2 Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1, 3QU, UK; 3 Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 63104; and 4 MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1, 3QU, UK
Received on May 27, 2004; accepted on June 17, 2004
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Abstract |
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Key words: end-effect/15N isotope incorporation / hyaluronan / NMR / triple-resonance
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Introduction |
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Relatively few oligo- or polysaccharide structures have been determined by crystallographic means, and there is no clear extrapolation from structures determined in a highly ordered and symmetrical crystal to that in solution, where a variety of rapidly interconverting conformations are expected. To determine these more dynamic structures, a solution technique such as nuclear magnetic resonance (NMR) is needed. Complete assignment of 13C nuclei in the hexamer (HA6) (Cowman et al., 1996) and 1H nuclei in the tetramer (HA4) (Livant et al., 1992
; Toffanin et al., 1993
) have been performed, although not without some discrepancies. Additional unique carbon nuclei assignments have been made for the ends of octasaccharides (Cowman et al., 1996
; Holmbeck et al., 1994
), and the dynamic motion at various positions along the chain assessed, demonstrating that end-effects are significant in HA6 (Cowman et al., 2001
). In combination with molecular dynamics simulations, a solution conformation based on average nuclear Overhauser effect (NOE) intensities at the glycosidic linkages has been calculated for the octamer (Holmbeck et al., 1994
). Scott and colleagues have also investigated higher-order structures of medium-weight and polymeric HA in solution using 13C natural-abundance NMR and have inferred tertiary structures (with intermolecular interactions) from chemical shift perturbations to the side chain carbonyl resonance (Scott and Heatley, 1999
, 2002
). Particular attention has been given to the presence in solution of the HN-COO hydrogen bond that these tertiary models require, which has been reported in dimethyl sulfoxide (Scott et al., 1981
) but refuted by studies in water (Cowman et al., 1984
; Sicinska et al., 1993
). In spite of this work, there is little sequence-specific information on the conformation of HA in aqueous solution. In consequence, averaged models have been produced (Holmbeck et al., 1994
), or some conformations have been singled out as a result of the particular modeling technique employed (Almond et al., 1998; Mikelsaar and Scott, 1994
), which may account for much of the disagreement. In addition, it is not trivial to relate properties observed in oligomers, which are dominated by end-effects, to those seen in the polymer.
The monosaccharide building blocks of complex carbohydrates are all very much alike, often differing only at stereocenters, and this produces severe resonance overlap, making acquisition of sequence-specific data extremely challenging (Homans, 1998). In sugars made up of repeated units, such as HA, this problem is further compounded, and it therefore becomes very difficult to collect data that can be used to perform structural analyses or test dynamic models (Almond et al., 2000
). The principal way to reduce the spectral overlap is to employ isotope enrichment of heteronuclei (13C, 15N) and use them to resolve resonances by correlating them in two or more dimensions, as has been employed in proteins to great success. However, there are no well-established methods for expression of labeled carbohydrates.
We have therefore developed a method for producing 15N- and 13C-isotopically-enriched HA oligosaccharides of defined lengths, which greatly enhances the scope of NMR in investigating the solution conformation of HA. These materials permit new experiments to be performed, allowing correlation of otherwise overlapping nuclei to those that are resolved. Of particular significance is the greater sensitivity of 15N nuclei to its environment in this system compared to 13C or 1H nuclei, giving unprecedented resolution of otherwise highly degenerate chemical shifts. In this work, we demonstrate that employment of 15N-labeling in this manner allows sequence-specific assignments to be made even in the middle of a HA decamer, and conclude that end-effects can be propagated through as many as three consecutive linkages, seen throughout the length of an octasaccharide. We also present data using 3D and triple-resonance techniques to measure chemical shifts in specific rings in a hexasaccharide, which provide information on changes in the local chemical environment along the length of the oligomer and are suggestive of subtle differences in average conformation. In spite of the common occurrence of nitrogen in sugar residues, the potential of the 15N nucleus has hitherto remained untapped in carbohydrate NMR. This work clearly shows its scope for combating chemical shift degeneracy in carbohydrates and may allow new methods in elucidating their structures to be developed.
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Results |
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The amide chemical shifts and assignments for each length of oligosaccharide are given in Table II. It is clear from these data and Figure 5 that as the oligomer length increases, the 15N chemical shift in the middle of an oligomer progressively decreases toward a minimum value (121.94 ppm). This value is only observed in oligosaccharides as long as or longer than HA10. There also appear to be more subtle trends in chemical shift at a given position (as seen for instance in the progression in 15N/1H values at the
ring across the series), but they are too small to be measured precisely from these experiments.
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Discussion |
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The chemical shift difference seen between HN and
HN of HA6/HA8/HA10, due to end-effects from the nonreducing terminal ring, is a displacement of
150 ppb in the 15N dimension (see Figure 5B). The small difference observed between
HN and
HN (
10 ppb 15N) also appears to arise from the nonreducing terminus end-effect (considering HA10 in Figure 6), but is an order of magnitude less, as might be expected because it is three rings away from the terminus (unlike the
HN group, which is adjacent to the terminal ring). The chemical shift difference observed at
HN, which is two rings away from the reducing terminus, is between these two values (
50 ppb 15N,
10 ppb 1H), suggesting that the magnitude of the perturbation in the 15N dimension is related to the distance from any end of an oligomer (a specific effect on the amide proton from the reducing terminus is apparently also felt). Furthermore, the chemical shift of the HA4
HN position, which at first sight seems to be anomalous, can now be seen to be the typical
HN HA6/HA8/HA10 position expected for a nonreducing terminal amide but with an additional perturbation from the reducing terminal end-effect (i.e., another
50 ppb 15N,
10 ppb 1H). There is no evidence that the
N peaks are split into distinct populations from the
- and ß-anomers, indicating that their different local chemical structure does not propagate significant chemical shift perturbations any further than the adjacent GlcA ring. In this light, perturbations from end-effects to the 15N chemical shift, which appear to be experienced at least as far as three rings, probably arise from long-range differential electrostatic fields (such as a variation in the pKa of the COO groups) and/or dynamic effects subtly affecting conformer distributions; their largely additive and distance-dependent nature further imply that in the case of the 15N nuclei, there is one dominant and simple factor present.
Because end-effects are clearly observed at both HN and
HN groups in HA6, it seems most likely that the variations in GlcNAc ring 1H and 13C chemical shifts arise from the same phenomena. The difference in H2 and C2 chemical shifts, for example, may report a slight change in the average orientation of the N-acetyl side chains with respect to their rings, which could not be detected from the 3JHNH2 coupling under the current resolution of the 1H,15N-HSQC spectra. Of particular interest is the comparison of the GlcNAc H4 chemical shifts, because the OH group on C4 is believed to contribute to the glycosidic conformation by making a hydrogen bond to the preceeding GlcA ring (see Almond et al., 1998b
). In this context, the difference observed at the H4 proton between
and
rings may reflect a different distribution of conformations of the nonreducing terminal ß(1
3) linkage relative to those seen in the middle of an oligomer, as predicted by molecular dynamics simulations (Almond et al., 1997
, 1998b
, 2000
).
Chain mobility at specific positions within HA oligosaccharides has been investigated by Cowman et al. (2001), using 13C NMR T1-relaxation measurements. In this study, the nonreducing terminus was found to be most mobile, followed by the reducing terminus, the penultimate residues, and finally the interior residues. These data are in good agreement with molecular dynamics simulations of hyaluronan tetra- and decasaccharides, which predicted the same relative mobilities within the chain due to the terminal rings being less constrained by having fewer intramolecular hydrogen bonds (Almond et al., 1998b
, 2000
). Significantly, this order of mobility corresponds well with the trend seen in this study in the 15N chemical shift; i.e. greater mobility correlates with greater chemical shift perturbations from the minimum value. Although the modeling studies indicate that the end-effects in HA oligomers primarily arise from differences in hydrogen bond networks, as the H4 chemical shift may reflect, the 15N chemical shift trend is unlikely to directly reflect a progressive change in the strength of the NH-COO hydrogen bond toward the middle of an oligomer, because there is very little change in the amide proton chemical shift along the chain.
In contrast to the 13C T1 relaxation measurements, which found that interior residues of HA6 have almost the same T1 as the polymer, these studies using 15N suggest that HA10 is the shortest length of oligosaccharide whose interior accurately reflects the properties of the polymer. In this light, extension of properties measured on the tetramer (which is manifestly dominated by end-effects) to the polymer may be problematic (Mikelsaar and Scott, 1994) and may account for some of the apparently contradictory data in the literature. The precise physical basis for the greater environmental sensitivity of 15N over 13C and 1H nuclei seen in HA is not clear at present, but is likely to be related to its different magnetic susceptibility and/or polarizability. This greater sensitivity is also seen in the clear difference in 15N chemical shifts in HA6 at the
and
rings, whereas there was no measurable variability in the adjacent carbonyl carbon nuclei (C7).
Previously, total assignment of all nuclei within HA oligosaccharides has only been achieved for HA4 (1H: Toffanin et al., 1993) and HA6 (13C: Cowman et al., 1996
) and not without some remaining disagreement (Livant et al., 1992
; Toffanin et al., 1993
). Employment of 15N- and 13C-isotopic labeling in HA oligomers indicates that it should now be possible, with appropriate 3D and triple-resonance experiments, to specifically assign and measure properties of ring 1H and 13C nuclei even in the interior of oligomers as long as HA10, a length clearly more relevant to understanding the polymer and proteinHA interactions. In addition, these samples will now allow new experiments to be performed to investigate the proposed interresidual hydrogen bonds on which various models of the solution conformation of HA rely.
The influence of end-effects in HA oligosaccharides on their structure and dynamics suggested by this study may provide a basis for understanding how oligosaccharides can display different biological properties from medium- and high-molecular-weight polymeric HA (Fieber et al., 2004; Ghatak et al., 2002
; Sugahara et al., 2003
; Termeer et al., 2000
). For example, not only do the free ends provide distinctive chemical environments to interact with, but their more dynamical nature may require different dynamics or tighter interaction networks to overcome a greater entropic penalty. In this regard, studies on the Link module of human TSG-6 have shown that HA8 is the minimum length of oligosaccharide that binds with maximal affinity (Kahmann et al., 2000
), although the structure indicates that the binding site could only accommodate five or six rings (Blundell et al., 2003
); it seems probable that the difference is due to end-effects. Isotopically-enriched HA oligosaccharides will clearly be of great use in ligand-binding studies and complex structure-determination by NMR, as we have already begun to demonstrate (Blundell et al., 2003
).
To the best of our knowledge, 15N-labeling in oligosaccharides has barely been studied before, and no 15N triple-resonance experiments have been performed. In this study, we found a greater sensitivity to its environment of 15N over 13C or 1H nuclei, giving rise to unprecedented chemical shift dispersion. By using 15N as a specific filter in correlated experiments, other nuclei can be resolved that are otherwise totally eclipsed, providing valuable sequence-specific information. Many other complex carbohydrates, such as the glycosaminoglycans heparin and chondroitin, the lactosaminoglycans, and many bacterial capsular polysaccharides, contain nitrogen, and employment of 15N-NMR techniques to these sugars may produce similar advances in our understanding of their structure and dynamics as has occurred in proteins.
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Materials and methods |
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Uniformly 15N- and 15N, 13C-labeled high Mr HA was produced by Escherichia coli K5 transfected with recombinant HA synthase from Pasturella multocida (Blundell et al., 2003; DeAngelis et al., 1998
), fermented at 30°C for 3 days in a modified M9 minimal medium supplemented with 15NH4Cl (0.55 g/L) and 13C6-D-glucose (1.6 g/L) as appropriate (both > 99 atom %, Spectra Stable Isotopes, Columbia, MD). Shed polymeric material in the supernatant was purified and quantified as described previously (DeAngelis et al., 2002
). This isotopically-labeled material was digested in the same manner as the unlabeled material.
Purification of HA oligosaccharides
Differing lengths of HA oligosaccharide were separated from the digest supernatant using anion exchange high-performance liquid chromatography on an AKTA system running UNICORN software. Digest supernatant was loaded onto a Sepharose-Q HR column (Amersham Biosciences, Little Chalfont, United Kingdom) equilibrated in MilliQ H2O at flow rate of 8 ml/min, and eluted with a gradient of 0 mM100 mM NaCl over 90 min. The column was regenerated by washing with 0.5 M NaCl and equilibrated with water. The eluent was monitored at 214 nm, and fractions from different runs were pooled according to their elution positions. Following lyophilization, each oligosaccharide was desalted using a Bio-Gel P2 column (BioRad) running in 50 mM HN4Ac, and then repeatedly lyophilized to remove residual HN4Ac. Oligosaccharide preparations were analyzed on a Mono-Q HR5/5 column (Amersham Biosciences), equilibrated in MilliQ H2O and eluting with a gradient of 0 mM100 mM NaCl over 40 min at a flow rate of 1 ml/min.
Fluorescent polyacrylamide gel electrophoresis
Purified oligosaccharides and the sample from the digestion reaction at 120 min were labeled with anthranilic acid according to the method of Anumula (1994). Excess free fluorophore was removed using a G-10 desalting column (Amersham Biosciences), running in 50 mM NH4Ac, and the tagged oligosaccharides were lyophilized. Following reconstitution in 10% (v/v) glycerol,
equimolar amounts of each oligosaccharide (
1 µg) and the digest sample were run at 4°C on a 27% native polyacrylamide gel (without stacking gel) in 192 mM glycine, 25 mM Tris, pH 8.4, running buffer. The gel was visualized using a UV transilluminator (Uvi Tec) fitted with a 430-nm bandpass blue filter (Andover, Salem, NH) over the camera, recording the negative image.
MALDI-TOF
Purified unlabeled and isotope-enriched HA oligosaccharides were analysed using an Ettan MALDI-ToF Pro version 2.0 mass spectrometer (Amersham Biosciences). Oligosaccharide sample solutions were prepared at 10 µg/ml in 50% (v/v) acetonitrile (ACN), 1% (w/v) trifluroacetic acid (TFA). Solutions for external calibration were composed of a mixture of 5 pmol/µl adrenocorticotrophic hormone fragment 1839 ([M-H] 2463.20 Da) and angiotensin II ([M-H] 1044.54 Da), also in 50% (v/v) ACN, 1% (w/v) TFA. Sample and calibrant solutions were mixed 1:1 with 2.5 mg/ml 2,5-dihydroxybenzoic acid (50% ACN [v/v], 1% TFA [w/v]) and spotted onto the running slides. Mass spectrometry was performed in the negative ion reflectron mode with a 20 kV accelerating potential.
Determination of oligosaccharide amounts
Accurate quantification of the amount of oligosaccharide in different samples was achieved by three independent methods. First, because the preparations had been shown to be >95% pure and very little water was present in a lyophilized pellet, measuring the dry mass with a sensitive balance was possible. Second, a small portion of unlabeled HA was allowed to continue enzymatic digestion for 24 h, resulting in the complete breakdown of the material to HA4, HA6, and a small amount of HA8. Known quantities of this exhaustive digest were run on the analytical FPLC column and, because the initial mass:volume ratio of polymeric HA before digestion was precisely known (10 mg/ml), the peaks represented the same ratio of HA in oligosaccharides (i.e., 10 mg/ml). By integrating the absorbance of the peaks, it was therefore possible to calculate the mean extinction coefficient for HA oligosaccharides at 214 nm as 600 ± 30 mAU per mg (irrespective of length); stock solutions made from dried material of measured mass and analyzed on the FPLC column were found to agree with this value within experimental error. Finally, the concentration of an oligosaccharide in an NMR tube was determined to ±10% by comparing the intensity of signal from the H8 methyl group at 2.0 ppm with that from a standard curve of GlcNAc samples of known concentration.
NMR spectroscopy
All samples were prepared from lyophilized oligosaccharides reconstituted in 10% (v/v) D2O, 0.02% (w/v) NaN3 and adjusted to pH 6.0 with 0.1 M HCl and NaOH. Samples of unlabeled HA4, HA6, HA8, and HA10 were prepared (5.0 ± 0.1 mM), each containing 0.3 mM 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) as an internal reference. Isotopically labeled samples were prepared in Shighemi tubes, giving concentrations of 11.8 ± 0.2 mM (15N-HA4), 10.8 ± 0.15 mM (15N-HA6), 4.3 ± 0.1 mM (15N-HA8), 2.3 ± 0.1 mM (15N-HA10), 0.1 mM (15N-HA12), and 7.2 ± 0.15 mM (15N-, 13C-HA6).
All spectra were recorded at 25°C and a proton frequency of 750 MHz unless otherwise stated. Homonuclear 1D spectra were recorded on the 5 mM unlabeled samples with an acquisition time of 113.66 ms (1024 complex points; all following numbers of points are complex). The 2D-TOCSY (mixing time 53.2 ms) spectrum was collected on the 5 mM HA6 sample, with acquisition times of 73.22 ms (t1, 512 points) and 113.66 ms (t2, 1024 points). Gradient-enhanced 1H,15N-HSQC data sets were recorded with acquisition times of 1088.00 ms (t1, 15N, 128 points) and 113.66 ms (t2, 1H, 1024 points) with the 15N carrier frequency set to 122.5 ppm.
Triple-resonance and 3D data sets were recorded with the 15N carrier frequency set to 122.0 ppm and with deliberate folding of the resonance in the 15N dimension to increase resolution. The 1H,15N-TOCSY-HSQC (600 MHz) and 1H,15N-NOESY-HSQC (600 ms mixing time) spectra were recorded with acquisition times of 21.31 ms (128 points) and 8.53 ms (64 points) (t1, 1H), 512.00 ms (16 points) and 819.20 ms (32 points) (t2, 15N) and 70.91 ms (512 points) and 113.66 ms (1024 points) (t3, 1H), respectively. Triple-resonance experiments were recorded with gradient enhancement. Normally, a constant time period is included to decouple the 15N and 13C nuclei, but in these experiments it was removed to allow for the long acquisition times associated with small molecules (resulting in the triplets seen in Figure 8). The HNCA spectrum (600 MHz) was collected with the 13C carrier frequency set to 45 ppm and with acquisition times of 31.78 ms (96 points) (t1, 13C), 743.62 ms (32 points) (t2, 15N), and 70.91 ms (512 points) (t3, 1H); this resulted in both C2 and C8 resonances being folded. The HNCO spectrum had acquisition times of 84.74 ms (64 points) (t1, 13C), 594.88 ms (32 points) (t2, 15N), and 56.83 ms (512 points) (t3, 1H) and the 13C carrier frequency set to 177.5 ppm.
Spectra were processed using NMRPipe (Delaglio et al., 1995) and analyzed with Sparky (Goddard and Kneller). Proton chemical shifts were referenced relative to internal DSS, and the hetereonuclei by indirect referencing (Wishart et al., 1995
).
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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