inc., New York). In order to reveal dependences of gene activity and LPS structure, we have now determined the composition of de-O-acylated LPS by electrospray ionization-Fourier transform ion cyclotron-mass spectrometry (ESI-FT-MS) and identified 11 different LPS molecules. We have isolated the major glycoforms after de-O- and de-N-acylation and obtained four oligosaccharides that differed in their carbohydrate structure and phosphate substitution. The main oligosaccharide accounted for approximately 70% of the total and had a molecular mass of 2516 Da according to ESI-FT-MS. The dodecasaccharide structure (glycoform I) as determined by NMR was consistent with MS and compositional analysis. One minor oligosaccharide (5%) of the same carbohydrate structure did not contain the 4'-phosphate of the lipid A. Two oligosaccharides contained the same phosphate substitution but differed in their carbohydrate structure, one (5%) which contained an additional beta-D-GlcN in 1-->7 linkage on a terminal heptose residue (glycoform II) which was N-acetylated in LPS. A minor amount of a molecule lacking the terminal L-alpha-D-Hep in the outer core but otherwise identical to the major oligosaccharide (glycoform III) could only be identified by ESI-FT-MS of the de-O-acylated LPS. The other oligosaccharide (20%) contained an alpha-Kdo-(2-->4)-[alpha-l-Rha-(1-->5)]-alpha-Kdo-(2-->4)-alpha-Kdo branched tetrasaccharide connected to the lipid A (glycoform IV). This novel inner core structure was accompanied by a truncation of the outer core in which the terminal disaccharide L-alpha-D-Hep-(1-->6)-alpha-D-Glc was missing. The latter structure was identified for the first time in LPS and revealed that changes in the inner core structure may be accompanied by structural changes in the outer core.

Carbohydrate Sequence↗
PubMed2005

Decorin core protein secretion is regulated by N-linked oligosaccharide and glycosaminoglycan additions.

Expression of decorin using the vaccinia virus/T7 expression system resulted in secretion of two distinct glycoforms: a proteoglycan substituted with a single chondroitin sulfate chain and N-linked oligosaccharides and a core protein glycoform substituted with N-linked glycans but without a glycosaminoglycan chain. In this report, we have addressed two distinct questions. What is the rate-limiting step in glycosaminoglycan synthesis? Is glycosylation with either N-linked oligosaccharides or glycosaminoglycan required for secretion of decorin? N-terminal sequencing of the core protein glycoform, the addition of benzyl-beta-d-xyloside, and a UDP-xylose: core protein beta-d-xylosyltransferase activity assay show that xylosylation is a rate-limiting step in chondroitin sulfate biosynthesis. Decorin can be efficiently secreted with N-linked oligosaccharides alone or with a single chondroitin sulfate chain alone; however, there is severely impaired secretion of core protein devoid of any glycosylation. A decorin core protein mutant devoid of N-linked oligosaccharide attachment sites will not be secreted by Chinese hamster ovary cells deficient in xylosyltransferase or by parental Chinese hamster ovary wild type cells if the xylosyltransferase recognition sequence is disrupted. This finding suggests that quality control mechanisms sensitive to an absence of N-linked oligosaccharides can be abrogated by interaction of the core protein with the glycosaminoglycan synthetic machinery. We propose a model of regulation of decorin secretion that has several components, including appropriate substitution with N-linked oligosaccharides and factors involved in glycosaminoglycan synthesis.

Animals↗
PubMed2006

Hyaluronan oligosaccharides induce matrix metalloproteinase 13 via transcriptional activation of NFkappaB and p38 MAP kinase in articular chondrocytes.

Hyaluronan exerts a variety of biological effects on cells including changes in cell migration, proliferation, and matrix metabolism. However, the signaling pathways associated with the action of hyaluronan on cells have not been clearly defined. In some cells, signaling is induced by the loss of cell-hyaluronan interactions. The goal of this study was to use hyaluronan oligosaccharides as a molecular tool to explore the effects of changes in cell-hyaluronan interactions and determine the underlying molecular events that become activated. In this study, hyaluronan oligosaccharides induced the loss of extracellular matrix proteoglycan and collagen from cultured slices of normal adult human articular cartilage. This loss was coincident with an increased expression of matrix metalloproteinase (MMP)-13. MMP-13 expression was also induced in articular chondrocytes by hyaluronan (HA) hexasaccharides but not by HA tetrasaccharides nor high molecular weight hyaluronan. MMP-13 promoter-reporter constructs in CD44-null COS-7 cells revealed that both CD44-dependent and CD44-independent events mediate the induction of MMP-13 by hyaluronan oligosaccharides. Electromobility gel shift assays demonstrated the activation of chondrocyte NFkappaB by hyaluronan oligosaccharides. NFkappaB activation was also documented in C-28/I2 immortalized human chondrocytes by luciferase promoter assays and phosphorylation of IKK-alpha/beta. The link between activation of NFkappaB and MMP-13 induction by HA oligosaccharides was further confirmed through the use of the NFkappaB inhibitor helenalin. Inhibition of MAP kinases also demonstrated the involvement of p38 MAP kinase in the hyaluronan oligosaccharide induction of MMP-13. Our findings suggest that hyaluronan-CD44 interactions affect matrix metabolism via activation of NFkappaB and p38 MAP kinase.

Animals↗
PubMed2006

Dietary fructo-oligosaccharides in healthy adults do not negatively affect faecal cytotoxicity: a randomised, double-blind, placebo-controlled crossover trial.

Fructo-oligosaccharides (FOS) are widely used in commercial food products. Most studies on FOS concern the health benefits, but some negative effects were recently reported concerning the faecal cytotoxicity and excretion of mucin-type oligosaccharides in combination with a Ca-restricted diet. The present study was performed to investigate whether these effects of FOS are observed in adults consuming a regular diet unrestricted in Ca. The study was a randomised, double-blind, placebo-controlled crossover trial, involving eleven healthy adults, who consumed 25-30 g FOS or maltodextrin (control) in a random order for 2 weeks in addition to their regular diet. Stools were collected for analysis of pH and SCFA (as markers of fermentation), for the assessment of faecal water cytotoxicity, and for the analysis of alkaline phosphatase activity (as a marker of epithelial cell turnover) and O-linked oligosaccharides (to estimate the excretion of mucin-type oligosaccharides). FOS consumption significantly altered bacterial fermentation (increased percentage of acetate, decreased percentage of butyrate) and tended to decrease stool pH. Furthermore, FOS consumption resulted in a significantly higher stool frequency and in significantly more complaints of flatulence. No significant differences between the control and FOS period were observed in the mean cytotoxicity of faecal water (37.5 (SEM 6.9)% v. 18.5 (SEM 6.9)%; P=0.084), in mean alkaline phosphatase activity (27.7 (SEM 2.9) v. 24.6 (SEM 3.2) U/g dry faeces; P=0.496) or in the mean excretion of mucin-type oligosaccharides (49.9 (sem 4.0) v. 53.5 (SEM 4.3) mg/g dry faeces; P=0.553). We conclude that dietary FOS in a dose up to 25-30 g/d altered the bacterial fermentation pattern but did not affect faecal cytotoxicity or the faecal concentration of mucin-type oligosaccharides in human adults consuming a regular diet.

Acetates↗
PubMed2002

Galacto-oligosaccharides stimulate the growth of bifidobacteria but fail to attenuate inflammation in experimental colitis in rats.

BACKGROUND: Galacto-oligosaccharides potentially attenuate colonic inflammation by two mechanisms: through beneficial effects on intestinal microflora and by increasing the colonic short-chain fatty acid concentration. The purpose of this study was to investigate the effects of galacto-oligosaccharides on the development of inflammation and on the growth of bifidobacteria in trinitrobenzene sulphonic acid (TNBS)-induced colitis, a model that has been shown to benefit from short-chain fatty acid administration and to be associated with alterations in the colonic microflora. METHODS: Rats were given daily either whey-derived or lactose-derived galacto-oligosaccharides (4 g kg(-1) day(-1), p.o.); starting 10 days before colitis induction, or dexamethasone (2 mg kg(-1) day(-1), s.c., a positive control), starting at colitis induction. Colon wet weight, macroscopic damage and myeloperoxidase activity were assessed 72 h after the induction of colitis. Faecal bifidobacteria were counted at the beginning of the study, and immediately before and 72 h after colitis induction. RESULTS: Galacto-oligosaccharides increased the colonic levels of bifidobacteria but also the levels of other bacterial species. Neither whey-derived nor lactose-derived galacto-oligosaccharides reduced the severity of inflammation. CONCLUSIONS: Galacto-oligosaccharides are able to modify gut microflora in severe TNBS-induced colitis, but unable to attenuate the inflammation.

Animals↗
PubMed2000

Nondigestible oligosaccharides.

Dietary carbohydrates range in molecular size from simple sugars to complex polymers with a degree of polymerization (DP) of up to 100,000 or more. Oligosaccharides are generally defined as carbohydrates from 2 to 20 monomeric units long. Oligosaccharides have been dietary staples since antiquity but have received much less attention than other carbohydrates such as simple sugars or dietary fiber. Recently, interest in oligosaccharides has increased not only because of properties that include sweetening ability and fat replacement, but also because of resistance to digestion in the upper gastrointestinal tract and fermentation in the large bowel. Thus, some oligosaccharides have functional effects similar to soluble dietary fiber such as enhancement of a healthy gastrointestinal tract, improvement of glucose control, and modulation of the metabolism of triglycerides. These oligosaccharides are the nondigestible oligosaccharides. These compounds are easily incorporated into processed foods and hold much promise as functional ingredients in nutraceutical products.

Chronic Disease↗
PubMed1985

Processing of the asparagine-linked oligosaccharides of secreted and intracellular forms of the vesicular stomatitis virus G protein: in vivo evidence of Golgi apparatus compartmentalization.

The structures of the asparagine-linked oligosaccharides of several variant forms of the vesicular stomatitis virus glycoprotein transiently expressed from cloned cDNAs have been determined. Glycopeptides isolated from forms of the G protein that reach the cell surface or that are secreted into the medium are virtually identical; they contain complex-type oligosaccharides whose nonreducing ends terminate in galactose and sialic acid residues. In contrast, forms of the G protein that remain intracellular possess oligosaccharides at intermediate stages in the processing pathway. One deletion mutant, delta 1473, codes for a protein that remains in the rough endoplasmic reticulum (Rose, J. K., and J. E. Bergmann, 1982, Cell, 30:753-762) and contains only high mannose-type oligosaccharides. Another mutant, delta 1554, codes for a glycoprotein that contains oligosaccharides of primarily two classes. One class is of the high mannose type and is similar to those found on the protein coded for by delta 1473. However, the major class contains biantennary and more highly branched complex-type oligosaccharides that terminate in N-acetylglucosamine rather than galactose or sialic acid residues. These data suggest that the protein coded for by delta 1554 migrates to the Golgi apparatus, but does not enter the more distal compartment(s) of the organelle which contains galactosyl- and sialyltransferases.

Animals↗
PubMed1987

Gaseous response to ingestion of a poorly absorbed fructo-oligosaccharide sweetener.

Fructo-oligosaccharides are naturally occurring sweet substances that are poorly absorbed and have the potential to be clinically useful nonnutritive sweeteners. Because most nonabsorbed carbohydrates are fermented yielding gas, we assessed flatulent symptoms and H2 excretion during ingestion of fructo-oligosaccharide (5 g tid) for 12 d. Ten subjects had significantly greater flatulence while taking the oligosaccharide than did five subjects taking sucrose (5 g tid). Breath H2 after 10 g fructo-oligosaccharide was similar to that of 10 g lactulose, suggesting near total malabsorption of the fructo-oligosaccharide. Although previous studies found a marked diminution in breath H2 after prolonged exposure to lactulose, breath H2 response increased by 50% after a 12-d period on the oligosaccharide and gaseous symptoms did not improve. We conclude that adaptation of colonic bacteria to carbohydrate malabsorption is variable and may depend upon quantity or nature of the carbohydrate.

Adult↗
PubMed1999

Gas production in human ingesting a soybean flour derived from beans naturally low in oligosaccharides.

BACKGROUND: Ingestion of soy products may cause excessive intestinal gas. This gas results from colonic bacterial fermentation of the indigestible oligosaccharides raffinose and stachyose, which are present in high concentrations in legumes. OBJECTIVE: The objective of the study was to compare gas production and gaseous symptoms in healthy volunteers after ingestion of 34 and 80 g soy flour made from either conventional soybeans or soybeans naturally low in indigestible oligosaccharides. DESIGN: In a double-blind, randomized, crossover protocol, breath hydrogen (an indicator of carbohydrate malabsorption), flatus frequency, and abdominal symptoms were assessed after subjects ingested the soy products and after 2 control meals (rice or lactose-hydrolyzed milk). RESULTS: The sum of breath-hydrogen concentrations for 8 h was significantly greater (P < 0.005) after 34 g conventional soy (60.4+/-9.4 ppm) than after low-oligosaccharide soy (34.3+/-8.1 ppm). Greater differences were observed with 80-g doses: 157.9+/-19.4 ppm after conventional soy and 50.8+/-6.8 ppm after low-oligosaccharide soy (P < 0.001). Flatus frequency (7.5+/-1.9 times/12 h) was significantly greater (P = 0.039) after ingestion of 80 g conventional soy than after the control, rice meal (3.2+/-0.8 times/12 h), whereas flatus frequency after the low-oligosaccharide soy meal (3.9+/-0.7 times/12 h) was comparable with that after the rice meal. There were no significant differences in the severity of other abdominal symptoms. CONCLUSION: Soy flour derived from low-oligosaccharide soybeans resulted in less gas production than that derived from conventional soybeans.

Adult↗
PubMed2000

Glycopeptide export from mammalian microsomes is independent of calcium and is distinct from oligosaccharide export.

Glycopeptides are exported from the endoplasmic reticulum to the cytosol of eukaryotic membranes in an ATP- and cytosol-requiring process (Romisch and Ali, 1997, Proc. Natl. Acad. Sci. USA,94, 6730-6734). Oligosaccharides of the polymannose-type are also exported from the endoplasmic reticulum of mammalian cells to the cytosol in an ATP-dependent fashion. These findings raise the strong possibility that the two substrate classes are transported by the same mechanism but the precise identity of the trans-location machinery for each substrate class has not been fully defined. Here we have investigated the mechanism by which a glycopeptide is exported from rat liver microsomes, and compare this to the export of free polymannose oligosaccharides. Using EGTA and the endoplasmic reticulum calcium mobilizing agents thapsigargicin and calcium ionophores A23187 and ionomycin, we show that glycopeptides, in contrast to oligosaccharides, are exported by a calcium-independent mechanism. On the other hand, Mg(2+)is required in the assay for the transport of glycopeptide from mammalian microsomes which is in common with oligosaccharide export. Deoxynojirimycin and castanospermine, inhibitors of ER glucosidases, when added to rat liver microsomes prior to loading with peptide that bears an N -glycosylation sequon, had no effect on the release of glucosylated glycopeptides from membranes, indicating that removal of the alpha-glucose units from the oligomannose glycan structure of the glycopeptide is not required for export. In contrast to oligosaccharides, where transport is efficiently inhibited, mannosides were without effect or only weak inhibitors of glycopeptide export. Taken together, these data suggest that glycopeptides are exported by a distinct mechanism from oligosaccharides of the polymannose-type and that the peptide moiety is an important structural determinant for glycopeptide export and capable of directing translocation of substrates to a specific transport pathway.

Animals↗
PubMed2000

Effect of proteasome inhibitors on the release into the cytosol of free polymannose oligosaccharides from glycoproteins.

Prompted by previous observations which suggested that the release of polymannose oligosaccharides shortly after the cotranslational N-glycosylation of proteins is a function of the ER-associated quality control system (Moore and Spiro (1994) J. Biol. Chem., 269, 12715-12721), we evaluated the effect which proteasome inhibitors have on the appearance of these free saccharide components. Employing as a model system castanospermine-treated BW5147 mouse T-lymphoma cells in which accelerated degradation of the T-cell receptor (TCR) alpha subunit takes place (Kearse et al. (1994) EMBO J., 13, 3678-3686), we noted that both lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal, but not leupeptin, brought about a rapid and substantial reduction in the release of free polymannose oligosaccharides into the cytosol during pulse-chase studies, while the oligosaccharides in the intravesicular compartment remained unchanged, as measured by streptolysin O permeabilization. This inhibition was furthermore selective in that it affected solely the components terminating in a single N-acetylglucosamine residue (OS-GlcNAc(1)) and not the oligosaccharides terminating in a di-N-acetylchitobiose sequence (OS-GlcNAc(2)), which reside primarily in the intravesicular compartment. Despite the quantitative effect of the proteasome inhibitors on the cytosolic oligosaccharides, the molar distribution of the triglucosyl OS-GlcNAc(1) species was unaffected. The decrease in cytosolic oligosaccharides brought about by proteasome inhibition was reflected in a pronounced increase in the stability of the TCRalpha subunit. Our findings suggest that the N-deglycosylation and proteasome mediated degradation are coupled events. On the basis of our data and those of others we propose that the quality control mechanism involves proteasomes associated with the cytosolic side of the endoplasmic reticulum acting in concert with a membrane situated N-glycanase. Such a complex by removing the carbohydrate units could facilitate the retrograde ER to cytosol translocation of glycoproteins.

Animals↗
PubMed1992

Structural heterogeneity in the Man8-13GlcNAc oligosaccharides from log-phase Saccharomyces yeast: a one- and two-dimensional 1H NMR spectroscopic study.

Previously, Man8-14GlcNAc oligosaccharides were isolated from highly purified Saccharomyces cerevisiae invertase and shown by one-dimensional 1H NMR spectroscopy and alpha 1,2-linkage-specific mannosidase digestion to constitute a homologous series of nearly homogeneous compounds, which appeared to define the intermediates in oligosaccharide core synthesis in yeast (Trimble, R.B. and Atkinson, P.H. (1986) J. Biol. Chem., 261, 9815-9824). To evaluate whether invertase oligosaccharides reflected global core processing of yeast glycans, the soluble glycoprotein pool of disrupted log-phase cells was digested with endo-beta-N-acetyl-glucosaminidase H and Man8-13GlcNAc were isolated by Bio-Gel P-4 chromatography. Although analysis of each size class by one-dimensional 400 MHz and two-dimensional 500 MHz phase-sensitive COSY 1H NMR spectroscopy revealed considerable structural heterogeneity in all but Man8GlcNAc, the major positional isomer in Man9-13GlcNAc (approximately 50%) was identical to that previously elucidated on invertase. The heterogeneity resided in four families of oligosaccharides: (i) Glc3Man9GlcNAc----Man8 GlcNAc trimming intermediates; (ii) alpha-mannosidase degradation products of the principal isomers; (iii) mannan elongation intermediates; (iv) core structures with the alpha 1,2-linked mannose usually removed by the processing alpha-mannosidase. The potential for the vacuolar alpha-mannosidase (AMS1 gene product) to generate heterogeneity in vitro was confirmed by isolating oligosaccharides from AMS1 and ams1 yeast strains in the presence of a Man13GlcNAc[3H]-ol marker (where GlcNAc[3H]-ol is N-acetylglucosamin [1-3H]itol). Degradation of the Man13GlcNAc[3H]-ol to Man9-12GlcNAc[3H]-ol occurred in the former, but not in the latter. A role for the vacuolar alpha-mannosidase in generating at least some heterogeneity in vivo was inferred from the 1H NMR spectrum of the AMS1 Man11GlcNAc pool, which showed more structural isomerism than seen in the spectrum of a comparable ams1 Man11GlcNAc preparation. Thus, the principal biosynthetic pathway of inner core mannan in Saccharomyces is defined by the Man8-13GlcNAc oligosaccharides found on external invertase, while structural heterogeneity in these size classes results from precursor processing in the endoplasmic reticulum, core extension in the Golgi and metabolic degradation in the vacuole.

Acetylglucosamine↗
PubMed1994

Advances in the use of biotinylated diaminopyridine (BAP) as a versatile fluorescent tag for oligosaccharides.

We recently described a novel fluorescent compound, 2-amino,6-amidobiotinyl-pyridine (BAP), that allows the tagging of oligosaccharides, their fractionation by reversed-phase HPLC with picomole scale detection, and the formation of functional neoglycoprotein equivalents with (strept) avidin for the detection of receptors and the generation of monospecific antibodies (Rothenberg et al., Proc. Natl Acad. Sci. USA, 90, 11939-11943, 1993). Here, we describe the enhancement of this approach by the following. (i) A simple one-step purification of BAP from its synthetic precursors and other reactants. (ii) Development of HPLC sizing column methods to quickly purify BAP-coupled oligosaccharides away from free BAP and other reactants. (iii) Development of anion-exchange and amine-adsorption HPLC procedures for the fractionation of BAP-oligosaccharide adducts by charge and size, respectively. (iv) Investigation of the affinity of BAP-oligosaccharides for (strept)avidin, confirming the formation of stable complexes. (v) The use of BAP for sensitive monosaccharide compositional analysis of glycoproteins. (vi) Formation of stable BAP adducts without reduction and its implications for the mechanism of adduct formation. These advances make available a multitude of techniques for the fractionation of BAP-coupled oligosaccharides based on several different physical parameters. Distinct species of BAP-coupled oligosaccharides can be isolated and subjected to detailed structural analysis. Such defined molecules form stable complexes with streptavidin that are effectively neoglycoproteins, which can be used in a variety of biological applications. Notably, all of these approaches require relatively inexpensive materials and conventional equipment available in most laboratories.

Amines↗
PubMed1995

The critical glycosylation site of human transferrin receptor contains a high-mannose oligosaccharide.

The human transferrin receptor (TfR) contains three N-linked oligosaccharides and glycosylation is required for the proper folding and function of the molecule. Earlier studies demonstrated that the oligosaccharide at Asn-727 is vital for the production of fully active TfR. The oligosaccharide(s) present at this site have been analysed using a combination of site-directed mutagenesis and chemical analysis. Wild-type TfR and mutants containing only the Asn-727 site or missing all three sites were transfected into mouse 3T3 cells and receptors were analysed by endo-N-acetylglucosaminidase H (Endo-H) digestion, SDS-PAGE and immunoblotting. These studies suggested that the Asn-727 site contains high-mannose or Endo-H-sensitive hybrid oligosaccharides. Glycosylation of Asn-727 found in the TfR purified from human placentae was analysed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and mass spectrometry following tryptic digestion, peptide purification via reverse-phase high-performance liquid chromatography (RP-HPLC) and peptide sequencing. HPAE-PAD showed the presence of a series of high-mannose oligosaccharides. Mass spectrometry confirmed these observations, but also showed the presence of an 80 Da anionic moiety on a fraction of the oligosaccharides.

3T3 Cells↗
PubMed1996

Structural characterization of novel oligosaccharides of cell-surface glycoproteins of Trypanosoma cruzi.

Affinity-purified glycopeptides were prepared from Trypanosoma cruzi using the carbohydrate-specific monoclonal antibody WIC29.26. These glycopeptides contain rhamnose, fucose, xylose, and galactose, in the ratio 1:1:2:3. A series of oligosaccharides was released from the glycopeptides by mild acid hydrolysis, while, in contrast, no oligosaccharides were released by either peptide N-glycosidase F or conventional base-catalyzed beta-elimination and reduction. This suggested the presence of a phosphodiester linkage between the carbohydrate and peptide, which was further supported by the detection of phosphothreonine in the glycopeptides. The mild acid liberated (MAL) fraction was resolved into two major acidic oligosaccharides (MAL-P1 and MAL-P2), two minor neutral oligosaccharides (MAL-P1b and MAL-P2b) and a neutral fraction (MAL-N1), consisting of Gal and Xyl monosaccharides. The MAL-P1 and MAL-P2 oligosaccharides proved to be hexa- and hepta-saccharides that shared a common xylose reducing terminus, but differed by one galactofuranose residue, and their negative charge was shown to be due to the presence of cyclic-phosphate attached to nonreducing terminal galactofuranose residues. The MAL-P1b and MAL-P2b oligosaccharides appeared to be nonphosphorylated versions of MAL-P1 and MAL-P2. Partial structures of MAL-P1 and MAL-P2 are suggested, based on compositional analyses, electrospray mass spectrometry, and tandem mass spectrometry before and after permethylation. The origin and significance of these unique trypanosomatid glycoconjugates is discussed.

Animals↗
PubMed1998

Determination of the structure of oligosaccharides prepared from acharan sulfate.

The fine structure of acharan sulfate, a recently discovered glycosaminoglycan isolated from Achatina fulica , was examined. This glycosaminoglycan has a major disaccharide repeating unit of -->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S(1--> (where GlcNpAc is N -acetylglucosamine, IdoAp is iduronic acid, and S is sulfate) making it structurally related to both heparin and heparan sulfate. Using heparin lyases prepared from Flavobacterium heparinum and a newly isolated heparinase from Bacteroides stercoris , the controlled enzymatic depolymerization of acharan sulfate was undertaken to prepare a mixture of oligosaccharides. Fractionation of this mixture of oligosaccharides by strong-anion-exchange high performance liquid chromatography afforded oligosaccharides that capillary electrophoresis established were sufficiently pure for structural characterization. Electrospray ionization mass spectrometry identified two series of oligosaccharides, one derived from acharan sulfate's major repeating unit and a second minor group of undersulfated oligosaccharides. Proton nuclear magnetic resonance spectroscopy established the structure of these two classes of oligosaccharides to be DeltaUAp2S(1-->[4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S (1-->]n4)- D-GlcNpAcalpha,beta (where n = 0,1,2,3 and DeltaUAp is 4-deoxy-alpha-L- threo -hex-4-enopyranosyluronic acid) and DeltaUAp(1-->[4)- alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp2S(1-->]m-D-GlcNpAcal pha,beta (where m = 1,2,3). These results suggest the presence of minor sequence variants in acharan sulfate containing unsulfated iduronic acid having the structure -->4)-alpha-D-GlcNpAc(1-->4)-alpha-L-IdoAp(1-->.

Animals↗