National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK
Accepted on July 27, 2000;
Abstract
The glycosaminoglycans heparin and heparan sulfate contain similar structural units in varying proportions providing considerable diversity in sequence and biological function. Both compounds are alternating copolymers of glucosamine with both iduronate- and glucuronate-containing sequences bearing N-sulfate, N-acetyl, and O-sulfate substitution. Protein recognition of these structurally-diverse compounds depends upon substitution pattern, overall molecular shape, and on internal mobility. In this review particular attention is paid to the dynamic aspects of heparin/heparan sulfate conformation. The iduronate residue possesses an unusually flexible pyranose ring conformation. This extra source of internal mobility creates special problems in rationalization of experimental data for these compounds. We present herein the solution-state NMR parameters, fiber diffraction data, crystallographic data, and molecular modeling methods employed in the investigation of heparin and heparan sulfate. Heparin is a useful model compound for the sulfated, protein-binding regions of heparan sulfate. The literature contains a number of solution and solid-state studies of heparin oligo- and polysaccharides for both isolated heparin species and those bound to protein receptors. These studies indicate a diversity of iduronate ring conformations, but a limited range of glycosidic linkage geometries in the repeating disaccharides. In this sense, heparin exhibits a well-defined overall shape within which iduronate ring forms can freely interconvert. Recent work suggests that computational modeling could potentially identify heparin binding sites on protein surfaces.
Key words: heparin/heparan sulfate/conformational analysis
Introduction
The glycosaminoglycans (GAGs) are linear polysaccharides with alternating uronic acid and hexosamine residues, in which a limited set of monosaccharide units gives rise to a number of complex sequences by variable substitution with O-sulfate, N-sulfate, and N-acetyl groups. GAGs usually exist as the O-linked side-chains of proteoglycans, displaying a set of physiological functions which is remarkably wide and as yet incompletely explored. They may act as structural components of connective tissue and the extracellular matrix, or as specific ligands in the relationship between the cell surface and its surroundings.
Table I presents a simplified summary of GAG structures. The galactosamine-containing GAGs are chondroitin-4-sulfate (chondroitin sulfate A), chondroitin-6-sulfate (chondroitin sulfate C), and dermatan sulfate, also known as chondroitin sulfate B. Glucosamine-containing GAGs include hyaluronan, heparin, and heparan sulfate. A closely related compound, keratan sulfate, contains no uronic acid but consists of a repeating poly-(N-acetyllactosamine) disaccharide sequence, usually with 6-O-sulfated GlcNAc and partially 6-O-sulfated Gal.
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In what sense can these linear polysaccharides display distinct conformational features? Unbranched, they cannot fold into anything resembling the tertiary structures of proteins, yet the specificity of their interactions with proteins suggests some level of structural definition. Lacking the variety of functional groups displayed by peptides, these anionic polysaccharides must provide a spatially defined pattern of sulfate and carboxylate substituents for molecular recognition to take place. All determinations of the conformation of heparin (both in solution and in the solid state) have indicated similar, well-defined molecular structures in terms of overall chain conformation, with additional subtlety arising from the conformational versatility of the pyranose ring of iduronic acid.
Theoretical and experimental approaches to conformation and molecular motion
The determination of three-dimensional structure in GAGs relies upon many of the same methods as those utilized in the investigation of other complex carbohydrates (Rao et al., 1998). Researchers generally employ NMR spectroscopy and molecular modeling to generate detailed solution structures for these polysaccharides, as many NMR parameters directly reflect molecular conformation. Spin-spin coupling constants (3JH,H) between vicinal protons monitor ring conformation while nuclear Overhauser effects (NOEs) between protons on adjacent monosaccharide residues provide information on glycosidic linkage geometries. Systematic energy calculations over all values of the glycosidic torsional angles
and
allow the prediction of favorable ring conformations and the comparison of theoretical and experimental NOEs (Figure 1). The recent book of Rao et al. (1998)
makes reference to many further strategies for applying NMR and molecular modeling to conformational analysis of complex carbohydrates.
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For polysaccharides in general, and glycosaminoglycans in particular, one must consider molecular motion and flexibility within a discussion of molecular conformation, as the frequency, amplitude, and geometry of such motions directly affect the measured NMR spectroscopic parameters.
A formal description of molecular motions generally requires the use of a reorientational correlation function, or equivalently, a spectral density function. These functions describe the way in which any "memory" of initial orientation changes as a result of overall molecular reorientation, internal motions, etc., for any given vector in the molecular framework. Thus, the reorientational correlation function represents a time course for the loss of such orientational memory. Its value of one at time zero must decay to a value of zero over time; its exact form will depend upon the details of the motion and on the vector selected. One defines correlation timethe quantity most frequently used in discussing typical timescales for molecular motionas the area under the correlation function. The spectral density function represents the frequency spectrum analogue of the reorientational correlation function; knowledge of one implies knowledge of the other.
When a rigid molecule tumbles isotropically in solution (approximating the behavior of a sphere), all possible vectors in the molecule behave equivalently. The correlation function is then typically a simple exponential decay and the spectral density is a Lorentzian function (Harris, 1986). For a rigid symmetric top molecule (approximating the behavior of an ellipsoid), the properties of a given vector will depend upon the reorientation rates about the axes parallel and perpendicular to the major top axis as well as on the angle between the major top axis and the vector (Harris, 1986
). Overall reorientation typically occurs on the picosecond (ps) to nanosecond (ns) timescale.
Researchers often utilize the model free approach of Lipari and Szabo (1982a,b) to discuss fast internal molecular motions. Here, internal motion quickly reduces the value of the correlation function from its initial value of one to a "plateau" value less than one. This model-free approach utilizes the extent of internal motions, rather than the exact detail of their type, to determine the plateau value obtained. The slower process of overall tumbling in solution subsequently effects a further loss in orientational correlation. The validity of this approach requires a much faster timescale for any internal motions than for overall reorientation. Some carbohydrate studies (Catoire et al., 1997) suggest that this is not always the case, in particular for changes in glycosidic linkage conformation across energy barriers between minima; small-amplitude changes in the same energy well, however, are predicted to be rapid (Rutherford et al., 1993
).
NMR spin relaxation studies serve as important tools for probing molecular motions because spin relaxation rates, such as 13C T1 and T2 values and 1H-1H cross relaxation rates directly relate to spectral density functions (Harris, 1986). Hadjuk et al. (1993)
have presented a brief and clear summary of the effect of picosecond timescale motions on NMR relaxation parameters in monosaccharides. Computer simulation methods such as molecular dynamics (McCammon and Harvey, 1987
) can help to rationalize experimental data. Unfortunately, current computer systems can simulate only a few nanoseconds, thereby limiting the range of motions that can be studied.
In the field of protein structure determination 1H-1H NOE and coupling constant data have traditionally been used to derive semi-quantitative distance and dihedral angle restraints (Wuthrich, 1986). These restraints are then used in a structure generation procedure that is based on either the distance geometry method (Wagner et al., 1993
) or a molecular dynamics simulation scheme (Brunger and Nilges, 1990
). Typically these structure generation methods seek to find a single structure that is consistent with the restraints. If such a restraint-based single structure generation procedure is applied to carbohydrates, where multiple conformations may exist and contribute to the NMR data, then there is a danger that a non-physically realistic or virtual conformation might be obtained (Cumming and Carver, 1987
). In analyzing GAG conformation and dynamics, the reorientation of a rigid body provides a starting point for the analysis, while rotation about the glycosidic linkage and the known flexibility of iduronate residues provide additional degrees of freedom. When such multiple conformations exist in solution the correct form of analysis depends upon the timescale of the conformer interchange. If changes in iduronate ring conformation or fluctuations in the torsion angles of glycosidic linkages consist of fast oscillations within a given energy well, then experience suggests that a Lipari-Szabo model superimposed on a rigid body description of overall reorientation is a good approximation. Conversely, slow oscillations complicate the analysisparticularly of 1H-1H NOE databy prohibiting a Lipari-Szabo approach. Analysis of NOE data in the presence of multiple solution conformations requires knowledge of the conformers themselves as well as some estimate of the timescale for their inter-conversion. For conformers that interconvert on a timescale much slower than molecular reorientation but much faster than NMR spin relaxation times (these typically range from milliseconds to seconds), the correct method of analysis requires computation of an averaged matrix of 1H-1H spin relaxation rate constants and its use in a multi-spin simulation of the NOE time course. Studies of a tetrasaccharide fragment from the cell wall polysaccharide of Streptococcus mitis J22 (Xu et al., 1996
) and the monosaccharide
-L-IdoA-OMe (Forster and Mulloy, 1994
) describe such an approach.
Analysis of 3JH,H values is simpler; the experimentally observed value is an average over all conformations present. This picture proves reliable unless the motions are sufficiently slow that they are comparable with the inverse of chemical shift differences (i.e., on the millisecond to second timescale) at which point NMR spectral line broadening and/or distinct spectra for each species might be found (Harris, 1986).
The conformational plasticity of -L-iduronic acid
The conversion of ß-D-glucuronate to -L-iduronate in vivo occurs after polymerization of the GAG chain; Hagner-McWhirter et al. (2000)
have described the specificity and mechanism of the C5 epimerase for heparin. This modification of the uronic acid residues, trivial as it may seem, profoundly affects the conformational and dynamic properties of the resulting polymer. All the D-galacto- and D-glucopyranose monosaccharide residues in GAGs are stable in the usual 4C1 chair conformation of their six-membered pyranose rings. As with most other L-hexapyranoses, one might expect L-iduronate to adopt a 1C4 chair as its most stable conformer. In fact, more than one solution conformation is accessible, and the equilibrium between them is readily altered. One can utilize proton NMR spectroscopy to track such ring geometries in some detail. Changes in the ring conformation not only alter the dihedral angles between vicinal hydrogen atoms, but also change the distances between hydrogens so that both spin-spin coupling constant (3JH,H) values and 1H-1H NOEs can effectively monitor ring shape. NMR studies of iduronate as a monosaccharide (Ferro et al., 1990
), or at the nonreducing end of oligosaccharides (Sanderson et al., 1987
; Ferro et al., 1990
), have given 3JH,H values which indicate a mixture of both the 1C4 and 4C1 chairs with an additional twisted conformation usually known as the 2S0 skew-boat (Sanderson et al., 1987
; Ferro et al., 1990
). The diagram in Figure 2 shows a conformational itinerary for the pyranose ring illustrating all the canonical forms: the two chairs, six boats, and six skew boats. Internal iduronate residues in heparin adopt an equilibrium between two of these forms, the 1C4 chair and the 2S0 skew-boat (Ferro et al., 1990
). Figure 3 shows these two forms superimposed so that the C4-O4 and C1-O1 bonds are similar in their orientations. This demonstrates that the two forms may interconvert with little disturbance to glycosidic linkages to neighboring residues in the polysaccharide chain. The balance of the chair to skew-boat equilibrium in internal iduronate residues depends both upon its own substitution with 2-O-sulfate and on the substitution of adjacent glucosamine residues (van Boeckel et al., 1987
).
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Molecular dynamics simulations of iduronate (as the methyl glycoside and as the central residue of a trisaccharide) indicate that the pyranose ring may explore part of this conformational space on a rapid time-scale; the observed 2S0 skew boat form may represent an ensemble average over several closely-related conformations. Predicted 1H-1H NOE and 3JH,H values for the ensemble average compare closely with those for a static 2S0 ring (Forster and Mulloy, 1994). Since the available coupling constant and NOE data could not distinguish between these two cases, it remains unclear whether extensive pseudorotation takes place in the methyl glycoside.
Studies of model compounds such as the neutral monosaccharide idose (Snyder and Serianni, 1986; Hadjuk et al., 1993
), or 1,2,3,4,6-penta-O-acetyl-
-D-idopyranose (Horita et al., 1997
), have shown that the interconversion rates between chair and skew-boat are slower than the rate of overall molecular reorientation.
Heparin
The NMR study of Gatti et al. (1979) indicated that the overall solution conformation of fully-sulfated, iduronate-containing regions of heparin might well resemble the earlier solid state structure of Atkins and Nieduszynski (1975)
. A later study confirmed this resemblance by analysis of inter-residue NOE measurements (Mulloy et al., 1993
). This study employed preparations of bovine lung heparin, which contains a high (>90%) proportion of the trisulfated disaccharide (Table I, Figure 4a). Similar studies have utilized a series of modified heparins, prepared from the same material, with systematically altered substitution patterns (Mulloy et al., 1994
). For all these heparin derivatives a distinctive pattern of intense inter-residue NOEs indicates relatively well-defined glycosidic linkages in iduronate containing sequences, as compared with those of other polysaccharides. (Mulloy et al., 1993
, 1994). The NOE data suggest occupation of a single area of low energy in the
,
map (see, for example, Figure 1). Surprisingly, all derivatives, regardless of sulfation pattern, exhibit similar glycosidic bond conformations but differ in the conformational equilibria of their iduronate residues. One heparin derivative in particular, de-N-sulfated, re-N-acetylated heparin, proved useful because internal iduronate residues exist almost entirely in the 1C4 conformation (consistent with the results of van Boeckel et al. (1987) for oligosaccharides in which IdoA2OSO3- is substituted at the 4-position by GlcNAc. Representation of this particular compound by a single molecular model allowed resolution of certain difficulties that had arisen in interpretation of NOE values. NOEs, like other relaxation phenomena, are a function of molecular motion (see Theoretical and experimental approaches to conformation and molecular motion). One cannot correctly predict the measured NOE values (even those within a single residue) assuming isotropic rotation for this system. However, assuming that heparin molecules behave as approximately cylindrical symmetric tops yields predicted NOEs close to experimental values (Forster et al., 1989
). Huckerby and Nieduszynski (1982)
predicted this motional anisotropy on the basis of 13C NMR relaxation parameters.
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Conformations of heparin oligosaccharides
Heterogeneity and polydispersity of heparin polysaccharide can complicate the analysis of NMR data, but the preparation of homogeneous heparin oligosaccharides can overcome this problem. Investigators have performed detailed NMR analyses on two such oligosaccharides, a tetrasaccharide (Mikhailov et al., 1996) and a hexasaccharide (Mikhailov et al., 1997
). One can estimate the overall conformation of the oligosaccharides using the Iterative Relaxation Matrix Approach (IRMA; Boelens et al., 1988
.) to interpret NOE data, followed by refinement using molecular mechanics. This method will, by its very nature, find one single conformation which best fits all the data; subsequent refinement of structures with solvent simulation gives some indication of the extent of internal mobility of the molecule. The glycosidic linkages have similar conformations to their equivalents in the polysaccharide.
An oligosaccharide of particular relevance to the therapeutic importance of heparin is the pentasaccharide at the heart of the naturally-occurring sequence with high affinity for antithrombin (Figure 4c). Ragazzi et al. (1987) have performed a molecular mechanics and proton NMR study of a corresponding synthetic pentasaccharide (Figure 4d) which identified several conformations with favorable energy. A dynamics study based on NMR relaxation measurements (Hricovini and Torri, 1995
) was consistent with anisotropic reorientation of the molecule and rapid internal motions.
Flexibility of heparinsummary
So far heparin has been presented as displaying both unusual mobility (in the iduronate ring) and unusual rigidity (in the glycosidic conformations and hence the overall molecular shape). Is heparin more or less flexible than other polysaccharides? Perhaps the answer is "differently flexible." Heparin and its derivatives appear to reorient as symmetric tops in solution, implying a rod-like shape. The glycosidic linkages in heparin appear relatively stiff, but the iduronate pyranose rings can adopt at least two conformations. Figure 3 shows how this ring form can change with only a modest effect on the geometry of linkages to the two neighboring residues. Flexibility of the iduronate pyranose ring need not alter the shape of the heparin polysaccharide chain. Thus far, the evidence gathered regarding this conformational change indicates that it is not rapid compared with overall molecular reorientation.
Heparan sulfate
Heparin is easy to obtain, easy to work with, and binds strongly to all or most of the proteins whose physiological ligand is cell-surface heparan sulfate (HS). HS has exactly the same component disaccharides as heparin but in different, and very much more variable, proportions (Figure 4a,b). The unsulfated GlcA-GlcNAc sequence is the most common, with shorter IdoA-containing, sulfated S-regions (Lyon and Gallagher, 1998) of two to nine disaccharides separated on average by sixteen to eighteen mixed or N-acetylated disaccharides. Clearly, the complex sequence patterns of HS do not arise at random (Lindahl et al., 1998
). A substantial proportion of the heparan sulfate chain consists of alternating GlcA-GlcNAc, with no sulfate substitution. Given that this sequence has not yet been found to directly influence protein binding, it may function as a spacer between the S-regions.
The most convenient model compound for unsulfated heparan is the capsular polysaccharide of E.coli K5. Hricovini et al. (1997) have collected carbon relaxation times at several fields for a low molecular weight sample of this polysaccharide and find that the data compare neither with a rigid isotropic motional model, nor with an isotropic model with rapid internal motion. Consideration of the molecule as a symmetric top, with additional rapid internal motions, improves the fit of predicted data to the experimental. Thus far there have been no reported studies of the glycosidic linkage geometries and overall conformation of this sequence.
One can consider the disaccharides maltose and cellobiose as models for the K5/heparan GlcNAc-GlcA and GlcA-GlcNAc linkages respectively. Researchers have identified more than one low-energy linkage conformation for both of these linkage types (Stevens and Sathyanarayama, 1989; Dowd et al., 1992a
,b). This would allow the S-regions of heparan sulfate to explore a greater variety of orientations with respect to the attached proteoglycan core than a rigid chain, and would allow two or more S-regions in a single HS chain more freedom to interact with different proteins. The interaction between heparan sulfate and the multimeric form of platelet factor 4 (PF4) is interesting in this regard. Stringer and Gallagher (1997)
have described a heparan sulfate fraction with high affinity for PF4, in which two S-regions are separated by a long unsulfated stretch. The length of this stretch of polysaccharide chain is sufficient to present S-regions to binding sites on opposite sides of the PF4 tetramer.
Conformations of heparin oligosaccharides bound to proteins
A large number of heparin-binding proteins has already been identified (Conrad, 1998) and the number continues to increase. Unfortunately, x-ray crystallographic studies exist for only a few heparin-protein complexes: heparin or heparin oligomers complexed with basic fibroblast growth factor (b-fgf; PDB codes 1BFB and 1BFC; Faham et al., 1996
), with acidic fibroblast growth factor (a-fgf; 1AXM and 2AXM; DiGabriele et al., 1998
), and with foot and mouth disease virus (1QQP; Fry et al., 1999
). An additional study features a synthetic heparin-like pentasaccharide (Figure 4e) complexed with both the active and latent members of an antithrombin dimer (1AZX; Jin et al., 1997
).
The heparin oligosaccharides bound to growth factors are all derived from multiples of the main trisulfated disaccharide repeat unit (Figure 4a). Faham et al. (1996) studied both a tetrasaccharide and hexasaccharide complexed with basic fibroblast growth factor. The conformation of the two iduronate rings in the hexasaccharide complex (1BFC; Faham et al., 1996
) is interesting; one adopts the 1C4 chair, and the other the 2S0 skew boat (see Figure 3). Lam et al. (1998)
have used this hexasaccharide/protein complex in a molecular modeling exercise to predict the structure of the multicomponent complex between b-fgf, heparin, and the high-affinity growth factor receptor.
In order to prepare the a-fgf/heparin complex, (1AXM, 2AXM) this study employs a decasaccharide (of which only 5 or 6 monosaccharide units are well-ordered in the crystal structure) sandwiched between two protein molecules. This 2:1 complex illustrates very well the double-sided nature of the heparin molecule, capable of almost identical interactions with two copies of a protein facing each other on either side of the heparin chain.
Fry et al. (1999) have determined the crystal structure of foot and mouth virus complexed with polydisperse heparin, as the large dimensions of the crystal can accommodate longer heparin chains than the small fibroblast growth factor proteins. Only five monosaccharide units give sufficiently well-defined electron density to yield coordinates in the PDB file (1QQP), with the outer two residues less well defined than the central three. Electron density for the central iduronate residue fits best to a mixture of 1C4 and 2S0 forms. One of the outer iduronates is in the 1C4 chair, while the other appears to adopt a 2,5B conformation (Figure 2). This is the first time experimental evidence for a boat form of iduronate has been reported.
The interaction between heparin and antithrombin differs from all other heparin-protein interactions described thus far. Interestingly, heparin, synthesized in mast cells and sequestered in their granules (Humphries et al., 1999; Forsberg et al., 1999
) contains a specific and unusual monosaccharide sequence with a high affinity for the plasma serine protease inhibitor antithrombin. The details of this sequence were first elucidated by Rosenberg and Lam (1979)
and by Lindahl et al. (1980)
. Heparin containing this sequence binds to antithrombin and causes a conformational change making it a much more efficient inhibitor of its target serine proteases. Choay et al. (1983)
have confirmed the structure of this sequence (Figure 4c) by synthesis of its central pentasaccharide (Figure 4d), thus enabling studies of its conformation and dynamics in solution as described above (Ragazzi et al., 1987
; Hricovini and Torri, 1995
). Other studies investigate antithrombin-tetrasaccharide complexes in the solution state (Figure 4f) (Hricovini et al., 1999
) and antithrombin-pentasaccharide complexes in the solid state (Figure 4e) (Jin et al., 1997
).
One can compare this crystal structure of an antithrombin/pentasaccharide complex (Jin et al., 1997) with an earlier molecular model prepared by homology modeling and docking calculations informed by biochemical data (Grootenhuis and van Boeckel, 1991
). The solid state study (Jin et al., 1997
) shows the pentasaccharide occupying the expected binding site, near the predicted orientation, but somewhat displaced; the heparin binding site of antithrombin can accommodate longer oligosaccharide sequences such as a recently studied heptasaccharide (Figure 4g) (Belzar et al., 2000
). The bound pentasaccharide has a conformation roughly similar to one of the conformations (designated "S1112") predicted for the pentasaccharide in Figure 4d (Ragazzi et al., 1987
); the iduronate residue adopts a conformation between the 2S0 skew-boat and 2,5B forms.
A substantial and complex NMR study of a tetrasaccharide (the methyl glycoside of the sequence AGA*I; Figure 4f) in solution and in the presence of antithrombin (Hricovini et al., 1999) provides a direct comparison of free and bound oligosaccharide. This study describes a distinct change in conformation at the AG glycosidic linkage on binding, and a stabilization of the 1C4 chair of the iduronate residue. Neither of these conformational features is seen in the pentasaccharide/antithrombin x-ray structure described above; however, the substantial differences in oligosaccharide structure may result in different binding modes within the long heparin binding site of antithrombin.
Figure 6 shows plots of the glycosidic dihedral angles of heparin oligosaccharides bound to fibroblast growth factors and to foot and mouth virus, together with values from NMR studies of heparin and of a heparin oligosaccharide. The similar glycosidic bond geometries of all these structures corroborates the idea that heparin has a well-defined conformation both in solution and in the solid state. The presence of skew-boat (1BFC), boat (1QQP), and intermediate (1AZX) ring forms of iduronate in crystals indicates the accessibility of these forms and lends weight to the idea that such conformations may exist in solution.
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Summary and conclusions
Heparin structures have now been reported in the solution and solid states as well as in complexes with a number of proteins. Analysis of these data indicates that the conformation of heparin is well-defined with similar glycosidic torsional angles found in almost all cases. Within this overall conformation, the pyranose rings of iduronate residues have significant flexibility. In solution they exist in a dynamic equilibrium, and when complexed with proteins can adopt several different forms. Studies of solution structure for heparin and its derivatives must take account of internal motion, and allow for averaging over several conformational states of NMR measurements such as NOEs. The relative timescales of internal and overall molecular motions must also be borne in mind. Variations in sulfate substitution affect the conformational equilibrium of the iduronate ring more than the overall shape of the heparin molecule.
There is, so far, less information available for the glucuronate-containing regions of heparan sulfate and heparin; glucuronate residues lack the flexibility of iduronate.
Molecular modeling exercises for heparin and heparan/protein complexes require care in the choice of strategy and parametrization of any force-field used. However, the well-defined shape of heparin molecules increases the value of predictive molecular modeling exercises.
Abbreviations
GAG, glycosaminoglycan; NOE, Nuclear Overhauser effect; a-fgf, acidic fibroblast growth factor FGF-I; b-fgf, basic fibroblast growth factor FGF-II
Footnotes
1 To whom correspondence should be addressed
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