Solution structure of two xenoantigens: {alpha}Gal-LacNAc and {alpha}Gal-Lewis X

Francisco Corzana2,3, Emmanuel Bettler2, Catherine Hervé du Penhoat2, Tatyana V. Tyrtysh4, Nicolai V. Bovin4 and Anne Imberty1,2

2Centre de Recherche sur les Macromolécules Végétales (Affiliated with Joseph Fourier University), BP 53, F-38041 Grenoble cedex 9, France; 3Departamento de Química, Universidad de La Rioja, Madre de Dios, 51, E-26006 Logroño (La Rioja), Spain; and 4Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul Miklukho-Maklaya 16/10, 17871 GSP-7, V-437, Moscow, Russia

Received on June 19, 2001; revised on August 31, 2001; accepted on August 31, 2001.


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Organ hyperacute rejection, a phenomenon occurring during discordant xenotransplantation, is due to the recognition of an oligosaccharide epitope by human xenoreactive natural antibodies. In addition to the {alpha}Gal(1-3)ßGal(1-4)GlcNAc trisaccharide, a fucosylated structure, {alpha}Gal-Lewis X, has been shown to be recognized by the antibodies. Both the trisaccharide and the tetrasaccharide have been synthesized by chemical methods. A complete nuclear magnetic resonance characterization of the two compounds has been performed, including the measurements of two-dimensional nuclear Overhauser effect spectroscopy data. Molecular dynamics simulations were run for several ns in the presence of explicit water molecules. The combination of experimental and theoretical approaches revealed the effect of an additional fucose residue on the conformational behavior of the xenoantigen. This branched fucose strongly rigidifies the N-acetyllactosamine. The effect on the {alpha}Gal(1-3)Gal fragment is less marked. In the presence of fucose, the terminal {alpha}Gal residue can still adopt two different conformations, but the equilibrium populations are modified.

Key words: /molecular dynamics/NMR/oligosaccharides/xenotransplantation


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Xenotransplantation—grafting of an organ from another species, generally from pig to human—is considered to be the main potential remedy for the shortage of donor organs (see review by Auchincloss and Sachs, 1998Go). The first barrier to be overcomed for transplanting a pig organ into a human is the hyperacute vascular rejection that occurs in the first minute (Cascalho and Platt, 2001Go). The antigen responsible for this hyperacute vascular rejection has been identified as the linear B oligosaccharide {alpha}Gal1-3ßGal1-4ßGlcNAc (Cooper et al., 1993Go) that is present on the endothelium of all mammals, except in higher apes and man (Galili et al., 1988Go; Oriol et al., 1999Go). On the other hand, antibodies against this epitope are present in all humans and higher apes (Galili et al., 1987Go; Samuelsson et al., 1994Go). Recognition of the {alpha}Gal epitope by these preformed antibodies activates both the pig cells and the human complement cascade, resulting in rapid thrombosis in the pig organ (Sandrin et al., 1993Go).

Much effort has been devoted to the conformational analysis of the {alpha}Gal1-3Gal disaccharide because this compound is the epitope recognized by human xenoreactive natural antibodies. In addition to an early nuclear magnetic resonance (NMR) study (Lemieux et al., 1980Go), energy maps of this linkage have been calculated with the help of the MM2 (Strotz and Cerezo, 1994Go; Bizik and Tvaroska, 1995Go) or MM3 programs (Imberty et al., 1995Go). Recently, the conformational study of an analogue, {alpha}Gal1-3ßGal1-4ßGlc-1NAc, has been performed by a combination of Monte Carlo calculations and NMR spectroscopy (Li et al., 1999Go). In all recent studies, the {alpha}Gal1-3Gal epitope has been predicted to be rather flexible because it adopts at least two different conformations in solution.

In addition to the linear {alpha}Gal1-3ßGal1-4ßGlcNAc epitope, present on glycolipids and glycoproteins of pig endothelial cells, a branched epitope, {alpha}Gal1-3ßGal1-4({alpha}Fuc1-3)ßGlcNAc ({alpha}Gal-Lex) has been characterized as a component of glycosphingolipids expressed in pig kidney (Bouhours et al., 1997Go, 1998). This tetrasaccharide has been revealed by immunostaining with a polyclonal antibody directed against {alpha}Gal1-3Gal1 determinants. Schematic representation of the oligosaccharides are given in Scheme 1.



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Scheme 1. Schematic representation of oligosaccharides 14 with the labeling of the torsion angles of interest.

 
The present study is dedicated to the comparison of the solution conformational behavior of these two oligosaccharides in the aim of defining their common conformational epitope recognized by cross-reacting antibodies. Both the linear trisaccharide and the branched tetrasaccharide have been synthesized and fully characterized by NMR. The conformational behavior of both compounds has been determined by molecular dynamics simulations in explicit water environment. The calculations were validated by comparing theoretical and experimental nuclear Overhauser effect spectroscopy (NOESY) data.


    Results
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
NMR data
Chemical shifts and coupling constants.
The 1H and 13C assignments for 2 and 4 were based on 2D correlation spectra (see Materials and methods), and these data have been collected in Tables I and II, respectively. There is very good agreement between the chemical shifts of 2 and 4 and the ones previously obtained for related oligosaccharides. 1H and 13C chemical shifts have been reported for compounds similar to trisaccharides 1 and 2, {alpha}Gal1-3ßGal-OR (where R = –(CH2)8CO2Me; Lemieux et al., 1980Go) and {alpha}Gal1-3ßGal1-4ßGlc-Nac (i.e., with the N-acetyl substituent at the anomeric carbon; Li et al., 1999Go). Partial proton data have been reported for compounds related to tetrasaccharide 3 and 4, {alpha}Gal1-3ßGal1-4[{alpha}Fuc1-3]ßGlcNAc-OH; Joziasse et al., 1993Go) and ßGal1-4[{alpha}Fuc1-3]ßGlcNAc-OR (where R = –(CH2)8CO2Me; Hindsgaul et al., 1982Go). The rotamer populations of the exocyclic groups of residues I and III of compounds 2 and 4 can be deduced from the fine structure of the H5/H6a,b crosspeaks (residue I, < 2 and ~ 5 Hz) and the 3JH5,H6,6' coupling constants (residue III, H5 – br t, 7.8 Hz), respectively. The former vicinal couplings (residue I) correspond to a 50/50 mixture of gg and gt populations, whereas the latter parameters (residue III) indicate a 50/50 mixture of tg and gt rotamers (Bock and Duus, 1994Go). Similar vicinal coupling constant data were reported for the {alpha}Gal1-3ßGal1-4ßGlcNAc trisaccharide (Li et al., 1999Go).


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Table I. 1H and 13Ca NMR data of a 6 mM solution of trisaccharide 2 in D2O at 298°K.
 

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Table II. 1H and 13Ca NMR data of a 8 mM solution of tetrasaccharide 4 in D2O at 298K.
 

NOESY data.
NOESY spectra were acquired for the trisaccharide 2 at 30 and 35°C with a 500-ms mixing time. Under these conditions the sign of the cross-peaks was opposite to those of the diagonal peaks (positive nOes, {omega}{tau}c < 1). Inspection of the rows containing the H1III signal on the diagonal revealed interglycosidic crosspeaks with H3II and H4II in a 2:1 ratio in agreement with nOe data reported earlier for the related {alpha}Gal1-3ßGal1-4ßGlcNAc trisaccharide (Li et al., 1999Go).

At 5°C overall tumbling of compound 4 was slow enough to yield measurable crosspeak volumes with the same sign as the diagonal peaks (negative nOes, {omega}{tau}c > 1) for all of the mixing times (100, 200, and 400 ms). However, at this temperature the anomeric signals displayed severe overlapping ({alpha}-sugars, H1III with H1IV; ß-sugars, H1I with H1II) and summed cross-peak volumes have been considered in both cases. The corresponding nOe build-up curves were linear in keeping with the theoretical ones (vide infra). The experimental NOESY cross-peak volumes for both the anomeric protons and strategic protons with signals in nonoverlapping regions have been collected in Table III (bold) and will be discussed below. An expansion of the NOESY spectrum acquired with a 200-ms mixing time has been given in Figure 1.


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Table III. Comparison between experimental (bold) 500 MHz NOESY data of aqueous solution (T 5°C) of tetrasaccharide 4 with theoretical NOESY data obtained from molecular dynamics of tetrasaccharide 3.
 


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Fig. 1. An expansion of the NOESY spectrum of {alpha}Gal-Lewis X (mixing time 200 ms, 5°C) with the low field signals on the diagonal (F1). Cross-peaks with the protons resonating at high field (F2) have been labeled (F1/F2). The observed inter-ring nOes effects are reported in Scheme 2.

 
Molecular dynamics simulations
Molecular dynamics trajectories of 3 ns were calculated for both oligosaccharides 1 and 3 in explicit water environment. During the simulation, trisaccharide 1 displays flexible behavior. Large ranges of the torsional angles are visited, specially for the {Psi} angles of both glycosidic linkages (Figure 2). The history of torsion angles during the simulation have been superimposed on the energy maps of each linkage that were previously calculated using the MM3 program (Imberty et al., 1995Go) (Figure 3). The ßGal1-4GlcNAc linkage explores the large low-energy plateau characteristic of two pyranose rings linked in a equatorial-equatorial configuration (Pérez et al., 2000Go). Variation ranges of 60° in {Phi} and 120° in {Psi} centered on this plateau are observed that correspond approximately to an energy window of 4–5 kcal/mol when calculated using the MM3 software. The {alpha}Gal1-3Gal linkage is more flexible, four discrete conformations are populated during the simulation, three of them being very different with respect to the {Psi} angle. They correspond to the trans, gauche, and gauche+ orientations of this torsion angle and have been labeled A, B, and C, respectively. Graphical representation of the lowest energy conformations of the trisaccharide are displayed in Figure 4. During the simulation, the trisaccharide starts from conformation B. After 500 ps, it makes short visits to conformation A, and after 1 ns stabilizes in this conformation during 200 ps. From there, it jumps for a single visit of 200 ps to conformation C, before returning to conformation B for the last ns. Another conformation, D, corresponds to a variation in the {Phi} angle and is visited only from conformation A.



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Fig. 2. Histories of the {Phi} and {Psi} torsion angles of the glycosidic linkages of trisaccharide 1 and tetrasaccharide 3 during the 3-ns molecular dynamics simulation in water environment.

 


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Fig. 3. Trajectories of the {Phi} and {Psi} torsion angles of the glycosidic linkages of trisaccharide 1 and tetrasaccharide 3 during the 3-ns molecular dynamics simulation in water environment superimposed on the MM3 energy map of each of the corresponding disaccharides.

 


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Fig. 4. Graphical representation of the main low-energy conformations of trisaccharide 1 and tetrasaccharide 3.

 
The conformational behavior of the tetrasaccharide 3 is quite different. Branching of a fucose residue on position 3 of GlcNAc resulted in a drastic stiffening of the adjacent ßGal1-4GlcNAc linkage. Because of the interactions between the Fuc and the ßGal residues, the Lewis X moiety of the molecule exists as a unique conformational family corresponding to that observed by X-ray crystallography (Pérez et al., 1996Go). As seen from Figures 2 and 3, the ßGal1-4GlcNAc linkage conformation exhibits variations of no more than 30° around a conformation centered at {Phi} = –80° and {Psi} = –120°. Although there is no direct interaction between the Fuc and the {alpha}Gal residues, the {alpha}Gal1-3Gal linkage flexibility is affected by the stiffening of the LacNAc backbone. The {alpha}Gal1-3Gal is still flexible, but it adopts predominantly the B conformation (Figure 4) and makes only a short visit into the A region, with no stabilization in this area and no visit to the gauche conformation. Due to the time scale of the simulation, it cannot be concluded that this particular conformation is not populated in solution. Nevertheless, it could be predicted that the presence of the fucose energetically favors the B conformers of the {alpha}Gal1-3Gal linkage and reduces its flexibility.

Comparison of experimental and theoretical NMR data
Theoretical NOESY were calculated from the molecular dynamics simulations trajectories and compared with the experimental data. For the {alpha}Gal-LacNAc trisaccharide, good agreement was obtained. The results do not differ significantly from those obtained by Li et al. (1999)Go and are therefore not presented further in this article. It could be confirmed that most of the flexibility of this molecule occurs from a conformational equilibrium between the two lowest-energy conformations at the {alpha}Gal1-3Gal linkage (conformations A and B in Figure 4). Other conformations can also occur in solution, but their population is not significant.

Comparison of theoretical and experimental NOESY for the {alpha}Gal-Lewis X is given in Table III. In general, the experimental NOESY volumes are smaller than the theoretical ones, a fact that can be explained by the rapid local motion of the methyl groups (NAc and Fucp). Because experimental and theoretical data have been obtained from tetrasaccharides 3 and 4, respectively, small differences are observed at the anomeric center of the GlcNAc residue: the experimental volumes involving H1I are expected to be slightly different from the theoretical ones because a supplementary relaxation pathway exists for this spin via the protons of the –CH2CH2CH2NH2 pendant group.

There is an excellent agreement between modeled and solution conformations for the Lewis X moiety. The strong nOe observed between H-5IV and H-2II confirms the interaction between the two nonbonded residues {alpha}Fuc and ßGal. As for the flexible part of the tetrasaccharide, that is, the {alpha}Gal1-3Gal linkage, no single conformation could explain the ensemble of NOESY data listed in Table III and represented in Figure 1. Of particular interest are the values of the H-1III/H-3II, H-1III/H-4II, and H-5III/H-3II interactions because they are very sensitive to the conformational equilibrium of the {alpha}Gal1-3Gal linkage. More precisely, conformation A of the tetrasaccharide is characterized by a short H-1III . . . H-3II distance, whereas conformation B displays a contact between H-1III and H-4II (data not quantified in Table III due to overlapping signals). The simulation shown in Figures 2 and 3 where the {alpha}Gal1-3Gal linkage is predicted to spend most of the time in conformation B while making several incursions of few tens of ps in energy minimum A allows for an excellent reproduction of these two experimentally observed NOESY values.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
The combination of experimental and theoretical approaches allowed for a precise determination of the effect of an additional fucose residue on the conformational behavior of the xenoantigen. This branched fucose strongly rigidifies the N-acetyllactosamine core but has a minor effect on the conformational behavior of the {alpha}Gal1-3Gal linkage. Nevertheless, the B conformation, which is adopted by 60% of the population for the trisaccharide, seems to be even more favored for the tetrasaccharide. Interestingly, the B conformation is also the one that is observed ({Phi} = 56°, {Psi} = 60°) for the {alpha}Gal1-3Gal linkage in the crystal structure of the blood group B trisaccharide (Otter et al., 1999Go).

In conclusion, design of a rigid inhibitor that would mimic the antigenic conformation of the {alpha}Gal1-3Gal epitope still represents a difficult problem. Due to the intrinsic flexibility of this linkage, it is difficult to predict the shape that will be recognized by an antibody. It is even possible that polyclonal antibodies may recognize different conformations of this molecule. Preformed xenoreactive antibodies are thought to arise during the first year of life, owing to stimulation of gut bacteria in the human host (Galili, 1993Go). It would be of special interest to identify the {alpha}Gal epitope present in these bacterial polysaccharides and to determine the conformational epitopes that are responsible for the early immunological event.

In addition, there have been several examples of protein carbohydrate interactions where the "bioactive" conformation of the oligosaccharide ligand, that is, the one observed in the protein binding site, does not correspond to the most populated one in solution but rather to a secondary minimum (Imberty et al., 1993Go; Imberty and Pérez, 2000Go). This has been exemplified recently in the crystal structures of E-selectin and P-selectin interaction with sialyl-Lewis X–containing ligand (Somers et al., 2000Go). It was previously pointed that {alpha}Gal-Lewis X shares some similarity to sialyl-LewisX (Joziasse et al., 1993Go). The previous study demonstrates another similarity with the present one: both molecules consist of a semiflexible nonreducing end, borne by the rigid Lewis X moiety.


    Materials and methods
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Synthesis
Trisaccharide 2 was obtained as described previously (Pazynina et al., 2001Go). Tetrasaccharide 4 was obtained by block synthesis 2 + 2 (see Scheme 2). Thioglycoside 8, used as glycosyl-donor, was obtained by {alpha}-galactosylation of 6 by tetra-O-benzylbromogalactose 5 in the presence of silver triflate followed by reprotection giving rise to thioglycoside 7. Glycosyl acceptor 12 was obtained by {alpha}-fucosylation of 4-OTr-substituted 1,6-anhydro-N-acetylglucosamine (Tyrtysh et al., 2000Go) by tri-O-benzylfucopyranosylbromide under the conditions of the Lemieux reaction followed by removal of trityl protection. By reaction of thioglycoside 8 with glycosyl acceptor 12 in the presence of NIS and TflOH, tetrasaccharide 13 was obtained in a yield of 59%. Under conditions of deprotection and peracetylation, this tetrasaccharide was converted to tetrasaccharide 14 followed by acetolysis to open the anhydro cycle. The oxazoline obtained in the acetolysis reaction along with 1-{alpha}OAc-derivative were glycosylated with HOCH2CH2CH2NHCOCF3 without purification. Peracetate of tetrasaccharide 15 was isolated in a yield of 43%, and 27% of 1-OAc- and 13% 1-{alpha}OH-derivatives were obtained as by-products. Deprotection of compound 12 led to the desired tetrasaccharide 4. The details of the synthesis will be published elsewhere.



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Scheme 2. Chemical synthesis of tetrasaccharide 4.

 
NMR spectroscopy
Approximately 4 (3.5) mg of tetrasaccharide 4 (trisaccharide 2), were lyophilized three times against D2O to exchange the hydroxyl hydrogens, dissolved in 0.75 ml of 99.96% D2O to afford a final concentration of ~8 mM (~6 mM), and then placed in a tube sealed under argon. NMR spectra were recorded on Varian 400 Inova (gHMQC and gHMBC), Varian 500 Unityplus (DQCOSY, total correlation spectroscopy [TOCSY] and NOESY) and a Bruker Avance 500 MHz (NOESY) spectrometers (double-resonance H/C gradient probe) at 25°C (COSYDQF, gHMQC, gHMBC, TOCSY). Regarding the 2D homonuclear and HMQC experiments, quadrature detection was achieved in the nonacquisition dimensions by the TPPI (Marion and Wüthrich, 1983Go) and the hypercomplex (States et al., 1982Go) methods, respectively. Phase-shifted squared sinebell apodization functions were applied in both dimensions and the first points were scaled. Data were zero-filled to 4096 and 2048 points in F2 and F1, respectively. A polynomial baseline correction was applied in F2 after Fourier transformation.

In the case of the tetrasaccharide 4, phase-sensitive NOESY experiments (Jeener et al., 1979Go; Macura and Ernst, 1980Go) were recorded with mixing times of 100, 200, and 400 ms at 5°C, whereas for the trisaccharide 2 NOESY data were recorded with a mixing time of 500 ms at both 30 and 35°C. The cross-peak volumes were evaluated by comparison to a crosspeak volume for two protons separated by a fixed distance (underlined in Table III).

Nomenclature and starting models
Schematic representation of the oligosaccharides are given in Scheme 1 together with the labeling of the torsion angles of interest. These latter have been defined as follows: {Phi} = {Theta}(O-5'{nabla}C-1'{nabla}O-1'{nabla}C-3) and {Psi} = {Theta}( C-1'{nabla}O-1'{nabla}C-3—C-4) for the 1–3 linkages and {Phi} = {Theta}(O-5'{nabla}C-1'{nabla}O-1'{nabla}C-4) and {Psi} = {Theta}( C-1'{nabla}O-1'{nabla}C-4—C-5) for the 1–4 linkages. Starting 3D structures were built with the Sybyl program (SYBYL), using residues from the Monosaccharides Database (www.cermav.cnrs.fr/databank/monosaccharides) and orientations of glycosidic linkages corresponding to the energy minima reported on the MM3 energy map (Imberty et al., 1995Go).

Simulation protocol
Simulations were performed using the AMBER-5.0 program package (Pearlman et al., 1991Go) together with the GLYCAM-93 parameters for carbohydrates (Woods et al., 1995Go). The molecules were hydrated in the Xleap module of AMBER by a periodic box of TIP3P waters, which was extended by 10 Å in each direction from the carbohydrate atoms and contained 1227 and 1291 water molecules for the tri- and tetrasaccharide, respectively. All simulations were run with the SANDER module of AMBER with SHAKE algorithm (Ryckaert et al., 1977Go) (tolerance = 0.0005 Å) to constrain covalent bonds involving hydrogens, using periodic boundary conditions, a 2 fs time step, a temperature of 300°K with Berendsen temperature coupling (Berendsen et al., 1984Go), a 9 Å cutoff applied to the Lennard-Jones interaction, and constant pressure of 1 atm. The nonbonded list was updated every 10 steps.

Equilibration was performed by first restraining the atoms of the oligosaccharide (water molecules were allowed to move) and running 1000 steps of minimization. After this initial minimization, all subsequent simulations were run by using the particle mesh Ewald method (Essmann et al., 1995Go) within AMBER with a cubic B-spline interpolation order and a 10–6 tolerance for the direct space sum cutoff. The first step was followed by 25 ps of dynamics with the position of the oligosaccharide fixed. Equilibration was continued with 25 kcal/(mol. Å) restraints placed on all solute atoms, minimization for 1000 steps, followed by 3 ps of molecular dynamics, which allowed the water to relax around the solute. This equilibration was followed by five rounds of 600 steps of minimization where the solute restraints were reduced by 5 kcal/(mol. Å) during each round. Finally, the system was heated from 100 to 300°K over 2 ps and the production run was initiated. The molecular dynamics trajectories were analyzed with the CARNAL module of AMBER.

Calculations of theoretical NMR data from molecular dynamics trajectories
Theoretical NOESY volumes were established from the averaged <r–3> and <r–6> interproton distance matrices of the molecular dynamics trajectories using the model-free approach of Lipari and Szabo (1982)Go. This approach requires a precise description of Brownian motion including the nature (isotropic or anisotropic, etc.) and the characteristic rotational correlation time(s) and both the amplitude and correlation time for internal motion. Strictly speaking, this formalism is only valid when internal motions are not coupled to overall motion, which is likely not the case with small sugars. However, inspite of this limitation, the Lipari-Szabo spectral densities have been widely applied to small oligosaccharides. Motional models can be established from experimental NMR data but, due to the small amount of sample available acquisition times, would be unreasonably long; a strategy based on hydrodynamic theory was adopted. Inspection of molecular models with favorable orientations of the glycosidic linkages revealed a slightly anisotropic shape that included a longer axis of roughly 15 Å (17 Å when the van der Waals distance between the outer atoms and the solvent is taken into account) and a shorter axis of approximately 9 Å. When the axial ratio is less than 2 the influence on NOESY data is very small (<2–3%) (Ejchart et al., 1992Go) so that the most simple isotropic model corresponding to a sphere of radius 7–8 Å was expected to be suitable for the tetrasaccharide 4. The rotational correlation time for a spherical molecule with a radius of 7.5 Å was established to be 0.86 ns from the molecular volume with the Stokes-Einstein-Debye relation as follows:

{tau}c = 4 {pi} a3 {eta}0 / 3 k T

where a is the molecular radius, {eta}0 the viscosity of the solvent (Matsunga and Nagashima, 1983Go), k the Boltzmann constant, and T the absolute temperature. This theoretical {tau}c value was adjusted to 0.88 ns to best fit the experimental data while using a correlation time of 50 ps ({tau}e) for internal motion. Three different values for the amplitude of internal motion (characterized by the order parameter S2ang, which varies from 1.0 for a rigid molecule to 0 for a totally flexible one) were implemented in the calculations corresponding to (1) 0.9 for the methine/methine interactions of the nonterminal sugars, I and II; (2) 0.85 for the interactions of the terminal sugars, III and IV; and (3) 0.7 for the interactions involving the pendant –CH2OH and –CH3 groups.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
E.B. was accorded a grant by the Xenotranplantation Project BIO4CT972242 of the Biotech program from European Union. The stay of F.C. in Grenoble was supported by Ministerio de Educación y Cultura. The authors are endebted to Drs. D. Bouhours and J.F. Bouhours (Nantes) for helpful discussion. T.T. was supported by Syntesome GmbH (Munich, Germany).


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
gHMBc; gHMQC; COSYDQF; DQCOSY; NMR, nuclear magnetic resonance; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy;


    Footnotes
 
1 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
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Bizik, F. and Tvaroska, I. (1995) Conformational analysis of disaccharide fragments of blood group determinants in solution by molecular modelling. Chem. Papers, 49, 202–214.[ISI]

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Galili, U., Shohet, S.B., Kobrin, E., Stults, C.L., and Macher, B.A. (1988) Man, apes, and Old World monkeys differ from other mammals in the expression of alpha-galactosyl epitopes on nucleated cells. J. Biol. Chem., 263, 17755–17762.[Abstract/Free Full Text]

Hindsgaul, O., Norberg, T., Le Pendu, J., and Lemieux, R.U. (1982) Synthesis of type 2 human blood-group antigen determinants. The H, X, and Y haptens and variations of the H type 2 determinants of probes for the combining site of the lectin I of Ulex europaeus. Carbohydr. Res., 109, 109–142.[CrossRef][ISI][Medline]

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