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PubMed1974

The copolymeric structure of pig skin dermatan suplhate. Characterization of D-glucuronic acid-containing oligosaccharides isolated after controlled degradation of oxydermatan sulphate.

Selective periodate oxidation of unsubstituted l-iduronic acid residues in copolymeric dermatan sulphate chains was followed by reduction-hydrolysis or alkaline elimination. By this procedure the glucuronic acid-containing periods were isolated in oligosaccharide form; general formula: [Formula: see text] Further degradation of these oligosaccharides with chondroitinase-AC yielded three types of products: (a) sulphated trisaccharide containing an unsaturated uronosyl moiety in the non-reducing terminal and a C(4) fragment in the reducing terminal, DeltaUA-GalNAc-(-SO(4))-R; (b) monosulphated, unsaturated disaccharide, DeltaUA-GalNAc-SO(4) when n is greater than or equal to 2; and (c) N-acetylgalactosamine with or without sulphate. Oligosaccharides containing a single glucuronic acid residue (n=1) comprised more than half of the glucuronic acid-containing oligosaccharides. The terminal N-acetylgalactosamine moiety of the shortest oligosaccharide was largely 4-sulphated, whereas higher oligosaccharides primarily contained 6-sulphated or unsulphated hexosamine moieties in the same position. Moreover, IdUA-SO(4)-containing oligosaccharides were encountered. These oligosaccharides were resistant to the action of chondroitinase-ABC.

Animals↗
PubMed1985

Comparison between 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin as inhibitors of oligosaccharide processing in intestinal epithelial cells.

The alpha-glucosidase inhibitor N-methyl-1-deoxynojirimycin (MDJN) inhibits the synthesis of N-linked complex oligosaccharides in rat intestinal epithelial cells to the same extent as reported previously for 1-deoxynojirimycin (DJN) [Saunier, Kilker, Tkacz, Quaroni & Herscovics (1982) J. Biol. Chem. 257, 14155-14161]. Analysis of each of the endo-beta-N-acetylglucosaminidase H (endo H)-sensitive oligosaccharides separated by h.p.l.c. with yeast glucosidase I, which specifically removes the terminal glucose residue from oligosaccharides containing three glucose residues, and with jack-bean (Canavalia ensiformis) alpha-mannosidase, indicates that both inhibitors cause the accumulation of a mixture of glucosylated oligosaccharides containing one to three glucose residues and seven to nine, and even possibly six, mannose residues. About 70% of the endo H-sensitive oligosaccharides formed in the presence of MDJN contain three glucose residues, compared with only about 20% of the corresponding oligosaccharides of the DJN treated cells. It is concluded that both compounds inhibit the formation of N-linked complex oligosaccharides by interfering with the processing glucosidases. These compounds are valuable in the study of the role of oligosaccharides in glycoproteins.

1-Deoxynojirimycin↗
PubMed1985

Hazelhurst-vesicular-stomatitis-virus G and Sindbis-virus E1 glycoproteins undergo similar host-cell-dependent variation in oligosaccharide processing.

We have examined and compared the host-cell-dependent glycosylation of the G glycoprotein of vesicular-stomatitis virus (Hazelhurst strain) and the E1 and E2 glycoproteins of Sindbis virus replicated by baby-hamster kidney, chicken-embryo fibroblast and mouse L929 monolayer cell cultures. The results of endo-beta-N-acetylglucosaminidase H digestion of viral proteins labelled with [3H]mannose or leucine and Pronase-digested glycopeptides labelled with [3H]mannose indicated that both the G protein and the E1 protein contained a similar mixture of endoglycosidase-resistant oligosaccharides of the complex acidic type and less extensively processed endoglycosidase-sensitive oligosaccharides of the neutral or hybrid type, with a relatively greater content of the endoglycosidase-sensitive oligosaccharides for virus replicated in the chicken as against hamster or mouse cells. A large fraction of the G protein and the majority of the E1 proteins from the mammalian host cells contained acidic-type oligosaccharides at both glycosylation sites, whereas most of the G and E1 glycoproteins from the avian host cells and essentially all of the E2 protein from all three host-cell types contained an acidic-type oligosaccharide at one site and neutral- or hybrid-type oligosaccharide at the other site. The relative increase in neutral- and hybrid-type oligosaccharides with five-mannose core structures observed for the G and E1 proteins of virus released from the avian host cells suggested that two specific steps in oligosaccharide processing (mediated by alpha-mannoside II and N-acetylglucosaminyltransferase I) were less efficient at one of the glycosylation sites of the vesicular-stomatitis-virus G protein and Sindbis-virus E1 protein in the avian as against mammalian host cells.

Acetylglucosaminidase↗
PubMed1986

Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits.

The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the alpha- and beta-subunits.

Cells, Cultured↗
PubMed1989

Neoglycolipids as probes of oligosaccharide recognition by recombinant and natural mannose-binding proteins of the rat and man.

Oligosaccharide recognition by three mammalian mannose-binding proteins was investigated by using as probes a series of structurally characterized neoglycolipids in t.l.c. binding assays. The neoglycolipids were derived from N-linked oligosaccharides of complex, high-mannose and hybrid types and from human milk oligosaccharides and simple di- and tri-saccharides. The three proteins, namely the recombinant carbohydrate-recognition domain of rat mannose-binding Protein A and the multi-subunit forms of rat and human serum mannose-binding proteins, were shown to have in common reactivity with oligosaccharide probes containing one or more non-reducing terminal N-acetylglucosamine residue(s). Substitution with galactose masks reactivity. The three proteins also bound to non-reducing terminal mannose residues in high-mannose-type oligosaccharides, non-reducing terminal fucose residues in the sequence Fuc alpha 1-4(Gal beta 1-3)GlcNAc and non-reducing terminal glucose residues in dextran oligomers; the recombinant binding domain gave consistently weaker binding. The relative reactivities with the various probes differ for each protein. Overall, the reaction patterns of the three mammalian proteins differ from that of the plant lectin concanavalin A, which showed preferential binding to the high-mannose type, weak binding to biantennary complex type and no binding to the fuco-oligosaccharide and simple oligosaccharide probes. As a group, the three mammalian proteins resemble bovine serum conglutinin and behave as lectins with rather broad sugar specificities directed at certain non-reducing terminal N-acetylglucosamine, mannose, glucose and fucose residues, but with subtle differences in fine specificities. These results illustrate the potential of neoglycolipids in studies of oligosaccharide recognition by natural and recombinant proteins of diverse biological systems.

Animals↗
PubMed2001

A comparative in vitro evaluation of the fermentation properties of prebiotic oligosaccharides.

AIMS: Comparison of in vitro fermentation properties of commercial prebiotic oligosaccharides. METHODS AND RESULTS: Populations of predominant gut bacterial groups were monitored over 24 h of batch culture through fluorescent in-situ hybridization. Short-chain fatty acid and gas production were also measured. All prebiotics increased the numbers of bifidobacteria and most decreased clostridia. Xylo-oligosaccharides and lactulose produced the highest increases in numbers of bifidobacteria whilst fructo-oligosaccharides produced the highest populations of lactobacilli. Galacto-oligosaccharides (GOS) resulted in the largest decreases in numbers of clostridia. Short-chain fatty acid generation was highest on lactulose and GOS. Gas production was lowest on isomalto-oligosaccharides and highest on inulin. CONCLUSIONS: The oligosaccharides differed in their fermentation characteristics. Isomalto-oligosaccharides and GOS were effective at increasing numbers of bifidobacteria and lactate whilst generating the least gas. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides comparative data on the properties of commercial prebiotics, allowing targeting of dietary intervention for particular applications and blending of oligosaccharides to enhance overall functionality.

Bacteria↗
PubMed1974

The role of a dolichol-oligosaccharide as an intermediate in glycoprotein biosynthesis.

Incubation of mouse myeloma microsomes with GDP-[(14)C]mannose results in the biosynthesis of [(14)C]mannose phosphoryl dolichol [Baynes, J. W., Hsu, A.-F. & Heath, E. C. (1973) J. Biol. Chem. 248, 5693-5704] and a [(14)C]mannose- and N-acetylglucosamine (GlcNAc)-containing oligosaccharide derivative of dolichol. Thus, [(14)C]mannose phosphoryl dolichol and [(14)C]mannose-labeled oligosaccharide pyrophosphoryl dolichol were isolated from incubation mixtures by solubilization in 2% (w/v) Triton X-100 and the lipids were separated from small molecules by gel filtration fractionation. After removal of radioactive protein from the preparation, the two lipid derivatives were separated quantitatively by fractionation on a concanavalin A-Sepharose column; [(14)C]mannose phosphoryl dolichol was not retained by the affinity resin but [(14)C]mannose-oligosaccharide pyrophosphoryl dolichol adsorbed to the gel and was eluted with alpha-methylmannoside.[(14)C]Mannose-oligosaccharide pyrophosphoryl dolichol appeared to be homogeneous when fractionated on DEAE-cellulose and in several thin-layer chromatographic systems. Treatment of [(14)C]mannose oligosaccharide pyrophosphoryl dolichol with 10% (w/v) NH(4)OH at 100 degrees for 1 hr resulted in the formation of a water-soluble radioactive oligosaccharide phosphate which was isolated and characterized as [Man](5) --> [GlcNAc --> GlcNAc --> P. Incubation of [(14)C]mannose-oligosaccharide pyrophosphoryl dolichol with myeloma microsomal preparations results in the transfer, presumably, of the entire oligosaccharide to endogenous protein. Kinetic studies indicate that the dolichol derivatives serve as intermediates in the glycosylation of protein as follows: [Formula: see text]

Animals↗
PubMed1988

Separation of positional isomers of oligosaccharides and glycopeptides by high-performance anion-exchange chromatography with pulsed amperometric detection.

High-performance anion-exchange (HPAE) chromatography under alkaline conditions (pH congruent to 13) has been found to efficiently separate neutral oligosaccharides (triose to undecaose) according to molecular size, sugar composition, and linkage of monosaccharide units. The method was able to resolve 1----3, 1----4, and 1----6 positional isomers of neutral oligosaccharides, which are defined as having the same number, type, sequence, and anomeric configurations of monosaccharides but differing in the linkage position of a single sugar. From correlating structural features of different oligosaccharides and retention times, we deduced that at least two factors are operative to determine the superior resolution of oligosaccharides by this type of chromatography: (i) the relative acidities of the hydroxyl groups and (ii) the accessibility of oxyanions of the oligosaccharides to the functional groups of the stationary phase. Splitting of peaks attributable to mutarotation was not observed. Reducing oligosaccharides were much more retained than their reduced counterparts. Linkage of Fuc(alpha 1-3) to GlcNAc of oligosaccharides markedly decreased retention times. Positional isomers of two branched monosaccharides, which differed by 1----6 and 1----4 linkages, were widely separated. The separation of 1----3 and 1----4 positional isomers of both tetrasaccharides and glycopeptides containing undecasaccharides demonstrated the significant improvement in resolution of HPAE compared to previous chromatographic methods by either reverse-phase or amine-bonded stationary phases. Picomole quantities of underivatized oligosaccharides have been detected by triple-pulse amperometric detection, which produced similar responses for a wide range of structures. Quantification of two triantennary glycopeptides from bovine fetuin by using either detector response or 1H NMR was comparable. The N-glycanase-catalyzed release of two 1----4 and 1----3 positional isomers of an undecasaccharide from a tryptic glycopeptide of bovine fetuin could be observed and quantified by direct injection of the enzyme mixture into the chromatograph.

Carbohydrate Conformation↗
PubMed1995

Expression of human thyrotropin in cell lines with different glycosylation patterns combined with mutagenesis of specific glycosylation sites. Characterization of a novel role for the oligosaccharides in the in vitro and in vivo bioactivity.

We used a novel approach to study the role of the Asn-linked oligosaccharides for human thyrotropin (hTSH) activity. Mutagenesis of Asn (N) within individual glycosylation recognition sequences to Gln (Q) was combined with expression of wild type and mutant hTSH in cell lines with different glycosylation patterns. The in vitro activity of hTSH lacking the Asn alpha 52 oligosaccharide (alpha Q52/TSH beta) expressed in CHO-K1 cells (sialylated oligosaccharides) was increased 6-fold compared with wild type, whereas the activities of alpha Q78/TSH beta and alpha/TSH beta Q23 were increased 2-3-fold. Deletion of the Asn alpha 52 oligosaccharide also increased the thyrotropic activity of human chorionic gonadotropin, in contrast to previous findings at its native receptor. The in vitro activity of wild type hTSH expressed in CHO-LEC2 cells (sialic acid-deficient oligosaccharides), CHO-LEC1 cells (Man5GlcNAc2 intermediates), and 293 cells (sulfated oligosaccharides) was 5-8-fold higher than of wild type from CHO-K1 cells. In contrast to CHO-K1 cells, there was no difference in the activity between wild type and selectively deglycosylated mutants expressed in these cell lines. Thus, in hTSH, the oligosaccharide at Asn alpha 52 and, specifically, its terminal sialic acid residues attenuate in vitro activity, in contrast to the previously reported stimulatory role of this chain for human chorionic gonadotropin and human follitropin activity. The increased thyrotropic activity of alpha Q52/CG beta suggests that receptor-related mechanisms may be responsible for these differences among the glycoprotein hormones. Despite their increased in vitro activity, alpha Q52/TSH beta, and alpha Q78/TSH beta from CHO-K1 cells had a faster serum disappearance rate and decreased effect on T4 production in mice. These findings highlight the importance of individual oligosaccharides in maintaining circulatory half-life and hence in vivo activity of hTSH.

Animals↗
PubMed1997

The phosphorylation pattern of oligosaccharides in secreted procathepsin D is glycosylation site-specific and independent of the expression of mannose 6-phosphate receptors.

Mammalian cells contain two types of mannose 6-phosphate receptors (MPR), MPRs 46 and 300, that contribute with variable efficiency to the sorting of individual lysosomal proteins. To evaluate the role of phosphorylated oligosaccharides for the sorting efficiency by either of the two receptors, the structure of phosphorylated oligosaccharides on lysosomal proteins escaping sorting in cells lacking MPR 46 and/or MPR 300 was analyzed. Procathepsin D was chosen as a model because it is sorted efficiently via MPR 300 and poorly via MPR 46 and contains a distinct and highly heterogenous mixture of phosphorylated oligosaccharides at either of its two N-glycosylation sites. Both MPRs 46 and 300 were found to have a minor but distinct preference for forms of procathepsin D and other lysosomal proteins containing oligosaccharides with two phosphomonoesters. However, the phosphorylation of oligosaccharides in procathepsin D and other lysosomal proteins that escape sorting in control cells or in cells lacking MPR 46 and/or MPR 300 was strikingly similar, and oligosaccharides with two phosphomonoesters represented the major oligosaccharide species. We conclude from these results that the position of the position of the phosphate groups, the structure of the underlying oligosaccharide, and/or the polypeptide backbone of lysosomal proteins have major roles in determining the affinity to MPRs.

Alkaline Phosphatase↗
PubMed1998

Role of asparagine-linked oligosaccharides in protein folding, membrane targeting, and thyrotropin and autoantibody binding of the human thyrotropin receptor.

The amino-terminal ectodomain of thyrotropin (TSH) receptor (TSHR) is heavily glycosylated with asparagine-linked (N-linked) oligosaccharides. The present studies were designed to evaluate how acquisition and processing of N-linked oligosaccharides play a role in the functional maturation of human TSHR. A glycosylation inhibitor tunicamycin, which inhibits the first step of N-linked glycosylation (acquisition of N-linked oligosaccharides), and a series of mutant Chinese hamster ovary (CHO)-Lec cells defective in the different steps of glycosylation processing were used. Inhibition of acquisition of N-linked oligosaccharides by tunicamycin treatment in CHO cells stably expressing TSHR produced nonglycosylated TSHR, which was totally nonfunctional. In contrast, all of the TSHRs synthesized in mutant CHO-Lec1, 2, and 8 cells (mannose-rich, sialic acid-deficient, and galactose-deficient oligosaccharides, respectively) bound TSH and produced cAMP in response to TSH with an affinity and an EC50 similar to those in TSHR expressed in parental CHO cells (CHO-TSHR; sialylated oligosaccharides). However, Lec1-TSHR and Lec2-TSHR were not efficiently expressed on the cell surface, whereas the expression levels of Lec8-TSHR and CHO-TSHR were essentially identical. All of the TSHRs expressed in CHO-Lec cells cleaved into two subunits. Finally, anti-TSHR autoantibodies from Graves' patients interacted with all of the TSHRs harboring different oligosaccharides to a similar extent. These data demonstrate that acquisition and processing of N-linked oligosaccharides of TSHR appear to be essential for correct folding in the endoplasmic reticulum and for cell surface targeting in the Golgi apparatus. We also show that complex type carbohydrates are not crucially involved in the interaction of TSHR with TSH and anti-TSHR autoantibodies.

Animals↗
PubMed2002

Hepatocyte growth factor/scatter factor binds to small heparin-derived oligosaccharides and stimulates the proliferation of human HaCaT keratinocytes.

Hepatocyte growth factor/scatter factor (HGF/SF) acts via a dual receptor system consisting of the MET tyrosine kinase receptor and heparan sulfate or dermatan sulfate proteoglycans. In optical biosensor binding assays, competition by oligosaccharides for binding of HGF/SF to immobilized heparin showed that disaccharides failed to compete, whereas tetrasaccharides inhibited HGF/SF binding (IC(50) 8 microg/ml). The inhibitory potency of the oligosaccharides increased as their length increased by successive disaccharide units, to reach a maximum (IC(50) 1 microg/ml) at degree of polymerization (dp) 10. In binding assays, HGF/SF was found to bind directly to oligosaccharides as small as dp 4, and the binding parameters were similar for oligosaccharides of dp 4-14 (k(a) 2.2-45.3 x 10(6) m(-1) s(-1), k(d) 0.033-0.039 s(-1), and K(d) 9-16 nm). In human keratinocytes, HGF/SF stimulated DNA synthesis, and this was dependent on a sustained phosphorylation of p42/44(MAPK). In chlorate-treated and hence sulfated glycosaminoglycan-deficient HaCaT cells, the stimulation of DNA synthesis by HGF/SF was almost abolished. Heparin-derived oligosaccharides from dp 2 to dp 24 were added together with HGF/SF to chlorate-treated cells to determine the minimum size of oligosaccharides able to restore HGF/SF activity. At restricted concentrations of oligosaccharides (4 ng/ml), HGF/SF required decasaccharides, whereas at higher concentrations (100 ng/ml) even tetrasaccharides were able to partly restore DNA synthesis. The results suggest that HGF/SF binds to a tetrasaccharide and that although this is sufficient to enable the stimulation of DNA synthesis, longer oligosaccharides are more efficient, perhaps by virtue of their ability to bind more easily other molecules.

Binding, Competitive↗
PubMed1992

Structures of high-mannose and complex oligosaccharides of mouse TSH and free alpha-subunits after in vitro incubation of thyrotropic tissue with TRH.

To determine whether incubation of mouse thyrotropic tissue with TRH in vitro influenced the oligosaccharide structure of TSH, thyrotropic tumor tissue or pituitary tissue was incubated in vitro with [3H]mannose or with [35S]sulfate and [3H]methionine, in the absence or presence of TRH for times up to 24 h. [3H]mannose-labeled oligosaccharides from intracellular TSH and free alpha-subunits were analyzed by paper chromatography, and were predominantly Man9GlcNAc and Man8GlcNAc units both in the absence and presence of TRH. The [35S]sulfate/[3H]methionine ratio in secreted molecules was greater for TSH than for free alpha-subunits; within TSH heterodimers the ratio was greater for beta-subunits than alpha-subunits. The [35S]/[3H] ratio was not altered in TSH or free alpha-subunits by TRH. Analyses of [3H]mannose-labeled charged oligosaccharides by HPLC anion-exchange chromatography revealed similar types of oligosaccharides present on TSH subunits and free alpha-subunits (having one or two sulfate residues, one or two sialic acid residues, or both a sulfate and a sialic acid residue). These charged oligosaccharides occurred in different proportions on TSH subunits compared to free alpha-subunits, and also differed depending on whether the tissue source was tumorous or nontumorous. The proportions of oligosaccharide unit types were not altered by TRH. Thus, while this study provided information concerning the high-mannose and complex oligosaccharides of mouse TSH, there was no evidence that short incubations of tissues with TRH in vitro caused modulation of TSH oligosaccharide structures.

Animals↗
PubMed1998

Degradation of misfolded endoplasmic reticulum glycoproteins in Saccharomyces cerevisiae is determined by a specific oligosaccharide structure.

In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal alpha1,2 glucose and the first alpha1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man8GlcNAc2 oligosaccharide. Reduced rate of degradation was mainly observed for misfolded CPY bearing Man6GlcNAc2, Man7GlcNAc2 and Man9GlcNAc2 oligosaccharides, whereas Man8GlcNAc2 and, to a lesser extent, Man5GlcNAc2 oligosaccharides supported degradation. These results suggest a role for the Man8GlcNAc2 oligosaccharide in the degradation process. They may indicate the presence of a Man8GlcNAc2-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae.

Base Sequence↗
PubMed1993

Biological roles of oligosaccharides: all of the theories are correct.

Many different theories have been advanced concerning the biological roles of the oligosaccharide units of individual classes of glycoconjugates. Analysis of the evidence indicates that while all of these theories are correct, exceptions to each can also be found. The biological roles of oligosaccharides appear to span the spectrum from those that are trivial, to those that are crucial for the development, growth, function or survival of an organism. Some general principles emerge. First, it is difficult to predict a priori the functions a given oligosaccharide on a given glycoconjugate might be mediating, or their relative importance to the organism. Second, the same oligosaccharide sequence may mediate different functions at different locations within the same organism, or at different times in its ontogeny or life cycle. Third, the more specific and crucial biological roles of oligosaccharides are often mediated by unusual oligosaccharide sequences, unusual presentations of common terminal sequences, or by further modifications of the sugars themselves. However, such oligosaccharide sequences are also more likely to be targets for recognition by pathogenic toxins and microorganisms. As such, they are subject to more intra- and inter-species variation because of ongoing host-pathogen interactions during evolution. In the final analysis, the only common features of the varied functions of oligosaccharides are that they either mediate 'specific recognition' events or that they provide 'modulation' of biological processes. In so doing, they generate much of the functional diversity required for the development and differentiation of complex organisms, and for their interactions with other organisms in the environment.

Glycoconjugates↗
PubMed1993

Novel Asn-linked oligosaccharides terminating in GalNAc beta (1-->4)[Fuc alpha (1-->3)]GlcNAc beta (1-->.) are present in recombinant human protein C expressed in human kidney 293 cells.

Recombinant human Protein C (rHPC), expressed in human kidney 293 cells, has a higher anticoagulant activity than plasma HPC, while its in vivo circulatory half-life is essentially unaltered compared to that of the natural protein. In seeking to elucidate the molecular basis for the improved efficacy of the recombinant antithrombotic drug, we focused on the carbohydrate moiety of rHPC. Protein C is a heavily post-translationally modified serine protease with four N-glycosylation sites. Glycosyl composition analysis of rHPC revealed a 5-fold higher fucose content and a 2-fold lower sialic acid content compared to plasma HPC. In addition, we found that rHPC contains N-acetylgalactosamine (2.6 mol GalNAc/mol rHPC) in its Asn-linked oligosaccharides, while plasma HPC is devoid of GalNAc. The Asn-linked oligosaccharides of rHPC were released by N-glycanase and separated into 25 fractions by high-pH anion-exchange chromatography. The most abundant oligosaccharides were structurally characterized by glycosyl composition and linkage analysis, in conjunction with 1H-NMR spectroscopy at 600 MHz. The structure of the major neutral oligosaccharide in rHPC was determined to be: [formula: see text] Two representatives of the sialylated oligosaccharides in rHPC are: [formula: see text] and [formula: see text] Thus, many of the Asn-linked oligosaccharides in rHPC were found to terminate in GalNAc beta (1-->4)GlcNAc beta (1-->.), in NeuAc alpha (2-->6)GalNAc beta (1-->4)GlcNAc beta (1-->.), and/or in GalNAc beta (1-->4)[Fuc alpha (1-->3)]GlcNAc beta (1-->.). Since the latter trisaccharide was first [Yan, S.B., Chao, B.Y. and Van Halbeek,H. (1992) J. Cell. Biochem., 16D, 151] observed in the Asn-linked oligosaccharides of rHPC derived from human kidney 293 cells, we propose to label the GalNAc beta-(1-->4)[Fuc alpha (1-->3)]GlcNAc beta (1-->.) terminal trisaccharide the PC-293 determinant. The PC-293-containing oligosaccharides may contribute to the higher anticoagulant activity of rHPC as compared to plasma HPC.

Asparagine↗
PubMed1995

Abnormal synthesis of dolichol-linked oligosaccharides in carbohydrate-deficient glycoprotein syndrome.

Carbohydrate-deficient glycoprotein syndrome (CDGS) is a rare metabolic disorder presenting in infancy with severe neurologic involvement and variable multisystemic abnormalities. Diagnosis relies upon the detection of abnormal serum glycoprotein isoforms on isoelectric focusing (IEF) gels. Carbohydrate structural analyses were performed on the N-linked oligosaccharides of serum alpha 1-antitrypsin (alpha-1AT) from two Danish children with classical type I CDGS. Following preparative gel electrophoresis of alpha-1AT isoforms, oligosaccharide charge and monosaccharide composition analyses revealed increased glycosylation heterogeneity in CDGS compared with normal alpha-1AT. CDGS alpha-1AT isoforms bore N-glycans co-migrating with monosialylated standards, while normal alpha-1AT oligosaccharides co-migrated with both mono- and disialylated standards. While the monosaccharide contents of normal alpha-1AT isoforms were relatively uniform, those of CDGS alpha-1AT isoforms varied widely, and many were relatively mannose enriched. The mannose-rich oligosaccharides of CDGS alpha-1AT were not typical oligomannose structures since they were not released by endo-beta-N-acetylglucosaminidase H (endo H) digestion. Metabolic labelling of CDGS fibroblasts with [3H]mannose showed lower than normal intracellular total mannose, free mannose and phosphorylated mannose species, as well as diminished [3H]mannose incorporation into dolichol-linked and protein-linked oligosaccharides. In addition, the glycans liberated from CDGS dolichol-linked oligosaccharides were significantly truncated compared with those from normal fibroblasts. These data suggest that our type I CDGS patients produce abnormal N-linked oligosaccharides due to impaired biosynthesis of dolichol-oligosaccharide precursors.

Congenital Disorders of Glycosylation↗
PubMed1994

Oligosaccharides from human milk block binding and activity of the Escherichia coli heat-stable enterotoxin (STa) in T84 intestinal cells.

Enterotoxin-producing Escherichia coli are major causes of pediatric diarrhea in developing countries. The heat-stable enterotoxin of Escherichia coli (STa) causes diarrhea by virtue of its ability to bind to and stimulate intestinal membrane-bound guanylate cyclase, generating cyclic GMP (cGMP). Previous work showed that a fucosylated oligosaccharide fraction of human milk was able to protect suckling mice from the secretory effects of STa, but the mechanism of the protection could not be determined. Oligosaccharide fractions from human milk were tested for their ability to block the biochemical effects of STa in T84 cells, a human colon carcinoma line responsive to the toxin. Total and fucosylated oligosaccharide fractions were found to inhibit STa-stimulated guanylate cyclase activity in T84 cell membranes and cGMP production in intact T84 cells by 60-80%. In addition, the total oligosaccharide fraction and the fucosylated oligosaccharide fraction inhibited 125I-STa binding significantly (17% and 27% inhibition, respectively). These findings demonstrate the protective activity of human milk oligosaccharides against STa in a human-derived cell line and show that the biochemical step blocked by oligosaccharides is STa-mediated stimulation of guanylate cyclase. This represents a novel mechanism by which human milk oligosaccharides protect against diarrhea.

Bacterial Toxins↗