Two-dimensional Homonuclear Hartmann-Hahn Research Articles

Structural Investigation of Cell Wall Mannan Antigen Obtained from Pathogenic Yeast <i>Candida zeylanoides</i>

Antigenicity of pathogenic yeast Candida zeylanoides NBRC 10326 was found to have similar to that of Candida guilliermondii, but it was slightly different, i.e. NBRC 10326 strain cells react strongly with the factor antibodies 1, 4, and 9, and react relatively weakly with factor antibody 5. We have investigated the structural characteristic of cell wall mannan of this strain using a non-degradable method, nuclear magnetic resonance analysis. The results obtained from NMR analysis, the two-dimensional homonuclear Hartmann-Hahn, indicated that C. zeylanoides mannan contains α-1, 3-linked and β-1,2-linked oligomannosyl side chains corresponding to C. guilliermondii specific antigenic factor 9. In addition, it was found that the mannan contains β-1,2-linked oligomannosyl side chains attached to phosphate groups corresponding to anitigenic factor 5, which were not found in C. guilliermondii mannan.

Distribution of antigenic oligomannosyl side chains in the cell wall mannans of several strains of Candida tropicalis

In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall mannans of the pathogenic yeast Candida tropicalis, the chemical structure of mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the mannans. The linear side chain Manalpha1-3Manalpha1-(2Manalpha1-)(n)2Man [n> or =2] is present in the mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the mannans from IFO 0199 and IFO 1647. The mannan of IFO 0589 is the only mannan with the branched side chains, Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man and Manalpha1-2Manalpha1-3[Manalpha1-6]Manalpha1-(2Manalpha1-)(n)2Man [n> or =2]. However, this mannan lacked the phosphate group and the beta-1,2-linked oligomannosyl side chain which are features of this group. The mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an alpha-1,3-linked mannose residue previously observed in Candida albicans.

Demonstration of the presence of alpha-1,6-branched side chains in the mannan of Candida stellatoidea.

A mild acetolysis of the mannans of Candida stellatoidea was performed after acetylation to yielded an alpha-1,6-branched mannohexaose, the presence of which had been predicted from the appearance of a specific H1-H2-correlated cross-peak in two-dimensional homonuclear Hartmann-Hahn spectroscopy. In this study, we found that the de-O-acetylation of a 4-O-acetyl group at the branching point, the 3,6-di-O-substituted mannose unit, of an acetylated oligosaccharide by sodium methoxide is significantly slower than that of other acetyl groups. We could separate the 4-O-acetylated branching oligosaccharide from linear isomer using high-performance liquid chromatography. Before and after the de-O-acetylation of the purified branching oligosaccharide, their 1H-NMR signals were sequentially assigned by means of the nuclear Overhauser effect. In the sequential NMR assignment study, we showed that the alpha-1,6-linked mannose unit is attached to the 3-O-substituted unit based on the presence of NOE cross-peak between H1 of the branching mannose unit and H6 of the 3-O-substituted mannose unit. An enzyme-linked immunosorbent inhibition assay of the reactivity of factor 4 serum to C. stellatoidea mannan by several oligosaccharides indicated that the alpha-1,6-branched oligosaccharide and the beta-1,2 linkage-containing oligosaccharides showed inhibitory activity. This result indicates that factor 4 serum, as well as factor 5 and 6 sera, contains antibodies against beta-1,2-linked mannose units which have been reported to participate in pathogenicity via cytokine production and/or adherence. From the assignment results of H1-H2-correlated cross-peaks of oligosaccharides and mannans, the molar ratio of the mannan side chains was proposed. In this study, we demonstrated that the epitope structure of the C. stellatoidea type I strains was the same as that of the C. albicans NIH B-792 (serotype B) strain.

1H-magnetic resonance spectroscopic observation of cultured malignant cells pharmacologically induced to different phenotypes.

We evaluated the 1H nuclear magnetic resonance spectra of malignant cells after the administration of drugs that cause morphologic changes. 1H spectra of a human lung adenocarcinoma cell line cultured with interferon gamma, dexamethasone, or sodium butyrate were obtained. The peaks were assigned by two-dimensional homonuclear Hartmann-Hahn spectroscopy spectra of the cells and their perchloric acid extracts. Differential spectra were used to evaluate relative changes in the peaks. In the control culture, choline/phosphocholine peaks were increased in the cell-growth phase, and the 1.26-ppm peak was increased in the confluent state. Treatment by interferon gamma and dexamethasone induced reproducible changes in the peaks of differential spectra corresponding to 1.26 ppm, choline/phosphocholine, and glutamate/glutamine. Dexamethasone treatment broadened lipid peaks. Changes after treatment with sodium butyrate were obscure. Microscopically, cells were induced to morphologically different phenotypes by each drug. Cells induced to exhibit morphologically different phenotypes present different 1H spectra.

Characterization of alpha-1,6-mannosyltransferase responsible for the synthesis of branched side chains in Candida albicans mannan.

A particulate insoluble fraction from Candida albicans NIH B-792 (serotype B) strain cells was obtained as the residue after extracting a 105000 x g pellet of cell homogenate with 1% Triton X-100. Incubation of this fraction with a mannopentaose, Man alpha 1-->3Man alpha 1-->2Man alpha 1-->Man alpha 1-->2Man, in the presence of GDP-mannose and Mn2+ at pH 6.0 gave a branched mannohexaose, [sequence: see text] 6 the structure of which was identified by means of sequential off assignment. However, the enzyme fraction obtained from Candida parapsilosis gave Man alpha 1-->2Man alpha 1-->3Man alpha 1-->2Man alpha 1-->2 Man alpha 1-->2Man under the same conditions. These results demonstrate the finding that the structural difference in the mannans of these two species is due to the presence of alpha-1.6-linked branching mannose units in the C. albicans mannan [Shibata, N., Ikuta, K., Imai, T., Satoh, Y., Satoh, R., Suzuki, A., Kojima, C., Kobayashi, H., Hisamichi, K. & Suzuki, S. (1995) J. Biol. Chem. 270, 1113-1122]. The substrate-specificity study of the enzyme indicated that the structural requirement of the alpha-1,6-mannosyltransferase is Man alpha 1-->3Man alpha 1-->. The alpha-1,6-mannosyltransferase also transferred the alpha-1,6-linked branching mannose unit to the mannan of Saccharomyces cerevisiae. The transformation of the mannan was detected by the appearance of antigenic factor 4 using an enzyme-linked immunosorbent assay and two-dimensional homonuclear Hartmann-Hahn spectroscopy.

The effect of conformation on membrane permeability of an acyloxyalkoxy-linked cyclic prodrug of a model hexapeptide.

To determine the different conformations of the acyloxyalkoxy-linked cyclic prodrug 1 of the model hexapeptide 2 in solution and to investigate the relationship between these solution conformations and the cellular permeability characteristics of this prodrug. Two-dimensional Homonuclear Hartmann-Hahn spectroscopy, Rotating-Frame Overhouser effect spectroscopy, circular dichroism and molecular dynamics simulations were used to find the solution conformers of cyclic prodrug 1. Our spectroscopic findings suggest that cyclic prodrug 1 exhibits a major and a minor conformer in solution. The major conformer appears to have a well-defined secondary structure, which involves a beta-turn and 4-->1 intramolecular hydrogen bond, creating a compact structure with a reduced average hydrodynamic radius compared to the model hexapeptide 2. The increased ability of cyclic prodrug 1 to permeate membranes compared to the model hexapeptide 2 could be due to reduction in the average hydrodynamic radius of the molecule facilitating paracellular flux and/or the reduction in the hydrogen bonding potential facilitating transcellular flux.

Existence of Branched Side Chains in the Cell Wall Mannan of Pathogenic Yeast, Candida albicans: STRUCTURE-ANTIGENICITY RELATIONSHIP BETWEEN THE CELL WALL MANNANS OF CANDIDA ALBICANS AND CANDIDA PARAPSILOSIS

Isolation of side chain oligosaccharides from mannans of Candida albicans NIH B-792 (serotype B) and Candida parapsilosis IFO 1396 strains has been conducted by acetolysis under mild conditions. Structural study of these oligosaccharides by 1H and 13C NMR and methylation analyses indicated the presence of novel branched side chains with the following structures in C. albicans mannan. [sequence: see text] It was observed that the H-1 proton chemical shifts of the second and the third mannose units from the reducing terminus in each oligosaccharide are shifted upfield by substitution with an alpha-linked mannose unit at position 6 of the 3-O-substituted mannose unit. An agglutination inhibition assay between factor 4 serum and cells of Candida stellatoidea IFO 1397 lacking the beta-1,2-linked mannose unit, with oligosaccharides obtained from these mannans, indicated that only the branched oligosaccharides were active. This finding suggests that the branched oligosaccharides correspond to the epitope of antigenic factor 4. The presence of the branched structure in other mannans was detected by the characteristic H-1-H-2-correlated cross-peak of the alpha-1,2-linked mannose unit connected with the 3,6-di-O-substituted one by two-dimensional homonuclear Hartmann-Hahn spectroscopy.

Expression of α-1,3 linkage-containing oligomannosyl residues in a cell-wall mannan of Candida tropicalis grown in yeast extract-Sabouraud liquid medium under acidic conditions

We investigated the cell-wall mannan obtained from Candida tropicalis IFO 1647 strain cells grown in yeast extract-Sabouraud medium at pH 3.0 by two-dimensional homonuclear Hartmann-Hahn spectroscopy. The results indicate that the phosphate group and the side chains containing a β-1,2-linked mannopyranose unit decreased compared to those of mannan from cells grown under conventional conditions (pH 5.9) with concomitant expression of α-1,3 linkage-containing oligomannosyl side chains. The results of acetolysis of these mannans indicated that the presence of α-1,3-linked mannopyranose unit existed in side chains corresponding to pentaose and hexaose, Man pα1-3Man pα1-2Man pα1-2Man pα1-2Man, and Man pα1-2Man pα1-3Man pα1-2Man pα1-2Man pα1-2Man, in the mannan from cells grown at pH 3.0.

Structural study of a cell-wall mannan of Saccharomyces kluyveri IFO 1685 strain. Presence of a branched side chain and beta-1,2-linkage.

Acetolysis of the cell-wall mannan of Saccharomyces kluyveri under mild conditions, gave fragments with 1-6 mannose residues. The structures of mannopentaose and mannohexaose were determined to be [Formula; see text] respectively, by two-dimensional homonuclear Hartmann-Hahn spectroscopy and a sequential NMR assignment method that combines 1H-13C correlated spectroscopy, relayed coherence transfer spectroscopy, 1H-detected heteronuclear multiple-bond connectivity and methylation analysis. The H1 proton chemical shift of a neighboring alpha-1,2-linked mannose unit of the 3-O-substituted structure was shifted upfield by the addition of a mannose unit to the adjacent 3-O-substituted unit by an alpha-1,6 linkage. The characteristic H1--H2-correlated cross-peak of the alpha-1,3-linked mannose unit substituted by a beta-1,2 linkage, beta 1-->2Man alpha 1-->3, in the mannan of S. kluyveri, as also found by two-dimensional homonuclear Hartmann-Hahn spectroscopy in the mannan of Candida guilliermondii, a pathogenic yeast in man.

Conformation of deltorphin-II in membrane environment studied by two-dimensional NMR spectroscopy and molecular dynamics calculations.

Two-dimensional homonuclear Hartmann-Hahn spectroscopy and NOESY (nuclear Overhauser effect and exchange spectroscopy) 1H-NMR techniques have been used to obtain complete proton resonance assignments and to perform a conformational investigation of deltorphin-II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2), a naturally occurring delta-selective opioid peptide, in the membrane-mimetic micelles of perdeuterated dodecylphosphocholine. This was done in order to examine conformational characteristics that would be closely related to the selectivity towards the delta-opioid receptor. With the use of the proton-proton distances derived from NOESY measurements, 50 possible three-dimensional structures were generated by means of distance-geometry calculations, and 25 of them were subjected to the molecular-dynamics simulations of 10 ps, which were energetically constrained for the NOE interproton distances. Most of the possible conformers simulated showed a common feature such that the conformation of deltorphin-II is characterized by the S-shaped back-bone structure in which the turn conformation of the Val-Val-Gly-NH2 sequence is located under the helically folded conformation of the N-terminal Tyr-D-Ala-Phe-Glu sequence. The possible relationship between this conformational characteristic and the delta-opioid-receptor selectivity has been discussed.

Complete structure of the adhesin receptor polysaccharide of Streptococcus oralis ATCC 55229 (Streptococcus sanguis H1).

This report describes the determination of the complete primary structure of the adhesin receptor polysaccharide of Streptococcus oralis ATCC 55229 (previously characterized as Streptococcus sanguis H1), a Gram-positive bacteria implicated in dental plaque formation. The polysaccharide was isolated from S. oralis ATCC 55229 cells after deproteination, enzymatic hydrolysis, and ion exchange chromatography. It was shown to consist of rhamnose, galactose, glucose, glycerol, and phosphate, in molar ratios of 2:3:1:1:1. Sequence and linkage assignments of the glycosyl residues were obtained by methylation analysis followed by gas-liquid chromatography and electron-impact mass spectrometry. 31P NMR spectroscopy revealed that phosphate was present in a diester, connecting glycerol to one of the galactosyl residues. High-performance liquid chromatography of a partial acid hydrolysate of the polysaccharide confirmed this finding by showing galactose 6-phosphate and glycerol 1-phosphate. The structural determination was completed by the combination of two-dimensional homonuclear Hartmann-Hahn and NOE experiments and heteronuclear [1H,13C] and [1H,31P] multiple-quantum coherence experiments. Thus, the adhesin receptor polysaccharide of S. oralis ATCC 55229 was found to be a polymer composed of hexasaccharide repeating units that contain glycerol linked through a phosphodiester to C6 of the alpha-galactopyranosyl residue and are joined end-to-end through galactofuranosyl-beta(1-->3)-rhamnopyranosyl linkages: [formula: see text] This structure is novel among bacterial cell surface polysaccharides in general and specifically among those implicated in dental plaque formation.

Nondestructive Analyses of Residue Types in β-d-Glucan Derivatives by Two-Dimensional Homonuclear Hartmann-Hahn Spectroscopy

Nondestructive Analyses of Residue Types in β-d-Glucan Derivatives by Two-Dimensional Homonuclear Hartmann-Hahn Spectroscopy

Structure chacterization by two-dimensional NMR spectroscopy, of two marine triterpene oligoglycosides from a pacific sponge of the genus Erylus

The isolation and characterization of two novel triterpene glycosides from a sponge of the genus Erylus, collected at a depth of 500 m in the South of New Caledonia, are described. The structures are characterized by the presence of a branched oligosaccharide chain, composed of three ( 1) and four (2) D-galactopyranose units, respectively. Analysis of the oligosaccaride structures was achieved by { 1H, 1H} correlation spectroscopy, two-dimensional homonuclear Hartmann-Hahn, and 1H-detected { 1H, 13C} one bond (HMQC) and multiple-bond (HMBC) shift correlation NMR experiments. The novel lanostane derived aglycone features a rare 14-carboxyl group and a 24-methylene, 25-methyl side chain.

A structural study of calcium-binding equine lysozyme by two-dimensional 1H-NMR

Since 1H-NMR spectra of the calcium bound form (holo) and the calcium free form (apo) of equine lysozyme have an overall similarity, the folded structure of apo equine lysozyme seems to be similar to the holo structure at 25 degrees C and pH 7.0, even at low ionic strengths except for subtle conformational change. However, calcium titration experiments showed that a number of resonances change by a slow exchange process. The changes saturated at one calcium ion per one lysozyme molecule, and no more change was observed by further addition of calcium ions. This shows that just one calcium ion binds to equine lysozyme. To make assignments for these changed proton resonances, two-dimensional 1H-NMR studies, correlated spectroscopy (COSY), two-dimensional homonuclear Hartmann-Hahn spectroscopy (HOHAHA) and nuclear Overhauser effect spectroscopy (NOESY) were carried out. A structural model of equine lysozyme based on the crystal structure of human lysozyme was estimated and used to assign some resonances in the aromatic and beta-sheet regions. It was possible to use some proton signals as a probe to determine the specific conformational change induced by calcium ions. The calcium binding constant KCa was estimated from calcium titration experiments in which changes in the proton signal were monitored. The log KCa value was found to be on the order of 6-7, which is in agreement with the calcium binding constant determined by fluorescence probes. This means that the protons are affected by specific calcium binding.

Conformational Analysis of the Tachykinins in Solution: Substance P and Physalaemin

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of beta-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.

Structure of a streptococcal adhesin carbohydrate receptor.

Interactions between complementary protein and carbohydrate structures on different genera of human oral bacteria have been implicated in the formation of dental plaque. The carbohydrate receptor on Streptococcus sanguis H1 (one of the primary colonizing species) that is specific for the adhesin on Capnocytophaga ochracea ATCC 33596 (a secondary colonizer) has been isolated from the streptococcal cell wall, purified, and structurally characterized. The hexasaccharide repeating unit of the polysaccharide was purified by reverse-phase, amino-bonded silica, and gel permeation high performance liquid chromatography. Earlier studies established that the repeating unit was a hexasaccharide composed of rhamnose, galactose, and glucose in the ration of 2:3:1, respectively. In the present study, determination of absolute configuration by gas chromatography of the trimethylsilyl (+)-2-butyl glycosides revealed that the rhamnose residues were of the L configuration while the hexoses were all D. 252Californium plasma desorption mass spectrometry of the native, the acetylated and the reduced and acetylated hexasaccharide determined that the molecular mass of the native hexasaccharide was 959, and that the 2 rhamnose residues were linked to each other at the nonreducing terminus of the linear molecule. Methylation analysis revealed the positions of the glycosidic linkages in the hexasaccharide and showed that a galactose residue was present at the reducing end. The structural characterization of the hexasaccharide was completed by one and two dimensional 1H and 13C NMR spectroscopy. Complete 1H and 13C assignments for each glycosyl residue were established by two-dimensional (1H,1H) correlation spectroscopy, homonuclear Hartmann-Hahn, and (13C,1H) correlation experiments. The configurations of the glycosidic linkages were inferred from the chemical shifts and coupling constants of the anomeric 1H and 13C resonances. The sequence of the glycosyl residues was determined by a heteronuclear multiple bond correlation experiment. These data show that the structure of the hexasaccharide repeating unit derived from the cell wall polysaccharide of S. sanguis H1 is: alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----3)-alpha-D-Galp- (1----3)-beta-D-Galp-(1----4)-beta-D-Glcp-(1----3)-alpha/beta-D-Gal.

Conformational studies on the N‐linked carbohydrate chain of bromelain

1H- and 13C-NMR assignments for the carbohydrate part of the glycopeptide alpha-D-Man-(1----6)-[beta-D-Xyl-(1----2)]-beta-D-Man-(1----4)-beta-D- GlcNAc-(1----4)-[alpha-L-Fuc-(1----3)]-beta-D-GlcNAc-(1----N)-Asn approximately, derived from the proteolytic enzyme bromelain (EC 3.4.22.4), have been obtained using homo- and heteronuclear correlation spectroscopy, two-dimensional homonuclear Hartmann-Hahn and nuclear Overhauser enhancement experiments. A conformational model for the carbohydrate chain, deduced from the NMR data and consistent with hard-sphere exo-anomeric calculations shows that the rotamer population about the C-5--C-6 bond of beta-Man is restricted to the P omega = 180 rotamer, mainly.

Two-Dimensional NMR Spectral Study (at 500 MHz) of a Glycooctapeptide Derived from Glycophorin AM

ABSTRACT One-dimensional and two-dimensional spectral data are presented for the N -terminal glycooctapeptide derived from the transmembrane glycoprotein, glycophorin AM. Two-dimensional homonuclear Hartmann-Hahn spectroscopy (2D-HOHAHA) and two-dimensional rotating frame nuclear Overhauser enhancement spectroscopy (2D-ROESY) were used to assign many of the resonances present in the spectra, with specific emphasis placed upon the oligosaccharide units. Not only were intra residue NOE's detected, but also inter residue NOE's involving two linked carbohydrates [β-Gal(1-->3)α-GalNAc] and also between the α-GalNac-->Thr linkage pair (between GalNAc H-1 and Thr Hβ) indicating restricted motion about this glycosidic bond. Moreover, the H-1/H-2 cross-peaks in the 2D-ROESY spectra appear to be well resolved indicating some chemical shift differences for H-1 and H-2 of the three linkage GalNAc residues found in this glycooctapeptide.

Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study.

1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP.

Refinement of the solution structure of the RNA-DNA hybrid 5′-[r(GCA)d(TGC)] 2 Combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution conformation of the self-complementary RNA-DNA hybrid hexamer 5′-[r(GCA)d(TGC)] 2 is investigated by NMR spectroscopy and restrained molecular dynamics. The 1H-NMR spectrum is assigned in a sequential manner using two-dimensional homonuclear Hartmann-Hahn and nuclear Overhauser enhancement spectroscopy. From the latter a set of 178 approximate interproton distance restraints are determined and used as the basis of a structure refinement by restrained molecular dynamics. Eight independent calculations are carried out, four from a classical A-type geometry and four from a classical B-type one. Convergence is achieved to very similar A-type structures with an average atomic root mean square difference between them of 1.0±0.2 Å. The converged structures exhibit variations in helical parameters similar to those found previously for the analogue RNA hexamer 5′-r(GCAUGC) 2 [(1988) Biochemistry 27, 1735–1743].