Herman van Halbeek

Structural analysis of xyloglucan oligosaccharides by 1H-n.m.r. spectroscopy and fast-atom-bombardment mass spectrometry.

A method to determine rapidly the identities and proportions of the oligosaccharide repeating-units in plant cell-wall xyloglucans by 1D 1H-n.m.r. spectroscopy was developed. Six of the most commonly found xyloglucan oligosaccharide subunits (including three subunits that had not been fully characterized previously) were prepared by endo-(1----4)-beta-D-glucanase digestion of xyloglucans from various plant species. The oligosaccharides were reduced to the corresponding oligoglycosyl-alditols, purified, and characterized by glycosyl composition and linkage analysis, 1H-n.m.r. spectroscopy, and f.a.b.-mass spectrometry. Correlations between the 1H-n.m.r. spectra and the structures of the oligoglycosyl-alditols can be used to identify oligoglycosyl-alditols derived from xyloglucans of unknown structure. The identities and relative amounts of the oligosaccharide subunits of xyloglucans isolated from tamarind seed and rapeseed hulls were determined on this basis.

The solution conformation of sialyl-α(2→6)-lactose studied by modern NMR techniques and Monte Carlo simulations

SummaryWe present a comprehensive strategy for detailed characterization of the solution conformations of oligosaccharides by NMR spectroscopy and force-field calculations. Our experimental strategy generates a number of interglycosidic spatial constraints that is sufficiently large to allow us to determine glycosidic linkage conformations with a precision heretofore unachievable. In addition to the commonly used {1H,1H} NOE contacts between aliphatic protons, our constraints are: (a) homonuclear NOEs of hydroxyl protons in H2O to other protons in the oligosaccharide, (b) heteronuclear {1H,13C} NOEs, (c) isotope effects of O1H/O2H hydroxyl groups on13C chemical shifts, and (d) long-range heteronuclear scalar coupling across glycosidic bonds.We have used this approach to study the trisaccharide sialyl-α(2→6)-lactose in aqueous solution. The experimentally determined geometrical constraints were compared to results obtained from force-field calculations based on Metropolis Monte Carlo simulations. The molecule was found to exist in 2 families of conformers. The preferred conformations of the α(2→6)-linkage of the trisaccharide are best described by an equilibrium of 2 conformers with Φ angles at −60° or 180° and of the 3 staggered rotamers of the Ω angle with a predominantgt conformer. Three intramolecular hydrogen bonds, involving the hydroxyl protons on C8 and C7 of the sialic acid residue and on C3 of the reducing-end glucose residue, contribute significantly to the conformational stability of the trisaccharide in aqueous solution.

N-glycosylation site mapping of human serotransferrinby serial lectin affinity chromatography, fast atom bombardment-mass spectrometry, and 1H nuclear magnetic resonance spectroscopy

This report describes the N-glycosylation site mapping of human serotransferrin (h-STF). Reduced and S-carboxymethylated h-STF was digested with trypsin or chymotrypsin. Glycopeptides in the proteolytic digests were isolated by serial concanavalin A (Con A), Sambucus nigra agglutinin (SNA), and Phaseolus vulgaris leukoagglutinin (LPHA) affinity chromatography and subjected to preliminary analysis by 1H NMR spectroscopy. The glycopeptide fractions were then individually digested with N-glycanase. One part of the digest of each fraction was analyzed by fast atom bombardment-mass spectrometry (FAB-MS) to identify the peptide sequences of the glycosylation sites. The other part was used to isolate the oligosaccharide by the corresponding lectin affinity chromatography and to characterize the structures of the isolated oligosaccharides by 1H NMR spectroscopy and FAB-MS. The oligosaccharides in the Con A-bound fraction were shown to have bi-alpha(2-->6)-sialyl, diantennary structures. The SNA-bound fraction was shown to contain trisialyl, triantennary structures. Di- and triantennary oligosaccharides were found to occur on each of the two N-glycosylation sites of h-STF (Asn413 and Asn611) in the ratio of approximately 85:15. The SNA-bound glycopeptides were further fractionated by LPHA affinity chromatography. Two different oligosaccharides were characterized, namely, a trisialyl 2,4-triantennary and a trisialyl 2,6-triantennary glycan. The ratio of 2,4-triantennary vs 2,6-triantennary oligosaccharides attached to glycosylation site Asn413 was found to be approximately 5:1, whereas the two isomeric triantennary oligosaccharides were found to be attached to glycosylation site Asn611 in the ratio approximately 1:1.

Location of the O-acetyl substituents on a nonasaccharide repeating unit of sycamore extracellular xyloglucan

The locations of the O-acetyl substituents on the major nonasaccharide repeating unit of the xyloglucan isolated from sycamore extracellular polysaccharides were determined by a combination of analytical methods, including f.a.b.-m.s. and 1H-n.m.r. spectroscopy. The O-2-linked-beta-D-galactosyl residue of the nonasaccharide was found to be the dominant site of O-acetyl substitution. Both mono-O-acetylated and di-O-acetylated beta-D-galactosyl residues were detected. The degree of O-acetylation of the beta-D-galactosyl residue, was estimated by 1H-n.m.r. spectroscopy to be 55-60% at O-6, 15-20% at O-4, and 20-25% at O-3. 1H-n.m.r. spectroscopy also indicated that approximately 50% of the beta-D-galactosyl residues are mono-O-acetylated, 25-30% are di-O-acetylated, and 20% are not acetylated.

NMR developments in structural studies of carbohydrates and their complexes

Abstract Notable recent developments in NMR methods used to study carbohydrate structure include the application of tailored homo- and heteronuclear (multi-) selective two- and three-dimensional techniques, improved measurements of long-range 13C-1H scalar couplings, and the observation of hydroxyl proton signals used to detect intramolecular hydrogen bonds in aqueous solution. In addition, technological advances in 13C isotopic labeling of carbohydrates are creating new ways of measuring NMR conformational parameters. The detailed study of a given oligosaccharide by NMR spectroscopy typically yields a number of constraints that are not all compatible with the existence of a single rigid structure, implying that oligosaccharides are, to a greater or lesser extent, dynamic with respect to torsional vibrations around each glycosidic bond. Consequently, the past two years have witnessed a vast increase in the number of efforts aimed at the measurement of NMR parameters directly related to the flexibility of carbohydrates, including 1H and 13C relaxation rates, homo- and heteronuclear cross-relaxation rates, and global and local correlation times. Improvements in the quality and quantity of obtainable NMR data are continuing to inspire molecular modelers to re-evaluate their previous analyses and interpretations. Furthermore, these NMR methods are now being used to investigate the structures of carbohydrates in complexes with proteins (antibodies, lectins) and the orientation and restrained dynamics of carbohydrates covalently attached to model membranes.

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein)

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479–81; Hessionet al., (1987) Science 237:1479–84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547–53]. We report here that the Man6(7)GlcNAc2-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2–2 μM, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man9GlcNAc2-R the most inhibitory of those tested [Muchmoreet al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man5GlcNAc2-R and Man6GlcNAc2-R. Man7GlcNAc2-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).

Preparation, purification, and structural characterization of linear oligogalacturonides. An FAB-mass spectrometric and NMR spectroscopic study

Many plants respond to microbial attack by accumulating small lipophilic molecules, called phytoalexins, that have antibiotic activity lm3. Phytoalexins are absent in healthy plants. Molecules (and other stimuli) that signal plants to begin the process of phytoalexin synthesis are called elicitors4,5. A number of different biotic elicitors (e.g., complex carbohydrates isolated from fungal and plant cell walls) and abiotic elicitors (e.g., heavy metal salts and UV light) are known. We are interested specifically in carbohydrate elicitors that originate either from the cell wall of the host plant (“endogenous” elicitors) or the wall of the invading microbe6-9. The endogenous carbohydrate elicitors are released from the plant cell wall by pectic-degrading enzymes secreted by the invading microorganism’0-‘2. It has been demonstrated with in vitro bio-assays that oligogalacturonides [(l + 4) linked oligomers of cw-o-galacturonic acid] of dp 9 to 15 derived by partial acid hydrolysis of plant cell walls and citrus pectin have high elicitor activity8.9,‘3. Complete structural and conformational characterization of these elicitors has not been reported; in particular, the correlation between the size of the oligosaccharides and their biological activity is poorly understood. We report the preparation and purification of milligram quantities of oligogalacturonides of dp 6 to 16 (see Fig. 1). As previously described9y13, partial acid hydrolysis of citrus pectin produces oligogalacturonides in a dp range from monomer to, at least, a hexadecamer. The crude hydrolysis mixtures have been sub-divided by low-resolution anion-exchange chromatography using successive gradient steps into fractions (A, B, and C) with increasing average dps. Fraction C

Synthesis of (±)-2,3-methanoproline: A novel inhibitor of ethylene biosynthesis

Abstract The title compound, 2-aza-bicyclo[3.1.0]hexane-1-carboxylic acid (2) was prepared by treatment of N-benzyloxycarbonyl-2, 3-dehydroproline tert-butyl ester with diazomethane followed by photolysis of the resulting pyrazoline and deprotection. Its N-acetyl-N′-methyl amide, a peptide mimic, was synthesized and the structure of was confirmed by X-ray diffraction studies. NMR spectroscopy was also used to examine the effect of the cyclopropane ring on its conformation. This 2, 3-methanoamino acid (2) was found to be a weak inhibitor of ethylene biosynthesis in cucumber cotyledon strips and germinating squash seeds. The data show that 2 probably inhibits the conversion of 1-aminocyclopropanecarboxylic acid to ethylene in these tissues.

Measurement of inter-glycosidic 13C-1H coupling constants in a cyclic β(1→2)-glucan by 13C-filtered 2D {1H,1H}ROESY

SummaryA method for measuring three-bond 13C-1H scalar coupling constants across glycosidic bonds in a cyclic β(1→2)-glucan icosamer is presented. This oligosaccharide molecule, with its high degree of symmetry, represents a particular challenge for NMR spectroscopy to distinguish inter-residue from intra-residue heteronuclear coupling effects. Chemically equivalent H2 protons in adjacent glucosyl residues are distinguished on the basis of their different through-space, dipolar interactions with the anomeric protons (H1). The strong NOE contact between anomeric (H1) and aglyconic (H2′) protons permits the selective observation of the inter-residue heteronuclear couplings 3JC1H2′ and 3JC2′H1 in a natural-abundance 13C-ω1-half-filtered {1H,1H} ROESY experiment.

A novel sialylhexasaccharide from human milk: Purification by affinity chromatography on immobilized wheat germ agglutinin

A sialylhexasaccharide fraction (S-5) of human milk was obtained as described by A. Kobata and V. Ginsburg [(1972) Arch. Biochem. Biophys. 150, 273-281] and labeled by reduction with NaB[3H]4. When subjected to affinity chromatography on immobilized wheat germ agglutinin (WGA), a single component representing 60% of the S-5 fraction was retarded by the column. The asialo derivative of the WGA-retarded oligosaccharide had a higher affinity for the WGA column than the native sialyloligosaccharide. The neutral hexaose was identified as lacto-N-neohexaose by sequential exoglycosidase digestions in combination with gel filtration analyses of digestion products. Enzymatic removal of the nonsialylated branch of the intact sialyloligosaccharide by jack bean beta-galactosidase and beta-N-acetylhexosaminidase resulted in a single sialyl[3H]tetraose which was identified as sialyltetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcO[3H]) by cochromatography with authentic standard and specific antibody binding. Independent evidence for the structure of the sialylhexasaccharide was obtained by 500-MHz1H NMR spectroscopy of the WGA-purified oligosaccharide before and after neuraminidase digestion. The structural data are consistent with the following, previously undescribed, sialylhexaose in human milk: (formula; see text).