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Using a series of relevant substrates, connective tissue of the snail Lymnaea stagnalis was shown to contain β1‐2 xylosyltransferase (β2Xyl‐T), β1‐2 N‐acetylglucosaminyltransferase I (β2GlcNAc‐T I), and β1‐2 N‐acetylglucosaminyltransferase II (β2GlcNAc‐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 GlcNAcβ1‐2Manα1‐6(GlcNAcβ1‐2Manα1‐3)Manβ1‐R [where R = ‐4GlcNAcβ1‐4GlcNAc 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 GlcNAcβ1‐2Manα1‐6(GlcNAcβ1‐2Manα1‐3)(Xylβ1‐2)Manβ1‐R. Substrate specificity studies focused on connective tissue β2Xyl‐T show that the minimal structure requirements are fulfilled in GlcNAcβ1‐2Manα1‐3Manβ1‐O‐(CH2)7CH3. The enzyme activity can therefore be characterized as UDP‐Xyl: GlcNAcβ1‐2Manα1‐3Manβ‐R (Xyl to Manβ) β1‐2 xylosyltransferase. In substrate‐specificity studies directed to connective tissue β2GlcNAc‐T I, it could be demonstrated that the enzyme is active towards acceptors having at the minimum a Manα1‐3Manβ‐R sequence, and that introduction of a βXyl residue at C2 of βMan totally abolishes the enzyme activity. Xylose‐containing oligosaccharides are not acceptors for β2GlcNAc‐T I. In combination with the substrate specificity of β2Xyl‐T, this shows that in snail connective tissue β2GlcNAc‐T I must act before β2Xyl‐T. The connective tissue β2GlcNAc‐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.

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.

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 Pl -4 linkage to the terminal GlcNAc residue of GlcNAcP-R. The albumin gland enzyme was partially purified by affinity chromatography on UDP-hexanolamine-Sepharose 4B. Using GlcNAcPl -2Manal-6(GlcNAcP1-2Manal-3)Man~l-4GlcNAcP1-4GlcNAc or GlcNAcPlOMe as substrates, the enzyme showed an absolute requirement for MnZ+ 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 GlcNAcPlOMe, and towards oligosaccharides or glycopeptides with a non-reducing terminal P-GlcNAc unit, but cannot act on GlcNAcal-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 GlcNAcPl -R as the terminal non-reducing unit, and for connective tissue N-acetylgalactosaminyltransferase with GlcNAcPl-OMe as acceptor. In all instances, products with GalNAc PI -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 :GlcNAcP-R Pl -4 N-acetylgalactosaminyltransferase, and is probably involved in the biosynthesis of N,N'-diacetyllactosediamine-containing glycoproteins, like hemocyanin, in the snail L. stugnalis.

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.

No abstract

Three glycoprotein N-glycans, namely. a disialylated diantennary carbohydrate chain linked to Asn. a monoslalylated, fucosylated diantennary glycopeptide with bisecting N-acetylglucosamine, and a tetrasialylated. fucosylated tetra-antennary oligosaccharide, have been investigated by twodimensional NOE and HOHAHA spectroscopy in 'H,O as solvent. The amide protons of all iv-acetylglucosamme 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 Ri-acetylglucosamine residues in HOHAHA spectra. These results have as main implication that several biologically important large glycans, will not become amenable for conformational studies by multidimensional NMR in 'H,O solution.