Structures of enzymically derived oligosaccharides from sorghum glucuronoarabinoxylan.

Oligosaccharides derived from alkali-extracted sorghum glucuronoarabinoxylan by digestion with a combination of (1-->4)-beta-D-arabinoxylan arabinofuranohydrolase (AXH) and endo-(1-->4)-beta-D-xylanase (Xyl I), both from Aspergillus awamori, were purified by size-exclusion chromatography followed by preparative high-performance anion-exchange chromatography. Structural studies including monosaccharide analysis, methylation analysis, 1H NMR spectroscopy, and mass spectrometry were carried out, resulting in the characterisation of four novel oligosaccharides, namely, alpha-D-GlcpA-(1-->2)-beta-D-Xyl p-(1-->4)-beta-D-Xyl p-(1-->4) -beta-D-Xyl p, alpha-D-GlcpA-(1-->2)-beta-D-Xyl p-(1-->4) [alpha-L-Araf-(1-->3)]-beta-D-Xyl p-(1-->4)-beta-D-Xyl p, alpha-D-GlcpA-(1-->2)-beta-D- Xyl p-(1-->4)-beta-D-Xyl p-(1-->4)[alpha-L-Araf-(1-->2)-alpha)-alpha- L-Araf-(1-->3)]-beta-D-Xyl p-(1-->4)-beta-D-Xyl p, and alpha-D-GlcpA-(1-->2) -beta-D-Xyl p-(1-->4)[alpha-L-Araf-(1-->3] beta-D-Xyl p-(1-->4)[alpha-L-Araf-(1-->2)-alpha-L-Araf-(1-->3)] -beta-D-Xyl p-(1-->4)-beta-D-Xyl p. The various oligosaccharides identified provide additional insight into the structure of sorghum glucuronoarabinoxylan. Furthermore, novel data were generated with respect to the substrate specificity of AXH and Xyl I towards glucuronoarabinoxylans in general.

Dimers of a GFG hexasaccharide occur in apple fruit xyloglucan.

Apple fruit xyloglucan is predominantly built up from XXXG, XXFG, and XLFG units (G = beta-D-Glcp-, X = alpha-D-Xylp-(1-->6)-beta-D-Glcp-, L = beta-D-Galp-(1-->2)-alpha-D-Xylp-(1-->6)-beta-D-Glcp-, F = alpha-L-Fucp-(1-->2)-beta-D-Galp-(1-->2)-alpha-D-Xyl p-(1-->6)-beta-D-Glcp-). However, small amounts of oligosaccharides with a less heavily branched glucan backbone also occur. Structural analysis of two such oligosaccharides, isolated from a xyloglucan preparation digested with endoglucanase i.v., using a combination of FAB mass spectrometry and 1H NMR spectroscopy, afforded the identification of GFG and a dimer of GFG. The finding of the dodecasaccharide GFGGFG as a structural element of apple fruit xyloglucan is most unusual.

In the biosynthesis of N-glycans in connective tissue of the snail Lymnaea stagnalis of incorporation GlcNAc by beta 2GlcNAc-transferase I is an essential prerequisite for the action of beta 2GlcNAc-transferase II and beta 2Xyl-transferase.

Using a series of relevant substrates, connective tissue of the snail Lymnaea stagnalis was shown to contain beta 1-2 xylosyltransferase (beta 2Xyl-T), beta 1-2 N-acetylglucosaminyltransferase I (beta 2GlcNAc-T I), and beta 1-2 N-acetylglucosaminyltransferase II (beta 2GlcNAc-T II) activities. These enzymes are probably involved in the biosynthesis of the N-linked carbohydrate chains, like those present in hemocyanin. The products formed by incubation of GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-R [where R = -4GlcNAc beta 1-4GlcNAc or O-(CH2)7CH3] with UDP-Xyl and connective tissue microsomes have been purified and characterized by 1H-NMR spectroscopy in conjunction with methylation analysis to be GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)(Xyl beta 1-2)Man beta 1-R. Substrate specificity studies focused on connective tissue beta 2Xyl-T show that the minimal structure requirements are fulfilled in GlcNAc beta 1-2Man alpha 1-3Man beta 1-O-(CH2)7CH3. The enzyme activity can therefore be characterized as UDP-Xyl:Glc-NAc beta 1-2Man alpha 1-3Man beta-R (Xyl to Man beta) beta 1-2 xylosyltransferase. In substrate-specificity studies directed to connective tissue beta 2GlcNAc-T I, it could be demonstrated that the enzyme is active towards acceptors having at the minimum a Man alpha 1-3Man beta-R sequence, and that introduction of a beta Xyl residue at C2 of beta Man totally abolishes the enzyme activity. Xylose-containing oligosaccharides are not acceptors for beta 2GlcNAc-T I. In combination with the substrate specificity of beta Xyl-T, this shows that in snail connective tissue beta 2GlcNAc-T I must act before beta 2Xyl-T. The connective tissue beta 2GlcNAc-T II activity follows the earlier established biosynthetic routes. Based on the substrate specificities of the various connective tissue glycosyltransferases known so far, and the structures isolated from L. stagnalis hemocyanin, a partial biosynthetic scheme for N-glycosylation in snail connective tissue is proposed.

A reinvestigation towards the conformation of methyl alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranoside by a combined ROE and molecular dynamics analysis.

Conformational analysis of alpha-D-Man p-(1-->6)-alpha-D-Man p1-OMe, by a combination of extensive molecular dynamics calculations in water and ROE buildup series, afforded two main minima, namely, phi/psi = 95/-178 and phi/psi = 140/-185. Transitions between these minima are observed, which have not previously been demonstrated using other approaches. In contrast to literature data for the glycosidic linkage, describing equal populations of both the gg and the gt rotamers, it was found that the gg conformer is present to ca. 96%. The non-reducing mannosyl unit showed approximately a 1:1 ratio for the gg:gt equilibrium, in accordance with earlier reports.

Identification of a novel UDP-GalNAc:GlcNAc beta-R beta 1-4 N-acetylgalactosaminyltransferase from the albumen gland and connective tissue of the snail Lymnaea stagnalis.

Both the albumen gland, one of the female accessory sex glands, and connective tissue of the freshwater snail Lymnaea stagnalis contain N-acetylgalactosaminyltransferase activity, capable of transferring GalNAc from UDP-GalNAc in beta 1-4 linkage to the terminal GlcNAc residue of GlcNAc beta-R. The albumin gland enzyme was partially purified by affinity chromatography on UDP-hexanolamine-Sepharose 4B. Using GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc or GlcNAc beta 1-OMe as substrates, the enzyme showed an absolute requirement for Mn2+ with an optimum concentration of 12.5-50 mM. The optimal pH was approximately pH 7.0. The enzyme activity was independent of the Triton X-100 concentration in the range 0.25-2.5%, and no activation effect was found. The more labile connective tissue microsomal enzyme, subjected to the same optimization procedure, gave comparable results. Both enzyme activities have similar substrate specificities towards GlcNAc or GlcNAc beta 1-OMe, and towards oligosaccharides or glycopeptides with a non-reducing terminal beta-GlcNAc unit, but cannot act on GlcNAc alpha 1-OMe. Saccharides with non-reducing terminal Gal or GalNAc residues, and free GalNAc, Gal or Glc residues are not acceptors. Product analysis was carried out for albumen gland N-acetylgalactosaminyltransferase and four acceptors having GlcNAc beta 1-R as the terminal non-reducing unit, and for connective tissue N-acetylgalactosaminyltransferase with GlcNAc beta 1-OMe as acceptor. In all instances, products with GalNAc beta 1-4-linked to GlcNAc were obtained, showing that the connective tissue and the albumen gland activities are probably from one enzyme. This enzyme activity can be identified as UDP-GalNAc:GlcNAc beta-R beta 1-4 N-acetylgalactosaminyltransferase, and is probably involved in the biosynthesis of N,N'-diacetyllactosediamine-containing glycoproteins, like hemocyanin, in the snail L. stagnalis.

Accumulation of fructose polymers in transgenic tobacco.

Fructan, a polyfructose molecule, is a storage compound in a limited number of plant species. Usually these species accumulate fructan with a low degree of polymerization (DP) and most of these plants have properties which preclude their use as a fructan source. With the eventual aim of allowing the accumulation of high DP fructans in non-fructan storing plants, we have investigated whether carbohydrate flow in the plant cell can be directed to produce this polymer. For this purpose the SacB gene from Bacillus subtilis, which encodes levansucrase, was modified and introduced into tobacco plants. Transgenic plants containing the sacB gene accumulate fructans. The size and properties of this fructan are similar to fructan produced by Bacillus subtilis, and is stable in plants. Although the level of fructan accumulation in the transgenic tobacco plants ranged from 3-8 percent of the dry weight, no levansucrase mRNA or protein could be detected in these plants. Extension of this work should permit the production of this high molecular weight biopolymer in crop plants for applications in food and non-food products.

The potency of amide protons for assignments of NMR spectra of carbohydrate chains of glycoproteins, recorded in 1H2O solutions.

Three glycoprotein N-glycans, namely, a disialylated diantennary carbohydrate chain linked to Asn, a monosialylated, fucosylated diantennary glycopeptide with bisecting N-acetylglucosamine, and a tetrasialylated, fucosylated tetra-antennary oligosaccharide, have been investigated by two-dimensional NOE and HOHAHA spectroscopy in 1H2O as solvent. The amide protons of all N-acetylglucosamine and sialic acid residues could readily be assigned. The large chemical-shift dispersion of the amide resonances of the N-acetylglucosamine residues, allowed the unambiguous assignment of every N-acetyl methyl signal, via strong NOEs. Subspectra could be obtained of all N-acetylglucosamine residues in HOHAHA spectra. These results have as main implication that several biologically important large glycans will now [corrected] become amenable for conformational studies by multidimensional NMR in 1H2O solution.