Characterization of oligosaccharides from the urine of loco-intoxicated sheep

Two major oligosaccharides were isolated by preparative HPLC from the urine of a locoweed-fed sheep. Analysis by gas-liquid chromatography and mass-spectrometry indicated compositions of (Man)4(GlcNAc)2 and (Man)5(GlcNAc)2, respectively. Structures were determined by digestion with alpha-D-mannosidase and endo-beta-N-acetylglucosaminidases D and H, and comparison of the products by HPLC with synthetic standards, and oligosaccharides isolated from human mannosidosis urine. Incubation with an exo-beta-N-acetylglucosaminidase was without effect.     

A general approach to desalting oligosaccharides released from glycoproteins

Desalting of sugar samples is essential for the success of many techniques of carbohydrate analysis such as mass spectrometry, capillary electrophoresis, anion exchange chromatography, enzyme degradation and chemical derivatization. All desalting methods which are currently used have limitations: for example, mixed-bed ion-exchange columns risk the loss of charged sugars, precipitation of salt by a non-aqueous solvent can result in co-precipitation of oligosaccharides, and gel chromatography uses highly crosslinked packings in which separation of small oligosaccharides is difficult to achieve. We demonstrate that graphitized carbon as a solid phase extraction cartridge can be used for the purification of oligosaccharides (or their derivatives) from solutions containing one or more of the following contaminants: salts (including salts of hydroxide, acetate, phosphate), monosaccharides, detergents (sodium dodecyl sulfate and Triton X-100), protein (including enzymes) and reagents for the release of oligosaccharides from glycoconjugates (such as hydrazine and sodium borohydride). There is complete recovery of the oligosaccharides from the adsorbent which can also be used to fractionate acidic and neutral glycans. Specific applications such as clean-up of N-linked oligosaccharides after removal by PNGase F and hydrazine, desalting of O-linked glycans after removal by alkali, on-line desalting of HPAEC-separated oligosaccharides and -eliminated alditols prior to electrospray mass spectrometry, and purification of oligosaccharides from urine are described.     

Interference with glycosylation of glycoproteins. Inhibition of formation of lipid-linked oligosaccharides in vivo.

Influenza-virus-infected cells were labelled with radioactive sugars and extracted to give fractions containing lipid-linked oligosaccharides and glycoproteins. The oligosaccharides linked to lipid were of the 'high-mannose' type and contained glucose. In the glycoprotein fraction, radioactivity was associated with virus proteins and found to occur predominantly in the 'high-mannose' type of glycopeptides. In the presence of the inhibitors 2-deoxy-D-glucose, 2-deoxy-2-amino-D-glucose (glucosamine), 2-deoxy-2-fluoro-D-glucose and 2-deoxy-2-fluoro-D-mannose incorporation of radiolabelled sugars into lipid- and protein-linked oligosaccharides was decreased. Kinetic analysis showed that the inhibitors affected first the assembly of lipid-linked oligosaccharides and then protein glycosylation after a lag period. During inhibition by deoxyglucose and the fluoro sugars lipid-linked oligosaccharides were formed that contained oligosaccharides of decreased molecular weight. No such aberrant forms were found during inhibition by glucosamine. In the case of inhibition by deoxyglucose it was shown that the aberrant oligosaccharides were not transferred to protein. Inhibition of formation of lipid-linked oligosaccharides by deoxyglucose and fluoro sugars was antagonized by mannose, in which case oligosaccharides of normal molecular weight were formed. The inhibition by glucosamine was reversed by its removal from the medium. The reversible effects of these inhibitors exemplify their usefulness as tools in the study of glycosylation processes.     

The carbohydrate moieties of human urinary ribonuclease UL

Ribonuclease UL purified from pooled human urine contains approximately 20.7% of neutral sugar and 7.8% of aminosugar. All sugars were quantitatively released as oligosaccharides on hydrazinolysis. The oligosaccharides were converted to tritium-labeled oligosaccharides on reduction with NaB3H4. The radioactive oligosaccharide fraction was separated into a neutral and an acidic fraction on paper electrophoresis. All oligosaccharides in the acidic fraction could be converted to neutral oligosaccharides with the release of one sialic acid residue by sialidase digestion. Both fractions were shown to be mixtures of more than fourteen oligosaccharides by gel permeation chromatography. Structural studies on these oligosaccharides involving sequential exoglycosidase digestion in combination with methylation analysis revealed that ribonuclease UL contains sialylated and non-sialylated mono, bi-, tri-, and tetraantennary complex type sugar chains with N-acetyllactosamine outer chains, and tri- and tetraantennary complex type sugar chains with various numbers of Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains. An important finding was that all sialic acid residues in the acidic oligosaccharides only occur as the Sia alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 group. Both fucosylated and non-fucosylated trimannosyl cores were found among the asparagine-linked sugar chains of ribonuclease UL.     

Influence of synthetic sheep urine on the microbial biomass,activity and community structure in two pastures in the Scottish uplands

The impact of urine on the microbial biomass, activity and community structure was compared in the soil beneath two pastures in the Scottish uplands; Fasset, a natural Agrostis capillaris–Festuca ovina–Galium saxatile grassland and Strathfinella, a semi-natural grassland, improved with fertiliser addition. Community level physiological profiles (CLPP) were used to characterise the microbial communities. The utilisation of sugars, oligosaccharides, alcohols, carboxylic acids, long chain aliphatic acids, acidic, basic and neutral amino acids, amide N, phenolic acids and long chain aliphatic acids was used to compare the soils and the impact of synthetic urine addition. In the untreated soils, the utilisation of all the substrates decreased from the first week in May through to October. Averaged over all times and urine treatment, the potential utilisation of all substrates except for phenolic acids, long chain aliphatic acids and carboxylic acids was greater in the improved and more intensively grazed Strathfinella site. When averaged over all sample times, urine increased the utilisation of sugars, oligosaccharides, basic amino acids and amide N and the increases were greater in the unimproved, less intensively grazed, Fasset soil than that at Strathfinella. The effect of urine tended to be greatest during the period between 2 and 5 weeks after urine addition when utilisation of alcohols, acidic and neutral amino acids was also increased. Microbial biomass C in the control soils was 155.9 and 112.7 g C m⁻² at Fasset and Strathfinella, respectively. Values did not change significantly with time and were unchanged by the addition of urine. However, urine addition significantly increased basal respiration rates at Fasset and decreased them at Strathfinella. Urine also increased bacterial numbers in both soils, but had no consistent effect on fungi or yeasts. The significance of these findings for studies of soil microbial community structure and activity in grazed upland grasslands is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glucosylated free oligosaccharides are biomarkers of endoplasmic- reticulum alpha-glucosidase inhibition

The inhibition of ER (endoplasmic reticulum) alpha-glucosidases I and II by imino sugars, including NB-DNJ (N-butyl-deoxynojirimycin), causes the retention of glucose residues on N-linked oligosaccharides. Therefore, normal glycoprotein trafficking and processing through the glycosylation pathway is abrogated and glycoproteins are directed to undergo ERAD (ER-associated degradation), a consequence of which is the production of cytosolic FOS (free oligosaccharides). Following treatment with NB-DNJ, FOS were extracted from cells, murine tissues and human plasma and urine. Improved protocols for analysis were developed using ion-exchange chromatography followed by fluorescent labelling with 2-AA (2-aminobenzoic acid) and purification by lectin-affinity chromatography. Separation of 2-AA-labelled FOS by HPLC provided a rapid and sensitive method that enabled the detection of all FOS species resulting from the degradation of glycoproteins exported from the ER. The generation of oligosaccharides derived from glucosylated protein degradation was rapid, reversible, and time- and inhibitor concentration-dependent in cultured cells and in vivo. Long-term inhibition in cultured cells and in vivo indicated a slow rate of clearance of glucosylated FOS. In mouse and human urine, glucosylated FOS were detected as a result of transrenal excretion and provide unique and quantifiable biomarkers of ER-glucosidase inhibition.     

Structural insights into rice BGlu1 beta-glucosidase oligosaccharide hydrolysis and transglycosylation

The structures of rice BGlu1 β-glucosidase, a plant β-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 Å and 1.55 Å resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) β-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long β-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa β-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-β-glucanase/β-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/β-II β-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.     

Granulocytic differentiation of HL-60 cells is associated with increase of poly-N-acetyllactosamine in Asn-linked oligosaccharides attached to human lysosomal membrane glycoproteins

HL-60 cells were induced to differentiate into granulocytic cells by dimethyl sulfoxide, and structures of Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins (lamp-1 and lamp-2) were elucidated before and after differentiation. Lamp-1 and lamp-2 were immunoprecipitated from the cells after labeling with radioactive sugars, and glycopeptides were prepared. The structures of glycopeptides obtained after serial lectin-affinity chromatography were elucidated by endo-beta-galactoside and methylation analysis. Glycopeptides bound to tomato lectin-Sepharose were found to be tetraantennary oligosaccharides that contain two or three poly-N-acetyllactosaminyl chains, of which one side chain contains three or more N-acetyllactosaminyl repeats, whereas those bound to Datura stramonium agglutinin-Sepharose were found to be tetraantennary oligosaccharides containing one or two short poly-N-acetyllactosaminyl side chains. Glycopeptides that were not bound to concanavalin A, tomato lectin, or D. stramonium agglutinin were found to be triantennary oligosaccharides with a negligible amount of poly-N-acetyllactosaminyl side chains. Comparison of Asn-linked oligosaccharides from undifferentiated and differentiated HL-60 cells reveals the following features. First, the number of Asn-linked oligosaccharides containing poly-N-acetyllactosaminyl side chains increases dramatically with a concomitant decrease in less complex Asn-linked oligosaccharides after differentiation. Second, the number of poly-N-acetyllactosaminyl side chains per Asn-linked oligosaccharides increases significantly. These increases in poly-N-acetyllactosamine were associated with increased activity of UDP-GlcNAc:beta-D-Gal-beta 1----3-N-acetylglucosaminyltransferase "extension enzyme," a key enzyme in the formation of poly-N-acetyllactosamines. Furthermore, the increased amount of poly-N-acetyllactosamine in lamp-1 and lamp-2 resulted in longer half-lives of lamp-1 and lamp-2 in differentiated HL-60 cells. These results suggest strongly that the differentiation of HL-60 cells into more phagocytic cells is associated with an increase in the complexity of Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins, which in turn may play a role in stabilizing lysosomes.     

Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors

We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan (beta 4-glucuronic acid-beta 3-N-acetylglucosamine (HA)) oligosaccharides. Potential medical applications for HA oligosaccharides (approximately 10-20 sugars in length) include killing cancerous tumors and enhancing wound vascularization. Previously, the lack of defined oligosaccharides has limited the exploration of these sugars as components of new therapeutics. The Pasteurella multocida HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (approximately 1-100 sugars/s), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and N-acetylglucosamine transferase). The two resulting enzymes were purified and immobilized individually onto solid supports. The two types of enzyme reactors were used in an alternating fashion to produce extremely pure sugar polymers of a single length (up to HA20) in a controlled, stepwise fashion without purification of the intermediates. These molecules are the longest, non-block, monodisperse synthetic oligosaccharides hitherto reported. This technology platform is also amenable to the synthesis of medicant-tagged or radioactive oligosaccharides for biomedical testing. Furthermore, these experiments with immobilized mutant enzymes prove both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.     

A defect in exodegradative pathways provides insight into endodegradation of heparan and dermatan sulfates

Within cells, dermatan sulfate (DS) and heparan sulfate (HS) are degraded in two steps. The initial endohydrolysis of these polysaccharides is followed by the sequential action of lysosomal exoenzymes to reduce the resulting oligosaccharides to monosaccharides and inorganic sulfate. Mucopolysaccharidosis (MPS) type II is a lysosomal storage disorder caused by a deficiency of the exoenzyme iduronate-2-sulfatase (I2S). Consequently, partially degraded fragments of DS and HS have been shown to accumulate in the lysosomes of affected cells and are excreted in the urine. Di- to hexadecasaccharides, isolated from the urine of a MPS II patient using anion exchange and gel filtration chromatography, were identified using electrospray ionization-tandem mass spectrometry (ESI-MS/MS). These oligosaccharides were shown to have non-reducing terminal iduronate-2-sulfate residues by digestion with recombinant I2S. A pattern of growing oligosaccharide chains composed of alternating uronic acid and N-acetylhexosamine residues was identified and suggested to originate from DS. A series of oligosaccharides consisting of hexosamine/N-acetylhexosamine alternating with uronic acid residues was also identified and on the basis of the presence of unacetylated hexosamine; these oligosaccharides are proposed to derive from HS. The presence of both odd and even-length oligosaccharides suggests both endo-beta-glucuronidase and endo-N-acetylhexosaminidase activities toward both glycosaminoglycans. Furthermore, the putative HS oligosaccharide structures identified indicate that heparanase activities are directed toward regions of both low and high sulfation, while the N-acetylhexosaminidase activity acted only in regions of low sulfation in this polysaccharide.     

Towards understanding the interaction between oligosaccharides and water molecules

Complex carbohydrates are implicated in many important biological processes, and have a strong interaction with water. This close interplay with molecular water through multiple hydroxyls may be an integral part of their emergent structure and dynamics, as selected during evolution. Using molecular dynamics simulations with explicit water the interactions at the linkages within a variety of oligosaccharides are investigated and contrasted, in order to establish correlations between linkage orientation, sugar epimerization, and water interaction. In particular, interactions at alpha linkages, and between mannose and glucose residues, that are common in oligosaccharides are considered. Sugars joined by alpha linkages at the 2-, 3-, and 6-position were found to interact via a combination of weak hydrogen-bonds and water-bridges, which is dependent on the epimerization state of the sugars. Due to their three-dimensional structure, they are also likely to interact with noncontiguous sugar residues in an oligosaccharide, which can lead to ordered structures through the exclusion of water. On the other hand, beta linkages (to 3- and 4-position) maintain strong hydrogen-bonds, have a limited ability to be involved in water-bridges, and predominantly interact with the directly attached sugars. Therefore, sequences of alpha-linked sugars form compact, branched structures that have conformational flexibility, and beta linkages form extended, relatively rigid structures, suitable for structural molecules, and at the termini of protein bound oligosaccharides. These results provide further tentative ties between chemical structure, water interactions, and the emergent form and function of specific sugars and linkages in oligosaccharides.     

Structures, function, and transformational changes of the sugar chains of glycohormones

Human chorionic gonadotropin (hCG), human luteinizing hormone, human thyroid-stimulating hormone, and human follicle-stimulating hormone are closely related family of proteins which share a common alpha-subunit. However, their sugar moieties are quite different. hCG contains five acidic asparagine-linked sugar chains. These five sugar chains are derived by sialylation from three neutral oligosaccharides: two biantennary (N-1 and N-2) and one monoantennary (N-3) complex-type oligosaccharides. Although hCG purified from the urine of pregnant women is more enriched in sialylated sugar chains than that purified from placenta, the molar ratio of N-1, N-2, and N-3 of these two hCGs are the same (1:2:1). Comparative study of the sugar moieties of the alpha- and beta-subunits of hCG revealed that alpha contains 1 mol each of N-2 and N-3, while beta contains 1 mol each of N-1 and N-2. This specific distribution of oligosaccharides at the four asparagine loci of the hCG molecule is now helping us to consider the functional role of the sugar moiety of glycohormones. hCG is produced not only by the trophoblast but also by various trophoblastic diseases. The hCGs purified from the urine of patients with hydatidiform mole contain the same oligosaccharides as normal hCG. However, those from the urine of choriocarcinoma patients contain five additional neutral oligosaccharides. In contrast, hCGs from invasive-mole patients contain three of the five oligosaccharides, specifically found in choriocarcinoma hCGs. The malignant transformational change of the sugar moiety of hCG can be explained by an increase of a fucosyltransferase, which forms the Fuc alpha 1----6GlcNAc group and by ectopic expression and subsequent modification of N-acetylglucosaminyltransferase IV. The appearance of tumor-specific sugar chains of hCG has been used to develop a new diagnostic method for invasive mole and choriocarcinoma.     

Comparison of methods of immobilization to enzyme-linked immunosorbent assay plates for the detection of sugar chains.

The immobilization of carbohydrates for solid-phase assays, including enzyme-linked immunosorbent assay (ELISA), is difficult because they are hydrophilic. We developed four new methods for the immobilization of oligosaccharides. ELISA plates were first coated with methyl vinyl ether-maleic anhydride copolymer (MMAC) and an excess of active anhydride groups was introduced. They were subsequently reacted, in four different ways, to bind oligosaccharides. In method 1, the anhydride groups were reacted with hydrazide groups, in the presence of adipic acid dihydrazide, and then coupled to the reducing ends of sugar chains by reductive amination. In method 2, the anhydride groups were reacted with p-aminophenyl glycoside obtained by reduction with p-nitrophenyl glycoside. In method 3, the anhydride groups were reacted with 1, 6-hexamethylenediamine. Aminooxy groups were coupled to the amino groups introduced and then aminooxyacetic acid with carbodiimide and ligated to oligosaccharides by oxime formation. In method 4, stereospecifically aminated oligosaccharides reacted with the anhydride groups. We compared, in solid-phase assays systems, the ability of lectins to detect oligosaccharides immobilized with either one of these four new methods or one of the two methods previously described. Detection of sugars with lectins is useful because, in most cases, they recognize sugars stereospecifically. The immobilization method should therefore be carefully selected to avoid changing the configuration and substitution in C-1.     

Structures of the major oligosaccharides from a human rectal adenocarcinoma glycoprotein

Four oligosaccharides in the reduced form were isolated from RMG (a mucin-type glycoprotein from a human rectal adenocarcinoma). They were 1) Sia alpha s2 leads to 6GalNAc-ol; 2) Sia alpha 2 leads to 6(Gal beta 1 leads to 3)GalNAc-ol; 3) Sia alpha 2 leads to 6(GlcNAc beta 1 leads to 3)GalNAc-ol; and 4) Sia alpha 2 leads to 6(GalNAc alpha 1 leads to 3)GalNAc-ol. The amounts of oligosaccharides 1, 2, 3, and 4 corresponded to 27, 5, 11, and 8% of the total N-acetylgalactosaminitol produced on alkaline borohydride treatment of RMG. To determine the structures of oligosaccharides 2, 3, and 4, a mixture of the three was subjected to methylation analysis which revealed that the N-acetylgalactosaminitol was substituted at both C-3 and C-6 and other sugars at the nonreducing ends. Desialized oligosaccharides were prepared, and the structures were deduced by analysis of the permethylated sugars on gas-liquid chromatography/mass spectrometry. Anomeric configurations were determined by exoglycosidase digestions except for galactose which was analyzed by chromium trioxide oxidation.     

Synthesis of Oligosaccharides by β-Fructofuranosidase in Biphasic Medium Containing Organic Solvent as Bulk Phase

The effect of four organic solvents on β-fructofuranosidase mediated synthesis of oligosaccharides from sucrose were investigated. Amongst the solvents examined, butyl acetate proved to be the best for oligosaccharide synthesis. Starting with the equivalent of 44.6 g/L of sucrose, 247 U of enzyme and 91.6% (by vol.) of butyl acetate results in the production of 8.8 g/L of oligosaccharides within 30 min, with trisaccharides constituting more than 60% of the oligosaccharides. The efficiency for conversion of sucrose to oligosaccharides is greater than 19%, and this exceeds the 11.6% (in 24 h) previously achieved with 1271 U of the same enzyme in aqueous medium. Use of butyl acetate as the bulk phase therefore modifies the reaction environment in favour of enhanced and accelerated rate of oligosaccharide synthesis by this β-fructofuranosidase.     

Structure and location of asparagine-linked oligosaccharides in the Fc region of a human immunoglobulin D

Seven kinds of asparagine-linked oligosaccharides were bound to the Fc region of a human immunoglobulin D(NIG-65). The oligosaccharides quantitatively released from four species of glycopeptides by digestion with almond glycopeptidase, were separated by Bio-Gel p-4 column chromatography and were purified further by thin-layer chromatography. The sugars were identified with GC-MS following the permethylation of respective oligosaccharide. To Asn-68(NIG-65 Fc numbering (1)), two kinds of high-mannose-type oligosaccharides were bonded. To Asn-159, a kind of hybride-type and two kinds of bisected complex-type oligosaccharides were attached. From Asn-210, four kinds of bisected complex-type oligosaccharides were isolated.     

Synthesis of fluorescent oligosaccharides for covalent attachment to living cells

Asparagine-linked oligosaccharides were liberated from glycoproteins by hydrazinolysis. The treatment resulted in de-N-acetylation of the amino sugars. After isolation of the oligosaccharides free amino groups were labeled with fluorescein isothiocyanate and remaining amino groups reacetylated. The fluorescent oligosaccharides were used to label living cells. They were converted to hydrazine derivatives and covalently attached to cell surface oligosaccharides, which had been treated with periodate or neuraminidase and galactose oxidase. This enabled the visualization of the attached oligosaccharides at the external aspect of the plasma membrane by fluorescence microscopy.     

Glycosylation of the two <Emphasis Type="Italic">O</Emphasis>-glycosylated domains of human MUC2 mucin in patients transposed with artificial urinary bladders constructed from proximal colonic tissue

Transposition of intestinal segments is frequently used for bladder reconstruction. Following transposition, bowel segments continue to produce mucus and a correlation between excessive mucus production and complications such as urinary tract infection or catheter blockage has been observed for a long time. However, no information is currently available on the change of mucin expression and glycosylation under these abnormal conditions. In this study, the variable number tandem repeat region and the irregular repeat domain of human MUC2 were isolated as two glycopeptide populations after reduction and trypsin digestion followed by gel chromatography from urine of patients transposed with urinary bladders. After alkaline borohydride treatment, the oligosaccharides released from the whole MUC2 mucin and the two glycosylated domains were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 60 different glycans were identified, mainly based on sialylated core 3 structures. Some core 1, 2 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly α2–6 linked to the N-acetylgalactosaminitol and sulphate residues exclusively 3-linked to galactose. No expression of blood group A and B or Sda/Cad determinants was observed. Similar patterns of glycosylation were found in the tandem repeat region and the irregular repeat domain and the level of expression of the major oligosaccharides were in the same order of magnitude. The most interesting feature of this study was that sialyl-Tn antigen, which is considered as a tumour antigen, was the oligosaccharide most highly expressed. This result suggests that mucins from intestinal transposed segments are abnormally glycosylated.     

Evidence for the presence of complex carbohydrates in Candida albicans cell wall glycoproteins

In order to test the hypothesis that cell wall glycoproteins of Candida albicans contained non-mannan oligosaccharides, the sugar composition of cell wall extracts and fractions of cell wall extracts was examined by means of fluorophore-assisted carbohydrate electrophoresis (FACE). In addition to the expected mannose, glucose, and N-acetyl-glucosamine, this analysis showed the presence of galactose, N-acetyl-galactosamine, fucose, and sialic acid and two unknown sugars. These sugars are also associated with complex oligosaccharides of mammalian glycoproteins. Presence of fucosylated cell wall components was further demonstrated by lectin-blotting analysis of cell wall extracts. Besides their structural role, complex carbohydrate structures on the surface of C. albicans may represent additional motifs through which interactions of this fungus with host cells and tissues could be established.     

Oligosaccharides from placenta: early diagnosis of feline mannosidosis

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.