Glycobiology Advance Access originally published online on June 15, 2005
Glycobiology 2005 15(10):1008-1015; doi:10.1093/glycob/cwi091
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Dynamic properties of biologically active synthetic heparin-like hexasaccharides
2 Grupo de Carbohidratos, Instituto de Investigaciones Químicas, CSIC, Américo Vespucio 49, Isla de la Cartuja, 41092 Sevilla, Spain; 3 Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava, Slovakia; 4 Unitat de RMN, Serveis Cientificotècnics, Universitat de Barcelona, Parc Científic de Barcelona, Josep Samitier 1-5, 08028-Barcelona, Spain; and 5 Institute for Chemical and Biochemical Research "G. Ronzoni", via G. Colombo 81, 20133 Milan, Italy
1 To whom correspondence should be addressed; e-mail: pedro.nieto{at}iiq.csic.es
Received on March 29, 2005; revised on May 16, 2005; accepted on June 7, 2005
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Abstract |
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A complete study of the dynamics of two synthetic heparin-like hexasaccharides, D-GlcNHSO3-6-SO4-
-(1
4)-L-IdoA-2-SO4--(14)-D-GlcNHSO3-6-SO4--(14)-L-IdoA-2-SO4--(14)-D-GlcNHSO3-6-SO4--(14)-L-IdoA-2-SO4--1iPr (1) and 4)-L-IdoA-2-SO4--(14)-D-GlcNHAc-6-SO4--(14)-L-IdoA--(14)-D-GlcNHSO3--(14)-L-IdoA-2-SO4--1iPr (2), has been performed using 13C-nuclear magnetic resonance (NMR) relaxation parameters, T1, T2, and heteronuclear nuclear Overhauser effect (NOEs). Compound 1 is constituted from sequences corresponding to the major polysaccharide heparin region, while compound 2 contains a sequence never found in natural heparin. They differ from each other only in sulphation patterns, and are capable of stimulating fibroblast growth factors (FGFs)-1 induced mitogenesis. Both oligosaccharides exhibit a remarkable anisotropic overall motion in solution as revealed by their anisotropic ratios (
/||), 4.0 and 3.0 respectively. This is a characteristic behaviour of natural glycosaminoglycans (GAG) which has also been observed for the antithrombin (AT) binding pentasaccharide D-GlcNHSO3-6-SO4--(14)-D-GlcA-ß-(14)-D-GlcNHSO3-(3,6-SO4)--(14)-L-IdoA-2-SO4--(14)-D-GlcNHSO3-6-SO4--1Me (3) (Hricovíni, M., Guerrini, M., Torri, G., Piani, S., and Ungarelli, F. (1995) Conformational analysis of heparin epoxide in aqueous solution. An NMR relaxation study. Carbohydr. Res., 277, 11–23). The motional properties observed for 1 and 2 provide additional support to the suitability of these compounds as heparin models in agreement with previous structural (de Paz, J.L., Angulo, J., Lassaletta, J.M., Nieto, P.M., Redondo-Horcajo, M., Lozano, R.M., Jiménez-Gallego, G., and Martín-Lomas, M. (2001) The activation of fibroblast growth factors by heparin: synthesis, structure and biological activity of heparin-like oligosaccharides. Chembiochem, 2, 673–685; Ojeda, R., Angulo, J., Nieto, P.M., and Martin-Lomas. M. (2002) The activation of fibroblast growth factors by heparin: synthesis and structural study of rationally modified heparin-like oligosaccharides. Can. J. Chem,. 80, 917–936; Lucas, R., Angulo, J., Nieto, P.M., and Martin-Lomas, M. (2003) Synthesis and structural studies of two new heparin-like hexasaccharides. Org. Biomol. Chem., 1, 2253–2266) and biological data (Angulo, J., Ojeda, R., de Paz, J.L., Lucas, R., Nieto, P.M., Lozano, R.M., Redondo-Horcajo, M., Giménez-Gallego, G., and Martín-Lomas, M. (2004) The activation of fibroblast growth factors (FGFs) by glycosaminoglycans: influence of the sulphation pattern on the biological activity of FGF-1. Chembiochem, 5, 55–61). Fast internal motions observed for the less sulphated compound 2, as compared with 1, may be related to their different behavior in stimulating FGF1-induced mitogenic activity. Key words: anisotropy / FGF interaction / heparan sulphate / heparin / NMR relaxation
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Introduction |
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Heparan sulphate glycosaminoglycans (GAG) are involved in molecular recognition events that occur at the cellular surface and in the extracellular matrix. These events are related to important biological functions, such as cell growth and differentiation, blood coagulation, viral infection, or angiogenesis (Conrad, 1998
). Among their biological functions, the regulation of biological activities of antithrombin (AT III) and fibroblast growth factors (FGFs) are probably the two most extensively studied. FGFs comprise a family of more than twenty proteins from which FGF1 (acidic FGF) and FGF2 (basic FGF) are the best known examples (Casu and Lindahl, 2001). These FGFs exert their biological functions through a heparan sulphate–mediated interaction with specific membrane receptors (fibroblast growth factor receptors [FGFRs]) (Faham et al., 1998). Experimental evidences indicate a close relationship between the biological activity of these systems and the GAG sulphation pattern, which are considered as an essential factor in the control of the selectivity of these processes. More specifically, in the FGF case, it has been proposed that the specificity relies on changes on the flexibility of the GAG chain modulated by the substitution. In fact, variations of biological activities of different heparin-like molecules have been attributed to changes in flexibilities of carbohydrate chains as a result of their different substitution patterns (Hricovíni et al., 2002; Raman et al., 2003; Hricovíni, 2004). In any case, the minimal structural requirements for the activation of FGF by GAGs have not been unambiguously determined. Crystalographic structures of binary GAG–FGF1 (DiGabriele et al., 1998) and GAG–FGF2 (Faham et al., 1996) complexes and of ternary FGF1–GAG–FGFR (Pellegrini et al., 2000) and FGF2–GAG–FGFR (Schlessinger et al., 2000) complexes show diverse spatial arrangements of the involved molecules, which may involve FGF cis or trans dimerization.
Recently, we have been investigating the molecular basis of this interaction using synthetic oligosaccharides with different sizes and sulphation patterns by comparing their capacity to stimulate FGF1-mediated mitogenic activity (Angulo et al., 2004). These results showed that, as expected, (Casu and Lindahl, 2001) the length of the GAG chain has a dramatic influence on the activity at the hexasaccharide and octasaccharide level. Although hexasaccharide 1 was able to induce FGF1 activity, it needed much higher concentrations to reach the activation level of its homologue octasaccharide. Surprisingly, hexasaccharide 2 was better activator for mitogenesis, showing the values of half-maximum activation concentration and maximum activating levels similar to synthetic octasaccharides, and even as high as natural heparin. The ability of 2 to efficiently induce FGF1-mediated mitogenesis demonstrated that the dimerization of two FGF molecules, previously proposed as the first step of the activation mechanism, is not an essential requisite for the FGF1 activation because the three dimensional arrangement of the sulphate groups in 2 is not adequate for trans aggregation, and its size hardly permits cis dimerization. Previous studies on the solution conformation of these synthetic oligosaccharides (de Paz et al., 2001; Ojeda et al., 2002; Lucas et al., 2003) demonstrated that the sulphation pattern does not seem to influence considerably their solution structure, which display a helical shape similar to that observed for natural heparan sulphate GAGs. It is because of this helical structure, that the sulphate groups in 2 are oriented towards the same face of the molecule preventing FGF trans dimerization.
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According to these results, the different sulphation patterns of hexasaccharides 1 and 2 have a remarkable influence on the stimulation of FGF1-induced mitogenic activity (Angulo et al., 2004
). The difference in the activity profiles of 1 and 2 could, therefore, be attributed to the different spatial arrangement of their electrostatic potentials (Angulo et al., 2004) as a result of the different sulphation patterns. But other factors might also be involved. Beyond the necessary three-dimensional complementarity between ligand and receptor, the interaction of these oligosaccharides with FGF1 may also depend on the dynamics of the interacting species.
Owing to relationships between dynamics and relaxation rates, 13C-NMR relaxation data can be used for quantification of molecular motion, which is a function of the overall shape of the molecule together with the extent and frequency of internal motions. In the case of heparin and its derivatives, those factors are mainly: the motions around the glycosidic linkages, the conformational flexibility of the iduronate rings together with a marked cylindrical shape (Hricovíni et al., 2002). This persistent shape causes a non-isotropic global motion which is reflected in the NMR relaxation data, such as T1, T2, and NOEs which in addition to the other factors depend on the orientation of the internuclear vector relative to the main molecular axis. Owing to these features heparin, chemically modified heparin and fragments of the natural polysaccharide, display a characteristic high anisotropy factor.
In this article, the dynamics of two synthetic hexasaccharides 1 and 2 are analyzed by performing 13C-NMR relaxation measurements in order to characterize the possible influence of the specific sulphation pattern on the flexibility of the oligosaccharide. As both compounds have all the sulphate groups needed to make a complete set of interactions with FGF1 and also have similar three-dimensional structure but induce different levels of FGF1-mediated activity they constitute an excellent case for addressing the role of the dynamics on the interaction. Thus, the final aim of this study is to get further insight into the different factors that regulate the heparan sulphate-FGF molecular recognition process.
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Results |
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The 1H and 13C high-resolution NMR spectra of 1 and 2 have been previously assigned using 1-D and 2-D NMR methods (de Paz et al., 2001
; Ojeda et al., 2002). Chemical shifts were influenced by the presence of electronegative groups, and the observed values were in agreement with those recently reported for various heparin derivatives (Yates et al., 2000). Signal overlap was observed in the NMR spectra because of the fact that these hexasaccharides consist of three structurally similar disaccharide units. The overlap was especially noticeable in the spectrum of hexasaccharide 1 (having the structure of the regular region of heparin) which did not allow a reliable determination of relaxation rates and NOEs for several signals. Experimental 13C T1, 13C T2, and 1H-13C NOEs for 1 and 2, determined at three different magnetic field strengths, see Figure 1, are presented in Tables I and II. Spin-lattice relaxation times varied with both the field strength and the position within the molecule. This is well seen primarily in 2 where carbons in both terminal residues showed longer longitudinal relaxation (e.g. 410–440 ms at 18.7 T) than those in the central residues (e.g. 360–380 ms at 18.7 T) indicating a non-isotropic motion of this molecule in aqueous solution. Similar trends appear to hold also for 1, although the experimental data for 1 were not as precisely determined as those for 2 as a result of the above mentioned signal overlapping.
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Previous relaxation data for heparin-derived oligosaccharides showed that saccharides consisting from more than four residues have non-isotropic tumbling (Hricovíni and Torri, 1995; Mikhailov et al., 1996). This evidence is compatible with preliminary data for 1 and 2 obtained from the analysis of the proton longitudinal cross-relaxation rate constants (
ij) (de Paz et al., 2001; Ojeda et al., 2002). The comparison of ij, determined at different magnetic fields, indicated that the cross-relaxation across the fixed distances within the glucosamine rings was influenced by non-isotropic overall motional properties of the molecule. For example, H1–H2 = –0.15 s–1 in the non-reducing end terminal glucosamine residue whereas H2–H4 = –0.039 s–1 what is compatible with variations in carbon relaxation times as well as with observations in a structurally similar pentasaccharide (Hricovíni and Torri, 1995).
A more quantitative description of the overall and the internal motions of these hexasaccharides has now been performed based on the model-free approach. Computed motional parameters, overall correlation times (
and ||), internal motion correlation times (e), and the generalized order parameters (S2) for 1 and 2 are listed in Table III and compared with those obtained for different sulphated saccharides. The computed values for the overall correlation times, ^ and ||, were 2.6 ns and 0.64 ns, respectively for 1 and 1.7 ns and 0.5 ns, respectively for 2. These values, as well as e, are slightly longer than those determined for the antitrombin binding pentasaccharide 3 (Hricovíni and Torri, 1995) though the axial ratios for both 1 and 2 are comparable to those for 3. This type of behaviour is also compatible with those observed for sulphated polysaccharides (Mulloy et al., 1993; Hricovíni and Torri, 1995). The computed 13C T1, 13C T2, and 1H-13C NOE values for 2, based on the derived motional parameters, are listed in Table IV. The averaged (computed for each monosaccharide unit) experimental values are listed for comparison as well. In general, the match between the experimental and the computed data is very good with the difference being smaller than 20 ms in most cases. The inspection of the derived parameters presented in Table V for molecules 2 and 3 further reveals that the order parameters decrease from the central residues (S2 = 0.91) towards both ends of the molecule. Similarly, computed e values differed along the saccharide chain being shorter at the terminal residues.
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Discussion |
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We have recently analyzed the three dimensional structures of hexasaccharides 1 and 2 (de Paz et al., 2001
; Ojeda et al., 2002). Both hexasaccharides displayed the characteristic helical arrangement of heparin and thus both molecules are appropriate models for the description of heparin structure. In this article, solution dynamics derived from NMR relaxation data of both oligosaccharides is discussed. The values of overall and internal motions are in general agreement with those reported for the AT binding pentasaccharide 3 (Tables III and V). The agreement between the theoretical and the measured values was better for hexasaccharide 2, than for the regular oligomer 1, owing to more consistent experimental data obtained for 2. In spite of some differences, however, the axial ratios (^/||) for oligosaccharides 1–3 fall within a similar range (Table III) indicating comparable rod-like molecular shape for all of them. These values imply a non-isotropic motional behaviour which will cause a dependence of the NMR relaxation data on the orientation of the considered vector with respect to the molecular axis. The e and S2 values derived for the individual monosaccharide units in 2 decreased from the central residue towards both ends (Table V). Thus as expected, the terminal residues undergo more pronounced internal motions than the internal residues. The same observation has been reported for 3 based on 13C relaxation data (Hricovíni et al., 1995). These data are compatible also with proton cross-relaxation rates (NOE) which considerably differed within the oligosaccharide chain as the consequence of the non-isotropic molecular motion (de Paz et al., 2001; Ojeda et al., 2002).
The existence of an asymmetrical molecular shape is the first obvious conclusion that can be deduced from the non-isotropic description of the molecular tumbling calculated from NMR relaxation measurements. However, the opposite conclusion is not necessarily correct. There are several examples of carbohydrates whose relaxation properties have been satisfactorily interpreted by isotropic models although their shapes would allow presuming a non-isotropic molecular motion. This was the case for some pentasaccharides reported in the literature (Mäler et al., 1996b; Rundlöf et al., 1999; Almond et al., 2001). In case of the Lacto-N-fucopentaose (LNF)-1 pentasaccharide, the computed anisotropy ratio from 13C-NMR relaxation data (^/||
1.4) differed from that obtained from hydrodynamics (^/||1.8) (Rundlöf et al., 1999). The contradictions between the higher expected anisotropy and the reduced values observed were explained by the effect of extensive internal motions at the reducing end of the molecule (Rundlöf et al., 1999). In the case of heparin polysaccharides the anisotropy factor (^/||) has been found to be particularly high suggesting a relatively rigid overall shape due to a weaker effect of flexibility upon the overall molecular shape in these molecules (Table III). Hexasaccharides 1 and 2, as well as pentasaccharide 3 (Hricovíni and Torri, 1995), show this behaviour with axial ratios of /|| of 4, 3, and 3.5, respectively, indicating a similar dynamic coherent with a low influence of fast internal motions in the non-isotropic behaviour. This result is apparently in contradiction with the characteristic flexibility of the iduronate rings that experience an extensive conformational equilibrium (Hricovíni et al., 2002). However, the analysis of the three-dimensional structures reveals that the conformational change of the iduronate rings has a very small influence upon the global molecular shape. Therefore, it can be deduced that the basis of the peculiar motional behaviour of the heparin are encoded in the reduced flexibility of their backbone given by the glycosidic linkage energetic landscape.
The descriptors of internal motions (see S2 and e in Table III) of 1 and 2 indicate the presence of some degree of flexibility at the glycosidic linkages connecting the terminal residues with their neighbouring units as well as the effect of iduronate residues conformational equilibrium. In addition, the difference in internal motions and in the axial ratio between 1 and 2 also indicates that the different sulphation patterns could influence dynamics to some extent, being 2 more flexible than 1. On one side, the iduronate ring conformational equilibrium on unit C, calculated from coupling constants (Angulo et al., 2003), is more extensive for 2 (1C4:2SO 55:45) than for 1 (1C4:2SO 69:31). On the other, replacement of two 6-O-sulphate groups by two hydroxyl groups and of N-sulphate by N-acetate in glucosamine residues might result in somewhat higher flexibility of 2 leading to lower axial ratio. In any case, computed S2 values were found to be still relatively high pointing out to limited internal motions even in hexasaccharide 2. Thus, partial flexibility in 2 seems to have only a limited effect on orientations of the sulphate groups which strongly influence the biological activity. Consequently, the behaviour of both molecules (1 and 2) as FGF1 activators well agrees with the above conclusions.
In summary, synthetic heparin-like hexasaccharides 1 and 2 seem to be appropriate structural models for larger GAGs, such as heparin/heparan sulphate also from the dynamical point of view. The application of the symmetric top model, in combination with the model-free approach, led to the proper description of the motional properties of both molecules and the derived parameters ( ^, ||, e, and S2) are in agreement with the experimental data. The derived dynamic values were found to be comparable with those reported for the structurally related AT binding pentasaccharide 3. Small differences in the axial ratio /|| between 1 and 2 reflect small differences in internal flexibility of these molecules. Compound 2 shows slightly higher flexibility than 1 as may be expected from its lower content of sulphate groups. This evidence is in agreement with molecular dynamics calculations (J. Angulo and P.M. Nieto, unpublished data). However, as these local motions are likely to be around a single minimum that limited flexibility should not have strong effect upon the orientation of the sulphate groups or into the strength of their interaction with positively charged amino acids in the binding site.
This study demonstrates the influence of the sulphation pattern of the GAG in their internal dynamics. This result considered together with the biological activity data and the similar three-dimensional structure of the two synthetic hexasaccharides (de Paz et al., 2001; Ojeda et al., 2002) support the importance of the GAGs flexibility on the selectivity of their interaction with this protein.
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Materials and methods |
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NMR
Hexasaccharides 1 and 2 were synthesised as previously described (de Paz et al., 2001
; Ojeda et al., 2002) and interchanged with D2O by several cycles of freeze-drying. The pH of the samples were adjusted close to 7 in the first cycle by adding small quantities of NaOD or DCl. The samples were dissolved in 99.99% D2O (5 mg of 1 to a total volume of 0.5 mL in a standard NMR tube, and 2.5 mg of 2 to 0.25 mL using a reduced volume NMR tube purchased from Shigemi, Hachioji-City, Japan). NMR relaxation experiments were recorded at 800, 600, and 500 MHz on Bruker Avance instruments (Bruker Avance, Rheinstetten, Germany). The temperature was set at 298K in all the experiments. 13C longitudinal (R1) and transversal (R2) relaxation rates and heteronuclear {1H}-13C NOE were measured using two dimensional 1H-13C correlation pulse sequences with minimum water suppression as the oligosaccharides were dissolved in D2O.
The inversion-recovery and spin-echo Carr-Purcell-Meiboom-Gill (CPMG) experiments were recorded using double insensitive nuclei enhanced by polarization transfer (INEPT) based experiments with sensitivity enhancement adapted to 13C from 15N sequences (Farrow et al., 1994). The heteronuclear NOE experiment was taken from Wagner (Dayie and Wagner, 1994) and optimized for 13C. The relaxation measurements were performed with suppression of the cross-correlation between the chemical shift anisotropy (CSA) and the dipolar relaxation mechanisms as it has been reported to contribute significantly to the overall relaxation rates (Hricovíni and Torri, 1995).
The relaxation data were used to calculate molecular motional properties by model-free analysis (Lipari and Szabo, 1982). For that, an expression for the correlation function was used that considered a prolate elipsoid molecular shape (symmetric top model) with internal motion together with the overall correlation time, the order parameter, and the internal correlation time.
For the R1 experiments, the inversion-recovery double INEPT based experiments consisted of a data matrix of 256 x 1024 data points, with 64 transients per increment, see Figure 2. Six inversion recovery delays (21, 63, 125, 185, 345, and 400 ms) were used for the construction of the decay curves, arranging the experiments randomly to avoid systematic errors. For the R2 experiments the same experimental setup was used, with 6 randomly arranged experiments differing in the length of the CPMG spin echo delays (10, 22, 32, 55, 74, and 106 ms). Some of the R1 and R2 experiments were repeated as a control. The cross-correlation between dipolar and chemical shift anisotropy relaxation mechanisms was cancelled in the R1 experiments by a train of 120–180° rectangular pulses at 15 KHz power separated by 5 ms each other. In the R2 experiments this was achieved by using a high power 180° pulse every four 13C spin echoes, these were achieved by a train of four 180° of 50 ms spaced 150 ms. {1H}-13C NOE were recorded using at least 64 scans of 1024 real points for each free-induction decay in T2 and 200 increments in T1. Steady-state NOE experiments were registered using a relaxation delay of 2 s and a saturation time of 3 s. The previous broad-band saturation was achieved by a train of 120° pulses separated by a delay of 5 ms. The control experiments were recorded with exactly the same relaxation delay than the {1H}-13C NOE and both were run simultaneously. For all the experiments, the raw data were zero-filled, multiplied by an adequate squared sine bell function before Fourier transform and baseline corrected. The same processing procedure was used for all the spectra within the same set of experiments.
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The peak volumes were integrated using manufacturer software XWINNMR, using the same region of each peak within the same set of experiments. Each volume was measured three times and the average value was used for the calculations (Viles et al., 2001). The R1 and R2 relaxation rates were determined by nonlinear fitting of the evolution of the peak volumes as a function of the length of the inversion recovery or the spin-echo delay (t) using a two-parameters (Io and R1,2) exponential decay function, I(t) = Io exp(–R1,2 t).
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Acknowledgments |
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This research was supported by the Ministry of Science and Technology (Grant BQU2002-0374). We thank The Ministry of Education, Fundación Ramón Areces and Fundación Francisco Cobos for fellowships to J.-L.d.P., J.A., R.L., and R.O., respectively. We thank the NMR Service of the Barcelona Scientific Park for 800 and 600 MHz instrument time allocation.
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Abbreviations |
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AT, antithrombin; CPMG, Carr-Purcell-Meiboom-Gill; CSA, chemical shift anisotropy; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; GAG, glycosaminoglycans; INEPT, insensitive nuclei enhanced by polarization transfer; LNF, Lacto-N-fucopentaose; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect
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References |
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