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.
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Abstract |
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Key words: /molecular dynamics/NMR/oligosaccharides/xenotransplantation
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Introduction |
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Much effort has been devoted to the conformational analysis of the 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., 1980
), energy maps of this linkage have been calculated with the help of the MM2 (Strotz and Cerezo, 1994
; Bizik and Tvaroska, 1995
) or MM3 programs (Imberty et al., 1995
). Recently, the conformational study of an analogue,
Gal1-3ßGal1-4ßGlc-1NAc, has been performed by a combination of Monte Carlo calculations and NMR spectroscopy (Li et al., 1999
). In all recent studies, the
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 Gal1-3ßGal1-4ßGlcNAc epitope, present on glycolipids and glycoproteins of pig endothelial cells, a branched epitope,
Gal1-3ßGal1-4(
Fuc1-3)ßGlcNAc (
Gal-Lex) has been characterized as a component of glycosphingolipids expressed in pig kidney (Bouhours et al., 1997
, 1998). This tetrasaccharide has been revealed by immunostaining with a polyclonal antibody directed against
Gal1-3Gal1 determinants. Schematic representation of the oligosaccharides are given in Scheme 1.
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Results |
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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, 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
Gal1-3ßGal1-4ßGlcNAc trisaccharide (Li et al., 1999
).
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, c > 1) for all of the mixing times (100, 200, and 400 ms). However, at this temperature the anomeric signals displayed severe overlapping (
-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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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 Gal-LacNAc trisaccharide, good agreement was obtained. The results do not differ significantly from those obtained by Li et al. (1999)
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
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 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 Fuc and ßGal. As for the flexible part of the tetrasaccharide, that is, the
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
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
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.
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Discussion |
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In conclusion, design of a rigid inhibitor that would mimic the antigenic conformation of the 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, 1993
). It would be of special interest to identify the
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., 1993; Imberty and Pérez, 2000
). This has been exemplified recently in the crystal structures of E-selectin and P-selectin interaction with sialyl-Lewis Xcontaining ligand (Somers et al., 2000
). It was previously pointed that
Gal-Lewis X shares some similarity to sialyl-LewisX (Joziasse et al., 1993
). 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.
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Materials and methods |
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In the case of the tetrasaccharide 4, phase-sensitive NOESY experiments (Jeener et al., 1979; Macura and Ernst, 1980
) 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: =
(O-5'
C-1'
O-1'
C-3) and
=
( C-1'
O-1'
C-3C-4) for the 13 linkages and
=
(O-5'
C-1'
O-1'
C-4) and
=
( C-1'
O-1'
C-4C-5) for the 14 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., 1995
).
Simulation protocol
Simulations were performed using the AMBER-5.0 program package (Pearlman et al., 1991) together with the GLYCAM-93 parameters for carbohydrates (Woods et al., 1995
). 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., 1977
) (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., 1984
), 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., 1995) within AMBER with a cubic B-spline interpolation order and a 106 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 <r3> and <r6> interproton distance matrices of the molecular dynamics trajectories using the model-free approach of Lipari and Szabo (1982). 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 (<23%) (Ejchart et al., 1992
) so that the most simple isotropic model corresponding to a sphere of radius 78 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:
c = 4
a3
0 / 3 k T
where a is the molecular radius, 0 the viscosity of the solvent (Matsunga and Nagashima, 1983
), k the Boltzmann constant, and T the absolute temperature. This theoretical
c value was adjusted to 0.88 ns to best fit the experimental data while using a correlation time of 50 ps (
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.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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Berendsen, H.J.C., Postma, J.P.M., and van Gunsteren, W.V. (1984) Molecular dynamics with coupling to an external bath. J. Chem. Phys., 81, 36843690.[CrossRef][ISI]
Bizik, F. and Tvaroska, I. (1995) Conformational analysis of disaccharide fragments of blood group determinants in solution by molecular modelling. Chem. Papers, 49, 202214.[ISI]
Bock, K. and Duus, J.O. (1994) A conformational study of hydroxymethyl groups in carbohydrates investigated by 1H NMR spectroscopy. J. Carbohydr. Chem., 13, 513543.[ISI]
Bouhours, D., Liaigre, J., Lemoine, J., Mayer-Posner, F., and Bouhours, J.F. (1998) Two novel isoneolacto-undecaglycosylceramides carrying Gal1>3Lewisx on the 6-linked antenna and N-acetylneuraminic acid
2>3 or Galactose
1>3 on the 3-linked antenna, expressed in porcine kidney. Glycoconj. J., 15, 10011016.[CrossRef][ISI][Medline]
Bouhours, D., Liaigre, J., Naulet, J., Maume, D., and Bouhours, J.F. (1997) A novel glycosphingolipid expressed in pig kidney: Gal13Lewis(x) hexaglycosylceramide. Glycoconj. J., 14, 2938.[CrossRef][ISI][Medline]
Cascalho, M. and Platt, J.L. (2001) The immunological barrier to xenotransplantation. Immunity, 14, 437446.[CrossRef][ISI][Medline]
Cooper, D.K., Good, A.H., Koren, E., Oriol, R., Malcolm, A.J., Ippolito, R.M., Neethling, F.A., Ye, Y., Romano, E., and Zuhdi, N. (1993) Identification of alpha-galactosyl and other carbohydrate epitopes that are bound by human anti-pig antibodies: relevance to discordant xenografting in man. Transpl. Immunol., 1, 198205.[CrossRef][Medline]
Ejchart, A., Dabrowski, J., and von der Lieth, C.W. (1992) Solution conformation of mono- and difucosyllactoses as revealed by rotating-frame NOE-based mapping and molecular mechanics and molecular dynamics calculations. Magn. Res. Chem., 30, S105S114.[ISI]
Essmann, U., Perera, L., Berkowitz, M.L., Darden, T., Lee, H., and Pedersen, L.G.J. (1995) A smooth particle mesh ewald method. Chem. Phys., 103, 85778593.[CrossRef]
Galili, U. (1993) Interaction of the natural anti-Gal antibody with -galactosyl epitopes: a major obstacle for xenotransplantation in humans. Immunol. Today, 14, 480482.[CrossRef][ISI][Medline]
Galili, U., Clark, M.R., Shohet, S.B., Buehler, J., and Macher, B.A. (1987) Evolutionary relationship between the natural anti-Gal antibody and the Gal alpha 13Gal epitope in primates. Proc. Natl Acad. Sci. USA, 84, 13691373.[Abstract]
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, 1775517762.
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, 109142.[CrossRef][ISI][Medline]
Imberty, A. and Pérez, S. (2000) Structure, conformation and dynamics of bioactive oligosaccharides: theoretical approaches and experimental validations. Chem. Rev., 100, 45674588.
Imberty, A., Bourne, Y., Cambillau, C., Rougé, P., and Pérez, S. (1993) Oligosaccharide conformation in protein-carbohydrate complexes. Adv. Biophys. Chem., 3, 71118.
Imberty, A., Mikros, E., Koca, J., Mollicone, R., Oriol, R., and Pérez, S. (1995) Computer simulation of histo-blood group oligosaccharides. Energy maps of all constituting disaccharides and potential energy surfaces of 14 ABH and Lewis carbohydrate antigens. Glycoconj. J., 12, 331349.[ISI][Medline]
Jeener, J., Meier, B.H., Bachmann, P., and Ernst, R.R. (1979) Investigation of exchange processes by two-dimensional NMR spectroscopy. J. Chem. Phys., 71, 45464553.[CrossRef][ISI]
Joziasse, D.H., Schiphorst, W.E., Koeleman, C.A., and Van den Eijnden, D.H. (1993) Enzymatic synthesis of the alpha 3-galactosyl-Lex tetrasaccharide: a potential ligand for selectin-type adhesion molecules. Biochem. Biophys. Res. Commun., 194, 358367.[CrossRef][ISI][Medline]
Lemieux, R.U., Bock, K., Delbaere, L.T.J., Koto, S., and Rao, V.S.R. (1980) The conformations of oligosaccharides related to the ABH and Lewis human blood group determinants. Can. J. Chem., 58, 631653.[ISI]
Li, J., Ksebati, M.B., Zhang, W., Guo, Z., Wang, J., Yu, L., Fang, J., and Wang, P.G. (1999) Conformational analysis of an -galactosyl trisaccharide epitope involved in hyperacute rejection upon xenotransplantation. Carbohydr. Res., 315, 7688.[CrossRef][ISI][Medline]
Lipari, G. and Szabo, A. (1982) Model-free approach to the interpretation of Nuclear Magnetic Resonance relaxation in macromolecules. 1. Theory and range of validity. J. Am. Chem. Soc., 104, 45464559.[ISI]
Macura, S. and Ernst, R.R. (1980) Elucidation of cross relaxation in liquids by two-dimensional NMR spectroscopy. Mol. Phys., 41, 95117.[ISI]
Marion, D. and Wüthrich, K. (1983) Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem. Biophys. Res. Commun., 113, 967974.[ISI][Medline]
Matsunga, N. and Nagashima, A. (1983) Transport of liquid and gazeous D2O over a wide range of temperature and pressure. J. Phys. Chem. Ref. Data, 12, 933966.[ISI]
Oriol, R., Candelier, J.J., Taniguchi, S., Balanzino, L., Peters, L., Niekrasz, M., Hammer, C., and Cooper, D.K.C. (1999) Major carbohydrate epitopes in tissues of domestic and African wild animals of potential interest for xenotransplantation research. Xenotransplantation, 6, 7889.
Otter, A., Lemieux, R.U., Ball, R.G., Venot, A.P., Hindsgaul, O., and Bundle, D.R. (1999) Crystal state and solution conformation of the B blood group trisaccharide -L-Fucp-(12)-[
-D-Galp-(13)]-ß-D-Galp-OCH3. Eur. J. Biochem., 259, 295303.
Pazynina, G.V., Tyrtysh, T.V., and Bovin, N.V. (2001) Synthesis of histo blood-group antigens A and B (type 2), xenoantigen Gal1-3Galß1-4GlcNAc, and related type 2 backbone oligosaccharides in spacered form. Carbohydr. Lett., in press.
Pearlman, D.A., Case, D.A., Caldwell, J.C., Seibel, G.L., Singh, C.U., Weiner, P., and Kollman, P.A. (1991) AMBER. San Franscico, CA, University of California.
Pérez, S., Gautier, C., and Imberty, A. (2000). Oligosaccharide conformations by diffraction methods. In B. Ernst, G. Hart, P. Sinay (eds.), Oligosaccharides in chemistry and biology: a comprehensive handbook. Wiley/VCH, Weinheim, pp. 9691001.
Pérez, S., Mouhous-Riou, N., Nifantev, N.E., Tsvetkov, Y.E., Bachet, B., and Imberty, A. (1996) Crystal and molecular structure of a histo-blood group antigen involved in cell adhesion: the Lewis X trisaccharide. Glycobiology, 6, 537542.[Abstract]
Ryckaert, J.P., Cicotti, G., and Berendsen, H.J.C. (1977) Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. J. Comp. Phys., 23, 327341.
Samuelsson, B.E., Rydberg, L., Breimer, M.E., Backer, A., Gustavsson, M., Holgersson, J., Karlsson, E., Uyterwaal, A.C., Cairns, T., and Welsh, K. (1994) Natural antibodies and human xenotransplantation. Immunol. Rev., 141, 151168.[ISI][Medline]
Sandrin, M.S., Vaughan, H.A., Dabkowski, P.L., and McKenzie, I.F. (1993) Anti-pig IgM antibodies in human serum react predominantly with Gal(alpha 13)Gal epitopes. Proc. Natl Acad. Sci. USA, 90, 1139111395.[Abstract]
Somers, W.S., Tang, J., Shaw, G.D., and Camphausen, R.T. (2000) Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLex and PSGL-1. Cell, 103, 467479.[ISI][Medline]
States, D.J., Haberkorn, R.A., and Ruben, D.J. (1982) A two-dimensional nuclear Overhauser experiment wtih pure absorption phase in four quadrants. J. Magn. Reson., 48, 286292.[ISI]
Strotz, C.A. and Cerezo, A.S. (1994) Use of a general purpose force-field (MM2) for the conformational analysis of the disaccharide -D-galactopyranosyl-(13)-ß-D-galactopyranose. J. Carbohydr. Chem., 13, 235247.[ISI]
Tyrtysh, T.V., Byramova, N.E., and Bovin., N.V. (2000) 1, 6-Anhydro-N-acetyl-ß-D-glucosamine in the oligosaccharide synthesis: I. Synthesis of 3-acetate and 3-benzoate of 1, 6-anhydro-N-acetyl-ß-D-glucosamine via the 4-O-trityl derivative. Russ. J. Bioorgan. Chem., 26, 414418.
Woods, R.J., Dwek, R.A., Edge, C.J., and Fraser-Reid, B. (1995) Molecular mechanical and molecular dynamical simulations of glycoproteins and oligosaccharides. 1. GLYCAM_93 parameter development. J. Phys. Chem., 99, 38323846.[ISI]