Structural studies of 4-O-acetyl-α-N-acetylneuraminyl-(2→3)-lactose, the main oligosaccharide in echidna milk

The main oligosaccharide (50%) in the milk of the Australian echidna (Tachyglossus aculeatus) has been identified unequivocally as 4-O-acetyl-α-N-acetylneur-amínyl-(2→3)-lactose. The 4-O-acetyl substituent of the sialic acid residue was characterised by g.l.c.-m.s. of the isolated (after mild, acid hydrolysis) and trimethyl-silylated/esterified sialic acid, and by m.s. (after derivatisation) and 500-MHz, ¹H-n.m.r. spectroscopy of the intact oligosaccharide. Information about the glycosidic bonds was obtained by methylation analysis and 500-MHz, ¹H-n.m.r. spectroscopy. This animal species is the third one known to produce 4-O-acetylated sialic acid.     

Determination of the d and l configuration of neutral monosaccharides by high-resolution capillary g.l.c.

Capillary g.l.c. on SE-30 of the trimethylsilylated (-)-2-butyl glycosides of d and l monosaccharides gives multiple peak patterns, which can be used for the assignment of the absolute configurations. (-)-2-Butyl glycosides can be prepared from monosaccharides or their methyl glycosides; consequently, for the analysis of oligo- or poly-saccharides, hydrolysis as well as methanolysis can be applied. Provided that the peaks of the (-)-2-butyl glycosides do not completely overlap, mixtures of monosaccharides can be analysed directly, as illustrated for the constituents of the cell-wall lipopolysaccharide from Salmonella typhimurium LT-2.     

Complete enzymic synthesis of the mucin-type sialyl Lewis x epitope, involved in the interaction between PSGL-1 and P-selectin

Sialyl Lewis x (sLe(x)) is an established selectin ligand occurring on N- and O-linked glycans. Using a completely enzymic approach starting from p-nitrophenyl N-acetyl-alpha-D-galactosaminide (GalNAc(alpha1-pNp as core substrate, the sLe(x)-oligosaccharide Neu5Ac(alpha2-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)[Gal(bet a1-3)]GalNAc(alpha1-pNp, representing the O-linked form, was synthesized in an overall yield of 32%. In a first step, Gal(beta1-3)GalNAc(alpha1-pNp was prepared in a yield of 52% using UDP-Gal and an enriched preparation of beta3-galactosyltransferase (EC 2.4.1.122) from rat liver. UDP-GlcNAc and a recombinant affinity-purified preparation of core 2 beta6-N-acetylglucosaminyltransferase (EC 2.4.1.102) fused to Protein A were used to branch the core 1 structure, affording GlcNAc(beta1-6)[Gal(beta1-3)]GalNAc(alpha1-pNp in a yield of >85%. The core 2 structure was galactosylated using UDP-Gal and purified human milk beta4-galactosyltransferase 1 (EC 2.4.1.38) (yield of >85%), then sialylated using CMP-Neu5Ac and purified recombinant alpha3-sialyltransferase 3 (EC 2.4.99.X) (yield of 87%), and finally fucosylated using GDP-Fuc and recombinant human alpha3-fucosyltransferase 6 (EC 2.4.1.152) produced in Pichia pastoris (yield of 100%). Overall 1.5 micromol of product was prepared. MALDI TOF mass spectra, and 1D and 2D TOCSY and ROESY 1H NMR analysis confirmed the obtained structure.     

N-glycans of recombinant human acid alpha-glucosidase expressed in the milk of transgenic rabbits

Pompe disease is a lysosomal glycogen storage disorder characterized by acid alpha-glucosidase (GAA) deficiency. More than 110 different pathogenic mutations in the gene encoding GAA have been observed. Patients with this disease are being treated by intravenous injection of recombinant forms of the enzyme. Focusing on recombinant approaches to produce the enzyme means that specific attention has to be paid to the generated glycosylation patterns. Here, human GAA was expressed in the mammary gland of transgenic rabbits. The N-linked glycans of recombinant human GAA (rhAGLU), isolated from the rabbit milk, were released by peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The N-glycan pool was fractionated and purified into individual components by a combination of anion-exchange, normal-phase, and Sambucus nigra agglutinin-affinity chromatography. The structures of the components were analyzed by 500 MHz one-dimensional and 600 MHz cryo two-dimensional (total correlation spectroscopy [TOCSY] nuclear Overhauser enhancement spectroscopy) (1)H nuclear magnetic resonance spectroscopy, combined with two-dimensional (31)P-filtered (1)H-(1)H TOCSY spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and high-performance liquid chromatography (HPLC)-profiling of 2-aminobenzamide-labeled glycans combined with exoglycosidase digestions. The recombinant rabbit glycoprotein contained a broad array of different N-glycans, comprising oligomannose-, hybrid-, and complex-type structures. Part of the oligomannose-type glycans showed the presence of phospho-diester-bridged N-acetylglucosamine. For the complex-type glycans (partially) (alpha2-6)-sialylated (nearly only N-acetylneuraminic acid) diantennary structures were found; part of the structures were (alpha1-6)-core-fucosylated or (alpha1-3)-fucosylated in the upper antenna (Lewis x). Using HPLC-mass spectrometry of glycopeptides, information was generated with respect to the site-specific location of the various glycans.     

Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of tetrasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (1) and octasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were beta-(1-->4)-connected to the internal N-acetyl-beta-D-glucosamine residues by using bovine milk beta-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.     

Oligosaccharides isolated from Agave vera cruz

The structures of naturally occurring and enzymically synthesized oligosaccharides, consisting of fructose and glucose residues and having d.p. 3–8, in the stem of Agave vera cruz have been investigated by using methylation analysis, mass spectrometry, and p.m.r. spectroscopy. The naturally occurring trisaccharides were identified as 1-kestose and neokestose, and the tetrasaccharides as nystose and at least one other related to neokestose. The higher fractions consist of mixtures of (branched) oligosaccharides related to 1-kestose, neokestose, or 6-kestose as basic structures. The enzymically synthesized trisaccharide was identified as 1-kestose, and the tetrasaccharides as nystose. The higher fractions consist of mixtures of linear oligosaccharides related to 1-kestose and neokestose.     

THE CELL WALL (THECA) OF TETRASELMIS STRIATA (CHLOROPHYTA): MACROMOLECULAR COMPOSITION AND STRUCTURAL ELEMENTS OF THE COMPLEX POLYSACCHARIDES

Isolated cell walls (thecae) from the scaly flagellate green alga Tetraselmis striata Butcher contain the unusual 2-keto-sugar acids 3-deoxy-manno-2-octulosonic acid (Kdo), 3-deoxy-5-O-methyl-manno-2-octulosonic acid (5OMeKdo), and 3-deoxy-lyxo-2-heptulosaric acid (Dha). In addition, galacturonic acid, galactose, gulose, and arabinose are present. EDTA-extraction yielded an insoluble fraction that retains the shape of the cell walls and contains no 2-keto-sugar acids. Methylation analysis demonstrated the presence of terminal hexose, GalA, Dha, and Kdo as well as 2-substituted hexose, 4-or 8-substituted Kdo, and 4,8-disubstituted Kdo. However, most of the carbohydrate material (about 60%) was not methylated. Periodate oxidation of the cell wall preparation showed the presence of 2-substituted Gul, 4- or/and 5-substituted and 7- or/and 8-substituted Kdo, which is in agreement with the methylation analysis. Again, a significant amount of carbohydrate material was not degraded, indicating complex substitution patterns. Oligosaccharides were generated by partial hydrolysis and fractionated using gel permeation chromatography and high-pH anion-exchange chromatography. Oligosaccharides contained either GalA and Kdo, or Gal, Kdo, Dha, and Gul, respectively. The structure of a GalA and Kdo containing disaccharide was established using ¹ H NMR spectroscopy.