Glycosyl Linkage Research Articles

Structural characterisation of xyloglucan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Linkage analysis of a xyloglucan from the extracellular medium of suspension cultures of Nicotiana plumbaginifolia showed mostly 4-Glc p and 4,6-Glc p, terminal Xyl p and 2-Xyl p, and terminal Ara f, along with ∼ 10% (w/w) O-acetyl groups, equivalent to ∼ 0.28 mol acetyl per mol of glycosyl residue. Methylation with methyl trifluoromethanesulfonate under neutral conditions, followed by re-methylation with CD 3I under basic conditions, and conversion into partially methylated alditol acetates showed that O-acetyl groups were primarily attached to C-6 of ∼ 44% of the 4-Glc p backbone not substituted with Xyl p residues and to C-5 of ∼ 15% of the terminal Ara f residues. These positions of the O-acetyl groups were confirmed by 1H-NMR. Oligosaccharides generated by digestion of native xyloglucan with endo-(1 → 4)- β-glucanase were separated by a combination of gel-filtration chromatography and anion-exchange HPLC, and analysed by glycosyl linkage analysis and by electrospray ionisation-mass spectrometry (ESI-MS). The major oligosaccharide subunits were Glc 4Xyl 2 and Glc 5Xyl 2, of which 50–60% are substituted with one terminal Ara f residue attached to 0–2 of a Xyl p residue, and a further 20–25% are substituted with two terminal Ara f residues attached to 0–2 of the Xyl p residues. ESI-MS showed that many of the oligosaccharide subunits carried one, two and, occasionally three O-acetyl groups.

Beta-glucan synthesis in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing beta-(1-->6) linkages

Bradyrhizobium japonicum synthesizes periplasmic cyclic beta-(1-->3),beta-(1-->6)-D-glucans during growth in hypoosmotic environments, and evidence is growing that these molecules may have a specific function during plant-microbe interactions in addition to osmoregulation. Site-directed Tn5 mutagenesis of the DNA region upstream of ndvB resulted in identification of a new gene (ndvC) involved in beta-(1--> 3), beta-(1-->6)-glucan synthesis and in nodule development. The predicted translation product was a polypeptide (ca. 62 kDa) with several transmembrane domains. It contained a sequence characteristic of a conserved nucleoside-sugar-binding motif found in many bacterial enzymes and had 51% similarity with a beta-glucanosyltransferase from Candida albicans. B. japonicum carrying a Tn5 insertion in ndvC resulted in synthesis of altered cyclic beta-glucans composed almost entirely of beta-(1--> 3)-glycosyl linkages. The mutant strain was only slightly sensitive to hypoosmotic growth conditions compared with the ndvB mutant, but it was severely impaired in symbiotic interactions with soybean (Glycine max). Nodulation was delayed by 8 to 10 days, and many small nodule-like structures apparently devoid of viable bacteria were formed. This finding suggests that the structure of the beta-glucan molecule is important for a successful symbiotic interaction, and beta-glucans may have a specific function in addition to their role in hypoosmotic adaptation.

Pectic polysaccharides from xylem-differentiating zone of Cryptomeria japonica

Pectin was extracted from the cell walls of xylem-differentiating zones of sugi ( Cryptomeria japonica) with the chelating agent, trans-1,2-cyclohexanediamine- N, N, N′, N′-tetraacetic acid at 20° and Na 2CO 3 solution at 1° and 20°. The extracted pectin fractions were purified by anion-exchange chromatography and digested with pure endo-polygalacturonase from Aspergillus niger. The hydrolysates were then fractionated by gel permeation chromatography into three or four fractions. Sugar composition and glycosyl linkage composition analyses of each fraction showed that the void volume fractions contained 2- and 2,4-linked rhamnosyl, 5-linked arabinosyl, terminal galactosyl and 4-linked galacturonic acid residues, indicating that they were rhamnogalacturonan I. The fractions obtained from the included volume had rare sugar residues, such as apiosyl, 2-O- methyl- l-fucosyl , traces of 3-O- methyl- l-fucosyl , 2-O- methyl- d-xylosyl residues aceric acid and thiobarbituric acid assay-positive sugar residues, showing that these fractions contained rhamnogalacturonan II-like polysaccharides. Several linear oligosaccharides composed of (1 → 4)-linked α- d-galacturonic acid residues were purified from the lowest M r regions and some oligogalacturonides were characterized by methylation analysis, FAB-mass spectrometry and NMR.

A study on the in vivo digestibility of retrograded starch

The digestibility of different forms of starch was examined in an ileostomy model. Six otherwise healthy ileostomists were fed a controlled polysaccharide-free diet for four days, on three of which a test starch was added at breakfast. 50 g starch was fed as either whole or homogenized chick peas or as a retrograded starch gel. A readily digestible wheat starch biscuit was used as a control. Ileostomy effluent was collected every 2 hours over a 16 hour period and a final collection made at 24 hours after the test meal. The monosaccharide composition and glycosyl linkages of the residual carbohydrate in the 2 hour peak period following the test meal was determined. Following consumption of the starch gel, poly- and oligo-saccharides from mucin and starch were identified in the effluent. At the peak of effluent production following the test meal, the average ratio of starch polysaccharide to mucin was 1:0.4. Of the 50 g of starch consumed, 7% of the starch escaped digestion in this fraction. Following consumption of cooked, cooled chick peas, which were fed whole or homogenized, polysaccharides deriving from starch, mucin and the cell wall were detected in the effluent. It was estimated from comparison of the composition of the food and effluent that 14% and 16% of the ingested starch in the form of homogenized and whole chick peas had escaped digestion in the small intestine. Linkage analysis showed the chemical structure of the starch escaping digestion after feeding the whole and homogenized chick peas was similar to that obtained after feeding the starch gel.

Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N-acetylglucosamine transferase (RfaK).

A lipooligosaccharide (LOS) mutant of Neisseria meningitidis serogroup B strain NMB (immunotype L3,7,9) was identified in a Tn916 (tetM) mutant bank by loss of reactivity with monoclonal antibody 3F11, which recognizes the terminal Galbeta1-->4GlcNAc epitope in the lacto-N-neotetraose moiety of the wild-type LOS structure. The mutant, designated 559, was found to express a truncated LOS of 3.0 kDa. Southern and PCR analyses demonstrated that there was a single intact Tn916 insertion (class I) in the mutant 559 chromosome. Linkage of the LOS phenotype and the Tn916 insertion was confirmed by transformation of the wild-type parent. Nucleotide sequence analysis of the region surrounding the transposition site revealed a 1,065-bp open reading frame (ORF). A homology search of the GenBank/EMBL database revealed that the amino acid sequence of this ORF had 46.8% similarity and 21.2% identity with the alpha1,2 N-acetylglucosamine transferase (RfaK) from Salmonella typhimurium. Glycosyl composition and linkage analysis of the LOS produced by mutant 559 revealed that the lacto-N-neotetraose group which is attached to heptose I (HepI) and the N-acetylglucosamine and glucose residues that are attached to HepII in the inner core of the parental LOS were absent. These analyses also showed that the HepII residue in both the parent and the mutant LOS molecules was phosphorylated, presumably by a phosphoethanolamine substituent. The insertion of nonpolar and polar antibiotic resistance cartridges into the parental rfaK gene resulted in the expression of LOS with the same mobility as that produced by mutant 559. This result indicated that the inability to add the lacto-N-neotetraose group to the 559 LOS is not due to a polar effect on a gene(s) downstream of rfaK. Our data indicate that we have identified the meningococcal alpha1,2 N-acetylglucosamine transferase responsible for the addition of N-acetylglucosamine to HepII. We propose that the lack of alpha-chain extension from HepI in the LOS of mutant 559 may be due to structural constraints imposed by the incomplete biosynthesis of the LOS inner core.

Tetrasodium P1,P4-Bis(5'-adenosyl)tetraphosphate Dodecahydrate

The title compound, 4Na+.C20H24N10O19P4-.12H2O, was found to be the dodecahydrate. The two ribose rings adopt the 3′-endo envelope conformation, with the glycosyl linkage anti. The exocyclic side chains have the g conformation. The crystal structure is stabilized by the interaction involving the Na+ ions and the hydrogen bonds.

Structural analysis of the carbohydrate moiety of arabinogalactan-proteins from stigmas and styles of Nicotiana alata

Arabinogalactan-proteins (AGPs) from the female reproductive tissues (stigmas and styles) of Nicotiana alata were isolated from the saturated ammonium sulfate supernatant of buffer-soluble extracts by precipitation with the β-glucosyl Yariv reagent, followed by gel-filtration chromatography under dissociating conditions. The AGPs had characteristics typical of other AGPs: a high proportion of carbohydrate (95%) with a high ratio of Gal p to Ara f (2:1), and a low protein content (5%) with high levels of alanine, serine, and hydroxyproline. The AGPs consisted of a major species which was almost neutral, and a minor species which was more negatively charged. Sedimentation equilibrium experiments showed that the purified AGPs had a weight-average molecular weight of 143 kD. Linkage analysis showed that the AGPs contained a highly branched backbone of 3-, 6-, and 3,6-linked Gal p residues, bearing terminal Gal p and terminal Ara f residues. Analysis by one-dimensional and two-dimensional 1H and 13C NMR spectroscopy confirmed the presence of these glycosyl linkage types, and showed a high mobility of the terminal Ara f residues consistent with their location on the periphery of the molecules. This analysis represents the most complete 1H assignment for AGP molecules in solution. No difference in the carbohydrate analyses was found between AGPs isolated separately from stigmatic or stylar tissue, or between AGPs isolated from stigmas and styles of plants of different self-incompatibility genotypes.

Enzymatic cyclization of 1,N 6-etheno-nicotinamide adenine dinucleotide

The ADP-ribosyl cyclase from Aplysia californica catalyzed the transformation of 1,N 6-etheno-nicotinamide-adenine dinucleotide ( 2) into a novel cyclic nucleotide, 3. The newly formed glycosyl linkage is attached onto the N-1 position of the etheno-adenine ring corresponding to the N-7 position of the adenine nucleus. This alternative mode of cyclization gives more versatility in the biosynthesis of novel analogs of cADPR ( 1).

The structure of a novel polysaccharide produced by Bradyrhizobium species within soybean nodules

Certain strains of Bradyrhizobium japonicum and B. elkanii produce a polysaccharide within the root nodules of their legume host, soybean. These nodule polysaccharides (NPSs) were isolated and characeterized. The NPS produced by B. elkanii strains proved to be identical in glycosyl composition and linkages to the extracellular polysaccharide (EPS) of this species indicating that the NPS and EPS for B. elkanii have identical structures (W.F. Dudman, Carbohydr. Res., 66 (1978) 9–23), ▪ However, the structure of the NPS from B. japonicum proved to be quite different from that of its EPS. Methylation analysis of this NPS showed that it consists of 3-linked Gal, 3-linked Rha, 2,4-linked Rha, 4-linked Rha, and terminal 2- O-methyl GLcA in a 1:1:1:1:1 ratio. Stereochemical configurations of the glycosyl residues were determined by the preparation and analysis of trimethylsilyl (Me 3Si) (−)-2-butylglycosides. NMR spectroscopy (both 1H and 13C) showed that the Gal residue is α-linked, while all the other glycosyl residues are β-linked. Oligosaccharides produced by periodate oxidation-Smith degradation were purified, as were oligosaccharides produced by partial acid hydrolysis. Characterization of the Smith degradation products by methylation analysis, NMR spectroscopy, electrospray-mass spectrometry, and characterization of the partial acid hydrolysate oligsaccharides showed that the repeating oligosaccharide unit of the NPS has the structure, ▪.

Extracellular polysaccharide of Erwinia chrysanthemi

The extracellular polysaccharide (EPS) produced by E. chrysanthemi pv. zeae strain SR260, a strain pathogenic to corn, has a branched hexasaccharide repeat unit consisting of a glucomannorhamnan backbone [ → 3)-β- d- Glcp-(1 → 4)-α- d- Manp-(1 → 3)-α- l- Rhap-(1 → ] and a trisaccharide side-chain [ α- l- Rhap-(1 → 3)-α- l- Rhap-(1 → 4)?- GlcAp-(1 → ] attached to O-3 of the mannose in the backbone [1]. All the anomeric configurations, except that of the rhamnosyl-glucosyluronic linkage in the side chain, were unambiguously assigned by a combination of 1 and 2D NMR spectroscopy. The tentative assignment of the rhamnosyl-glucosyluronic glycosyl linkage as α- l is now proven.

Mannan glycopeptide elicits p-coumaroylamino acids in Ephedra distachya cultures

A mannan glycopeptide (Con A-II) which elicits the accumulation of p-coumaroylamino acids in Ephedra distachya cells was purified ca 20-fold from commercial yeast extract by ethanol precipitation, DEAE-cellulose, CM cellulose, gel filtration and Con A-Sepharose chromatography, consecutively. There was no significant elicitor activity in the glucan fraction. The elicitor active fraction (Con A-II) was partially hydrolysed with 2 M TFA and the hydrolysate chromatographed on Sephadex G-25 followed by ethanol precipitation. The activity was found in the 60% ethanol soluble but 80% ethanol insoluble fraction (MGP) whose M r on gel filtration HPLC was nearly homogeneous ( ca 4600). The GC-mass spectral analysis of the partially O-methylated alditol acetate and 13C NMR spectroscopy revealed that MGP contained terminal-(25.4%), α-1,2-(33.6%), α-1,6-(20.6%) and α-1,2,6-(20.4%) mannan glycosyl linkages. MGP elicitor also contained 17.4% (by weight) of peptide which was composed of 10 kinds of amino acid. Ser and Thr comprised 45 molar% of the total amino acid content. Hydrazinolysis of MGP revealed that the mannan moiety was possibly linked to Ser and Thr of the peptide through O-glycosidic bonds. Enzymatic degradation of MGP using α-mannosidase and proteinase K suggested that both mannan and peptide moieties were essential for elicitor activity.

Structure and conformation of 5-hydroxymethyl-2'-deoxycytidine, C10H15N3O5

The deoxyribose ring in the title compound adopts a C(3')-endo envelope conformation (3E), with the glycosyl linkage anti [χ = 195.8 (1)°]. The pseudorotational parameters are P = 10.2 (2)° and τ m = 32.1 (2)°. The exocyclic side chain at C(5') has the t conformation [γ = 174.6 (2)°]. The hydroxymethyl group side chain at C(5) is on the opposite side of the pyrimidine plane as is O(4') of the furanose ring

Arabinogalactan for hepatic drug delivery.

Arabinogalactan, a polysaccharide from the tree Larix occidentalis, has been purified and its biological and physical properties described. Intravenous injection of radiolabeled arabinogalactan (4 mg/kg) in rats resulted in 52.5% of the dose being present in the liver, while prior injection of asialofetuin (100 mg/kg) reduced hepatic radioactivity to 3.54%. Gel chromatography indicates arabinogalactan is a single species of 19 kDa, while light scattering gave a molecular weight of 40 kDa. Glycosyl linkage analysis of arabinogalactan is consistent with a highly branched structure comprising a backbone of 1,3-linked galactopyranose connected by 1,3-glycosidic linkages, comprised of 3,4,6-,3,6-, and 3,4- as well as 3-linked residues. In the carbon-13 NMR spectra, the major resonances of arabinogalactan are assigned to beta-galactopyranose, beta-arabinofuranose, and beta-arabinopyranose. Arabinogalactan produced no adverse reactions in single intravenous dose (mouse, 5000 mg/kg) and repeat dose toxicity studies (rats, 500 mg/kg/day, 90 days). When tritiated arabinogalactan was injected, radioactivity cleared from the liver with a half-life of 3.42 days. Arabinogalactan has properties that make it suitable as a carrier for delivering diagnostic or therapeutic agents to hepatocytes via the asialoglycoprotein receptor.

3'-Azido-2',3'-dideoxy-5-hydroxymethyluridine

In the title compound, C 10 H 13 N 5 O 5 , the furanose ring adopts a C(2')-endo envelope conformation ( 2 E), with the glycosyl linkage anti [χ=219.0 (2) o ]. The pseudorotational parameters are P=159.2 (3) o and τ m =34.3 (2) o . In the deoxyribose ring, the side chain on C(5') is partially disordered and exhibits two distinct conformations, g + [γ=58.6 (5) o ] and g - [γ=-58.2 (9) o ]. The hydroxymethyl side chain at C(5) is on the same side of the pyrimidine plane as O(4') of the furanose ring

Ruminal digestion and glycosyl linkage patterns of cell wall components from leaf and stem fractions of alfalfa, orchardgrass, and wheat straw

Samples of alfalfa, orchardgrass, and wheat straw were hand-separated into leaf and stem fractions that were subjected to in situ ruminal fermentation for various lengths of time to assess the rate and extent of degradation of cell wall neutral monosaccharides, uronic acids, acetyl groups, and hydroxycinnamic acids. A second objective was to measure the glycosyl linkage patterns of leaf and stem fractions of substrates before and after ruminal fermentation. Samples were fermented for 0, 6, 12, 24, 48, 96, and 192 h in each of two ruminally cannulated steers. In situ disappearance data were fitted to a first-order exponential equation to estimate the following substrate parameters: insoluble, potentially digestible fraction (fd), indigestible fraction (fi), and fractional rate constant of degradation of the potentially digestible fraction (k). Leaves contained larger concentrations of crude protein and smaller concentrations of cell wall components than did stem fractions. Estimates of fi were 7.3, 39.2, 22.1, 49.3, 27.7, and 36.3 for dry matter disappearances for alfalfa leaf, alfalfa stem, orchardgrass leaf, orchardgrass stem, wheat straw leaf, and wheat straw stem, respectively. Averaged across substrates, estimates of fi for arabinose, galactose, glucose, xylose, uronic acids, acetyl groups, and p-coumaric acid were 16.5, 11.4, 14.8, 31.2, 12.8, 25.3, and 22.6% in leaf fractions and 29.5, 19.9, 37.5, 56.2, 35.0, 52.4, and 44.6% in stem fractions. Rates of digestion of all monomeric components except galactose and xylose were greater (P < .05) for alfalfa than for orchardgrass or wheat straw. Differences in digestibility of cell wall components from leaf and stem fractions were greater in alfalfa and orchardgrass than in wheat straw. Glycosyl linkage analysis indicated that xylans in leaf and stem fractions of alfalfa, orchardgrass leaf, and wheat straw stem that resisted degradation had a lower degree of substitution with acid-labile constituents (i.e., other monosaccharides) than was found in original substrates. Different rates and extents of digestion of leaf and stem fractions of forages explain part, but not all, of the observed differences in digestibilities of cell wall monomers by ruminants.

Indolocarbazoles. 4. Synthetic studies towards staurosporine and tjipanazoles: Reactions of indolocarbazole with glycals

In an attempt to construct the unique N,N′-bidentate glycosyl linkage found in the Staurosporine 1 class of natural products, the first example of an acid catalyzed 2,6-condensation of an activated pyran with indolocarbazole 4 is reported. In pursuit of the real system, a variety of glycals were reacted with indolocarbazole 4. Formation of novel unexpected products ( B/C) with 1,3-connections along with the expected product ( A) and its' synthetic transformation to a potentially useful intermediate 24 are detailed.

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.

Xyloglucan glucosyltransferase in Golgi membranes from Pisum sativum (pea)

Cell membranes from etiolated Pisum sativum (pea) tissues were separated by ultracentrifugation on linear sucrose density gradients and assayed for membrane marker and glycosyltransferase activity. Membrane fractions were shown to incorporate glucose from UDP-D-[14C]glucose into polysaccharides with glycosyl linkages consistent with synthesis of xyloglucan. A combined assay using g.c., radiogas proportional counting and m.s. was employed to determine the identities of 14C-labelled glycosyl residues and the glycosyl linkages between them. In glucan synthase I assays, membrane fractions enriched for Golgi membranes showed 14C incorporation into 4- and 4,6-glucose residues, with minor incorporation into 3-glucose residues. In glucan synthase II assays, all 14C incorporation was into 3- and 3,4-glucose. There was a shift in glycosyl linkage of 14C incorporation from predominantly 4-glucose at low UDP-glucose concentration to predominantly 3- and 3,4-glucose at high UDP-glucose concentrations. Mn2+ stimulated incorporation of radioactivity into 4,6-glucose residues characteristic of xyloglucan polysaccharides. Addition of exogenous UDP-xylose to assay mixtures stimulated incorporation into 4,6-glucose, with a maximum at 15 microM UDP-xylose.

Structure of ten free N-glycans in ripening tomato fruit. Arabinose is a constituent of a plant N-glycan.

The concentration-dependent stimulatory and inhibitory effect of N-glycans on tomato (Lycopersicon esculentum Mill.) fruit ripening was recently reported (B. Priem and K.C. Gross [1992] Plant Physiol 98: 399-401). We report here the structure of 10 free N-glycans in mature green tomatoes. N-Glycans were purified from fruit pericarp by ethanolic extraction, desalting, concanavalin A-Sepharose chromatography, and amine-bonded silica high performance liquid chromatography. N-Glycan structures were determined using 500 MHz 1H-nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectrometry, and glycosyl linkage methylation analysis by gas chromatography-mass spectrometry. A novel arabinosyl-containing N-glycan, Man alpha 1-->6(Ara alpha 1-->2)Man beta 1-->4GlcNAc beta 1-->4(Fuc alpha 1-->3)GlcNAc, was purified from a retarded concanavalin A fraction. The location of the arabinosyl residue was the same as the xylosyl residue in complex N-glycans. GlcNAc[5']Man3(Xyl)GlcNAc(Fuc)GlcNAc and GlcNAc[5']Man2GlcNAc(Fuc)GlcNAc were also purified from the weakly retained fraction. The oligomannosyl N-glycans Man5GlcNAc, Man6GlcNAc, Man7GlcNAc, and Man8GlcNAc were purified from a strongly retained concanavalin A fraction. The finding of free Man5GlcNAc in situ was important physiologically because previously we had described it as a promoter of tomato ripening when added exogenously. Mature green pericarp tissue contained more than 1 microgram of total free N-glycan/g fresh weight. Changes in N-glycan composition were determined during ripening by comparing glycosyl and glycosyl-linkage composition of oligosaccharidic extracts from fruit at different developmental stages. N-Glycans were present in pericarp tissue at all stages of development. However, the amount increased during ripening, as did the relative amount of xylosyl-containing N-glycans.

Isolation and characterisation of a rhamnogalacturonan II from red wine

A complex polysaccharide originating from grape was purified from a red wine by size-exclusion and ion-exchange chromatography. This polysaccharide contained 2- O-methylfucose, rhamnose, fucose, 2- O-methylxylose, arabinose, apiose, galactose, galacturonic and glucuronic acids, and thiobarbituric acid assay-positive material, presumably 3-deoxy- d- manno-2-octulosonic acid (Kdo) and 3-deoxy- d- lyxo- 2-heptulosaric acid (Dha). Methylation structural analysis showed that this polysaccharide is a typical rhamnogalacturonan II (RG-II), a structurally complex pectic polysaccharide that has been isolated previously from the primary cell walls of sycamore. Red wine RG-II presented discrete differences from previously described RG-II's, a greater richness in uronic acids and some additional glycosyl linkages, which might be related to the pattern of degradation of grape-wall pectic substances by endogenous pectinolytic enzymes after pressing the berries and subsequent fermentation to wine.