Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane.

Two forms of protein-membrane anchor have been described for the externally disposed glycoproteins of eukaryotic plasma membranes; namely, the hydrophobic transmembrane polypeptide and the complex glycosylphosphatidylinositol (G-PI) moiety. The chemical structures of the major species of G-PI anchors found on a single variant surface glycoprotein (VSG) of the parasitic protozoan Trypanosoma brucei were determined by a combination of nuclear magnetic resonance spectroscopy, mass spectrometry, chemical modification, and exoglycosidase digestions. The G-PI anchor was found to be heterogeneous with respect to monosaccharide sequence, and several novel glycosidic linkages were present. The results are pertinent to the mechanism of the biosynthesis of G-PI anchors.

The use of two-dimensional correlated spectroscopy to obtain new assignments in the high-resolution 1H nuclear magnetic resonance spectrum of the biantennary complex oligosaccharide isolated from human serum transferrin by hydrazinolysis.

The asialo biantennary complex type oligosaccharide from human serum transferrin was isolated by hydrazinolysis, a method which results in the quantitative release of the intact oligosaccharide free of all amino acids. The 1H-NMR chemical shifts of the previously assigned anomeric and H-2 protons from the peripheral residues of the glycopeptide are identical to the corresponding values for the reduced oligosaccharide. The chemical shift of GlcNAc-1 H-1 proton in the reduced oligosaccharide was assigned by selective deuteration. Proton J connectivities were determined using two-dimensional 1H-1H correlated high resolution NMR spectroscopy. Twelve new assignments were made within the central envelope of the NMR spectrum and a further six were tentatively proposed. The ability to assign proton resonances in this way should allow further conformational studies of the oligosaccharide using nuclear Overhauser effects between the relevant assigned protons on different saccharide residues (Homans, S.W., Dwek, R.A., Fernandes, D.L. and Rademacher, T.W. (1982) FEBS Lett. 150, 503-506).

The analysis of coupling networks in a complex oligosaccharide mixture derived from the Fc region of rabbit immunoglobulin G using 1H-1H correlated NMR spectroscopy combined with double quantum NMR spectroscopy.

In glycoproteins, even for those containing a single glycosylation site, diversity is manifest in the occurrence of a family of structurally-related yet distinct oligosaccharides. To date this 'microheterogeneity' is universal in mammalian glycoproteins. A method is described, using 1H-1H correlated and double quantum nuclear magnetic resonance NMR spectroscopy, for the assignment of proton resonances within a mixture of complex-type oligosaccharides derived from the Fc region of rabbit immunoglobulin G. The ability to assign resonances in heterogeneous populations will be of importance in the chemical shift analysis of the 1H-NMR spectra of glycopeptides since these cannot generally be separated on the basis of their carbohydrate sequence. The resulting assignments will be necessary before conformational studies on glycopeptides using nuclear Overhauser effects can be made.

Nuclear magnetic resonance study of a deoxyoligonucleotide duplex containing a three base bulge.

The three-dimensional structures of a DNA oligonucleotide containing three extra unpaired adenosine residues (dGCCAGGAAATGGAC+dGTCCGACCTGGC) and that of the perfect duplex analogue (dGCCAGGTCGGAC+dGTCCGACCTGGC) have been studied in solution by 1H and 13C nuclear magnetic resonance. All non-exchangeable aromatic and H-1', H-2', H-2" sugar protons were assigned using standard assignment pathways for B-DNA. All cross-peaks within these pathways were present for the perfect duplex molecule as would be expected for a right-handed A or B-form duplex. However, a few cross-peaks which would be expected in the standard case are extremely weak in the nuclear Overhauser enhancement spectroscopy (NOESY) spectrum of the bulged duplex even at long mixing times (250 ms). For example, almost no cross-relaxation is observed between the H-6 proton of C22 and the H-1' of A21, directly across from the three base bulge. Yet the continuity of assignment pathways through the three base bulge argues against any discontinuous "looping out" of one or more of the extra adenosine residues. Double quantum-filtered correlated spectroscopy experiments demonstrate very little deviation from south sugar conformations for residues at or near the bulge. The perfect duplex contains three A.T basepairs as expected, resulting in three very intense T imino-AH2 cross-peaks in the H2O NOESY experiment. In contrast, only two such intense cross-peaks are observed in the same experiment using the bulged duplex sample. Assignments of the two T imino peaks using one-dimensional NOEs are consistent with disruption of the T.A base-pair immediately 3' to the bulge; this is consistent with our earlier observation of chemical reactivity at a T 3' to an An or Tn bulge. We also find evidence of disruption of the G.C base-pair immediately 5' to the bulge.

Residual dipolar couplings as new conformational restraints in isotropically 13C-enriched oligosaccharides.

We report the measurement of residual dipolar couplings for l3C-enriched NeuNAcalpha2-3Galbeta1-4Glc in a dilute liquid-crystalline medium. These couplings provide long-range conformational restraints that hitherto have not been available for oligosaccharides. We utilise these restraints in dynamical simulated annealing calculations, which support current models of the solution behaviour of the trisaccharide.

Solution conformation of the biantennary N-linked oligosaccharide of human serotransferrin using 1H NMR nuclear Overhauser effect measurements.

The conformation in solution of the biantennary complex type oligosaccharide unit derived from human serotransferrin has been investigated using 1H-1H Nuclear Overhauser Effect (NOE) measurements at 300 MHz. From quantitation of the NOE, the alpha(1-3) antenna is shown to exist in a preferred solution conformation with respect to the mannosyl-chitobiose core. The flexibility of the alpha(1-6) arm, together with the absence of NOE data between this arm and the core, indicates that, in contrast to the alpha(1-3) arm the alpha(1-6) arm has no preferred conformation with respect to the core.

Residual 2H quadrupolar couplings in weakly aligned carbohydrates.

We report a novel two-dimensional NMR pulse scheme for the 1H-detected observation of 2H in isotopically 13C, 2H-enriched carbohydrates. This scheme is used for the indirect observation of residual quadrupolar couplings in 13C, 2H-enriched methyl-beta-D-glucopyranoside weakly aligned in a dilute lyotropic liquid-crystalline medium comprising 20% (w/v) dihexanoyl-phosphatidylcholine/dimyristoyl-phosphatidylcholine (1:3 mol/mol) in D2O. The observed residual quadrupolar couplings are substantially larger than residual dipolar one-bond 13C-1H couplings under the same experimental conditions. These quadrupolar couplings are thus a useful alternative to dipolar couplings for the structural analysis of small molecules that align very weakly in dilute liquid-crystalline media. Moreover, since the quadrupolar coupling constant is very uniform throughout endocyclic deuterons of the carbohydrate, these data suggest that adoption of a single average value of this parameter in 2H relaxation studies on the glycan moieties of glycoproteins and glycopeptides is a valid assumption.

Novel GPI structures of the membrane anchor of acetylcholinesterase from the electric organ of Torpedo californica.

The structure of the glycan moiety of the glycosylphosphatidylinositol (GPI) membrane anchor from Torpedo californica electric organ acetylcholinesterase was solved using nuclear magnetic resonance (NMR), methylation analysis, and chemical and enzymic microsequencing. Two structures were found to be present: Glc alpha 1-2 Man alpha 1-2 Man alpha 1-6 Man alpha 1-4 GlcN alpha 1-6myo-inositol, and Glc alpha 1-2 Man alpha 1-2 Man alpha 1-6 (GalNAc beta 1-4) Man alpha 1-4 GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule.

Homonuclear three-dimensional NMR methods for the complete assignment of proton NMR spectra of oligosaccharides--application to Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc.

Two new homonuclear three-dimensional NMR techniques are described for the simplification of proton resonance assignment in oligosaccharides, namely HOHAHA-COSY and ROESY-COSY. The former technique is of value in the resonance assignment of gluco-configuration monosaccharide residues, whereas the latter is more suited to resonance assignment of galacto-configuration monosaccharide residues. The value of these techniques is illustrated by application to the proton resonance assignment of the pentasaccharide Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3 Gal beta 1-4Glc, a compound which exhibits a variety of assignment problems due to severe cross-peak overlap in conventional COSY or HOHAHA spectra.

A molecular mechanical force field for the conformational analysis of oligosaccharides: comparison of theoretical and crystal structures of Man alpha 1-3Man beta 1-4GlcNAc.

A molecular mechanical force field is described for the conformational analysis of oligosaccharides. This force field has been derived by the addition of new parameters to the AMBER force field and is compatible with simulations of proteins. This new parametrization is assessed by comparison of the theoretically predicted conformations of Man alpha 1-3Man beta 1-4GlcNAc with the corresponding crystal structure. Molecular dynamics simulation data are presented for this structure both in vacuo and with the explicit inclusion of water molecules. While the former demonstrate significant torsional oscillations about glycosidic linkages at physiological temperature, in the latter these oscillations are highly damped due to the stabilizing influence of a "cage" of solvent-solvent and solvent-solute hydrogen bonds.

Restrained vs free dynamics simulations of oligosaccharides: application to solution dynamics of biantennary and bisected biantennary N-linked glycans.

We have examined the dynamic behavior in solution of the biantennary glycan GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1- 4GlcNAc beta 1-4GlcNAc, and its "bisected" analogue GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)(GlcNAc beta 1- 4)Man beta 1-4GlcNAc beta 1-4GlcNAc, by use of both free dynamics simulations and restrained dynamics simulations using distance restraints derived from 1H NMR rotating frame Overhauser effect measurements. Data resulting from each type of simulation are compared with experimental data and are critically evaluated. Both methods suggest that most glycosidic linkages exhibit significant torsional oscillations in solution, and the dynamic behavior of certain linkages, notably Man alpha 1-6Man and Man alpha 1-3Man, were found to be restricted by the presence of the bisecting GlcNAc residue. The average structures so obtained were found to agree closely with those predicted in previous investigations where torsional oscillations about glycosidic linkages were not considered. In particular, the characteristic chemical shift perturbations induced by the bisecting GlcNAc residue could be explained in terms of the dynamic differences between the two glycans.

Proton resonance assignments in oligosaccharides containing multiple monosaccharide residues of the same type.

A technique is described for assisting the resonance assignment process in oligosaccharide proton NMR spectra, where multiple residues of the same type generate extreme resonance overlap in the spectrum. The approach involves the modification of a conventional HOHAHA experiment with constant-time acquisition in t1, which effectively proton decouples the C-1 protons of residues whose resonances overlap, thus affording a significant increase in effective resolution in that dimension. For a sufficiently long spin-lock time, complete one-dimensional subspectra are obtained essentially free of cross talk from adjacent resonances. Further simplification of the assignment process is illustrated by incorporation of the constant-time modification into a three-dimensional HOHAHA-HOHAHA experiment.

Application of restrained minimization, simulated annealing and molecular dynamics simulations for the conformational analysis of oligosaccharides.

The purpose of the present study was to determine the confidence with which the small number of 1H NMR nuclear Overhauser effect (NOE) distance constraints measurable across glycosidic linkages in oligosaccharides could be used for solution conformational analysis. This was assessed by use of these constraints in restrained molecular mechanical minimization of the tetrasaccharide Gal beta 1----4(Fuc alpha 1----3)Glc-NAc beta 1----3Gal, a model compound of the Lewis-X antigenic determinant. This presents a particularly severe test case in view of extreme resonance overlap and a dearth of inter-residue distance constraints. It is concluded that these constraints, when used in conventional restrained minimization, result in the generation of 'virtual conformations' and local minima about glycosidic linkages. However, these restraints are nevertheless found to be useful in the initial stages of a conformational analysis strategy involving restrained minimization combined with dynamical simulated annealing to define more accurately the global minimum energy configuration, together with molecular dynamics simulation to explore conformational mobility about this minimum. Theoretical ROE values calculated over the time course of the MD simulation, using a formalism appropriate for the time scale of the internal motion, are compared with those obtained experimentally in the oligosaccharide.

Reducing the overlap problem in the proton NMR spectra of oligosaccharides by application of pseudo-four-dimensional homonuclear HOHAHA-HOHAHA-COSY.

A new homonuclear NMR experiment is described for the assignment of the proton NMR spectra of oligosaccharides, namely HOHAHA-HOHAHA-COSY (where HOHAHA is homonuclear Hartmann-Hahn spectroscopy and COSY is correlated spectroscopy). While this technique may formally be thought of as a four-dimensional NMR experiment, by use of selective pulses it is demonstrated that the analogous pseudo-four-dimensional experiment is a valuable improvement over conventional three-dimensional HOHAHA-COSY, in that the degree of resonance overlap is markedly reduced by the dispersion of resonances into a fourth effective dimension. The technique is demonstrated by application to the biantennary nonasaccharide Gal beta 1-4-GlcNAc beta 1-2Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man-beta 1-4GlcNAc beta 1-4GlcNAc.

Parasite glycoconjugates: towards the exploitation of their structure.

The parasitic protozoa express many unusual complex carbohydrates at the cell surface in the form of glycoproteins and glycophospholipids. In several cases, such molecules have been shown to be involved in parasite survival, infectivity and host-cell recognition. The carbohydrate chains of these glycoconjugates are often highly immunogenic, and can in some cases elicit protective immunity. The immunogenicity of some parasite glycans is a function of their unusual chemical structure as compared with mammalian glycans. This suggests differences in the glycosylation pathways between host and parasite due to their evolutionary distance. This article describes how a combination of biophysical and biochemical techniques can be used to determine the primary and three-dimensional structures of parasite carbohydrate and how this information might be exploited towards the development of new selective chemotherapeutic agents and synthetic vaccines.

Identification of the defect in lipophosphoglycan biosynthesis in a non-pathogenic strain of Leishmania major.

The major macromolecule on the surface of the protozoan parasite, Leishmania major, is a complex lipophosphoglycan (LPG), which is anchored to the plasma membrane by an inositol-containing phospholipid. A defect in LPG biosynthesis is thought to be responsible for the avirulence of the L. major strain LRC L119 in mice. In order to identify the nature of this defect we have characterized two truncated forms of LPG, which are accumulated in this strain, by one- and two-dimensional 500-MHz 1H NMR spectroscopy, two-dimensional heteronuclear 1H-31P NMR spectroscopy, methylation analysis, and exoglycosidase digestions. The structures of these glycoinositolphospholipids, termed GIPL-4 and -6, are as follows: [formula: see text] The glycan moieties of GIPL-4 and -6 are identical to the anchor region of LPG, which is also substituted with a Glc-1-PO4 residue in approximately 60% of the structures. However, instead of being capped with chains of phosphorylated oligosaccharide repeat units, both glycan moieties terminate in Man alpha 1-PO4, suggesting that the defect in LPG biosynthesis is in the transfer of galactose to this residue to form the disaccharide backbone of the first repeat unit. These results indicate that the phosphoglycan moiety of LPG is essential for intracellular survival of the parasite and have implications for LPG biosynthesis.

Solution dynamics of the oligosaccharide moiety of ganglioside GM1: comparison of solution conformations with the bound state conformation in association with cholera toxin B-pentamer.

The solution dynamics of the oligosaccharide moiety of ganglioside GM1 have been determined by use of a combination of 1H rotating frame Overhauser effect measurements and restrained molecular dynamics simulations. It is found that the Galbeta1-3 and NeuNAc moieties which are primarily recognized by cholera toxin both exhibit considerable torsional flexibility about their respective glycosidic linkages. A comparison with the bound state conformation of the ganglioside in association with cholera toxin B-pentamer, shows that a low energy conformation of the oligosaccharide, which closely approximates the global minimum, is selected upon binding.