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Immunomodulatory effects of breast milk oligosaccharides.

Breast milk oligosaccharides are excreted in urine in amounts that suggest that they may exist in the circulation at levels compatible with a physiological function. Some oligosaccharides have structural similarity to cellular adhesion molecules and may influence adhesion of cells in breast fed infants. In this study, breast milk oligosaccharides were purified and incubated in assays of cell adhesion. They were found to inhibit neutrophil adhesion to stimulated vascular endothelial cells in a dose dependent fashion. In contrast they enhanced platelet-neutrophil complex formation. These results indicate that breast milk oligosaccharides may play a physiological role in modulating cellular adhesion in vivo.

Absorption↗

Differentiation-associated expression of beta-N-acetylgalactosaminylated N-linked oligosaccharides in mammary epithelial cells.

Not only mammalian pituitary glycoprotein hormones but also many glycoproteins from a variety of animal species have been shown to contain N-linked oligosacchardies with the GalNAcbeta1 --> 4GlcNAc structure. Two types of beta-1,4-GalNAcT were found; one transfers N-acetylgalactosamine to acceptor oligosaccharides, which is stimulated by the hormone peptide and the other simply transfers sugar without such activation. In the case of bovine mammary membrane glycoproteins, the expression of beta-N-acetylgalactosaminylated N-linked oligosaccharides was associated with the functional differentiation of the epithelial cells. In contrast, the expression level of such oligosaccharides was much reduced in glycoprotein samples from human breast tumors compared with those from the unaffected regions. These results strongly suggest that the beta-N-acetylgalactosaminylation is one which is regulated under cellular differentiation and dedifferentiation of the mammary gland. Whether or not beta-N-acetylgalactosaminylated N-linked oligosaccharides have unique functions in addition to clearance of the hormone from the circulation remains to be elucidated.

Acetylgalactosamine↗

Health benefits of non-digestible oligosaccharides.

Non-digestible oligosaccharides are complex carbohydrates of the non-a-glucan type which, because of the configuration of their osidic bonds, resist hydrolysis by salivary and intestinal digestive enzymes. In the colon they are fermented by anaerobic bacteria. Among the non-digestible oligosaccharides, the chicory fructooligosaccharides occupy a key position and, in most european countries, they are recognised as natural food ingredients. The other major products are the short chain fructooligosaccharides and galactooligosaccharides obtained by enzymatic synthesis using sucrose and lactose as substrates respectively, the soybean oligosaccharides, the xylooligosaccharides produced by partial hydrolysis of xylans and polydextrose or pyrodextrins prepared by a chemical treatment of carbohydrates. The most well known effect of most non-digestible oligosaccharides, and in particular of the fructooligosaccharides, is the selective stimulation of the growth of Bifidobacteria thus modifying significantly the composition of the colonic microbiota. Such a modification, which has clearly been demonstrated in human volunteers, is meant to be benificial in part because it is accompanied by a significant reduction in the number of bacteria reported to have pathogenic potential. Within the framework of research and development of "functional foods", such an effect justifies a "functional claim" for fructooligosaccharides namely "bifidogenesis". They are also typical "prebiotics". Besides their bifidogenic effect, the chicory fructooligosaccharides have additional nutritional properties on digestive physiological parameters like colonic pH and stool bulking which justify their classification as dietary fibers. Moreover, in experimental models, it has also been reported that they improve the bioavailability of essentiel minerals and that they reduce serum triglyceridemia by lowering hepatic lipogenesis. Such effects demonstrate interactions between the chicory fructooligosaccharides and key functions in the body but their significance for humans still need to be proven before being used to justify additional claims.

Bifidobacterium↗

Characterization of N-linked oligosaccharides of human urinary kallikrein molecules.

The micro-heterogeneity due to varied N-linked oligosaccharides of both active- and pro-types of human urinary kallikrein (HUK) in normal subjects and some patients were investigated by the methods of serial lectin affinity chromatography and crossed affino-immunoelectrophoresis. In the case of both types of normal HUK, the species carrying tri- and/or tetra-antennary oligosaccharide(s), corefucosylated bi-antennary oligosaccharide(s), and bi-antennary oligosaccharides containing outer galactose residues and an N-acetylglucosamine residue linked beta 1,4 to a beta-linked mannose residue (bisecting N-acetylglucosamine residue) amounted to approximately 36, 33 and 17% of the total of each type of HUK, respectively. On the other hand, in some diseases, i.e. essential hypertension, Bartter's syndrome and acute pancreatitis, alterations of the chromatographic and electrophoretic patters were observed and are assumed to correspond to glycosylation changes in each HUK molecule.

Chromatography, Affinity↗

Heteronuclear NMR studies of 13C-labeled yeast cell wall beta-glucan oligosaccharides.

The structures of uniformly 13C-labeled beta-glucan octa- and undeca-oligosaccharides enzymatically prepared by the yeast cell wall glucanosyl transferase of Candida albicans were characterized by using a combination of HCCH-COSY, HCCH-TOCSY, and HMBC experiments. The oligosaccharide structures indicate that the cell wall glucanosyl transferase cleaves two glucosyl units from the reducing end of the initial linear beta(1-->3) penta-oligosaccharide and subsequently transfers the remainder to another oligosaccharide at the nonreducing end via a beta(1-->6) linkage. These results indicate that the combined action of cell wall glucanase and glucanosyl transferase activities could not only introduce intrachain beta(1-->6) linkages within a single glucan strand, but also result in cross-linking of two initially separate glucan strands with concurrent introduction of intrachain beta(1-->6) linkages. Since isolated fungal membranes only synthesize linear beta(1-->3) glucan strands, wall-associated enzymes probably participate in the assembly of the final wall glucan structure during cell growth and division.

Candida albicans↗

Effect of a phospho-oligosaccharidic putative insulin messenger on insulin release in rats.

The phospho-oligosaccharide extracted from rat liver and supposed to act as the insulin second messenger inhibits glucose-stimulated insulin release. In the present study, this phospho-oligosaccharide was found not to affect D-[U-14C]glucose oxidation and 45Ca net uptake, but to inhibit insulin release evoked by either D-glucose or 2-ketoisocaproate in isolated rat islets. The relative extent of the latter inhibition was unaffected by either the concentration of D-glucose or the presence of dibutyryl-cyclic AMP, forskolin or glucagon in the incubation medium. At variance with the inhibitory effect of clonidine, that of the phospho-oligosaccharide was resistant to both blockade of alpha 2-adrenergic receptors or pre-treatment with the toxin of Bordetella pertussis. It is speculated, therefore, that such a phospho-oligosaccharide might interfere with a distal event in the insulin secretory sequence.

Animals↗

Novel N-linked oligo-mannose type oligosaccharides containing an alpha-D-galactofuranosyl linkage found in alpha-D-galactosidase from Aspergillus niger.

Structures of oligosaccharides from Aspergillus niger alpha-D-galactosidase [EC 3.2.1.22] were studied. Purified alpha-D-galactosidase was treated with N-glycosidase F, and six kinds of oligosaccharides were isolated by gel chromatography and anion-exchange chromatography. The structures of the oligosaccharides were determined by 1H-NMR and compositional analysis to be Man5GlcNAc2, Man6GlcNAc2, Man9GlcNAc2, GlcMan9GlcNAc2, GalMan4GlcNAc2 and GalMan5GlcNAc2. From mild acid hydrolysis, methylation analysis and ROESY spectral analysis, it was ascertained that the galactosyl residue in two oligosaccharides was in the furanose form and was bound to mannose at the nonreducing end with an alpha 1-2 linkage (GalfMan4GlcNAc2 and GalfMan5GlcNAc2).

Aspergillus niger↗

Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UK delta GS1) by changing culture conditions of a lymphoblastoid cell line Namalwa KJM-1 adapted to serum-free medium.

Pro-UK delta GS1 was designed as a long-life and thrombin-resistant derivative of pro-urokinase (pro-UK) by deleting the growth factor domain of pro-UK and introducing a glycosylation site near the thrombin cleaving site for thrombin-resistance using site-directed mutagenesis. An expression plasmid for pro-UKDGS1, pIH1UK delta GS1SEd1-5 was constructed and introduced into Namalwa KJM-1, a lymphoblastoid cell line adapted to serum-free medium, and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UK delta GS1 producer (resistant to 200 nM of MTX), clone 2-9, was selected and used for further studies. Under the conventional conditions, i.e. at 37 degrees C in serum-free ITPSGF medium (based on RPMI-1640 medium), the oligosaccharide structure of pro-UK delta GS1 produced by clone 2-9 mainly consisted of fucose (Fuc)-containing biantennary complex-type oligosaccharide. Addition of dexamethasone (Dex), changed the carbohydrate contents in the media, and a shift down of incubation temperature caused a change in oligosaccharide structure of pro-UK delta GS1 from mainly Fuc-containing biantennary to mainly Fuc-containing tri- and tetraantennary complex-type oligosaccharide. The modulated pro-UK delta GS1 showed superior in vivo activity for a canine femoral thrombosis formed by inserting a copper-coil.

Amino Acid Sequence↗

Mitogenic activity of acidic fibroblast growth factor is enhanced by highly sulfated oligosaccharides derived from heparin and heparan sulfate.

The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sulfated hexasaccharide [IdoUA,2S-GlcNS,6S]2-[GlcUA-GlcNS,6S] the structural repetitive unit of lung heparin chains. On a mass basis, the effect of both heparin and oligosaccharide are equivalent whereas on a molar basis, heparin, which contains about seven hexasaccharide repeats, is more efficient. On the other hand, a pentasulfated tetrasaccharide or di- and tri-sulfated disaccharides are much less effective in potentiating aFGF activity than the hexasaccharide. If the growth factor is pre-incubated with the hexasaccharide at pH 7.2 and then exposed to pH 3.5 the 306/345 nm fluorescence ratio is similar to that of native aFGF indicating that the oligosaccharide stabilizes a native conformation of the protein. Heparan sulfates extracted from various mammalian tissues were also able to potentiate aFGF mitogenic activity. On a mass basis they were in general less efficient than heparin; however, heparan sulfate prepared from medium conditioned by 3T3 fibroblasts is more efficient than heparin both on a mass and molar basis. A highly sulfated oligosaccharide isolated after digestion of pancreas heparan sulfate with heparitinase I is more active than the intact molecule, reaching a potentiating effect equivalent to that of lung heparin, whereas an N-acetylated oligosaccharide isolated after nitrous acid degradation is inactive. These data suggest that the mitogenic activity of aFGF is primarily potentiated by interacting with highly sulfated regions of heparan sulfates chains.

Animals↗

Increased expression of highly branched N-linked oligosaccharides terminating in N-acetylglucosamine residues in neoplastic and sclerodermal chicken fibroblasts.

Although neoplastic cells often show a shift towards the expression of larger N-linked oligosaccharides compared to their normal counterparts, little consideration has been given to the possibility that these changes might be a more general phenomenon characteristic of certain neoplastic and non-neoplastic proliferative disorders. Terminal N-acetylglucosamine (GlcNAc) cluster antigen (TGCA) is an immunoreactive epitope(s) of highly branched N-linked oligosaccharides terminating in GlcNAc residues. Here we have compared the expression of this antigen in normal, neoplastic and sclerodermal chicken fibroblasts by immunomorphological methods. TGCA was detectable in only a few, if any, fibroblasts of normal chicken skin or those cultured from chicken embryos. In contrast, the antigen appeared in 15 to 30% of chicken embryo fibroblasts transformed with avian sarcoma viruses and about 50% of neoplastic fibroblasts of both Rous sarcoma virus-induced fibrosarcomas and carcinogen-induced transplantable fibrosarcomas. Significantly, TGCA was also found in most activated fibroblasts in the skin of chickens with hereditary scleroderma. These results indicate that increased expression of highly branched N-linked oligosaccharides terminating in GlcNAc residues is characteristic of both neoplastic and sclerodermal chicken fibroblasts. Investigation of this phenomenon may thus provide insight into biochemical pathways involved in neoplastic transformation and pathogenesis of a number of non-neoplastic proliferative connective tissue disorders such as scleroderma. Moreover, changes in the expression of TGCA-positive oligosaccharides (or their modified biochemical counterparts in mammalian species) may have considerable value for diagnosis of several connective tissue diseases.

Acetylglucosamine↗

Basement membrane invasion by first trimester human trophoblast: requirement for branched complex-type Asn-linked oligosaccharides.

Trophoblast cells of normal first trimester human placenta share with malignant tumor cells the ability for significant cellular proliferation and invasion of basement membranes. Because tumor cell metastasis in vivo and invasion of basement membranes in vitro have recently been shown to require the expression of -GlcNAc beta 1-6 Man alpha 1-6 Man beta 1-branched complex type Asn-linked oligosaccharides in tumor cell surface glycoproteins, we decided to determine if such structures were also necessary for invasion by trophoblast cells. We report here that invasive first trimester trophoblasts express leukoagglutinin-reactive beta 1-6 branched Asn-linked oligosaccharides on their surface. Moreover, basement membrane invasion by trophoblast was significantly inhibited by pretreating the cells with swainsonine, a non-toxic inhibitor of Golgi alpha-mannosidase II which blocks beta 1-6 branching of Asn-linked oligosaccharides. The first trimester trophoblast cells pretreated with swainsonine attached more avidly to the amnion basement membrane and to an extracellular matrix (ECM) preparation compared to control non-treated erophoblast cells. Swainsonine treatment did not inhibit secretion of gelatinase or plasminogen activator activities by trophoblast cells. These results suggest that expression of beta 1-6 branched oligosaccharides in trophoblast cells may be functionally important for the implantation and placentation processes by reducing cell adhesion to ECM and thereby facilitating trophoblast cell invasion.

Alkaloids↗

Oligosaccharides and glycoconjugates in human milk: their role in host defense.

Human milk contains an extremely high concentration of complex carbohydrates, especially oligosaccharides, the third most abundant solid constituent of human milk. The value of human milk nutrients to infants is now widely recognized, and a role for the secretory antibodies of human milk in the defense of the infant is generally accepted. However, a function for nonimmunoglobulin milk protective factors, many of them non-nutrients, in providing for the defense of the nursling is only now beginning to be appreciated. Prominent among postulated defense agents are the milk oligosaccharides and glycoconjugates. Their complex carbohydrate structures are thought to be assembled by the same enzymes, the glycosyltransferases, that synthesize the cell surface glycoconjugates often used as receptors by pathogens. Some milk oligosaccharides and glycoconjugates may protect the nursing infant by acting as receptor homologs, inhibiting the binding of enteropathogens to their host receptors. Ongoing research is linking specific carbohydrate structures with protection against specific pathogens. Current information regarding the composition, protective activities, and protective mechanisms of the milk glycolipids, glycoproteins, mucins, glycosaminoglycans, and oligosaccharides is reviewed.

Female↗

Oligosaccharide and creatine supplementation on glucose and urea nitrogen in blood and serum creatine kinase in basketball athletes.

The effects of oligosaccharide and creatine (Cr) supplementation on glucose, lactic acid and urea nitrogen levels in blood and activity of serum creatine kinase (CK) were explored. Twenty CUBA male athletes were divided into 4 groups: group A (supplementation of Cr alone), group B (supplementation of oligosaccharide), group C (supplementation of oligosaccharide and Cr) and group D (placebo control group). By using orthogonal L4 table (2(3)), the experiment was performed. There were factors including oligosaccharide (carbohydrate, CHO), Cr and their correlation. Each factor had two levels: supplementation and no-supplementation. The results showed that the supplementation of CHO or Cr alone, combined supplementation of CHO and Cr could significantly reduce the glucose, urea nitrogen levels in blood and serum CK activity after competition in the athletes. Moreover, the effects of combined supplementation of CHO and Cr were more satisfactory. It was concluded that supplementation of CHO and Cr could promote the recovery of physical performance and athletic abilities after athletics in basketball athletes.

Athletes↗

Structural characterization of neutral oligosaccharide mixtures through a combination of capillary electrochromatography and ion trap tandem mass spectrometry.

A CEC/ESI-MS/MS combined system has been developed for the separation and on-line structural analysis of neutral oligosaccharides. Various types of isomeric oligosaccharides were first successfully separated by CEC using polar monolithic columns, while the on-line tandem mass spectrometry has been explored to differentiate and elucidate the structures of isomeric oligosaccharides. The experimentally obtained tandem spectra usually provide sequence, branching, and linkage information. Oligosaccharide isomers with a different monomeric composition and branching showed different patterns of glycosidic linkage cleavage (B- and Y-ion series), allowing us to deduce their sequence and branching points. Isomers with different linkages were distinguished by identifying cross-ring fragment ions (A-ion series). While (1-->4) linkages yielded dominant (0,2)A ions, (1-->6) linkages showed an extensive and complete cross-ring cleavage series: (0,2)A, (0,3)A, and (0,4)A ions. Although the anomeric configurations and monosaccharide identification are rarely obtained from tandem MS, the relevant mixture components can be completely resolved with high-efficiency CEC columns featuring a polar functionality.

Carbohydrate Conformation↗

In vitro evaluation of the prebiotic activity of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel.

The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).

Bacteria↗

Mutational studies on endo-beta-N-acetylglucosaminidase D which hydrolyzes core portion of asparagine-linked complex type oligosaccharides.

Endo-beta-N-acetylglucosaminidase D (Endo D) produced by Streptococcus pneumoniae hydrolyzes the di-N-acetylchitobiose structure in the core of complex-type asparagine-linked oligosaccharides, and has a molecular weight of 180 kDa. A truncated Endo D of 102 kDa in which 134 N-terminal amino acids and 599 C-terminal amino acids were deleted, still retained the enzymatic activity. The truncated Endo D has specificity indistinguishable from the intact enzyme, and also acted on the core structure of asparagine-linked oligosaccharides attached to intact IgG. Because of its lower molecular weight, the truncated enzyme may be useful as a tool for protein deglycosylation. The entire region of the truncated Endo D had 32% sequence identity to endo- beta-N-acetylglucosaminidase BH (Endo BH) from Bacillus halodurans, which acted on high-mannose type oligosaccharides. Chimeric constructs of the truncated Endo D and Endo BH showed no activity. Glutamic acid 324 (E 324) in Endo D is conserved in Endo BH and Endo M, and is an essential amino acid in Endo M. Mutation of E324 abolished Endo D activity. The specificity of Endo D for complex type oligosaccharides is probably defined by multiple domains in the Endo D structure.

Amino Acid Sequence↗

Chromatography of permethylated oligosaccharide alditols.

A new chromatographic method which enables the separation of permethylated oligosaccharide alditols has been developed. The method entails chromatography on precoated silica gel plates using benzene-methanol (16:1, v/v or 10:1 v/v) as developing solvent. Separations of disaccharides were obtained on the basis of glycosidic linkage and anomeric configuration; the method can accomodate oligosaccharides containing up to 15 glycose units. The combined use of thin-layer chromatography and gas-liquid chromatography provides a powerful approach for the characterization of oligosaccharides. Retention indices are given of permethylated oligosaccharide alditols on a fused-silica capillary column bonded with DB-1.

Chemical Phenomena↗

Structural characterization of oligosaccharides by high-performance liquid chromatography, fast-atom bombardment-mass spectrometry, and exoglycosidase digestion.

A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5-1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequence and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will allow almost the complete structure of small amounts of oligosaccharides to be determined.

Chemical Phenomena↗